Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.     

Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat

Background

Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.

Methodology and Principal Findings

Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.

Conclusions

Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.     

Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively. Levels of 8-OHdG and AP sites were markedly elevated 16-72 h following MCAO in the frontal cortex, representing the peri-infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT- and TUNEL-positive cells began to be detectable 4-8 h following MCAO in the caudate-putamen and parietal cortex and reached maximal levels at 72 h. PANT- and TUNEL-positive cells were also detected 16-72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single-strand breaks and DNA fragmentation. In contrast, levels of PANT-positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri-infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.     

Autophagy in myocardium of murine hearts subjected to ischemia followed by reperfusion

Autophagy in myocardium has been thought to be cardioprotective, but its extent after transient or prolonged myocardial ischemia remains unclear. Accordingly, we characterized its magnitude in myocardium of murine hearts subjected to ischemia with or without reperfusion. Ten-week-old transgenic GFP-LC3 mice and C57Bl6 mice were subjected to coronary ligation for 1 or 4 h followed by 24 h of reperfusion (1HTL, 4HTL) or to 24 h of persistent ligation (24HPL). Their hearts were analyzed by fluorescence microscopy, electron microscopy, and by Western blotting. Fluorescent GFP-LC3 dots indicative of autophagy were absent in infarct zones and reduced markedly in the peri-infarct zones compared with dots in sham controls (p ≤ 0.05). The LC3-II/LC3-I ratio indicative of autophagy did not increase in LV homogenates from hearts following ischemia. Phosphorylation of ribosomal protein S6 increased in LV homogenates in hearts from mice subjected to 4HTL and 24HPL (p ≤ 0.05). Virtually no autophagic cells recognizable by electron microscopy were evident in infarct or peri-infarct zones. Autophagy is virtually absent within 24 h in the center of zones of infarction and is decreased significantly in the peri-infarct zones compared with that in normal hearts.     

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.     

Neuroprotective Mechanism of Taurine due to Up-regulating Calpastatin and Down-regulating Calpain and Caspase-3 during Focal Cerebral Ischemia

Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover, taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.     

Role of sodium/hydrogen exchanger isoform 1 in microglial activation and proinflammatory responses in ischemic brains

Our recent study reveals that Na?/H? exchanger isoform 1 (NHE-1) mediates H? extrusion during "respiratory bursting", which is important for microglial activation. In the present study, we further investigated whether NHE-1 plays a role in proinflammatory activation of microglia in vivo using a mouse model of transient focal cerebral ischemia and reperfusion (I/R). Activated microglial cells were identified by their expression of two microglial marker proteins (CD11b and Iba1) as well as by their transformation from a "ramified" to an "amoeboid" morphology. An immediate increase in activated microglial numbers was detected in the ipsilateral ischemic core area of NHE-1?/? brains at 1 hour (h) I/1 h R, which gradually decreased during 6-24 h I/R. This was followed by a sharp rise in microglial activation in the peri-infarct area and an increase in proinflammatory cytokine formation at 3 day after I/R. Interestingly, HOE 642 (a potent NHE-1 inhibitor) -treated or NHE-1 heterozygous (NHE-1?/?) mice exhibited less microglia activation, less NADPH oxidase activation, or a reduced proinflammatory response at 3-7 day after I/R. Blocking NHE-1 activity also significantly decreased microglial phagocytosis in vitro. In contrast, astrogliosis formation in the peri-infarct area was not affected by NHE-1 inhibition. Taken together, our results demonstrate that NHE-1 protein was abundantly expressed in activated microglia and astrocytes. NHE-1 inhibition reduced microglial proinflammatory activation following ischemia.     

SM-31900, a novel NMDA receptor glycine-binding site antagonist,reduces infarct volume induced by permanent middle cerebral artery occlusion in spontaneously hypertensive rats

The purpose of this study was to investigate the effect of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900), an antagonist with high selectivity and affinity for the NMDA receptor glycine-binding site, on the cerebral infarct volume in a permanent middle cerebral artery occlusion (MCAo) model, which was constructed by electrocoagulation of a unilateral middle cerebral artery distal to the olfactory tract using spontaneously hypertensive rats (SHRs). To investigate the dose-response characteristics and the therapeutic time window of SM-31900 in this MCAo model, we conducted three experiments, in which the administration of SM-31900 was started 5min (experiment I), 30min (experiment II), or 60min (experiment III) after MCAo, respectively. In all the studies, SM-31900 was administered by intravenous bolus injection followed by continuous intravenous infusion to obtain a steady-state level of this compound in blood immediately after its administration. The treatment with SM-31900 was continued until 24h after MCAo, at which time the cerebral infarct volume was measured. In experiment I, SM-31900 significantly reduced the infarct volume by 37% at a dosage of 0.38mg/kg bolus followed by 1.5mg/kg/h continuous infusion (0.38mg/kg+1.5mg/kg/h). In experiment II, the neuroprotective effect of SM-31900 was also significant, with a 25% reduction in infarct volume at a dosage of 0.38mg/kg+1.5mg/kg/h, and a 40% reduction at 1.5mg/kg+6.0mg/kg/h. Furthermore, even in experiment III, SM-31900 exerted a significant neuroprotective effect, with a 20% reduction at 1.5mg/kg+6.0mg/kg/h. These studies revealed that SM-31900 can exert a neuroprotective effect when it is administered up to at least 60min after the onset of ischemia in the MCAo model, an animal model of stroke, indicating that SM-31900 is a good candidate for treating acute brain ischemia.     

IKs protects from ventricular arrhythmia during cardiac ischemia and reperfusion in rabbits by preserving the repolarization reserve

Introduction

The function of the repolarization reserve in the prevention of ventricular arrhythmias during cardiac ischemia/reperfusion and the impact of ischemia on slowly activated delayed rectifier potassium current (I_Ks) channel subunit expression are not well understood.

Methods and Results

The responses of monophasic action potential duration (MAPD) prolongation and triangulation were investigated following an L-768,673-induced blockade of I_Ks with or without ischemia/reperfusion in a rabbit model of left circumflex coronary artery occlusion/reperfusion. Ischemia/reperfusion and I_Ks blockade were found to significantly induce MAPD90 prolongation and increase triangulation at the epicardial zone at 45 min, 60 min, and 75 min after reperfusion, accompanied with an increase in premature ventricular beats (PVBs) during the same period. Additionally, I_Ks channel subunit expression was examined following transient ischemia or permanent infarction and changes in monophasic action potential (MAP) waveforms challenged by β-adrenergic stimulation were evaluated using a rabbit model of transient or chronic cardiac ischemia. The epicardial MAP in the peri-infarct zone of hearts subjected to infarction for 2 days exhibited increased triangulation under adrenergic stimulation. KCNQ1 protein, the α subunit of the I_Ks channel, was downregulated in the same group. Both findings were consistent with an increased incidence of PVBs.

Conclusion

Blockade of I_Ks caused MAP triangulation, which precipitated ventricular arrhythmias. Chronic ischemia increased the incidence of ventricular arrhythmias under adrenergic stimulation and was associated with increased MAP triangulation of the peri-infarct zone. Downregulation of KCNQ1 protein may be the underlying cause of these changes.     

Acute connexin43 temporal and spatial expression in response to ischemic stroke

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.     

Docosahexaenoic Acid Signaling Modulates Cell Survival in Experimental Ischemic Stroke Penumbra and Initiates Long-Term Repair in Young and Aged Rats

Background

Docosahexaenoic acid, a major omega-3 essential fatty acid family member, improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia. We hypothesize that DHA elicits neuroprotection by inducing AKT/p70S6K phosphorylation, which in turn leads to cell survival and protects against ischemic stroke in young and aged rats.

Methods and Results

Rats underwent 2 h of middle cerebral artery occlusion (MCAo). DHA, neuroprotectin D1 (NPD1) or vehicle (saline) was administered 3 h after onset of stroke. Neurological function was evaluated on days 1, 2, 3, and 7. DHA treatment improved functional recovery and reduced cortical, subcortical and total infarct volumes 7 days after stroke. DHA also reduced microglia infiltration and increased the number of astrocytes and neurons when compared to vehicle on days 1 and 7. Increases in p473 AKT and p308 AKT phosphorylation/activation were observed in animals treated with DHA 4 h after MCAo. Activation of other members of the AKT signaling pathway were also observed in DHA treated animals including increases in pS6 at 4 h and pGSK at 24 h. DHA or NPD1 remarkably reduced total and cortical infarct in aged rats. Moreover, we show that in young and aged rats DHA treatment after MCAo potentiates NPD1 biosynthesis. The phosphorylation of p308 AKT or pGSK was not different between groups in aged rats. However, pS6 expression was increased with DHA or NPD1 treatment when compared to vehicle.

Conclusions

We suggest that DHA induces cell survival, modulates the neuroinflammatory response and triggers long term restoration of synaptic circuits. Both DHA and NPD1 elicited remarkable protection in aged animals. Accordingly, activation of DHA signaling might provide benefits in the management of ischemic stroke both acutely as well as long term to limit ensuing disabilities.     

Human Placenta-Derived Adherent Cell Treatment of Experimental Stroke Promotes Functional Recovery after Stroke in Young Adult and Older Rats

Background

Human Placenta-Derived Adherent Cells (PDAC®) are a novel mesenchymal-like cell population derived from normal human placental tissue. PDA-001 is a clinical formulation of PDAC® developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in young adult (2–3 month old) and older rats (10–12 months old).

Methods

To evaluate efficacy and determine the optimal number of transplanted cells, young adult Wistar rats were subjected to MCAo and treated 1 day post MCAo with 1×10⁶, 4×10⁶ or 8×10⁶ PDA-001 cells (i.v.), vehicle or cell control. 4×10⁶ or 8×10⁶ PDA-001 cells were also tested in older rats after MCAo. Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. Young adult rats were sacrificed 56 days after MCAo, older rats were sacrificed 29 days after MCAo, and lesion volumes were measured using H&E. Immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed.

Results

In young adult rats, treatment with 4×10⁶ PDA-001 cells significantly improved functional outcome after stroke (p<0.05). In older rats, significant functional improvement was observed with PDA-001 cell therapy in both of the 4×10⁶ and 8×10⁶ treatment groups. Functional benefits in young adult and older rats were associated with significant increases in the number of BrdU immunoreactive endothelial cells, vascular density and perimeter in the ischemic brain, as well as significantly increased synaptophysin expression in the ischemic border zone (p<0.05).

Conclusion

PDA-001 treatment significantly improved functional outcome after stroke in both young adult and older rats. The neurorestorative effects induced by PDA-001 treatment may be related to increased vascular density and synaptic plasticity.     

Therapeutic window of bradykinin B2 receptor inhibition after focal cerebral ischemia in rats

Following cerebral ischemia bradykinin/kinin B(2) receptors mediate inflammatory responses resulting in edema formation and secondary brain damage. However, the therapeutic window for B(2) receptor inhibition determining its potential clinical use has not been investigated so far. The aim of the current study was therefore to investigate the effect of delayed B(2) receptor inhibition on morphological and functional outcome following experimental stroke. Rats were subjected to 90 min of middle cerebral artery occlusion (MCAo) by an intraluminal filament. Animals received 0.9% NaCl or 1.0mg/kg/day Anatibant (LF 16-0687 Ms), a selective bradykinin B(2) receptor antagonist, for 3 days beginning at different time points after MCAo: 1, 2.5, 4.5, or 6.5h (n=10 per group). Neurological recovery was examined daily, infarct volume on day 7 after MCAo. Animal physiology was not influenced by B(2) receptor inhibition. Significant improvement of functional outcome was observed when treatment was delayed up to 4.5h after ischemia (p<0.05 versus vehicle). Inhibition of B(2) receptors during ischemia, i.e. when the inhibitor was given 1h after MCAo, reduced infarct volume in the basal ganglia and in the cortex by 49% (p<0.05) and 26% (p<0.05), respectively. Inhibition of B(2) receptors at later time points (2.5, 4.5, or 6.5 after MCAo) reduced penumbral damage, i.e. cortical infarction, by 19-26% (p<0.05). In conclusion, the current study shows that the therapeutic window of B(2) receptor inhibition extends for up to 6.5h after MCAo. Our data therefore suggest that inhibition of kinin B(2) receptors represents a treatment strategy for ischemic stroke which may warrant clinical validation.     

Focal cerebral ischaemia induces corticotropin releasing factor (CRF) vascular immunoreactivity in rat occluded hemisphere

Corticotropin-releasing factor (CRF) induces the dilatation of cerebral blood vessels and increases cerebral blood flow (CBF). CRF receptor antagonists reduce ischaemic damage in the rat. In the present study, the expression of CRF around cerebral vessels has been investigated in the rat. No CRF immunoreactivity was identified around pial or intracerebral vessels in the absence of cerebral ischaemia. Four hours after middle cerebral artery occlusion (MCAo), intensely CRF-positive blood vessels were evident on the ischaemic cortical surface and in the peri-infarct and infarct zone. Increased CRF immunoreactivity was also detected in swollen axons in subcortical white matter, caudate nucleus and lateral olfactory tract of the ipsilateral hemisphere, consistent with the failure of axonal transport. These data provide morphologic support for a role of CRF in the pathophysiology of cerebral ischaemia.     

Complement insufficiency limits efficacy in a xenograft model of hyperacute rejection for cancer therapy

Hyperacute rejection (HAR) is a rapid immunological response to an organ xenotransplant caused by recognition of endothelial galactose(α1,3)galactose (αGal) epitopes and complement-mediated cell lysis by host anti-αGal antibody (‘natural antibody’). The αGal epitope is synthesised by a galactosyl transferase ((α1,3)GT) which humans lack. Because human cells transduced with (α1,3)GT are sensitised to natural antibody/complement-mediated lysis in human serum, delivery of (α1,3)GT to tumour vasculature in patients is a potential therapeutic strategy, by mimicking the pathophysiology of organ rejection. We therefore sought to develop an animal model of HAR for cancer therapy. Nude/(α1,3)GT knock-out mice allowed the growth of human tumour xenografts and the use of ecotropic retrovirus producer cells to generate expression of αGal on tumour vasculature. Lysis of αGal-positive murine endothelial cells with rabbit complement in conjunction with murine anti-αGal antibody in vitro was used to define the conditions necessary for HAR. However, tumour growth retardation and destruction of αGal-positive tumour endothelium were minimal after their administration, despite sera retaining post hoc cytolytic activity with fresh complement. The major limitation of this experimental system, of relevance to other therapeutic approaches, results from the use of a xenograft, in which additional xenoreactivities lead to complement insufficiency. Development of a tractable preclinical model in which to evaluate HAR for cancer therapy requires a syngeneic experimental system.     

Propofol Protects Against Focal Cerebral Ischemia via Inhibition of Microglia-Mediated Proinflammatory Cytokines in a Rat Model of Experimental Stroke

Ischemic stroke induces microglial activation and release of proinflammatory cytokines, contributing to the expansion of brain injury and poor clinical outcome. Propofol has been shown to ameliorate neuronal injury in a number of experimental studies, but the precise mechanisms involved in its neuroprotective effects remain unclear. We tested the hypothesis that propofol confers neuroprotection against focal ischemia by inhibiting microglia-mediated inflammatory response in a rat model of ischemic stroke. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. Propofol (50 mg/kg/h) or vehicle was infused intravenously at the onset of reperfusion for 30 minutes. In vehicle-treated rats, MCAO resulted in significant cerebral infarction, higher neurological deficit scores and decreased time on the rotarod compared with sham-operated rats. Propofol treatment reduced infarct volume and improved the neurological functions. In addition, molecular studies demonstrated that mRNA expression of microglial marker Cd68 and Emr1 was significantly increased, and mRNA and protein expressions of proinflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 were augmented in the peri-infarct cortical regions of vehicle-treated rats 24 h after MCAO. Immunohistochemical study revealed that number of total microglia and proportion of activated microglia in the peri-infarct cortical regions were markedly elevated. All of these findings were ameliorated in propofol-treated rats. Furthermore, vehicle-treated rats had higher plasma levels of interleukin-6 and C-reactive protein 24 h after MCAO, which were decreased after treatment with propofol. These results suggest that propofol protects against focal cerebral ischemia via inhibition of microglia-mediated proinflammatory cytokines. Propofol may be a promising therapeutic agent for the treatment of ischemic stroke and other neurodegenerative diseases associated with microglial activation.     

Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice

Heme oxygenase-1 (HO-1, HSP32) is an early gene that is responsive to an array of pathological conditions including, but not limited to, hypoxia and cerebral ischemia. HO-1 cleaves the heme molecule and produces carbon monoxide (CO) and biliverdin (an antioxidant) and is essential for iron homeostasis. The purpose of this study was to investigate, using transgenic (Tg) mice, whether overexpression of HO-1 in the brain augments or attenuates cellular injury caused by ischemic stroke. Homozygous HO-1 Tg mice that overexpress HO-1 under the control of the neuron-specific enolase promoter (characterized previously) were used. Under halothane anesthesia and normothermic conditions, wild-type nontransgenic (nTg; n = 22) and HO-1 Tg (n = 24) mice were subjected to middle cerebral artery occlusion (MCAo). Six hours after induction of ischemia, Tg and nTg mice developed infarcts that were 39 +/- 6 and 63 +/- 9 mm3, respectively (p < 0.01). No significant difference between the two strains was observed in the values of brain edema (11.3 +/- 4% in Tg vs. 14.6 +/- 5% in nTg; p < 0.1). At 24 h after MCAo, Tg mice exhibited significant neuroprotection as determined by the stroke volumes (41 +/- 2 mm3 in Tg vs. 74 +/- 5 mm3 in nTg; p < 0.01) and values of ischemic cerebral edema (21 +/- 6% in Tg vs. 35 +/- 11% in nTg; p < 0.01). Data suggest that neuroprotection in Tg mice was, at least in part, related to the following findings: (a) constitutively up-regulated cyclic GMP and bcl-2 levels in neurons; (b) inhibition of nuclear localization of p53 protein; and (c) antioxidant action of HO-1, as detected by postischemic neuronal expression of ferritin, and decreases in iron staining and tissue lipid peroxidation. We suggest that pharmacological stimulation of HO-1 activity may constitute a novel therapeutic approach in the amelioration of ischemic injury during the acute period of stroke.     

Extract EGb 761 Pretreatment Limits Ubiquitin Positive Aggregates in Rabbit Spinal Cord Neurons after Ischemia-Reperfusion

1. Ubiquitin immunohistochemistry was used for investigation of time dependent changes of ubiquitin in the nerve cells reacting to ischemic/reperfusion damage. In the rabbit spinal cord ischemia model a period of 30 min ischemia followed by 24 and 72 h of reperfusion caused neuronal degeneration selectively in the ventral horn motor neurons as well as interneurons of the intermediate zone.2. Ubiquitin aggregates were accumulated in the neurons of lamina IX and the neurons of intermediate zone destined to die 72 h after 30 min of the spinal cord ischemia.3. The activation of ubiquitin hydrolytic system is related to a defective homeostasis and could trigger different degenerative processes. Having in mind this, we used EGb 761 to rescue the motor neurons and interneurons against ischemia/reperfusion damage. Our results show that after 30 min of ischemia and 24 or 72 h of reperfusion with EGb 761 pre-treatment for 7 days the vulnerable neurons in the intermediate zone and lamina IX exhibit marked elevation of ubiquitin–positive granules in the cytoplasm, dendrites and nuclei. Abnormal protein aggregates have not been observed in these cells.4. The rabbits were completely paraplegic after 30 min of ischemia and 24 or 72 h of reperfusion. However, after 7 days EGb 761 pre-treatment, 30 min of ischemia and 24 or 72 h of reperfusion the animals did not show paraplegia.5. Evaluated ubiquitin–positive neurons of the L₅–L₆ segments showed significant decrease in number and significant increase of density after 30 min of ischemia followed by 24 h and mainly 72 h of reperfusion. Ubiquitin immunohistochemistry confirmed the protective effect of EGb 761 against ischemia/reperfusion damage in the rabbit spinal cord.     

Possible Involvement of PTEN Signaling Pathway in the Anti-apoptotic Effect of Electroacupuncture Following Ischemic Stroke in Rats

As a traditional therapeutic method, electroacupuncture (EA) has been adopted as an alternative therapy for stroke recovery. Here, we aimed to evaluate whether EA therapy at points of Quchi (LI11) and Zusanli (ST36) alleviated neuronal apoptosis by PTEN signaling pathway after ischemic stroke. A total of 72 male Sprague–Dawley rats were randomized into three groups, including sham group, MCAO group, and EA group. EA was initiated after 24 h of reperfusion for 3 consecutive days. At 72 h following ischemia/reperfusion, neurological deficits, infarct volumes, and TUNEL staining were evaluated and the PTEN pathway-related proteins together with apoptosis-related proteins were detected. The results indicated that EA treatment significantly decreased cerebral infarct volume, neurological deficits and alleviated proportion of apoptotic cells in cerebral ischemic rats. Furthermore, EA significantly up-regulated the phosphorylation levels of PDK1, Akt(Thr308), GSK-3β, and down-regulated the phosphorylation levels of PTEN, Akt(Ser473) in the peri-infarct cortex. EA treatment significantly reduced the up-regulation of caspase-3, cleaved-caspase-3, Bim, and reversed the reduction of Bcl-2 induced by the ischemic stroke. These findings suggest that EA treatment at points of Quchi (LI11)- and Zusanli (ST36)-induced neuroprotection might involve inhibition of apoptosis via PTEN pathway.