Intracerebroventricular BDNF infusion may reduce cerebral ischemia/reperfusion injury by promoting autophagy and suppressing apoptosis.

Here, it was aimed to investigate the effects of intracerebroventricular (ICV) Brain Derived Neurotrophic Factor (BDNF) infusion for 7 days following cerebral ischemia (CI) on autophagy in neurons in the penumbra. Focal CI was created by the occlusion of the right middle cerebral artery. A total of 60 rats were used and divided into 4 groups as Control, Sham CI, CI and CI + BDNF. During the 7-day reperfusion period, aCSF (vehicle) was infused to Sham CI and CI groups, and BDNF infusion was administered to the CI + BDNF group via an osmotic minipump. By the end of the 7th day of reperfusion, Beclin-1, LC3, p62 and cleaved caspase-3 protein levels in the penumbra area were evaluated using Western blot and immunofluorescence. BDNF treatment for 7 days reduced the infarct area after CI, induced the autophagic proteins Beclin-1, LC3 and p62 and suppressed the apoptotic protein cleaved caspase-3. Furthermore, rotarod and adhesive removal test times of BDNF treatment started to improve from the 4th day, and the neurological deficit score from the 5th day. ICV BDNF treatment following CI reduced the infarct area by inducing autophagic proteins Beclin-1, LC3 and p62 and inhibiting the apoptotic caspase-3 protein while its beneficial effects were apparent in neurological tests from the 4th day.

Melatonin Attenuates Cerebral Ischemia/Reperfusion Injury through Inducing Autophagy.

INTRODUCTION: The aim of this study was to investigate how melatonin administration for 3 days or 7 days following cerebral ischemia (CI) injury would affect autophagy and, therefore, survival in neurons of the penumbra region. Moreover, it was also aimed at determining how this melatonin treatment would affect the neurological deficit score and rotarod and adhesive removal test durations. METHODS: Focal CI (90 min) was achieved in a total of 105 rats utilizing a middle cerebral artery occlusion model. After the start of reperfusion, the groups were treated with melatonin (10 mg/kg/day) for 3 days or 7 days. In all groups, neurological deficit scoring, rotarod, and adhesive removal tests were executed during reperfusion. Infarct areas were determined by TTC (2,3,5-triphenyltetrazolium chloride) staining at the end of the 3rd and 7th days of reperfusion. Beclin-1, LC3, p62, and caspase-3 protein levels were assessed using Western blot and immunofluorescence methods in the brain tissues. Moreover, penumbra areas were evaluated by transmission electron microscopy (TEM). RESULTS: Following CI, it was observed that melatonin treatment improved the rotarod and adhesive removal test durations from day 5 and reduced the infarct area after CI. It also induced autophagic proteins Beclin-1, LC3, and p62 and suppressed the apoptotic protein cleaved caspase-3. According to TEM findings, melatonin treatment partially reduced the damage in neurons after CI. CONCLUSION: Melatonin treatment following CI reduced the infarct area and induced the autophagic proteins Beclin-1, LC3, and p62 by inhibiting the apoptotic caspase-3 protein. The functional reflection of melatonin treatment on neurological test scores was became significant from the 5th day onward.

Minimally Invasive Surgery for Evacuating the Intracerebral Hematoma in Early Stages Decreased Secondary Damages to the Internal Capsule in Dog Model of ICH Observed by Diffusion Tensor Imaging.

BACKGROUND: Diffusion tensor imaging was used to observe the effects of performing early minimally invasive surgery (MIS) on internal capsule in dog model of intracerebral hemorrhage (ICH). METHODS: Twenty-five male dogs were selected to prepare an ICH model, and then they were randomly distributed into a model control (MC) group (5 dogs) or an MIS group (20 dogs). In the MIS group, the intracerebral hematoma was evacuated by stereotactic minimally invasive procedures over 6 hours (5 dogs), 12 hours (5 dogs), 18 hours (5 dogs), or 24 hours (5 dogs) after successful induction of ICH. The same procedure was performed in the MC group but without evacuating the hematoma. All the animals were sacrificed within 2 weeks after the hematoma was surgically evacuated. The neurologic deficit score and diffusion tensor imaging (DTI) were observed before and after the MIS. The perihematomal blood-brain barrier (BBB) permeability and the brain water content (BWC) were measured 2 weeks after the hematoma was surgically evacuated. RESULTS: The DTI demonstrated that integrity of the internal capsule restored largely after surgery and the fractional anisotropy (FA) values of the internal capsule on the hematoma side increased significantly as compared with those in the MC group or those before surgery in the same group. The postoperative ratios of FA values of each MIS subgroup increased compared with the MC group and those before surgery in the same subgroup before operation. The neurologic deficit score, the perihematomal BBB permeability, and the BWC of each MIS subgroup decreased significantly compared with those of the MC group. The 6-12-hour group displayed a more favorable result. CONCLUSIONS: Performing the MIS in the early stage (6-12 hours) after ICH could decrease the secondary damages to the internal capsule so as to promote the recovery of motor function. The optimal time window for MIS should be within 6-12 hours after onset of ICH.

Real-Time Objective Evaluation of the Ischemic Stroke through pH-Responsive Fluorescence Imaging.

A rapid and comprehensive assessment of ischemic stroke (IS) is critical for clinicians to take the most appropriate treatment. Currently, IS assessment is mainly carried out by computed tomography and magnetic resonance imaging in combination with observing the clinical symptoms and inquiring about contraindications. However, they cannot diagnose pathological conditions and judge the microenvironment in real-time. Near-infrared fluorescence imaging has advantages for IS imaging, such as high sensitivity, high spatiotemporal resolution, and straightforward real-time operation. Herein, a pH-responsive fluorescent liposomal probe (BOD@Lip) is prepared for in vivo real-time visualization of the degree of IS based on the different acid microenvironments in the progression of the disease. The fluorescence imaging with BOD@Lip shows the degree of IS, and the correlation between fluorescence signals and the neurological deficit scores is established for the first time. This work provides a new method to objectively evaluate the degree of IS through a visualized route and a new insight into the relationship between the acidic microenvironment and the progression of IS.

The Effects of Vagus Nerve Stimulation on Animal Models of Stroke-Induced Injury: A Systematic Review.

Ischemic stroke is one of the leading causes of death worldwide, and poses a great burden to society and the healthcare system. There have been many recent advances in the treatment of ischemic stroke, which usually results from the interruption of blood flow to a particular part of the brain. Current treatments for ischemic stroke mainly focus on revascularization or reperfusion of cerebral blood flow to the infarcted tissue. Nevertheless, reperfusion injury may exacerbate ischemic injury in patients with stroke. In recent decades, vagus nerve stimulation (VNS) has emerged as an optimistic therapeutic intervention. Accumulating evidence has demonstrated that VNS is a promising treatment for ischemic stroke in various rat models through improved neural function, cognition, and neuronal deficit scores. We thoroughly examined previous evidence from stroke-induced animal studies using VNS as an intervention until June 2022. We concluded that VNS yields stroke treatment potential by improving neurological deficit score, infarct volume, forelimb strength, inflammation, apoptosis, and angiogenesis. This review also discusses potential molecular mechanisms underlying VNS-mediated neuroprotection. This review could help researchers conduct additional translational research on patients with stroke.

An Ultrasound Model to Predict the Short-Term Effects of Endovascular Stent Placement in the Treatment of Carotid Artery Stenosis.

Purpose: The present study aimed to explore the predictive ability of an ultrasound linear regression equation in patients undergoing endovascular stent placement (ESP) to treat carotid artery stenosis-induced ischemic stroke. Methods: Pearson's correlation coefficient of actual improvement rate (IR) and 10 preoperative ultrasound indices in the carotid arteries of 64 patients who underwent ESP were retrospectively analyzed. A predictive ultrasound model for the fitted IR after ESP was established. Results: Of the 10 preoperative ultrasound indices, peak systolic velocity (PSV) at stenosis was strongly correlated with postoperative actual IR (r = 0.622; P < 0.01). The unstable plaque index (UPI; r = 0.447), peak eccentricity ratio (r = 0.431), and plaque stiffness index (ß; r = 0.512) moderately correlated with actual IR (P < 0.01). Furthermore, the resistance index (r = 0.325) and the dilation coefficient (r = 0.311) weakly correlated with actual IR (P < 0.05). There was no significant correlation between actual IR and the number of unstable plaques, area narrowing, pulsatility index, and compliance coefficient. In combination, morphological, hemodynamic, and physiological ultrasound indices can predict 62.39% of neurological deficits after ESP: fitted IR = 0.9816 - 0.1293ß + 0.0504UPI - 0.1137PSV. Conclusion: Certain carotid ultrasound indices correlate with ESP outcomes. The multi-index predictive model can be used to evaluate the effects of ESP before surgery.

Bone marrow-derived mesenchymal stem cell transplantation attenuates overexpression of inflammatory mediators in rat brain after cardiopulmonary resuscitation.

Emerging evidence suggests that bone marrow-derived mesenchymal stem cell transplantation improves neurological function after cardiac arrest and cardiopulmonary resuscitation; however, the precise mechanisms remain unclear. This study aimed to investigate the effect of bone marrow-derived mesenchymal stem cell treatment on expression profiles of multiple cytokines in the brain after cardiac arrest and cardiopulmonary resuscitation. Cardiac arrest was induced in rats by asphyxia and cardiopulmonary resuscitation was initiated 6 minutes after cardiac arrest. One hour after successful cardiopulmonary resuscitation, rats were injected with either phosphate-buffered saline (control) or 1 × 106 bone marrow-derived mesenchymal stem cells via the tail vein. Serum S100B levels were measured by enzyme-linked immunosorbent assay and neurological deficit scores were evaluated to assess brain damage at 3 days after cardiopulmonary resuscitation. Serum S100B levels were remarkably decreased and neurological deficit scores were obviously improved in the mesenchymal stem cell group compared with the phosphate-buffered saline group. Brains were isolated from the rats and expression levels of 90 proteins were determined using a RayBio Rat Antibody Array, to investigate the cytokine profiles. Brain levels of the inflammatory mediators tumor necrosis factor-α, interferon-γ, macrophage inflammatory protein-1α, macrophage inflammatory protein-2, macrophage inflammatory protein-3α, macrophage-derived chemokine, and matrix metalloproteinase-2 were decreased ≥ 1.5-fold, while levels of the anti-inflammatory factor interleukin-10 were increased ≥ 1.5-fold in the mesenchymal stem cell group compared with the control group. Donor mesenchymal stem cells were detected by immunofluorescence to determine their distribution in the damaged brain, and were primarily observed in the cerebral cortex. These results indicate that bone marrow-derived mesenchymal stem cell transplantation attenuates brain damage induced by cardiac arrest and cardiopulmonary resuscitation, possibly via regulation of inflammatory mediators. This experimental protocol was approved by the Institutional Animal Care and Use Committee of Fujian Medical University, China in January 2016 (approval No. 2016079).

Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice.

Cerebral ischemia injury is a clinical, frequently occurring disease, which causes a heavy burden on society and families. It has been demonstrated that cytosolic phospholipase A2α (cPLA2α) is significant in neurological injury caused by ischemic brain injury, and inhibition of cPLA2α may reduce stroke injury. In the present study, the role of cPLA2α was investigated in a mouse model of middle cerebral artery occlusion and/or reperfusion (MCAO/R) using an effective cPLA2α inhibitor and adenoviruses­mediated RNA interference. The most effective recombinant adenovirus encoding cPLA2α small interfering RNA (pAd­siRNA­cPLA2α) was constructed and selected. MCAO/R surgery is used to construct the model of focal ischemic brain damage in mice. Adenoviruses­mediated RNA interference targeting cPLA2α was administered by stereotactic surgery 2 h before the MCAO/R. The expression/activity of cPLA2α and cPLA2α­derived injurious lipid mediators was assessed. pAd­siRNA­cPLA2α­treated animals (RNA interference; RNAi group) were compared with pAd-siRNA-control-treated animals (negative group) with regard to neurological deficit, motor function, pathological changes, apoptosis, and infarct volume. The RNAi group animals reduced the expression level of cPLA2α, as determined by western blotting and reverse transcription­quantitative polymerase chain reaction, the improvement of locomotor function was evaluated by rotarod test, and the decrease of apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end­labeling staining. The decreased infarct areas were evaluated by 2,3,5­triphenyltetrazolium chloride staining. The expression levels of prostaglandin E2, leukotrienes B4, lysophosphatidylcholine and free fatty acids were reduced in the RNAi group when compared with the negative control group. Thus, the data indicates that the expression level of cPLA2α was effectively controlled by pAd­siRNA­cPLA2α treatment. pAd­siRNA­cPLA2α treatment, in reducing the levels of inflammatory factors, neurological deficit and tissue damage, represents an effective potential therapeutic strategy. pAd­siRNA­cPLA2α reduces cPLA2α expression levels with long­term efficacy, thereby improving functional deficits and effectively attenuating ischemic brain damage. Thus, pAd­siRNA­cPLA2α shows potential value for therapeutic evaluation in ischemic brain damage.

Sameerpannag Ras Mixture (SRM) improved neurobehavioral deficits following acute ischemic stroke by attenuating neuroinflammatory response.

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemic stroke is one of the leading causes of death and long-term disability worldwide. Unfortunately, due to the failure of most of drugs in clinical trials recently, attentions have moved towards the traditional system of medicines including Ayurveda. In Ayurveda, Sameerpannag Ras (SR) is a mineral and metallic origin based formulation which has been used for the treatment of arthritis and chronic systematic inflammatory disorder. The current study was designed to investigate the neuroprotective effects of Sameerpannag Ras Mixture (SRM), on the neurobehavioral dysfunction and associated neuroinflammation, induced by transient Internal Carotid Artery Occlusion (ICAO) in mice model. MATERIALS AND METHODS: In the present study, mice were treated with Sameerpannag Ras Mixture (SRM) at the dose of 40mg/kg body weight by oral gavages for 3 and 7days respectively, twice a day, after the induction of ICAO for 90min followed by reperfusion. The efficacy of SRM was examined by scoring neurological behavioral deficit using the standard neurological deficit score (NDS), grip strength and rotarod performance tests at different time intervals of post-ICAO. RESULTS: Post-ischemic treatment with Sameerpannag Ras Mixture (SRM) at 40mg/kg significantly reduced Neurological Deficit Score and improved the motor coordination at different time intervals post-ICAO. The analysis of RT-qPCR data showed that transient cerebral ischemia could induce the inflammatory response genes in the affected striatal region of ICAO group, as compared to sham group, on day3 and day7 post-ICAO. Interestingly, SRM treatment showed marked improvement in the ischemia-induced neurobehavioral deficits by attenuating ischemia-induced neuroinflammatory response at both gene and protein level. CONCLUSION: The present study suggests that Sameerpannag Ras Mixture (SRM) treatment ameliorates behavioral outcomes after mild ischemia through the suppression of a number of inflammatory response genes involved in neuronal damage. This is the first report of the molecular mechanism underlying the significant neuroprotective action of the Ayurvedic drug, Sameerpannag Ras Mixture (SRM), using a mild stroke in mice model.

Blockage of cytosolic phospholipase A2 alpha by monoclonal antibody attenuates focal ischemic brain damage in mice.

The purposes of the current study were to investigate the effects of a monoclonal antibody (mAb) on cytosolic phospholipase A2 alpha (cPLA2α) in mice with cerebral ischemia-reperfusion (IR) injury and to ascertain the potential mechanisms of those effects. This study evaluated whether the use of anti-cPLA2α mAb could reduce stroke injury in a mouse model of cerebral IR injury. The expression/activity of cPLA2α and cPLA2α- derived proinflammatory lipid mediators such as prostaglandin E2 (PGE2), leukotriene B4 (LTB4), lysophosphatidylcholine (LPC), and free fatty acids (FFA) was assessed. This study also evaluated neurological deficits, motor function, pathological changes, apoptosis, and the area of infarction in the injured mice. Mice treated with anti-cPLA2α mAb recovered neurological function and their condition improved, apoptosis in the brain decreased and infarct volume decreased, and expression of cPLA2α, 5-LOX, COX-2, FFA, LPC, PGE2, and LTB4 was attenuated. Our findings indicate that cPLA2α plays a key role in cerebral IR injury and that treatment with anti-cPLA2α mAb after cerebral IR injury helps to reduce levels of proinflammatory cytokines, alleviate tissue damage, and reduce levels of deleterious lipid mediators. Thus, anti-cPLA2α mAb treatment has the potential to treat ischemic brain damage.

Serum C-reactive protein, fibrinogen and D-dimer in patients with progressive cerebral infarction.

OBJECTIVE: Progressive cerebral infarctions increase mortality and functional disability through mechanisms which have yet to be completely understood. The goal of this study was to explore the dynamic changes of serum C-reactive protein (CRP), fibrinogen (FIB) and D-dimer (D-D) in order to better characterize progressive cerebral infarction. METHODS: The amount of serum CRP, FIB and D-D was measured in 82 patients with progressive cerebral infarction by taking samples from the internal carotid artery (progressive group), and in 186 patients with non-progressive cerebral infarction (non-progressive group) by using an automatic biochemical analyzer during the next day (day 1), day 3, day 7, and day 14 after being admitted to hospital. Carotid vascular ultrasound and neurological deficit score (National Institutes of Health Stroke Scale, NIHSS) were also recorded. RESULTS: Carotid stenosis ratio was significantly higher in the progressive group than in the non-progressive group (P < 0.01) on admission. In the progressive group, CRP increased significantly on day 3, followed by a decline on day 7 and day 14, but was significantly higher than those in the non-progressive group (P < 0.01). The levels of FIB and D-D increased in the progressive group more than those in the non-progressive group on day 3, day 7, and day 14 (P < 0.01). The progressive group patients' NIHSS score gradually increased after admission, which was opposite to the non-progressive group patients whom followed a downward trend. The difference between these two groups was significant (P < 0.01). CONCLUSION: Observing changes of CRP, FIB and D-D may contribute to early identification and timely treatment of progressing ischemic strokes.

Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke.

The temporal dynamics of neuronal autophagy and apoptosis in the ischemic penumbra following stroke remains unclear. Therefore, in this study, we investigated the dynamic changes in autophagy and apoptosis in the penumbra to provide insight into potential therapeutic targets for stroke. An adult Sprague-Dawley rat model of permanent ischemic stroke was prepared by middle cerebral artery occlusion. Neuronal autophagy and apoptosis in the penumbra post-ischemia were evaluated by western blot assay and immunofluorescence staining with antibodies against LC3-II and cleaved caspase-3, respectively. Levels of both LC3-II and cleaved caspase-3 in the penumbra gradually increased within 5 hours post-ischemia. Thereafter, levels of both proteins declined, especially LC3-II. The cerebral infarct volume increased slowly 1-4 hours after ischemia, but subsequently increased rapidly until 5 hours after ischemia. The severity of the neurological deficit was positively correlated with infarct volume. LC3-II and cleaved caspase-3 levels were high in the penumbra within 5 hours after ischemia, and after that, levels of these proteins decreased at different rates. LC3-II levels were reduced to a very low level, but cleaved caspase-3 levels remained high 72 hours after ischemia. These results indicate that there are temporal differences in the activation status of the autophagic and apoptotic pathways. This suggests that therapeutic targeting of these pathways should take into consideration their unique temporal dynamics.

Immediate effects of scalp acupuncture with twirling reinforcing manipulation on hemiplegia following acute ischemic stroke: a hidden association study.

Data mining has the potential to provide information for improving clinical acupuncture strategies by uncovering hidden rules between acupuncture manipulation and therapeutic effects in a data set. In this study, we performed acupuncture on 30 patients with hemiplegia due to acute ischemic stroke. All participants were pre-screened to ensure that they exhibited immediate responses to acupuncture. We used a twirling reinforcing acupuncture manipulation at the specific lines between the bilateral Baihui (GV20) and Taiyang (EX-HN5). We collected neurologic deficit score, simplified Fugl-Meyer assessment score, muscle strength of the proximal and distal hemiplegic limbs, ratio of the maximal H-reflex to the maximal M-wave (Hmax/Mmax), muscle tension at baseline and immediately after treatment, and the syndromes of traditional Chinese medicine at baseline. We then conducted data mining using an association algorithm and an artificial neural network backpropagation algorithm. We found that the twirling reinforcing manipulation had no obvious therapeutic difference in traditional Chinese medicine syndromes of "Deficiency and Excess". The change in the muscle strength of the upper distal and lower proximal limbs was one of the main factors affecting the immediate change in Fugl-Meyer scores. Additionally, we found a positive correlation between the muscle tension change of the upper limb and Hmax/Mmax immediate change, and both positive and negative correlations existed between the muscle tension change of the lower limb and immediate Hmax/Mmax change. Additionally, when the difference value of muscle tension for the upper and lower limbs was > 0 or < 0, the difference value of Hmax/Mmax was correspondingly positive or negative, indicating the scalp acupuncture has a bidirectional effect on muscle tension in hemiplegic limbs. Therefore, acupuncture with twirling reinforcing manipulation has distinct effects on acute ischemic stroke patients with different symptoms or stages of disease. Improved muscle tension in the upper and lower limbs, reflected by the variation in the Hmax/Mmax ratio, is crucial for recovery of motor function from hemiplegia.

Early-stage minimally invasive procedures decrease perihematomal endothelin-1 levels and improve neurological functioning in a rabbit model of intracerebral hemorrhage.

INTRODUCTION: To determine the effects of minimally invasive surgery (MIS) at various stages after intracerebral hemorrhage (ICH) on perihematomal endothelin (ET)-1 levels and neurological functioning. METHODS: Sixty rabbits were randomly distributed into a model control group (MC group, 30 rabbits) or a MIS group (MI group, 30 rabbits). An ICH model was established in all animals. In the MI group, ICH was evacuated by MIS at 6, 12, 18, 24, and 48 hours (six rabbits at each time point) after the ICH was established. The animals in the MC group underwent the same procedures for ICH evacuation, but with a sham operation without hematoma aspiration. All the animals were sacrificed 7 days after the ICH was established. Neurological deficit scores were determined, and the perihematomal brain tissue was removed to determine the ET-1 levels, blood-brain barrier (BBB) permeability, and brain water content (BWC). RESULTS: The neurological deficit scores, perihematomal ET-1 levels, BBB permeability, and BWC all decreased significantly in the MI group compared to the MC group. Performing the MIS for evacuating the ICH at 6 hours resulted in the most remarkable decreases in these indices, followed by a significant difference observed at 12 hours within the MI subgroups. CONCLUSIONS: Performing MIS at 6-12 hours after ICH resulted in the most significant decreases in neurological deficit scores, ET-1 levels, BBB permeability, and brain edema. The optimal time window for performing MIS for ICH evacuation might be within 6-12 hours after hemorrhage.

Neuroprotective efficacy of subcutaneous insulin-like growth factor-I administration in normotensive and hypertensive rats with an ischemic stroke.

The aim of this study was to test the insulin-like growth factor-I (IGF-I) as a neuroprotective agent in a rat model for ischemic stroke and to compare its neuroprotective effects in conscious normotensive and spontaneously hypertensive rats. The effects of subcutaneous IGF-I injection were investigated in both rat strains using the endothelin-1 rat model for ischemic stroke. Motor-sensory functions were measured using the Neurological Deficit Score. Infarct size was assessed by Cresyl Violet staining. Subcutaneous administration of IGF-I resulted in significantly reduced infarct volumes and an increase in motor-sensory functions in normotensive rats. In these rats, IGF-I did not modulate blood flow in the striatum and had no effect on the activation of astrocytes as assessed by GFAP staining. In hypertensive rats, the protective effects of IGF-I were smaller and not always significant. Furthermore, IGF-I significantly reduced microglial activation in the cortex of hypertensive rats, but not in normotensive rats. More detailed studies are required to find out whether the reduction by IGF-I of microglial activation contributes to an impairment IGF-I treatment efficacy. Indeed, we have shown before that microglia in hypertensive rats have different properties compared to those in control rats, as they exhibit a reduced responsiveness to ischemic stroke and lipopolysaccharide.

Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: rescue effect of mild hypothermia.

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.