Chapter IV

Azadirachta indica inhibits oxidative damage and

apoptosis to prevent cerebral ischemia-reperfusion

injury in rats
 Chapter IV 2012

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4.1 Introduction
Millions of people in developed countries are affected by the long-lasting disability of
stroke (Hicks and Jolkonnen, 2009; Yousuf et al., 2009). Stroke may be caused due to
hemorrhage in brain or restriction to the blood flow in the major artery of brain. Cerebral
ischemia, the major type of stroke, results due to restricted blood supply to the parts of
brain. The major pathobiological mechanisms of ischemia/reperfusion (IR) injury include
excitotoxicity, oxidative stress, inflammation, and apoptosis (Ozbal et al., 2008; Yousuf et
al., 2009). Vascular reperfusion subsequent to transient occlusion results in worsening the
damage resulting in more free radicals generation and subsequent depletion in antioxidant
contents of cells (Kelly et al., 2008; Wu et al., 2008). A majority of ischemic cell deaths in
the brain has been attributed to necrotic processes, especially in the ischemic core region.
However, the roles of apoptotic mechanisms have been suggested in the penumbral regions
of ischemic or post-ischemic brain (Racay et al., 2009). An increase in pro-apoptotic
proteins that promote cell death may be one of the mechanisms underlying post ischemic-
reperfusion apoptotic cell death (Broughton et al., 2009). Mitochondria of ischemic cells
succumb to great demand of maintaining energy supply to cells during hypoxic-
hypoglycemic conditions (Preston and Webster, 2004). As a result, Cytochrome C gets
released from mitochondria and binds with Apaf-1 to form an oligomer which binds and
activates the initiator Caspase-9. Activated Caspase-9 in turn, binds with procaspase-3
which is finally cleaved to release the effectors Caspase-3 to execute apoptotic neuronal cell
death (Brahma et al., 2009; Végran et al., 2011).
The middle cerebral artery occlusion (MCAO) model of cerebral ischemia has much
importance in preclinical testing, in improving the understanding of deleterious insult in the
middle cerebral artery region of the brain and to study the potential efficiency of therapeutic
interventions (Dhanuka et al., 2001). In recent times, there has been a global increase in
natural product research and many plants have shown immense potential for therapeutic
uses. Azadirachta indica L. (Meliaceae), commonly known as neem, is widely used in
Indian System of Ayurvedic Medicine for its immunomodulator, antipyretic, vasodialator,
hypoglycemic, and diuretic properties (Biswas et al., 2002; Akihisa et al., 2009; Bhowmik
et al., 2010). Azadirachta seed is a good source of neem oil and having many limonoids that
may have medicinal values. Few reports showed that the Azadirachta seed also contains
antidiabetic, antihyperlipaemic, antihaemorrhagic, anti-inflammatory, and antioxidant
properties (Biswas et al., 2002; Bhowmik et al., 2010).

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As the role of A. indica is not well known in I/R injuries, therefore, the present study was
undertaken to evaluate the neuronal protective potential of A. indica seed extract (ASE) in
MCAO induced focal cerebral I/R model in rats. In this study, we examined in vitro free
radical scavenging properties of ASE and its anti-oxidative and anti-apoptosis potential in
MCAO model of cerebral ischemia.

4.2 Materials and methods

Azadirachta seed extract and chemicals are described in materials and methods (Chapter
III). Antioxidant potential, HPTLC fingerprinting, induction of ischemia, behavioral study
(FT, SMA, grip strength and rota-rod), infarct volume assessment, mitochondrial injury,
biochemical estimations (TBARS, NO, GSH, GR, GPx, catalase), ELISA (Caspase-3 and
9), immunohistochemistry of Caspase-3 were performed as given in materials and methods
(Chapter III).

4.2.1 Experimental design

The rats (16 weeks old; 260 - 280 g) were divided into six groups. The first group served as
Sham and vehicle (water) was given orally for 15 days (n = 16). The second group was the
vehicle (water) pretreated cerebral ischemic group (MCAO) in which ischemia was induced
for 2 h followed by reperfusion for 22 h (n = 20). The third to fifth groups were pretreated
with ASE (125, 250 and 500 mg/kg orally once daily for 15 days) followed by
ischemia/reperfusion (ASE125 + MCAO, n = 10; ASE250 + MCAO, n = 10; and ASE500 +
MCAO, n = 20; groups, respectively). After the last dose of ASE, i.e. on 16th day,
ischemia/reperfusion was induced. The sixth group served as sham-operated drug control
pretreated with ASE alone (500 mg/kg orally once daily for 15 days) (ASE500 + Sham; n =
6). After reperfusion, the rats were assessed for neurobehavioral activities and then
sacrificed. The brain was taken out to dissect the striatum, hippocampus and frontal cortex
for biochemical estimations.

4.3 Results

4.3.1 Total phenolic and flavonoid contents and antioxidant status

The ASE contains significantly high amount of flavonoids and polyphenols (Fig. 4.1). The
total flavonoid content (TFC) of ASE was significantly high as compared to the total
phenolic content (TPC). The TFC and TPC were expressed as quercetin

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Fig 4.1: Antioxidant property: Evaluation of antioxidant properties of ASE. (A) Standard curves of
ascorbic acid, gallic acid and quercetin. (B) TAP and RP of ASE at different conc. were determined
using the standard curve of ascorbic acid. TPC and TFC of ASE were determined by using standard
curves of gallic acid and quercetin, respectively. DPPH• and NO• free radical scavenging activities of
different concentration of ASE were determined by ascorbic acid standard curve (C and D). Results
show moderate free radical scavenging ability of extract, interestingly with high RP with means ±
S.D. Each experiment was performed in triplicate.

equivalent (QE) and gallic acid equivalents (GAE) respectively (Fig. 4.1A). The TFC and
TPC were calculated as 101.1 ± 7.7 mg QE/g extract and 59.6 ± 6.0 mg GAE/g extract,
respectively (Table 4.1). Total antioxidant activity (TAP) of ASE was increased with the
increasing concentration of the extracts and a significant change was observed at 10 to 100
µg of the extract (Fig. 4.1B). For the concentrations 10 to 100 µg of ASE, the absorbance
was between 0.04-0.149. However, the total antioxidant activity of ASE was less than the
positive control, ascorbate (Fig. 4.1A).

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The ASE has shown considerable amount of reducing activity (126.2 ± 20.0 mg AsAE/g
extract). The reducing power (RP) of the extract was increased with the increasing
concentration, and a significant absorbance (0.08 – 0.281) was observed at concentration of
10 to 100 µg of ASE (Fig. 4.1B). Free radical scavenging activities of ASE in the range of
20-200 µg was assessed by the DPPH• and NO• assays. IC 50 values of the positive control
ascorbic acid were 3.2 and 4.5 μg/ml for DPPH• and NO• free radicals (Fig. 4.1C). Fig.
4.1D illustrates a significant decreased concentration of free radicals which was due to the
scavenging ability of the extract. The results show that ASE has moderate DPPH• and NO•
scavenging activity with an IC 50 value of 171.0 and 176.0 μg/ml, respectively.

4.3.2 Correlation and regression analysis

Table 4.1 shows highly significant correlation coefficient for phenolic contents and total
flavonoids with TAP, RP, DPPH• and NO• scavenging (0.951 & 0.962, 0.917 & 0.951,
0.897 & 0.929 and 0.922 & 0.948, respectively; p < 0.001). Regression analysis for
phenolic and flavonoid contents with TAP, RP, DPPH• and NO• scavenging gives
significant RSQ (r2) values in the range of 0.806-0.905 and 0.863-0.925, respectively (p <
0.001). The slope of ASE showed higher significant value for flavonoid content with
DPPH• scavenging (6.719) and NO• scavenging (6.935) compared to TAP and RP.
However, phenolic content shows comparatively lower slopes in each in vitro analysis.

4.3.3 Fingerprint of ASE

The chromogram showed sharp and well defined peaks with maximum area at wavelength
248 nm (Fig. 4.2A); hence, it was selected for fingerprinting analysis. The fingerprinting
analysis showed the presence of 11 peaks at different R f values, i.e. 0.15 (1.6%), 0.25
(0.6%), 0.34 (1.2%), 0.40 (6.2%), 0.50 (3.5%), 0.67 (28.5%), 0.74 (2.4%), 0.83 (5.0%), 0.89
(21.2%), 0.92 (9.8%) and 0.94 (20.1%) in the chromatogram (Fig. 4.2B).

4.3.4 Behavioral outputs

The motor activity has shown no neurological deficits in sham group rats, while in MCAO
group, the neurological deficits were severe at 22 h after reperfusion. In flexion test, the
ischemic rats (MCAO group) showed persistent circling movements with significant severe
paw flections. Whereas in ASE-pretreated MCAO group, less persistent circling and
comparatively less severe paw flections with decreased postural disturbances was observed.
In spontaneous motor activity (SMA) test, ischemic animals spend most of the time in the
center of the cage with posture curved towards the paretic side. A marked decrease (p <

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0.01) in behavioral efficiency was observed in MCAO group as compared to Sham. The
ASE500 + MCAO group rats moved around in the cage and explored their environment
more efficiently as compared to MCAO group (p < 0.01). A marked improvement (p <
0.01) in behavioral outputs was observed in ASE pretreated groups as compared to MCAO
group (Fig. 4.3A and B).

Table 4.1: Correlation and regression of total phenolic and flavonoid contents from ethanolic
Azadirachta seed extract (ASE) with total antioxidant potential (TAP), reducing power (RP) and free
radical scavenging (DPPH· and NO·).

ASE TAP RP DPPH· NO·

Contents (30.29 ± 5.46 (126.24 ± 19.98 (25.97 ± 2.95 (27.97 ± 3.82
mg AsAE/g mg AsAE/g mg AsAE/g mg AsAE/g
Extract) Extract) Extract) Extract)

TPC r = 0.951 r = 0.917 r = 0.897 r = 0.922

(59.63 ± 5.95 r2 = 0.905 r2 = 0.841 r2 = 0.806 r2 = 0.849
mg GAE/g Slope = 0 738 Slope = 1.318 Slope = 1.519 Slope = 1.577
Extract) p < 0.001 p < 0.001 p < 0.001 p < 0.001

TFC r = 0.962 r = 0.951 r = 0.929 r = 0.948

2 2 2
(101.1 ± 7.69 r = 0.925 r = 0.905 r = 0.863 r2 = 0.899
mg QE/g Slope = 3.189 Slope = 5.844 Slope = 6.719 Slope = 6.935
Extract) p < 0.001 p < 0.001 p < 0.001 p < 0.001

*The pair(s) of variables with positive correlation coefficients and p < 0.01 tends to increase
together. AsAE- ascorbic acid equivalent; GAE- gallic acid equivalent and QE- quercetin equivalent.

In a grip strength test, a significant (p < 0.01) weak grip strength was observed in MCAO
group as compared to Sham group. Whereas the ASE pre-treatment has attenuated the
weakened grip strength significantly in all ASE + MCAO groups when it was compared to
MCAO group (Fig. 4.3C). Fig. 4.3D shows a significant (p < 0.01) depletion in muscle
coordination in MCAO group as compared to Sham group. Treatment with ASE was
effective in partial recovery of muscular in- coordination in ASE treated MCAO group as
compared to MCAO group.

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Fig. 4.2: HPTLC fingerprint of ASE: The chromogram shows sharp and well defined peaks with
maximum area at wavelength 248 nm (A). The fingerprinting analysis shows the presence of 11
peaks at different R f values i.e. at 0.15 (1.62%), 0.25 (0.59%), 0.34 (1.23%), 0.40 (6.24%), 0.50
(3.47%), 0.67 (28.47%), 0.74 (2.42%), 0.83 (4.95%), 0.89 (21.16%), 0.92 (9.78%) and 0.94
(20.06%) in chromatogram (B).

4.3.5 ASE reduced mitochondrial injury

TTC staining of the brain sections obtained from MCAO rats showed reproducible and
readily detectable lesions in the areas that are supplied by the MCA at 22 h after the
reperfusion. The lesions were present in hippocampus, lateral striatum and the overlying
cortex. ASE (125, 250 and 500 mg/kg, p.o.) reduced the infarct volume as compared to the
MCAO group (Fig. 4.4A). Mitochondrial injury was assessed as percentage loss with
respect to sham group (MCAO, 49.30%; ASE 125 - 500, 47.32, 44.82 and 27.61%,
respectively). The data represents the maximum loss in MCAO group with respect to sham
operated control group (Fig. 4.4B).

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Fig. 4.3: Behavioral deficits: The effect of Azadirachta (ASE) pretreatment on flexion test (FT; A)
and spontaneous motor activity (SMA; B) in MCAO rats is presented in above figure. A significant
motor deficit was observed in MCAO group animals as compared to Sham (S) group. Treating the
animals with ASE followed by MCAO has protected the motor deficit. Muscular grip strength (C) and
motor coordination skills (D) were significantly altered in MCAO group rats as compared to sham
group rats. ASE treatment has shown significant protection in ASE pretreated group as compared to
#
MCAO group. Significance were ascertained as p < 0.01 vs. Sham; * p < 0.05 and **
p < 0.01 vs.
MCAO.

4.3.6 Effect of ASE on the contents of TBARS, GSH and NO

The effect of ASE on the contents of TBARS, GSH and NO was measured to demonstrate
the oxidative damage in striatum, hippocampus and cortex of MCAO rats. The NO was
estimated in the form of nitrite. A significant increase (p < 0.01) in TBARS and nitrite level
was observed in MCAO group animals as compared to Sham group animals. Rats of ASE
pretreated MCAO group (ASE 500 + MCAO) exhibited significant attenuation (p < 0.01) in
TBARS and nitrite concentrations in comparison to MCAO group rats. The lower doses
(125 and 250 mg/kg) has shown significant decrease in TBARS in striatum and

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Fig. 4.4: The brain infarct: The effect of Azadirachta pretreatment on the brain infarct size was
evaluated by TTC stain after MCAO for 2 h and reperfusion of 22 h. Representative photographs
showing the brain sections stained with 0.1% TTC (A). To account all damage (visible and diffused),
measurement of mitochondrial injury (B) of various groups are presented. MCAO group produced a
significant infarct (white area) compared to contra-lateral side (red). Here, the altered texture of
MCAO rat brain is noted due to ischemic-reperfusion injury, whereas ASE pretreated group showed
a significant reduction in tissue damage at two higher dosages (250 and 500 mg/kg) of ASE as
compared to MCAO group (B). Values are expressed as mean ± SEM. *p < 0.05 and **p < 0.01, vs.
MCAO.

hippocampus only. The lower dose (125 mg/kg) has not shown any significant alterations in
the level of nitrite. ASE alone pretreated Sham group (ASE500 + Sham) showed no
significant changes in TBARS and nitrite level as compared to Sham group (Fig. 4.5A and

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C). The GSH level in striatum, hippocampus and cortex was found to be depleted
significantly (p < 0.01) in MCAO group as compared to Sham group. ASE treatment has
restored the level of GSH significantly (p < 0.01) in ASE500 + MCAO group as compared
to MCAO group (Fig. 4.5B). However, other ASE pretreated MCAO groups along with
ASE500 + Sham group exhibited no significant changes in GSH level as compared to
MCAO and Sham groups, respectively.

4.3.7 ASE augmented the activities of antioxidant enzymes (GR, GPx and Catalase)
Ischemia-reperfusiojn injury in MCAO rats reduced the antioxidant enzymes activity
significantly (p < 0.01). The activities of glutathione reductase (GR), glutathione peroxidase
(GPx) and catalase (CAT) were found to be restored by the administration of ASE (500
mg/kg) prior to MCAO (p < 0.01). The ASE control group (ASE 500 + Sham) has shown
no significant difference from the Sham group (Table 4.2).

4.3.8 ASE ameliorated histological changes in MCAO rats

Fig. 4.6 shows the histological changes after 2 h of occlusion and 22 h of reperfusion.
Sections of the brain passing through the striatum, hippocampus and cortex of sham,
MCAO and ASE500 + MCAO groups were examined. Sections of MCAO group have
shown edematous tissue and presence of numerous vacuolated spaces with heavy neuronal
loss (p < 0.01). The corresponding area in the sections of ASE500 + MCAO group showed
partial neuronal loss (p < 0.01) and the presence of intact neurons in between the vacuolated
spaces. The section of the sham group showed normal neurons with no pathological change.

4.3.9 ASE inhibited Caspase-3 and Caspase-9 activation

Apoptotic status in penumbral cortex was estimated in terms of Caspase activity (Fig. 4.7).
MCAO group (4.7B and E) has shown significant expression of Caspase-3 in cortex region
as compared to the sham group (4.7A and D). The percentage of Caspase-3 positive cells
has been reduced significantly by the administration of ASE (p < 0.05) [4.7G]. Moreover,
higher activities of Caspase-3 (115.8%) and Caspase-9 (118.4%) were observed as
compared to sham group (4.7H and I). The treatment with ASE at 500 mg/ kg has
significantly attenuated the activation of Caspase-3 (26.7%, p < 0.05) and Caspase-9
(31.2%, p < 0.01).

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Fig. 4.5: Effect of ASE on TBARS, GSH and NO contents: Effect of ASE treatment on the
contents of TBARS (A), GSH (B) and NO (C) in the striatum, hippocampus and cerebral cortex in
MCAO rats. NO is represented in terms of nitrite. The contents of TBARS and NO were significantly
increased while reduced GSH content was decreased in MCAO group as compared to sham group
(#p < 0.01, MCAO vs. Sham group). ASE treatment has restored the levels of TBARS, GSH and NO

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significantly in ASE 500 + MCAO group only as compared to MCAO group (*p < 0.05; **p < 0.01 vs.
MCAO group) in all the three tissues. Values are expressed as mean ± SEM.

4.4 Discussion

The present work emphasizes on anti-ischemic activity of ASE by virtue of its free radical
scavenging property. Previously, it has been reported that Azadirachta seed contains
Azadirachtin, nimbin, salanin, and gedunin as a major limonoids (Akihisa et al., 2009;
Ghimeray et al., 2009; Jang et al., 2010). The HPTLC of ASE showed the presence of other
limonoids, flavonoids and polyphenols that may play important role in neuroprotection by
free radical scavenging activity. The present findings show high total antioxidant property
(TAP) and reducing power (RP) values and moderate free radical scavenging activity for
DPPH• and NO• of ASE attributed to its moderate phenolic and flavonoid contents (Table
4.1). This is in well conjuncture with previous reports showing that extract containing
significant amount of phenolics and flavonoids have good antioxidant activity (Lee et al.,
2007; Rao et al., 2010).

DPPH• radical scavenging property is considered as a good in vitro model used widely to
assess the antioxidant efficacy. In the radical form, DPPH• disappears on reduction by an
antioxidant compound and becomes a stable diamagnetic molecule (Rao et al., 2010). ASE
exhibited appreciable scavenging activity, however it was not at the level of ascorbic acid
but there was a significant correlation between DPPH• radical scavenging activity and
polyphenolic content. IC 50 value of ASE indicates that it has a moderate proton donating
ability that could serve as free radical inhibitor or scavenger, acting possibly as primary
antioxidants (Noh et al., 2002; Rao et al., 2010).

Our data demonstrate that the ASE inhibits nitrite formation (IC 50 - 176 µg/ml) by directly
competing with oxygen in the reaction with nitric oxide. These oxy-radicals are toxic to the
tissues and are responsible for various deleterious responses (Yin et al., 2007). Our results
indicate that ASE contains significant amounts of flavonoids (101.1 ± 7.7 mg QE/g extract).
In the present study, the anti-free radical efficiency of ASE was positively correlated with
total phenolic and flavonoids contents (Table 4.1). Thus, these primary but important in
vitro findings made a basis to assess the efficacy of ASE in acute ischemic injury.

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Table 4.2: Antioxidant enzymatic evaluation of ASE in MCAO rats. The activities of enzymes
glutathione reductase (GR), glutathione peroxidase (GPx) and Catalase (CAT) were estimated in
striatum, hippocampus and cortex of rat brain.

Groups Brain GR (nmoles GPx (nmoles CAT (nmoles

Parts NADPH oxidized/ NADPH oxidized/ H 2 O 2 consumed/
min/ mg protein) min/ mg protein) min/ mg protein)
Sham Sm 368.28 ± 7.25 272.50 ± 11.16 106.23 ± 12.95
Hs 337.03 ± 22.56 258.74 ± 12.72 70.24 ± 4.97
Cx 370.22 ± 12.54 314.65 ± 23.08 71.38 ± 9.42
MCAO Sm 84.98 ± 4.99# 100.33 ± 13.95# 37.26 ± 4.41#
(-76.93%) a (-63.18%) a (-64.93%) a
Hs 115.02±11.97# 87.63 ± 2.55# 26.28 ± 2.06#
(65.87%) a (-66.13%) a (-62.58%) a
Cx 156.18±6.84# 115.61 ± 4.73# 56.83 ± 4.41#
(57.81%) a (-63.26%) a (-20.38%) a
ASE 500 Sm 247.94±15.87* 161.15 ± 7.40* 68.94 ± 5.62*
+ MCAO (191.76%)b (60.62%) b (85.02%) b
Hs 224.08±21.12* 169.03 ± 4.11* 53.91 ± 6.63*
(94.81%) b (92.90%) b (105.13%) b
Cx 238.33± 15.82* 198.84 ± 10.00* 70.69 ± 5.71*
(52.60%) b (71.99%) b (24.37%) b
ASE 500 Sm 345.23 ± 6.88 278.86 ± 16.98 105.49 ± 3.09
+ Sham (-6.26%)a (2.33%)a (-0.70%)a
Hs 360.87± 42.41 264.18 ± 7.59 67.61 ± 6.96
(7.07%)a (2.10%)a (-3.74%)a
Cx 408.04 ± 28.53 337.47 ± 15.75 84.65 ± 7.58
a a
(10.22%) (7.25%) (18.59%)a

$
Values are expressed as mean ± SEM (n=6). #p < 0.01 vs. Sham group; *p < 0.01 vs. MCAO
group. Values in parentheses show the percentage increase or decrease with respect to their control
(a vs. Sham; b vs. MCAO)

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Fig. 4.6: Histological findings: Effect of ASE treatment on hematoxylin and eosin (H & E) staining
in striatum and cortical brain sections (A-C and G-I) and cresyl violet (CV) stained CA1 region of
hippocampus (D-F) of Sham, MCAO and ASE-treated MCAO (ASE500 + MCAO) groups.
Photomicrograph (A, D and G) of the brain sections of sham group animal has shown uniform
distribution of cells, normal neurons with the characteristic conical outlines (shown by bold arrow)
with no abnormal features. Photomicrograph (40x) (B, E and H) of the brain sections around infarct
area in MCAO group has shown a focal area of maculation, alteration in neuronal morphology
(shown by triangle) and neuronal loss (shown by bold wing). However, few normal neurons were
also observed around infarct area (B and H). ASE-treated MCAO group (ASE500 + MCAO) has
shown partial neuronal loss (C, F and I) as compared to MCAO group. No significant loss of neurons
was observed in ASE treated Sham groups (ASE500 + Sham) as compared to Sham group. The
loss of intact neurons was counted in each region of brain (J). The MCAO group has shown
maximum loss (71.72%) in striatum than hippocampus (33.10%) and cortex (46.92%). ASE at higher
dose (500 mg/kg) significantly protected the neurons in each region (p < 0.01). The Sham group has
shown basal level of neuronal loss.

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Fig. 4.7: Effect of ASE on activity of Caspase-3 and 9: The effect of highest dose of ASE (500
mg/kg b.wt.) on Caspase-3 expression (A-F) and activities of Caspase-3 (H) and Caspase-9 (I) were
evaluated. MCAO group has shown significant activation of Caspase-3 (H) and Caspase-9 (I) as
compared to Sham (#p < 0.01 vs. sham). ASE treatment significantly attenuated the activity of these
apoptotic markers (*p < 0.05; **p < 0.01 vs. MCAO; H and I). Immunohistochemistry of Caspase-3
(A-F) has shown up regulation of Caspase-3 in MCAO group (shown by black arrowheads). Sham
group has shown normal cells with no activated Caspase-3. ASE treatment significantly attenuated
the apoptosis (G). Many healthy neurons (as shown by arrowhead along with lesser number of
Caspase-3 immunoreactive cells (black arrowhead) were observed in this group. Photomicrographs
at lower magnification (10x; A - C) have shown cortical tissues in each group. Higher magnification
(100x; D - F) was used to analyze the immunoreactive cells qualitatively. Cell counting were
performed in five random optical field in penumbral cortex and repeated thrice. Values are
expressed as mean ± SEM.

Ischemic reperfusion after MCAO results the membrane disintegration, failure of ion
channels, loss of ATP, damage of mitochondrial and other macromolecules (Andrabi et al.,
2004; MacDougall and Muir, 2011). This ultimately results into a catastrophe that causing
the cell to succumb to oxidative insult. Neurological severity thus resulted was reported to
be highest in MCAO group (Yousuf et al., 2009; Khan et al., 2009). We also observed a

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high severity score in MCAO group that was found to be less in ASE treated group as
compared to MCAO group. The damage to striatum and cortex area in the brain manifests
the alteration in motor skills of animals (Moran et al., 1995, Salim et al., 2003). Treatment
of rats with ASE has ameliorated these alterations in rota rod coordination and grip strength.
Neurological severity and behavioral deficits resulted in ischemic-reperfusion injury has
been attributed to loss of cellular integrity and functions. One of the important cell
organelles, mitochondria remains under great stress during ischemia and damage of
mitochondrial integrity leads to accumulation of ROS and a spreading cell loss (Preston and
Webster, 2004). The MCAO group in the present study has shown higher infarct volume
while three doses of ASE have reduced the infarction significantly. These findings suggest
that ASE could salvage the rat brain from ischemic-reperfusion injury. Since ASE has
shown a moderate antioxidant activity in vitro therefore, we further investigated its effect on
ischemia induced oxidative stress in rat brain.

Membrane and other macromolecules damaged from ischemic injury start a chain of
reaction to generate free radicals, leading to further damage (Wu et al., 2008; Khan et al.,
2009; MacDougall and Muir, 2011). However, by virtue of their free radical scavenging
properties, ASE was found to be effective in ameliorating the oxidative stress successfully
by lowering LPO and nitrite contents and restoring GSH level in MCAO rats. However,
lower doses of ASE did not show any significant changes in GSH contents after ischemia,
suggesting that higher dose (500 mg/kg) could reach the optimum level to counter the ROS
mediated damage in the brain. Further, higher dose of ASE has shown the restoration of
antioxidant enzymes (GR, GPx and CAT) activities in MCAO brain. Morphologically, ASE
pretreated group showed less neuronal loss and less inflammation when compared with
MCAO group in discrete areas of the brain. This infers that ASE being potent antioxidant
neutralized the free radicals generated after ischemia-reperfusion, thus inhibiting the
progression of cell death in penumbral region.

Moreover, the cell death in the brain after ischemic reperfusion injury occurs due to
necrosis in ischemic core and due to apoptosis in penumbra. A shift in the balance between
pro- and anti-apoptotic protein that promote cell death may be one of the mechanism
underlying post ischemic-reperfusion apoptotic cell death (Broughton et al., 2009; Végran
et al., 2011). An acute phase of hypoxia-hypoglycemia is developed after ischemia, due to

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Fig. 4.8: Mechnaism of protection afforded by ASE: The representative photograph shows the
overall mechanism of protection afforded by ASE. The ischemia-reperfusion results in reactive
oxygen species (ROS) generation and mitochondrial injury. With the burden of increasing oxidants,
the cells become depleted of endogenous antioxidant store, which ultimately aggravate the injury.
Finally cells succumb to death under the huge stress. Mitochondrial injury also results in Caspase-3
activation. Therefore, neurons in penumbra region of cortex display a mixed population of necrotic
and apoptotic cells. The ASE being potent antioxidant, scavenged the ROS generated after I/R
injury and thus protect the endogenous antioxidant store. Moreover, ASE also found to inhibit the
Caspase-3 & 9 activation. Thus, in this present study, ASE was proved to be protective against I/R
injury.
which mitochondria have come under great stress of providing energy required to the cell
(Preston and Webster, 2004). Therefore, in the present study, we evaluated the inhibitory
role of ASE in mitochondrial intrinsic apoptotic pathway and measured the activities of
caspase-3 &9. Activation of Caspase-3 and 9 is reported as one of the major pathways
associated with stroke-induced apoptotic neuronal cell death and might be activated by free
radical generation (Brahma et al., 2009; Végran et al., 2011). Oral treatment with ASE (500
mg/kg) has shown strong apoptosis inhibiting property, especially in intrinsic mitochondrial
pathway. ASE (500 mg/kg) inhibited the Caspase-3 and 9 significantly. Moreover, the
expression of activated Caspase-3, which was up-regulated in ischemic group (MCAO),
was down-regulated with the treatment of ASE in ASE 500 + MCAO group. Our findings
are in well conjunction with other reports showing that herbal products protect the brain
from ischemic-reperfusion insult in MCAO model (Yousuf et al., 2009; Khan et al., 2009).

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This suggests that ASE is not only a potent antioxidant, but also an excellent Caspase
inhibitor. Recently, azadirachtin, the major insecticidal limonoid of Azadirachta seed has
shown to possess good anti-inflammatory activity (Thoh et al., 2010; 2011). Other major
limonoids, nimbin possesses antioxidant, anti-inflammatory and antidiabetic activities
(Biswas et al., 2002; Kumar et al., 2010; Bhowmik et al., 2010), while deoxygedunin
possess neurotrophic activity (Jang et al., 2010). These reports in congruence with our
results suggest the effectiveness of ASE in neurodegenerative diseases that may make it
preferable pharmacological candidate for further study.

In conclusion, the findings of this study suggest that the neuroprotective effect of ASE is
mediated by the inhibition of neurological deficits and oxidative damage by virtue of its
polyphenolic and flavonoids contents followed by the inhibition of apoptotic responses as
caspase activation. Further understanding the mechanism of neuroprotection of A. indica
seed extract will provide an avenue to explore its biomolecules for the potential use in
pharmaceutical formulations.

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