Yuchuan Ding

Upper limb ischemic preconditioning prevents recurrent stroke in intracranial arterial stenosis

Objective: This study aims to evaluate protective effects of brief repetitive bilateral arm ischemic preconditioning (BAIPC) on stroke recurrence in patients with symptomatic atherosclerotic intracranial arterial stenosis (IAS). Methods: A total of 68 consecutive cases with symptomatic IAS, diagnosed by imaging, were enrolled in this prospective and randomized study. All patients received standard medical management. Patients in the BAIPC group (n = 38) underwent 5 brief cycles consisting of bilateral upper limb ischemia followed by reperfusion. The BAIPC procedure was performed twice daily over 300 consecutive days. Incidence of recurrent stroke and cerebral perfusion status in BAIPC-treated patients were compared with the untreated control group (n = 30). Results: In the control group, incidence of recurrent stroke at 90 and 300 days were 23.3% and 26.7%, respectively. In the BAIPC group, incidence of recurrent stroke was reduced to 5% and 7.9% at 90 and 300 days (p < 0.01), respectively. The average time to recovery (modified Rankin Scale score 0–1) was also shortened by BAIPC. Cerebral perfusion status, measured by SPECT and transcranial Doppler sonography, improved remarkably in BAIPC-treated brain than in control (p < 0.01). Conclusion: This study provides a proof-of-concept that BAIPC may be an effective way to improve cerebral perfusion and reduce recurrent strokes in patients with IAS. Further investigation of this therapeutic approach is warranted as some patients were excluded after randomization.

Forced, not voluntary, exercise effectively induces neuroprotection in stroke.

Previous treadmill exercise studies showing neuroprotective effects have raised questions as to whether exercise or the stress related to it may be key etiologic factors. In this study, we examined different exercise regimens (forced and voluntary exercise) and compared them with the effect of stress-only on stroke protection. Adult male Sprague-Dawley rats (nxa0=xa065) were randomly assigned to treatment groups for 3xa0weeks. These groups included control, treadmill exercise, voluntary running wheel exercise, restraint, and electric shock. Levels of the stress hormone, corticosterone, were measured in the different groups using ELISA. Animals from each group were then subjected to stroke induced by a 2-h middle cerebral artery (MCA) occlusion followed by 48-h reperfusion. Infarct volume was determined in each group, while changes in gene expression of stress-induced heat shock proteins (Hsp) 27 and 70 were compared using real-time PCR between voluntary and treadmill exercise groups. The level of corticosterone was significantly higher in both stress (Pxa0<xa00.05) and treadmill exercise (Pxa0<xa00.05) groups, but not in the voluntary exercise group. Infarct volume was significantly reduced (Pxa0<xa00.01) following stroke in rats exercised on a treadmill. However, the amelioration of damage was not duplicated in voluntary exercise, even though running distance in the voluntary exercise group was significantly (Pxa0<xa00.01) longer than that of the forced exercise group (4,828 vs. 900xa0m). Furthermore, rats in the electric shock group displayed a significantly increased (Pxa0<xa00.01) infarct volume. Expression of both Hsp 27 and Hsp 70 mRNA was significantly increased (Pxa0<xa00.01) in the treadmill exercise group as compared with that in the voluntary exercise group. These results suggest that exercise with a stressful component, rather than either voluntary exercise or stress alone, is better able to reduce infarct volume. This exercise-induced neuroprotection may be attributable to up-regulation of stress-induced heat shock proteins 27 and 70.

Cerebral metabolism after forced or voluntary physical exercise.

The pathophysiology of stroke, a leading cause of morbidity and mortality, is still in the process of being understood. Pre-ischemic exercise has been known to be beneficial in reducing the severity of stroke-induced brain injury in animal models. Forced exercise with a stressful component, rather than voluntary exercise, was better able to induce neuroprotection. This study further determined the changes in cerebral metabolism resulting from the two methods of exercise (forced versus voluntary). Adult male Sprague-Dawley rats were randomly assigned to 3 groups: the control group (no exercise), the forced treadmill exercise group, and the voluntary running wheel exercise group. In order to measure the extent of cerebral metabolism in animals with different exercise regimens, mRNA levels and protein expression of glucose transporter 1 and glucose transporter 3 (GLUT-1 and GLUT-3), phosphofructokinase (PFK), lactate dehydrogenase (LDH), and adenosine monophosphate kinase (AMPK) were measured utilizing real-time reverse transcription polymerase chain reaction (PCR) analysis as well as Western blot analysis. Phosphorylated AMPK activity was also measured using an ELISA activity kit, and hypoxic inducible factor (HIF)-1α was measured at transcription and translation levels. The data show that the forced exercise group had a significant (p < 0.05) increase in cerebral glycolysis, including expressions of GLUT-1, GLUT-3, PFK, LDH, phosphorylated AMPK activity and HIF-1α, when compared to the voluntary exercise and the control groups. Our results suggest that the effects of different exercise on HIF-1α expression and cerebral glycolysis may provide a possible reason for the discrepancy in neuroprotection, with forced exercise faring better than voluntary exercise through increased cerebral metabolism.

Pre-ischemic exercise reduces matrix metalloproteinase-9 expression and ameliorates blood-brain barrier dysfunction in stroke.

Exercise reduces ischemia and reperfusion (I/R) injury in the rat stroke model. We investigated whether pre-ischemic exercise ameliorates blood-brain barrier (BBB) dysfunction in stroke by reducing matrix metalloproteinase (MMP)-9 expression and strengthening basal lamina. Adult male Sprague-Dawley rats were subjected to a 30 min exercise program on a treadmill 5 days a week for 3 weeks. Stroke was induced by a 2-h middle cerebral artery (MCA) occlusion using an intraluminal filament in the exercised and non-exercised groups. Brain infarction was measured and neurological deficits were scored. BBB dysfunction was determined by examining brain edema and Evans Blue extravasation. Expression of collagen IV, the major component of basal lamina essential for maintenance of the endothelial permeability barrier, was quantitatively detected by Western blot and immunocytochemistry. Ex vivo techniques were used to compare collagen IV-labeled vessels in response to ischemic insult. Temporal relationship of expression of MMP-9 and its endogenous inhibitor, the tissue inhibitors of metalloproteinase-1 (TIMP-1), was determined by real-time PCR for mRNA and Western blot for protein during reperfusion. Brain edema and Evans Blue leakage were both significantly (P<0.01) reduced after stroke in the exercised group, in association with reduced brain infarct volume and neurological deficits. Western blot analysis indicated that exercise enhanced collagen IV expression and reduced the collagen loss after stroke. Immunocytochemistry demonstrated that collagen IV-labeled vessels were significantly (P<0.01) increased in exercised rats. In the ex vivo study, after exercised brains were incubated with ischemic brain tissue, a significantly (P<0.01) higher level of collagen IV-labeled vessels was observed as compared with non-exercised brains following the same treatment. The ex vivo study also revealed a key role of MMP-9 in exercise-strengthened collagen IV expression against I/R injury. TIMP-1 protein levels were significantly (P<0.01) increased by exercise. Our results indicate that pre-ischemic exercise reduces brain injury by improving BBB function and enhancing basal lamina integrity in stroke. This study suggests that the neuroprotective effect of physical exercise is associated with an imbalance of MMP-9 and TIMP-1 expression.

Neuroprotective Effect of Acute Ethanol Administration in a Rat With Transient Cerebral Ischemia

Background and Purpose— Ethanol consumption is inversely associated with the risk of ischemic stroke, suggesting a neuroprotective effect. In a rat model of transient cerebral ischemia, we identified ethanol as a possible treatment for acute ischemic stroke. Methods— Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 hours. Five sets of experiments were conducted: to determine the dose–response effect of ethanol on brain infarction and functional outcome; to determine whether combining ethanol and hypothermia produces synergistic neuroprotection; to determine the therapeutic windows of opportunity for ethanol in stroke; to test whether ethanol promotes intracerebral hemorrhage in a hemorrhagic or ischemic stroke or after administration of thrombolytics; and to test the affect of ethanol on hypoxia-inducible factor-1&agr; protein expression. Results— Ethanol at 1.5 g/kg reduced infarct volume and behavioral dysfunction when administered at 2, 3, or 4 hours after middle cerebral artery occlusion. The protective effect of ethanol was not improved when paired with hypothermia. Ethanol did not promote cerebral hemorrhage in hemorrhagic or ischemic stroke in combination with recombinant tissue-type plasminogen activator or urokinase. Ethanol treatment (1.5 g/kg) increased protein levels of hypoxia-inducible factor-1&agr; at 3 hours postreperfusion. Conclusions— Ethanol exerts a strong neuroprotective effect when administered up to 4 hours after ischemia, increases expression of hypoxia-inducible factor-1&agr;, and does not promote intracerebral hemorrhage when used with thrombolytics. Ethanol is a potential neuroprotectant for acute ischemic stroke.

Pericyte-mediated vasoconstriction underlies TBI-induced hypoperfusion

OBJECTIVESnEndothelin-1 is a 21-amino acid peptide that together with specific receptors, A (ETrA) and B (ETrB) is induced following traumatic brain injury (TBI) and has been closely linked to regulation of cerebral vasospasm, oxidative stress, and hypoperfusion. Specific endothelin receptor antagonists have been shown to ameliorate early evidence of neuronal cell injury, activation of microglial cells, and hypoperfusion following TBI. The exact mechanism involved in TBI-induced hypoperfusion is still unclear; however, it is thought that endothelin-1 engagement of ETrA is primarily responsible for changes in blood flow. In this study we question the role of the microvascular pericyte in endothelin-1-mediated pathophysiology in TBI.nnnMETHODSnPericyte expression of endothelin-1, ETrA, and ETrB was examined in primary culture and in sham and impacted rat brain. Adult male rats were also given intracerebroventricular injections of ETrA (BQ-123) before being subjected to TBI using a closed head acceleration impact model.nnnRESULTSnPrimary pericytes express both endothelin-1 and its receptors ETrA and ETrB. Following TBI, the number of alpha-smooth muscle actin (SMA) positive pericytes located in microvessels is significantly increased by 4u2005hours post-traumatic impact. Increases in pericyte expression of alpha-SMA correlated with evidence of a reduction in both arteriolar and capillary diameter. Capillary endothelin-1, ETrA, and ETrB transcript and protein was also increased. Increased endothelin-1 expression was seen by 2-4 hours post-impact. Upregulation of receptors was observed by 4-8 hours and maximum by 24 hours. ETrA antagonists decreased the number of alpha-SMA(+) pericytes as well as changes in microvascular diameter.nnnCONCLUSIONnThese results suggest that decreased vasoconstriction following TBI may be due to an endothelin-1-induced pericyte-mediated regulation of microvessel blood flow following TBI. Furthermore, results suggest that ETrA antagonists ameliorate trauma induced hypoperfusion, in part, by inhibiting endothelin-1-mediated upregulation of alpha-SMA in pericytes.

Preischemic Induction of TNF-α by Physical Exercise Reduces Blood-Brain Barrier Dysfunction in Stroke

This study explores the neuroprotective action of tumor necrosis factor-α (TNF-α) induced during physical exercise, which, consequently, reduces matrix metalloproteinase-9 (MMP-9) activity and ameliorates blood—brain barrier (BBB) dysfunction in association with extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Adult male Sprague—Dawley rats were subjected to exercise on a treadmill for 3 weeks. A 2-h middle cerebral artery occlusion and reperfusion was administered to exercised and nonexercised animals to induce stroke. Exercised ischemic rats were subjected to TNF-α inhibition and ERK1/2 by TNF-α antibody or UO126. Nissl staining of coronal sections revealed the infarct volume. Evans blue extravasation and water content evaluated BBB function. Western blot was performed to analyze protein expression of TNF-α, ERK1/2, phosphorylated ERK1/2, the basal laminar protein collagen IV, and MMP-9. The activity of MMP-9 was determined by gelatin zymography. Tumor necrosis factor-α expression and ERK1/2 phosphorylation were upregulated during exercise. Infarct volume, brain edema, and Evans blue extravasation all significantly decreased in exercised ischemic rats. Collagen IV production increased in exercised rats and remained high after stroke, whereas MMP-9 protein level and activity decreased. These results were negated and returned toward nonexercised values once TNF-α or ERK1/2 was blocked. We concluded that preischemic, exercise-induced TNF-α markedly decreases BBB dysfunction by using the ERK1/2 pathway.

Peripheral thermal injury causes blood–brain barrier dysfunction and matrix metalloproteinase (MMP) expression in rat

Mortality after serious systemic thermal injury may be linked to significant increases in cerebral vascular permeability and edema due to blood-brain barrier (BBB) breakdown. This BBB disruption is thought to be mediated by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs). The gelatinases, MMP-2 and MMP-9, digest the endothelial basal lamina of the BBB, which is essential for maintaining BBB integrity. The current study investigated whether disruption of microvascular integrity in a rat thermal injury model is associated with gelatinase expression and activity. Seventy-two adult Sprague-Dawley rats were anesthetized and submerged horizontally, in the supine position, in 100 degrees C (37 degrees C for controls) water for 6 s producing a third-degree burn affecting 60-70% of the total body surface area. Brain edema was detected by calculating water content. Real time PCR, Western blot, and zymography were used to quantify MMP mRNA, protein, and enzyme activity levels. Each group was quantified at 3, 7, 24, and 72 h post thermal injury. Brain water content was significantly increased 7 through 72 h after burn. Expression of brain MMP-9 mRNA was significantly increased as early as 3 h after thermal injury compared to controls, remained at 7 h (p<0.01), and returned to control levels by 24 h. MMP-9 protein levels and enzyme activity began to increase at 7 h and reached significant levels between 7 and 24 h after thermal injury. While MMP-9 protein levels continued to increase significantly through 72 h, enzyme activity returned to control level. The increase in MMP-9 expression and activity, associated with increased BBB permeability following thermal injury, indicates that MMP-9 may contribute to observed cerebral edema in peripheral thermal injury.

Role of tumor necrosis factor-α and matrix metalloproteinase-9 in blood-brain barrier disruption after peripheral thermal injury in rats: Laboratory investigation

OBJECTnA relationship has been found between peripheral thermal injury and cerebral complications leading to injury and death. In the present study, the authors examined whether tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-9 (MMP-9) play a causative role in blood-brain barrier (BBB) disruption after peripheral thermal injury.nnnMETHODSnThirty-two male Sprague-Dawley rats were subjected to thermal injury. One hour later, 8 rats were injected with TNF-alpha neutralizing antibody, and 8 were injected with doxycycline, an inhibitor of the MMP family proteins; 16 rats did not receive any treatment. Brain tissue samples obtained 7 hours after injury in the treated animals were examined for BBB function by using fluorescein isothiocyanate-dextran and by assessing parenchymal water content. Protein expression of basement membrane components (collagen IV, laminin, and fibronectin) was quantified on Western blot analysis, and MMP-9 protein expression and enzyme activity were determined using Western blot and gelatin zymography. Thermally injured rats that did not receive treatment were killed at 3, 7, or 24 hours after injury and tested for BBB functioning at each time point. Histological analysis for basement membrane proteins was also conducted in untreated rats killed at 7 hours after injury. Results of testing in injured rats were compared with those obtained in a control group of rats that did not undergo thermal injury.nnnRESULTSnAt 7 hours after thermal injury, a significant increase in the fluorescein isothiocyanate-dextran and water content of the brain was found (p < 0.05), but BBB dysfunction was significantly decreased in the rats that received TNF-alpha antibody or doxycycline (p < 0.05). In addition, the components of the basal lamina were significantly decreased at 7 hours after thermal injury (p < 0.01), and there were significant increases in MMP-9 protein expression and enzyme activity (p < 0.05). The basal lamina damage was reversed by inhibition of TNF-alpha and MMP-9, and the increase in MMP-9 protein was reduced in the presence of doxycycline (p < 0.05). The authors found that MMP-9 enzyme activity was significantly increased after thermal injury (p < 0.01) but decreased in the presence of either TNF-alpha antibody or doxycycline (p < 0.01).nnnCONCLUSIONSnThe dual, inhibitory activity of both TNF-alpha and MMP-9 in brain injury suggests that a TNF-alpha and MMP-9 cascade may play a key role in BBB disruption. These results offer a better understanding of the pathophysiology of burn injuries, which may open new avenues for burn treatment beyond the level of current therapies.

Effect of remote ischemic postconditioning on an intracerebral hemorrhage stroke model in rats

Abstract Background and purpose: While recent studies suggest that remote ischemic postconditioning (RIP) therapy may be of benefit to patients with acute ischemic stroke, RIP’s effects on intracerebral hemorrhage (ICH) still remains unclear. In the present study, the use of RIP in a rat model ICH was investigated to elucidate any potential beneficial or detrimental effects as determined by motor testing, blood brain barrier integrity, and brain water content, as well as aquaporin-4 (AQP-4) and matrix metalloproteinase-9 (MMP-9) expression. Methods: ICH was induced in Sprague–Dawley rats and they were randomized into either a control (nu200a=u200a24) or RIP treatment (nu200a=u200a24) group. RIP was performed by repetitive, brief occlusion and release of the bilateral femoral arteries. Functional outcome in each group was assessed by neurologic deficits on vibrissae-elicited forelimb placing test and a 12-point outcome scale. At 72 hours, brain blood volume, water content, blood–brain barrier (BBB) permeability, and protein expression of AQP-4 and MMP-9 were determined. Results: This collagenase model yielded well-defined striatal hematomas. Vibrissae-elicited forelimb placement was significantly (P<0·01) affected by ICH. However, there was no significant difference between the RIP and control groups at either 24 or 72 hours. A 12-point neurological deficit score also failed to differentiate between the RIP and control. There were no significant differences between the two groups in cerebral blood volumes, brain water content, Evans blue extravasations, and expressions of AQP-4 and MMP-9. Conclusions: Although RIP did not show a beneficial effect in our ICH model, treatment with RIP did not exacerbate ICH.