Nasrul Hoda

Remote Ischemic Perconditioning Is Effective Alone and in Combination With Intravenous Tissue-Type Plasminogen Activator in Murine Model of Embolic Stroke

Background and Purpose— Remote ischemic conditioning is cardioprotective in myocardial infarction and neuroprotective in mechanical occlusion models of stroke. However, there is no report on its therapeutic potential in a physiologically relevant embolic stroke model (embolic middle cerebral artery occlusion) in combination with intravenous tissue-type plasminogen activator (tPA). Methods— We tested remote ischemic perconditioning therapy (RIPerC) at 2 hours after embolic middle cerebral artery occlusion in the mouse with and without intravenous tPA at 4 hours. We assessed cerebral blood flow up to 6 hours, neurological deficits, injury size, and phosphorylation of Akt (Serine473) as a prosurvival signal in the ischemic hemisphere at 48 hours poststroke. Results— RIPerC therapy alone improved the cerebral blood flow and neurological outcomes. tPA alone at 4 hours did not significantly improve the neurological outcome even after successful thrombolysis. Individual treatments with RIPerC and intravenous tPA reduced the infarct size (25.7% and 23.8%, respectively). Combination therapy of RIPerC and tPA resulted in additive effects in further improving the neurological outcome and reducing the infarct size (50%). All the therapeutic treatments upregulated phosphorylation of Akt in the ischemic hemisphere. Conclusions— RIPerC is effective alone after embolic middle cerebral artery occlusion and has additive effects in combination with intravenous tPA. RIPerC may be a simple, safe, and inexpensive combination therapy with intravenous tPA.

Attenuation by Nardostachys jatamansi of 6-hydroxydopamine-induced parkinsonism in rats: Behavioral, neurochemical, and immunohistochemical studies

Parkinsons disease (PD) is one of the commonest neurodegenerative diseases, and oxidative stress has been evidenced to play a vital role in its causation. In the present study, we evaluated whether ethanolic extract of Nardostachys jatamansi roots (ENj), an antioxidant and enhancer of biogenic amines, can slow the neuronal injury in a 6-OHDA-rat model of Parkinsons. Rats were treated with 200, 400, and 600 mg/kg body weight of ENj for 3 weeks. On day 21, 2 microl of 6-OHDA (12 microg in 0.01% in ascorbic acid-saline) was infused into the right striatum, while the sham-operated group received 2 microl of vehicle. Three weeks after the 6-OHDA injection, the rats were tested for neurobehavioural activity and were sacrificed after 6 weeks for the estimation of lipid peroxidation, reduced glutathione content, the activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase, quantification of catecholamines, dopaminergic D2 receptor binding and tyrosine hydroxylase expression. The increase in drug-induced rotations and deficits in locomotor activity and muscular coordination due to 6-OHDA injections were significantly and dose-dependently restored by ENj. Lesioning was followed by an increased lipid peroxidation and significant depletion of reduced glutathione content in the substantia nigra, which was prevented with ENj pretreatment. The activities of glutathione-dependent enzymes, catalase and superoxide dismutase in striatum, which were reduced significantly by lesioning, were dose-dependently restored by ENj. A significant decrease in the level of dopamine and its metabolites and an increase in the number of dopaminergic D2 receptors in striatum were observed after 6-OHDA injection, and both were significantly recovered following ENj treatment. All of these results were exhibited by an increased density of tyrosine hydroxylase immunoreactive (TH-IR) fibers in the ipsilateral striatum of the lesioned rats following treatment with ENj; 6-OHDA injection had induced almost a complete loss of TH-IR fibers. This study indicates that the extract of Jatamansi might be helpful in attenuating Parkinsonism.

The indoleamine 2,3-dioxygenase pathway controls complement-dependent enhancement of chemo-radiation therapy against murine glioblastoma

BackgroundIndoleamine 2,3-dioxygenase (IDO) is an enzyme with immune-suppressive properties that is commonly exploited by tumors to evade immune destruction. Anti-tumor T cell responses can be initiated in solid tumors, but are immediately suppressed by compensatory upregulation of immunological checkpoints, including IDO. In addition to these known effects on the adaptive immune system, we previously showed widespread, T cell-dependent complement deposition during allogeneic fetal rejection upon maternal treatment with IDO-blockade. We hypothesized that IDO protects glioblastoma from the full effects of chemo-radiation therapy by preventing vascular activation and complement-dependent tumor destruction.MethodsTo test this hypothesis, we utilized a syngeneic orthotopic glioblastoma model in which GL261 glioblastoma tumor cells were stereotactically implanted into the right frontal lobes of syngeneic mice. These mice were treated with IDO-blocking drugs in combination with chemotherapy and radiation therapy.ResultsPharmacologic inhibition of IDO synergized with chemo-radiation therapy to prolong survival in mice bearing intracranial glioblastoma tumors. We now show that pharmacologic or genetic inhibition of IDO allowed chemo-radiation to trigger widespread complement deposition at sites of tumor growth. Chemotherapy treatment alone resulted in collections of perivascular leukocytes within tumors, but no complement deposition. Adding IDO-blockade led to upregulation of VCAM-1 on vascular endothelium within the tumor microenvironment, and further adding radiation in the presence of IDO-blockade led to widespread deposition of complement. Mice genetically deficient in complement component C3 lost all of the synergistic effects of IDO-blockade on chemo-radiation-induced survival.ConclusionsTogether these findings identify a novel mechanistic link between IDO and complement, and implicate complement as a major downstream effector mechanism for the beneficial effect of IDO-blockade after chemo-radiation therapy. We speculate that this represents a fundamental pathway by which the tumor regulates intratumoral vascular activation and protects itself from immune-mediated tumor destruction.

Endothelial PFKFB3 Plays a Critical Role in Angiogenesis

Objective—Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3) is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. Approach and Results—Under hypoxic conditions or after treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. In addition, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated protein kinase B was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. Conclusions—The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis.Objective Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3), is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo.Objective— Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3) is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. Approach and Results— Under hypoxic conditions or after treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. In addition, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated AKT was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. Conclusions— The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis.

Remote Limb Perconditioning and Postconditioning Will It Translate Into a Promising Treatment for Acute Stroke

Preconditioning is an adaptive process of endogenous protection in which small, sublethal doses of a harmful agent protect the organism against a later lethal dose of the same agent. The basic principle is evident in human thought and literature, encapsulated in Nietzsche’s statement, “what does not kill me makes me stronger.”1 Ischemic preconditioning, the use of short episodes of sublethal ischemia to protect against a later episode of lethal ischemia, is one of the most effective cardioprotectants known.2 Moreover, ischemic conditioning is protective in organ ischemia of multiple animal species and effective even when applied after the onset of ischemia. Ischemic conditioning targets ischemia-reperfusion injury. Depending on the temporal relation of the sublethal ischemia to the lethal ischemia, the ischemic stimulus can be applied before ischemia and vessel occlusion (preconditioning), during the ischemia and before reperfusion (perconditioning), or after the lethal ischemic episode, during reperfusion (postconditioning; Figure 1). Preconditioning has limited clinical application and is best suited to clinical situations of predictable ischemia, such as coronary artery bypass grafting or carotid endarterectomy; perconditioning and postconditioning are ideally suited to treatment of acute ischemic stroke because they can be applied during or after the ischemic period. Conventional preconditioning and postconditioning require occluding and releasing the artery supplying the target organ. Although percutaneous interventions are commonplace in the treatment of myocardial infarctions and more stroke patients are receiving intra-arterial interventions, accessing a coronary or cerebral artery is not always feasible, practical, nor safe. Figure 1. Schematic of the temporal relation of the remote conditioning stimulus to the episode of ischemia. One of the most significant breakthroughs in the ischemic conditioning field came with the discovery that ischemic conditioning can also be performed at a distance or remote from the ischemic target organ. Transient, sublethal mesenteric, renal, and limb ischemia all protect against …

Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke

BackgroundMinocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans.MethodsFive groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed.ResultsThe model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P < 0.0001) and also improved neurologic score (P < 0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P = 0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P < 0.0001).ConclusionIn a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

Comparative analysis of the neurovascular injury and functional outcomes in experimental stroke models in diabetic Goto-Kakizaki rats

Diabetes worsens functional outcome and is associated with greater hemorrhagic transformation (HT) after ischemic stroke. We have shown that diabetic Goto-Kakizaki (GK) rats develop greater HT and neurological deficit despite smaller infarcts after transient middle cerebral artery occlusion (MCAO) with the suture model. However, the impact of (1) the duration of ischemia/reperfusion (I/R); (2) the method of ischemia; and (3) acute glycemic control on neurovascular injury and functional outcome in diabetic stroke remained unanswered. Wistar and GK rats were subjected to variable MCAO by suture or embolus occlusion. A group of GK rats were treated with insulin or metformin before stroke with suture occlusion. In all groups, infarct size, edema, HT occurrence and severity, and functional outcome were measured. Infarct size at 24h was smaller in GK rats with both suture and embolic MCAO, but expanded with longer reperfusion period. Edema and HT were increased in GK rats after 90min and 3h occlusion with the suture model, but not in the embolic MCAO. Neurological deficit was greater in diabetic rats. These findings suggest that diabetes accelerates the development of HT and amplifies vascular damage in the suture model where blood flow is rapidly reestablished. Acute metformin treatment worsened the infarct size, HT, and behavior outcome, whereas insulin treatment showed a protective effect. These results suggest that the impact of ischemia/reperfusion on neurovascular injury and functional outcome especially in disease models needs to be fully characterized using different models of stroke to model the human condition.

Remote ischaemic conditioning-a new paradigm of self-protection in the brain.

Remote ischaemic conditioning (RIC) triggers endogenous protective pathways in distant organs such as the kidney, heart and brain, and represents an exciting new paradigm in neuroprotection. RIC involves repetitive inflation and deflation of a blood pressure cuff on the limb, and is safe and feasible. The exact mechanism of signal transmission from the periphery to the brain is not known, but both humoral factors and an intact nervous system seem to have critical roles. Early-phase clinical trials have already been conducted to test RIC in the prehospital setting in acute ischaemic stroke, and in subarachnoid haemorrhage for the prevention of delayed cerebral ischaemia. Furthermore, two small randomized clinical trials in patients with symptomatic intracranial atherosclerosis have shown that RIC can reduce recurrence of stroke and have neuroprotective activity. RIC represents a highly practical and translatable therapy for acute, subacute, and chronic neurological diseases with an ischaemic or inflammatory basis. In this Review, we consider the principles and mechanisms of RIC, evidence from preclinical models and clinical trials that RIC is beneficial in neurological disease, and how the procedure might be used in the future in disorders such as vascular cognitive impairment and traumatic brain injury.

Comparative Analysis of Different Methods of Ischemia/Reperfusion in Hyperglycemic Stroke Outcomes: Interaction with tPA

Acute hyperglycemia (HG) exacerbates reperfusion injury and aggravates tissue plasminogen activator (tPA)-induced hemorrhagic transformation (HT). Previous experimental hyperglycemic stroke studies employed very high blood glucose levels and exclusively used suture occlusion model to induce ischemia. Only few studies evaluated HG in embolic stroke and mostly involving the use of 10-fold higher dose of tPA than that is used in patients. However, the interaction between acute HG and low (human) dose tPA in different experimental models of stroke has never been reported. We first tested the impact of the severity of acute HG on stroke outcome. Building upon our findings, we then compared the impact of mild acute HG on neurovascular injury in rats subjected to suture or thromboembolic occlusion with and without low dose tPA. We assessed cerebral blood flow, neurobehavioral outcomes, infarction, hemorrhage, and edema. tPA did not change the infarct size in either control or hyperglycemic animals when compared to no tPA groups. HG increased HT and worsened functional outcomes in both suture and embolic occlusion models. The combination of HG and tPA exacerbated the vascular injury and worsened the neurological deficits more than each individual treatment in both models. Our findings show that the interaction between HG and even low dose tPA has detrimental effects on the cerebrovasculature and functional outcomes independent of the method of reperfusion.

Combined therapy with COX-2 inhibitor and 20-HETE inhibitor reduces colon tumor growth and the adverse effects of ischemic stroke associated with COX-2 inhibition

20-Hydroxyeicosatetraenoic acid (20-HETE), Cyp4a-derived eicosanoid, is a lipid mediator that promotes tumor growth, as well as causing detrimental effects in cerebral circulation. We determined whether concurrent inhibition of cyclooxygenase-2 (COX-2) and 20-HETE affects colon tumor growth and ischemic stroke outcomes. The expression of Cyp4a and COXs and production of 20-HETE and PGE2 were determined in murine colon carcinoma (MC38) cells. We then examined the effects of combined treatment with rofecoxib, a potent COX-2 inhibitor, and HET0016, a potent Cyp4a inhibitor, on the growth and proliferation of MC38 cells. Subsequently, we tested the effects of HET0016 plus rofecoxib in MC38 tumor and ischemic stroke models. Cyp4a and COXs are highly expressed in MC38 cells. Respectively, HET0016 and rofecoxib inhibited 20-HETE and PGE2 formation in MC38 cells. Moreover, rofecoxib combined with HET0016 had greater inhibitory effects on the growth and proliferation of MC38 cells than did rofecoxib alone. Importantly, rofecoxib combined with HET0016 provided greater inhibition on tumor growth than did rofecoxib alone in MC38 tumor-bearing mice. Prolonged treatment with rofecoxib selectively induced circulating 20-HETE levels and caused cerebrovascular damage after ischemic stroke, whereas therapy with rofecoxib and HET0016 attenuated 20-HETE levels and reduced rofecoxib-induced cerebrovascular damage and stroke outcomes during anti-tumor therapy. Thus these results demonstrate that combination therapy with rofecoxib and HET0016 provides a new treatment of colon tumor, which can not only enhance the anti-tumor efficacy of rofecoxib, but also reduce rofecoxib-induced cerebrovascular damage and stroke outcomes.