Adam Institoris

Diazoxide and dimethyl sulphoxide prevent cerebral hypoperfusion-related learning dysfunction and brain damage after carotid artery occlusion

Chronic cerebral hypoperfusion, a mild ischemic condition is associated with advancing age and severity of dementia; however, no unanimous therapy has been established to alleviate related neurological symptoms. We imposed a permanent, bilateral occlusion of the common carotid arteries of rats (n=18) to create cerebral hypoperfusion. A mitochondrial ATP-sensitive K+ channel opener diazoxide (DZ, 5 mg/kg) or its solvent dimethyl sulphoxide (DMSO) were administered i.p. (0.25 ml) on five consecutive days after surgery. Sham-operated animals (n=18) served as control for the surgery, while nontreated rats were used as control for the treatments. Three months after the onset of cerebral hypoperfusion, the rats were tested in a hippocampus-related learning paradigm, the Morris water maze. Subsequently, the animals were sacrificed and neurons, astrocytes and microglia were labeled with immunocytochemistry in the dorsal hippocampus. DMSO and diazoxide dissolved in DMSO restored cerebral hypoperfusion-related learning dysfunction and prevented cyclooxygenase-2-positive neuron loss in the dentate gyrus. Cerebral hypoperfusion led to reduced astrocyte proliferation, which was not clearly affected by the treatment. Microglia activation was considerably enhanced by cerebral hypoperfusion, which was completely prevented by diazoxide dissolved in DMSO, but not by DMSO alone. We conclude that diazoxide can moderate ischemia-related neuroinflammation by suppressing microglial activation. Furthermore, we suggest that DMSO is a neuroprotective chemical in ischemic conditions, and it must be considerately used as a solvent for water-insoluble compounds in experimental animal models.

Protection against Recurrent Stroke with Resveratrol: Endothelial Protection

Despite increased risk of a recurrent stroke following a minor stroke, information is minimal regarding the interaction between injurious mild cerebral ischemic episodes and the possible treatments which might be effective. The aim of the current study was to investigate recurrent ischemic stroke and whether resveratrol, a nutritive polyphenol with promising cardio- and neuro- protective properties, could ameliorate the associated brain damage. Experiments in adult rats demonstrated that a mild ischemic stroke followed by a second mild cerebral ischemia exacerbated brain damage, and, daily oral resveratrol treatment after the first ischemic insult reduced ischemic cell death with the recurrent insult (P<0.002). Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. The protection observed with resveratrol treatment could not be explained by systemic effects of resveratrol treatment including effects either on blood pressure or body temperature measured telemetrically. Investigation of resveratrol effects on the blood-brain barrier in vivo demonstrated that resveratrol treatment reduced blood-brain barrier disruption and edema following recurrent stroke without affecting regional cerebral blood flow. Investigation of the mechanism in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against an in vitro ‘ischemia’ resulting in improved viability against oxygen and glucose deprivation (39.6±6.6% and 81.3±9.5% in vehicle and resveratrol treated cells, respectively). An inhibition of nitric oxide synthesis did not prevent the improved cell viability following oxygen glucose deprivation but SIRT-1 inhibition with sirtinol partially blocked the protection (P<0.001) suggesting endothelial protection is to some extent SIRT-1 dependent. Collectively, the results support that oral resveratrol treatment provides a low risk strategy to protect the brain from enhanced damage produced by recurrent stroke which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature.

Depolarization of Mitochondria in Endothelial Cells Promotes Cerebral Artery Vasodilation by Activation of Nitric Oxide Synthase

Objective—Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. Approach and Results—Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca2+]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca2+]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. Conclusions—Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca2+]i as well as by phosphoinositide-3 kinase-protein kinase B–induced eNOS phosphorylation. Both mitochondrial reactive oxygen species–dependent and –independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Mitochondrial Dynamics Associated with Oxygen-Glucose Deprivation in Rat Primary Neuronal Cultures

Our objective was to investigate the mitochondrial dynamics following oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. We documented changes in morphology, protein expression, and DNA levels in mitochondria following OGD and examined the roles of mitochondrial fission [dynamin-related protein 1 (Drp1), fission protein-1 (Fis1)] and fusion [mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy-1 protein (OPA1)] proteins on mitochondrial biogenesis and morphogenesis. We tested the effects of two Drp1 blockers [15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2) and Mitochondrial Division Inhibitor (Mdivi-1)] on mitochondrial dynamics and cell survival. One hour of OGD had minimal effects on neuronal viability but mitochondria appeared condensed. Three hours of OGD caused a 60% decrease in neuronal viability accompanied by a transition from primarily normal/tubular and lesser number of rounded mitochondria during normoxia to either poorly labeled or small and large rounded mitochondria. The percentage of rounded mitochondria remained the same. The mitochondrial voltage-dependent anion channel, Complex V, and mitoDNA levels increased after OGD associated with a dramatic reduction in Drp1 expression, less reduction in Mfn2 expression, an increase in Mfn1 expression, with no changes in either OPA1 or Fis1. Although PGJ2 increased polymerization of Drp1, it did not reduce cell death or alter mitochondrial morphology following OGD and Mdivi-1 did not protect neurons against OGD. In summary, mitochondrial biogenesis and maintained fusion occurred in neurons along with mitochondrial fission following OGD; thus Mfn1 but not Drp1 may be a major regulator of these processes.

The effect of pre- and posttreatment with diazoxide on the early phase of chronic cerebral hypoperfusion in the rat

Diazoxide has been identified as a mitochondrial, ATP-dependent K(+) channel opener, and a potentially neuroprotective compound under ischemic conditions. We set out to characterize the consequences of various treatment strategies with diazoxide in a rat model of chronic cerebral hypoperfusion. Cerebral hypoperfusion was induced by permanent, bilateral occlusion of the common carotid arteries (2VO, n = 36), sham-operated rats serving as controls (SHAM, n = 29). Diazoxide or its vehicle was administered i.p. daily (5 x 0.5 mg/kg/0.25 ml) or as a bolus injection (5 mg/kg/0.25 ml) before surgery or daily after surgery (5 x 0.5 mg/kg/0.25 ml). Spatial learning performance was assessed 1 week after 2VO in the Morris maze. Hippocampal pyramidal cell loss was assessed on cresyl violet-stained sections, while glial reactivity was labeled immunocytochemically. Daily or bolus pretreatment with diazoxide significantly improved 2VO-related learning impairment, whereas posttreatment was ineffective. The number of CA1 pyramidal neurons was reduced by 2VO, which was prevented by repeated or bolus pretreatment with diazoxide. Astrocyte proliferation and microglial activation were enhanced by posttreatment with diazoxide in the hippocampus CA1 area of 2VO animals as compared with SHAM. These data demonstrate that the neuroprotective effect exerted by diazoxide depends on the time of administration with respect to the onset of ischemia; pretreatment but not posttreatment with the compound has proved to be neuroprotective in chronic cerebral hypoperfusion. Thus, pretreatment with diazoxide offers therapeutical prospects for the treatment of cerebral ischemia.

Changes in pro-oxidant and antioxidant enzyme levels during cerebral hypoperfusion in rats

Chronic cerebral hypoperfusion is a mild ischemic condition associated with a cognitive decline which is prevalent during senescence or Alzheimers disease. Its experimental animal model compromises permanent occlusion of the common carotid arteries (2VO) in rats, which results in neuronal damage and microglia activation. Various mechanisms, including oxidative stress, have been proposed to be involved in this process. Accordingly, we set out to characterize the changes induced in the expressions of several pro-oxidant and antioxidant enzymes in cerebral hypoperfusion. Male Wistar rats were exposed to 2VO (n=30) or sham operation (n=33), while a third group served as absolute control (naive, n=16). Tissue samples from the hippocampus and frontal cortex were taken 1 and 3 days, 1 and 2 weeks and 3, 6 and 12 months following surgery. Western blot analysis was applied to determine the expressions of cyclooxygenase-2 (COX-2), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS, respectively) and manganese superoxide dismutase (MnSOD). During the early phase of hypoperfusion, the COX-2 and eNOS enzyme levels increased in both the hippocampus and the frontal cortex, indicating the presence of excitotoxicity and vascular reactions caused by ischemia, while the expressions of nNOS, iNOS and MnSOD were less affected. There were significant reductions in most of the investigated enzyme levels 2 weeks and 3 months after 2VO induction, which may be a sign of neuronal loss. One year following 2VO onset, the eNOS expression was upregulated, which may strengthen the adaptation of the brain to cerebral ischemia.

Diazoxide and dimethyl sulphoxide alleviate experimental cerebral hypoperfusion-induced white matter injury in the rat brain

Aging and dementia are accompanied by cerebral white matter (WM) injury, which is considered to be of ischemic origin. A causal link between cerebral ischemia and WM damage has been demonstrated in rats; however, few attempts appear to have been made to test potential drugs for the alleviation of ischemia-related WM injury. We induced cerebral hypoperfusion via permanent, bilateral occlusion of the common carotid arteries of rats. A mitochondrial ATP-sensitive potassium channel opener diazoxide (5 mg/kg) or its solvent dimethyl sulphoxide (DMSO) was administered i.p. (0.25 ml) on 5 consecutive days after surgery. Sham-operated animals served as control for surgery, and non-treated rats as controls for treatments. Thirteen weeks after surgery, the animals were sacrificed and astrocytes and microglia were labeled immunocytochemically in the internal capsule, the corpus callosum and the optic tract. The astrocytic proliferation was enhanced by cerebral hypoperfusion in the optic tract, and reduced by diazoxide in DMSO, but not by DMSO alone in the corpus callosum. After carotid artery occlusion, microglial activation was enhanced two-fold in the corpus callosum and four-fold in the optic tract. DMSO decreased microglial activation in the optic tract, while diazoxide in DMSO, but not DMSO alone, restored microglial activation to the control level in the corpus callosum. In summary, the rat optic tract appeared to be particularly vulnerable to ischemia, while the effect of diazoxide was restricted to the corpus callosum. We conclude that diazoxide dissolved in DMSO can moderate ischemia-related neuroinflammation by suppressing glial reaction in selective cerebral WM areas.

Effects of early aging and cerebral hypoperfusion on spreading depression in rats

Cortical spreading depression (CSD) is a feature of stroke pathophysiology. As stroke incidence increases with age, we have examined the effects of early aging and chronic cerebral hypoperfusion on CSD in rats. Three groups were studied: Young, 2-month-old animals; Middle-aged-2VO, subjected to 8 months of bilateral carotid occlusion from 2-month-of-age; and Middle-aged-SHAM, sham-operated. At 2- and 10-month-of-age for the Young and Middle-aged groups, recurrent CSD were induced under halothane anesthesia, by sustained application of 1 M KCl to the cortex for 2 h. Propagating CSD (i.e., cortical EEG, direct current potential) and associated laser Doppler blood flow changes were recorded anteriorly. Susceptibility to CSD and event duration were both decreased by early aging (frequency: 21±0.5 and 6±0.5 CSD/h; duration: 139±7 and 63±8 s; in Young and Middle-aged-SHAM, respectively). There was also a tendency for CSD-associated hyperemia to be reduced in the Middle-aged-2VO group (8.9±2.1 vs. 32.8±12.6% × min in Young). These data suggest reduced sensitivity of the cortex to CSD elicitation with early aging, and a less responsive cerebrovascular system with chronic hypoperfusion.

Impaired vascular responses of insulin-resistant rats after mild subarachnoid hemorrhage

Insulin resistance (IR) impairs cerebrovascular responses to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after subarachnoid hemorrhage (SAH) have not been described in IR. We hypothesized that IR worsens vascular reactions after a mild SAH. Hemolyzed blood (300 μl) or saline was infused (10 μl/min) into the cisterna magna of 11-13-wk-old ZO (n = 25) and Zucker lean (ZL) rats (n = 25). One day later, dilator responses of the basilar artery (BA) and its side branch (BA-Br) to acetylcholine (ACh, 10(-6) M), cromakalim (10(-7) M, 10(-6) M), and sodium nitroprusside (10(-7) M) were recorded with intravital videomicroscopy. The baseline diameter of the BA was increased both in the ZO and ZL rats 24 h after the hemolysate injection. Saline-injected ZO animals showed reduced dilation to ACh (BA = 9 ± 3 vs. 22 ± 4%; and BA-Br = 23 ± 5 vs. 37 ± 7%) compared with ZL rats. Hemolysate injection blunted the response to ACh in both the ZO (BA = 4 ± 2%; and BA-Br = 12 ± 3%) and ZL (BA = 7 ± 2%; and BA-Br = 11 ± 3%) rats. Cromakalim (10(-6) M)-induced dilation was significantly reduced in the hemolysate-injected ZO animals compared with the saline control (BA = 13 ± 3 vs. 26 ± 5%; and BA-Br = 28 ± 8 vs. 44 ± 9%) and in the hemolysate-injected ZL rats compared with their saline control (BA = 24 ± 4 vs. 32 ± 4%; but not BA-Br = 39 ± 6 vs. 59 ± 9%). No significant difference in sodium nitroprusside reactivity was observed. Western blot analysis of the BA showed a lower baseline level of neuronal nitric oxide synthase expression and an enhanced cyclooxygenase-2 level in the hemolysate-injected ZO animals. In summary, cerebrovascular reactivity to both endothelium-dependent and -independent stimuli is severely compromised by SAH in IR animals.