Endothelial Cell Dysfunction and Injury in Subarachnoid Hemorrhage.

In the brain, vascular endothelial cells conserve blood viscosity, control blood flow, and form the interface between central nervous system and circulating blood. Clinical outcome after aneurysmal subarachnoid hemorrhage is linked to early brain injury, cerebral vasospasm, and other causes of delayed cerebral ischemia. The cerebral vasculature remains a unique target for therapies since it becomes rapidly disrupted after subarachnoid hemorrhage, and damage to the blood vessels continues into the delayed injury phase. The current failure of therapies to improve clinical outcome warrants a re-evaluation of current therapeutic approaches. The mechanisms of endothelial cell injury and blood-brain barrier breakdown are critical to the pathway of cerebral injury, and an improved understanding of these mechanisms may lead to novel therapeutic targets. This review provides an update on the current understanding of endothelial cell injury following aneurysmal subarachnoid hemorrhage, including blood-brain barrier dysfunction.

The Role of Thromboinflammation in Delayed Cerebral Ischemia after Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) is a major determinant of patient outcome following aneurysmal subarachnoid hemorrhage. Although the exact mechanisms leading to DCI are not fully known, inflammation, cerebral vasospasm, and microthrombi may all function together to mediate the onset of DCI. Indeed, inflammation is tightly linked with activation of coagulation and microthrombi formation. Thromboinflammation is the intersection at which inflammation and thrombosis regulate one another in a feedforward manner, potentiating the formation of thrombi and pro-inflammatory signaling. In this review, we will explore the role(s) of inflammation and microthrombi in subarachnoid hemorrhage (SAH) pathophysiology and DCI, and discuss the potential of targeting thromboinflammation to prevent DCI after SAH.

Efficient specification of interneurons from human pluripotent stem cells by dorsoventral and rostrocaudal modulation.

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs, and their malfunction, resulting in failure to intricately regulate neural circuit balance, is implicated in brain diseases such as Schizophrenia, Autism, and Epilepsy. During early development, GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers, and migrate to their target sites where they form local synaptic connections. In this study, using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH, Wnt, and FGF8), we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly, modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6-expressing GABAergic interneurons and showed migratory properties. These human MGE-derived neurons generated GABA, fired action potentials, and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well-contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus, we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine, developmental studies, disease modeling, bioassay, and drug screening.

Expression of cholinergic, insulin, vitamin D receptors and GLUT 3 in the brainstem of streptozotocin induced diabetic rats: effect of treatment with vitamin D3.

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B(max) of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D3 receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D3 and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D3 treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D3 in managing neurological disorders associated with diabetes.

The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats.

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M(1) receptor showed a significant decrease and muscarinic M(3) receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M(1), M(3) receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M(1), M(3) receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes.

Role of curcumin in the prevention of cholinergic mediated cortical dysfunctions in streptozotocin-induced diabetic rats.

Diabetes exacerbates neuronal injury mediated through neurotransmitters deregulation in cerebral cortex. Our study analyzed the neuroprotective effect of curcumin to prevent cortical dysfunction associated with diabetes. Our study revealed decreased gene expression of muscarinic M1, insulin receptor, SOD, choline acetyl transferase and increased gene expression of muscarinic M3, α7-nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 in cerebral cortex of diabetic rats. Curcumin and insulin treatment reversed this altered parameters to near control. Immunohistochemistry studies of muscarinic M1 and M3 confirmed the gene expression at protein level. Decreased novel arm entry of diabetic rats in Y-maze test, improved in treatment group. These results suggest that cholinergic dysfunction, impaired glucose transport and oxidative stress contributes to learning and memory deficits in diabetes and further suggest that antioxidant curcumin has potential therapeutic role in preventing and/or delaying the diabetic complications associated with brain.

Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats.

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). alpha7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and alpha7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.

Behavioral deficit and decreased GABA receptor functional regulation in the cerebellum of epileptic rats: effect of Bacopa monnieri and bacoside A.

In the present study, the effects of Bacopa monnieri and its active component, bacoside A, on motor deficit and alterations of GABA receptor functional regulation in the cerebellum of epileptic rats were investigated. Scatchard analysis of [(3)H]GABA and [(3)H]bicuculline in the cerebellum of epileptic rats revealed a significant decrease in B(max) compared with control. Real-time polymerase chain reaction amplification of GABA(A) receptor subunits-GABA(Aalpha1), GABA(Aalpha5,) and GABA(Adelta)-was downregulated (P<0.001) in the cerebellum of epileptic rats compared with control rats. Epileptic rats exhibit deficits in radial arm and Y-maze performance. Treatment with B. monnieri and bacoside A reversed these changes to near-control levels. Our results suggest that changes in GABAergic activity, motor learning, and memory deficit are induced by the occurrence of repetitive seizures. Treatment with B. monnieri and bacoside A prevents the occurrence of seizures thereby reducing the impairment of GABAergic activity, motor learning, and memory deficit.

Cholinergic, dopaminergic and insulin receptors gene expression in the cerebellum of streptozotocin-induced diabetic rats: functional regulation with Vitamin D3 supplementation.

The study was to find out the effect of Vitamin D3 supplementation on preventing the altered gene expression of cholinergic, dopaminergic, insulin receptors and GLUT3 gene expression in cerebellum of diabetic rats. Radioreceptor binding assays and gene expression were done in the cerebellum of male Wistar rats. Rota rod has been used to evaluate motor coordination. Our results showed a significantly increased gene expression of dopamine D2, muscarinic M1, M3, alpha7 nicotinic acetylcholine, insulin receptors, acetylcholine esterase, GLUT3 and Vitamin D receptor in the cerebellum of diabetic rats. There was a down-regulation of dopamine D1 receptor. Total dopamine receptor showed a decreased and total muscarinic, muscarinic M1 and M3 receptors showed an increased binding parameter, B(max). Rota rod experiment showed a significant decrease in the retention time on the rotating rod in diabetic while treatment improved retention time near to control. Vitamin D3 and insulin treatment markedly recovered the altered gene expression and binding parameters to near control. Our study showed Vitamin D3 functional regulation through dopaminergic, cholinergic and insulin receptors and glucose transport mechanism through GLUT3 in the cerebellum of diabetic rats which play a major role in neuroprotection in diabetes which has clinical application.

Enhanced NMDAR1, NMDA2B and mGlu5 receptors gene expression in the cerebellum of insulin induced hypoglycaemic and streptozotocin induced diabetic rats.

Glucose homeostasis in humans is an important factor for the functioning of the nervous system. A decrease in glucose content below a minimal level or hypoglycemia is dangerous for cells of the central and peripheral nerve system. In the present study we showed the effects of insulin induced hypoglycaemia and streptozotocin induced diabetes on the cerebellar glutamate receptor subunits and glutamate transporter. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. We found an up regulation in NMDA receptor number and gene expression of N-methyl-d-aspartic acid (NMDA(R1)), NMDA(2B), metabotrophic glutamate 5 (mGlu(5)) glutamate receptor subunits in experimental rats. The glutamate content was shown to be increased with decreased glutamate aspartate transporter (GLAST) gene expressions indicating lower reuptake of glutamate. The enhanced gene expression of NMDA(R1), NMDA(2B), mGlu(5) glutamate receptors were confirmed by immunohistochemistry studies. At the second messenger level, the IP3 content and IP3 receptors were enhanced in the cerebellum of both hypoglycaemic and diabetic rats increased. The present study showed that the enhanced glutamate content activates NMDA receptors, increasing the inositol triphosphate (IP3) content which mediates Ca(2+) overload in cells causing cell damage and neurodegeneration. Our results also showed that the enhanced glutamate receptor activity were more prominent in hypoglycaemic group compared to diabetic group. Further the neurodegeneration by the up regulation of glutamate receptor activity causing motor dysfunction was demonstrated by the Rotarod test. Thus our results suggest that enhanced NMDA receptor mediated neurodegeneration affect the motor learning and memory ability of an individual.

Acetylcholine and muscarinic receptor function in cerebral cortex of diabetic young and old male Wistar rats and the role of muscarinic receptors in calcium release from pancreatic islets.

We investigated acetylcholine esterase (AChE) activity, acetylcholine and muscarinic M1, M3 receptors kinetics in the cerebral cortex of young and old streptozotocin induced and insulin treated diabetic rats. The role of muscarinic receptors in intracellular calcium release from pancreatic islets was studied in vitro. Wistar rats of 7 and 90-weeks old were used. All studies were done in cerebral cortex. AChE assay was done by spectrophotometric method. Radioreceptor binding assays were done for Acetylcholine, Muscarinic M1 and M3 receptors using specific ligands. Calcium imaging was done using fluo4-AM in pancreatic cells. Ninety-weeks old control rats showed significantly decreased Vmax and increased Km for AChE compared to 7-weeks old control rats. An increased Vmax observed in both 7 and 90-weeks old diabetic groups with significant decrease in Km. Scatchard analysis using specific agonists showed significant decrease in the B (max) and K (d) of acetylcholine and muscarinic M1 receptors in 90-weeks old control rats compared to 7-weeks old control. Binding studies for M3 receptors showed no significant change compared to 7-weeks old control. Acetylcholine, muscarinic M1 and M3 receptor number significantly increased in 90-weeks old diabetic rat groups compared to their respective controls. Insulin treatment significantly reversed the binding parameters to near control compared to diabetic group. In vitro studies showed that acetylcholine through muscarinic M1 and M3 receptors' stimulated calcium release from the pancreatic islets. Thus our studies suggest that Insulin signaling play an important part in differentially regulating pancreatic cholinergic activity, and the diabetes mediated cortical dysfunctions with age.

Decreased GABAA receptors functional regulation in the cerebral cortex and brainstem of hypoxic neonatal rats: effect of glucose and oxygen supplementation.

Hypoxia in neonates can lead to biochemical and molecular alterations mediated through changes in neurotransmitters resulting in permanent damage to brain. In this study, we evaluated the changes in the receptor status of GABA(A) in the cerebral cortex and brainstem of hypoxic neonatal rats and hypoxic rats supplemented with glucose and oxygen using binding assays and gene expression of GABA(Aalpha1) and GABA(Agamma5). In the cerebral cortex and brainstem of hypoxic neonatal rats, a significant decrease in GABA(A) receptors was observed, which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with glucose and oxygen showed a reversal of the GABA(A) receptors, andGABA(Aalpha1) and GABA(Agamma5) gene expression to control. Glucose acts as an immediate energy source thereby reducing the ATP-depletion-induced increase in GABA and oxygenation, which helps in encountering anoxia. Resuscitation with oxygen alone was less effective in reversing the receptor alterations. Thus, the results of this study suggest that reduction in the GABA(A) receptors functional regulation during hypoxia plays an important role in mediating the brain damage. Glucose alone and glucose and oxygen supplementation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.

Neuroprotective role of curcumin in the cerebellum of streptozotocin-induced diabetic rats.

AIMS: Chronic hyperglycaemia in diabetes involves a direct neuronal damage caused by intracellular glucose which leads to altered neurotransmitter functions and reduced motor activity. The present study investigated the effect of curcumin in the functional regulation of muscarinic and alpha7 nicotinic acetylcholine receptors, insulin receptors, acetylcholine esterase and Glut3 in the cerebellum of streptozotocin (STZ)-induced diabetic rats. MAIN METHODS: All studies were done in the cerebellum of male Wistar rats. Radioreceptor binding assays were done for total muscarinic, M(1) and M(3) receptors using specific ligands, and the gene expression was also studied using specific probes. KEY FINDINGS: Our results showed an increased gene expression of acetylcholine esterase, Glut3, muscarinic M1, M3, alpha7 nicotinic acetylcholine and insulin receptors in the cerebellum of diabetic rats in comparison to control. Scatchard analysis of total muscarinic, M1 and M3 receptors showed an increased binding parameter, B(max) in diabetic rats compared to control. Curcumin and insulin inhibited diabetes-induced elevation in the gene expression of acetylcholine esterase, Glut3, insulin and cholinergic receptors in the cerebellum of diabetic rats. SIGNIFICANCE: Our studies suggest that curcumin plays a vital role in regulating the activity of cholinergic and insulin receptors and mechanism of glucose transportation through Glut3, which results in normalizing the diabetes-mediated cerebellar disorders. Thus, curcumin has a significant role in a therapeutic application for the prevention or progression of diabetic complications in the cerebellum.