METABOTROPIC GLUTAMATE RECEPTOR 1α :
MODULATION OF SOMAN- INDUCED STATUS EPILEPTICUS

LEONG AI LIN
B.Sc, The University of Melbourne

A THESIS SUBMITTED FOR THE DEGREE OF
MASTERS OF SCIENCE
DEPARTMENT OF PHARMACOLOGY
NATIONAL UNIVERSITY OF SINGAPORE
2003


ACKNOWLEDGEMENT

“Great wisdom is lessons learnt from many men’s acts of foolishness.”

We learn everyday and sad to say usually through mistakes. In the pursuit of this MSc
degree, I made mistakes, I corrected them and most importantly I learnt. All these
would be impossible if not for Assoc. Professor Sim Meng Kwoon and Dr. Tang Feng
Ru. Many thanks for their expert guidance, patient teachings and kind understanding.

I am very grateful to all the staff of DSO National Laboratories with whom I spent my
2½ years. Many thanks especially to Mr. Loke Weng Keong for giving me the chance
to work in DSO and providing me his ever- ready- help in many areas of my study. I
would like to thank members of the Pharmacology and Toxicology Group, Emily,
Doris, Yong Teng, Alicia and ex-members, Cynthia and Jing Ping who made my life
in the group very happy and memorable.

Last but not least, my sincere thanks to my family and friends for all their support
during the ‘darkest days’ of my life.

i


TABLE OF CONTENTS
Page
List of Figures

iv

List of Tables

v

List of Abbreviations

vi

Summary of Thesis

viii

Chapter 1: Introduction

1

Chapter 2: The Animal Model
2.1

2.2


2.3

Materials and Methods
2.1.1

Animals

5

2.1.2

Recordings of electroencephalogram

5

2.1.3

Intoxication by soman

6

2.1.4

Perfusion and cell histology

7

2.1.5


Neuropathology assessment

7

2.2.1

Seizure and EEG recordings

8

2.2.2

Histology Examination

9

Results

Discussion

15

Chapter 3: Metabotropic Glutamate Receptors Expression during SISE
3.1

Overview

3.2

Materials and Methods

3.2.1

3.3

Immunohistology staining

22

Effect of SISE on mGluR1α expression

23

Results
3.3.1

3.4

18

Discussion

26

Chapter 4: Post Treatment of SISE
4.1

Overview

4.2


Materials and Methods

28
ii


4.3

4.4

4.2.1

Drugs

32

4.2.2

Intracereboventricular (icv) administration
of mGluR1 antagonist

32

4.3.1

Neuroprotective activity of diazepam

34

4.3.2


Neuroprotective activity of LY367385

36

Results

Discussion

39

Chapter 5: Neurotransmitters and SISE
5.1

Overview

5.2

Materials and Methods
5.2.1

5.3

42
Microdialysis fiber implantation
and perfusion

46

5.2.2


Analysis of amino acids

47

5.2.3

Statistical analysis

47

5.3.1

Chromatographic separation

48

5.3.2

Effects of LY367385 on extracellular

Results

glutamate and GABA
5.3.2.1

Delivery via icv

5.3.2.2


Delivery via reverse
microdialysis

5.4

Discussion

50
52
56

References

60

Appendix A

A-1

iii


LIST OF FIGURES
Figure

Title

Page

2.1


Phases of electroencephalogram pattern changes observed in
rats intoxicated with soman

10

2.2

Cresyl violet staining at piriform cortex of vehicle (saline)treated rat, and soman- treated rats

11

2.3

Pathology in the piriform cortex 1 day after soman-induced
SE visualized by cresyl violet stain

12

2.4

Cell quantification at layer II and III of the piriform cortex at
different time intervals after soman intoxication

14

3.1

Immunoreactivity for mGluR1α in layer III of the piriform
cortex.


24

3.2

mGluR1α immunoreactivity in the piriform cortex at
different time intervals after soman intoxication.

25

4.1

Chemical structure of LY367385.

31

4.2

Neuroprotective effect of diazepam administrated
intramuscularly 20 minutes post seizure onset.

35

4.3

Cresyl violet staining at piriform cortex of SISE rats treated
with combinations of LY367385 and diazepam.

38


5.1

Resolution and detection of glutamate and GABA by HPLC

49

5.2

Time course of changes in glutamate output induced by
SISE and SISE + post- treatment with LY367385 and
diazepam

51

5.3

Effects of reverse microdialysis of LY367385 on the release
of glutamate and GABA from the piriform cortex

54

5.4

Time course of changes in glutamate and GABA output
induced by reverse microdialysis of 5mM LY367385 in the
piriform cortex of SISE rats

55

iv



LIST OF TABLES
Table

Title

Page

4.1

Neuroprotective effect of drug combination treatment on
SISE

37

5.1

Pathways utilizing glutamate as a transmitter

43

v


LIST OF ABBREVIATIONS
2-PAM

Pralidoxime-2-chloride


3HPG

3-hydroxyphenylglycine

AC

Adenyl cyclase

ACh

Acetylcholine

AChE

Acetylcholinesterase

ACSF

Artificial cerebo- spinal fluid

AIDA

(RS)-1-aminoindan-1,5-dicarboxylic acid

AMN

Atropine methyl nitrate

AMPA


α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

BSA

Bovine serum albumin

CA

Cornu ammonis

CBPG

(S)-(+)-2-(3’-carboxybicyclo[1.1.1]pentyl)- glycine

ChE

Cholinesterase

CV

Cresyl violet

DHPG

3, 5 dihydroxyphenylglycine

EAA

Excitatory amino acid


ED50

50% Effective dose

EEG

Electroencephalogram

EPSP

Excitatory postsynaptic potential

GA

Tabun

GABA

γ- aminobutyric acid

GB

Sarin

GD

Soman

GTP


Guanine triphosphate

HPLC

High pressure liquid chromatography

i.m.

Intramuscular

i.p.

intraperitoneally

IAA

Inhibitory amino acid

icv

Intracerebroventricular

iGluR

ionotropic glutamate receptor

IOD

integrated optical density


IP3

inositol-1,4,5-trisphosphate

LD50

50% lethal dosage
vi


LY367385

(+)-2-methyl-4-carboxyphenylglycine

mGluR

Metabotropic glutamate receptor

NMDA

N-methyl-D-aspartate

OD

optical density

OP

Oraganophosphorus


OPA

O-phthalaldehyde

PB

Phosphate buffer

PBS

Phosphate buffered saline

PBSTx

Phosphate buffered saline with 0.1% Triton X

PI

Polyphosphoinositide

PKC

Protein kinase C

PLC

Phospholipase C

sc


Subcutaneous

SE

Status epilepticus

SISE

Soman-induced status epilepticus

TBS

Tris- buffered saline

VX

O-ethyl-S-[2-(diisopropylamino)ethyl]
methylphosphonothioate

vii


SUMMARY OF THESIS
Soman

(pinacolyl

methylphosphonofluoridate),

a


potent

irreversible

acetylcholinesterase (AChE) inhibitor, induces status epilepticus (SISE) in rats during
severe intoxication. Subsequent neurodegeneration in the limbic structures is a result
of hyperexcitability of the epileptic brain and is thought to be mediated by activation
of glutamate receptors. The aim of this study was to focus on the role played by
metabotropic glutamate receptor (mGluR) and the effect of selective blockage of
mGluR1α as a post- treatment of SISE.

In this current work, an initial effort was concentrated on the establishment of an in
vivo animal model to facilitate the study of post-SISE treatment drugs. An intoxication
regime was drawn up to include a pre-treatment combination of pyridostigmine
bromide and atropine methyl nitrate (AMN) administered 30 minutes prior to a
convulsive dose of 176µg/ kg (equivalent to 1.6LD50 ) soman injected subcutaneously
into Wistar rats, followed by injection of pralidoxime-2-chloride (2-PAM) and an
additional dose of AMN. Pathological symptoms such as mastication, motor
convulsion and salivation were exhibited within minutes of exposure. A histological
examination of the brain sections revealed a progressive neuronal loss throughout a
one- month time course after intoxication. Piriform cortex, amygdala and hippocampus
are the most vulnerable structures. Immunohistological staining for mGluR1α in the
piriform cortex showed a 3-fold upregulation of expression from basal level at hour 8
post-SISE.

Activation of Group I mGluR produces long-lasting epileptiform discharges in the
hippocampus (Merlin and Wong, 1997) and intracerebroventricular (icv) injection of 3,
5-dihydroxyphenylglycine (DHPG) into rats induced seizure and subsequent neuronal
viii



damage (Camon et al., 1998). To investigate if such an activation of mGluR1α in our
SISE model is neurotoxic, a selective mGLuR1α antagonist, LY367385, was used. Icv
administration of 4- 400 nmole of LY367385 at 15 minutes after seizure onset did not
terminate seizure activity nor showed any neuroprotection. Cell density, regardless of
dosage tested, was not significantly different from that of soman-intoxicated untreated
animals.

In another experiment, a combination of diazepam and LY367385 was tested.
LY367385 (200 nmole, icv) was given at 15 minutes after seizure onset, followed by
intramuscular injection of 2 mg/kg diazepam 5 minutes later. Though seizure
termination was not observed in this group, such a combination improved the
percentage of cell survival in layer II and III of the piriform cortex by more than 2.5
and 3.5 times, respectively, as compared to untreated rats or rats that received only
diazepam (2 mg/kg) treatment. At a higher dose of combination --- 400 nmole of
LY367385 and 4 mg/kg of diazepam, three out of six rats displayed lowered amplitude
EEG spikes within 20 - 30 minutes after drug treatment and seizure activity was
altogether terminated by the fourth hour. These three animals did not suffer any
neuropathology and their cell density in the piriform remained unchanged from the
control level.

In a subsequent study, reverse microdialysis of 5 mM LY367385 solution into the
piriform cortex during SISE increased the release of the inhibitory amino acid GABA
by 300- fold and reduced the excitatory amino acid glutamate to 10% of basal level.
However, this mode of local administration to the piriform cortex was not effective in

ix



terminating seizure activity. Cell densities in layer II and III of the piriform cortex
were observed to be 70% and 40 %, respectively, that of non-intoxicated rats.

In conclusion, activation of mGluR1α during SISE leads to neurotoxicity, and
blockage of the receptor by the antagonist LY367385 modulates the release of
glutamate and GABA contributing to the anti-epileptic and neuroprotective activity
when used in combination with diazepam.

x


Chapter 1

Introduction
Organophosphorus (OP) nerve agents are highly toxic chemical weapons. They are
traditionally classified into the G agents, such as GA (tabun, ethyl, N,Ndimethylphosphoramidocyanide), GB (sarin, isopropylmethylphosphonofluoridate)
and GD (soman, pinacolyl methylphosphonofluoridate), and the V agents, exemplified
by VX (O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate). They are
believed to be stockpiled in military arsenal of several countries. To date, no largescale military deployment of a nerve agent has occurred during war, although indirect
evidence exists that the Iraqi military used tabun against Kurdish villagers in 1988 as
well as during the Iraq-Iran War. More recently was the infamous 1995 Tokyo Subway
sarin attack launched by a Japanese terrorist cult, Aum Shinrikyo, killing 13 people
and resulting in more than 5500 casualties(Nozaki et al., 1995). Nerve agents acquired
their name because they affect the transmission of nerve impulses in the nervous
system when absorbed through the skin and via respiration.

The G- agents are liquids at moderate temperature and humidity so that the term ‘nerve
gas’ is a misnomer, but the vapour pressure of the G-agents make them significant
inhalation hazards. Sarin is highly volatile compared to tabun and soman. The physical
characteristics of G-agents permit their evaporation and dispersion over several hours

under temperate climate conditions and, thus, they are also termed as nonpersistent. On
the other hand, the V-agents are persistent. They can cause cascualties by both
inhalation and absorption through the skin. As an oily and non-volatile liquid, VX may

1


remain in place for weeks or longer after dispersion (see review in Somani, et al.,
1992).

Following absorption, the OP nerve agent will irreversibly inhibit the enzyme
acetylcholinesterase (AChE), by phosphorylating a serine hydroxyl group in the active
site of the enzyme, resulting in the cessation of acetylcholine (ACh) hydrolysis.
Rapidly, the huge excessive accumulation of ACh will overstimulate the cholinergic
system leading to clinical manifestations that can be divided into three groups:
muscarinic (postganglionic parasympathetic receptors), nicotinic (at preganglionic
sympathetic and parasympathetic terminals and neuromuscular junction) and central.
Muscarinic symptoms included bronchorrhea, salivation, constriction of the pupil of
the eye (miosis), abdominal colic and bradycardia. Increased activity at the nicotinic
receptors can produce pallor, tachycardia, hypertension, muscle fasciculation and later
paralysis. In the central nervous system (CNS), symptoms include restlessness, tremor,
convulsion, seizure and respiratory depression (see review in Marrs, et al., 1996). As
nerve agent poisoning in humans is rare, all the above clinical effects and lethal dose
studies are collective observations from experimental poisoning in animals. In cases
where subjects survived the acute intoxication, morphologic changes in the brain may
occur.

Earlier views were of the opinion that OP nerve agents are neurotoxic (Petras, J. M.
1981). However McDonough et al., (1987) showed that neuronal damage only
occurred in convulsing animals and not in non-convulsing ones when soman was

directly microinjected into the amygdala. Hence, neuronal damage was a result of
seizure activity.

2


The longer seizure activity lasts, the harder it becomes to stop. Application of
anticholinergic drugs that target muscarinic receptors like caramiphen, scopolamine
and dexetimide were effective in terminating soman-induced seizures when given
shortly after (2.5 - 10 min) seizure onset, but displayed no anticonvulsant activity when
treatment was delayed for 40 min (Shih, et al., 1999). These results added to the
established hypothesis that prolonged seizure, though initiated by hyperactivation of
cholinergic drive, was maintained by non-cholinergic systems. This advanced episode
is often referred as status epilepticus (SE). With soman-induced SE (SISE) lasting for
several hours, neuropathology was severe. The vulnerable brain regions are the
piriform cortex, hippocampus, frontal cortex, entorinal cortex, amygdaloid complex
(mainly the limbic structures), and several thalamic nuclei (McLeod et al., 1984; Baze,
1993; McDonough et al., 1998).

Continual efforts have been made to arrest seizure before the propagation of
epileptogenesis reaches a devastating state that results in wide-spread neuronal
damage. Hence, the development and use of CNS depressant drugs like
benzodiazepines (diazepam, lorazepam, and midazolam) are recommended for the
treatment of SE (Marrs et al., 1996).

Research of anticonvulsants has not been limited to the development of drugs for
increasing inhibitory transmission to dampen the hyperexcitability of neurons during
SE, novel ways of decreasing excitatory transmission were also reported. Since the
maintenance of SE is under the control of non- cholinergic systems, one very likely
candidate would be the glutamatergic neurotransmission system. As detailed in the


3


latter chapters agonists or antagonists at the glutamate receptors can bring about
facilitation or inhibition of excitatory postsynaptic potentials (EPSP).

The aim of this study was to focus on the role played by metabotropic glutamate
receptor (mGluR) and the effect of selective blockage of mGluR1α as a post- treatment
of SISE.

The results demonstrate that neuropathology could be detected as early as 8 hours of
SISE. At the same time- point, we found that expression of mGluR1α was upregulated. Post treatment with a combination of diazepam and a selective mGluR1α
antagonist (LY367385) confers neuroprotection in some animals by stopping seizure
activity. Reverse microdialysis of LY367385 reduced glutamate and increased GABA
output in the piriform cortex suggesting that modulation of neurotransmitter release
may contribute to neuroprotective activity.

4


Chapter 2
The Animal Model
The effects of soman had been studied using various animal species, from small
laboratory animals like mice, rats and guinea pigs to bigger mammals such as cats,
dogs, pigs, horses and non-human primates. Setting up an easy-to-manipulate in vivo
animal model to achieve reproducible and reliable results is critical to this study.
Therefore, the Wistar rat strain, which we have a plentiful supply from Laboratory
Animal Centre, National University of Singapore, was chosen.


2.1 Materials and Methods
2.1.1 Animals
Male Wistar rats of body weight 200-300g were housed five per cage (38cm x 20cm x
15cm) with wood chips bedding and held quarantined for evidence of disease for a
minimum of 3 days before usage in experiments. Commercial certified rodent ration
and tap water were provided ad libitum. The Animal Holding Unit was maintained at
21±2oC with 60±10% relative humidity. The rats were on a 12-hour light/ dark full
spectrum lighting cycle with no twilight. The animal experiments were carried out
according to International Guiding Principles For Animal Research adapted from
WHO Chronicle, 39(2): 51-56, 1985.

2.1.2 Recordings of electroencephalographic patterns
Two cortical stainless steel connectors (female ends; Elfa EB) were positioned at
stereotaxic coordinates relative to bregma of AP-7, L±2 mm; (Paxinos and Watson,
1982) and secured with dental cement. The male ends of the connectors were attached

5


directly to the lead wire ends of a TA10C-F40 telemetry transmitter of computerized
EEG system (Data Sciences International). The EEG signal was continuously digitized
and stored to disk at a frequency of 250Hz with a filter cutoff set at 100Hz. During
EEG recordings, all animals were housed in individual plastic cages that allowed free
movement with the exception of the recording leads attached to the connectors on the
top of the head. Baseline EEG activity was monitored for 30 minutes.

2.1.3 Intoxication by soman
The 50% lethal dosage (LD50) of 100 ppm soman in ice-cold (0.9% sNaCl) saline
(synthesized by Center for Chemical Defence, DSO National Laboratories, Singapore)
was determined to be 110µg/kg when injected via subcutaneously into Wistar rats. In

our animal model, a dose of 1.6LD50 (176µg/kg) was adopted. The intoxication
method used was similar to Ballough et al., (1995). A pretreatment regimen was
included to reduce the acute lethality. An intramuscular (i.m.) dose of 0.13mg/kg of
pyridostigmine bromide and either 4mg/kg or 8mg/kg of AMN were given 30 minutes
before soman administration. Immediately following soman, an i.m. injection of
pralidoxime-2-chloride (2-PAM) (25mg/kg) was made. Control animals (n = 3) were
subjected to the same pre-treatment but received saline in place of soman. Animals
were observed for 4 hours and at that time surviving animals were given 5ml of 5%
glucose in saline intraperitoneally (i.p.). They were randomly assigned to be
euthanised at Hour 8 (n = 6), Day 1 (n = 6), Week 1 (n = 4) or Week 4 (n = 6).

2.1.4 Perfusion and cell histology
At the specified times, survivors were anesthetized by a 3.3ml/kg i.p. dose of analeptic
mixture of hypnorm (Jassen Pharmaceutica, Begium): dormicum® (Roche,

6


Switzerland): water (1:1:2 ratio) and perfused transcardially with 15 minutes of saline
followed by 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4) for 30
minutes. The brains were removed and post-fixed in the same fixative overnight at
4oC. Additional cryoprotection treatment was carried out in 30% sucrose in 0.1M PB at
4oC for at least 48 hours. 40-µm thick frozen sections were cut serially in a coronal
plane by a Leica CM1800 cryostat set at –18oC and mounted onto Vector-bond (Vector
Laboratories, USA) coated glass slides for cresyl violet (CV) staining (0.8% CV in
0.1M acetate buffer, pH 3.5) for cell histology.

2.1.5 Neuropathology quantification
Four sections (Bregma AP–2.8, -3.6, -3.8, -4.3 mm, according to Paxinos and Watson,
1982) were chosen to determine the level of neuronal loss in the piriform cortex.

Quantification was carried out with a computerized image analysis (Image Pro Plus,
Media Cybernetics, USA). Sections were viewed through a microscope (Leica DMLB)
connected via videocamera to a PC monitor. Cell quantification in the piriform cortex
was expressed as a percentage of total area occupied by the cells in layer II and III
respectively over the total section area of 312108

m2 in the image at 100x

magnification.

7


2.2

Results

2.2.1 Seizure and EEG recording
Soman- intoxicated rats displayed a progression of toxic signs, typically classified into
5 stages:
i)

reduced movement, dazed

ii)

mastication, muscle fasciculations

iii)


tremor, straub tail

iv)

clonic jerks

v)

generalized convulsions; tonic spasms; facial and forepaw clonus

The seizure onset was observed to begin 7-19 min after soman exposure. However, the
present intoxication protocol led to a high mortality rate 80%. Animals died within the
first 3 hours usually due to respiratory distress. In an attempt to improve the survival
rate, an additional dose (4mg/kg, i.m.) of AMN was given together with 2-PAM, and
an almost 3 fold increase of survivors (up to 57%) was achieved.

EEG changed rapidly within a few minutes after soman intoxication (Fig 2.1). Baseline
monitoring characterised by low amplitude, regular frequency turned into high
amplitude rhythmic epileptiform spikes to mark the initiation of seizure activity.
Following these trains of high amplitude peaks, the next phase of EEG activity, which
the animals were found to be in for the longest period of time as showed in Fig 2.1C,
exhibited a pattern consisting of more stable amplitude spikes with the appearance of
flat periods (1-3s) interrupting the seizure discharge. Seizure activity continued
unabated for the duration of the experiment (at least 4 hours after seizure onset).

8


2.2.2 Histology examination
Light microscopic examination from samples obtained from rats euthanized at various

time points after SISE revealed neuropathology was observed 8 hours post exposure.
By 24 hours, the severity of damage could be observed at various brain areas. Fig 2.2
showed the progression of neuropathology at the piriform cortex. Decreases in cell
density at the limbic structures, hippocampus, amygdaloid complex and subcortical
structures including the olfactory cortex were also observed. At higher magnification
in piriform cortex (Fig 2.3B), shrunken and condensed nuclei in the pyramidal neurons
along with swollen neurons were detected. Vacuolations of neutrophil were observed
in some samples. By week 1 and week 4, lost neurons were not replaced and massive
gliosis characterized by astrocytes and microglial cells filled the neurodengenerated
tissues. Remaining neurons were scarce in numbers. These findings were consistent
with the neuropathology observed by McDonough et al., (1998). Neuropathology was
only confined to rats that experienced prolonged SE, while non-convulsing rats
retained their cell morphology and density similar to that of saline-treated controls.

Cell quantification was performed on 40 m CV stained sections of the piriform
cortex, a brain region which invariantly suffered the most severe damage after SISE.
The piriform cortex is the largest area of the mammalian olfactory cortex and is
divided into an anterior and a posterior part. It is usually described as a three- layered
structure. Layer I, which is the superficial layer, contains a small number of neurons,
dendrites and fiber systems. Layer II is a compact layer of cell bodies comprised of
semilunar cells and pyramidal cells. Layer III displays a decreasing gradient of
pyramidal cells but contains large sized multipolar cells and a dense network of basal
dendrites extending from pyramidal cells in Layer II (see review in Haberly, 1998).

9


baseline

(A)


Seizure onset

(B)

Prolong seizure lasting for more than 30 min

(C)

Fig.2.1 Phases of electroencephalogram pattern changes observed in
rats intoxicated with soman. (A) Baseline EEG was obtained before
receiving pretreatment and soman insult. (B) 10 minutes after soman
administration, high amplitude rhythmic spikes marked the onset of
seizure. (C) Stable trains of high amplitude spikes interrupted by flat
periods during status epilepticus.

10


A

B

III
II
I

C

D


E

Fig. 2.2 Cresyl violet staining at piriform cortex of (A) vehicle (saline)- treated rat, and
soman- treated rats at (B) 8 hours, (C) 1 day , (D) 1 week , (E) 4 weeks (100x mag).
Massive neuronal degeneration occurred in layer II and III and gliosis at Week 4 as
compare to control animal tissue.

11


(A)

(B)

Fig. 2.3 Pathology in the piriform cortex 1 day after SISE visualized with
cresyl violet stain. (A) The piriform cortex in saline-treated animals showed
numerous cells with regular cell morphology. (B) In contrast in animals that
experienced SISE, significant neuronal loss was evident. Note swollen cell
(arrow) with condensed nucleus.
12


Cell quantification to show progressive neuronal loss in the piriform cortex throughout
the one-month time-course was shown in Fig 2.4. The most pronounced decrease was
found on day 1 after soman insult, as cell density in layer III fell to half of that of
controls. By week 4, remaining cells were less than 30% of normal cell density.

13



16%
14%

Mean cell density

12%

*

*

10%

*

*

*

8%

*

*

6%

*
4%

2%
0%
control

n=3

h8

d1

n=6

n=6

w1

n=4

w4

n=6

Fig. 2.4 Cell quantification at Layer II ( ) and III ( )of the piriform cortex at
different time intervals after soman intoxication. (Mean cell density is the total
intact cell area expressed as a percentage of the section area in the image; see
Materials and Methods (page 7) for details.) Values are means + SEM.
*Significantly different (p<0.05) from the corresponding control values.

14