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3.1 Subjects and surgery: The experiments were carried out with male
Charles Foster rats of age 12-14 weeks and weight around 180-200 grams at the
beginning of the experiment. The rats were individually housed in polypropylene
cages (30 cm x 20 cm x 15 cm) with drinking water and food (Hindustan Liver
Limited, India) ad libitum on commercial laboratory food pellets. All rats were
kept in an ambient environment temperature of 23 ± 1 oC from birth and the animal
room was artificially illuminated with 12:12 hours Light: Dark cycle, changed at
07.00 hours and 19:00 hours IST.
Rats were chosen, as they are very well suited for neurophysiological
experiments because neurological developments in them are similar to human
development. The primitive cortex in rats is in a greater proportion and is more
superficial, so cortical electrodes can record the changes in primitive cortex, where
the stress affecting the mood is more pronounced. Moreover, the extensive sleep-
wake recordings could be done easily on the rats due to their nocturnal activity and
easy availability unlike the human subjects.
For the common grounding, midline Frontal stainless steel screw electrode,
1 mm in diameter, and two other similar screw electrodes were used for cortical
EEG. Four stainless steel loop electrodes, insulated, except at the tip (two for EOG
and two for EMG), were also used. Their socket contacts had earlier been prepared
to a seven-pin amphitronic connector. Screw and loop electrodes were connected
and soldered to the free pins of the connector, connected with thin flexible wires.
The EEG and grounding screw electrodes were kept free; however, four pins of
EOG and EMG electrodes were fixed in the amphitronics connector with the help
of dental acrylic, well before implantation. Screw electrodes were connected and
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Experimental Design and Methods Applied
fixed to the socket contact by dental acrylic after fixing them on the skull. Such
separate connectors were used for each of the experimental animals for the
recording of electrophysiological signals. The procedure of electrodes
implantation in rats has been presented in Figures-3.1(a - d).
(a) (b)
(c) (d)
Figure-3.1: (a) The exposed skull of the anesthetized rat with positions
marked for attaching the screw electrodes. Two positions marked on bilateral
frontal region for EEG and one on the midline frontal region for grounding
electrodes. (b) Exposed skull with three screw electrodes. (c) After the fixation
of screws, two loop electrodes for EOG (left and right outer canthus muscles
respectively) and two loop electrodes for EMG (left and right cervical muscles
respectively) were sutured to keep them in position. (d) A rat after electrode
implantation.
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Experimental Design and Methods Applied
3.2.1 Heat stress model: The stress was produced in the rats, by subjecting
them in the Biological Oxygen Demand (BOD) incubator (Oceania, India) at
preset temperature of 38±1oC and relative humidity 45-50% (Sharma et al., 1998),
simulated with the environmental conditions of Varanasi (India) in the months of
May and June.
Acute heat stress: Rats were subjected to the BOD incubator for continuous
four hours of heat exposure from 8.00 a.m. to 12.00 p.m. for a single day, just
before the recording of electrophysiological signals.
Chronic heat stress: Rats were subjected to the BOD incubator for one hour
daily for 21 days of chronic heat exposure from 8.00 a.m. to 9.00 a.m. and
electrophysiological signals were recorded on 22nd day.
Control: Respective control groups of rats were placed in the incubator at room
temperature (23±1oC) and whole procedure was followed exactly similar to that of
their stressed groups.
Table – 3.1
Day Experimental protocol for acute stress group
1 Implantation of electrode measurement of body weight and body
temperature
2-7 Recovery and habituation to experimental chamber.
8 1. Measurement of body weight and the body temperature before stress
2. Animal subjected to the experimental chamber at high ambient
temperature (38±1oC) for 4 hours.
3. Measurement of core temperature after the stress.
4. Continuous recording of EEG, EOG and EMG for 4 hours.
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Experimental Design and Methods Applied
Table 3.2
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were made on the walls of test chamber for proper ventilation. The continuous
four hours of recordings of EEG, EOG and EMG were performed from 12.00 hour
to 16.00 hours IST (Indian Standard Time) on the recording day for chronic and
acute heat stressed rats through the 8 channels Electroencephalograph (EEG -8,
Recorders & Medicare Systems, India). The paper recordings were performed at
the chart speed of 7.5 mm/sec.
The recording setup along with the snapshot of running window have been
presented in Figure-3.2 and 3.3 and the parameters of amplifier setting for
different electrophysiological signals are given in Table-3.3.
Figure-3.2: The whole setup with the recording cage holding the rat whose
electrodes were connected to the EEG machine. The EEG machine was
connected to the AD card, which is fixed inside the computer. The analog
paper recording as well as, the digital recordings on the computer monitor, can
be seen.
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Experimental Design and Methods Applied
Some examples of two minutes recordings of raw EEG signals have been given in
Figure-3.4.
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The digitized data was collected, stored and processed with the help of data
acquisition system (ADLiNK, 8112HG, NuDAQ, Taiwan) and processing
software (Visual Lab.-M, Version 2.0c, Blue Pearl Laboratory, USA). The
recordings were done with the sampling frequency of 256 Hz and selected data
were stored in hard disk in small segments (approximately 2 minutes) in separate
data files. Further, for ease of wavelet processing, recorded signals for all three
states were split into an epoch of two seconds length.
Two seconds long processed EEG signals for the three sleep
states - AWAKE, SWS and REM have been shown in Figure-3.5. Some examples of
two seconds epochs of unprocessed EEG recordings for AWAKE, SWS sleep and
REM conditions have been presented in Figures-3.6 (a-c). Figure-3.7 depicts
recordings of unprocessed sleep-EEG with its corresponding EMG and EOG signals
with baseline drift unadjusted.
Amplitude (mv)
Amplitude (mv)
for AWAKE, SWS sleep and REM conditions have been shown in Figure-3.4.(a)
0.15
AWAKE data
0.6
SWS data
0.15
REM data
with its corresponding EMG and EOG signals with Baseline drift unadjusted.
0.05 0.2 0.05
0 0 0
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Experimental Design and Methods Applied
(a) AWAKE
(b) SWS
(c) REM
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corresponds to the linear frequency of 128 Hz. The above procedure of subband
coding is repeated for further decomposition. This decomposition halves the time
resolution since only half the number of samples now characterizes the entire
signal. However, this operation doubles the frequency resolution since the
bandwidth of the signal now spans only half the previous frequency band,
effectively reducing the uncertainty in the frequency by half.
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Experimental Design and Methods Applied
signal and categorizing into delta, theta, alpha, and beta frequency bands, powers
of all the four bands of AWAKE, REM and SWS were computed for each subject
belonging to stress group and control group, which have also been shown through
plots generated by Matlab (ref. Figures: 4.1-4.9). Graphs explained how frequency
changes with time for stress and control groups. The program is so written that
after calculating wavelet coefficients, frequencies and their powers, 4 basic plots
other than many subplots are generated projecting significant information
regarding time, frequency, and power. The first plot is a 3-D plot of time, scale,
and coefficients along X, Y, and Z axis respectively (Figure-4.3, Chapter-4). The
second plot contains four 3-D subplots each showing powers of the four frequency
bands separately with respect to time and frequency (Figures: 4.4 – 4.9). The third
plot (2-D) consisting of 4 subplots for delta, theta, alpha, and beta with time
[1, 512] along X-axis and power (≈ 10 -6 V2/10-7 V2) along Y – axis
(Figures: 4.10b – 4.12b). The fourth plot shows changes in power w.r.t. time for all
frequencies between 0 to 30 Hz in one epoch for AWAKE, SWS and REM
(Figure-4.14). Further, changes observed due to stress in EEG frequencies and
powers of all the four hours of continuous recording have been plotted and
discussed.
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Experimental Design and Methods Applied
0.0
-0.2
-0.4
-0.6
-0.8
EEG
EMG
EOG
60 61 62
Time (s) 0.2
0.0
-0.2
-0.4
-0.6
EEG
EMG
EOG
100 200 300 400 500
Time (Samples)
0.0
-0.2
-0.4
-0.6
EEG
EMG
EOG
100 200 300 400 500
Time (Samples)
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(1)
Relative Band power is calculated for the four frequency bands on EEG (Fig 3.9
shows the power spectral density of a given frequency band) [8] [11] .Relative
band power is calculated by dividing the total power spectral density of a given
band to the total power spectral density of the entire frequency range. This relative
band power implies how much a particular frequency band constitutes in the
complete frequency band. This forms the basis of deciding whether a particular
frequency constitutes a large or small portion of the frequency band, which forms
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Experimental Design and Methods Applied
the fuzzy sets for ANFIS. Apart from the relative band power total band power
was also extracted for the four frequency bands for the given data. Total band
power changes from control to Acute/Chronic state. The EOG and EMG activity is
also evaluated.
(2)
Where N represents the sample length or the time window for the signal x.
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Experimental Design and Methods Applied
3.7 Fuzzy Logic Application for sleep stage and stress level
determination
A Matlab program has been written to implement ANFIS for sleep stage
classification and analyze the results. ANFIS has been chosen because visual
inspection data was present to do the supervised learning. A sugeno type fuzzy
inference system is created first with 6 inputs and one output. The six inputs were
Alpha band power, Beta band power, Theta Band power, Delta band power, EOG
activity and EMG activity. Sleep stages classification has earlier been attempted
without using polygraphs but using only EEG signals. It dealt mostly with
identifying certain wave patterns and spindle and k-complexes detection to
classify sleep stages and the results were not very accurate but presence of other
event markers like EOG and EMG reinforces the sleep classification capabilities.
Figure 3.10 shows the ANFIS structure where six inputs to the Mamdani type
system can be seen with one output which represents the sleep stage. Also as far as
various methods used in AND and OR, min and max method is chosen. For
implication min method is chosen because it gives the best results in normal cases.
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Experimental Design and Methods Applied
For aggregation max method is used and for defuzzification Centroid method,
most commonly used method, is used.
Regarding membership function for inputs the gbellmf function is used for
representing each fuzzy set low and high while for output triangular membership
function are used. We have chosen the Gbell membership function because of its
nature that it can take any values in any of its variables and it is differentiable all
throughout its range. Most of the membership function suffers this limitation e.g. a
triangular membership function has zero slopes at some points and is non-
differentiable while its variables cannot take certain values. Figure 3.11 shows a
generalized bell membership function which can take any variable for its
parameters a, b and c.
The output of the system is properly quantized to analyze the results. The training
has been done for 1000 epochs and system achieved satisfactory results with
learning algorithm converging in about 100 epochs.
After the sleep stages have been classified for each stress level, heat stress
classification is done separately for all the three stages because physiological
changes happens differently in each of the sleep stages. To analyze the changes in
total band power which happens predominantly when the subject is subjected to
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Experimental Design and Methods Applied
thermal stress for each sleep stage. A measure which evaluates the difference of
the total band power for each input is coded. Since, these changes happen only in
the EEG, EOG and EMG data are not required.
Figure 3.12 shows a Mamdani type fuzzy inference system which has been applied
for tress level classification. tAlpha, tBeta, tTheta and tDelta are the changes in the
band power of respective frequency bands. The membership function for inputs
and outputs are of the form Triangular. The AND and OR methods are min and
max respectively. Implication and aggregation method are min and max
respectively. Defuzzification method is centroid as used for sleep stage
classifications as well.
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Experimental Design and Methods Applied
The next section deals with the sleep stage detection and stress level detection
where use of fuzzy for these purposes has been expounded well.
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