Experimental Stroke In Mice Research Articles

The SWELL1 Channel Promotes Ischemic Brain Damage by Mediating Neuronal Swelling and Glutamate Toxicity.

Cytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume-regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl- influx-dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA-approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease.

Direct neuronal protection by the protease-activated receptor PAR4 antagonist ML354 after experimental stroke in mice.

Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuroinflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcomes were assessed. Abundance of target genes, proteins, and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting, and immunofluorescence. Stroke up-regulated PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduced infarct size, apoptotic markers, macrophage accumulation, and interleukin-1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis, and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

X-irradiated umbilical cord blood cells retain their regenerative effect in experimental stroke

Although regenerative therapy with stem cells is believed to be affected by their proliferation and differentiation potential, there is insufficient evidence regarding the molecular and cellular mechanisms underlying this regenerative effect. We recently found that gap junction-mediated cell–cell transfer of small metabolites occurred very rapidly after stem cell treatment in a mouse model of experimental stroke. This study aimed to investigate whether the tissue repair ability of umbilical cord blood cells is affected by X-irradiation at 15 Gy or more, which suppresses their proliferative ability. In this study, X-irradiated mononuclear (XR) cells were prepared from umbilical cord blood. Even though hematopoietic stem/progenitor cell activity was diminished in the XR cells, the regenerative activity was surprisingly conserved and promoted recovery from experimental stroke in mice. Thus, our study provides evidence regarding the possible therapeutic mechanism by which damaged cerebrovascular endothelial cells or perivascular astrocytes may be rescued by low-molecular-weight metabolites supplied by injected XR cells in 10 min as energy sources, resulting in improved blood flow and neurogenesis in the infarction area. Thus, XR cells may exert their tissue repair capabilities by triggering neo-neuro-angiogenesis, rather than via cell-autonomous effects.

Prediction of Stroke Outcome in Mice Based on Noninvasive MRI and Behavioral Testing.

Prediction of poststroke outcome using the degree of subacute deficit or magnetic resonance imaging is well studied in humans. While mice are the most commonly used animals in preclinical stroke research, systematic analysis of outcome predictors is lacking. We intended to incorporate heterogeneity into our retrospective study to broaden the applicability of our findings and prediction tools. We therefore analyzed the effect of 30, 45, and 60 minutes of arterial occlusion on the variance of stroke volumes. Next, we built a heterogeneous cohort of 215 mice using data from 15 studies that included 45 minutes of middle cerebral artery occlusion and various genotypes. Motor function was measured using a modified protocol for the staircase test of skilled reaching. Phases of subacute and residual deficit were defined. Magnetic resonance images of stroke lesions were coregistered on the Allen Mouse Brain Atlas to characterize stroke topology. Different random forest prediction models that either used motor-functional deficit or imaging parameters were generated for the subacute and residual deficits. Variance of stroke volumes was increased by 45 minutes of arterial occlusion compared with 60 minutes. The inclusion of various genotypes enhanced heterogeneity further. We detected both a subacute and residual motor-functional deficit after stroke in mice and different recovery trajectories could be observed. In mice with small cortical lesions, lesion volume was the best predictor of the subacute deficit. The residual deficit could be predicted most accurately by the degree of the subacute deficit. When using imaging parameters for the prediction of the residual deficit, including information about the lesion topology increased prediction accuracy. A subset of anatomic regions within the ischemic lesion had particular impact on the prediction of long-term outcomes. Prediction accuracy depended on the degree of functional impairment. For the first time, we developed and validated a robust tool for the prediction of functional outcomes after experimental stroke in mice using a large and genetically heterogeneous cohort. These results are discussed in light of study design and imaging limitations. In the future, using outcome prediction can improve the design of preclinical studies and guide intervention decisions.

Inhibition of EphA4 reduces vasogenic edema after experimental stroke in mice by protecting the blood-brain barrier integrity

Cerebral vasogenic edema, a severe complication of ischemic stroke, aggravates neurological deficits. However, therapeutics to reduce cerebral edema still represent a significant unmet medical need. Brain microvascular endothelial cells (BMECs), vital for maintaining the blood-brain barrier (BBB), represent the first defense barrier for vasogenic edema. Here, we analyzed the proteomic profiles of the cultured mouse BMECs during oxygen-glucose deprivation and reperfusion (OGD/R). Besides the extensively altered cytoskeletal proteins, ephrin type-A receptor 4 (EphA4) expressions and its activated phosphorylated form p-EphA4 were significantly increased. Blocking EphA4 using EphA4-Fc, a specific and well-tolerated inhibitor shown in our ongoing human phase I trial, effectively reduced OGD/R-induced BMECs contraction and tight junction damage. EphA4-Fc did not protect OGD/R-induced neuronal and astrocytic death. However, administration of EphA4-Fc, before or after the onset of transient middle cerebral artery occlusion (tMCAO), reduced brain edema by about 50%, leading to improved neurological function recovery. The BBB permeability test also confirmed that cerebral BBB integrity was well maintained in tMCAO brains treated with EphA4-Fc. Therefore, EphA4 was critical in signaling BMECs-mediated BBB breakdown and vasogenic edema during cerebral ischemia. EphA4-Fc is promising for the treatment of clinical post-stroke edema.

Extensive complex neocortical movement topography devolves to simple output following experimental stroke in mice.

The neocortex encodes complex and simple motor outputs in all mammalian species that have been tested. Given that changes in neocortical reorganization (and corresponding corticospinal output) have been implicated in long term motor recovery after stroke injury, there remains a need to understand this biology in order to expedite and optimize clinical care. Here, changes in the neocortical topography of complex and simple movement outputs were evaluated in mice following experimental middle cerebral artery occlusion (MCAo). Neocortical motor output was defined using long-duration parameters of intracortical microstimulation (LD-ICMS) based on area and spatial coordinates of separate motor output types to build upon our recent report in uninjured mice. LD-ICMS test sites that elicited complex (multi-joint) movement, simple (single skeletal joint) movement, as well as co-elicited FORELIMB + HINDLIMB responses were detected and recorded. Forelimb reaching behavior was assessed using the single pellet reaching (SPR) task. At 6 weeks post-surgery, behavioral deficits persisted and neocortical territories for separate movements exhibited differences in neocortical area, and spatial location, and differed between MCAo-Injured animals (i.e., the MCAo group) and Sham-Injured animals (i.e., the Control group). MCAo-Injury reduced neocortical area of complex movements while increasing area of simple movements. Limited effects of injury were detected for spatial coordinates of neocortical movements. Significant positive correlations were detected between final SPR performance and either area of complex retract or area of co-occurring FORELIMB + HINDLIMB sites.

The GHB analogue HOCPCA improves deficits in cognition and sensorimotor function after MCAO via CaMKIIα

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA’s effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA’s selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.

Abstract WP211: Mir-34a Modulates Outcomes After Experimental Stroke In Mice

Ischemic stroke is one of the leading causes of death worldwide and causes significant morbidity among survivors. Studies have shown correlations between small noncoding RNAs (microRNAs, miRNAs) and stroke. We have previously reported that miR-34a depletion decreases infarct volume and attenuates neurological deficits in stroke mice. To further investigate the role of miR-34a in stroke, we utilized inducible miR-34a overexpression (miR-34a TG ) mice. Transient middle cerebral artery occlusion (tMCAO) was performed on 6-month old doxycycline-induced miR-34a TG mice compared to wild-type (WT) controls. We confirmed that miR-34a TG mice had a ~5-fold increase of miR-34a in plasma at 24 hours post-stroke as detected by real-time PCR compared with WT naive controls (p < 0.0001) and WT-stroke controls (p < 0.0001). Following ischemia due to 60 min tMCAO, overexpression of miR-34a significantly exacerbated neurological deficit scores (p = 0.0035) and increased infarct volume compared to WT control mice at 24 hours of reperfusion (p = 0.002). Systemic delivery of a locked nucleic acid (LNA)-modified miR-34a inhibitor, antagomir-34a, in stroke mice substantially reduced infarct volume (p = 0.0021) and improved neurological deficits (p = 0.0057) at 24 hours post-stroke. Our post-stroke behavioral study revealed that antagomir-34a significantly improved functional outcomes including motor function assessed by accelerated rotarod tests at 2 weeks post-stroke (p = 0.0046) and memory deficits assessed by Morris Water Maze tests at 4 weeks post-stroke (p = 0.0012). These data demonstrate that miR-34a plays an important role in modulating acute stroke outcomes and suggest that miR-34a may be a promising therapeutic target for stroke therapy.

Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

Infarct growth precedes cerebral thrombosis following experimental stroke in mice

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.

Expression of miR-200c corresponds with increased reactive oxygen species and hypoxia markers after transient focal ischemia in mice

Embolic stroke results in a necrotic core of cells destined to die, but also a peri-ischemic, watershed penumbral region of potentially salvageable brain tissue. Approaches to effectively differentiate between the ischemic and peri-ischemic zones is critical for novel therapeutic discovery to improve outcomes in survivors of stroke. MicroRNAs are a class of small non-coding RNAs regulating gene translation that have region- and cell-specific expression and responses to ischemia. We have previously reported that global inhibition of cerebral microRNA-200c after experimental stroke in mice is protective, however delineating the post-stroke sub-regional and cell-type specific patterns of post-stroke miR-200c expression are necessary to minimize off-target effects and advance translational application. Here, we detail a novel protocol to visualize regional miR-200c expression after experimental stroke, complexed with visualization of regional ischemia and markers of oxidative stress in an experimental stroke model in mice. In the present study we demonstrate that the fluorescent hypoxia indicator pimonidazole hydrochloride, the reactive-oxygen-species marker 8-hydroxy-deoxyguanosine, neuronal marker MAP2 and NeuN, and the reactive astrocyte marker GFAP can be effectively complexed to determine regional differences in ischemic injury as early as 30 min post-reperfusion after experimental stroke, and can be effectively used to distinguish ischemic core from surrounding penumbral and unaffected regions for targeted therapy. This multi-dimensional post-stroke immunofluorescent imaging protocol enables a greater degree of sub-regional mechanistic investigation, with the ultimate goal of developing more effective post-stroke pharmaceutical therapy.

The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke

The precise mechanisms underlying the beneficial effects of regulatory T (Treg) cells on long-term tissue repair remain elusive. Here, using single-cell RNA sequencing and flow cytometry, we found that Treg cells infiltrated the brain 1 to 5weeks after experimental stroke in mice. Selective depletion of Treg cells diminished oligodendrogenesis, white matter repair, and functional recovery after stroke. Transcriptomic analyses revealed potent immunomodulatory effects of brain-infiltrating Treg cells on other immune cells, including monocyte-lineage cells. Microglia depletion, but not Tcell lymphopenia, mitigated the beneficial effects of transferred Treg cells on white matter regeneration. Mechanistically, Treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. Increasing Treg cell numbers by delivering IL-2:IL-2 antibody complexes after stroke improved white matter integrity and rescued neurological functions over the long term. These findings reveal Treg cells as a neurorestorative target for stroke recovery.

Abstract P736: Memantine, an NMDA Antagonist and Aging Alter the Thalamic Gliosis in Experimental Stroke in Mice

Secondary injury in the thalamus has been observed following cortical stroke in rodents and humans and is associated with worsened recovery. Interruption of this progressive injury reflects an important therapeutic goal. However, the mechanisms whereby primary cortical infarction leads to remote injury in distant regions of brain are not well defined. We used a mouse model of cortical stroke (which demonstrates delayed thalamic injury) to define the time course of thalamic gliosis and neuronal injury and then test the potential of delayed memantine treatment (an NMDA receptor antagonist) to attenuate this secondary injury. Methods: Cortical infarction was induced by permanent occlusion of the distal middle cerebral artery (pdMCAO) in male C57BL/6J mice (young and aged) and CCR2-RFP mice. Brain infarct, cell-specific injury, and gliosis were measured by cresyl violet, Fluoro-jade C (FJC), TTC, FACS, and immunofluorescence. In young mice, memantine was injected at 4 and 24 hours post-stroke (100 and 50 mg/kg, ip). Brains were evaluated at post-stroke day 3 and 14 (PSD3 and PSD14). Results: At PSD3, the primary infarct was restricted to the cortex of the MCA territory, with no infarct detected in the thalamus of young mice. However, by PSD 14, neurons in the ipsilateral thalamus exhibited significant injury (FJC positive, condensed pyknotic nuclei). Gliosis was first detectable in the ipsilateral thalamus at PSD3 and progressively increased to PSD14 (anti-GFAP and Iba1). Infiltration of peripheral-derived monocytes was determined to be one source of the activated microglia in the thalamus (CCR2-RFP reporter mice, n=3). Interestingly, pdMCAO mice allowed to recover for two years demonstrated persistent astrogliosis (cortex and thalamus), though microgliosis was no longer evident (n=2). Aged mice subjected to pdMCAO also demonstrated gliosis in thalamus at PSD14, albeit to a lesser extent than young mice (n=5 each age). Finally, delayed treatment with memantine resulted in significantly attenuated gliosis and neuronal loss in the thalamus at PSD14 (young mice, n=9 each). Conclusions: These results further define gliosis in the mechanism of secondary injury and importantly demonstrate attenuation of secondary injury by delayed NMDA receptor antagonism.

Poncirin suppresses lipopolysaccharide (LPS)-induced microglial inflammation and ameliorates brain ischemic injury in experimental stroke in mice.

BackgroundBased on accumulating evidence, excessive activation of microglia-mediated inflammatory responses plays an essential role in ischemic stroke. Poncirin (Pon) exerts anti-hyperalgesic, anti-osteoporotic and anti-tumor effects on various diseases. However, the roles of Pon in microglial activation and the underlying mechanism have not been elucidated. This study aimed to explore whether Pon inhibits lipopolysaccharide (LPS)-induced microglial neuroinflammation and protects against brain ischemic injury in experimental stroke in mice.MethodsPrimary microglia cells were prepared from the cerebral cortices of 1- to 2-day-old C57BL/6J mice. Murine BV2 cells and primary microglia were stimulated with LPS and the effects of a non-cytotoxic concentration of Pon on LPS-stimulated pro-inflammatory factors were measured using real-time PCR and enzyme-linked immunosorbent assays (ELISAs). Western blot analyses were used for mechanistic studies. In an in vivo study, 8-week-old male C57BL/6J mice were subjected to focal cerebral ischemia through middle cerebral artery occlusion (MCAO). Pon (30 mg/kg, i.p.) or the same volume of saline was administered after the MCAO model was established, and the infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. We also evaluated animal behaviours, the expression of pro-inflammatory cytokines and microglial activation in the ischemic hemisphere.ResultsPon prevented the release of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6 and tumor necrosis factor-alpha (TNF-α) in both BV2 cells and primary microglia stimulated with LPS. The inhibitory effects of Pon were associated with the regulation of the ERK1/2, JNK and nuclear factor kappa B (NF-κB) signaling pathways. In mice that underwent MCAO, Pon administration decreased the lesion size and improved neurological deficits. Furthermore, Pon attenuated the production of inflammatory cytokines mainly by restraining microglial activation after ischemic stroke.ConclusionsBased on the findings from the present study, Pon provides neuroprotection through its anti-inflammatory effects on microglia and it may be a useful treatment for ischemic stroke.

Improved reperfusion following alternative surgical approach for experimental stroke in mice.

Background: Following ischemic stroke, recanalisation and restoration of blood flow to the affected area of the brain is critical and directly correlates with patient recovery. In vivo models of ischemic stroke show high variability in outcomes, which may be due to variability in reperfusion. We previously reported that a surgical refinement in the middle cerebral artery occlusion (MCAO) model of stroke, via repair of the common carotid artery (CCA), removes the reliance on the Circle of Willis for reperfusion and reduced infarct variability. Here we further assess this refined surgical approach on reperfusion characteristics following transient MCAO in mice. Methods: Mice underwent 60 min of MCAO, followed by either CCA repair or ligation at reperfusion. All mice underwent laser speckle contrast imaging at baseline, 24h and 48h post-MCAO. Results: CCA ligation reduced cerebral perfusion in the ipsilateral hemisphere compared to baseline (102.3 ± 4.57%) at 24h (85.13 ± 16.09%; P < 0.01) and 48h (75.04 ± 12.954%; P < 0.001) post-MCAO. Repair of the CCA returned perfusion to baseline (94.152 ± 2.44%) levels and perfusion was significantly improved compared to CCA ligation at both 24h (102.83 ± 8.41%; P < 0.05) and 48h (102.13 ± 9.34%; P < 0.001) post-MCAO. Conclusions: Our findings show CCA repair, an alternative surgical approach for MCAO, results in improved ischemic hemisphere perfusion during the acute phase.

Improved reperfusion following alternative surgical approach for experimental stroke in mice

Background: Following ischemic stroke, recanalisation and restoration of blood flow to the affected area of the brain is critical and directly correlates with patient recovery. In vivo models of ischemic stroke show high variability in outcomes, which may be due to variability in reperfusion. We previously reported that a surgical refinement in the middle cerebral artery occlusion (MCAO) model of stroke, via repair of the common carotid artery (CCA), removes the reliance on the Circle of Willis for reperfusion and reduced infarct variability. Here we further assess this refined surgical approach on reperfusion characteristics following transient MCAO in mice. Methods: Mice underwent 60 min of MCAO, followed by either CCA repair or ligation at reperfusion. All mice underwent laser speckle contrast imaging at baseline, 24h and 48h post-MCAO. Results: CCA ligation reduced cerebral perfusion in the ipsilateral hemisphere compared to baseline (102.3 ± 4.57%) at 24h (85.13 ± 16.09%; P < 0.01) and 48h (75.04 ± 12.954%; P < 0.001) post-MCAO. Repair of the CCA returned perfusion to baseline (94.152 ± 2.44%) levels and perfusion was significantly improved compared to CCA ligation at both 24h (102.83 ± 8.41%; P < 0.05) and 48h (102.13 ± 9.34%; P < 0.001) post-MCAO. Conclusions: Our findings show CCA repair, an alternative surgical approach for MCAO, results in improved ischemic hemisphere perfusion during the acute phase.

Improved reperfusion following alternative surgical approach for experimental stroke in mice

Background: Following ischemic stroke, recanalisation and restoration of blood flow to the affected area of the brain is critical and directly correlates with patient recovery. In vivo models of ischemic stroke show high variability in outcomes, which may be due to variability in reperfusion. We previously reported that a surgical refinement in the middle cerebral artery occlusion (MCAO) model of stroke, via repair of the common carotid artery (CCA), removes the reliance on the Circle of Willis for reperfusion and reduced infarct variability. Here we further assess this refined surgical approach on reperfusion characteristics following transient MCAO in mice. Methods: Mice underwent 60 min of MCAO, followed by either CCA repair or ligation at reperfusion. All mice underwent laser speckle contrast imaging at baseline, 24 h and 48 h post-MCAO. Results: CCA ligation reduced cerebral perfusion in the ipsilateral hemisphere compared to baseline (102.3 ± 4.57%) at 24 h (85.13 ± 16.09%; P &lt; 0.01) and 48 h (75.04 ± 12.954%; P &lt; 0.001) post-MCAO. Repair of the CCA returned perfusion to baseline (94.152 ± 2.44%) levels and perfusion was significantly improved compared to CCA ligation at both 24 h (102.83 ± 8.41%; P &lt; 0.05) and 48 h (102.13 ± 9.34%; P &lt; 0.001) post-MCAO. Conclusions: Our findings show CCA repair, an alternative surgical approach for MCAO, results in improved ischemic hemisphere perfusion during the acute phase.

Simultaneous alterations of oligodendrocyte-specific CNP, astrocyte-specific AQP4 and neuronal NF-L demarcate ischemic tissue after experimental stroke in mice

Ischemic stroke not only affects neurons, but also glial and vascular elements. The development of novel neuroprotective strategies thus requires an improved pathophysiological understanding of ischemia-affected cell types that comprise the ‘neurovascular unit’ (NVU). To explore spatiotemporal alterations of oligodendrocytes, astrocytes and neurons after experimental ischemic stroke, we applied a permanent middle cerebral artery occlusion model in mice for 4 and 24 h. Using fluorescence microscopy, the oligodendrocyte marker 2′,3′-cyclic nucleotide phosphodiesterase (CNP), the neuronal neurofilament light chain (NF-L) and the astroglial aquaporin-4 (AQP4) were analyzed in regional relation to one another.Immunofluorescence intensities of CNP and NF-L were simultaneously increased in the ischemic neocortex and striatum. AQP4 immunoreactivity was decreased in the ischemic striatum, which represents the initial and potentially strongest affected site of infarction. The more distant ischemic neocortex and infarct border zones exhibited areas with alternately increased or decreased AQP4 immunoreactivity, leading to an increase of fluorescence intensity in total.Further, deformed CNP-immunopositive processes were found around axonal spheroids, indicating a combined affection of oligodendrocytes and neurons due to ischemia. Importantly, altered AQP4 immunosignals were not limited to the ischemic core, but were also detectable in penumbral areas. This applies for CNP and NF-L also, since altered immunosignals of all three markers coincided regionally at both time points.In conclusion, the present study provides evidence for a simultaneous affection of oligodendrocytes, astrocytes and neurons after experimental focal cerebral ischemia. Consequently, CNP, AQP4 and NF-L immunofluorescence alterations can be utilized to identify ischemia-affected tissue. The simultaneity of the described alterations further strengthens the concept of interdependent NVU components and distinguishes NF-L, CNP and AQP4 as highly ischemia-sensitive elements. Consequently, future therapeutic approaches might influence stroke evolution via strategies simultaneously addressing both neuronal and glial functions.

Absence of MCP-induced Protein 1 Enhances Blood-Brain Barrier Breakdown after Experimental Stroke in Mice.

Focal cerebral ischemia can cause blood–brain barrier (BBB) breakdown, which is implicated in neuroinflammation and progression of brain damage. Monocyte chemotactic protein 1–induced protein 1 (MCPIP1) is a newly identified zinc-finger protein that negatively regulates inflammatory signaling pathways. We aimed to evaluate the impact of genetic MCPIP1 deletion on BBB breakdown and expression of BBB-related matrix metalloproteinases (MMPs) and tight junction proteins after cerebral ischemia/reperfusion (I/R) using MCPIP1-deficient (MCPIP1–/–) mice. Transient middle cerebral artery occlusion was induced in the MCPIP1–/– mice and their wild-type littermates for 2 h followed by reperfusion for 24 h. The degree of BBB breakdown was evaluated by injection of fluorescein isothiocyanate (FITC)-dextran. Quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were performed to compare the expression of MMPs and claudin-5 and zonula occludens-1 (ZO-1). MCPIP1 deficiency in mice resulted in enhanced leakage of FITC-dextran, increased expression of MMP-9/3, and reduced expression of claudin-5 and ZO-1 in the brain compared to that seen in their wild-type littermates subjected to cerebral I/R. These results demonstrate that absence of MCPIP1 exacerbates cerebral I/R-induced BBB disruption by enhancing the expression of MMP-9/3 and the degradation of claudin-5 and ZO-1, providing novel insights into the mechanisms underlying BBB breakdown after cerebral ischemia/reperfusion