Madhur Ray

Dose dependence and therapeutic window for the neuroprotective effects of curcumin in thromboembolic model of rat

Curcumin (diferuloylmethane), an active ingredient of turmeric, obtained from the powdered rhizomes of Curcuma longa Linn., has been traditionally recognized for treatment of several diseases. To evaluate the potential clinical use of curcumin, we determined the dose dependence of its effects in the therapeutic window and of the neuroprotective efficacy in a cerebral thromboembolic model of the rat. Rats were subjected to occlusion of the middle cerebral artery (MCAo) by a thrombus and treated with different doses of curcumin or the vehicle at 4h after ischemia. The animals were assessed after 24h for motor performance and neurological deficit. The rats were sacrificed immediately afterwards for evaluation of infarct, edema volume, estimation of nitrate and nitrite levels, neutrophil infiltration and levels of GSH and glutathione peroxidase (GSH-Px) in brain tissue. Curcumin reduced in a dose-dependent manner the ischemia-induced cerebral infarct and edema volume and attenuated neurological deficits observed after 24h. Curcumin reduced post-ischemic brain neutrophil infiltration, nitrate and nitrite levels and ameliorated the loss of GSH-Px and tends to increase the GSH levels but not significantly in the brain tissue. Neuronal levels of reactive oxygen species, peroxynitrite, and nitric oxide were lowered and in brain cryosections inducible nitric oxide synthase expression were significantly inhibited after treatment with curcumin. The present study is the first evidence of effectiveness of curcumin when given 4h post-ischemia in the rat thromboembolic stroke models, as it reduces infarct volume, ameliorates the sensory motor function and significantly attenuated the nitrosative stress.

Reduction in oxidative stress and cell death explains hypothyroidism induced neuroprotection subsequent to ischemia/reperfusion insult.

Hypometabolic state following hypothermia is known to protect tissues from ischemic injury. Hypothyroidism produces a hypometabolic state. The present study was undertaken to investigate the protective effects of hypothyroidism following cerebral ischemia and to ascertain the underlying mechanism. Euthyroid (E) and hypothyroid (H) animals were exposed to a 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion (I/R). Specific enzymatic methods and flowcytometry were used to assess the quantitative changes of molecules involved in neuronal damage as well as in protection. As compared to euthyroid ischemic reperfused (E + I/R) rats, H + I/R rats had insignificant neurological deficit, and smaller area of infarct. H + I/R rats had significantly lower markers of oxidative stress, and lactate dehydrogenase (LDH) activity (a marker for necrosis). Natural antioxidant activity (particularly superoxide dismutase) and integrity of mitochondria (membrane potential) were maintained in H + I/R group but not in E + I/R group. The number of neurons undergoing apoptosis significantly lower in hypothyroid ischemic rats as compared to euthyroid ones. These results suggest that hypothyroid animals face ischemia and reperfusion much better compared to euthyroid animals. A possible explanation could be the decreased oxidative stress and maintained antioxidant activity that finally leads to a decrease in necrosis and apoptosis. These observations may suggest strategies to induce brain-specific downregulation of metabolism that may have implications in the management of strokes in human beings.

Neuroprotective efficacy and therapeutic window of curcuma oil: in rat embolic stroke model

BackgroundAmong the naturally occurring compounds, turmeric from the dried rhizome of the plant Curcuma longa has long been used extensively as a condiment and a household remedy all over Southeast Asia. Turmeric contains essential oil, yellow pigments (curcuminoids), starch and oleoresin. The present study was designed for investigating the neuroprotective efficacy and the time window for effective therapeutic use of Curcuma oil (C. oil).MethodIn the present study, the effect of post ischemic treatment of C.oil after ischemia induced by occlusion of the middle cerebral artery in the rat was observed. C.oil (500 mg/kg body wt) was given 4 hrs post ischemia. The significant effect on lesion size as visualized by using diffusion-weighted magnetic resonance imaging and neuroscore was still evident when treatment was started 4 hours after insult. Animals were assessed for behavioral deficit scores after 5 and 24 hours of ischemia. Subsequently, the rats were sacrificed for evaluation of infarct and edema volumes and other parameters.ResultsC.oil ameliorated the ischemia induced neurological functional deficits and the infarct and edema volumes measured after 5 and 24 hrs of ischemia. After 24 hrs, immunohistochemical and Western blot analysis demonstrated that the expression of iNOS, cytochrome c and Bax/Bcl-2 were altered after the insult, and antagonized by treatment with C.oil. C.oil significantly reduced nitrosative stress, tended to correct the decreased mitochondrial membrane potential, and also affected caspase-3 activation finally apoptosis.ConclusionHere we demonstrated that iNOS-derived NO produced during ischemic injury was crucial for the up-regulation of ischemic injury targets. C.oil down-regulates these targets this coincided with an increased survival rate of neurons.

Curcuma oil modulates the nitric oxide system response to cerebral ischemia/reperfusion injury

The antioxidant activity of C.oil in cerebral stroke has been reported earlier. We have attempted here to clarify the mechanisms underlying the neuroprotection against experimental cerebral ischemia by Curcuma oil (C.oil), isolated from the rhizomes of Curcuma longa. C.oil (250 mg/kg i.p.) was given 30 min before focal ischemia in rats caused by occlusion of the middle cerebral artery (1h of occlusion, 24h of reflow). Ischemia, leads to elevation in [Ca(2+)] this sets into motion a cascades of ischemic injury which was attenuated by C.oil. C.oil reduced post-ischemic brain neutrophil infiltration in the ischemic area, controlled tissue NOx levels and the neuronal levels of nitric oxide, peroxynitrite and reactive oxygen species when measured after 24h of reflow. Double immunofluorescence staining analysis and Western immunoblot analysis with C.oil treatment showed that the expression of nitric oxide synthase (NOS) isoforms were decreased significantly compared to the untreated ischemia group. Ischemia is associated with increased in TUNEL (TdT-mediated dUTP nick-end labeling) positive cells in brain sections indicating DNA fragmentation. The C.oil treated group showed a significant decrease in numbers of apoptotic cells compared to the untreated ischemia group, as seen in the flowcytometric analysis of the neurons. Results of immunohistochemistry and Western immunoblot indicate that C.oil suppressed the elevated protein level of Bax, and aided mitochondrial translocation and activation of Bcl-2 by altered mitochondrial membrane potential. It also inhibits the cytosolic release of apoptogenic molecules like cytochrome c, inhibits the activation of caspase-3 and the expression of p53 ultimately inhibiting apoptosis. Our observations suggest that high levels of NO generated by NOS isoforms are partially responsible for exacerbating the neuronal damage induced by MCAo by intraluminal filament.

Studies of free radical generation by neurons in a rat model of cerebral venous sinus thrombosis

The role of free radicals in the pathogenesis of arterial stroke is well documented but not in venous stroke. The aim of the study was to investigate the possible role of free radicals in the pathogenesis of cerebral venous sinus thrombosis (CVST). For inducing CVST in Sprague-Dawley rats, a cranial window was made to expose the superior sagittal sinus (SSS). On the exposed sinus, a strip of filter paper soaked with 40% ferric chloride was applied. In the control rats 0.9% saline was used instead of ferric chloride. After induction of sinus thrombosis, clinical evaluations were done on days 1, 2 and 7 for neurological deficit, weight of thrombus and brain lesion volume. In neuronal-rich cell preparations flow cytometric estimations were done at different time points. In the study group on sequential follow-up, there was spontaneous recanalization of SSS as well as a significant decrease in brain lesion volume. An insignificant improvement in neurological deficit was also observed. In the controls, there was no neurological deficit or evidence of infarction. Neuronal free radical levels were significantly increased in the study group on day 1 compared to controls, but on follow-up free radicals levels decreased. It is concluded that the free radicals increase in the early stage of venous stroke and may be important in its pathogenesis.

The neuronal apoptotic death in global cerebral ischemia in gerbil: Important role for sodium channel modulator†

Global ischemia was induced in gerbil by bilateral occlusion of the common carotid arteries for 5 min. Sodium ionophore monensin or sodium channel blocker tetrodotoxin (TTX) was administered at doses of 10 μg/kg, i.p., 30 min before ischemia induction; the dose was repeated after 22 hr. Subsequently, brain infarct occurred, determined at 24 hr after occlusion. Large, well‐demarcated infarcts were observed in both hemispheres, an important observation because it critically influences the interpretation of the data. Because nitric oxide (NO) production is thought to be related to ischemic neuronal damage, we examined increases in Ca2+ influx, which lead to the activation of nitric oxide synthase (NOS). Then we evaluated the contributions of neuronal NOS, endothelial NOS, and inducible NOS to NO production in brain cryosections. The cytosolic release of apoptogenic molecules like cytochrome c and p53 were confirmed after 24 hr of reflow. TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labeling) labeling detected the apoptotic cells, which were confirmed in neuron‐rich cell populations. After 24 hr, all the ischemic changes were amplified by monensin and significantly attenuated by TTX treatment. Additionally, the nesting behavior and histological outcomes were examined after 7 day of reflow. The neuronal damage in the hippocampal area and significant decrease in nesting scores were observed with monensin treatment and reduced by TTX pretreatment after day 7 of reflow. To our knowledge, this report is the first to highlight the involvement of the voltage‐sensitive Na+ channel in possibly regulating in part NO system and apoptosis in a cytochrome c–dependent manner in global ischemia in the gerbil, and thus warrants further investigation.

Cerebral venous sinus thrombosis: Developing an experimental model

To develop a simple experimental model for cerebral venous sinus thrombosis in rat, 24 male Sprague-Dawley rats (14 experimental and 10 controls) were studied. After anesthesia, a 1.5 mm x 10 mm cranial window was made to expose superior sagittal sinus (SSS). A filter paper strip soaked in 40% ferric chloride topically applied for 5 min to induce SSS thrombosis in experimental group. Five of these rats underwent autopsy at 24 h after rota rod study, other 9 rats underwent MRI after 24 h followed by autopsy at 36-48 h. On MRI, T2 hyperintense lesions were seen in all 9 rats of study group but none in controls. On autopsy, TTC staining revealed changes in 7 out of 14 experimental rats but in none of the controls. The rota rod study results revealed an insignificant difference in the experimental and control group. There was no spontaneous death in experimental or control group during the study. The present experimental model is simple, easy to carry out and results in TTC staining changes in 50% and T2 hyperintensity on MRI in all which could be due to SSS thrombosis.

Design, synthesis and biological evaluation of new ionone derivatives as potential neuroprotective agents in cerebral ischemia

A new series of ionone derived allylic alcohols have been evaluated for anti-ischemic activity. Out of them, 12f and 13b decreased infarct volume to 23.98+/-4.7 mm3 and 93.98+/-24.8 mm3 as compared to ischemic group.