Background
Intracerebral hemorrhage (ICH) is an intracranial rupture of small blood vessels in the brain parenchyma with persistent increased blood pressure. Hypertension is the most common cause of ICH [
1]. Primary ICH, with no underlying vasculopathy, is associated with about 10–15% of all stroke cases, whereas in Oriental population, the figures are as up to 30–50% [
2‐
5]. Despite of the previous understanding of the mechanisms associated with ICH, there is no FDA approved treatment for ICH till date. ICH injury occurs in two different steps. First the leakage of blood in brain parenchyma elevates intracranial pressure that causes mechanical shear and damage to the neurons and glial cells [
6]. Secondly, products of erythrocyte lysis and damaged microglia trigger the inflammatory cascade and oxidative stress, causing secondary damage associated with ICH [
7]. Oxidative stress and neuronal cell damage associated cognitive decline are the most important implications in the pathogenesis of ICH [
8‐
10]. In the delayed phase of ICH, noxious proteases like matrix metalloproteinase (MMP-9) which are released pathologically from neutrophils and activated microglia [
11] cause structural damage to the blood brain barrier [
12] leading to the brain edema. Release of erythrocyte lysis product like iron, which catalyzes the production of free radicals and reactive oxygen species (ROS), is the key mediator of inflammatory cascade leading to cell death and perihematomal edema [
13,
14]. An increase in oxidative stress saturates like reduced glutathione (GSH), which is the main antioxidant molecule in brain, along with other endogenous antioxidants like superoxide dismutase (SOD), catalase etc. Although endogenous antioxidants can scavenge and neutralize the free radicals. Hence, neuroprotection in ICH needs a combined approach of decreasing different byproducts of secondary brain damage and restoring the antioxidant machinery in the brain. ICH leads to neuroinflammation and mitochondrial dysfunction, which are key features of chronic neurodegenerative diseases. These conditions leads to increased oxidative stress by excessive release of harmful reactive oxygen and nitrogen species (ROS and RNS), which further promote neuronal damage and subsequent inflammation resulting in a feed-forward loop of neurodegeneration. The cytokine tumor necrosis factor-α (TNF-α), a master regulator of the immune system, plays an important role in the propagation of inflammation due to the activation and recruitment of immune cells via its receptor [
15].
Animal model of ICH have been used to study the pathophysiology and treatment of ICH, including the microballoon model, the bacterial COL injection model and the autologous blood injection model. In the COL injection model, the hemorrhage size is controllable which was induced by small vessel breakdown. This model also can mimic the onset of spontaneous intraparenchymal bleeding and the expansion of continuous bleeding in ICH patients. In the past several years, previous studies have proven that our modified COL IV injection model is a reliable and reproducible model of ICH in rat [
16].
Antioxidants and anti-inflammatory drugs have been evaluated for potential pharmacotherapeutic activity in different models of ischemic and subarachnoid hemorrhage [
17‐
19] but no effective treatment method has been resolved out. So, agents that can strengthen endogenous antioxidant machinery and have direct ROS scavenging properties are the pressing need of the hour against ICH associated oxidative damage. RSV, a natural polyphenol found in grapes and red wine, could be a phytochemical of choice for ICH related oxidative damage. It has been shown to possess potent anti-oxidative, anticancer, anti-inflammatory and antiapoptotic effects in animal and clinical studies [
20‐
22]. Moreover, recent evidences indicate angiogenic and protective effects of RSV in variety of in-vivo and in-vitro ischemic model [
23‐
25]. Furthermore, accumulating evidences indicates potent neuroprotective and cardioprotective properties of RSV [
26‐
28]. With this background, the current study was designed to explore pharmacotherapeutic potential of RSV in experimental paradigm of ICH.
Discussion
ICV-COL model is a reliable and reproducible ICH model. It has been previously studied that COL injection in brain increases blood brain barrier permeability and brain edema which further leads to cognitive and behavioral deficits in animals [
44,
45]. Furthermore, basal lamina destruction and erythrocytic lysis induced by COL leads to increased generation of free radicals, pro-inflammatory cytokines, neuroinflammation, neuronal cell death and oxidative stress as seen in secondary injury during ICH.
Movement and functional disorders are seen in about 0.08% of the stroke patient causing temporary to permanent disability [
46]. The mechanism leading to stroke related functional disorders is not clear, but probably it is the delayed phase hematoma expansion induced damage to striatal neurons [
47] and resulting dopamine loss in the basal ganglia. Strial neurons act as relay centre for neuronal connections in basal ganglia. This hypothesis can be further corroborated by the fact that levodopa treatment improves functional recovery after experimental stroke [
48]. Moreover, brain herniation and oxido-inflammation caused by delayed ischemia in hemorrhagic stroke can cause focal damage and loss of function in particular brain region. Hypokinetic movement disorders are well reported in unilateral or bilateral infarction in striatum and lentiform nucleus [
49,
50]. In our study, we performed neurological scoring by using a battery of tests like horizontal bar test, forelimb flexion, righting reflex, cylinder test, spontaneous motility [
31]. Actophotometer test and rotarod test were done to assess motor impairments. We found that ICV COL administration produced significant motor and neurological defects, probably caused by acute caudate vascular lesion and oxido-inflammatory cerebral injury. Antioxidants have been shown to attenuate neuromotor deficits associated with various neurodegenerative diseases in different animal models [
51‐
53]. RSV restored these motor and neurological deficits owing to its inhibitory effect on oxido-inflammatory cascade involved in secondary brain injury after ICH.
Free radical generation and oxidative stress are the two most important factors in the ICH related brain injury. In the present study, an increase in oxidative stress is indicated by increased malondialdehyde and nitrite levels along with decreased endogenous antioxidants like catalase, superoxide dismutase and reduced glutathione levels. Vital organs like brain are more vulnerable to ischemic damage and oxidative stress due to its paucity of antioxidant defenses, uninterrupted oxygen demand and high lipoic myelin and iron content. Moreover, several compounds with antioxidant properties have been demonstrated to reduce stroke-related brain damage in animal models. Furthermore, delayed cerebral ischemia due to hematoma obstruction activates calcium dependent nitric oxide synthase which leads to increase in nitric oxide and ROS generation, leading to blood brain barrier disruption. This oxido-nitrosative stress leads to major neurological deficits like dementia, pain and depression in COL treated rats. In the present study, we found that RSV attenuated oxidative stress in the brain of COL treated rat by virtue of its strong antioxidant potential. RSV has been found to show antioxidant property by virtue of its free radical scavenging activity to protect hippocampal neuronal cells against toxicity induced by nitric oxide [
54]. Moreover, RSV up regulates heme-oxygenase 1 (HO1), an endogenous anti-oxidant protecting against neuronal cell death [
55].
Microglia is activated within minutes after ICH to clear hematoma and cell debris by phagocytosis. However, emerging evidences indicate that activated microglia contribute to hemorrhage related cell damage by releasing different inflammatory cytokines including TNF-α. TNF-α has been shown to increase after ICH in different in-vivo and in-vitro studies [
56‐
58]. The evidence is further strengthened by the fact that inhibition of microglia decreases ICH related brain damage [
59]. Plasma TNF-α level has been shown to have direct correlation with brain edema in ICH patients [
60]. In the present study, inflammatory cytokine generation as evident by elevated levels of TNF-α, was inhibited by RSV (10 & 20 mg/kg). Our observation is supported by the study done by Bi XL in which RSV inhibits nitric oxide and TNF-α production by lipopolysaccharide-activated microglia has also been reported to inhibit TNF-α both in-vivo and in-vitro [
61].
Physical disability, cognitive impairment and social isolation are the common factors leading to post stroke depression, anhedonia, anxiety and mood disorders and are seen in about one third of the stroke patients [
62‐
64]. In the present study, post stroke depression was evaluated using forced swim test. COL-induced post stroke depression is evident by increase in immobility time in force swim test on 14th day after COL administration in ICV-COL group. Increase oxidative stress and inflammatory cytokines in COL-induced ICH leads of depressive symptom. Antioxidant and anti-inflammatory agents from natural resources have been tried successfully in the amelioration of depressive behavior in different animal models [
65‐
68]. Moreover, extensive data on anti-depressant action of RSV has been reported [
69‐
71]. RSV has also been reported to inhibit monoamine oxidase activity (MAO) in vitro [
72] and to up regulate serotonin, dopamine and nor-epinephrine in a mice model of depression [
73]. Moreover, RSV has been shown to regulate HPA-axis and provide beneficial effect in ischemic stroke associated depression [
70]. In our study RSV at highest dose (20 mg/kg) restores the increased in immobility time in FST which is supported by the study done by Xu et al., [
73] where
trans-RSV led to a dose-dependent reduction in the immobility period.
As ICH leads to progressive memory deterioration and cognitive decline, MWM test was used to assess memory function. Increased escape latency (time spent to locate hidden platform) in repeated trials demonstrated memory deficit. Mean distance travelled was significantly reduced in RSV treated group as compared to the COL treated rats. Our observation is supported by the study in which RSV improves cognition and reduces oxidative stress in rats with vascular dementia [
74]. Previous studies have potentiated the role of neuroinflammation and microglial activation in memory deterioration and cognitive decline. RSV improved memory and cognition in ICV colchicine-induced cognitive impairment [
75]. RSV also inhibits LPS challenged microglial nitric oxide production by inhibiting NF-κB as evident by an in-vitro study [
61,
76]. Moreover, RSV has shown potent anti-inflammatory and neuroprotective effects in different models of ischemic stroke [
23,
77,
78].
Central post stroke pain (CPSP) is a pain syndrome common among 2–8% of stroke patients [
79]. Neuropathic pain is mainly associated with brain lesion or disease of somatosensory system [
80]. The mechanism of CPSP is still ambiguous but thought to be involvement of neuroinflammation induced activation of cell surface purinergic receptors [
81]. P
2X
7 receptor, a type of purinergic receptors is widely involved in pro-inflammatory receptors effects in central nervous system [
82]. Subsequent release of inflammatory cytokines like IL-1β has been proved to sensitize and destroy nerve terminals, resulting in neuropathic pain [
83]. ICV COL reduced pain threshold in von Frey hair and Randall-Selitto by releasing inflammatory cytokines like TNF-α and IL-1β. There was a significant increase in paw withdrawal threshold in RSV administered animals as compared to the COL treated animals [
84,
85].
Acknowledgement
Research grants sanctioned by SERB, Department of Science & Technology (grant no SB/FT/LS-284/2012), All India Council of Technical Education (11-25/RIFD/CAYT/POL- II/2013-14) and University Grants Commission (20-29(12)/2012(BSR), New Delhi to Dr. Anurag Kuhad are gratefully acknowledged. Junior Research Fellowship sanctioned by University Grants Commission, New Delhi to Mr. Navdeep Singh is also gratefully acknowledged.