PhDThesis Moraru Liviu

UNIVERSITY OF PATRAS
SCHOOL OF MEDICINE
DEPARTMENT OF MEDICAL PHYSICS

NATIONAL TECHNICAL UNIVERSITY OF ATHENS
DEPARTMENT OF ELECTRICAL AND COMPUTER
ENGINEERING

NATIONAL TECHNICAL UNIVERSITY OF ATHENS
DEPARTMENT OF MECHANICAL ENGINEERING

INFORMATION DOMAIN ANALYSIS OF
PHYSIOLOGICAL SIGNALS:
APPLICATIONS ON THE CARDIAC AND
NEURAL SYSTEMS OF RATS AND
MONKEYS

PhD Thesis

Liviu Moraru

Interdepartmental Program of Postgraduate Studies in
BIOMEDICAL ENGINEERING

UNIVERSITY OF PATRAS, GREECE
J ULY, 2006

THREE-MEMBER ADVISORY COMMITTEE
Professor Anastassios Bezerianos Main Supervisor
Professor Nicolas Pallikarakis Member of the Advisory Committee
Professor Dimitris Koutsouris Member of the Advisory Committee

SEVEN-MEMBER EXAMINATION COMMITTEE
Professor Anastassios Bezerianos Main Supervisor
Professor Nicolas Pallikarakis Member of the Advisory Committee
Professor Dimitris Koutsouris Member of the Advisory Committee
Professor George Kostopoulos Member of the Examination Committee
Professor Anastassios Bountis Member of the Examination Committee
Professor Spiros Fotopoulos Member of the Examination Committee
Associate Professor . Hiladakis Member of the Examination Committee

Acknowledgements
I would like to express my gratitude to Professor Anastasios Bezerianos for the
opportunity to do this work and for his supervision and advice. He showed me
different ways to approach a research problem and the need to be persistent to
accomplish any goal. Without his continuous support, this work would have never
been possible.
I am grateful to Professor Nicolas Pallikarakis and Professor Dimitris Koutsouris
for their participation in the advisory committee. A special thanks goes to Professor
Nicolas Pallikarakis for the support offered during my time spent with the
European Postgraduate Program in Biomedical Engineering.
Many thanks go to all my friends and colleagues, especially to Laura Cimponeriu
for the inspirational discussions and for her support in the early stages of my
research.
During a six months research fellowship I had the great chance to meet Professors
Phillipe Lefevre and Marcus Missal from Catholic University of Louvain La Neuve
(UCL). I wish to thank them for all the help provided during my stay in Belgium.
I express particular gratitude to Professor Rodica Strungaru, from my home
Politehnica University of Bucharest Romania, who was the initiator of my
postgraduate studies.
I would like to acknowledge the financial support offered by the State Scholarships
Foundation (IKY) (20002004) and Marie Curie Fellowship at Control Training
Site UCL Belgium (February 2005 J uly 2005).

Table of Contents

LIST OF FIGURES
ACKNOWLEDGMENTS
ABSTRACT
CHAPTER 1: INTRODUCTION...........................................................................1
1.1 MOTIVATION AND BACKGROUND .....................................................................1
1.1.1 General Overview.....................................................................................1
1.1.2 Information Theory and Nonlinear Dynamics in Analyzing Cardiac and
Neural Signals....................................................................................................2
1.2 ORGANIZATION OF THE THESIS.........................................................................4
1.3 CONTRIBUTIONS AND PUBLICATIONS................................................................6

CHAPTER 2: PHYSIOLOGICAL BACKGROUND..........................................9
2.1 CARDIOVASCULAR SYSTEM..............................................................................9
2.1.1 Cardiac Control .........................................................................................9
2.1.2 The Neural Regulation of the Cardiovascular System............................12
2.1.3 Heart Rate Variability.............................................................................15
2.1.3.1 Physiological Mechanisms of Heart Rate Variability......................15
2.1.3.2 Clinical Implications and the Use of Heart Rate Variability...........18
2.2 THE CENTRAL NERVOUS SYSTEM (CNS)........................................................19
2.2.1 Organization of the CNS.........................................................................20
2.2.2 Brain Electrical Activity.........................................................................22
2.2.3 Neural Oscillations..................................................................................25
2.2.4 Neural Responses During Visual Pursuit................................................26
2.3 BRAIN INJ URY EFFECTS ON PHYSIOLOGICAL SIGNALS....................................27
2.3.1 Cardiac Responses to Hypoxia...............................................................28
2.3.2 Neural Responses During Hypoxia.........................................................29
2.3.3 Ischemic Preconditioning Effects...........................................................30

CHAPTER 3: ANALYSIS OF PHYSIOLOGICAL TIME SERIES................33
3.1 ASSESSMENT OF HEART RATE VARIABILITY...................................................33
3.1.1 Linear Methods of Assessing Heart Rate Variability.............................33
3.1.1.1 Time Domain Analysis of Heart Rate Variability...........................33
3.1.1.2 Frequency Domain Measures of Heart Rate Variability..................35
3.1.2 Insight Into the Nonlinear Dynamics of HRV........................................38
3.1.2.1 Information Theoretical Methods....................................................40
3.1.2.2 Geometrical Methods of Heart Rate Variability Analysis...............43
3.1.2.3 Other Approaches............................................................................45
3.2 ANALYSIS OF BRAIN ELECTRICAL ACTIVITY ..................................................46
3.2.1 General Approaches................................................................................46
3.2.2 New Approaches of Brain Oscillations...................................................48
3.2.2.1 Empirical Mode Decomposition Technique....................................48
3.2.2.2 Phase Synchronization Analysis......................................................51
3.2.3 Analysis of Anticipatory Pursuit in Behaving Monkey..........................52

CHAPTER 4: ANALYSIS OF ELECTROPHYSIOLOGICAL SIGNALS IN
RESPONSE TO BRAIN INJURY........................................................................55
4.1 THE EFFECTS OF ISCHEMIC PRECONDITIONING ON THE HRV RESPONSE TO
TRANSIENT GLOBAL ISCHEMIA.............................................................................55
4.1.1 Introduction.............................................................................................55
4.1.2 Experimental Protocol ............................................................................58
4.1.3 Data Analysis..........................................................................................59
4.1.3.1 Data Acquisition and Preprocessing................................................59
4.1.3.2 Linear and Nonlinear Methods of Analysis.....................................61
4.1.3.3 Statistical Analysis...........................................................................61
4.1.4 Results and Discussions..........................................................................61
4.1.5 Conclusion..............................................................................................69
4.2 A NEW APPROACH TO SYNCHRONIZATION ANALYSIS OF BRAIN ELECTRICAL
SIGNALS................................................................................................................69
4.2.1 Introduction.............................................................................................69
4.2.2 Coupled Oscillators Approach to Synchronization Analysis..................70
4.2.3 Experimental Protocol ............................................................................73
4.2.4 Results and Discussions..........................................................................74
4.2.5 Conclusions.............................................................................................76

CHAPTER 5: ANALYSIS OF NEURAL ACTIVITY IN SUPPLEMENTARY
EYE FIELDS DURING ANTICIPATORY EYE MOVEMENTS ...................77
5.1 INTRODUCTION................................................................................................77
5.2 MATERIALS AND METHODS ............................................................................79
5.3 RESULTS AND DISCUSSIONS............................................................................82
5.4 CONCLUSION...................................................................................................86

CHAPTER 6: CONCLUSIONS AND FURTHER WORK...............................87

BIBLIOGRAPHY .91

List of Figures

Fig. 2.1 The heart. The autorhythmic cells controlling the pace of cardiac
contractions are located in the SA node. Signals generated by the SA node
travel to the AV node and down the AV bundle and
branches......10
Fig. 2.2 Functional Organisation of the ANS .14
Fig. 2.3 Schematic representation of opposing feedback mechanisms which,
in addition to central integration, subserve neural control of the
cardiovascular system. Baroreceptive and vagal afferent fibers from the
cardiopulmonary region mediate negative feedback mechanisms (exciting the
vagal outflow and inhibiting the sympathetic outflow), whereas positive
feedback mechanisms are mediated by sympathetic afferent fibers (exciting
the sympathetic outflow and inhibiting the vagal outflow) (from Malliani et
al. 1998) .....15
Fig. 2.4 One second EEG recordings and its components ...23
Fig. 3.1 Schematic representation of normal ECG trace, with waves, and RR
intervals labeled ...34
Fig. 3.2 Example of power spectral quantification of representative baseline
RRI signal in rats. The LF (0.195-0.6 Hz) and HF (0.6-2.5 Hz) bands are
defined. The peak centered around 1 Hz reflects respiratory sinus arrhythmia
(RSA)...37
Fig. 3.3 Poincare plot of the representative baseline RRI signal in rats. The
parameters SD1 and SD2 are defined as standard deviation of the distance of
each RR(i) from the lines y=x and y=-x+2*RRI
m
where RRI
m
is the mean of
all RRIs.......44
Fig. 3.4 EMD of two component signal, (a) sum of two components, (b) lower
and upper envelopes and their mean, (c) the first IMF and (d) the first
residual (after Oonincx and Hermand, 2004).50
Fig. 3.5 Cross-correlation method; example of a cross-correlation function
computed between the velocity of the eye and the spike density
waveform..53

Fig. 4.1 Experimental design protocol of asphyxia-cardiac arrest. Continuous
digital ECG is taken from each phase of the experiment. Two group of rats
were studied; preconditioned rats (n=5) and non-preconditioned rats (n=5).
For the non-preconditioned rats an extended baseline was conducted in place
of the one hour of preconditioning and recovery prior to asphyxia...59
Fig. 4.2 Mean RRI during baseline, 30 min after ROSC (1), 60 min after ROSC
(2), 90 min after ROSC (3), 120 min after ROSC (4) and 150 min after
ROSC (5)62
Fig. 4.3 Standard deviation (SD) of the beat-to-beat RRI signals during baseline,
30 min after ROSC (1), 60 min after ROSC (2), 90 min after ROSC (3), 120
min after ROSC (4) and 150 min after ROSC (5).62
Fig. 4.4 LF (a), HF (b), LF/HF (c) indices quantified from the power spectrum
estimation of RRI signals during baseline, 30 min after ROSC (1), 60 min
after ROSC (2), 90 min after ROSC (3), 120 min after ROSC (4) and 150
min after ROSC (5). P<0.05 compared with baseline within each group; *
P<0.05 compared to control group.63
Fig. 4.5 Approximate Entropy (ApEn) of RRI signals during baseline, 30 min
after ROSC (1), 60 min after ROSC (2), 90 min after ROSC (3), 120 min
after ROSC (4) and 150 min after ROSC (5).64
Fig. 4.6 SD1 (a), SD2 (b), SD1/SD2 (c) indices quantified from Poincare plots
of RRI signals during baseline, 30 min after ROSC (1), 60 min after ROSC
(2), 90 min after ROSC (3), 120 min after ROSC (4) and 150 min after
ROSC (5)65
Fig. 4.7 Difference in Neurodeficit Score between non-preconditioned and
preconditioned rats at 24 hours (n=5 each). * P<0.05 compared to control
group..66
Fig. 4.8 Synchronization index versus coupling strength. Computations are
performed considering the natural phase variables (dashed line) and the
phases corresponding to the first intrinsic modes (solid line)71
Fig. 4.9 Illustration of the EMD method applied to the time series of x variable
of the response system in the unidirectionally coupled configuration of two
Lorenz systems...72
performed considering the natural phase variables (dashed line) and the
phases corresponding to the first intrinsic modes (solid line)73
Fig. 4.11 Illustration of the EMD method applied to experimental EEG data..75
Fig. 4.12 The time evolution of the synchronization index over the whole
recording period75

Fig. 5.1. Example of an animal pursuing a moving target spot back-projected
on the tangent screen. Each trial was initiated by the appearance of a target
during which the monkeys had to saccade to that initial stimulus position...80
Fig. 5.2. Measures of saccade parameters and neuronal activity. From top to
bottom: eye position (Ev), vertical eye velocity (v), spike density (Spd) and
individual spikes (vertical lines)81
Fig. 5.3. Typical anticipatory eye movements, the corresponding neural
activity and the correlations between them in the preferred direction (normal
correlations)82
Fig. 5.4. Differentials spike density and eye velocity respectively and the
differential correlation coefficient..83
Fig. 5.5. First and last four values of the correlation coefficient for the early
anticipatory pursuit interval...84
Fig. 5.6. Plots of correlation coefficients computed from random selections of
trials in the preferred direction during the early anticipatory pursuit84
Fig. 5.7. Histogram of the optimal time lag for early and late anticipatory
response ...85

Abstract
Extraction of physiological and clinical information hidden in biosignals, such as
cardiac and neural signals, is an important and fascinating field of research.
Noninvasive assessment of the physiological parameters of a patient enables to
study the physiology and pathophysiology of the investigated system, with minimal
interference and inconvenience. This approach may also help to assess
noninvasively the clinical condition of the patient.
The primary focus of this study is therefore to extend the arsenal of research tools
for the noninvasive investigation of the neural and cardiac systems.
The approaches developed in this work concern two major directions:
The first direction relies on the analysis of cardiac and neural responses during
hypoxia. Hypoxia-ischemia remains a great challenge to the researchers, since it
triggers complex responses at different levels in the organism. The functional
recovery depends on a number of factors among which the state of autonomic
nervous system (ANS) regulation plays an important role. Two different
applications were considered in this framework. The first application studied the
effect of global ischemic preconditioning on the heart rate variability (HRV)
response to the asphyxia insult. Using linear (time and frequency domain) and
nonlinear (approximate entropy and parameters of Poincare plots) measures, we
evaluated the dynamic time course of the HRV response to the asphyxia insult and
the effect of preconditioning on the autonomic neurocardiac control. Our results
show for the first time that global ischemic preconditioning influences the HRV
response to the asphyxia injury. The neuroprotective effect of preconditioning
translates into a faster recovery of the basal HRV and the autonomic modulation of
the heart. For the preconditioned group, at about 90 min after the asphyxic insult,
the autonomic neural balance (measured by LF/HF ratio) appears fully recovered.
Another application addressed the problem of phase synchronization analysis of
EEG signals during monitoring of recovery process following brain injury episode.
The concept of phase synchronization offers a new perspective on the
understanding and quantification of the dynamical interactions established among
coupled systems. In this thesis, we present a new approach for the identification of
the degree of interaction between two complex dynamical systems from
experimental data analysis. We use the empirical-mode-decomposition (EMD)
technique to decompose the output signals into a number of elementary orthogonal
modes with well defined instantaneous attributes (IMFs).
The second direction addressed the problem of correlations between anticipatory
pursuit eye movements and the neural response in the Supplementary Eye Fields
(SEF) of the Macaque monkey. Anticipatory pursuit is a smooth movement of the
eye occurring before the appearance of an expected moving target. The expectation
of the subject is based on a subjective estimation of the probability that the target
will move in a given direction. Recently, it has been suggested that the SEF could
play a role in using past experience to guide anticipatory pursuit. This hypothesis is
currently being tested at the single neuron level. In the behaving monkey, it has
been shown that electrical microstimulation in the SEF can facilitate smooth
pursuit initiation towards a moving target, suggesting that activation of the SEF
might change the internal gain of the smooth pursuit pathway. In this study, we
favored anticipatory responses in monkeys by using a cognitive cue, which
produces a different anticipatory pursuit response than the one observed in previous
studies, based on repetition.

H

.

.
.

.

:

.

.

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(heart rate variability - HRV)
. (
) (
Poincare)
HRV -
.
HRV
.

HRV .
90 ,
(
.

.

.

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(empirical-mode-decomposition EMD)

(instantaneous attributes IMFs).

(Supplementary Eye Fields SEF) Macaque.

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.

- 1 -
Chapter 1: Introduction
1.1 Motivation and Background
1.1.1 General Overview
Physiological systems exhibit complex dynamics at multiple spatial and temporal
scales. A variety of mathematical methods have been developed to characterize
complex rhythms that are observed in physiological systems. Much of the current
understandings of how physiological systems work has followed from the use of
linear models, based on the apparent proportionality of the relationship between
stimuli and physiological response over a narrow range. The interaction of different
phenomena that were studied in isolation from noisy environment, was viewed as
additive, i.e. the behaviour of a complex system was believed to be a linear
superposition of the behaviours of the systems elements. However, it has been
realized that linear models are not capable to explain the rich temporal behaviour
including non-stationarity, abrupt changes, periodic and aperiodic variability and
emergent phenomena of life systems. Such behaviour is displayed by nonlinear
dynamical systems. It was soon realized that physiological systems are inherently
complex, and often far more complex than usually appreciated.
Many physiological systems are still incompletely understood; hence accurate
analytical models cannot be developed. In such circumstances, understanding their
complex dynamics in normal and pathological conditions relies on the power of
signal analysis and information theory.

Chapter 1

- 2 -
1.1.2 Information Theory and Nonlinear Dynamics in Analyzing Cardiac and
Neural Signals
Information theory is a branch of mathematics that overlaps into communications
engineering, biology, medical science, sociology, and psychology. The theory is
devoted to the discovery and exploration of mathematical laws that govern the
behavior of data as it is transferred, stored, or retrieved.
Information theory was new and exciting to a great many psychologists starting
with 1950s. The basic works of Wiener [Wiener, 1948] and Shannon [Shannon,
1949] in the late 1940s had an almost immediate impact on psychology. In 1949,
Miller and Frick showed how response sequences could be analyzed in
informational terms, and Garner and Hake [Garner et al. 1951] presented a method
of multivariate informational analysis that had a generality far beyond its original
application to absolute judgments. Starting from this point, the methods and
measures of information theory and non-linear dynamics have found extensive
applications to the study of dynamical behaviour of physiological time series,
complementing the linear analysis tools. In some areas, such as heart diseases and
brain functioning, information theory and nonlinear dynamics have changed the
conventional view of health and disease (i.e., based on the classical concept of
homeostasis [Cannon, 1929]). In this sense, the term of dynamical disease has been
introduced [Mackey et al, 1977] in order to describe qualitative changes in the
temporal pattern of physiological variables associated to malfunction of
physiological systems.
Hypoxia-ischemia is a great challenge to the researchers, since it triggers complex
responses at different levels in the organism. The functional recovery depends on a
number of factors among which the state of autonomic nervous system (ANS)
regulation plays an important role [Miao-Kun et al. 1999].
The analysis of heart rate variability (HRV) can provide valuable insights into the
neural control of the heart, giving rise to a new discipline: 'neurocardiology', which
studies the interactions between the neural system and the heart. HRV has been
extensively studied to understand and quantify the ANS functioning, being of
particular importance for the diagnosis and prognosis of pathological conditions,
like sudden infant death syndrome, heart failure or sleep apnea.
Introduction

- 3 -
A recent approach motivated by the need to develop a rigorous measure of the
degree of disorder (or complexity) of the EEG signal in brain injury has been
considered by [Bezerianos et al. 2003]. The EEG signal complexity has been
studied by means of the correlation dimension D2 [Casdagli et al. 1997, Lehnertz et
al 1998, Stam et al 1999]. A basic requirement for applying the tools of nonlinear
dynamics (chaos theory) to experimental data is the stationary of the time series.
This suggests that the time series is representative of a unique and stable attractor
and is statistically invariant over different time intervals. Also, for the evaluation
chaotic measures like D2, long-time recordings are required. Generally, these
measures are noise sensitive and their usefulness decreases especially in the case of
additive noise [Abarbanel 1996, Basar 1998]. Unfortunately, the EEG data are
noisy and the stationarity requirement is not fulfilled; therefore, other methods
have to be explored. [Bezerianos et al. 2003] postulate that entropy analysis will
provide a quantitative measure of the degree of disorder in the brain rhythm at
various times in brain injury and recovery.
An important application of the tools of nonlinear dynamics is the study of brain
activity at different levels of neuronal organization, from the cellular level up to
large scale neuronal networks [Schiff et al.1996, Tass et al. 1998, van Quyen et al.
1998, Arnhold, et al. 1999, Rodriguez et al. 1999, Neiman et al. 1999, 2002].
Rhythmical phenomena in the electrical activity of the brain can be often
understood within the framework of the coupled oscillators theory. This formalism
has provided explanation for a number of observed synchronization phenomena
and coherent behavior in neuronal ensembles. In addition, techniques derived from
synchronization theory have found useful applications in the analysis of brain
electrical activity [Tass et al. 1998, Mormann et al 2000, Quiroga et al. 2000]. Non-
invasive brain recordings are typically multichannel measurements of electric
(electroenchephalogram, EEG) or magnetic brain activity
(magnetoenchephalogram, MEG) which reveal the coexistence of broad-band
activity and rhythms of different frequencies.
Despite the shortcomings associated to the application of nonlinear time series
methods to univariate signals generated from physiological systems with less
known structure, it is commonly accepted today that nonlinear physiological
signals analysis is able to provide new and relevant information as long as the
Chapter 1

- 4 -
limitations of the techniques are taken into consideration and the results are
interpreted carefully.

1.2 Organization of the Thesis
The dissertation is organized as follows:
Chapter 2 provides a general overview on the two physiological systems
considered in this work. We begin with a short review of the autonomic nervous
system, cardiovascular system and central nervous system. Next we present the
physiological signals we will further analyze in our experiments. First, we analyze
the beat to beat cardiac rhythm variability in more common terms called heart rate
variability (HRV). We further investigate the neural responses during two different
situations: hypoxia and visual pursuit.
Chapter 3 briefly presents an overview of the main concepts of linear and
nonlinear methods and their application to time series analysis in physiology and
medicine. Measures and technique developed within the framework of linear and
nonlinear dynamics are presented. The one selected and detailed here describe and
quantify fundamental features of physiological signal during various conditions, eg
hypoxia-asphyxia.
Chapter 4 considers the problem of cardiac and neural responses to hypoxia.
Ischemic preconditioning has been used as a strategy to prevent cell death in
various organs, including the brain and the heart. Investigation of the effects of
ischemic preconditioning mostly employed models with reduced complexity, such
as cell cultures, tissue slices or perfused organ preparations. Although such models
can provide valuable insight into the protective mechanism of preconditioning, the
functional (re)organization of the control mechanisms at the level of the living
organism cannot be assessed. A first application was to evaluate the effect of global
ischemic preconditioning on the heart rate variability (HRV) response to the
asphyxia insult. The data consisted of 4 hours RR interval measurements recorded
in five preconditioned and five non-preconditioned Wistar rats. Using linear (time
and frequency domain) and nonlinear (approximate entropy and parameters of
Poincare plots) measures, we evaluated the dynamic time course of the HRV
Introduction

- 5 -
response to the asphyxia insult and the effect of preconditioning on the autonomic
neurocardiac control. Both the linear and non-linear parameters indicate a faster
recovery of the baseline HRV corresponding to the preconditioned groups, though
only the spectral analysis identifies a statistically significant difference between the
two groups. For the preconditioned group, at about 90 min after the asphyxic insult,
the autonomic neural balance (measured by LF/HF ratio) appears fully recovered.
The small variation of the rest of the parameters indicates the necessity of further
investigation including the design of a larger study with a higher statistical power.
Our results show for the first time that global ischemic preconditioning influences
the HRV response to the asphyxia injury. The neuroprotective effect of
preconditioning translates into a faster recovery of the basal HRV and the
autonomic modulation of the heart.
The second application addressed the problem of phase synchronization analysis of
EEG signals during monitoring of recovery process following a brain injury
episode. The concept of phase synchronization offers a new perspective on the
understanding and quantification of the dynamical interactions established among
coupled systems. In the present study, we present a new approach for the
identification of the degree of interaction between two complex dynamical systems
from experimental data analysis. We use the empirical-mode-decomposition
(EMD) technique to decompose the output signals into a number of elementary
orthogonal modes with well defined instantaneous attributes (IMFs). We
demonstrate by numerical simulations that the quantification of the phase
synchronization of the intrinsic oscillatory modes provides a reliable indicator of
the degree of interaction between coupled dynamical systems.
Chapter 5 presents an application of correlations between anticipatory pursuit eye
movements and the neural response in the Supplementary Eye Fields (SEF) of the
Macaque monkey. Anticipatory movements are actions starting before the
occurrence of likely sensory events. In an experimental setting, this can be studied
by having subjects pursue a moving dot of light appearing on a visual display. If
the visual stimulus moves consistently in the same direction during a few
presentations, subjects anticipate that it will likely continue to do so during the next
presentation and the eyes will start to move in advance of the beginning of the next
stimulus appearance. Anticipatory pursuit is a smooth movement of the eye
Chapter 1

- 6 -
occurring before the appearance of an expected moving target. The expectation of
the subject is based on a subjective estimation of the probability that the target will
move in a given direction. Anticipatory pursuit has been extensively studied at the
behavioral level. However, the neural mechanisms allowing this important
behavior are unknown. Recently, it has been suggested that the SEF could play a
role in using past experience to guide anticipatory pursuit. This hypothesis is
currently being tested at the single neuron level. In the behaving monkey, it has
been shown that electrical microstimulation in the SEF can facilitate smooth
pursuit initiation towards a moving target, suggesting that activation of the SEF
might change the internal gain of the smooth pursuit pathway In this study, we
favored anticipatory responses in monkeys by using a cognitive cue, which
produces a different anticipatory pursuit response than the one observed in pervious
studies, based on repetition.

1.3 Contributions and Publications
This section briefly outlines the main contributions of this thesis and the list of
publications and presentations resulted from the work developed under the
framework of this research:
1. A study of autonomic cardiovascular responses in a brain hypoxia and
ischemia experiment using linear and non-linear methods of analysis
(publications 3, 6, 8 and 9 in the list below and Chapter 4.1)
2. A study of the ischeming preconditioning effects on heart rate variability.
Using linear (time and frequency domain) and nonlinear (approximate
entropy and parameters of Poincare plots) measures, we evaluated the
dynamic time course of the HRV response to the asphyxia insult and the
effect of preconditioning on the autonomic neurocardiac control
(publications 2, 7 in the list below and Chapter 4.1)
3. A new approach for the problem of phase synchronization analysis of EEG
signals during monitoring of recovery process following a brain injury
episode. We used empirical mode decomposition (EMD) technique for the
identification of the degree of interaction between two complex dynamical
Introduction

- 7 -
systems from experimental data analysis (publication 1, 5 below and
Chapter 4.2)
4. Study of correlations between anticipatory pursuit of eye movements and
the neural response in the Supplementary Eye Fields of a behaving monkey
using a cognitive cue (publication 4 and Chapter 5)
The research presented in this thesis contributed to the following publications in
international journals and conference proceedings:
1. L. Moraru, L. Cimponeriu, S. Tong, N. Thakor, A. Bezerianos, A New
Approach For Phase Synchronization Analysis Of Brain Electrical Signals,
in Int. Journal of Sci Res. 2006, vol 16 (in press)
2. L. Moraru, S. Tong, A. Malhotra, R. Geocadin, N. Thakor and A.
Bezerianos: The Ischemic Preconditioning Effects on the HRV after
Transient Global Ischemia, Med Eng Phys. 2005 J ul;27(6):465-73
3. L. Moraru, L. Cimponeriu, S. Tong, N. Thakor, A. Bezerianos:
Characterization of Heart Rate Variability Changes Following Asphyxia in
Rats., Methods of Information in Medicine Journal, , 2004, vol.43(1):118-
121.
4. L. Moraru, A. Bezerianos, M. Missal, P. Lefvre: Correlations Between
Neural Activity in Supplimentary Eye Field and Anticipatory Eye
Movements, submitted to VII International Symposium on Biological and
Medical Data Analysis, 2006. Thessaloniki, Greece
5. L. Moraru, L. Cimponeriu, S. Tong, N. Thakor, A. Bezerianos: Neural
Phase Synchronization During An Asphyxia Experiment In Rats, in IFMBE
Proceedings of the X
th
Mediterranean Conference on Medical and
Biological Engineering, MEDICON 2004, J uly 31- August 5, Naples, Italy
6. L. Moraru, R. Strungaru, A.Bezerianos: Heart Rate Variability Analysis on
Small Handheld, Mobile Computing Device, in the Proceedings of
BIOSIGNAL 2004 Conference, 23-25 J une 2004, Brno, Czech Republic,
ISSN 1211-412X, pp.88-90.
7. L. Moraru, R. Strungaru, S. Tong, N.V. Thakor, A. Bezerianos: The
Ischemic Preconditioning Effects on the Cardiac Autonomic Control in Rats
Chapter 1

- 8 -
Following Transient Global Ischemia, in the Proceedings of MEDINF2003
Conference, October 9 - 11, 2003, Craiova, Romania, ISSN: 1454-6876,
Vol.5, Sup.3, pp. 180:183.
8. L. Moraru, L. Cimponeriu and A. Bezerianos: Heart Period Dynamics
Following an Asphyxia Experiment in Rats, in the Proceedings of the 14th
International Conference on Digital Signal Processing DSP2002, J uly 1-3,
2002 in Santorini, Greece
9. L. Moraru, L. Cimponeriu and A. Bezerianos: Autonomic Cardiovascular
Responses in a Brain Hypoxia and Ischemia Experiment, in the
Proceedings of the 3rd Symposium on Biomedical Engineering, 27 August-
01 September 2002 in Patras, Greece

- 9 -
Chapter 2: Physiological Background
The motive behind the detailed physiological background in this chapter is two
fold. Firstly, it describes the, physiological mechanisms which give rise to the
various signals used in this study. Secondly, an adequate understanding of the
factors controlling these control systems is necessary for a proper interpretation
of the signal analysis results in this work.
The two major physiological systems considered here are the:
1. Cardiovascular system, and
2. Central nervous system.

2.1 Cardiovascular System
Cardiovascular signals are the first signal group used in this study.
Electrocardiogram (ECG), heart rate variability (HRV) and blood pressure (BP)
signals are the three most important representatives of cardiovascular signals.
The cardiovascular system's primary task is the adequate oxygen perfusion to the
cells in multi-cellular systems, such as the human body. This requires a method of
oxygen transport from the point of oxygen uptake to the individual cells. While
the blood has the means to bind oxygen, it is the heart that maintains the initial
task of circulating the blood through the vessels.

2.1.1 Cardiac Control
The most characteristic feature of cardiac control is the rhythmical occurrence of
heart beats. The cardiac musculature consists of two types of muscle cells (or
Chapter 2
- 10 -
fibers): (i) cells that initiate and conduct impulses, and (ii) cells that, besides
conducting, respond to stimuli by contracting. Initiation of the inherent
rhythmicity of the heart (auto-rhythmicity) originates from the sinoatrial node
(SA node) (Figure 2.1), which is the physiological pacemaker of the heart.

Fig. 2.1 The heart. The autorhythmic cells controlling the pace of cardiac
contractions are located in the SA node. Signals generated by the SA node travel
to the AV node and down the AV bundle and branches

From there excitation of the heart normally proceeds through both atria to the
atrioventricular node (AV node) to eventually reach the ventricular muscle via the
bundle of His and its two branches which terminate in the Purkinje fibre network.
Although, due to its autonomy, the heart is able to beat entirely on its own,
adaptation of cardiac activity to the changing needs of the organism is largely
dependent on participating cardiac nerves. Control of cardiac activity originates
in the circulatory centres of the medulla oblongata and pons, via sympathetic and
parasympathetic nerves, so it is large degree dependent upon intact cardiac
nerves.
When analysing cardiac signals, one often is inclined to believe that
chronotropism (frequency at which the heart beats, as determined by pacemaker
impulse initiation) is the factor actually measured. However, it is only the direct
factor out of several others indirectly acting on the heart beat, which include
dromotropism or bathmotropism (strength of contraction).
Physiological Background
- 11 -
Chronotropism is lowered by the right branch of the vagus (negative chronotropic
effect) and raised by the sympathetic fibres (positive chronotropic effect).
Simultaneous stimulation of both nerves usually results in vagus dominance.
While the ventricles are exclusively under sympathetic influence, control of the
atria is the product of both sympathetic and parasympathetic activity. Vagotomy
results in heart beat acceleration and sympathectomy reduces heart rate. Since the
auto-rhythmically beating heart is considerably higher than resting heart rate,
vagal dominance may be presumed at rest. HRV signals thus reflect the overall
balance of the two ANS branches activity.
Dromotropism (conduction speed, especially in the AV node) is influenced by the
left branch of the vagus which retards AV conduction time, and the sympathetic
nerves which accelerate transmission of stimuli in the AV node (negative and
positive dromotropic effect, respectively). Extensive vagal stimulation, however,
can lead to a transient AV block.
The afferent input of the carotid and aortic baroreceptors continuously modify the
efferent activity cardiac vagus nerves and thus heart rate. The baroreceptor loop
is a reflex loop, and although strong, is interrupted centrally each time inspiration
occurs. The neural activity in the medulla which initiates inspiration inhibits
cardiac vagal discharge causing the phenomenon of respiratory sinus arrhythmia.
However, interruption of vagal motor discharge is not the only reason for such
phenomena as cardiac sympathetic branches show a rhythmic activity too. It has
been shown in ventilated animal experiments that this rhythmic activity is not
attributable to afferent discharges of pulmonary stretch receptors (Hexing-Breuer
reflex) and seems to originate from a central respiratory rhythm.
At rest, carotid and aortic chemoreceptors show a sparse activity and influence
heart-rate to a much lesser degree than baroreceptors. However, their reflex
activity can be powerful and include vasoconstriction with overall blood pressure
increase, hyperpnia (increase in depth of breathing, with or without increased
respiratory rate) and bradycardia.
An important reflex to be mentioned in this context is the diving reflex. When
activity of central respiratory neurons is abolished by nasal or laryngeal
stimulation, the effects of chemoreceptor reflex activity are fully pronounced.
Chapter 2
- 12 -
Thus, as in the seal, diving causes marked slowing of heart beats accompanied
with intense vasoconstriction. This maintains adequate perfusion of vital organs
while others switch to anaerobic metabolism.

2.1.2 The Neural Regulation of the Cardiovascular System
The cardiovascular system is regulated by a complex balance of neural, humoral
and metabolic factors. The autonomic nervous system (ANS) modulates the cardiac
pacemaker and provides beat-to-beat regulation of cardiovascular system.
The autonomic nervous system is traditionally described as a motor system that
provides control over visceral functions critical for homeostasis. This system is
responsible for extrinsic regulation of cardiac muscle, smooth muscles (e.g.
vascular smooth muscles) and all glandular secretions (e.g. hormones). However,
the autonomic nervous system is not strictly an efferent (motor) nervous system.
Almost all visceral nerve bundles have sensory fibres intermixed with motor
fibres (approximately 50% of all axons in the splanchnic nerves and 20% of all
axons in the vagus nerves). These sensory fibres carry information from receptors
to the central nervous system. This information is, in turn, integrated and relayed
by multi-neuronal pathways to the brain and/or spinal cord and eventually
modulates the autonomic motor outflow. Thus, the ANS provides all the
pathways for negative feedback control systems. The autonomic nervous system
is composed of three different divisions: the Sympathetic, Parasympathetic and
Enteric Nervous Systems. The efferent cardiac nerves, via which the function of
the heart can be influenced are the parasympathetic fibres of the vagus nerve
(cholinergic) and sympathetic nerves.
The motor portion of sympathetic and parasympathetic systems is made up of two
sets of serially connected neurons, known as pre-ganglionic and post-ganglionic
neurons (Figure 2.2). The cell bodies of pre-ganglionic neurons originate in the
central nervous system and synapse in the peripheral nervous system on second
order neurons called ganglion or post-ganglionic neurons. It is at these synapses
that the information transmitted along the nerve fibres can be blocked, for
sympathetic and parasympathetic nerves either collectively or differentially
(Figure 2.2) [Despopoulos and Silbernagl, 1991].
- 13 -
The two branches of the ANS are quite distinct in both anatomy and physiology.
The cell bodies of the sympathetic pre-ganglionic fibres are in the lateral horn of
thoracic and lumbar spinal cord and terminate either in the paravertebral
ganglionic chain, in the cervical or abdominal ganglia or in the so-called terminal
ganglia. Within the ganglion, transmission of the stimulus to the postganglionic
fiber is cholinergic, the transmitter substance being acetylcholine. Sympathetic
pre-ganglionic neurons are largely myelinated (i.e. faster conduction time) and
conduct impulses at speeds of 1 to 20 meters per second. Since most sympathetic
ganglia lie far from the end-organ, their post-ganglionic axons are relatively long.
This makes them more vulnerable to axonopathy which occurs in diseases such as
diabetic neuropathy.
Parasympathetic pre-ganglia fibers run from the brain stem with the cranial
nerves to muscles and glands in the head (cranial nerves III, VII, IX) and to the
organs in thorax and abdomen (vagus or 10th cranial nerve). The parasympathetic
ganglia are situated close to, or even within on the wall of the organ they
innervate; thus the axons of these ganglion cells are shorter than those arising
from sympathetic ganglion cells.
Most organs are innervated by fibers from both the sympathetic and
parasympathetic divisions of the autonomic nervous system (Table 2.1). The
response of the two types of fibers may be either antagonistic (as in the heart) or
almost parallel (in salivary glands).
Table 2.1: Effects of Parasympathetic vs Sympathetic Stimulation [73]
Innervated Organ Parasympathetic Nerves Sympathetic Nerves
Heart muscle
Reduction of Heart-rate and
Contraction Strength
Increase of Heart-rate and
Contraction Strength
Blood Vessels Constriction Dilation
Fat
Cells
Lipolysis (free fatty acids
production) decreased
Lipolysis
increased
Insulin Secretion increased Secretion decreased
Bronchi Constriction Dilation

Chapter 2
- 14 -

Fig. 2.2 Functional Organisation of the ANS

The response of the two types of fibre is mostly antagonistic. Functionally, both
divisions act synergically on the controlled organ, i.e. the desired response is
brought by simultaneous stimulation of one division and stimulation reduction of
- 15 -
the other division, and vice versa. However, such functionality assumes identical
control time delays. Thus, control by sympathetic and parasympathetic fibres may
also be viewed as sequential. A dynamic (fast and short-term) parasympathetic
branch may provide the initial response which is then followed by a less dynamic
(slower and long-term) sympathetic response, as research indicates is the case in
cardiac control. Parasympathetic control thus often dominates regulation of end
organs innervated by both divisions of the autonomic nervous system (e.g. blood
pressure response in cardiac control).

Fig. 2.3 Schematic representation of opposing feedback mechanisms which,
in addition to central integration, subserve neural control of the cardiovascular
system. Baroreceptive and vagal afferent fibers from the cardiopulmonary
region mediate negative feedback mechanisms (exciting the vagal outflow and
inhibiting the sympathetic outflow), whereas positive feedback mechanisms are
mediated by sympathetic afferent fibers (exciting the sympathetic outflow and
inhibiting the vagal outflow) (from Malliani et al. 1998)

2.1.3 Heart Rate Variability
2.1.3.1 Physiological Mechanisms of Heart Rate Variability
Various cardiovascular variables, such as heart rate and blood pressure, fluctuate
from one beat to another. Heart Rate Variability has become the conventionally
accepted term to describe variations of both instantaneous heart rate and RR
intervals. In order to describe oscillation in consecutive cardiac cycles, other terms
have been used in the literature, for example cycle length variability, heart period
variability, RR variability and RR interval tachogram, and they more appropriately
emphasize the fact that it is the interval between consecutive beats that is being
analysed rather than the heart rate per se. However, these terms have not gained as
wide acceptance as HRV.
Chapter 2
- 16 -
Beat-to-beat fluctuation in heart rate partly reflects the interplay between various
perturbations of cardiovascular function and the response of the cardiovascular
regulatory systems to these perturbations and also initially raised behavior. The
changes in heart rate behavior may be either exogenous or endogenous. Continuous
changes in sympathetic and parasympathetic neural impulses exhibit changes in
heart rate and cause oscillation around the mean heart rate.
Although cardiac automaticity is intrinsic to various pacemaker tissues, heart rate
and rhythm are largely under the control of the autonomic nervous system. The
parasympathetic influence on heart rate is mediated via release of acetylcholine by
the vagus nerve, while the sympathetic influence on heart rate is mediated by
release of epinephrine and norepinephrine. Under resting conditions, vagal tone
prevails [Levy 1971] and variations in heart period are largely dependent on vagal
modulation [Chess et al 1975]. The vagal and sympathetic activities constantly
interact. As the sinus node is rich in acetylcholinesterase, the effect of any vagal
impulse is brief because the acetylcholine is rapidly hydrolyzed. Parasympathetic
influences exceed sympathetic effects probably via two independent mechanisms: a
cholinergically induced reduction of norepinephrine released in response to
sympathetic activity, and a cholinergic attenuation of the response to a adrenergic
stimulus.
The RR interval variations present during resting conditions represent a fine tuning
of the beat-to-beat control mechanisms [Akselrod et al. 1985, Saul et al. 1990].
Vagal afferent stimulation leads to reflex excitation of vagal efferent activity and
inhibition of sympathetic efferent activity [Schwartz et al. 1973]. The opposite
reflex effects are mediated by the stimulation of sympathetic afferent activity
[Malliani 1982]. Efferent vagal activity also appears to be under tonic restraint by
cardiac afferent sympathetic activity [Cerati et al. 1991]. Efferent sympathetic and
vagal activities directed to the sinus node are characterized by discharge largely
synchronous with each cardiac cycle which can be modulated by central (e.g.
vasomotor and respiratory centres) and peripheral (e.g. oscillation in arterial
pressure and respiratory movements) oscillators. Sympathetic excitations have been
suggested to correspond to RR interval fluctuation at around 0.1 Hz frequency
[Malliani et al. 1991, Pagani et al. 1997]. However, most evidence does not support
the notion that low frequency spectral power detects changes of sympathetic nerve
- 17 -
activity [Koh et al. 1994, Hopf et al. 1995, Saul et al. 1990, Kingwell et al. 1994].
The phenomenon of sympatho-vagal balance in heart rate variability analysis can
also be questioned [Eckberg 1997].
One fluctuation loop affecting heart rate variability is the vasomotor part of the
baroreflex loop, which is responsible for arterial pressure oscillations [Madwed et
al. 1989], causing low frequency fluctuation. Several other factors, such as
peripheral vascular resistance and thermoregulation, are suggested to cause very
low frequency oscillation [Rosenbaum et al. 1968, Kitney 1975], but the relevance
of these suggestions can be questioned. In addition, rapid control systems of
pressoreceptors and chemoreceptors maintain the cardiovascular homeostasis by
altering the heart rate through small frequent adjustments [Ravenswaaij et al.
1993].
A relatively well known event that causes oscillations in heart rate is respiration.
Heart rate fluctuation is related to respiration due to the inspiratory inhibition of
vagal tone. The inspiratory inhibition is evoked primarily by central impulses from
the medullary and cardiovascular center [Davidson et al. 1976]. This
parasympathetically mediated fluctuation can be abolished by atropine or
vagotomy [Akselrod et al. 1985, McCabe et al. 1985, Raczkowska et al. 1983,
Pomeranz et al. 1985]. RR interval fluctuation in relation to respiration is used as a
noninvasive index of vagal nerve excitation in humans [Hayano et al. 1991,
Eckberg 1983, Kollai et al. 1990]. However, respiration related high-frequency
heart rate fluctuation has been shown to be a somewhat imperfect index of vagal
activity [Kollai et al. 1990]. There are situations in which high frequency changes
of RR intervals may not reflect changes in vagal modulation at all (Brown et al.
1993), but can be explained by the kinetics of sino-atrial node responses to
acetylcholine [Saul et al. 1991]. This respiration caused fluctuation occurs at both
high and low frequencies [Koh et al. 1994].
Heart rate fluctuation is also a result of various factors, which are often difficult to
discern from total behaviour, which combine different wave forms. Thus, by
studying heart rate variability, we have an opportunity to study the cardiac dynamic
behaviour influenced by a variety of endogenous and exogenous factors. It is
possible to obtain information about the nature of the perturbations to which the
cardiovascular system is exposed as well as the regulatory responses to these
Chapter 2
- 18 -
perturbations. Since the process is dynamic and nonlinear, the usefulness of
studying the behaviour of fluctuations rather than static averages is acknowledged.

2.1.3.2 Clinical Implications and the Use of Heart Rate Variability.
Although it may not be immediately obvious, one extremely important point is that
beat to beat cardiovascular variability is normal physiology, and furthermore, the
loss of variability has been correlated with disease states. Power spectral analysis
provide a measure of the sympatho-vagal balance and important results have been
provided in a number of important pathologic conditions, spanning from
myocardial ischemia and infarction, heart failure, hypertension, diabets, obesity,
and chronic alchool abuse.
Temporal fluctuations in cardiovascular signals were noted in ancient times, but
their possible significance has been overlooked for a long time. The clinical
relevance of heart rate variability was first appreciated in 1965 when [Hon et al.
1965] noted that fetal distress was preceded by alterations in interbeat intervals
before any appreciable change occurred in the heart rate itself. The clinical
importance of HRV became apparent in the late 1980s [Saul et al. 1987] when it
was confirmed that HRV was a strong and independent predictor of mortality
following an acute myocardial infarction.
Initially, heart rate variability measurements were based on simple measurements
of RR intervals in studies on diabetics [Murray et al. 1975]. Later, the HRV has
been recognized as a powerful risk stratifier for adverse cardiac events in a number
of normal and pathological conditions, such as: aging [Tsuji et al 1994],
arrhytmogenesis/sudden death [Dougherty et al 1992], heart failure [Ho et al.
1997], coronary artery disease [Rich et al. 1988], post-myocardium infarction
[Wolf et al. 1978], sleep apnea [Ivanov et al. 1996].
Although HRV has been the subject of numerous clinical studies investigating a
wide spectrum of cardiological and non-cardiological diseases and clinical
conditions, a general consensus of the practical use of HRV in adult medicine has
been reached only in two clinical scenarios. Depressed HRV can be used as a
predictor of risk after acute MI and as an early warning sign of diabetic neuropathy.
It has been known that cardiovascular autonomic diabetic neuropathy is
- 19 -
associated with a loss of heart rate variability [Persson et al. 1983].These patients
have a poor cardiovascular prognosis, with a 5-year mortality greater than 50 %
[Ewing et al. 1976]. The detection of autonomic dysfunction may be used as
markers of pathology, particularly to study the benefits of therapeutic
interventions. The analytic methods based on nonlinear dynamics have developed
to the point where they can make valuable contributions not only to the
understanding of cardiac rhythms but also they may aid in the identification of
patients with various pathologies and who may be at risk for arrhythmia [van
Leeuwen et al. 2000].
The range of clinical applications continues to expand as more specific and
sensitive indicators are discovered. Although nonlinear methods could
complement the linear analysis, broad clinical trials are necessary which link such
measures with the outcome of intervention. There are many sources of
nonlinearity in cardiovascular regulation. One of the earliest was revealed by
[Rosenblueth et al. 1934] and refers to the interaction between sympathetic and
parasympathetic innervations of the sinus node. The existence of nonlinear
mechanisms raised the possibility that irregular-appearance of heart rate cannot
be fully described by linear techniques and that nonlinear measures may reveal
clinically relevant aspects of heart rate dynamics. However, uniform
interpretation criteria have not been established in these situations, at least
partially due to insufficient understanding of physiology and pathophysiology of
HRV in this age group [Patzak et al 2000].

2.2 The Central Nervous System (CNS)
The central nervous system is the most complex physiological system in this
study. It is also the least understood. The CNS consists of the brain and spinal
cord. Its function is to connect various cell groups within the body and to co-
ordinate their activities. Thus the role of the CNS is to scan, evaluate and process
the information received and to respond with efferent impulses. It resembles a
communication system and is the primary cause of correlations in physiological
activities.

Chapter 2
- 20 -
2.2.1 Organization of the CNS
The CNS consists of the brain and spinal cord, which are located in the dorsal body
cavity. The brain is surrounded by the cranium, and the spinal cord is protected by
the vertebrae. The brain is continuous with the spinal cord at the foramen magnum.
The brain can be divided into four interconnected areas: brainstem, diencephalons,
limbic and neocortex. The complexity of structure, cellular organization and
function increases from the lower, simpler areas such as the brainstem to the most
complex, the neocortex. The cerebral cortex is the outer most layer of the cerebral
hemispheres of the brain, and mediates all conscious activity including planning,
problem solving, language and speech. It is also involved in perception and
voluntary motor activity.
The functions of the CNS may be broken down into specific functions, somewhat
misleadingly suggesting independence. One of these specialised functions is to
adjust muscle movement in response to external factors and to provide purposeful
movement (the so-called motor system). Another function of the CNS is to
receive sensory signals and process them to conscious feelings and perceptions
(sensory centres). Finally, a third part of the CNS controls the function of inner
organs (the vegetative nervous system).
Current practice divides the cortex into sensory, motor and associative regions.
The motor pathways are pathways which originate in the brain or brainstem and
descend down the spinal cord to control the a-motor neurons. These large neurons
in the ventral horns of the spinal cord send their axons out via the spinal roots and
directly control the muscles. The motor pathways can control posture, reflexes, and
muscle tone, as well as the conscious voluntary movements that we think of when
we hear "motor system". The most famous pathway is the so called "pyramidal
system", which begins with the large pyramidal neurons of the motor cortex,
travels through the pyramids of the brainstem, (somewhere in here there is a
coincidence), and finally ends on or near the a-motor neurons. The somatosensory
system includes multiple types of sensation from the body - light touch, pain,
pressure, temperature, and joint and muscle position sense (also called
proprioception). However, these modalities are lumped into three different
pathways in the spinal cord and have different targets in the brain.
- 21 -
Parts of the body from which sensory information is received is mapped onto
sensory cortical fields. Similarly, parts of the body which receive motor
commands are represented in the motor cortical fields. This mapping is called
somatotopic representation. Unspecific or so-called associative areas are fields to
which currently no specific sensory or motor function has been attributed.
This subdivision of the cortex, however, is not a rigid representation. Many
functions of the CNS, such as sleep cycles, speech, motivation and emotions
required contribution of multiple areas of the brain. For instance, speech cannot
be controlled without involvement of the thalamus. Equally, a lesion of the motor
area does not render subjects immobile (apart from fine-tuned motor actions).
Thus, specific functional areas are considered to be primarily involved with a
particular function.
Architecturally, the cortex is divided into 6 main layers, although layer boundaries
are not very obvious in routine sections. Each layer has different role and vary in
relative thickness among cortical regions (e.g., a sensory region has a thick internal
granule layer; a motor area has a thick internal pyramidal cell layers, and it is
characterised by the type of neuronal cells and nerve fibres it contains. Density
and degree of presence of the layers varies greatly across the brain. There is,
however, a certain match between the functional division of the brain and its
cyto-architecture (form and structure of neurons).
Two neuron types predominate in the cortex the pyramidal cells and the granule
cells. The pyramidal cell are conical cell body (>30 m in diameter) with apical
and basal dendrites and an axon that leaves the base of the cell to enter white
matter. Pyramidal cells vary in size. They are the output cells of the cerebral cortex.
The granule cell are small, round cell body (<10 m in diameter). They serve as
interneurons, receiving input from cortical afferent fibers and synapsing on output
neurons (pyramidal cells) of the cortex.
Layer V of the neocortex contains large pyramidal cells. The axons of these cells
may extend to sub-cortical regions such as brainstem and spine. Similar to its
axons, pyramidal dendrites are aligned vertically with respect to the cortex
surface. They form synapses with mostly excitatory axons from other contra- and
ipsilateral regions of the brain. The vertical alignment of axons and dendrites is
considered to be the basis for the superposition of electric field potentials,
Chapter 2
- 22 -
generated by postsynaptic potentials. The vertical structure is also consistent with
the somatotopic representation of the cortex.

2.2.2 Brain Electrical Activity
Electrical impulses in the brain are a way brain cells communicate and work
together. An electroencephalogram (EEG) is a recording, from the scalp, of the
electrical activity of the brain over a short period of time. It provides valuable non-
invasive insight into functional state of the brain and its different levels of arousal.
The EEG was introduced in 1929 by Hans Berger (1873-1941), who published his
observations after performing human scalp recordings. Bergers discovery initiated
numerous investigations attempting to understand the nature and origin of the
electrical potentials produced by the brain [Thompson et al. 1974]. Electrical
activity originates from chemical events that occur on the cellular level when
neurons receive and process information transmitted from other nerve cells in the
form of electrical impulses or action potentials. These neuro-electric phenomena
may be recorded with the continuous electroencephalogram (EEG) when the
subject is at rest and not involved in a designated task, or with the time specific
event-related brain potentials (ERPs) during cognitive tasks.
When such a recording is obtained from electrodes placed directly on the surface of
the brain - usually during neurosurgery - the measure is called the
electrocorticogram (ECoG). Under normal conditions, frequency and amplitude
depend on the subject, location of measurement and degree of brain alertness.
An EEG contains a series of wave forms that are classified into various
frequencies. Traditional time domain EEG spectra are separated into fundamental
bands qualitatively based on the shape and range of frequency for clinical
applications. These generally occur within the limits of 0.1 to 35 Hz and include
alpha, beta, delta and theta waves.
EEG analysis often focuses on frequency, amplitude, shape and distribution of
waves contained in the signal. The following is a summary of the EEG signals
division into different frequency ranges and some diagnostic interpretation
(Figure 2.4).

- 23 -

Fig. 2.4 One second EEG recordings and its components

1. The rhythm originates in a group of subcortical cells that induce the rhythmic
activity of the neurons in the cerebral cortex [Andersen et al 1968] and it is
characterized by a frequency raging from 8 to 13 Hz. Approximately 95% of
humans produce a clearly identifiable alpha rhythm when awake with the eyes
closed. During wakefulness brain activity of this frequency range is normally
distributed over the posterior skull regions. Visual and other stimuli map cause
attenuation of EEG activity (-blockage). EEG activity in the frequency band
over central regions of one or both sides (sec Figure 2.4) is normally referred to
as the rhythm and is not affected by any ocular movements.
Chapter 2
- 24 -
The resting EEG is characterized by two dominant frequencies: alpha and beta. It is
believed that rhythmic activity of the cortex, in particular the rhythm, is
induced mainly by sub-cortical regions, especially by the thalamus. There are
indications of thalamic pacemaker regions. Their activity, in turn, is modulated
by reticular structures, such as during sleep cycles.
2. The frequency range includes all frequencies above 13Hz with low
amplitudes rarely exceeding that of 30mV. They can exist simultaneously
throughout the cortex at various frequencies but are most common to the frontal
and central head regions in nearly all healthy adults. Localized bursts of 40Hz
oscillations are characteristic prior voluntary movement, such as wrist or finger
extensions, and beta synchronization appears at approximately 20Hz after
movement [Pfurtscheller et al. 1992]. If the rhythm resembles the or rhythm
in the distribution sense, then it is often referred to as an or variant. During
so-called blockage, waves often replace the rhythm, with a frequency of
approximately 20 Hz and lower amplitude than during activity (desynchronised
EEG). These EEG patterns express an increase in alertness.
3. Gamma waves belongs to the frequency range approximately 2680 Hz. Gamma
rhythms appear to be involved in higher mental activity, including perception,
problem solving, fear, and consciousness.
4. The and frequencies range are approximately between 4-7 Hz and 0-3 Hz,
respectively. Delta rhythms consist of low frequency, high amplitude waveforms
recorded between 1 to 4 Hz with amplitude ranges commonly from 20-30mV. They
are associated with periods of unconsciousness typically appearing in cerebral
monitoring during sleep, coma, or after convulsive seizure. Theta waves measure
from 4 to 7.5Hz and are low to moderate amplitudes. They play a vital role in
conditions of drowsiness and sleep in all ages and may be linked to the emotional
processes in children [Niedermeyer 1993]. During alertness, and in a healthy adult
subject, these rhythms are not clearly visible or almost absent. However; an
increase of and activity is often observed during deep sleep, anaesthesia and
hyperventilation.
- 25 -
5. So-called DC-Potentials (direct current potentials) can be measured if directly
coupled EEG amplifiers are used. These very slow potentials may provide more
detailed information and have become increasingly used in EEG experiments.

2.2.3 Neural Oscillations
Recently, oscillatory EEG activity has been discussed in relation with functional
neuronal mechanisms. In this regard, it is of major interest to investigate how brain
electric oscillations get synchronized in pathological or physiological brain states
(e.g., epileptic seizures, sleep-wake stages, etc.), or by external and internal
stimulation (event related potentials (ERP) or evoked potentials (EP)). This issue
can be addressed by applying methods of systems analysis to the EEG signals,
because changes in EEG activity occur in temporal relation to triggering events,
and could be thought of as transitions from disordered to ordered states (or vice
versa).
The concept of neural oscillations is close to the concept of brain waves. However,
the latter reflect the summed activity of populations of neurons, and the former
refers to more invasive recording techniques such as single-unit recordings with
extracellular electrodes, intracellular recordings of neuronal potentials and
recordings of local field potentials (LFPs) using electrodes directly contacting the
brain. Oscillatory activity observed in LFP and EEG recordings obtained under
different conditions in a wide variety of animal groups and reveals the ubiquitous
existence of synchronization in groups of neurons. Neuronal oscillations are quite
typical for brain cells and neural ensembles. They occur at different frequency
ranges, in different brain areas, and some type of oscillations have been related to
particular behaviors.
In the beginning of 1990s neuronal oscillations became a hot topic in
Neuroscience, when the studies of the visual system of the brain by [Gray 1994]
appeared to support neural binding hypothesis. According to this idea, synchronous
oscillations in neuronal ensembles bind neurons representing different features of
an object. For example, when a person looks at a tree, visual cortex neurons
representing the tree trunk and those representing the branches of the same tree
would oscillate in synchrony to form a single representation of the tree.
Chapter 2
- 26 -
Oscillatory activity is a basic feature of neuronal wetware. Both the active
properties of neuronal membranes as well as recurrent synaptic connections tend to
support the generation of oscillatory activity. Indeed, neural oscillations are
ubiquitous from the ganglia of early invertebrates to the human brain. The potential
functional roles of oscillations are diverse but contentious. For example, the
hippocampal theta rhythm has been implicated in sequence encoding and temporal
compression. In the visual system gamma has been argued to underlie the object
segmentation and feature binding. In the parietal and motor system coherent
beta/gamma oscillations were related to intentions.
Oscillations have been reported in the motor system, somatosensory cortex
Lebedev et al. 1995] and in premotor cortex [Lebedev et al. 2000]. [Murthy et al.
1992] described motor cortical oscillations in monkey cortex when the monkeys
performed motor acts that required significant attention (retrieval of raisins from
unseen locations). Similar oscillations were observed in motor cortex during
periods of immobility by other research groups. In the premotor cortex, 20-40 Hz
oscillations are often observed during periods of attentive immobility, and they
typically disappear during movements.
Oscillations recorded from multiple cortical areas can become synchonized and
form a large-scale network, whose dynamics and functional connectivity can be
studied by means of spectral analysis [Fingelkurts et al. 2003], Granger causality
[Brovelli et al, 2004], and operational synchrony [Fingelkurts et al. 2001, 2005].

2.2.4 Neural Responses During Visual Pursuit
Anticipatory movements are motor responses starting before the occurrence of
likely sensory events. In an experimental setting, this can be studied by having
subjects pursue a moving dot of light appearing on a visual display. If the visual
stimulus moves consistently in the same direction during a few presentations,
subjects anticipate that it will likely continue to do so during the next presentation
and the eyes will start to move in advance of the beginning of the next stimulus
appearance. Anticipatory pursuit is a smooth movement of the eye occurring before
the appearance of an expected moving target. The expectation of the subject is
based on a subjective estimation of the probability that the target will move in a
- 27 -
given direction. Anticipatory pursuit has been extensively studied at the behavioral
level. However, the neural mechanisms allowing this important behavior are
unknown. Recently, it has been suggested that the SEF could play a role in using
past experience to guide anticipatory pursuit. This hypothesis is currently being
tested at the single neuron level. Indeed, it has been shown that neurons in the SEF
are active during smooth pursuit in the absence of saccades [Heinen 1995],
especially when predictable changes in target motion occur [Heinen et al. 1997]. In
the behaving monkey, it has been shown that electrical microstimulation in the SEF
can facilitate smooth pursuit initiation towards a moving target, suggesting that
activation of the SEF might change the internal gain of the smooth pursuit pathway
[Missal et al. 2001]. Anticipatory

responses may be favored by using a cognitive
cue, which produces a different anticipatory pursuit response than the one observed
in pervious studies, based on repetition.

2.3 Brain Injury Effects on Physiological Signals
Hypoxic-ischemic disturbances are the result of either respiratory or cardiac
insufficiency, alone or in combination. Severe cardiac contractile dysfunction due
to either major cardiac malformations or severe hypoxemia leads to cerebral
hypoperfusion and loss of cerebrovascular regulation. In response to hypoxic-
ischemic insults, circulatory rearrangement of the cardiac output occurs, with
shunting of blood flow away from the liver, kidneys, gut, lungs, and skeletal
muscle into the heart, brain, and adrenal glands of the infant. With progression of
the hypoxic-ischemic state, the heart rate, blood pressure, and cardiac output
decrease substantially; systemic metabolic acidosis increases partly because of the
production of lactic acid [Shalaka et al. 2004].
There are many different types of bodily trauma that can bring about asphyxiation.
One set of causes is the physical prevention of the circulation of air to and from the
lungs. This can be brought on by drowning, chocking, constriction of the chest or
abdomen, and the inhalation of vomit, to name a few. Another cause is breathing in
low oxygen environments. Finally, sleep apnea, contact with a pulmonary agent, or
a seizure that stops breathing, will also all lead to asphyxiation.

Chapter 2
- 28 -
2.3.1 Cardiac Responses to Hypoxia
Hypoxia is a potent stressor that elicits complex compensations in respiration,
hemodynamics, and cardiovascular autonomic

regulations. During hypoxia, the
body attempts to maintain an adequate blood flow and supply

of oxygen to the
heart and brain, while decreasing blood supply

to other organs and mobilizing
blood from the splanchnic circulation.

Consequently, mean systemic arterial blood
pressure (P
SA
)

is normally maintained or slightly elevated during hypoxia,

whereas
heart rate (HR) tends to decrease [Campen et al 2004].
Although there is a consistent increase in pulmonary

ventilation in response to
decreased inspired PO
2
, the cardiovascular

autonomic changes may vary depending
on the experimental settings,

such as type and severity of hypoxic exposure, subject
species,

associated hypercapnia or hypocapnia, and method and depth of

anesthesia.
From the ethical and technical viewpoints,

it is difficult to obtain substantial levels
of hypoxia in humans.

In small animals, it is also difficult to maintain a consistent

level of eucapnia to eliminate the effects of hypercapnia or hypocapnia.

Furthermore, to detect autonomic responses that could developrapidly during acute
hypoxia, a high time resolution and applicability

to nonstationary data are required
for autonomic assessment [Yasuma et al 2000].
The responses in cardiovascular variables and their oscillation might reflect the
functional consequences of the systematic

defensive responses to the threat of
hypoxia of vital organs.

The observed tonic cardiovascular responses to hypoxia are
consistent

with the suppressed cardiac vagal activity and the augmented vasomotor

sympathetic activity. During advancing hypoxia, the maintenance of systemic
oxygenation is critical for the organism; consequently,

a redistribution of blood
flow to the vital organs is necessary

for survival. For this purpose, such circulatory
adjustments as sinus tachycardia to increase cardiac output and an elevation in
blood pressure, presumably suitable for the redistributionof blood flow, need to
occur.
The autonomic responses to progressive hypoxia involve tonic controls and phasic
modulations of cardiovascular

variables. The former may be characterized by the
suppressed cardiac

vagal activity and the probable augmented sympathetic activity,

which lead to sinus tachycardia and elevated blood pressure. Thelatter may be
- 29 -
characterized by the progressive reduction in respiratory

vagal modulation of heart
rate and the transient augmentationin low-frequency sympathetic modulation of
bloodpressure.

2.3.2 Neural Responses During Hypoxia
Hypoxic-anoxic injuries result when there is a substantial (partial, or hypoxic) or a
complete (total, or anoxic) lack of oxygen supplied to the brain. The brain
consumes about 20% of the body's total oxygen. 90% of the brain's total energy is
used to send electrochemical impulses and maintain the neurons' ability to send
these impulses. Much like the base of a pyramid on which everything else rests,
oxygen is necessary to metabolize glucose which is used to provide the energy for
all living cells.
Since oxygen is required for normal brain functioning, this diminished oxygen
supply to the brain may produce profound cognitive (thinking), physical
(movement), and affective (emotional) impairments which may be slow to recover.
If oxygen is not available, a cascade-effect of problems occurs. Oxygen and
glucose are responsible, either directly or indirectly, for a variety of chemical
reactions which are involved in the production of important chemical-like brain
neurotransmitters (e.g., dopamine, norepinephrine, and serotonin). Such
neurotransmitters act to regulate the brain's many complex functions. One
particular neurotransmitter, acetylcholine (Ach), seems to play a direct role in
memory.
A hypoxic-anoxic injury, also known as HAI, occurs when that flow is disrupted,
essentially starving the brain and preventing it from performing vital biochemical
processes. Hypoxic refers to a partial lack of oxygen; anoxic means a total lack. In
general, the more complete the deprivation, the more severe the harm to the brain
and the greater the consequences. HAI can be caused by a variety of disease
processes and injuries.
Anoxic anoxia: also called high-altitude sickness occurs when there is not enough
oxygen in the air to be absorbed by the body and used. This can occur at high
altitudes, where the air is thinner than at sea level, but is extremely unusual
otherwise.
Chapter 2
- 30 -
Anemic anoxia: occurs when there is not enough blood or haemoglobin. Acute
hemorrhage, chronic anemia, carbon monoxide poisoning are common causes of
this type of injury. Acute hemorrhages (bleeding) can occur due to gunshot
wounds. Chronic anemia occurs when there are persistent low red blood cells or
haemoglobin.
Ischemic anoxia (hypoxic-ischemic injury HII): - is the most common type of
injury, and occurs when there is not enough cerebral blood flow to carry blood to
the brain. The injury can be localized (such as ischemic strokes) or generalized
(circulatory collapse secondary to cardiac arrhythmias or cardiac arrest). This type
of injury causes general, diffuse damage to the cerebral cortex and cerebellum.
Areas of the brain that are very sensitive to lack of oxygen include the
hippocampus (a region critical for memory), border-zone areas of the cerebral
cortex (the parieto-occipital and fronto-parietal regions), cerebellum, basal ganglia,
and spinal cord (thoracic region) [Groswasser et al 1989].

2.3.3 Ischemic Preconditioning Effects
Although there is no current approved intervention to reduce damages or
complications following cardiac arrest, animal studies have been shown that
sublethal transient global ischemia (ischemic preconditioning) induce
neuroprotection against subsequent lethal ischemic insult. Ischemic
preconditioning (IP) is a phenomenon where brief periods of mild hypoxic
conditions prior to a severe insult enhance the ischemic tolerance in a variety of
organ systems, including the heart and brain.
For example, in heart, the IP protects from subsequent sustained coronary
occlusion both in the region where preconditioning was elicited and in remote
virgin myocardium [Przyklenk et al. 1993]. At the level of brain, previous studies
in vivo indicated that preconditioning with sublethal ischemic insults separated by
long time latency periods, protected tissues from a subsequent lethal insult
[Kitagawa et al. 1990]. Shorter latency periods between the conditioning and test
insults have been found to be protective in brain slices [Prez-Pinzn et al. 1996].
Although there is evidence that IP protects both the heart and the brain, the
intensity of ischemic insults and the latency between the insults determine the
- 31 -
degree of protection. In brain, however, IP paradigms are not well defined.
Investigation of the effects of ischemic preconditioning mostly employed models
with reduced complexity, such as cell cultures, tissue slices or perfused organ
preparations. Although such models can provide valuable insight into the protective
mechanism of preconditioning, the functional (re)organization of the control
mechanisms at the level of the living organism cannot be assessed. Hypoxia-
ischemia remains a great challenge to the researchers, since it triggers complex
regulation plays an important role [Sun 1999].

- 33 -
Chapter 3: Analysis of Physiological Time
Series
3.1 Assessment of Heart Rate Variability
Heart rate variability (HRV) represents one of the most promising markers for the
quantification of the relationship between the autonomic nervous system and
cardiovascular mortality. The apparently easy derivation of this measure has
popularized its use, since its measurements are non-invasive and easy to perform,
have relatively good reproducibility and provide prognostic information on patients
with heart disease [Kleiger et al. 1987, Casolo et al. 1989, Hayano et al. 1990, Van
Hoogenhuyze et al. 1991, Bigger, Jr. et al. 1992, Bigger, Jr. et al. 1996, Huikuri et
al. 1998]. As many commercial devices now provide automated measurement of
HRV, the cardiologist has been provided with a seemingly simple tool for both
research and clinical studies. However, the significance and meaning of the many
different measures of HRV are more complex than generally appreciated and there
is a potential for incorrect conclusions and for excessive or unfounded
extrapolations.

3.1.1 Linear Methods of Assessing Heart Rate Variability
3.1.1.1 Time Domain Analysis of Heart Rate Variability
Variations in heart rate may be evaluated by a number of methods. One of the
simplest analyses to perform are the time domain measures. With these methods
Chapter 3
- 34 -
either the heart rate at any point in time or the intervals between successive normal
complexes are determined. In a continuous electrocardiographic (ECG) record,
each QRS complex is detected, and the so-called R-to-R (RR) intervals or the
instantaneous heart rate is determined. Simple timedomain variables that can be
calculated include the mean and variance of the RR interval (as presented in Figure
3.1), the mean heart rate, the difference between the longest and shortest RR
interval, the difference between night and day heart rate, etc. The most widely used
time domain index is the average heart rate, which is easy to calculate over a
suitable length of time. The calculations of other different time domain indices
naturally require precise timing of R waves. Other timedomain measurements that
can be used are variations in instantaneous heart rate secondary to respiration, tilt,
Valsalva manoeuvre, or secondary to phenylephrine infusion. These differences
can be described as either differences in heart rate or cycle length.

Fig. 3.1 Schematic representation of normal ECG trace, with waves, and RR
intervals labeled

Time domain analysis can be performed on short electrocardiogram segments
(lasting from 0.5 to 5 minutes) or on 24-hour electrocardiographic recordings.
Beat-to-beat or short term variability represents fast changes in heart rate while
long-term variability indices mainly reflect slower fluctuation of RR intervals.
From a series of instantaneous heart rates or cycle intervals, particularly those
recorded over longer periods, traditionally 24 h, more complex statistical time-
domain measures can be calculated. These measures can be divided into two
classes: (a) those derived from direct measurements of the RR intervals or
instantaneous heart rate (standard deviation of beat-to-beat RR interval differences
Linear and nonlinear methods for physiological time-series analysis
- 35 -
within the time window is one short term variability index frequently used), and (b)
those derived from the differences between RR intervals such as standard deviation
of all the RR intervals or the difference between maximum and minimum RR
interval length, within the window. These variables may be derived from analysis
of the total electrocardiographic recording or may be calculated using smaller
segments of the recording period. The latter method allows comparison of HRV to
be made during varying activities, e.g. rest, sleep, etc. The measures should be
compared within segments of similar length, and so the durations of the recordings
used to determine the above indexes values (and similarly other HRV measures)
should be standardized. Usually, short-term 5-min recordings and nominal 24h
long-term recordings seem to be appropriate options.
The most commonly used index is the standard deviation of all normal-to-normal
RR intervals (SDRR) over a 24h period, which is the square root of variance. Since
variance is mathematically equal to total power of spectral analysis, SDRR reflects
all the cyclic components responsible for variability in the period of recording. This
index is probably also the best known heart rate variability index. [Kleiger et al.
1987] estimated RR interval standard deviation over a 24h period as a predictor of
mortality in postmyocardial infarction patients. This estimate reflects primarily the
very low frequency fluctuation in heart rate behavior, not the heart rate fluctuations
in segments with a duration of < 1 minute, because these fast fluctuations of RR
intervals drown under the slower waves.
The most commonly used measures derived from interval differences include: (i)
RMSSD - which is the square root of the mean squared differences of successive
RR intervals, (ii) NN50 - which is a measure of the instantaneous difference over
50 ms between two consecutive normal-to-normal RR intervals [Ewing et al.
1984], and (iii) pNN50 which is the proportion derived by dividing NN50 by the
total number of RR intervals. All the time domain measure indices could be
affected by artifacts and outliers, and these measures therefore require data from
which artifacts and ectopic beats have been carefully eliminated.

3.1.1.2 Frequency Domain Measures of Heart Rate Variability
Various spectral methods [Kay et al 1981] for the analysis of the tachogram have
been applied since the late 1960s. Since the introduction of spectral analysis as a
Chapter 3
- 36 -
method for studying heart rate variability [Akselrod et al. 1981, Bloomfield 1976],
an increasing number of investigators have utilized this method. Power spectral
density (PSD) analysis provides the basic information of how power (i.e. variance)
distributes as a function of frequency. The main advantage of spectral analysis of
signals is the possibility to study their frequency-specific oscillations. Spectral
analysis involves decomposition of the series of sequential RR intervals into a sum
of sinusoidal functions of different amplitudes and frequencies. The result can be
displayed with the magnitude of variability as a function of frequency (power
spectrum). The power spectrum reflects the amplitude of the heart rate fluctuations
present at different oscillation frequencies.
Independent of the method employed, only an estimate of the true PSD of the
signals can be obtained by proper mathematical algorithms. Methods for the
calculation of PSD may be generally classified as non-parametric and parametric.
In most instances, both methods provide comparable results.
The power spectrum can be divided into different spectral bands and the powers in
these bands is further calculated. The spectrum is usually divided into three or four
different bands, depending on the major frequency bands. Because of the important
differences in the interpretation of the results, the spectral analyses of short- and
long-term electrocardiograms should always be strictly distinguished. The
boundaries of the most commonly used frequency bands are as follows: ultra low
frequency (ULF) which contains all frequencies < 0.0033 Hz, very low frequency
(VLF) from 0.0033 0.04 Hz, low frequency (LF) from 0.04 0.15 Hz and high
frequency (HF) from 0.15 to 0.4 Hz. The boundaries that should be used in
physiological studies have been recommended by European Society of Cardiology
and the North American Society of Pacing and Electrophysiology [Task Force,
1996]. These recommendations are based on a suggested, but only partly proved,
physiological background of heart rate variability.

Prior to the frequency domain analysis, the unevenly sampled RR sequences
resulted after the QRS detection algorithm, have to be converted into an uniformly
spaced time-series using a resampling method followed by a cubic spline
interpolation.
The resampling frequency must be sufficiently high to avoid aliasing. (Nyquist
frequency to be outside of the frequency range of interest) [Task Force, 1996].
- 37 -
Power spectral density can be estimated using Welchs averaged periodogram
method. Each segment of RRI is divided into overlapping sections of 50% of their
length. Each section is further windowed with a Hamming window and for each,
modified periodograms are computed and averaged. The quantification of the
power spectrum was achieved by defining the LF and HF bands and computing the
energy in each band as the percentage of the total energy.
For the HRV analysis in rats, different studies used various selections of the LF and
HF bands, showing controversial results. In our study we defined the range 0.195-
0.6 Hz for LF band, and 0.6-2.5 Hz for HF band [Aubert et al. 1999] (Figure 3.2).

Fig. 3.2 Example of power spectral quantification of representative baseline
RRI signal in rats. The LF (0.195-0.6 Hz) and HF (0.6-2.5 Hz) bands are
defined. The peak centered around 1 Hz reflects respiratory sinus arrhythmia
(RSA).

Additionally, the evaluation of neurocardiac function using spectral analysis of
heart rate has been described in a limited number of studies. Most of these studies
investigated the response to pharmacological interventions [Just et al. 2000]. The
representation of LF and HF is computed in normalized units (n.u.) to emphasize
the controlled and balanced behaviour of the two branches of the autonomic
nervous system [Malliani et al. 1991].
Another index employed is the ratio between the LF power and HF power, the
LF/HF index. The LF/HF ratio has been associated with the so-called sympatho-
vagal balance but the physiological basis for the use of this parameter is unclear
[Eckberg, 1997].
Chapter 3
- 38 -
A number of animal and human experiments with pharmacological blockade of the
ANS have revealed that the power in the LF and HF ranges reflects the HR
modulation of sympathetic and parasympathetic components. This view has been
further supported by the fact that HRV is dramatically reduced in denervated
human hearts [Dureau et al. 1997]. As a measure of vagal activity, spectral analysis
of the high-frequency component has been widely employed. Some investigators
have used the ratio of the low-to-high frequency spectra as an index of
parasympathetic-sympathetic balance [Paggani et al 1986]; however, this remains
controversial [Eckberg 1997] because of our lack of complete understanding of the
low frequency component (which seems to be affected by centrally generated
brainstem rhythms, baroreceptor feedback influences, as well as both sympathetic
and vagal input).
The analysis of heart rate power spectra has also proven useful in assessing
autonomic function in patients with condition associated with autonomic failure
such as diabetes or hypertension. The observation of the beat-to-beat variability of
the heart rate (and other cardiovascular variables) has been shown to provide a
measure by which autonomic nervous system function can be assessed, providing a
tool for characterizing autonomic dysfunction in a variety of disease states.
When analysing stationary short-term recordings, more experience and theoretical
knowledge exists on the physiological interpretation of the frequencydomain
measures compared to the timedomain measures derived from the same
recordings. However, many time- and frequency-domain variables measured over
the entire 24-h period are strongly correlated with each other. These strong
correlations exist because of both mathematical and physiological relationships.

3.1.2 Insight Into the Nonlinear Dynamics of HRV
Non-linear phenomena are certainly involved in the genesis of HRV, being
determined by complex interactions of haemodynamic, electrophysiological and
humoral variables, as well as by autonomic and central nervous regulations. It has
been speculated that analysis of HRV based on the methods of non-linear dynamics
might elicit valuable information for the physiological interpretation of HRV and
for the assessment of the risk of sudden death. There is increasing evidence to
- 39 -
suggest that the heart is not a periodic oscillator under normal physiologic
conditions [Babyloyantz et al. 1988, Kaplan et al. 1987, Goldberger et al 1987],
and the commonly employed moment statistics of heart rate variability may not be
able to detect subtle, but important changes in heart rate time series. Therefore
several new analysis method of heart rate behaviour, motivated by nonlinear
dynamics and chaos theory, have been developed to quantify the dynamics of heart
rate fluctuations [Goldberger et al. 1987, Pincus 1991, Yamamoto et al. 1991].
Heart rate variability reflects the complex interactions of many different control
loops of the cardiovascular system. In relation to the complexity of the sinus node
activity modulation system, a more predominantly nonlinear behavior has to be
assumed. Thus, the detailed description and classification of dynamic changes
using time and frequency measures is often not sufficient [Cimponieriu et al.
2000]. Experimentally observed sequences of heart periods are short, noisy and
often nonstationary. This favours those measures of complexity able to quantify the
system dynamics from rather short time series.
Although in principle these techniques have been shown to be powerful tools for
characterization of various complex systems, no major breakthrough has yet been
achieved by their application to bio-medical data including HRV analysis. It is
possible that integral complexity measures are not adequate to analyse biological
systems and thus, are too insensitive to detect the non-linear perturbations of RR
interval which would be of physiological or practical importance. However, no
systematic study has been conducted to investigate large patient populations using
these methods.
At present, the non-linear methods represent potentially promising tools for HRV
assessment, but standards are lacking and the full scope of these methods cannot be
assessed. Advances in technology and the interpretation of the results of non-linear
methods are needed before these methods are ready for physiological and clinical
studies.
We have introduced a method derived from the symbolic dynamics transformation
to distinguish between different states of the HRV dynamics. The first step of this
approach is the transformation of the time series into symbol sequences with
symbols from a given alphabet. Some detailed information is lost in this process,
Chapter 3
- 40 -
but the coarse dynamic behaviour is retained. Transformations into symbols have
to be chosen on a context- dependent basis.
The sequence of heart periods can be analyzed with the help of entropy measures
such as Shannon entropy or renormalized entropy [Aubert et al.1999, Troncoso et
al.1995]. These measures used in conjunction with the concept of symbolic
dynamics proved to be useful in detection of patients at high risk for sudden
cardiac death [Pagani et al. 1986].
Non-linear methods may provide more appropriate tools to investigate the
interaction between the various regulatory systems of the heart rate, due to the
intrinsic nonlinearity of physiological systems.

3.1.2.1 Information Theoretical Methods
Approximate Entropy Analysis
Approximate entropy is a measure and parameter that quantifies the regularity or
predictability of time series data. It has been developed for time series to classify
complex systems that include both deterministic chaotic and stochastic processes
[Pincus 1991, Pincus et al 1994, Pincus et al. 1992]. Reduced complexity of heart
rate dynamics has been found in sick neonates and in patients with postoperative
complications after cardiac surgery [Pincus et al. 1992, Fleisher et al. 1993]. The
obvious advantage of this method is its capability to discern changing complexity
from a relatively small amount of data. This makes the approximate entropy
measure applicable to a variety of contexts. This measure cannot certify chaos.

Approximate entropy (ApEn) is a statistic that can be used as a measure to quantify
the complexity (or irregularity) of a signal [Pincus, 1991]. We will summarize first
the definition of ApEn, followed by the physiological interpretations.
Given a time series (e.g., R-R tachogram) with N data points u(1), u(2), . . . , u(N),
sequences of vectors x(1), . . . , x(N- m + 1) are formed by defining

x(i) = [u(i), u(i + 1), . . . , u(i + m - 1)], i=1,N-m+1 (3.1)

- 41 -
The parameter m, the number of components in each vector, has to be fixed. In
nonlinear dynamics theory this would be interpreted as an "m-dimensional state
space reconstruction". Next, the distance d[x(i), x(j)] between two vectors x(i) and
x(j) (j=1,N-m+1) is defined as the maximum difference of all their scalar
components as:

d[x(i),x(j)]=max[ | u( i +k- 1) - u( j +k- 1)|], k=1,2,...,m (3.2)

The "correlation sum" of vector x(i) is:

1 m - N
r ] x(j) d[x(i), such that 1 m - N j of no.
) (
+
+
= r C
m
i
(3.3)

The parameter r acts like a filter value: within resolution r, the numerator counts
the number of vectors that are approximately the same as a given reference vector
x(i). The quantity ) (r C
m
i
is called the correlation sum because it quantifies the
summed (or global) correlation of vector x(i) with all other vectors.
Next, the mean logarithmic correlation sum of all vectors is defined as:

+
=
+
=
1
1
) ( log
1
1
) (
m N
i
m
i
m
r C
m N
r (3.4)

and ApEn is represented as:

) ( ) ( (u) N) r, ApEn(m,
1 m
r r
m+
= , m 1 (3.5)

ApEn(m=2, r=0.2SD, N) (u) measures the logarithmic frequency with which
vectors with two components that are close (within resolution r=0.2SD)
remain close when the number of vector components is increased by one. This
is the key to a measure of irregularity: small values of ApEn indicate
regularity (predictability), and large values imply substantial fluctuations or
irregularity (randomness) in a time series u. Impairment of the function of the
Chapter 3
- 42 -
autonomic nervous system during hypoxia should lead to regularization and/or
diminished variability of the heart rate [Chaffin et al. 1995].

Symbolic Dynamics Analysis
Different techniques of symbolic dynamics for data analysis are further presented.
In a first step, the record has to be transformed into a symbolic string. In doing so
one loses a certain amount of detailed information, but some of the invariant,
robust properties of the dynamics are retained.
We can distinguish static transformations, where the transformation is based on a
few thresholds and dynamic ones, where step-to-step difference of data points
adjacent in time is considered.
An example of a static transformation using four symbols is explained in equation
3.6.

<
+ <
+ >
=

) 1 ( : 3
) 1 ( : 2
) 1 ( : 1
) 1 ( : 0
) (
a x
x a
a x
a x
x s
i
i
i
i
i i
(3.6)
The time series of the beat to beat intervals x
1
,x
2
,x
N
is transformed into the
symbol sequence s
1
,s
2
,.s
N
, s
i
={0,1,2,3}. The transformation into symbols
refers to three given levels where denotes the mean beat-to-beat interval and a is
a special parameter that we have chosen 0.05; we tested several values of a from
0.05 to 0.08, however the resulting symbol sequences differed not significantly.
The second approach was to transform the data segments using a dynamical
transformation, where the step-to-step differences in the sequence are taken into
account. We consider the kind of difference between two adjacent measurement
values:

=

otherwise
x x
s
i i
i
: 0
: 1
1
(3.7)
This method extracts solely dynamic properties of the R-R series, disregarding all
information influenced by the absolute values of the R-R intervals, e.g., mean R-R
- 43 -
interval, R-R standard deviation, and other measures of R-R interval variability
[Mezzacappa et al 1994].
Another symbol transformation could be defined as:
<
< <
< >
>
=

m x x abs x x
m x x abs x x
m x x abs x x
m x x abs x x
x s
i i i i
i i i i
i i i i
i i i i
i i
) ( & : 3
) ( & : 2
) ( & : 1
) ( & : 0
) (
1 1
1 1
1 1
1 1
(3.8)
where m is a threshold defined by the difference between two successive R-R
intervals and the value was selected to be 1ms. This transformation preserve the
dynamical information whereas the threshold m acts as a filtering component.

3.1.2.2 Geometrical Methods of Heart Rate Variability Analysis
Geometrical methods present RR intervals in geometric patterns and various
approaches have been used to derive measures of heart rate variability from them.
The triangular index is a measure where the length of RR intervals serves as the x-
axis of the plot and the number of each RR interval length serves as the y-axis. The
length of the base of the triangle is used and approximated by the main peak of the
RR interval frequency distribution diagram. Triangular interpolation approximates
the RR interval distribution by a linear function and the baseline width of this
approximation triangle is used as a measure of the heart rate variability index
[Malik et al 1991, Farrell et al. 1992]. This triangular index had a high correlation
with the standard deviation of all RR intervals, but it is highly insensitive to
artifacts and ectopic beats, because they are left outside the triangle. This reduces
the need for preprocessing of the recorded data.
Another geometrical measure is the Poincar plot, a diagram (scattergram) in which
each RR interval is plotted as a function of the previous RR interval. Poincar plots
can be interpreted visually and also quantitatively [Huikuri et al. 1998, Tulppo et
al. 1996]. Instantaneous beat-to-beat variability of data and continuous long-term
variability of RR intervals can be calculated. In addition to the present quantization,
which can be classified as geometrical, the Poincar plot also gives a useful visual
scheme of the RR data by representing qualitatively with graphic means the kind of
RR variations included in the recording. The shape of the plot can be used to
Chapter 3
- 44 -
classify the signal into one of several classes [Woo et al. 1994, Schechtman et al.
1993], and the irregular shapes quanfied from Poincar plots may then be classified
as nonlinear.
Visual inspection of the Poincare plot has been largely used in the analysis of
HRV. The Poincare plot is a diagram where each RRI(i) is plotted as a function of
RRI(i-t), where t is a predefined delay. Typically, t=1 has been used for the RRI
signal. The length and width of the Poincare plot have been suggested as indicative
of the levels of long- and respectively short-term variability. The Poincare plot may
be analyzed quantitatively by calculating the standard deviations of the distances of
the RRI(i) to the lines y = x and y = -x + 2*RRI
m
, where RRI
m
is the mean of all
RRI(i) (Figure 3.3).

Fig. 3.3 Poincare plot of the representative baseline RRI signal in rats. The
parameters SD1 and SD2 are defined as standard deviation of the distance of
each RR(i) from the lines y=x and y=-x+2*RRI
m
where RRI
m
is the mean of all
RRIs.

These standard deviations are referred to as SD1 and SD2, respectively. SD1 is
related to the instantaneous beat-to-beat variability of the data, while SD2 describes
the longer-term variability of RRI [Tulppo et al. 1996, Brennan et al. 2001,
Korhonen et al. 2001]. The ratio SD1/SD2 may also be computed to describe the
relationship between these components. This analysis does not require any pre-
- 45 -
processing and minimal assumptions of stationarity of the data, and is hence
especially attractive.

3.1.2.3 Other Approaches
Among others parameters which have were used to measure non-linear properties
of HRV we noted the 1/f scaling of Fourier spectra [Goldberger & West 1987,
Goldberger 1996, Goldberger et al. 1987]. Spectral analysis alone cannot
distinguish a chaotic process either, but some investigators have suggested that a
particular spectral pattern (one in which the power density is inversely related to
frequency) is highly suggestive of a nonlinear or chaotic process. However, the
diagnostic value of this 1/f pattern has also been questioned [Pool 1989, since
wrinkle fluctuations occur in the human heart rate dynamics, which have many of
the characteristics of nonlinear dynamics and deterministic chaos.
For data representation plots, singular value decomposition and attractor
trajectories have been used. By evaluating the Haussdorff correlation dimension D,
evidence of the chaotic nature of cardiac activity can be obtained [Berg et al.
1984, Eckmann et al 1985]. Haussdorff dimension D is a measure of the
complexity of the system: the lower the value of D, the more coherent the
dynamics. D = 1 presents periodic oscillations. If D has non-integer values greater
than two, it defines a chaotic behaviour [Grassberger et al 1983b, Berg et al. 1984,
Eckmann et al. 1985, Mayer-Kress 1987]. Although D is a convenient measure,
because it does not require the system to be stationary, it unfortunately always
involves a potentially large error of estimation. Therefore, instead of using D, it is
more convenient to evaluate the correlation dimension D2 from a time series with
the help of the existing algorithms [Grassberger et al. 1983a, 1983b, Eckmann &
Ruelle 1985].
For other quantitative descriptions Lyapunov exponents and Kolmogorov entropy
indexes have been used. The Lyapunov exponent [Wolf et al. 1985, Eckmann et al.
1985] is a quantitative measure of separation the trajectories that diverge widely
from their initial close positions. The major limitation in their calculation is that the
currently available algorithms require large amounts of data and long computing
times. Also, the system must remain stable over the recording time, but biologic
systems seldom remain stable. Kolmogorov entropy K was also proposed to
Chapter 3
- 46 -
analyse the nonlinearity of HRV, which may be estimated by a procedure
[Grassberger et al. 1983b, Eckmann et al. 1985] close to the one used for
dimension D. K-entropy is related to the sum of the positive Lyapunov exponents
[Eckmann et al. 1985]. The above methods can be evaluated quantitatively and are
diagnostic of chaos, whereas spectral analysis, time autocorrelation function and
Poincar plot construction are qualitative methods.
Whether the various nonlinear methods detect chaotic behaviour is an important
academic issue, but from the practical point of view, it is important to know
whether they are applicable for clinical purposes. The prognostic accuracy and
clinical applicability of these measures are not well known.

3.2 Analysis of Brain Electrical Activity
3.2.1 General Approaches
The conventional approach to analyzing EEG rhythm is to obtain power spectra
and delineate power in different spectral bands [Levy et al. 1990, Williams et al
1985]. Alternatively, an algorithm that identifies dominant frequencies in EEG was
shown to be more responsive to recovery patterns of brain rhythm following
ischemic injury [Goe et al. 1996]. Clinically, it may be useful to obtain a single
measure of such a recovery response as a diagnostic monitoring tool. Distance
metrics, spectral and cepstral distance, have been proposed to determine the
differences in the spectra of normal and injured brain[ Kong et al 1999]. However,
such distance measures require comparison with base line, and in clinical
situations, particularly unanticipated cardiac arrest, such base-line data are usually
not available. Moreover, the distance metrics are not sensitive to the rapidly
changing signal statistics (nonstationarities) of the recovering EEG.
A recent approach motivated by the need to develop a rigorous measure of the
degree of disorder (or complexity) of the EEG signal in brain injury has been
considered by [Bezerianos et al. 2003]. The EEG signal complexity has been
studied by means of the correlation dimension D2 [Casdagli et al. 1997, Lehnertz et
al 1998, Stam et al 1999]. A basic requirement for applying the tools of nonlinear
dynamics (chaos theory) to experimental data is the stationary of the time series.
This suggests that the time series is representative of a unique and stable attractor
- 47 -
and is statistically invariant over different time intervals. Also, for the evaluation
chaotic measures like D2, long-time recordings are required. Generally, these
measures are noise sensitive and their usefulness decreases especially in the case of
additive noise [Abarbanel 1996, Basar 1998]. Unfortunately, the EEG data are
noisy and the stationarity requirement is not fulfilled; therefore, other methods
have to be explored. [Bezerianos et al. 2003] postulate that entropy analysis will
provide a quantitative measure of the degree of disorder in the brain rhythm at
various times in brain injury and recovery.
An important application of the tools of nonlinear dynamics is the study of brain
activity at different levels of neuronal organization, from the cellular level up to
large scale neuronal networks [Schiff et al.1996, Tass et al. 1998, van Quyen et al.
1998, Arnhold, et al. 1999, Rodriguez et al. 1999, Neiman et al. 1999, 2002].
Numerous theoretical studies have been devoted to the understanding of the inter-
cellular coupling mechanisms in conjunction with the intrinsic properties of the
neurons account for experimentally observed coherent, rhythmical behavior in
neuronal populations. [Pinsky et al. 1994]. This knowledge could further illuminate
on the functional role of interaction between different populations in brain
information processing, both in normal and pathological conditions. The ultimate
goal of the research in this field is to translate theoretical knowledge into novel
noninvasive diagnostic tools (e.g., synchronization tomography [Tass et al.
2003]) and therapeutic techniques (e.g., demand-controlled desynchronizing deep
brain stimulation based on stochastic phase resetting [Tass 2000]).
Rhythmical phenomena in the electrical activity of the brain can be often
understood within the framework of the coupled oscillators theory. This formalism
has provided explanation for a number of observed synchronization phenomena
and coherent behavior in neuronal ensembles. In addition, techniques derived from
synchronization theory have found useful applications in the analysis of brain
electrical activity [Tass et al. 1998, Mormann et al 2000, Quiroga et al. 2000]. Non-
invasive brain recordings are typically multichannel measurements of electric
(EEG) or magnetic brain activity (MEG) which reveal the coexistence of broad-
band activity and rhythms of different frequencies.

Chapter 3
- 48 -
3.2.2 New Approaches of Brain Oscillations
Aside from revealing and quantifying interdependencies from physiological signals
analysis, a close related but less explored problem is that of identifying directional
coupling between the underlying systems. This problem becomes of particular
relevance in the context of studying effective connectivity in the brain (the
influence one system exerts over another either directly or indirectly). All the
above mentioned techniques of data analysis cannot detect exchange in information
or asymmetric relationships between two systems.
This problem has been recently approached from the viewpoint of information
theory. If the presence of interaction is established or physiologically motivated,
directional interdependencies can be described in terms of information transfer
[Granger 1969; Schreiber 2000; Palus et al. 2001]. Another approach is based on
the notion of dynamical interdependence [Schiff et al.1996], i.e. on existence of a
functional relationship between the points in the (reconstructed) phase spaces of
two systems; this relationship can be asymmetrical. The latter approach is tightly
related to the notion of generalized synchronization and can be also described in
terms of mutual predictability [Quiroga et al. 2000].
Recently, the problem of identification of asymmetric relations between interacting
dynamical systems has been approached from the viewpoint of coupled oscillators
theory. It has been reported that direction of interaction in weakly interacting
oscillator systems can be identified [Rosemblum et al. 2002]. This approach to
analysis of directionality of interaction is explicittly based on the assumption that
experimentalists deal with weakly interacting self-sustained oscillators. In this
particular, but pretty often encountered case the direction of coupling can be
efficiently quantified.

3.2.2.1 Empirical Mode Decomposition Technique
As introduced by [Huang et al. 1998], the empirical mode decomposition (EMD)
technique is capable of adaptively decomposing signals into oscillating intrinsic
components. The EMD is an adaptive decomposition technique with which any
complex signal can be decomposed into a definite number of high frequency and
- 49 -
low frequency components by means of a process called sifting. These components
are called intrinsic mode functions (IMF). These IMFs have well defined
instantaneous attributes and are defined as functions which (1) have the same
number of zero-crossings and extrema; and (2) the mean value of the upper and the
lower envelopes is equal to zero.
The key innovation embodied in the method is the introduction of the IMF, which
is based on the local natural properties of the signal and which gives a subsequent
meaning to the concept of instantaneous frequency. An IMF can be best defined as
a hidden oscillation mode that is embedded in the data series. Suppose x(t) is the
signal to be decomposed. The sifting process is conducted by first constructing the
upper and lower envelope of x(t) by connecting its local maxima and local minima
through a cubic spline. The mean of the two envelopes is then computed and
subtracted from the original time history. The difference between the original time
history and the mean value is called the first IMF if it satisfies the following two
conditions: (1) have the same numbers of zero crossing and extrema and (2) are
symmetric with respect to the local mean. The first condition is similar to the
narrow-band requirement for a stationary Gaussian process. The second condition
modifies a global requirement to a local one, and is necessary to ensure that
instantaneous frequency will not have unwanted fluctuations as induced by
asymmetric waveforms. The difference between x(t) and imf
1
is then treated as a
new time history and subjected to the same sifting process, giving the second IMF.
The EMD procedure continues until the residue becomes so small that it is less
than a predetermined value of consequence, or the residue becomes a monotonic
function. The original time history x(t) is finally expressed as the sum of the IMF
components plus the final residue:
=
+ =
n
i
i
t r t imf t x
1
) ( ) ( ) (
(3.9)
where r(t) is the residue of the decomposition.
The phase of each imf
i
(t) has been obtained using the Hilbert transform:

t d
t t
t imf
V P t H
i
i
imf
) ( 1
. . ) (
, (3.10)
Chapter 3
- 50 -
where P.V. stands for the Cauchy principal value for integral. The analytic signal is
constructed to yield an amplitude function A(t) and a phase function ) (t ,
) (
) ( ) ( * ) ( ) (
t i
i imf i i
i
i
e t A t H i t imf t

= + =
(3.11)

The EMD technique is described in Figure 3.4.

Fig. 3.4 EMD of two component signal, (a) sum of two components, (b)
lower and upper envelopes and their mean, (c) the first IMF and (d) the first
residual (after Oonincx and Hermand, 2004)

The decomposition of the signal into IMFs is performed as follows (Figure 3.4):
1. Identify the positive peaks (maxima) and negative peaks (minima) of the original
signal.
2. Construct the lower and the upper envelopes of the signal by the cubic spline
method.
3. Calculate the mean values by averaging the upper envelope and the lower
envelope.
4. Subtract the mean from the original signal to produce the first intrinsic mode
function imf1 component.
5. Calculate the first residual component by subtracting imf1 from the original
signal. This residual component is treated as new data and is subjected to the same
processes described above to calculate the next IMF.
- 51 -
6. Repeat the steps above until the final residual component becomes a monotonic
function and no more IMFs can be extracted.

3.2.2.2 Phase Synchronization Analysis
Phase synchronization is the process by which two or more cyclic systems (or
subsystems) tend to oscillate with a repeating sequence of relative phase angles.
The stability of phase relations (or phase delay, or cross phase) between two
oscillations of the same frequency at different sites of the cortex is called phase
synchronization by several authors [Rodriguez et al. 1999; Varela et al. 2001; Gong
et al. 2003; Jansen et al. 2003, Pilovski et al. 2001]. The circular statistics of phase
delays is known as phase coherence and it rates the variability of phase delays.
Because of the widespread use of the circular statistics of phase delays, this
parameter is often equated with the term called phase synchronization. In contrast
to the coherence function, amplitude information has no influence on phase
coherence. Other parameters like entropy measures [Tass et al. 1999] are also
applied to evaluate the stability of phase delays. Different methods were introduced
to study synchronization of ongoing oscillations. Estimation methods for
instantaneous phase, which are performed on the basis of analytic signals or via
Hilbert filters [Arnold et al.2002] do not require the stationarity of phase dynamics
(e.g., stationarity within a sliding window) [Schack et al 2005].
The evaluation of phase synchronization is based on the calculation of
instantaneous phases. Widespread approaches used for this procedure are first the
Hilbert transform [Tass et al. 1998] and second the convolution with a complex
wavelet [Lachaux et al. 2000; Gong et al. 2003]. Both methods were compared and
found fundamentally equivalent for the study of neuroelectric signals [Quiroga et
al. 2002].

The degree of synchronization between each corresponding imf
i
s of bivariate EEG
signals, can be measured by a statistic on the distribution of their phase difference:

= ))) ( ) ( ( exp(
1
1 2
t t i
N
R
i i i
(3.12)

Chapter 3
- 52 -
R
i
takes on values in the range from 0 (non phase synchronization) to 1 (perfect
synchronization). The phase synchronization analysis of the dominant intrinsic
modes can provide a useful indicator of the degree of interaction between coupled
dynamical systems.

3.2.3 Analysis of Anticipatory Pursuit in Behaving Monkey
Anticipatory movements are actions starting before the occurrence of likely sensory
events. In an experimental setting, this can be studied by having subjects pursue a
moving dot of light appearing on a visual display. If the visual stimulus moves
consistently in the same direction during a few presentations, subjects anticipate
that it will likely continue to do so during the next presentation and the eyes will
start to move in advance of the beginning of the next stimulus appearance.
Anticipatory pursuit is a smooth movement of the eye occurring before the
appearance of an expected moving target. The expectation of the subject is based
on a subjective estimation of the probability that the target will move in a given
direction. Anticipatory pursuit has been extensively studied at the behavioural
level. However, the neural mechanisms allowing this important behaviour are
unknown. Recently, it has been suggested that the SEF could play a role in using
past experience to guide anticipatory pursuit. This hypothesis is currently being
tested at the single neuron level. In the behaving monkey, it has been shown that
electrical microstimulation in the SEF can facilitate smooth pursuit initiation
towards a moving target, suggesting that activation of the SEF might change the
internal gain of the smooth pursuit pathway. In our study, we favoured anticipatory
responses in monkeys by using a cognitive cue, which produces a different
anticipatory pursuit response than the one observed in pervious studies, based on
repetition.
The hypothesis that the SEF contains neurons active before the anticipatory pursuit
movements motivated us to search for correlations between anticipatory pursuit
velocity and the corresponding spike density activity.
To determine whether the instantaneous activity of burst neurons is related to the
instantaneous velocity of the eye, it is computed the cross-correlation function
between the velocity of the eye and the spike density waveform. The cross-
correlation method relies on the basic assumption that the spike density function is
- 53 -
related to the velocity of the eye [Missal et al. 2000]. The value of the correlation
will increase and reach a maximum when the time delay between the two signals is
equal to zero. If n is the number of data points in the signal, the result of that
procedure is a cross-correlation function with 2*n -1 elements. The maximum of
the cross-correlation function is a good indicator of the dynamic correlation
between the eye velocity and spike density function.

Fig. 3.5 Cross-correlation method; example of a cross-correlation function
computed between the velocity of the eye and the spike density waveform.

Figure 3.5A shows an example of a crosscorrelation function between eye velocity
and spike density. The maximum correlation was 0.97. The resulting cross-
correlation function allows to determine the delay of a neurons activity with
respect to the saccade because the position of the peak in the crosscorrelation
function depends on the number of steps necessary to bring the signals into
register. In Fig. 3.5A, the position of the peak of the correlation function is at
sample 120, whereas the middle of the function is at sample 112 (n = 112). Figure
3.5B shows the two signals before (left) and after the spike density was shifted with
Chapter 3
- 54 -
respect to eye velocity by 16 ms (right). The cross-correlation method gives
therefore a quantitative measure of the dynamic correlation between eye velocity
and spike density and an alternative method of measuring burst latency. This
estimation of burst latency is similar to the dynamic lead time proposed by
Cullen and Guitton (1997).

- 55 -
Chapter 4:
Analysis of Electrophysiological Signals in
Response to Brain Injury
Due to the multi-disciplinary nature of the research, this chapter is divided into two
sections, describing its two aspects. The first section presents an application of
HRV signal analysis following an asphyxia- hypoxia experiment in rats; the second
one consists in neural signal analysis for the same experiment.
The experimental data analysed in this chapter has been provided courtesy of Prof.
Nitish Thakor, Dept. of Biomedical Engineering, Johns Hopkins University School
of Medicine.

4.1 The Effects of Ischemic Preconditioning on the HRV Response
to Transient Global Ischemia
4.1.1 Introduction
About 70 000 persons per year are successfully resuscitated after cardiac arrest in
both hospital and community settings in the United States. The cardiovascular
complications and neurological recovery after successful resuscitation from cardiac
arrest largely influences the morbidity and mortality in these patients [Longstreth et
al. 1983].
Although there is no current approved intervention to reduce damages or
complications following cardiac arrest, animal studies have been shown that
Chapter 4
- 56 -
sublethal transient global ischemia (ischemic preconditioning) induce
neuroprotection against subsequent lethal ischemic insult. Ischemic
preconditioning (IP) is a phenomenon where brief periods of mild hypoxic
conditions prior to a severe insult enhance the ischemic tolerance in a variety of
organ systems, including the heart and brain.
For example, in heart, the IP protects from subsequent sustained coronary
occlusion both in the region where preconditioning was elicited and in remote
virgin myocardium [Przyklenk et al. 1993]. At the level of brain, previous studies
in vivo indicated that preconditioning with sublethal ischemic insults separated by
long time latency periods, protected tissues from a subsequent lethal insult
[Kitagawa et al. 1990]. Shorter latency periods between the conditioning and test
insults have been found to be protective in brain slices [Prez-Pinzn et al. 1996].
Although there is evidence that IP protects both the heart and the brain, the
intensity of ischemic insults and the latency between the insults determine the
degree of protection. In brain, however, IP paradigms are not well defined.
Investigation of the effects of ischemic preconditioning mostly employed models
with reduced complexity, such as cell cultures, tissue slices or perfused organ
preparations. Although such models can provide valuable insight into the protective
mechanism of preconditioning, the functional (re)organization of the control
mechanisms at the level of the living organism cannot be assessed. Hypoxia-
ischemia remains a great challenge to the researchers, since it triggers complex
regulation plays an important role [Miao-Kun et al. 1999].
The analysis of heart rate variability (HRV) can provide valuable insights into the
neural control of the heart, giving rise to a new discipline: 'neurocardiology', which
studies the interactions between the neural system and the heart. HRV has been
extensively studied to understand and quantify the ANS functioning, being of
particular importance for the diagnosis and prognosis of pathological conditions,
like sudden infant death syndrome, heart failure or sleep apnea. The purpose of the
present animal model study was to evaluate the effect of global ischemic
preconditioning on the dynamic time course of heart rate variability response to the
asphyxia insult using linear and nonlinear measures of HRV. Simple linear time
Information domain analysis on rats electrical response to injury
- 57 -
domain statistics, such as standard deviation (SD) and root mean square of
successive R-R-interval differences (RMSSD) have been widely employed in
quantification of the overall variability of the heart rate. With respect to the
characterization of HRV response to global ischemic preconditioning, in a recent
report it has been shown that time domain parameters of short-term HRV are
sensitive indicators of short asphyxic episodes [Boardman et al. 2002]. Heart rate
spectral analysis quantifies the frequency content of the variation of heart rate,
providing markers of the cardiac autonomic regulation, i.e. the sympatho-vagal
balance [Lombardi et al. 1996, Mezzacappa et al. 1994].
Given the intrinsic nonlinear nature of the complex regulating mechanisms, a
detailed description and classification of the dynamical changes of HRV becomes
inaccessible if only linear methods of analysis are employed [Wessel et al. 2000,
Cimponeriu et al. 2000, Bezerianos et al. 1999]. However, there is no agreement,
which parameters are the most relevant for the analysis of HRV during asphyxia
and recovery after asphyxia-cardiac arrest.
The non-linear interaction between the various regulatory systems of the heart rate
gives rise to clinically useful concepts of variability and regularity. Impairment of
the function of these feedback systems by hypoxia should lead to regularization
and/or diminished variability of the heart rate [Chaffin et al. 1995]. Experimentally
observed sequences of heart periods are short, noisy and often nonstationary. This
favors those measures of complexity able to quantify the system dynamics from
rather short time series. The sequence of heart periods can be analyzed with the
help of entropy measures such as Shannon entropy or renormalized entropy
[Schafer et al. 1995, Voss et al. 1996]. In the present study, we use the approximate
entropy (ApEn) measure for quantification of regularity from short time sequences
of R-R intervals. ApEn displays the ability to detect differences in heart period
dynamics assessing the complex properties of the signal [Pincus 1991]. Another
tool employed in the analysis of HRV response to asphyxia insult involves the
quantification of the Poincare maps. This analysis is especially attractive because it
does not require any pre-processing and asks for minimal assumptions of data
stationarity.

Chapter 4
- 58 -
4.1.2 Experimental Protocol
General Surgical Procedures
Ten male Wistar rats ranging in weight from 250-350g were anesthetized with 5%
halothane and intubated with a 14G catheter. Femoral vessels were cannulated to
monitor mean arterial pressure (MAP), sample of arterial blood gases and for fluid
and drug delivery. Blood gases were taken at 20, 30 and 40 minutes in
preconditioning group to assure O2 saturation was approximately 80%. The rectal
temperature of the animal was maintained at 37.0 0.5
o
C. A more detailed
description of the animal model can be found in [Geocadin et al. 2000]. The
protocol was approved by the Animal Care and Use Committee of the Johns
Hopkins Medical Institutions.

Preconditioned Rats
Ten minute baseline and 5 minute washout were followed by thirty minutes 10%
fraction of inspired oxygen (FI O2) and twenty five minutes recovery. Washout
was again conducted for five minutes followed by global asphyxia, which was
induced by air occlusion for 5 minutes. Cardiac arrest was observed with asystole
and non-pulsatile MAP<10 mmHg. Cardiopulmonary resuscitation (CPR) was
provided after asphyxia by reinstating ventilation (100% O2) at a frequency of 60
strokes/min (in comparison to 40 strokes/min in the baseline), epinephrine injection
(0.005 mg/kg) flushed by 0.5 cc saline and chest compression, until return of
spontaneous circulation (ROSC).

Non-preconditioned Rats
The experiment protocol is similar to preconditioning rats except for the following:
an extended baseline was conducted in place of the one hour of preconditioning
and recovery prior to asphyxia. The experiment design is shown in Fig. 4.1.
- 59 -

Fig. 4.1 Experimental design protocol of asphyxia-cardiac arrest. Continuous
digital ECG is taken from each phase of the experiment. Two group of rats were
studied; preconditioned rats (n=5) and non-preconditioned rats (n=5). For the
non-preconditioned rats an extended baseline was conducted in place of the one
hour of preconditioning and recovery prior to asphyxia.

4.1.3 Data Analysis
4.1.3.1 Data Acquisition and Preprocessing
The ECG was recorded continuously before the insult, during the insult, and after
insult for 4 h and was digitized using the data acquisition system (hardware: DI700,
software: Windaq, DATAQ Instruments Inc., Akron OH). A sampling frequency of
250 Hz and 12 bit A/D conversion was used. The high- and low-pass filter cutoff
frequencies were set at 0.3 and 100 Hz, respectively. Power line noise was
eliminated by using a 60 Hz notch filter.
In order to increase the localization resolution of the R-wave and hence to enhance
the heart rate series accuracy, off-line preprocessing of the ECG signals included
Chapter 4
- 60 -
resampling at 1000 Hz followed by a cubic spline interpolation. For the purpose of
our study, this technique restored the original dynamics of HRV with sufficient
accuracy for a proper estimation of the HRV parameters considered here.
Beat-to-beat RRI (R-to-R-interval) signal was constructed off-line, as a series of
time difference between the successive heartbeats. We analyzed RRI segments of 5
min each, extracted from the 4 h continuous recordings including baseline and
recovery following the asphyxic injury. Therefore, our RR interval time series were
derived from long-term ECG recordings and small number of ectopic beats are
common is such recordings.
Two potential sources of error have to be eliminated: (1) A QRS complex could be
missed or an extra RR interval detected; (2) Disturbances due to either abnormal
impulse formation or impaired conduction give rise to non-sinus beats. These
ectopic beats produce short beat-to-beat intervals, followed by a compensatory
delay. Both disturbances induce a sharp transient peak in the tachogram, and can
lead to major errors in the analysis of HRV.
For the removal of ectopic beats, two linear filters can be applied to correct for data
points which fall outside of a certain range: a percentage filter will replace any
deviation that deflects more than 20% of the median of a predefined segment by the
mean value of the five preceding and the five succeeding values, or the filter used
in the present analysis which corrects for deviations with a deflection larger than
three standard deviations (3 SD) from the mean value.
=
=
+
=
5
0 ; 5
) (
10
1
i
i i
i old new
RR RR
(4.1)

The assumption that underlies this correction is that the RR interval does not
oscillate abruptly from the mean value.
From all 10 rats, 5 min segments of RRI were selected for further analysis,
corresponding to the following distinct phases: baseline, 30 min after ROSC (1), 60
min after ROSC (2), 90 min after ROSC (3), 120 min after ROSC (4), 150 min
after ROSC (5).
- 61 -
The data have been detrended in order to eliminate the baseline fluctuations and to
ensure the quasi-stationarity in the wide sense, necessary for further analysis.

4.1.3.2 Linear and Nonlinear Methods of Analysis
Using linear (time and frequency domain) and nonlinear (approximate entropy and
parameters of Poincare plots) measures, we evaluated the dynamic time course of
the HRV response to the asphyxia insult and the effect of preconditioning on the
autonomic neurocardiac control. These methods were described in details in
Chapter 3.

4.1.3.3 Statistical Analysis
HRV parameters analyzed during different situations were plotted as mean and
standard error of mean (SEM).
Pearson's linear correlation coefficient R was computed between parameters, in
order to reveal their interrelationships (Table 4.1, 4.2).

The time courses of the estimated parameters for each group of subjects (non-
preconditioned and preconditioned group) were analyzed using repeated-measures
analysis of variance and post hoc comparisons by Dunnetts test, comparing the
estimated parameter values for each phase to baseline values. Between the groups,
data at each time point were compared by using Students unpaired t-test. A level
of p<0.05 was selected in order to consider the differences significant.

4.1.4 Results and Discussions
The mean RRI does not discriminate the response to the asphyxia insult for the two
groups non-preconditioned and respectively preconditioned (Fig 4.2). However, an
initial common reduction of the mean RRI with respect to baseline values (0.157
0.023 and 0.129 0.019 s) appears to characterize the response to the asphyxia
insult. At 30 min after ROSC, mean RRI was 0.124 0.022 s (p=0.14 compared to
baseline) and 0.119 0.004 s (p=0.23 compared to baseline) for the non-
preconditioned group and the preconditioned group, respectively. For the
Chapter 4
- 62 -
preconditioned group, the basal mean RRI fully recovers approximately 150 min
after ROSC (0.124 0.004 s, p=0.67 compared to baseline, p=0.07 when compared
between groups).

Fig. 4.2 Mean RRI during baseline, 30 min after ROSC (1), 60 min after
ROSC (2), 90 min after ROSC (3), 120 min after ROSC (4) and 150 min after
ROSC (5).

RR variability (SD) peaks 30 min after ROSC (0.0115 0.007 s, p=0.09 and
0.0111 0.006 s, p=0.06 compared to baseline, respectively) and recovered to the
basal level in the preconditioned group 150 min after ROSC (0.018 0.0003 s,
p=0.9) (Fig 4.3).

Fig. 4.3 Standard deviation (SD) of the beat-to-beat RRI signals during
baseline, 30 min after ROSC (1), 60 min after ROSC (2), 90 min after ROSC
(3), 120 min after ROSC (4) and 150 min after ROSC (5).
- 63 -
The quantification of the power spectrum of RRI signals showed marked
differences before and after asphyxia for the two groups (Fig. 4.4).

Fig. 4.4 LF (a), HF (b), LF/HF (c) indices quantified from the power
spectrum estimation of RRI signals during baseline, 30 min after ROSC (1), 60
min after ROSC (2), 90 min after ROSC (3), 120 min after ROSC (4) and 150
min after ROSC (5). P<0.05 compared with baseline within each group; *
P<0.05 compared to control group.

Heart rate variability in the respiratory frequency range significantly increased
immediately after asphyxia compared to the baseline (Fig. 4.4b), whereas in the
low frequency range decreased (Fig. 4.4a). During the recovery process there is a
significant difference between non-preconditioned and preconditioned group for
the LF and HF bands. In the LF band, the values at 90 min and 120 min after
ROSC were 8.7 3.6 and 6.4 2.8 n.u. (p<0.01 compared to baseline) for the non-
preconditioned group, while for the preconditioned group were 27 11.2 and 31
11 n.u. (p>0.9 compared to baseline, p<0.05 when compared between groups). For
the control group in the HF band the values after 90 min and 120 min after ROSC
were 62 7 and respectively 61 5.6 n.u. (p<0.05 compared to baseline) for the
non-preconditioned group; for the preconditioned group these values were 30.8
4.8 and 35.5 7.8 n.u. (p=0.3 and p=0.6 compared to baseline, p<0.05 when
compared between groups). In other words, spectral analysis distinguishes a
differentiate response in the two groups. In the preconditioned rats, 90 minutes
after ROSC the LF/HF balance recovers to the baseline level, while in the control
group this ratio reflects the increased relative contribution of the HF spectral
activity (Fig. 4.4c).
Chapter 4
- 64 -
Quantification of the ApEn reports similar recovery patterns of response to
asphyxia insult (Fig 4.5).

Fig. 4.5 Approximate Entropy (ApEn) of RRI signals during baseline, 30
min after ROSC (1), 60 min after ROSC (2), 90 min after ROSC (3), 120 min
after ROSC (4) and 150 min after ROSC (5).

The baseline values were 1.52 0.04 and 1.56 0.019 followed by a reduction at
30 min after ROSC to 1.25 0.33 (p=0.1 compared to baseline) for the non-
preconditioned group and 1.25 0.37 (p=0.06 compared to baseline) for the
preconditioned group, respectively. Preconditioned rats appears to have a faster
return to baseline value at 90 min after ROSC with values of 1.57 0.007 (p=0.98
compared to baseline) while for the non-preconditioned group these values were
1.47 0.09 (p=0.84 compared to baseline, p=0.06 when compared between
groups).

Figure 4.6 shows the SD1 and SD2 indices quantified from Poincare plots of RRI
signals and their ratio. At 30 min after ROSC we observed an initial common
increase of SD1 and SD2 parameters with respect to baseline values. For the non-
preconditioned group SD1 was 1.7*10
-3
0.7*10
-3
in baseline vs. 7.0*10
-3

2.5*10
-3
(p=0.11) and SD2 was 2.0*10
-3
0.9*10
-3
-3

2.6*10
-3
s (p=0.17), while for the preconditioned group SD1 was 0.7*10
-3

0.05*10
-3
-3
3.5*10
-3
(p=0.06) and SD2 was 1.0*10
-3

0.2*10
-3
-3
3.2*10
-3
(p=0.08). At 150 min after ROSC these
parameters returned to baseline values.
- 65 -

Fig. 4.6 SD1 (a), SD2 (b), SD1/SD2 (c) indices quantified from Poincare
plots of RRI signals during baseline, 30 min after ROSC (1), 60 min after ROSC
(2), 90 min after ROSC (3), 120 min after ROSC (4) and 150 min after ROSC
(5).

Different HRV parameters were highly correlated despite their different theoretical
background (Table 4.1, 4.2). We remark a strong correlation (p<0.05) between SD,
SD1, SD2 and ApEn.

Table 4.1 Pearsons correlation coefficients between different HRV parameters in
preconditioned rats
Pearsons
R
SD LF
power
HF
power
ApEn LF/H
F
SD1 SD2 SD1/SD2
Mean RRi -0.34 0.67 -0.22 0.60 0.56 -0.40 -0.36 -0.66
SD -0.83
*
0.88
*
-0.87
*
-0.83
*
0.99
*
0.99
*
0.46
LF power -0.73 0.96
*
0.95
*
-0.87
*
-0.83
*
-0.81
*
HF power -0.85
*
-0.86
*
0.89
*
0.88
*
0.52
ApEn 0.98
*
-0.90
*
-0.88
*
-0.71
LF/HF -0.87
*
-0.83
*
-0.79
SD1 0.99
*
0.57
SD2 0.48
* p<0.05, two-tailed Students t-test
Chapter 4
- 66 -
Table 4.2 Pearsons correlation coefficients between different HRV parameters in
non-preconditioned rats
Pearsons
R
SD LF
power
HF
power
ApEn LF/HF SD1 SD2 SD1/SD2
Mean RRi -0.67 0.72 -0.82
*
0.83
*
0.69 -0.56 -0.52 -0.18
SD -0.49 0.78 -0.89
*
-0.49 0.96
*
0.93
*
0.52
LF power -0.89
*
0.72 0.99
*
-0.40 -0.53 -0.004
HF power -0.86
*
-0.89
*
0.69 0.75 0.25
ApEn 0.71 -0.83
*
-0.87
*
-0.28
LF/HF -0.40 -0.54 -0.006
SD1 0.89
*
0.68
SD2 0.31
* p<0.05, two-tailed Students t-test

Neurodeficit scoring (NDS) was conducted to verify neurological and behavioral
ability through multiple tests. This examination corresponded to that used in a
clinical setting following cardiac arrest. The test was conducted at twenty-four
hours following global asphyxia, a time point found by [Geocadin et al 2000] to
show statistically significant differences in cohorts with varying degrees of injury.
The preconditioned rats (n=5) demonstrated an NDS of 80 12.7 while the non-
preconditioned cohort (n=5) had an NDS of 34.5 17.7 (Fig. 4.7) showing a
significant difference between groups (p<0.05).

Fig. 4.7 Difference in Neurodeficit Score between non-preconditioned and
preconditioned rats at 24 hours (n=5 each). * P<0.05 compared to control group.

This study shows that the changes in HRV after transient global ischemia can be
distinguished and quantified using the parameters employed here. The time profile
- 67 -
of HRV response supports the hypothesis of a potential beneficial effect of
preconditioning on postischemic functional recovery.
Although parameters such as the mean RRI, SD, ApEn, SD1 and SD2 did not
showed statistically significant differences between the two conditions, the
direction of the differences was consistently favorable to the preconditioned group.
They displayed similar trends within both groups having a statistical tendency (p
between 0.05 and 0.1) in the preconditioned group to discriminate between baseline
and 30 min after ROSC. For the non-preconditioned group only ApEn displayed a
statistical tendency to discriminate between baseline and 30 min after ROSC.
SD1/SD2 parameter failed to discriminate between the groups and phases of the
experiment.
The increase in mean heart rate corresponding to both groups just after ROSC
could be assigned to the resumption of airflow at termination of asphyxia according
to the experimental protocol. Additionally, for the non-preconditioned group, the
mean heart rate remained increased at 150 min after ROSC suggesting only a
partial recovery compared with the preconditioned group, which showed a full
mean RRI recovery.
The amount of variability in the respiratory frequency range significantly
increased, whereas in the low-frequency range it tended to decrease immediately
after asphyxia. This could be related to the fact that immediately after asphyxia the
frequency of the mechanical ventilation was set at 60 strokes/min (comparing to 40
strokes/min in the baseline). Previous studies reported that the increase in
respiratory frequency results in a significantly decrease in R-R interval power at
both respiratory and low frequencies [Brown et al. 1993]. In our study, the
relatively increase in the normalized HF band may be a consequence of a more
severe decrease in the absolute LF power compared to the decrease in HF power.
Additionally, the autonomic neural balance measured by LF/HF ratio appears fully
recovered at about 90 min after the asphyxic insult, while for the control group at
150 min after ROSC it remains decreased. This finding is supported by other
studies which found that ischemic preconditioning attenuates cardiac sympathetic
nerve injury during myocardial ischemia [Miura et al. 2001].
Chapter 4
- 68 -
Among the estimated parameters, the ApEn was an approach capable of assessing
complex properties, sensitive to the changes following the global ischemia (Fig.
4.5). A small value of ApEn decreases immediately after asphyxia which indicates
a higher regularity of the heart rate while the total amount of variability (SD) is
larger. These results show that immediately following the insult, the heart rhythm
appears to lack the complexity associated with the normal nonlinear feedback
control.
The quantitative Poincare plot analysis does not require preprocessing, it asks for
minimal assumptions of data stationarity and the computation of the parameters
SD1 and SD2 is very efficient.
The fact that the nonlinear analysis parameters (ApEn, SD1, SD2) failed to
significantly discriminate between the two groups during the late recovery phase
could be attributed to the loss of nonlinearity of the heart rhythms following the
ischemic injury.
The changes in HRV response to transient global ischemia were identified both by
linear and non-linear methods. The overall HRV decreased after asphyxia
compared to the baseline but after 90 min most of the parameters indicate the
recovery process following the asphyxia injury. For the preconditioned group,
almost all parameters indicate a faster return to the baseline values, showing a
better recovery of the cardiac autonomic control, in agreement with the evolution
of the NDS.
Among the parameters evaluated in our study, several were found closely
correlated. This has been observed from the analysis of the interrelationships
between parameters using the Pearson linear correlation coefficients. The results
shown in Table 4.1, 4.2 explain the quantitative similar or distinct time course of
the parameters.
The main limitation of this study, inherent to such experiments as the one
considered here was the small sample size. This may account for the lack of
statistical power. We need to emphasize that the present work is a pilot study
needed to provide for the first time to our knowledge, a description of the effects of
ischemic preconditioning on the HRV response to transient global ischemia.

- 69 -
4.1.5 Conclusion
The precise mechanism underlying the neuroprotective effect of sublethal ischemic
insults (ischemic preconditioning) is still unknown. Our results show for the first
time that global ischemic preconditioning influences the HRV response to asphyxia
injury. During the different phases of the experiment, HRV changes show an
important difference in the recovery process after transient global ischemia in non-
preconditioned and preconditioned rats. The protection against neuronal
degradation and cell death offered by ischemic preconditioning may underlie the
faster recovery of the basal HRV and the autonomic modulation of the heart.
However, HRV is an indirect measure of the input to the cardiovascular system and
makes very difficult any inference about the exact neuroprotection mechanism of
preconditioning. The HRV is a potential tool for monitoring the recovery process
after transient global ischemia. The results are promising, although further
validation studies in larger groups of animals would be needed for more accurate
discrimination between groups and phases of the experiment and for safer
conclusions.

4.2 A New Approach to Synchronization Analysis of Brain
Electrical Signals
4.2.1 Introduction
In the study of complex dynamical systems such as the brain, one of the primary
concerns is the characterization and quantification of interdependencies between
different subsystems from experimental data analysis. Cognitive acts require the
integration of numerous functional areas widely distributed over the brain, and in
constant interaction with each other. Several widely employed methods for analysis
of these interactions are the cross-correlation, coherence [Clifford-Carter 1987] and
mutual information [Roulston, 1999].
Nonlinear dynamics theory has provided a number of useful tools for the analysis
of interdependencies. Developed within the theoretical framework of coupled
dynamical systems, the concept of phase synchronization offers a new perspective
Chapter 4
- 70 -
on the understanding and quantification of the dynamical interactions established
among coupled systems and has been succesfully applied for analysing brain
electrical signals [Lachaux et al. 1999]. Transient phase synchronization of EEG
oscillations was reported to be essential for conscious brain activity [Varela et al.
2001, Rodriguez et al. 1999].
The empirical mode decomposition (EMD) technique has been used in medical
sciences, e.g. in achieving artefact reduction in electrogastrograms [Liang et al.
2000] or for deriving the respiratory sinus arrhythmia from the heartbeat time series
[Balocchi et al 2004]. This method is best suited for nonlinear and nonstationary
signals with components which overlap in both time and frequency, thus being
impossible to be separated by any non-adaptive filtering techniques.
The purpose of this study is to present a new approach for the analysis of phase
synchronization of dominant intrinsic oscillatory modes derived from left-right
parietal EEG signals using the empirical mode decomposition method (EMD)
[Huang et al. 1998]. Numerical simulation results indicate that the quantification of
the phase synchronization between the dominant intrinsic oscillatory modes
provides a reliable indicator of the degree of interaction between the coupled
dynamical systems.
As an application, we used the empirical mode decomposition (EMD) technique to
decompose the EEG signals into a number of intrinsic mode functions (IMFs) with
well defined instantaneous frequency. Here, instantaneous phase has been
computed by applying the Hilbert transform on the dominant oscilatory
components identified by EMD. Phase correlation have been further quantified
using a suitable defined synchronisation index. Our results indicate the potential
benefit of phase synchronization analysis of IMFs obtained from left-right parietal
EEG signals for monitoring the recovery process following a brain-injury episode.

4.2.2 Coupled Oscillators Approach to Synchronization Analysis.
For the beginning, we consider for analysis two coupled (phase coherent) Rossler
oscillators in a unidirectional (driver-response) configuration. The governing
equations of the two oscillators are given by:
- 71 -
1
1 1 1
1
1 1 1
1
1 1
dx
= - y -z
dt
dy
= x +0.15y
dt
dz
= 0.2+(x -10)z
dt

2
2 2 2 1
2
2 2 2
2
2 2
dx
= - y -z +x
dt
dy
= x +0.15y
dt
dz
= 0.2+(x -10)z
dt
(4.2)

where
1
= 0.97 and
2
= 0.99 and is strength of coupling. The coupling strengh
is varied in a certain range [0.05, 0.25]. We have chosen the parameters of the
Rossler system so that it is in the so-called phase coherent regime (i.e. the x-y
projection of the trajectory of the chaotic system is shaped like an annulus) with
well defined phase variable. The rotation angle arctan(y / x) = can be defined as the
phase, which increases almost uniformly (i.e. the oscillator has a coherent phase
dynamics). Alternatively, the phase of each intrinsic component (corresponding to
intrinsic time scales) is computed by employing the EMD analysis on the x
1,2

variables.

performed considering the natural phase variables (dashed line) and the phases
corresponding to the first intrinsic modes (solid line).

For each coupling strength value within the explored range, we quantify the phase
synchronization considering for analysis both natural phases and the phases
corresponding to the first intrinsic modes. The results are presented in Fig.4.8. It
can be observed that the synchronization degree of the first intrinsic modes
Chapter 4
- 72 -
provides useful information on the degree of coupling between the two dynamical
systems.
Another point of interest in our study concerned the performance of this analysis on
more complex dynamical systems (in what respects the phase synchronization
behaviour), such as two unidrectionally coupled Lorenz systems. The model reads:

1
1 1
1
1 1 1 1
1
1 1 1
dx
= -10(x -y )
dt
dy
= -x z +28x -y
dt
dz
= x y -(8/3)z
dt

2
2 2 1
2
2 2 2 2
2
2 2 2
dx
= -10(x -y )+x
dt
dy
= -x z +28x -y
dt
dz
= x y -(8/3)z
dt
(4.3)

The definition of the natural phase variable for the Lorenz system can be
introduced in a similar way as for the Rossler system. The phase can be defined by:
c
2 2
c
z-y
= arctan( )
x +y -x
(4.4)

with a suitable defined rotation center (x
c
, y
c
).

Fig. 4.9 Illustration of the EMD method applied to the time series of x
variable of the response system in the unidirectionally coupled configuration of
two Lorenz systems.

- 73 -
The EMD analysis allows the decomposition into intrinsic oscillatory modes,
which can be visualized in Fig. 4.9. We focused again on the analysis of the phase
synchronization of the smallest time scale intrinsic modes (first modes)
corresponding to the x variable, in comparison to the natural phase variables. As it
can be observed in Fig. 4.10, a monotonic increase of synchronization index with
increasing coupling strength is reflected by the intrinsic oscillatory modes, in
contrast with results obtained from the analysis of the natural phases.

performed considering the natural phase variables (dashed line) and the phases
corresponding to the first intrinsic modes (solid line).

To conclude this section, the synchronization analysis of the intrinsic oscillatory
modes offers useful information about the effective coupling the strength between
the two interacting systems, acting at intrinsic time scale of the modal components.

The conventional approach for analysis of EEG signals is to obtain power spectra
and delineate power in different spectral bands. Alternatively, a method that
analyzes the dominant frequencies in EEG was shown to be more responsive to
recovery patterns of brain rhythm following ischemic injury [Goel et al. 1996].
However, if the signal is characterized by a broadband spectrum without displaying
dominant spectral components, an adaptive decomposition based on the local
Chapter 4
- 74 -
properties of the signal would be necessary. The EEG signal complexity has been
extensively studied using information measures like Shannon and Tsallis entropy,
able to detect transient or time-varying changes in brains recovery process after
asphyxia [Bezerianos et al. 2003].
In the present experimental setting, adult male Wistar rats were assigned to graded
asphyxia of 5 min that resulted in cardiac arrest. The mean duration of
cardiopulmonary resuscitation (CPR) was 54.7 12 s. A description of the animal
model can be found in [Geocadin et al. 2000]. Two channels of EEG were recorded
using subdermal needle electrodes placed in the right and left parietal areas during
4h of experiment including baseline, asphyxia and recovery period. The EEG
signals were sampled at 250Hz and filtered using a band-pass filter (0.150 Hz).
The entire data set was divided into overlapping segments of 16 s with 25%
overlap between adjacent segments. On each segment we applied the empirical
mode decomposition method and obtained a number of intrinsic modes, each
corresponding to a proper rotation in the complex plane of the analytic signal.

4.2.4 Results and Discussions
The analysis has been performed using a sliding temporal window (w= 16s, 25%
overlap) on the bivariate segment of data recorded during baseline, asphyxia and
recovery period. Phase synchronization indices have been computed from the first
three corresponding imf
i
s obtained by means of the EMD technique. Figure 4.11
illustrates the imf
i
s corresponding to a single 16s EEG segment extracted from the
baseline. The first trace represents the original EEG signal followed by the imf
i
s
components.
Figure 4.12 shows the evolution of the synchronization indices for the three imf
i
s,
i=1..3, during the 1.5 hours recording time including baseline (15 min), asphyxia (5
min) and recovery period. It can be observed that, the phase synchronization of the
corresponding IMFs components extracted from bivariate EEG signals allows the
detection of injury-related changes during an asphyxia experiment in rats.

- 75 -

Fig. 4.11 Illustration of the EMD method applied to experimental EEG data.

Fig. 4.12 The time evolution of the synchronization index over the whole
recording period.
Chapter 4
- 76 -
The recovery period is characterized by the recovery of the baseline phase
synchronization degree. This forwards this method as a possible indicator for the
recovery of the brain electrical activity after asphyxia.

4.2.5 Conclusions
In the present study, we introduced a new approach for the identification of the
degree of interaction between two complex dynamical systems from experimental
data analysis. We use the empirical-mode-decomposition (EMD) technique to
decompose the output signals into a number of elementary orthogonal modes with
well defined instantaneous attributes (IMFs). We show that the quantification of
the phase synchronization between the dominant intrinsic oscillatory modes
provides a reliable indicator of the degree of interaction between coupled
dynamical systems acting at the time scale of the intrinsic modes. As an
application, we showed that the phase synchronization analysis of IMFs obtained
from left-right parietal EEG signals can provide a new tool for monitoring the
recovery process following a brain-injury episode. The approach can be further
extended to the analysis of multivariate phase synchronization of EEG signals.

- 77 -
Chapter 5: Analysis of Neural Activity in
Supplementary Eye Fields During
Anticipatory Eye Movements
5.1 Introduction
In a fast changing visual environment, it is vital to rapidly detect moving predators
or preys and to anticipate their future actions. The visual system of primates has
evolved under these constraints. It is believed that the use of biological movement
as an orienting stimulus in behavioral and electrophysiological studies will
significantly alter our understanding of anticipation and prediction in general.
Smooth pursuit eye movements will be used as a tool to investigate anticipation
and prediction of actions [Missal et al 2001].
Until recently, neuroscientists have developed stimuli as simple as possible to
investigate particular brain functions, e.g. a single moving dot of light on a dark
background. Such a simple stimulus is easy to produce and give rise to better
controlled behavioral and neuronal responses. This approach has proved extremely
successful in the characterization of a basic behavioral repertoire and the
identification of a set of fundamental neuronal pathways in the visual system.
However, it is obvious that in a natural environment primates do not often pursue
single dots but try to follow with the eyes the movement of other animals in order
to predict their action and to take a decision. The neural encoding of visual
information and its subsequent use to plan a movement is likely to be different
when subjects view a biologically relevant action, like human locomotion, that is a
Chapter 5

- 78 -
more natural time-dependent input to the visual system [van Steveninck et al.
1997]. We hypothesize that neuronal responses in high level sensori-motor areas
will show less variability during biological action viewing and planning of the
matching anticipatory motor response.
Biological movements are complex. However, a simplified version of a human
walker can be obtained by attaching a light source to the major joints of the subject,
creating a point-light walker [Johansson 1973]. [Cutting 1978] proposed a simple
algorithm to generate a synthetic walker on a computer screen. Moreover, motion
capture systems are currently available to record movements of a human subject or
an animal and later display them during experiments. The psychophysics of
biological motion and the brain areas involved in its processing are currently being
investigated by different groups in the world. However, to our knowledge,
biological motion has never been considered to study anticipatory and predictive
responses. There is neuropsychological evidence that there may be a specialized
system for action recognition [Lassonde et al 1993; Vaina et al 1990]. We
hypothesize that predictive capabilities of the oculomotor system can be better
understood if the driving stimulus is biologically relevant action. Biological
adaptability is the origin of cognitive functions. At the electrophysiological level,
we will record the activity of populations of neurons in the supplementary eye
fields of the frontal lobe during biological action viewing and anticipation.

Anticipatory movements are actions starting before the occurrence of likely sensory
events. In an experimental setting, this can be studied by having subjects pursue a
moving dot of light appearing on a visual display. If the visual stimulus moves
consistently in the same direction during a few presentations, subjects anticipate
that it will likely continue to do so during the next presentation and the eyes will
start to move in advance of the beginning of the next stimulus appearance.
Anticipatory pursuit is a smooth movement of the eye occurring before the
appearance of an expected moving target. The expectation of the subject is based
on a subjective estimation of the probability that the target will move in a given
direction. Anticipatory pursuit has been extensively studied at the behavioral level.
However, the neural mechanisms allowing this important behavior are unknown.
Analysis of Neural Activity in Supplementary Eye Fields during Anticipatory Eye Movements

- 79 -
Recently, it has been suggested that the SEF could play a role in using past
experience to guide anticipatory pursuit. This hypothesis is currently being tested at
the single neuron level. Indeed, it has been shown that neurons in the SEF are
active during smooth pursuit in the absence of saccades [Heinen 1995], especially
when predictable changes in target motion occur [Heinen et al. 1997]. In the
behaving monkey, it has been shown that electrical microstimulation in the SEF
can facilitate smooth pursuit initiation towards a moving target, suggesting that
activation of the SEF might change the internal gain of the smooth pursuit pathway
[Missal et al. 2001]. In this study, we favored anticipatory

responses in monkeys by
using a cognitive cue, which produces a different anticipatory pursuit response than
the one observed in pervious studies, based on repetition.

5.2 Materials and Methods
One male rhesus monkey (Macaca mulatta) was used in this study. The monkey
was seated in a primate chair with head restrained for the duration of the sessions.
Behavioural paradigms, visual displays and data storage were performed under the
control of a real time program running on a laboratory PC system. The targets were
presented via a computer-controlled analog oscilloscope, which back-projected
light spots on a 90x90
o
translucent screen placed 40cm in front of the monkey
[Crandall et al. 1985]. The eye movement signals were obtained by placing the
head-restrained animal with an implanted scleral coil in a pair of orthogonally
aligned magnetic fields. The neural activity was recorded using one microelectrode
placed in the region of the SEF over the dorsal medial frontal cortex. The electrode
was lowered by a hydraulic drive system while the animal performed the pursuit
tasks. Single neurons were isolated online using a pattern discriminator. The pulse
signals produced by the discriminator and all other events were digitally stored for
off-line analysis. A more detailed description of the animal model and surgical
preparations can be found in [Missal et al. 2001].
Animals were trained to pursue a moving target spot back-projected on the tangent
screen. Each trial was initiated by the appearance of a target for 400ms during
Chapter 5

- 80 -
which the monkeys had to saccade to that initial stimulus position. After the animal
foveated the target, the fixation period lasted for 500 ms, during which the animals
had to maintain gaze within a square electronic window of 4 x 4 centered on the
target. At the end of the fixation period, the fixation point was turned off and
simultaneously a target appeared at a horizontal eccentric position. This eccentric
target moved at constant velocity. In figure 5.1 an example of the above procedure
is presented.

Fig. 5.1. Example of an animal pursuing a moving target spot back-projected
on the tangent screen. Each trial was initiated by the appearance of a target
during which the monkeys had to saccade to that initial stimulus position.

5.2.2 Data Analysis
Eye velocity was obtained by digital differentiation of the eye position signal and
the eye acceleration was obtained by differentiation of the eye velocity trace. The
onset of the pursuit movement was determined by using an acceleration threshold
(50
0
/s
2
). The offset of pursuit was the time when eye velocity returned to the value
observed at the time of pursuit initiation.
To estimate the neuronal firing rate, the discrete spike trains produced by the
discriminator, were transformed into a continuous function of time. To achieve
this, each spike was convolved with a Gaussian function. The spike density
function was defined as the sum of the individual Gaussian functions. For all the
trials in, and on a trial-by-trial basis, the eye velocity and the spike density were
averaged using a non-overlapping 50ms sliding window. The averaged data-points
were considered for further analysis.

- 81 -
The hypothesis that the SEF contains neurons active before the anticipatory pursuit
movements motivated us to search for correlations between anticipatory pursuit
velocity and the corresponding spike density activity.

Fig. 5.2. Measures of saccade parameters and neuronal activity. From top to
bottom: eye position (Ev), vertical eye velocity (v), spike density (Spd) and
individual spikes (vertical lines).

The cross-correlation method relies on the basic assumption that the spike density
function is related to the velocity of the eye [Missal et al. 2000]. The cross-
correlations have been computed between the averaged eye velocity data and
averaged spike density data respectively. Two approaches have been adopted. In
the first approach of the study, we considered the correlations between spike
density and eye velocities in the preferred direction, while in the second approach
we computed correlations between differential spike density and differential eye
velocities between preferred and unpreferred directions. For the eye velocity data,
data-points within 450ms-700ms (early anticipatory pursuit response) and 700ms-
900ms (late anticipatory pursuit response) have been considered.
The neuronal activity may lead or lag the movement of the eye. We took into
consideration a negative time-lag between 50 and 200 ms in the averaged spike
Chapter 5

- 82 -
density data. The optimal time lag leads to a maximum value of the correlation
coefficients. We also considered positive time-lags but the results showed lower
values in the correlation coefficients.

5.3 Results and Discussions
Typical anticipatory eye movements, the corresponding neural activity and the
correlations between them in the preferred direction (normal correlations) are
shown in Fig. 5.3.

Fig. 5.3. Typical anticipatory eye movements, the corresponding neural
activity and the correlations between them in the preferred direction (normal
correlations)

In Fig. 5.4 are plotted the differentials spike density and eye velocity respectively
as well as the differential correlation.
The spike density function (Fig. 5.3a) presents a peak around 1s corresponding to
the target movement onset and it is correlated with the peak eye velocity (Fig.

- 83 -
5.3b). Neural correlates of the anticipatory pursuit eye movements are presented in
Fig. 5.3c, 5.4c. The continuous line represents the correlation coefficient calculated
for the early anticipatory pursuit response, while the dotted line represents the
correlation coefficient calculated for the late anticipatory pursuit response. The
statistical significant values for the correlation coefficient are marked in the figure
with a star (*).

Fig. 5.4. Differentials spike density and eye velocity respectively and the
differential correlation coefficient

Fig. 5.5 shows an example for the calculation of the first and last four values of the
correlation coefficient for the early anticipatory pursuit interval.

To test the robustness for the correlation coefficient computing method, we
randomly selected subsets from the total numbers of trials in the preferred
direction, and we computed the correlation coefficients as described before. The
random selections were performed ten times and the corresponding correlation
coefficients are presented in Fig. 5.6.
Chapter 5

- 84 -

Fig. 5.5. First and last four values of the correlation coefficient for the early
anticipatory pursuit interval.

One-way analysis of variance (ANOVA) was performed for comparing the
correlation coefficients. The computed p-value of 0.8, supports the null hypothesis
and shows no significant differences between the ten correlation coefficients.

Fig. 5.6. Plots of correlation coefficients computed from random selections
of trials in the preferred direction during the early anticipatory pursuit


- 85 -
The reason for adopting the differential approach is based on the hypothesis that
the information coded by the neurons during the visual cue period, is the same for
both the preferred and the unpreferred directions. Thus, by subtracting
subsequently the spike densities corresponding to the unpreferred direction neurons
from the preferred direction ones, we eliminate the common response given by the
visual cue, enhancing thus the information coded in the planning phase
(anticipatory pursuit movements). The maximum correlation coefficient at 70ms,
may indicate the premotor delay activity.
To characterize the neural mechanisms allowing the anticipatory pursuit behaviour
we used in this study signals from 30 neurons. They can be classified into groups
which displayed similar trends for the correlation coefficients values. Among them
10 neurons displayed correlation coefficient values with a statistical significance.

Fig. 5.7. Histogram of the optimal time lag for early and late anticipatory
response

Chapter 5

- 86 -
In Fig. 5.7 is presented the histogram of the optimal time lag for early and late
anticipatory response using both differential and normal approach. For the early
anticipatory pursuit, the differential approach displayed slightly larger peaks for 60
and 180s response time lags.
The small sample size represents the main limitation of this study. Further work
may be required to validate the results obtained here.

5.4 Conclusion
The neural mechanisms allowing the anticipatory pursuit behaviour are unknown.
The differential approach may indicate more reliable results for computing the
correlation coefficients between spike densities and eye velocities. The maximum
correlation coefficient present at different time lags, indicate a variation in the
premotor delay.
Further validation studies in larger set of neurons would be needed for more
accurate discrimination between direct and differential approach, as well in the
calculation of the optimal time lag.

- 87 -
Chapter 6: Conclusions and Further Work
The aim of this thesis was to extract physiological and clinical information hidden
in biosignals such as cardiac and neural signals, making use of linear and nonlinear
methods.
Our analyses have primarily been focused on the important problem of cardiac and
neural responses to hypoxia. A first application was to evaluate the effect of global
ischemic preconditioning on the heart rate variability (HRV) response to the
asphyxia insult. The experimental data (heart rate variability time series derived
from the ECG measurements) was obtained from ten male Wistar rats, five of
which were subjected to a ischemic preconditioning process before the induced
global asphyxia and cardiac arrest. Our results show for the first time that global
ischemic preconditioning influences the HRV response to asphyxia injury. During
the different phases of the experiment, HRV changes show an important difference
in the recovery process after transient global ischemia in non-preconditioned and
preconditioned rats. The protection against neuronal degradation and cell death
offered by ischemic preconditioning may underlie the faster recovery of the basal
HRV and the autonomic modulation of the heart. However, HRV is an indirect
measure of the input to the cardiovascular system and makes very difficult any
inference about the exact neuroprotection mechanism of preconditioning. The HRV
is a potential tool for monitoring the recovery process after transient global
ischemia. The results are promising, although further validation studies in larger
groups of animals would be needed for more accurate discrimination between
groups and phases of the experiment and for safer conclusions.
Chapter 6
- 88 -
Neural changes following cardiac arrest were studied within the framework of
coupled oscillators theory. We have considered the problem of phase
synchronization of dominant intrinsic oscillatory mode functions (IMFs) with well
defined instantaneous frequency derived from left-right parietal EEG signals using
as a new approach the empirical mode decomposition method (EMD). Numerical
simulation results indicate that the quantification of the phase synchronization
between the dominant intrinsic oscillatory modes provides a reliable indicator of
the degree of interaction between the coupled dynamical systems. Instantaneous
phase has been computed by applying the Hilbert transform on the dominant
oscilatory components identified by EMD. Phase correlation have been further
quantified using a suitable defined synchronisation index. We show that the
quantification of the phase synchronization between the dominant intrinsic
oscillatory modes provides a reliable indicator of the degree of interaction between
coupled dynamical systems acting at the time scale of the intrinsic modes. Our
results indicate the potential benefit of using the phase synchronization analysis of
IMFs obtained from left-right parietal EEG signals as a new tool for monitoring the
recovery process following a brain-injury episode. This approach can be further
extended to the analysis of multivariate phase synchronization of EEG signals.
Another application in this thesis addressed the problem of neural activity in
supplementary eye fields (SEF) during anticipatory eye movements. Anticipatory
pursuit has been extensively studied at the behavioral level. However, the neural
mechanisms allowing this important behavior are unknown. Recently, it has been
suggested that the SEF could play a role in using past experience to guide
anticipatory pursuit. This hypothesis is currently being tested at the single neuron
level. In the behaving monkey, it has been shown that electrical microstimulation
in the SEF can facilitate smooth pursuit initiation towards a moving target,
suggesting that activation of the SEF might change the internal gain of the smooth
pursuit pathway. In this study, we favored anticipatory

responses in monkeys by
using a cognitive cue, which produces a different anticipatory pursuit response than
the one observed in pervious studies, based on repetition. The experimental data
used in this study was obtained from one male rhesus monkey (Macaca mulatta).
The hypothesis that the information coded by the neurons during the visual cue
period is the same for both the preferred and the unpreferred directions, determined
Conclusions and Further Work

- 89 -
us to try to eliminate the common response given by the visual cue by subtracting
subsequently the spike densities corresponding to the unpreferred direction neurons
from the preferred direction ones. In this way we enhanced the information coded
in the planning phase (anticipatory pursuit movements). The differential approach
may indicate more reliable results for computing the correlation coefficients
between spike densities and eye velocities. The maximum correlation coefficient
present at different time lags shows a variation in the premotor delay.
Within the context of neural synchronization, future work will focus on
investigation of cortical connectivity sub-serving real and imaginary rhythmic
finger tapping, from the analysis of multi-channel EEG scalp recordings. Our
future aim is to develop a new strategy for recognizing imagined movements in
EEG-based brain computer interface research.

- 91 -
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