Focal Cortical Ischemia Research Articles

Functional ultrasound imaging of stroke in awake rats

Anesthesia is a major confounding factor in preclinical stroke research as stroke rarely occurs in sedated patients. Moreover, anesthesia affects both brain functions and the stroke outcome acting as neurotoxic or protective agents. So far, no approaches were well suited to induce stroke while imaging hemodynamics along with simultaneous large-scale recording of brain functions in awake animals. For this reason, the first critical hours following the stroke insult and associated functional alteration remain poorly understood. Here, we present a strategy to investigate both stroke hemodynamics and stroke-induced functional alterations without the confounding effect of anesthesia, i.e., under awake condition. Functional ultrasound (fUS) imaging was used to continuously monitor variations in cerebral blood volume (CBV) in +65 brain regions/hemispheres for up to 3 hr after stroke onset. The focal cortical ischemia was induced using a chemo-thrombotic agent suited for permanent middle cerebral artery occlusion in awake rats and followed by ipsi- and contralesional whiskers stimulation to investigate on the dynamic of the thalamocortical functions. Early (0–3 hr) and delayed (day 5) fUS recording enabled to characterize the features of the ischemia (location, CBV loss), spreading depolarizations (occurrence, amplitude) and functional alteration of the somatosensory thalamocortical circuits. Post-stroke thalamocortical functions were affected at both early and later time points (0–3 hr and 5 days) after stroke. Overall, our procedure facilitates early, continuous, and chronic assessments of hemodynamics and cerebral functions. When integrated with stroke studies or other pathological analyses, this approach seeks to enhance our comprehension of physiopathologies towards the development of pertinent therapeutic interventions.

Functional ultrasound imaging of stroke in awake rats.

Anesthesia is a major confounding factor in preclinical stroke research as stroke rarely occurs in sedated patients. Moreover, anesthesia affects both brain functions and the stroke outcome acting as neurotoxic or protective agents. So far, no approaches were well suited to induce stroke while imaging hemodynamics along with simultaneous large-scale recording of brain functions in awake animals. For this reason, the first critical hours following the stroke insult and associated functional alteration remain poorly understood. Here, we present a strategy to investigate both stroke hemodynamics and stroke-induced functional alterations without the confounding effect of anesthesia, i.e., under awake condition. Functional ultrasound (fUS) imaging was used to continuously monitor variations in cerebral blood volume (CBV) in +65 brain regions/hemispheres for up to 3 hr after stroke onset. The focal cortical ischemia was induced using a chemo-thrombotic agent suited for permanent middle cerebral artery occlusion in awake rats and followed by ipsi- and contralesional whiskers stimulation to investigate on the dynamic of the thalamocortical functions. Early (0-3 hr) and delayed (day 5) fUS recording enabled to characterize the features of the ischemia (location, CBV loss), spreading depolarizations (occurrence, amplitude) and functional alteration of the somatosensory thalamocortical circuits. Post-stroke thalamocortical functions were affected at both early and later time points (0-3 hr and 5 days) after stroke. Overall, our procedure facilitates early, continuous, and chronic assessments of hemodynamics and cerebral functions. When integrated with stroke studies or other pathological analyses, this approach seeks to enhance our comprehension of physiopathologies towards the development of pertinent therapeutic interventions.

Neuroprotective Effects of Melittin Against Cerebral Ischemia and Inflammatory Injury via Upregulation of MCPIP1 to Suppress NF-κB Activation In Vivo and In Vitro

Melittin, a principal constituent of honeybee venom, exhibits diverse biological effects, encompassing anti-inflammatory capabilities and neuroprotective actions against an array of neurological diseases. In this study, we probed the prospective protective influence of melittin on cerebral ischemia, focusing on its anti-inflammatory activity. Mechanistically, we explored whether monocyte chemotactic protein-induced protein 1 (MCPIP1, also known as ZC3H12A), a recently identified zinc-finger protein, played a role in melittin-mediated anti-inflammation and neuroprotection. Male C57/BL6 mice were subjected to distal middle cerebral artery occlusion to create a focal cerebral cortical ischemia model, with melittin administered intraperitoneally. We evaluated motor functions, brain infarct volume, cerebral blood flow, and inflammatory marker levels within brain tissue, employing quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and western blotting. In vitro, an immortalized BV-2 microglia culture was stimulated with lipopolysaccharide (LPS) to establish an inflammatory cell model. Post-melittin exposure, cell viability, and cytokine expression were examined. MCPIP1 was silenced using siRNA in LPS-induced BV-2 cells, with the ensuing nuclear translocation of nuclear factor-κB assessed through cellular immunofluorescence. In vivo, melittin enhanced motor functions, diminished infarction, fostered blood flow restoration in ischemic brain regions, and markedly inhibited the expression of inflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α, and nuclear factor-κB). In vitro, melittin augmented MCPIP1 expression in LPS-induced BV-2 cells and ameliorated inflammation-induced cell death. The neuroprotective effect conferred by melittin was attenuated upon MCPIP1 knockdown. Our findings establish that melittin-induced tolerance to ischemic injury is intrinsically linked with its anti-inflammatory capacity. Moreover, MCPIP1 is, at the very least, partially implicated in this process.

Environmental enrichment enhanced neurogenesis and behavioral recovery after stroke in aged rats.

Age is identified as a significant prognostic factor for poorer outcome after stroke. However, environmental enrichment (EE) has been reported to promote functional recovery after ischemic stroke. The purpose of this study was to investigate whether environmental enrichment was beneficial to ischemic stroke in aged rats. Aged rats were randomly assigned as control rats, rats subjected to cerebral ischemia, and rats with cerebral ischemia treated with EE for 30 days. Focal cortical ischemia was induced by intracranial injection of endothelin-1 (ET-1). EE housing began one day after focal ischemia and was maintained for the whole experimental period. We used immunofluorescence staining to analyze the neurogenesis in the subventricular zone (SVZ) and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay to evaluate apoptosis. The expression of neuronal nuclei, glial fibrillary acidic protein (GFAP) and Iba-1 around the infarcted area were also measured by double immunohistochemistry. EE enhanced the proliferation of newborn neurons in the SVZ, as well as increased the long-term survival of newborn neurons. EE also exerted effects on inflammation after stroke. Furthermore, EE suppressed apoptosis and improved the motor functions after stroke in the aged rats. EE improved post-stroke recovery on the basis of enhancing neurogenesis in aged rats.

Depth-profile of impairments in endothelin-1 – induced focal cortical ischemia

The development of ischemic lesions has primarily been studied in horizontal cortical space. However, how ischemic lesions develop through the cortical depth remains largely unknown. We explored this question using direct current coupled recordings at different cortical depths using linear arrays of iridium electrodes in the focal epipial endothelin-1 (ET1) ischemia model in the rat barrel cortex. ET1-induced impairments were characterized by a vertical gradient with (i) rapid suppression of the spontaneous activity in the superficial cortical layers at the onset of ischemia, (ii) compartmentalization of spreading depolarizations (SDs) to the deep layers during progression of ischemia, and (iii) deeper suppression of activity and larger histological lesion size in superficial cortical layers. The level of impairments correlated strongly with the rate of spontaneous activity suppression, the rate of SD onset after ET1 application, and the amplitude of giant negative ultraslow potentials (∼−70 mV), which developed during ET1 application and were similar to the tent-shaped ultraslow potentials observed during focal ischemia in the human cortex. Thus, in the epipial ET1 ischemia model, ischemic lesions develop progressively from the surface to the cortical depth, and early changes in electrical activity at the onset of ET1-induced ischemia reliably predict the severity of ischemic damage.

Liraglutide Ameliorates Cerebral Ischemia in Mice via Antipyroptotic Pathways.

It was recently shown that pyroptosis, an inflammatory form of programmed cell death, is critically involved in the pathogenesis of ischemic stroke. Liraglutide (Lg) is a novel long-acting analog of glucagon-like peptide-1 that has potential protective effects against stroke. However, the relationship between Lg and pyroptosis in the brain is not well defined. In this study, we found that injection of Lg significantly improved the recovery of motor function, increased cerebral blood flow and ameliorated cerebral damage in a mouse model of focal cerebral cortical ischemia. Our results revealed that Lg treatment significantly reduced the levels of NLRP3, Caspase1, IL-1β and the pore-forming protein gasdermin D in microglial cells in vitro, suggesting that the neuroprotective effect of Lg may be achieved through the inhibition of pyroptosis. Furthermore, by using a specific inhibitor of NOD-like receptor protein 3 (NLRP3), we confirmed that the antipyroptotic mechanism of Lg may be mediated by NLRP3 in vivo. Our present study unveils a novel neuroprotective mechanism through which Lg alleviates ischemia by exerting NLRP3-dependent antipyroptotic effects.

Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice

Ischemic stroke can cause secondary myelin damage in the white matter distal to the primary injury site. The contribution of astrocytes during secondary demyelination and the underlying mechanisms are unclear. Here, using a mouse of distal middle cerebral artery occlusion, we show that lipocalin-2 (LCN2), enriched in reactive astrocytes, expression increases in nonischemic areas of the corpus callosum upon injury. LCN2-expressing astrocytes acquire a phagocytic phenotype and are able to uptake myelin. Myelin removal is impaired in Lcn2−/− astrocytes. Inducing re-expression of truncated LCN2(Δ2–20) in astrocytes restores phagocytosis and leads to progressive demyelination in Lcn2−/− mice. Co-immunoprecipitation experiments show that LCN2 binds to low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. Knockdown of Lrp1 reduces LCN2-induced myelin engulfment by astrocytes and reduces demyelination. Altogether, our findings suggest that LCN2/LRP1 regulates astrocyte-mediated myelin phagocytosis in a mouse model of ischemic stroke.

Secondary Degeneration of White Matter After Focal Sensorimotor Cortical Ischemic Stroke in Rats.

Ischemic lesions could lead to secondary degeneration in remote regions of the brain. However, the spatial distribution of secondary degeneration along with its role in functional deficits is not well understood. In this study, we explored the spatial and connectivity properties of white matter (WM) secondary degeneration in a focal unilateral sensorimotor cortical ischemia rat model, using advanced microstructure imaging on a 14 T MRI system. Significant axonal degeneration was observed in the ipsilateral external capsule and even remote regions including the contralesional external capsule and corpus callosum. Further fiber tractography analysis revealed that only fibers having direct axonal connections with the primary lesion exhibited a significant degeneration. These results suggest that focal ischemic lesions may induce remote WM degeneration, but limited to fibers tied to the primary lesion. These “direct” fibers mainly represent perilesional, interhemispheric, and subcortical axonal connections. At last, we found that primary lesion volume might be the determining factor of motor function deficits.

Comparative Enhancement of Motor Function and BDNF Expression Following Different Brain Stimulation Approaches in an Animal Model of Ischemic Stroke

Background Combinatory intervention such as high-frequency (50-100 Hz) excitatory cortical stimulation (ECS) given concurrently with motor rehabilitative training (RT) improves forelimb function, except in severely impaired animals after stroke. Clinical studies suggest that low-frequency (≤1 Hz) inhibitory cortical stimulation (ICS) may provide an alternative approach to enhance recovery. Currently, the molecular mediators of CS-induced behavioral effects are unknown. Brain-derived neurotrophic factor (BDNF) has been associated with improved recovery and neural remodeling after stroke and thus may be involved in CS-induced behavioral recovery. Objective To investigate whether inhibitory stimulation during RT improves functional recovery of severely impaired rats, following focal cortical ischemia and if this recovery alters BDNF expression (study 1) and depends on BDNF binding to TrkB receptors (study 2). Methods Rats underwent ECS + RT, ICS + RT, or noCS + RT treatment daily for 3 weeks following a unilateral ischemic lesion to the motor cortex. Electrode placement for stimulation was either placed ipsilateral (ECS) or contralateral (ICS) to the lesion. After treatment, BDNF expression was measured in cortical tissue samples (study 1). In study 2, the TrkB inhibitor, ANA-12, was injected prior to treatment daily for 21 days. Results ICS + RT treatment significantly improved impaired forelimb recovery compared with ECS + RT and noCS + RT treatment. Conclusion ICS given concurrently with rehabilitation improves motor recovery in severely impaired animals, and alters cortical BDNF expression; nevertheless, ICS-mediated improvements are not dependent on BDNF binding to TrkB. Conversely, inhibition of TrkB receptors does disrupt motor recovery in ECS + RT treated animals.

Compensatory neuromuscular junction adaptations of forelimb muscles in focal cortical ischemia in rats.

IntroductionUpper limb movements are affected frequently by brain ischemia (BI). Mechanisms involved in recovery and compensatory movements have developed several studies. However, less attention is given to skeletal muscles, where neuromuscular junction (NMJ) has an important role on muscle tropism and functional performance.MethodsAnimals were divided into two groups: control (C) and BI. Then, animals were skilled to perform single‐pellet retrieval task, following these procedures: habituation, shaping, and single‐pellet retrieval task. BI was induced using stereotaxic surgery in order to apply endothelin‐1 in motor cortex, representative of movements of dominant paw. Reaching task performance was evaluated by single‐pellet retrieval task 1 day before BI induction, 4 and 15 days after BI induction. After that, biceps, triceps, fingers flexor, and extensor muscles were extracted. NMJ was assessed in morphometric characteristics (total area, total perimeter, and feret). Muscle fiber cross‐sectional area and connective tissue percentage were also evaluated for characterization. Student's t test was used for comparisons between C and BI groups. Tau Kendall's correlation was applied among variables from BI group.ResultsAn increase in all NMJ morphometric parameters, as well as increase of atrophy and fibrosis in BI group compared with C. There was a high level of direct correlation between mean values of NMJ morphometry with percentage of success in reaching task in BI group.ConclusionBrain ischemia‐induced NMJ compensatory expansion, muscle atrophy, and fibrosis in forelimb muscles that are related to reaching performance.

Long-Term Motor Deficit and Diffuse Cortical Atrophy Following Focal Cortical Ischemia in Athymic Rats

Development of new stroke therapies requires animal models that recapitulate the pathophysiological and functional consequences of ischemic brain damage over time-frames relevant to the therapeutic intervention. This is particularly relevant for the rapidly developing area of stem cell therapies, where functional replacement of circuitry will require maturation of transplanted human cells over months. An additional challenge is the establishment of models of ischemia with stable behavioral phenotypes in chronically immune-suppressed animals to allow for long-term survival of human cell grafts. Here we report that microinjection of endothelin-1 into the sensorimotor cortex of athymic rats results in ischemic damage with a sustained deficit in function of the contralateral forepaw that persists for up to 9 months. The histological post-mortem analysis revealed chronic and diffuse atrophy of the ischemic cortical hemisphere that continued to progress over 9 months. Secondary atrophy remote to the primary site of injury and its relationship with long-term cognitive and functional decline is now recognized in human populations. Thus, focal cortical infarction in athymic rats mirrors important pathophysiological and functional features relevant to human stroke, and will be valuable for assessing efficacy of stem cell based therapies.

Abstract 158: Optogenetic Theta-band Activation of Pyramidal Neurons Elicits Peri-infarct Spreading Depolarizations in the ET-1 Rat Model of Focal Cortical Ischemia

Peri-infarct spreading depolarization (PIDs) frequently occur post ischemic stroke in animals and humans, propagating from the infarct core into the penumbra, and have been associated with worsened neurological outcome in patients. The etiology of PIDs is not well understood, complicating the development of targeted treatments. To examine the cellular origins of PIDs, we hypothesized that synchronous, frequency-specific, activation of specific neuronal subpopulation is necessary for PID occurrence. We transfected adult male Sprague Dawley rats with ChR2 either under the CamKII (AAV5.CAMKII.ArchT.GFP.WPRE.SV40) or CAG (AAV5.CAG.hChR2(H134R)-mCherry.WPRE.SV40) promoters into the somatosensory cortex to induce opsin transfection in pyramidal neurons or interneurons, respectively. Two weeks later, we assessed neuronal activity changes induced by focal cortical ischemia by inserting two electrodes ~2 mm apart in rostro-caudal direction. We then used a glass micropipette to inject 800 pmol of ET-1 into the primary somatosensory cortex in correspondence of rostral electrode, so as to examine the activity at the ‘core’ of the ischemic insult as well as from the peri-infarct zone, which coincided with the transfected area. We recorded baseline (pre-ischemic) local field potentials (LFPs) before injecting ET-1. Following ET-1 injection, we observed an average of five CSDs, with a mean amplitude of 57 mV and a mean duration of 1.2min, propagating from the infarct core to the peri-infarct tissue. In all subjects, the CSDs subsided within the first hour after the ischemic insult. In the second hour following stroke, we stimulated peri-infarct pyramidal neurons or interneurons at different frequency bands (theta, alpha, beta, low gamma, high gamma, and DC). Only theta band stimulation of the pyramidal neurons reliably triggered PIDs. Stimulating pyramidal neurons at higher frequencies or interneurons at any frequency did not elicit PIDs. Activation of peri-infarct zone pyramidal neurons in the theta band elicits cortical PIDs. The identification of a neuronal-specific and frequency-specific trigger for PIDs provides a roadmap for development of PID-targeted treatments in the acute stage of stroke.

Protein expression profiling in the hippocampus after focal cerebral ischemia injury in rats.

The aim in this study was to explore protein expression profiles in the rat hippocampus after induction of focal ischemia injury. Forty male Sprague Dawley rats were randomly divided into four equal groups after ischemia injury surgery: Control, Day 3, Day 7, and Day 14. Focal cortical ischemia was induced in thirty rats by photothrombosis of cortical microvessels. After surgery, the induction of ischemia was confirmed by infarct size measurement using staining by 2, 3, 5-triphenyltetrazolium chloride. To identify the differential expression of proteins between the diseased and control sides of the hippocampus, a comparative proteome analysis was performed using isobaric tags for relative or absolute quantitation coupled with 2D liquid chromatographytandem mass spectrometry analysis. 4,081 proteins were identified, 260 of which were non-redundant and showed differential expression between the three surgery groups and the control. Hierarchical cluster analysis indicated that the three surgical groups had markedly different expression patterns of these 260 proteins, including 160 upregulated and 80 downregulated proteins. A gene ontology analysis revealed 4,944 terms, among which myelin sheath and cell junction were the two most enriched items. In KEGG database pathway analysis, ribosome was the most abundant item. A Venn diagram showing the overlap of 25 of the differentially expressed proteins quantified from the four groups and results from the KEGG pathway analysis suggested that Epstein-Barr virus infection was the most abundant item. From the protein-protein interaction network, a total of 223 interactive proteins were predicted and used to construct a network. In conclusion, myelin sheath, cell junction, and Epstein–Barr virus infection were implicated in focal ischemia injury. Vimentin and albumin may be important proteins involved in focal ischemia injury.

Delayed Administration of the Glucagon-Like Peptide 1 Analog Liraglutide Promoting Angiogenesis after Focal Cerebral Ischemia in Mice

Glucagon-like peptide 1 (GLP-1) analogs administered before or after cerebral ischemia have been shown to provide neuroprotection. Here, we explored whether delayed administration of a GLP-1 analog, liraglutide, could improve long-term functional recovery and promote angiogenesis after stroke. In the present study, mice were established as a focal cerebral cortical ischemia model and were intraperitoneally administered liraglutide or normal saline (NS) daily for 14 consecutive days, starting 1 day after cerebral ischemia. The neurological deficits were evaluated using rotarod test. The microvessel density (MVD) and endothelial cell (EC) proliferation were assessed by immunohistochemical staining. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. Liraglutide significantly reduced infarct volume and improved the rotarod test scores, compared with mice treated with NS. Liraglutide also greatly increased the MVD and EC proliferation and simultaneously upregulated the expression of VEGF in the cerebral ischemic area. These results demonstrated that liraglutide promoted angiogenesis and long-term recovery of cerebral ischemia through increasing the expression of VEGF.

Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke

BackgroundChoroid plexus (CP) supports the entry of monocyte-derived macrophages (MDMs) to the central nervous system in animal models of traumatic brain injury, spinal cord injury, and Alzheimer’s disease. Whether the CP is involved in the recruitment of MDMs to the injured brain after ischemic stroke is unknown.MethodsAdult male C57BL/6 mice were subjected to focal cortical ischemia by permanent occlusion of the distal branch of the right middle cerebral artery. Choroid plexus tissues were collected and analyzed for Vcam1, Madcam1, Cx3cl1, Ccl2, Nt5e, and Ifnγ expression at different timepoints after stroke using qPCR. Changes of MDMs in CP and cerebrospinal fluid (CSF) at 1 day and 3 days after stroke were analyzed using flow cytometry. Infiltration of MDMs into CP and CSF were validated using β-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. CD115+ monocytes were isolated using a magnetic cell separation system from bone marrow of Cx3cr1-GFP or wild-type C57BL/6 donor mice. The freshly isolated monocytes or M2-like MDMs primed in vitro with IL4 and IL13 were stereotaxically injected into the lateral ventricle of stroke-affected mice to trace for their migration into ischemic hemisphere or to assess their effect on post-stroke recovery using open field, corridor, and active avoidance behavioral tests.ResultsWe found that CP responded to cortical stroke by upregulation of gene expression for several possible mediators of MDM trafficking and, concomitantly, MDMs increased in CP and cerebrospinal fluid (CSF). We then confirmed that MDMs infiltrated from blood into CP and CSF after the insult using β-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. When MDMs were directly administered into CSF following stroke, they homed to the ischemic hemisphere. If they had been primed in vitro prior to their administration to become M2-like macrophages, they promoted post-stroke recovery of motor and cognitive function without influencing infarct volume.ConclusionsOur findings suggest the possibility that autologous transplantation of M2-like MDMs into CSF might be developed into a new strategy for promoting recovery also in patients with stroke.

Chronic stress induced disruption of the peri-infarct neurovascular unit following experimentally induced photothrombotic stroke

How stress influences brain repair is an issue of considerable importance, as patients recovering from stroke are known to experience high and often unremitting levels of stress post-event. In the current study, we investigated how chronic stress modified the key cellular components of the neurovascular unit. Using an experimental model of focal cortical ischemia in male C57BL/6 mice, we examined how exposure to a persistently aversive environment, induced by the application of chronic restraint stress, altered the cortical remodeling post-stroke. We focused on systematically investigating changes in the key components of the neurovascular unit (i.e. neurons, microglia, astrocytes, and blood vessels) within the peri-infarct territories using both immunohistochemistry and Western blotting. The results from our study indicated that exposure to chronic stress exerted a significant suppressive effect on each of the key cellular components involved in neurovascular remodeling. Co-incident with these cellular changes, we observed that chronic stress was associated with an exacerbation of motor impairment 42 days post-event. Collectively, these results highlight the vulnerability of the peri-infarct neurovascular unit to the negative effects of chronic stress.

Regulation of therapeutic hypothermia on inflammatory cytokines, microglia polarization, migration and functional recovery after ischemic stroke in mice

Stroke is a leading threat to human life and health in the US and around the globe, while very few effective treatments are available for stroke patients. Preclinical and clinical studies have shown that therapeutic hypothermia (TH) is a potential treatment for stroke. Using novel neurotensin receptor 1 (NTR1) agonists, we have demonstrated pharmacologically induced hypothermia and protective effects against brain damages after ischemic stroke, hemorrhage stroke, and traumatic brain injury (TBI) in rodent models. To further characterize the mechanism of TH-induced brain protection, we examined the effect of TH (at ±33°C for 6h) induced by the NTR1 agonist HPI-201 or physical (ice/cold air) cooling on inflammatory responses after ischemic stroke in mice and oxygen glucose deprivation (OGD) in cortical neuronal cultures. Seven days after focal cortical ischemia, microglia activation in the penumbra reached a peak level, which was significantly attenuated by TH treatments commenced 30min after stroke. The TH treatment decreased the expression of M1 type reactive factors including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-12, IL-23, and inducible nitric oxide synthase (iNOS) measured by RT-PCR and Western blot analyses. Meanwhile, TH treatments increased the expression of M2 type reactive factors including IL-10, Fizz1, Ym1, and arginase-1. In the ischemic brain and in cortical neuronal/BV2 microglia cultures subjected to OGD, TH attenuated the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), two key chemokines in the regulation of microglia activation and infiltration. Consistently, physical cooling during OGD significantly decreased microglia migration 16h after OGD. Finally, TH improved functional recovery at 1, 3, and 7days after stroke. This study reveals the first evidence for hypothermia mediated regulation on inflammatory factor expression, microglia polarization, migration and indicates that the anti-inflammatory effect is an important mechanism underlying the brain protective effects of a TH therapy.

Chronic stress exposure following photothrombotic stroke is associated with increased levels of Amyloid beta accumulation and altered oligomerisation at sites of thalamic secondary neurodegeneration in mice

Exposure to severe stress following stroke is recognised to complicate the recovery process. We have identified that stress can exacerbate the severity of post-stroke secondary neurodegeneration in the thalamus. In this study, we investigated whether exposure to stress could influence the accumulation of the neurotoxic protein Amyloid-β. Using an experimental model of focal cortical ischemia in adult mice combined with exposure to chronic restraint stress, we examined changes within the contra- and ipsilateral thalamus at six weeks post-stroke using Western blotting and immunohistochemical approaches. Western blotting analysis indicated that stroke was associated with a significant enhancement of the 25 and 50 kDa oligomers within the ipsilateral hemisphere and the 20 kDa oligomer within the contralateral hemisphere. Stroked animals exposed to stress exhibited an additional increase in multiple forms of Amyloid-beta oligomers. Immunohistochemistry analysis confirmed that stroke was associated with a significant accumulation of Amyloid-beta within the thalami of both hemispheres, an effect that was exacerbated in stroke animals exposed to stress. Given that Amyloid-beta oligomers, most notably the 30–40 and 50 kDa oligomers, are recognised to correlate with accelerated cognitive decline, our results suggest that monitoring stress levels in patients recovering from stroke may merit consideration in the future.

Therapeutic potential of the novel hybrid molecule JM-20 against focal cortical ischemia in rats

Context: Despite the great mortality and morbidity of stroke, treatment options remain limited. We previously showed that JM-20, a novel synthetic molecule, possessed a strong neuroprotective effect in rats subjected to transient middle cerebral artery occlusion. However, to verify the robustness of the pre-clinical neuroprotective effects of JM-20 to get good prognosis in the translation to the clinic, it is necessary to use other experimental models of brain ischemia. Aims: To evaluate the neuroprotective effects of JM-20 following the onset of permanent focal cerebral ischemia induced in rats by thermocoagulation of blood into pial blood vessels of cerebral cortices. Methods: Ischemic lesion was induced by thermocoagulation of blood into pial blood vessels of primary motor and somatosensory cortices. Behavioral performance was evaluated by the cylinder testing for a period of 2, 3 and 7 days after surgery, and was followed by histopathological study in brain cortex stained with hematoxylin- eosin. Results: Ischemic injury resulted in impaired function of the forelimb evidenced by high asymmetry punctuation, and caused histopathological alterations indicative of tissue damage at cerebral cortex. JM-20 treatment (4 and 8 mg/kg) significantly decreased asymmetry scores and histological alterations with a marked preservation of cortical neurons. Conclusions: The effects of permanent brain ischemia were strongly attenuated by JM-20 administration, which expands and improves the current preclinical data of JM-20 as neuroprotector against cerebral ischemia, and strongly support the examination of its translation to the clinic to treat acute ischemic stroke.

Fluoxetine Enhances Neurogenesis in Aged Rats with Cortical Infarcts, but This is not Reflected in a Behavioral Recovery.

Age is associated with poor outcome and impaired functional recovery after stroke. Fluoxetine, which is widely used in clinical practice, can regulate hippocampal neurogenesis in young rodents. As the rate of neurogenesis is dramatically reduced during aging, we studied the effect of post-stroke fluoxetine treatment on neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ) of dentate gyrus (DG) and whether this would be associated with any behavioral recovery after the cortical infarct in aged rats. Aged rats were randomly assigned to four groups: sham-operated rats, sham-operated rats treated with fluoxetine, rats subjected to cerebral ischemia, and rats with ischemia treated with fluoxetine. Focal cortical ischemia was induced by intracranial injection of vasoconstrictive peptide, endothelin-1 (ET-1). Fluoxetine was administered in the drinking water for 3 weeks starting 1 week after ischemia at a dose of 18 mg/kg/day. Behavioral recovery was evaluated on post-stroke days 29 to 31 after which the survival rate and fate of proliferating cells in the SVZ and DG were assessed by immunohistochemistry. Apoptosis was measured with the TUNEL assay. The results indicated that chronic fluoxetine treatment after stroke enhanced the proliferation of newborn neurons in the SVZ, but not in SGZ, and it suppressed perilesional apoptosis. Fluoxetine treatment did not affect the survival or differentiation of newly generated cells in the SVZ i.e., the enhanced neurogenesis was not translated into a behavioral outcome.