Chapter VI

Zingerone protects brain against rat focal transient

cerebral ischemia: A behavioral, neurochemical

and histological alterations and down regulation of

Apaf-1, Bax, Caspase-3 and 9

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6.1 Introduction

Multifactorial nature of stroke is responsible for a number of pathological events involving

oxidative stress, inflammation and necroptosis. Focal transient cerebral ischemia produced

by middle cerebral artery occlusion (MCAO) method is a well established experimental

model that depicts the pathological consequences similar to human stroke (Liu and

McCullough, 2011; MacDougall and Muir, 2011; Onken et al., 2012). MCAO followed by

reperfusion results in the depletion of antioxidant store of the cell along with the apoptotic

insults in terms of Bcl-2, Bax, Apaf-1, Caspase-9 and Caspase-3. Under the huge burden of

depleted endogenous antioxidants and increased apoptosis, the neuronal cells succumb to

death via necroptosis and thus, make a basis of behavioral impairments (Li et al., 2009;

Abas et al., 2010). Recently, accumulating lines of evidence showed that dietary enrichment

with nutritional antioxidants could reduce brain damage and improve behavioral functions

(Cui et al., 2010; Qi et al., 2010; Guo et al., 2011). However, most of the works have been

dedicated to preventive potential of herbal products given prior to the MCAO. Here in, we

evaluated zingerone as a modulator given after the onset of ischemia-reperfusion in rats.

Zingiber officinale rhizome (Ginger), is widely used in Indian System of Medicine for

antioxidant (Oboh et al., 2012a), tyrosinase inhibitor (Kuo et al., 2005),

antiacetylcholineesterase (Oboh et al., 2012b) and antidiabetic (Shanmugam et al., 2011).

Zingerone, an alkaloid principle of Zingiber officinale rhizome, has been reported to inhibit

ascorbate/Fe2+

Chemicals and reagents, behavior parameters (FT, SMA, grip strength and rota-rod

muscular co-ordination), histological experiments (H&E and CV staining, TTC staining and

infarct volume), Biochemical parameters (LPO, GSH, nitrite, Na

-induced lipid peroxidation in rat (Reddy and Lokesh, 1992; Oboh et al.,

2012a) and to possess superoxide dismutase (SOD) like activity (Krishnakantha and

Lokesh, 1993). It is reported recently that zingerone can protect the mice brain from 6-

OHDA induced Parkinson’s disease (Kabuto et al., 2005). Therefore, we examined effects

of zingerone on ischemia-reperfusion induced-injury in MCAO rat brain, and its possible

implication on oxidative stress and apoptosis.

6.2 Materials and Methods

+-K+ ATPase, SOD,

catalase, mitochondrial injury), poly (ADP-ribose) polymerase [PARP] activity and

immunohistochemistry protocols (Bcl-2, Bax, Caspase-9, Caspase-3, and Apaf-1) are

described in materials and methods section (Chapter III).

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6.2.1 Experimental design

The rats (16 weeks old; 260-280 g) were divided into five groups. The first group served as

a sham (S) and water as vehicle was given two times in whole experiments (n = 12). The

second group was the vehicle treated middle cerebral artery occluded (MCAO) group in

which ischemia was induced for 2h followed by reperfusion for 22h (n = 16). The third to

fourth group were treated with zingerone (50 and 100 mg/kg b.wt. orally, at 5h and 12h

from the initiation of MCAO) followed by reperfusion (MCAO + Z 50; n = 16; and MCAO

+ Z 100; n = 16 groups, respectively). The fifth group (Sham + Z 100; n = 10) served as

sham-operated drug control treated with high dose of zingerone alone (100 mg/kg b.wt.

orally, at 5h and 12h). After reperfusion, the rats were assessed for neurobehavioral

activities and then sacrificed. The brains were taken out to dissect the hippocampus and

cortex for biochemical estimations.

6.3 Results

6.3.1 Improvement of behavioral deficits

A significant flexion test (FT) (p < 0.01), spontaneous motor activity (SMA) (p < 0.01) and

neurological deficits (ND) (p < 0.01) was observed in MCAO group as compared to sham

group (Fig.6.1A). Simultaneously, a significant decrease in grip strength (Fig. 6.1B) and

motor co-ordination (Fig. 6.1C) was observed in ischemic group as compared to sham

group rats (p < 0.01). Zingerone afforded a significant restoration in behavioral and

muscular strength as compared to MCAO group (p < 0.01). However, the higher dose of

zingerone (MCAO + Z 100) showed a significant improvement in terms of neurological

deficits (p < 0.05) and grip strength as compared to ischemic group (p < 0.01).

6.3.2 Infarct and mitochondrial injury

The MCAO group has shown maximum visible infarct (43.25%) 24 h after ischemia-

reperfusion (Fig. 6.2A and B). Administration of zingerone at both doses reduced the infarct

significantly (MCAO + Z 50; 22.62% and MCAO + Z 100; 12.93%; p < 0.01). The highest

dose of zingerone (Z 100) was found to be more protective than the lower dose (Z 50).

Further, to account for diffused cellular loss, mitochondrial injury was assessed in each

group. A high percentage of injury (56.99%) with respect to sham was observed in MCAO

group (Fig. 6.2C). The mitochondrial injury was reduced in zingerone administered groups

significantly (MCAO + Z 50, 38.13% and MCAO + Z 100, 25.64%; p < 0.01).

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Fig.6.1: Behavioral deficits: A significant neurological severity as depicted by flexion test (FT) (p <

0.01), spontaneous motor activity (SMA) (p < 0.01) and neurological deficits (ND) (p < 0.01) was

observed in MCAO group as compared to sham group (A). Simultaneously, a significant decrease (p

< 0.01) in grip strength (B) and motor coordination (C) was observed in ischemic group as compared

to sham group rats. Zingerone (MCAO + Z 50; p < 0.05 and MCAO + Z 100; p < 0.01) afforded a

significant restoration in behavioral and muscular strength as compared to MCAO group (p < 0.05).

However, only the higher dose of zingerone (MCAO + Z 100) has significantly improved the grip

strength as compared to ischemic group (p < 0.01). No significant difference was observed between

sham treated with higher dose of zingerone (Sham + Z 100) and sham group. Values are

represented as mean ± SEM and significance was ascertained as #p < 0.01 vs. sham and *p < 0.05;

**p < 0.01 vs. MCAO.

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Fig. 6.2: Effect of zingerone on cerebral infarct and mitochondrial injury. TTC-stained coronal

sections (A), assessment of infarct in rat brain by image j analysis software (B) and estimation of

mitochondrial injury (C) are shown in figure 6.2. A higher volume of infarction was observed in

MCAO group (43.25%). Zingerone treated groups has shown a decreased infarct volume (MCAO +

Z 50; 22.62% and MCAO + Z 100; 12.93%) in comparison to MCAO group (p < 0.01). A high

percentage of injury (56.99%) was observed in MCAO group which accounted for all visible and

diffused infarct. The zingerone administration at both doses successfully reduced the injury after

ischemia-reperfusion (p < 0.01). The data are shown as mean ± SEM and p values less than 0.05

were ascertained significant (*p < 0.01).

6.3.3 Histological changes

The edematous tissue with vacuolated morphology (shown by pointer) and pyknotic nuclei

(shown by wing) were observed in MCAO rat brain. The zingerone treatment (50 mg/kg

and 100 mg/kg) successfully reduced the injury and protected the normal architecture of the

brain (Fig. 6.3). Histological sections of ipsilateral cortex (H&E stained) and hippocampus

(CV stained) from MCAO group has shown large number of degenerated neurons (56.81%;

p < 0.01 and 36.40%; p < 0.01).

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The zingerone fed group showed reduced injury (MCAO + Z 50; 24.76% & 12.93% and

MCAO + Z 100; 21.97% & 10.95%; p < 0.01) in penumbral cortex (Fig. 6.3C - D) and

hippocampus (Fig. 6.3G - H).

Fig. 6.3: Zingerone and histological alterations. Histological sections exhibit severe loss of intact

neurons (Cortex; 56.81% and Hippocampus; 36.40%) in ischemic brain (B and F) (shown by black

wing) in comparison to sham group (A, E). Zingerone administered groups (C - D and G - H) showed

a decrease in neuronal loss in penumbral cortex (MCAO + Z 50; 24.76% and MCAO + Z 100;

21.97%) and in hippocampal CA1 region (MCAO + Z 50; 12.93% and MCAO + Z 100; 10.95%). The

intact neurons are indicated by black arrow. The loss of intact neurons in each group is represented

as percentage of total cells (I). Values are expressed as mean ± S.E.M and significance was

ascertained as #

A significant increase in TBARS level and decrease in Na

p < 0.01 vs. Sham; *p < 0.01 vs. MCAO (Magnification x20).

6.3.4 Effect of zingerone on oxidative stress +-K+ ATPase activity were

observed in MCAO group animals as compared to sham group animals (p < 0.01).

Zingerone treatment has reduced the level of TBARS and protected Na+ K+-ATPase activity

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significantly (p < 0.01) in both the groups (MCAO + Z 50 and MCAO + Z 100) as

compared to MCAO group (Table 6.1).

The GSH level and SOD activity in hippocampus and cortex were found to be depleted

significantly (p < 0.01) in MCAO group as compared to sham group. Zingerone treatment

has restored the level of GSH and protected SOD activity significantly (p < 0.01). However,

significant protection in terms of SOD activity was observed in cortex of both the groups

(MCAO + Z 50 and MCAO + Z 100) as compared to MCAO group (Table 6.1). The

MCAO group showed an elevated level of nitrite in hippocampus and cortical tissues. The

treatment with high dose of zingerone has attenuated the level of nitrite significantly (p <

0.01) in hippocampus as compared to MCAO group.

6.3.5 Inhibition of PARP activation

The results of ischemia-reperfusion showed a extensive DNA damage that cause an

increased activity of poly (ADP-ribose) polymerase (PARP). In the present study, an

increased PARP activity was observed in MCAO group (p <0.01) as compared to sham

group. The zingerone administration significantly restored the PARP activity at both the

doses (p<0.01) [Fig. 6.4].

Table 6.1: Oxidative stress: A significant increase (p < 0.01) in TBARS and nitrite level was

observed in MCAO group animals as compared to sham group animals. Zingerone treated MCAO

rats exhibited significant attenuation (p < 0.01) in TBARS contents in comparison to MCAO group

rats. The MCAO group treated with high dose of zingerone (MCAO + Z 100) showed a significant

decrease in nitrite level (p < 0.01) within hippocampus while zingerone at both doses significantly

attenuated the increase in nitrite level (p < 0.01) in cortical tissue. The GSH level, and Na+ K+-

ATPase and SOD activities in hippocampus and cortex were depleted significantly (p < 0.01) in

MCAO group as compared to sham group. Zingerone treatment has restored the level of GSH and

protected Na+ K+-ATPase activity significantly (p < 0.01) in both the groups (MCAO + Z 50 and

MCAO + Z 100) as compared to MCAO group. SOD activity was found to be significantly

augmented (p < 0.01) in cortical tissues only after zingerone administration. No significant difference

was observed in hippocampal tissues of zingerone administered groups and MCAO group. Further,

zingerone treated sham group (Sham + Z 100) exhibited no significant changes as compared to

sham groups in both tissues.

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The values in parentheses show the percentage increase or decrease with respect to their control

(avs. Sham; bvs. MCAO). #p<0.01 vs. Sham; *P<0.01 vs. MCAO; Cx: Cortex; Hs: Hippocampus

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Fig. 6.4: Inhibition of PARP. The MCAO group has shown high activity of PARP (p < 0.01)

suggesting the high extent of DNA damage due to ischemia-reperfusion. Both the doses of

zingerone (50 mg/kg and 100 mg/kg) have protected the PARP activity significantly (p<0.05). The

sham group has shown the basal level of activity. Values are represented as mean ± S.E.M. p <

0.05 was considered significant (#p < 0.01 vs. Sham; * p < 0.05 vs. MCAO).

6.3.6 Zingerone inhibits apoptosis (Bcl-2, Bax, Caspase-9, Caspase-3 and Apaf-1)

The ischemia-reperfusion resulted in the activation of apoptotic proteins (Bax, Caspase-9,

Caspase-3 and Apaf-1) and decrease in the expression of anti-apoptotic protein Bcl-2. We

observed negligible expression of Bcl-2 (7.64%) along with high expression of Bax

(48.54%), Caspase-9 (33.76%), Caspase-3 (38.04%) and Apaf-1 (45.94%) in penumbral

cortex region (p < 0.01). This shows that penumbral cortex region is the major site of

ischemia-reperfusion injury-induced apoptosis. Zingerone administrated doses (50 mg/kg

and 100 mg/kg) have restored the level of pro and anti-apoptotic proteins significantly (p <

0.01), suggesting its efficacy against mitochondrial intrinsic apoptosis pathway (Fig. 6.5

and 6.6).

6.4 Discussion

Oxidant stress and mitochondrial apoptosis have been implicated in the cell death observed

during ischemia-reperfusion (I/R), but the relationship between these contributing factors is

not clear. A substantial body of evidence indicates that ROS production occurs during I/R,

and that antioxidants confer protection against I/R-induced cell death (Vanden Hoek et al.,

1997; Levraut et al., 2003; Qi et al., 2010; Guo et al., 2011; Loor et al., 2011). However,

apoptosis has also been reported to be activated by I/R, and important in mediating I/R-

induced cell death (Hochhauser et al., 2003; Tanaka et al., 2004; Robin et al., 2007). It is

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Fig. 6.5: Effect of zingerone on apoptotic proteins (Caspase-9, Caspase-3 and Apaf-1). The

MCAO group (B, F, J, N, R and V) showing high expression of all the apoptotic proteins in

comparison to Sham control group (A, E, I, M, Q and U; p < 0.01). The zingerone treatment groups

(C-D, G-H, K-L, O-P, S-T and W-X), however has shown reduced expressions (Y; p < 0.01). The

Sham group has basal level of expressions (Y). Values are expressed as mean ± S.E.M and

significance was ascertained as #p < 0.01 vs. Sham; *p < 0.01 vs. MCAO.

conceivable that oxidant stress during I/R mediates cell death by activating a programmed

cell death pathway (Qin et al., 2004). The present study sought to examine how ischemia-

reperfusion contributes to cell death, and the relationship between ischemic stress and

protection afforded by zingerone.

Zingerone doses (50 and 100 mg/kg) used in this study ameliorated all neuropathological

consequences of ischemia, indicating its therapeutic potential. For all behavioral

parameters, a dose-dependent effect was served except for neurological deficit examination

and grip strength. Zingerone promoted an important protection against motor hyperactivity.

This paradoxical behavior may indicate a reduction of the loss of motor neurons in cortex in

vehicle treated ischemic rats in comparison to sham operated group. It is widely reported

that a brief period of cerebral ischemia induces neurological dysfunction in laboratory

animals, which is reflected in the deterioration of memory and motor functions due to a

more vulnerability of cortical neurons (Sun et al., 2011; Gaur and Kumar, 2011). In

behavior activity like rotarod (motor coordination test) and grip strength, zingerone treated

rats showed recovered motor ability after ischemia-reperfusion injury at 24 h. These

findings suggest that zingerone at these doses successfully prevented the brain specially the

sensory-motor cortex area from the ischemic insult.

The cerebral ischemia followed by reperfusion results in production of reactive oxygen

species (ROS), nitric oxide (NO), prostaglandins and inflammatory cytokines. These

mediators also extend the ischemic insult to distant region like hippocampus causing

diffused injury (Abdallah et al., 2011; Xuan et al., 2012). Histologically, MCAO group

showed edematous brain architecture with vacuolation and pyknotic nuclei in cortex and

hippocampus. The zingerone treated groups, however, protected the histological alterations,

thus formed a basis of behavioral improvements.

Zingerone also minimized the infarct volume in I/R rats even when given with the delay of 5h. The histological findings showed the less infarct area in zingerone treated ischemic

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Fig. 6.6: Effect of zingerone on Bcl-2 and Bax expressions. Ischemia-reperfusion (MCAO) group has shown up regulation of Bax (48.54%) and down regulation of Bcl-2 (7.64%) in penumbral cortex in comparison to sham (p < 0.01) [B & F and J & N]. The zingerone treatment groups (MCAO + Z 50 and MCAO + Z 100) augmented the expression of Bcl-2 (15.37% and 18.53%) and normalized the level of Bax (15.07% and 12.92%) [C - D, G - H, K - L and O – P], thus reduced the apoptotic burden (Q). Bcl-2/Bax ratio was found to be minimal in case of MCAO group (0.16%; p < 0.01) which has been restored up to some extent by zingerone administration (MCAO + Z 50; 1.00% and MCAO + Z 100; 1.43%; R). The sham group has shown basal level of Bcl-2 and Bax expression. Values are expressed as mean ± S.E.M and significance was ascertained as #

rats in comparison to vehicle treated ischemic rats. This is further ascertained by reduction in neuronal loss in cortical and hippocampal sections, suggesting that zingerone protected the neurons from acute ischemic insult not only in adjacent penumbral region, but also in distant

p < 0.01 vs. Sham; *p<0.05 and **p<0.01 vs. MCAO.

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hippocampus. In congruence to this, previous published reports have also shown the loss of hippocampal neurons and decrease in level of antioxidant molecules after I/R which successfully reverted by antioxidant supplementation (Abdallah et al., 2011; Jiao et al., 2011, Xuan et al., 2012). These results suggest that zingerone was able to reduce I/R insults thus protected the brain from further injury.

Cerebral ischemia caused the elevation in oxidative stress, depletion of endogenous antioxidants such as reduced glutathione (GSH), and decreased activities of free radical scavenging enzymes such as superoxide dismutase (SOD) (Sun et al., 2009; Abdallah et al., 2011; Zhang et al., 2011). Events early in reperfusion, most likely the ROS burst, appear to be responsible for triggering both the loss of plasma membrane integrity and the later release of cytochrome C (Loor et al., 2011). Once the plasma membrane integrity is lost, a cell becomes committed to death regardless of whether or not cytochrome C is later released. Our results also confirm the increased lipid peroxidation and decreased level of intracellular reduced glutathione and activity of Na+-K+ ATPase in ischemic rats. The findings indicate that ischemia followed by reperfusion resulted in increased production of ROS as a result of cellular death and macrophagic activity. This overproduction of ROS exhausted the intracellular antioxidant defense of neuronal cell and resulted in diminished GSH. The cellular antioxidant defense once succumb to free radical stress leading the membranous lipid peroxidation and distruption of Na+-K+ ATPase activity, leading to cell death. However, zingerone doses protected the neuron from oxidative stress. Zingerone treated group showed restoration of GSH level and improved activity of Na+-K+

At the cellular level, the lack of oxygen supply caused the depletion of adenosine triphosphate (ATP) due to anaerobic glycolysis. Such reduction in ATP concentration may impair the Na

ATPase after ischemia-reperfusion. Zingerone also significantly decreased the lipid peroxidation thus maintained the membranous integrity of neurons. In support of this, It has been reported that Zingiber officinales attenuated the memory impairment and oxidative stress induced by ischemia-reperfusion in rats via its free radical scavenging ability (Wattanathorn et al., 2011).

+ K+ ATPase activity, which is critical to the maintenance of cell membrane excitability and functions (Carletti et al., 2012). Wyse et al. (2000) have demonstrated that recovery of Na+ K+ ATPase activity in the hippocampus is responsible for neuroprotection induced by brain ischemic preconditioning. These results are consistent with a recent report which identified a decrease in Na+ K+ ATPase activity in cortex 1 h after the hypoxic-ischemic insult (Weis et al., 2011). Further, this enzyme is very susceptible to free radical reaction and lipid peroxidation because it is wedged in cell membrane and requires phospholipids for the maintenance of the activity (Ahmad et al., 2011; Khan et al., 2010). Since, zingerone has been administered at 5th and 12th h after MCAO and capsaicin at the start of MCAO; therefore, we were interested to investigate the energy deprivation and its effect on related biomolecules like Na+ K+ ATPase

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when therapeutic intervention was delayed. Our results have shown that zingerone is effective against the I/R-induced energy deprivation and protected the activity of Na+ K+

The Bcl-2 family of proto-oncogenes encodes specific proteins such as Bcl-2, Bcl-XL, and Bax that regulate apoptosis. The anti-apoptotic effect of Bcl-2 occurs by prevention of cytochrome C release into the cytoplasm (Nakka et al., 2008). Many studies have shown that over-expression of Bcl-2 can reduce ischemic brain injury in animal models of stroke (Okazaki et al., 2008; Xing et al., 2008). Activated Bax promotes cell death, unless it is bound by either Bcl-2 or Bcl-XL (Okuno et al., 2004). However, these two molecules can act independently to exert their effects on cell death (Nakka et al., 2008). Kim et al. (2011) has found strong induction of Bax protein 1 day after infarction in the ischemic hemisphere and it reached a peak on day 3. Compared with Bax expression, Bcl-2 protein expression was slightly reduced at day 1 and then abruptly increased at day 3 and was sustained at day 7. In a previous report using the same stroke model (Isenmann et al., 1998), Bax protein was in the cytoplasm of degenerating neurons in the center of the infarction between 4 h and 3 days after photothrombosis, and at the same time points, the levels of Bcl-2 and Bcl-XL proteins were markedly reduced in this region.

ATPase, thus maintaining the potential gradient across the membrane.

The present finding extends the work done by Loor et al. (2011) showing that oxidant stress increases in the matrix as well, suggesting that ischemic oxidants originate from the electron transport chain. It seems unlikely that each of the chemically diverse antioxidants was able to scavenge superoxide directly. More likely, some of these agents may have acted by protecting the redox status of proteins that were the targets of superoxide attack (Levraut et al., 2003; Robin et al., 2007; Loor et al., 2011). In agreement to this, we observed reduced activity of SOD in MCAO group. However, the zingerone treatment group has shown the augmented activities of SOD that may protect the brain from superoxide induced injury. Our finding has been supported by previous reports stating that zingerone has protective effect in 6-OHDA induced Parkinsonian mice and indicated that it activates SOD activity in brain and thus exerts antioxidant effect via superoxide scavenging (Kabuto et al., 2005).

Focal cerebral ischemia in rats triggers apoptotic cell death within the penumbral region. In the cytosol, Cytochrome C forms a complex with Apaf-1, which then binds to and activates Caspase-9. This triggers the activation of terminal caspases such as Caspase-3, which in turn lead to cell death (Kroemer and Reed, 2000; Ferrer, 2006; Broughton et al., 2009; Loor et al., 2011). Although the downstream cascade for the execution of apoptosis has now become clear, the mechanism by which the mitochondrial signaling pathway is activated during apoptosis is complex and appears to depend on the types of death stimuli and the cell types receiving such stimuli. In the present study, we observed that Apaf-1 was upregulated after I/R injury at 24 h, which was subsequently down regulated by zingerone.

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These findings suggest that Bcl-2 and Bax protein expressions switch over after ischemia to induce apoptosis (Kim et al., 2011). Interestingly, in the MCAO mouse model with preconditioning, Bcl-2 and Bcl-XL were increased after sub-lethal forebrain ischemia but Bax remained unchanged (Wu et al., 2003). These differential expressions of the Bcl-2 family were associated with animal modeling, such as preparing of preconditioning, and reproducibility of lethal lesion induction. The slight decrease in Bcl-2 expression within 1 day might also be attributed to post-ischemic protease activation (Panayiotidis et al., 2006).

In vitro cell-free studies showed that Bax can form pH- and voltage dependent ion-conducting channels in planar lipid bilayer membranes, and this channel-forming activity in mitochondrial membrane is likely involved in the regulation of mitochondrial membrane permeability (Antonsson et al., 1997; Schlesinger et al., 1997). Apoptosis-associated cytochrome C release appears to involve limited permeabilization of the outer mitochondrial membrane and maintenance of transmembrane potential. However, cytochrome C/Apoptosome-mediated caspase activation appears to result in swelling of the mitochondrial matrix and rupture of the inner and outer mitochondrial membranes (Martinou and Green, 2001; Zamzami and Kroemer, 2001). Thus, Caspase-dependent mitochondrial attack, which may result in permeability transition pore opening or generalized mitochondrial destruction, could facilitate Smac/DIABLO release from mitochondria downstream of cytochrome C efflux (Adrain et al., 2001). Cytochrome C forms a multi-protein complex known as the ‘Apoptosome’ and initiates the activation of Caspase cascade through Caspase-9.Once formed, the Apoptosome recruits and cleaves pro-Caspase-9 to activate Caspase-9, which then activates effector Caspase-3 and thus, leads to apoptosis.

Caspases are cysteine proteases that are constitutively expressed as zymogens or pro-Caspases and that are specifically activated during apoptotic stimuli (Chaitanya and Babu, 2008). Several previous studies reported that the role of Caspase-3 is as a key mediator of apoptosis in ischemic stroke (Le et al., 2002). Our results showed that activated caspase-3 has been highly expressed in penumbral cortex region at the end of 24 h after I/R injury. In congruence with the report that up regulation of Caspase-3 mRNA in rat brain occurs 1 h after the onset of focal ischemia, whereas Caspase-3 and its cleavage products in mouse brain are activated during early reperfusion 2 h after MCAO (Kim et al., 2011). We observed that zingerone given with delay of 3h reduced the level of activated Caspase-3. This is also supported by the fact that ischemic damage could be reduced by caspase inhibitor treatment even 9 h after MCAO (Kim et al., 2011). Further, Kim et al. (2011) observed a significant increase in the cleaved active form of caspase-3 from day 1 of operation until day 3, when the maximal intensity was observed. Also, this increased Caspase-3 expression was sustained at day 7. These findings are in agreement with an extended treatment window for caspase inhibition after stroke (Broughton et al., 2009).

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Fig. 6.7: Pictorial representation of ischemia-reperfusion induced cell death in brain and protection afforded by zingerone: The ischemia-reperfusion (I/R) resulted in depletion of endogenous antioxidants and leads to energy deficits and ionic imbalance. The ROS generated by I/R injury, led to cell membrane degeneration and also activates various endonucleases, peptidases and lipases. The mitochondrial stress occurred during ischemia resulted in aggrevation of ROS inside the cell and also increased the membrane permeability of mitochondria by more and more mitochondrial translocation of cytosolic Bax. This resulted in leakage of Cytochrome C (Cyt C) and Smac/Diablo proteins in cytosol, which ultimately led to the formation of Apoptosome. Finally Caspase-3 was recruited and apoptosis occurred. Penumbral cortex showed a wide variety of cell death including caspase dependent and Caspase-independent. The Endonuclease G (EndoG) activation led to the DNA fragmentation in nucleus which in turn led to PARP activation. Activated PARP utilizes nicotinamide adenine dinucleotide (NAD+) molecules to repair nick in DNA. This NAD+

molecule is formed inside cell by utilization of ATP thus, excessive use of NAD+

In addition, we observed the increased level of PARP in MCAO group which was restored by the administration of zingerone at both the dosages. It has been reported that I/R injury

led to depletion of ATP molecules and added to energy deficit and mitochondrial stress. PARP activation also resulted in release of apoptosis inducing factor (AIF) from mitochondria which resulted in Caspase-independent apoptotic cell death. Zingerone (Z) being potent antioxidant was able to reduce the oxidative stress inside cell thus checked the free radicals generations after I/R. We can say, here, that zingerone thus reduced the mitochondrial stress by inhibiting ROS. Further, zingerone downregulated the Bax, Caspase-9, Apaf-1 and Caspase-3 and upregulated Bcl-2, thus strongly prevented the mitochondrial apoptotic pathway in order to protect the brain after ischemia-reperfusion.

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contributes to the extensive damage to nuclear DNA that ultimately results in high expression of activated PARP (Ahmad et al., 2011). PARP, activated by cleaved DNA strands, utilizes nicotinamide adenine dinucleotide (NAD+) as its substrate and synthesizes long branched and negatively charged polymers of ADP-ribose which are covalently bound to chromatin associated proteins and to PARP itself. Re-synthesis of NAD+ leads to ATP depletion and necrotic cell death (Yu et al., 2002; Graziani and Szabo, 2005). There are also proposition that PARP induced NAD+ utilization may first release apoptosis inducing factor (AIF) from mitochondria and later, activate caspase independent mechanisms of apoptotic cell death (Yu et al., 2002). The catalytic function of PARP is thought to dominate in early necrotic cell death whereas delayed cell death primarily involves role of PARP as a substrate for caspases (Oliver et al., 1998; Herceg and Wang, 1999; Yu et al., 2002). In recent study, it has been reported that an increase in PARP levels at 30 min post-hypoxia occurred which dropped to baseline levels by 2 h. By 6 h PARP began to increase again and peaked at 12 h and remained elevated above normal levels up to 24 h post-hypoxia (Martin et al., 2005). Therefore, we were investigated the role of PARP along with the apoptotic changes in MCAO brain at the end of 24 h after I/R when zingerone was administered with the delay of 3 h after MCAO. We observed that the delayed administration of zingerone successfully ameliorated the I/R-induced injury and thus maintain the energy level of cell by inhibiting PARP activation. The present finding also supports the earlier reports that PARP inhibition has reduced the infarct size in both, adult and immature animals (Takahashi et al., 1999; Ducrocq et al., 2000; Hagberg et al., 2004). PARP and Caspase-3 activation, in the ischemic stroke induces apoptotic processes in the cortical region via mitochondria-mediated pathway.

It is obvious from the present study that zingerone possesses a good anti-apoptotic effect along with its remarkable antioxidant property that made the basis of protection from ischemic-reperfusion injury. Briefly, the administration of zingerone, could inhibit I/R induced cell death in rat brain, likely by augmenting anti-oxidant enzymes and Bcl-2 and down regulating pro-apoptotic molecules like Bax, Apaf-1 and Caspase-3.

In conclusion, targeting and preventing apoptosis in the penumbra seems to be a rational therapeutic goal for reducing cerebral infarct volume after stroke, and the present study may provide the basis for the design of therapeutic interventions afforded by zingerone using the intraluminal transient cerebral ischemia model. However, the expression of each apoptosis regulatory protein as well as the regional distribution and association with DNA fragmentation should be assessed further in terms of post-ischemic recovery in longer period to impart the deep understanding of the action of zingerone against I/R injury.