Paul Fu

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 (n = 24) or RIP treatment (n = 24) 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.

Synergetic Neuroprotection of Normobaric Oxygenation and Ethanol in Ischemic Stroke Through Improved Oxidative Mechanism

Background and Purpose— Normobaric oxygenation (NBO) and ethanol both provide neuroprotection in stroke. We evaluated the enhanced neuroprotective effect of combining these 2 treatments in a rat stroke model. Methods— Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 hours. Reperfusion was then established and followed by treatment with either (1) an intraperitoneal injection of ethanol (1.0 g/kg), (2) NBO treatment (2-hour duration), or (3) NBO plus ethanol. The extent of brain injury was determined by infarct volume and motor performance. Oxidative metabolism was determined by ADP/ATP ratios, reactive oxygen species levels, nicotinamide adenine dinucleotide phosphate oxidase activity, and pyruvate dehydrogenase activity. Protein expression of major nicotinamide adenine dinucleotide phosphate oxidase subunits (p47phox, gp91phox, and p67phox) and the enzyme pyruvate dehydrogenase was evaluated through Western immunoblotting. Results— NBO and ethanol monotherapies each demonstrated reductions as compared to stroke without treatment in infarct volume (36.7% and 37.9% vs 48.4%) and neurological deficits (score of 6.4 and 6.5 vs 8.4); however, the greatest neuroprotection (18.8% of infarct volume and 4.4 neurological deficit) was found in animals treated with combination therapy. This neuroprotection was associated with the largest reductions in ADP/ATP ratios, reactive oxygen species levels, and nicotinamide adenine dinucleotide phosphate oxidase activity, and the largest increase in pyruvate dehydrogenase activity. Conclusions— Combination therapy with NBO and ethanol enhances the neuroprotective effect produced by each therapy alone. The mechanism behind this synergistic action is related to changes in cellular metabolism after ischemia reperfusion. NBO plus ethanol is attractive for clinical study because of its ease of use, tolerability, and tremendous neuroprotective potential in stroke.

Neuroprotection by local intra-arterial infusion of erythropoietin after focal cerebral ischemia in rats

Abstract Objectives: The neuroprotective effect of erythropoietin has been demonstrated by ischemia and reperfusion models in adult and neonatal rodents. However, administration of high-dose erythropoietin has potential complications. The goal of this study was to determine whether local infusion of low dose erythropoietin offers neuroprotective effects after ischemia and reperfusion injury. Methods: Adult male Sprague-Dawley rats subject to middle cerebral artery occlusion were randomly divided into three groups: (1) sham group: the rats received the same procedure as the other two groups except that no suture was inserted; (2) vehicle group: intra-artery local infusion of saline was administered via middle cerebral artery after reperfusion; and (3) treatment group: 50 U/kg intra-artery local infusion of erythropoietin was administered via middle cerebral artery after reperfusion. Neurological deficit scores and infarct volume (determined by hematoxylin-eosin staining) were evaluated 48 hours after reperfusion. Apoptosis was measured through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of vascular endothelial growth factor and phosphorylated extracellular signal-regulated kinase were investigated by immunohistochemistry method. Results: The results show that intra-artery local infusion of erythropoietin, via the middle cerebral artery, significantly reduced neurological deficit scores, foot fault number, and the infarct volume at 48 hours after reperfusion. Significant reductions were also found in the number of positive cells stained by TUNEL assay within the ischemic core and penumbra. Furthermore, local infusion of erythropoietin increased the expression of phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Discussion: Local infusion of low-dose erythropoietin via the middle cerebral artery is shown to be neuroprotective against cerebral ischemia and reperfusion injury. The mechanism of neuroprotection may be associated with the increased expression of phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor.

Acute administration of ethanol reduces apoptosis following ischemic stroke in rats

In recent studies, acute ethanol administration appears to play a neuroprotective role during ischemic stroke. We sought to confirm these findings by identifying if ethanol-derived neuroprotection is associated with a reduction in apoptosis. Ethanol at 0.5 and 1.5 g/kg doses was given by intraperitoneal injections to Sprague-Dawley rats after 2h of middle cerebral artery (MCA) occlusion, followed by reperfusion. We quantified apoptotic cell death in each of the treatment groups with ELISA, and measured pro- and anti-apoptotic protein expression with Western blot analysis. Cell death was significantly increased in rats after ischemia and was subsequently significantly reduced by the administration of 1.5 g/kg of ethanol. We found that the 1.5 g/kg dose promoted the expression of pro-survival factors and decreased the expression of apoptotic proteins at 3h after reperfusion. This effect was maintained at 24h for Caspase-3 and apoptosis-inducing factor (AIF), although not for Bcl-2, Bcl-xL, and Bcl-2-associated X (Bax). Administration of 0.5 g/kg of ethanol was not as effective in regulating protein expression as the 1.5 g/kg dose. Our study suggests that administration of ethanol at a dose of 1.5 g/kg after stroke - which provides rat blood alcohol levels equivalent to the legal driving limit - produces a differential protein profile, with increased expression of anti-apoptotic proteins and decrease in pro-apoptotic factors. This results in a significant reduction of neuronal apoptosis and is neuroprotective in ischemia-reperfusion injury.

Clinical outcomes of fast MRI-based trombolysis in wake-up strokes compared to superacute ischemic strokes within 12 hours

Abstract Background: It is unknown whether thrombolysis is beneficial in patients with Wake-Up Ischemic Strokes (WUIS). This study compares the clinical outcomes of MRI-based intravenous thrombolysis in patients with hyperacute ischemic stroke presenting within 12 hours of symptom onset against WUIS patients receiving the same therapy. Methods: Patients presenting within 12 hours of acute stroke symptom onset and those with WUIS confirmed by CT, and without intracranial hemorrhage, were encouraged to perform an emergent brain MRI scan to confirm the diagnosis of hyperacute ischemic stroke [hyper-intense in diffusion-weighted imaging (DWI) and no hypo-intense change in T2-weighted imaging (T2WI) or FLAIR]. These patients then received intravenous thrombolytic therapy with tissue-type plasminogen activator (rt-PA). All patients were divided into either stroke presenting within 12 hours or WUIS. The clinical outcomes were assessed by the modified Rankin Scale (mRS) and the Barthal Index (BI) at baseline and at 90 days after the thrombolysis therapy. Results: A total of 427 patients presenting with stroke like symptoms were given a MRI scan. Of these, 240 patients had confirmed diagnosis of hyperacute ischemic stroke (WUIS, n = 68, 68/116 = 58·62% versus within 12 hour, n = 172, 172/311 = 55·3%). Altogether, 186 patients (138 in within 12 hours group, and 48 in WUIS group) received intravenous thrombolytic therapy with rt-PA. No significant differences were found in clinical outcomes between the two groups at the baseline and at 90 days after the thrombolysis therapy. Also, no difference was found in the incidence rate of secondary hemorrhage (including both of asymptomatic and symptomatic) and mortality rate between the two groups. Conclusion: Our study suggested that MRI-based intravenous thrombolysis is safe and effective in both of patients’ hyperacute stroke within 12 hours of symptom onset and WUIS.

Effects of comprehensive education protocol in decreasing pre-hospital stroke delay among Chinese urban community population

Abstract Background and purpose: Studies have shown that awareness of early stroke symptoms and the use of ambulances are two important factors in decreasing pre-hospital stroke delay. The purpose of this study is to evaluate a comprehensive educational stroke protocol in improving stroke response times. Method: Two urban communities in Beijing (population ≈50 000), matched in economic status and geography, were enrolled in this study. A comprehensive educational protocol, which included public lectures and distribution of instructive material for the community and its medical staff, was implemented from August 2008 to December 2010. Surveillance of new onset stroke in both communities was carried out during the same period. Pre-hospital delay time and percentage of patients using emergency medical services (EMS) were compared between the two communities. Results: After comprehensive educational protocol, we found that: (i) pre-hospital delay (time from stroke symptom onset to hospital arrival) decreased from 180 to 79 minutes, (ii) the proportion of patients arriving within three hours of stroke onset increased from 55·8% to 80·4%, (iii) pre-hospital delay of stroke patients with symptoms of paralysis, numbness, and speech impediments was decreased, and (iv) the proportion of stroke patients calling for EMS increased from 50·4% to 60·7%. Conclusion: The comprehensive educational stroke protocol was significantly effective in decreasing pre-hospital stroke delay.

Changes in spinal cord met-enkephalin levels and mechanical threshold values of pain after pulsed radio frequency in a spared nerve injury rat model

Abstract Objectives: The present study investigated changes in the met-enkephalin (M-ENK) levels in the spinal cord. We also determined the mechanical threshold value of pain in spared nerve injury (SNI) rats after applying pulsed radiofrequency (PRF) on L5 dorsal root ganglion (DRG). Methods: Sixty-four rats were divided into four groups: the normal group (n = 16), the control group (n = 16), the sham intervention group (n = 16), and the PRF group (n = 16). With exception for the normal group, the other three groups were treated with an established SNI model. After 7 days, PRF or sham intervention was applied on the right L5 DRG. The M-ENK levels in the spinal cord were examined by radioimmunoassay 24 hours after applying PRF or sham operation. Mechanical threshold values of pain were also tested 1 day before SNI procedure, 1 and 2 days after SNI procedure, and 2 and 24 hours after applying PRF or sham operation. Results: Twenty-four hours after treatment with PRF, M-ENK levels in spinal cord increased significantly, while no changes were detected in the sham intervention group. Hyperalgesia was found in rats 1–2 days after SNI procedure and was improved by PRF. This was demonstrated by an increased mechanical threshold of pain 2 and 24 hours after the PRF. The sham intervention group showed no change in the mechanical threshold of pain. Conclusion: This study demonstrates that applying PRF on the DRG can improve hyperalgesia and increase M-ENK levels in the spinal cord of SNI rats within 24 hours. These findings indicate that the endogenous M-ENK in the spinal cord is involved in the mechanism of PRF on the therapy of neuropathic pain.

At low doses ethanol maintains blood–brain barrier (BBB) integrity after hypoxia and reoxygenation: a brain slice study

Abstract Post-ischemia ethanol (EtOH) treatments have been shown to exhibit neuroprotective effects in stroke. However, the mechanisms underlying these effects and those on blood–brain barrier (BBB) integrity have yet to be elucidated. In the present study, we determined whether administering differing concentrations of EtOH alter the expressions of BBB integral proteins, including aquaporins-4 and -9 (AQP-4, AQP-9), matrix metallopeptidases-2 and -9 (MMP-2, MMP-9), zonula occludens-1 (ZO-1), and basal lamina (laminin). We employed an organotypic brain slice culture model that utilizes oxygen–glucose deprivation followed by reoxygenation (OGD/R). Brain slices were obtained from 10-day-old Sprague–Dawley rats and divided into the following five groups (n  =  8 subjects per group): (1) control, (2) hypoxia (OGD/R), no EtOH, (3) OGD/R and 10 mM EtOH, (4) OGD/R and 30 mM EtOH, and (5) OGD/R and 90 mM EtOH. To assess BBB integrity, levels of AQPs, MMPs, ZO-1, and laminin were determined by Western blot. Compared to control, OGD/R without EtOH significantly increased AQP-4, AQP-9, MMP-2, and MMP-9 levels, while decreasing ZO-1 and laminin levels. All EtOH concentration treatments (groups 3 through 5) significantly reduced the expressions of AQP-4, AQP-9, MMP-2, and MMP-9, compared to the OGD/R, non-alcohol treated slices. Furthermore, compared to the OGD/R without EtOH group, the 30 mM EtOH treatment significantly increased ZO-1 and laminin levels. In contrast, the 90 mM EtOH level neither enhanced the reduction in AQP and MMP levels nor increased ZO-1 or basal lamina expressions observed in the 30 mM treatment. In conclusion, at an optimal dose of 30 mM, EtOH improves the expressions of MMP-2, MMP-9, AQP-4, AQP-9, ZO-1, and basal laminin, previously altered by OGD/R. These effects may indicate a beneficial effect of EtOH on BBB integrity after stroke.

Leptin’s effect on accelerated fracture healing after traumatic brain injury

Abstract Objective: To investigate mechanisms behind the faster rehabilitation of limb fractures when associated with traumatic brain injury (TBI). Methods: New Zealand rabbits were divided into TBI group and sham-operation group for four studies as follows: (1) blood and cerebrospinal fluid (CSF) were drawn on days 1, 3, and 7 to demonstrate changes in serum leptin, growth hormone (GH), insulin-like growth factor 1 (IGF-1), and CSF leptin; (2) bone defection was created by drilling in the tibial bone and either leptin or normal saline was injected into rabbit’s cerebellomedullary cistern. X-ray was taken at 1 days, 2 weeks, and 5 weeks and evaluated by criteria to determine rate of bone healing; (3) FITC-labeled rabbit leptin was injected into TBI and sham-operation groups, and frozen sections of rabbit brain were observed to identify differences in central nervous system (CNS) leptin by fluorescence; (4) polymerase chain reaction (PCR) was used to evaluate the expression of leptin production by brain tissue. Results: Serum and CSF leptin, GH, and IGF-1 concentrations were found to be higher in the TBI group than the sham-operation group at days 1, 3, and 7 (P<0·05). CSF leptin of the TBI group was positively correlated with serum leptin on day 1 (P<0·05), and positively correlated with GH and IGF-1 on days 3 and 7 (P<0·05). X-ray criteria demonstrated that leptin administration caused significantly faster healing calluses at 3 and 5 weeks as compared to control animals (P<0·05). FITC-labeled leptin study demonstrated that TBI animals had stronger expression of leptin in the brain than sham-operated animals. However, PCR of brain tissue leptin showed no significant differences between TBI and sham-operated animals in the expression of leptin. Conclusions: Our study suggests that increased CSF leptin, likely from blood–brain barrier breakdown, combined with elevated serum GH and IGF-1 after TBI, leads to accelerated fracture healing.

Neuroprotection & Mechanism of Ethanol in Stroke and Traumatic Brain Injury Therapy: New Prospects for an Ancient Drug

Effective efforts to screen for agents that protect against the devastating effects of stroke have not produced viable results thus far. As a result this article reviews the possible role of ethanol as a neuroprotective agent in stroke and traumatic brain injury (TBI). Previous studies have associated ethanol consumption with a decreased risk of ischemic stroke, suggesting a neuroprotective mechanism. The translation of this clinical knowledge into basic science research with the goal of new therapy for acute stroke patients remains in its initial stages. In a recent study involving rats, we have shown that ethanol administration, in the correct dose after stroke onset, protects against ischemia-induced brain injury. The purpose of this paper is to discuss ethanols neuroprotective properties in stroke when consumed as a preconditioning agent, in TBI with a positive blood alcohol content, and finally in stroke treatment, with the goal of using post-ischemia ethanol (PIE) therapy to ameliorate brain damage in the future.