AIM:- The preset study has been designed to investigate the

antioxidant effect of HASPERIDIN on global cerebral ischaemia and

reperfused-induced cerebral injury.

INTRODUCTION:-
Stroke is a syndrome characterized by rapid onset of neurological injury due to
interruption of blood flow to the brain(Baker et al.,1998,Easton et al.,1998).Although mortality
from stroke has declined over the last decade but it still remains the third leading cause of the
death(Whisnant,1984;WHO,1992).Moreover,stroke is associated with higher incident of deficits
in sensory motor function and cognitive ability(Phipps,1991).It is estimated that stroke is
responcible for about 102,000 deaths annually in india,which represent 1.2% of the total death in
country (Anand et al.,2001). Hypertension has long been recognized as one of the most
important risk factor for stroke;however there is growing evidence that familial predisposition or
hereditary factor may play a significant role in the etiology of stroke(Diaz et al.,1986; Banerjee
wt al.,2001).

Brain injury following stroke develops from a complex cascade of cellular events that ultimately
leads to cerebral infraction(Lipton;1999;Small et al.,1999). Decreased blood flow leads to
reduction in phosphocreatinine and ATP which results in disruption of membrane ionic gradient
leading to neuronal depolarization(Katasura et al.,1994) and produces massive release of
glutamate from presynaptic nerve terminal. The released glutamate activated post synaptic N-
methyl-D-aspartate(NMDA) receptors leading to abnormally high Ca+2 promote the formation
of free radicals(Smdani et al.,1997).

Ca+2 overload-induced inhibition of mitochondrial electron transport chain and activation of

PLA2 are the main source of reactive oxygen species (ROS) formation during cerebral
ischemia(Drenth et al.,1976;Moskowitz et al.,1984). Reperfusion of ischemia brain may further
aggravate neuronal injury by increasing free radical formation(D ELZAPPO AND Hellen
beck.2000). Free radicals are demonstrated to promote lipid peroxidation(Kumar et al.,1987),
which ultimately results in altered plasma membrane integrity(Mead.1976). Neuronal plasma is
rich in polyunsaturated fatty acid and is particularly vulnerable to the cytotoxic effects of free
radicals(Siesjo.1992). moreover,level of antioxidant enzyme in brain is relatively
low(Bast,1994).

DNA nicking, a characteristic of oxidative injury to DNA can be detected within minutes of
cerebral ischemia. The ROS such as peroxynitrite (Epe.et al,1996), hydroxyl radical(Beesl et
al.,1979). Superoxide anion and to lesser extent nitric oxide (Delaney et al.,1997) are reported to
produce DNA nicking. ROS are also noted to activate lysosomalenzymes,which may contribute
to neuronal injury(Kalra et al.,1998;Ollinger and Brunk,1995). Additionally ,ROS mediated
damage to mitochondria release proapoptotic factor such as cytochrome C, caspane 9 and
apoptosis inducing factor,which may contribute to delayed cell death after cerebral ischemia and
reperfusion(Hirsch et al.,1998;Gottlieb,1999;Eldadaeh and Faden,2009;Fiskum,2000). The
increase in the generation of ROS and their involvement in neuronal damage has been
demonstrated in several experimental and clinical studies(Kirsch et al.,1987;Caoet
al.,1988;Kitagawa et al.,1990;Globus et al.,1995).

Therefore it is rational to investigate the effect of some exogenously administrated antioxidant

on ischemia and reperfusion-induced cerebral injury.

MATERIAL AND METHOD:-

BAL b\c mice of either sex weighing 23-27 g.maintained on standard laboratory diet(kisan feeds
ltd.mumbai,india) and tap water add libitum ,where employed in the present study,they were
housed in the departmental animal house and were exposed to a 12h cycle of light and dark. All
the animals used in study were naïve to the elevated plus-maze test. The experiments were
conducted in semi-sound proof laboratory.

GLOBAL CEREBRAL ISCHEMIA AND REPERFUSION:-

Mice are anaesthetized with chloral hydrate(400 mg/kg,ip). A middle ventral incision was made
in the throat. Right and left common carotid arteries were located and freed from surrounding
tissue and vagus nerve. A cotton thread was passed below each of the carotid artery. Global
cerebral ischemia was induced bypulling the ends of thread with constant weight. After 10 min.
of global cerebral ischemia, weight on the thread was removed to allow the reflow of blood
through carotid arteries. The incision was sutured back in layers(Himori et al.,1990). The sutured
area was cleaned with 70% ethanol and was sprayed with antiseptic dusting powder. After
completion of surgical percedure ,the animal was shifted individually to their home cages and
were allowed to recover. Body temperature of mice was maintained at 37’c by heated surgical
platform. All the surgical instruments used in the surgical procedure were sterilized by
incineration prior to use.

ASSESSMENT OF CEREBRAL INFARCT SIZE:-

At the end of 24 hr. reperfusion after the global cerebral ischemia animals were sacrificed by
spinal dislocation and the brain was removed. The brain was kept overweight at -4’c. frozen
brain was sliced into uniform of about 2-3 mm thickness. The slice s were incubated in 1%
triphenyltetrazolium chloride(TTC) at 37’c in 0.2 M buffer(pH7.4) for 20 min( Bochelen et
al.,1999), TTC is converted to red formazone pigment by NAD and dehydrogenase and therefore
stained the viable cell deep red. The infracted cells have lost the enzyme and cofactor and thus
remained unstained dull yellow. The brain slices placed over glass plate. A transparent plastic
grid with 100 squares in 1 cm2 was placed over it. Average area of each brain slice was
calculated bt counting the number of square on either sides. Similarly, numbers of squares falling
over non-stained dull yellow area also counted. Itfracted area was expressed as a percentage of
total brain volume. Whole brain slices were weighted. Infracted dull yellow part was dissected
out and weighted. Infracted size was expressed of total wet weight of brain.

SHORT TERM MEMORY EVALUATION USING ELEVATED PLUS MAZE:-

Plus-maze consisted of two open(16*5 cm) and two enclosed (16*5*12cm) arms, connected by
central platform(5*5cm). the apparatus was elevated to a weight of 25cm above the floor. A fine
line was drawn in the middle of the floor of each cnclosed arm. All the animals were given a
single trail on plus-maze. Each mouse was individually placed at the end of open arm facing
away from central platform of the maze. The time taken by the mouse to enter from open arm
with all the floor legs into the enclosed arm was taken as transfer latency time(TLT). In case the
animal didn’t enter the enclosed arm within 90 sec. , it was gently pushed into the enclosed arm
and a TLT of 90 sec. was assigned to it. The animal was allowed to explore the maze for an
assitional 10 sec. after the measurement of TLT (Itoh et al.,1990).

Animal was subject to global cerebral ischemis for 10 min followed by reperfusion for 24 hr.then
it was put to elevated plus-maze test. TLT measured on plus maze on first and second day serve
as index of learning or acquisition, whereas TLT recorded an the third day served as an index of
retrival or memory. Utmost care was taken not to change the relative location of plus maze with
respect to any object serving as visual clue in laboratory.

EXPERIMENTAL PROTOCOL:-
A total of three groups of animals were wmployed in the present study.

SHAM GROUP (group 1;n=6):-

Mice were subjected to surgical procedure and thread was passed below both carotid artires were
not occuladed. After 10 min. thread was removed and the animal were sutured back and allow to
recover for 24 hr.

CONTROL GROUP (group 2 n=6):-

Mice were subjected to 10 min. global cerebral ischemia followed by reperfusion for 24 hr.

HESPERIDIN TREATED GROUP (group 3 n=6):-

Group 3 comparised of mice injected hesperidin (40mg/kg,ip) 30 min. before subjecting the mice
to the global cerebral ishchemia.
EXTRACTION AND PREPARATION OF TEST SAMPLE:-
ISOLATION OF HESPERIDIN FROM ORNAGE PEEL:-

100gm of dried orange peel was refluxed with 400 ml of petroleum ether on water bath for 1
hour. Then contents were then filtered and marc obtained was dried at room temperature. Dried
marc was extracted with 400 ml of methanol under reflux for 3 hour. Filterate so obtained was
concentrated under reduced pressure to syrupy mass and hesperidin was crystallized from dilute
acetic acid solution.

The isolated fraction was suspended in aqueous solution of 1 % carboxy methyl cellulose by
vortexing for 15 min. followed by ultrasonication for a period of 20 min. just prior to
adminsteration.

DRUGS AND CHEMICALS:-

All chemicals used were a analar quantity. TTC was procured from lobachem. All drugs
solutions were freshly prepared before use.

RESULT:-
SHAM CONTROL HESPERIDIN
TREATED
CEREBRAL 6 45 25
INFARCT SIZE BY
(VOL.)
CEREBRAL 5 43 22
INFARCT SIZE BY
(WT.)
PLUS-MAZE (SEC.) 8 170 75

DISSCUSSION:-
Global cerebral ischemia and reperfusion model employed in the present study is reported to
stimulated the clinical situation of cerebral ischemia due to cardiac arrest of the drowing
(Alonsode lecina et al.,2001). Prolonged global cerebral ishchemia results in neuronal death
irrespective of post ischemia reperfusion (Neumar,2000). Therefore global cerebral ischemia of
short duration followed by reperfusion has been employed in present study. Hippocampal
neurons are highly susceptible to deleterious effects of the ischemia and reperfusion (JENKIN
ET AL.,1981). Hippocampal is involved in regulation of short term memory, therefore elevated
plus maze test hase been employed in the present study to evaluate the effect of global cerebral
ischemia and reperfusion on short term memory.

Administration of hesperidin prior to global cerebral ischemia has a attenuated ischemia and
reperfusion induced cerebral infract size and has prevented ischemia and reperfusion induced
impairment of short term memory. These observation suggest neuroprotective effect of
hesperidin. Free radicals are implicated in in global cerebral ischemia and reperfusion induced
neuronal injury (Cross et al.,1987,Globus et al,1995,Baker et al,1998).

Hesperidin and its metabolites are known to cross BBB in vivo as well as in vitro (Yodim et
al.,2003). Hesperidin show antioxidant activity by inhibiting nitric oxide synthase-2 (NOS-2),
which is actively involved in free radical generation (Olszanecki et al.2002). thus hesperidin
possibly act as neuroprotective agent.

SUMMARY AND CONCLUSION:-

On the basis of results of present study, following findings may be summarized.

1. Global cerebral ischemia of 10 min. followed by reperfusion for 24hr. produced neuronal
injury as indicated by cerebral infarct, impairment of short term memory, motor incoordination
and postural deficit.

2. Administration of hesperidin before cerebral ischemia and reperfusion is neuroprotective

because it has significantly reduced cerebral infarct size and has prevented global cerebral
ischemia and reperfusion induced impairment of short term memory, motor incoordination and
postural deficit.

In conclusion the present study indicates that research is required to be done in finding out the
components of plant extracts responsible for their neuroprotective effect and to further study the
potential of hesperidin as compound protective for stroke.