Transient Middle Cerebral Artery Occlusion Model Research Articles

IPSC-derived mesenchymal stem cells attenuate cerebral ischemia-reperfusion injury by inhibiting inflammatory signaling and oxidative stress

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebralartery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.

High-frequency repetitive transcranial magnetic stimulation protects against cerebral ischemia/reperfusion injury in rats: Involving the mitigation of ferroptosis and inflammation.

Repetitive transcranial magnetic stimulation (rTMS) has been found to attenuate cerebral ischemia/reperfusion (I/R) injury. However, its effects and mechanism of action have not yet been clarified. It has been reported that cerebral I/R injury is closely associated not only with ferroptosis but also with inflammation. Hence, the current study aimed to investigate whether high-frequency rTMS attenuates middle cerebral artery occlusion (MCAO)-induced cerebral I/R injury and further to elucidate the mediatory role of ferroptosis and inflammation. The protective effects of rTMS on experimental cerebral I/R injury were investigated using transient MCAO model rats. Neurological scores and pathological changes of cerebral ischemic cortex were assessed to evaluate the effects of rTMS on cerebral I/R injury. The involvement of ferroptosis and that of inflammation were examined to investigate the mechanism underlying the effects of rTMS. High-frequency rTMS remarkably rescued the MCAO-induced neurological deficits and morphological damage. rTMS treatment also increased the mRNA and protein expression of glutathione-dependent peroxidase 4, decreased the mRNA and protein levels of acyl-CoA synthetase long-chain family member 4 and transferrin receptor in the cortex. Moreover, rTMS administration reduced the cerebrospinal fluid IL-1β, IL-6, and TNF-α concentrations. These findings implicated that high-frequency rTMS alleviates MCAO-induced cerebral I/R injury, and the underlying mechanism could involve the inhibition of ferroptosis and inflammation. Our study identifies rTMS as a promising therapeutic agent for the treatment of cerebral I/R injury. Moreover, the mechanistic insights into ferroptosis and inflammation advance our understanding of it as a potential therapeutic target for diseases beyond cerebral ischemia stroke.

Intravenous Infusion of Mesenchymal Stem Cells Enhances Therapeutic Efficacy of Reperfusion Therapy in Cerebral Ischemia

Reperfusion therapy is a standard therapeutic strategy for acute stroke. Non-favorable outcomes are thought to partially result from impaired microcirculatory flow in ischemic tissue. Intravenous infusion of mesenchymal stem cells (MSCs) reduces stroke volume and improves behavioral function in stroke. One suggested therapeutic mechanism is the restoration of the microvasculature. The goal of this study was to determine whether infused MSCs enhance the therapeutic efficacy of reperfusion therapy following stroke in rats. First, to establish a transient middle cerebral artery occlusion (MCAO) model displaying approximately identical neurologic function and lesion volume as seen in permanent MCAO (pMCAO) at day 7 after stroke induction, we transiently occluded the MCA for 90, 110, and 120 minutes. We found that the 110-minute occlusion met these criteria and was used as the transient MCAO (tMCAO) model. Next, 4 MCAO groups were used to compare the therapeutic efficacy of infused MSCs: (1) pMCAO+vehicle, (2) tMCAO+vehicle, (3) pMCAO+MSC, and (4) tMCAO+MSC. Our ischemic model was a unique ischemic model system in which both pMCAO and tMCAO provided similar outcomes during the study period in the groups without MSC infusion groups. Behavioral performance, ischemic volume, and regional cerebral blood flow (rCBF) using arterial spin labeling-magnetic resonance imaging and histologic evaluation of microvasculature was performed. The behavioral function, rCBF, and restoration of microvasculature were greater in group 4 than in group 3. Thus, infused MSCs facilitated the therapeutic efficacy of MCA reperfusion in this rat model system. Intravenous infusion of MSCs may enhance therapeutic efficacy of reperfusion therapy.

Celecoxib Treatment Improves Neurologic Deficit and Reduces Selective Neuronal Loss and Glial Response in Rats after Transient Middle Cerebral Artery Occlusion.

Areas of selective neuronal loss (SNL) represent the first morphologic signs of damage in the penumbra region and are considered putative targets for ischemic stroke therapy. We performed a novel assessment of measuring the effects of the anti-inflammatory agent celecoxib by analyzing simultaneously the different neural populations (neurons, astrocytes, and microglia cells) in SNL and non-SNL areas. Rats were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and treated with celecoxib 1 and 24 hours after ischemia. Infarct volume measurements and triple immunostaining of neurons (neuronal nuclear antigen), microglia (ionized calcium-binding adaptor molecule 1), and astroglia were performed after 12 and 48 hours of reperfusion. Motor response was tested by standard behavioral assays at 3, 12, 24, and 48 hours. Confocal analysis revealed that the percentage of SNL areas, microglia densities, and glial activation increased at 48 hours of reperfusion. Celecoxib treatment improved the neurologic deficit, reduced the infarct volume by 50% after 48 hours of reperfusion, and resulted in a reduced percentage of SNL areas and microglia and astroglia reactivity after 48 hours of reperfusion. This study proves, for the first time, that celecoxib presents postischemic neuroprotective effects in a transient MCAO model, prevents or delays the presence of SNL areas, and reduces glial activation.

Abstract WMP78: Brain Ischemia Induces Diversified Neuroantigen-Specific T Cell Responses That Exacerbate Brain Injury

Background: Autoimmune responses can occur when antigens from the central nervous system (CNS) are presented to lymphocytes in the periphery or CNS. However, whether autoimmune responses emerge after brain ischemia remains controversial. Similarly, it is debated whether genesis of autoimmune responses can impact outcome of ischemic stroke. We hypothesized that that brain ischemia can diversify T cell responses against CNS antigens, and that expanded CNS antigen-specific T cells can promote ischemic brain injury. Methods: We quantified the presence and status of T cells from brain slices of ischemic patients. Using a mouse strain that harbor a transgenic T cell receptor (TCR) to a CNS antigen, the myelin oligodendrocyte glycoprotein (MOG 35-55 ) epitope (2D2), we determined the anatomic location and involvement of antigen presentation cells in the development of T cell reactivity after brain ischemia and how T cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion (MCAO) and photothrombotic stroke models were used in this study. Results: By coupling transfer of labeled myelin oligodendrocyte glycoprotein (MOG) 35-55 -specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG 97-108 and MOG 103-125 in transient MCAO and photothrombotic stroke models. This reactivity and T cell activation occur in the brain and not in the peripheral lymphoid organs after ischemia. Also, microglia act as antigen-presenting cells (APCs) that effectively present MOG antigen, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T cell activation and MOG-specific T cells can be seen in the brain of ischemic stroke patients. Conclusion: Our findings suggest that brain ischemia activates and diversifies T cell responses locally, which exacerbates ischemic brain injury.

Precision Stroke Animal Models: the Permanent MCAO Model Should Be the Primary Model, Not Transient MCAO.

An argument for preclinical stroke research to make more use of the permanent middle cerebral artery occlusion (MCAO) model, rather than transient MCAO, is presented. Despite STAIR recommending permanent MCAO as the primary model, preclinical stroke research has not been listened. In 2012, Hossmann reported that 64% of the treatment studies for MCAO used prompt transient MCAO models and only 36% of the studies used permanent MCAO or gradual transient MCAO (i.e., embolic stroke model). Then, in 2014 and 2015, 88% of published basic science studies on large vessel occlusion used the transient MCAO model. However, this model only represents 2.5-11.3% of large vessel stroke patients. Therefore, the transient MCAO model, which mimics stroke with reperfusion, does not accurately reflect the majority of clinical stroke cases. Thus, once again, the argument for studying permanent MCAO as a primary model is made and supported.

DhHP‑6 attenuates cerebral ischemia‑reperfusion injury in rats through the inhibition of apoptosis.

As a novel reactive oxygen species (ROS) scavenger, deuterohemin His peptide-6 (DhHP-6) has been demonstrated to prolong the lifespan of Caenorhabditis elegans and has also exhibited protective effects in myocardial ischemia-reperfusion injury. Whether similar effects occur during cerebral ischemia-reperfusion (CIR) injury remains to be elucidated. The present study evaluated the function of DhHP-6 and its underlying mechanisms in a middle cerebral artery occlusion (MCAO) model in rats. The focal transient MCAO model was implemented using the Longa method of ischemia for 2 h followed by reperfusion for 22 h in male Wistar rats. DhHP-6 was administered at the onset of reperfusion via intraperitoneal injection. The infarct volume, brain edema, brain apoptosis and neurological function were evaluated 24 h following stroke. To further determine the role of DhHP-6 in CIR injury, the levels of ROS and malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and the protein expression levels of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), cleaved caspase-3, cytochrome c, Bcl-2 and phosphorylated-Akt/Akt were measured in ischemic cortex tissues. The results demonstrated that DhHP-6 significantly improved infarct volume, brain edema and neurological deficits, and reduced the percentage of TUNEL-positive cells. The levels of ROS and MDA were decreased, whereas no significant changes in the activities of SOD, CAT and GSH-Px were observed. The levels of Bax, cleaved caspase-3, and cytochrome c were downregulated, whereas the levels of Bcl-2 and p-Akt/Akt were upregulated. The results of the present study indicated that DhHP-6 may offer therapeutic potential for cerebral ischemia. The neuroprotective effects of DhHP-6 maybe mediated by its anti-oxidative properties, anti-apoptotic activities, or activation of the phosphoinositide 3-kinase/Akt survival pathway.

Macrophage-Derived Angiopoietin-Like Protein 2 Exacerbates Brain Damage by Accelerating Acute Inflammation after Ischemia-Reperfusion.

Ischemic stroke is a leading cause of death and disability worldwide. Several reports suggest that acute inflammation after ischemia-reperfusion exacerbates brain damage; however, molecular mechanisms underlying this effect remain unclear. Here, we report that MAC-3-positive immune cells, including infiltrating bone marrow-derived macrophages and activated microglia, express abundant angiopoietin-like protein (ANGPTL) 2 in ischemic mouse brain in a transient middle cerebral artery occlusion (MCAO) model. Both neurological deficits and infarct volume decreased in transient MCAO model mice established in Angptl2 knockout (KO) relative to wild-type mice. Acute brain inflammation after ischemia-reperfusion, as estimated by expression levels of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor alpha (TNF)-α, was significantly suppressed in Angptl2 KO compared to control mice. Moreover, analysis employing bone marrow chimeric models using Angptl2 KO and wild-type mice revealed that infiltrated bone marrow-derived macrophages secreting ANGPTL2 significantly contribute to acute brain injury seen after ischemia-reperfusion. These studies demonstrate that infiltrating bone marrow-derived macrophages promote inflammation and injury in affected brain areas after ischemia-reperfusion, likely via ANGPTL2 secretion in the acute phase of ischemic stroke.

Delivery of Hypoxia-Inducible Heme Oxygenase-1 Gene for Site-Specific Gene Therapy in the Ischemic Stroke Animal Model.

To reduce side effects due to non-specific expression, the heme oxygenase-1 (HO-1) gene under control of a hypoxia-inducible erythropoietin (Epo) enhancer (pEpo-SV-HO-1) was developed for site-specific gene therapy of ischemic stroke. pEpo-SV-HO-1 was constructed by insertion of the Epo enhancer into pSV-HO-1. Dexamethasone-conjugated polyamidoamine (PAMAM-Dexa) was used as a gene carrier. In vitro transfection assays were performed in the Neuro2A cells. In vivo efficacy of pEpo-SV-HO-1 was evaluated in the transient middle cerebral artery occlusion (MCAO) model. In vitro transfection assay with the PAMAM-Dexa/pEpo-SV-HO-1 complex showed that pEpo-SV-HO-1 had higher HO-1 gene expression than pSV-HO-1 under hypoxia. In addition, pEpo-SV-HO-1 reduced the level of apoptosis more efficiently than pSV-HO-1 in Neuro2A cells under hypoxia. For in vivo evaluation, the PAMAM-Dexa/pEpo-SV-HO-1 complex was injected into the ischemic brain of the transient MCAO model. pEpo-SV-HO-1 increased HO-1 expression and reduced the number of apoptotic cells in the ischemic brain, compared with the pSV-HO-1 injection group. As a result, the infarct volume was more efficiently decreased by pEpo-SV-HO-1 than by pSV-HO-1. pEpo-SV-HO-1 induced HO-1 gene expression and therapeutic effect in the ischemic brain. Therefore, pEpo-SV-HO-1 may be useful for site-specific gene therapy of ischemic stroke.

Poly-arginine peptides reduce infarct volume in a permanent middle cerebral artery rat stroke model

BackgroundWe recently reported that poly-arginine peptides have neuroprotective properties both in vitro and in vivo. In cultured cortical neurons exposed to glutamic acid excitotoxicity, we demonstrated that neuroprotective potency increases with polymer length plateauing at R15 to R18 (R = arginine resides). In an in vivo study in rats, we also demonstrated that R9D (R9 peptide synthesised with D-isoform amino acids) administered intravenously at a dose of 1000 nmol/kg 30 min after permanent middle cerebral artery occlusion (MCAO) reduces infarct volume. Based on these positive in vitro and in vivo findings, we decided to examine the neuroprotective efficacy of the L-isoform poly-arginine peptides, R12, R15 and R18 when administered at a dose of 1000 nmol/kg 30 min after permanent MCAO in the rat.ResultsAt 24 h post-MCAO, there was reduced total infarct volume for R12 (12.8 % reduction) and R18 (20.5 % reduction), but this reduction only reached statistical significance for R18. Brain slice analysis revealed significantly reduced injury in coronal slices 4 and 5 for R18, and slice 5 for R12. The R15 peptide had no effect on infarct volume. Peptide treatment did not reveal any statistical significant improvement in functional outcomes.ConclusionWhile these findings confirm the in vivo neuroprotective properties of poly-arginine peptides, additional dose studies are required particularly in less severe transient MCAO models so as to further assess the potential of these agents as a stroke therapy.

Progesterone treatment in two rat models of ocular ischemia.

To determine whether the neurosteroid progesterone, shown to have protective effects in animal models of traumatic brain injury, stroke, and spinal cord injury, is also protective in ocular ischemia animal models. Progesterone treatment was tested in two ocular ischemia models in rats: a rodent anterior ischemic optic neuropathy (rAION) model, which induces permanent monocular optic nerve stroke, and the middle cerebral artery occlusion (MCAO) model, which causes transient ischemia in both the retina and brain due to an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. Visual function and retinal histology were assessed to determine whether progesterone attenuated retinal injury in these models. Additionally, behavioral testing and 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining in brains were used to compare progesterone's neuroprotective effects in both retina and brain using the MCAO model. Progesterone treatment showed no effect on visual evoked potential (VEP) reduction and retinal ganglion cell loss in the permanent rAION model. In the transient MCAO model, progesterone treatment reduced (1) electroretinogram (ERG) deficits, (2) MCAO-induced upregulation of glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP), and (3) retinal ganglion cell loss. As expected, progesterone treatment also had significant protective effects in behavioral tests and a reduction in infarct size in the brain. Progesterone treatment showed protective effects in the retina following MCAO but not rAION injury, which may result from mechanistic differences with injury type and the therapeutic action of progesterone.

Abstract T P229: Deletion of Voltage Gated Proton Channel in Microglial Cells is Neurovascular Protective After Ischemic Brain Injury

Despite the failure of antioxidant treatments in clinical trials, the undoubted role of reactive oxygen species (ROS) in neurovascular damage after ischemic stroke calls for a more targeted approach. ROS production by microglia, the primary resident immune cells in the brain, is a key event of this process in ischemic stroke. Voltage gated proton channel, Hv1, is localized primarily to microglia and sustains NADPH oxidase activity. Deletion of Hv1 is neuroprotective after permanent middle cerebral artery occlusion (MCAO). We hypothesized that Hv1-mediated microglial ROS generation is also critical for vascular integrity and contributes to reperfusion injury after transient ischemic stroke. The wildtype (WT) and Hv1 knockout (KO) rats (n=4) were subjected to permanent or 3/24 h transient MCAO. The neurological deficiency, infarct, hemorrhagic transformation, and edema ratio were assessed. We found that in both permanent and transient MCAO model, KO rats develop smaller infarct, less vascular injury, edema, and hemorrhagic transformation, resulting in better short-term functional outcome. These results suggest that deletion of microglial Hv1 channel is vasculoprotective after ischemia/reperfusion and the underlying mechanisms need to be further studied.

Efficacy of liposome-encapsulated hemoglobin in a rat model of cerebral ischemia.

Use of liposome-encapsulated hemoglobin (LEH) for oxygen delivery in the treatment of cerebral ischemia has been studied previously and its expected benefits confirmed. However, the relationship between the timing of administration and the efficacy of LEH in cerebral ischemia has not been studied in detail. We therefore investigated the therapeutic time window of LEH by using a rat model of cerebral ischemia, as well as evaluating the contribution of oxygen delivery to the efficacy of LEH. Dose-dependent effects and the therapeutic time window of LEH were studied using models of transient and permanent middle cerebral artery occlusion (MCAO), respectively, in SD rats. LEH was intravenously administered at 0.5 h after the onset of ischemia in the transient MCAO model and at 0.5, 2, 4, or 6 h in the permanent MCAO model. Efficacy of LEH treatment was evaluated using the infarct volume, which was examined with 2,3,5-triphenyltetrazolium chloride staining and estimated by integrating the unstained areas in serial sections of cerebral tissue. Effects of oxygen delivery by LEH were examined immunohistochemically with pimonidazole to stain for areas of low oxygen tension in the tissue. LEH treatment dose-dependently reduced the cerebral infarct volume, which was especially significant in the cortical region at doses of over 60 mg hemoglobin (Hb)/kg. In rats with permanent MCAO, LEH administration at a dose of 300 mg Hb/kg at 0.5 h and 2 h after the onset of cerebral ischemia significantly reduced cerebral infarct volume. Furthermore, immunohistochemical staining with pimonidazole showed that the areas of cerebral tissue that were hypoxic and had abnormal histological structure were reduced after LEH treatment. These results indicated that LEH is efficacious in the treatment of cerebral infarction secondary to MCAO and that oxygen delivery to ischemic cerebral tissues by LEH administered early after the onset of cerebral ischemia contributes to this effect.

Contribution of Hypothermia and CB1 Receptor Activation to Protective Effects of TAK-937, a Cannabinoid Receptor Agonist, in Rat Transient MCAO Model

BackgroundCannabinoid (CB) receptor agonists are expected to alleviate ischemic brain damage by modulating neurotransmission and neuroinflammatory responses via CB1 and CB2 receptors, respectively. In a previous study, TAK-937, a novel potent and selective CB1 and CB2 receptor agonist, was shown to exert significant cerebroprotective effects accompanied by hypothermia after transient middle cerebral artery occlusion (MCAO) in rats. Sustained hypothermia itself induces significant neuroprotective effects. In the present studies, we examined the relative contribution of hypothermia and CB1 receptor activation to the cerebroprotective effects of TAK-937.Methodology/Principal FindingsUsing a multichannel brain temperature controlling system we developed, the brain temperature of freely moving rats was telemetrically monitored and maintained between 37 and 38°C during intravenous infusion of TAK-937 (100 µg/kg/h) or vehicle for 24 h after 2 h MCAO. AM251, a selective CB1 receptor antagonist, was administered intraperitoneally at 30 mg/kg 30 min before starting intravenous infusion of TAK-937 (100 µg/kg/h) for 24 h. Rats were sacrificed and their brains were isolated 26 h after MCAO in both experiments. When the hypothermic effect of TAK-937 was completely reversed by a brain temperature controlling system, the infarct-reducing effect of TAK-937 was attenuated in part, but remained significant. On the other hand, concomitant AM251 treatment with TAK-937 completely abolished the hypothermic and infarct-reducing effects of TAK-937.Conclusions/SignificanceWe conclude that the cerebroprotective effects of TAK-937 were at least in part mediated by induction of hypothermia, and mainly mediated by CB1 receptor activation.

Therapeutic effects of tetramethylpyrazine nitrone in rat ischemic stroke models

Free radical-mediated neuronal cell damage is an important pathological process in ischemic stroke. We have previously reported a novel dual-functional agent, 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of tetramethylpyrazine armed with anitrone moiety. In this report, we further evaluate TBN'stherapeutic parameters in a rat middle cerebral artery occlusion (MCAO) model. Its abilities to cross the blood-brain barrier, scavenge free radicals, and inhibit Ca(2+) influx were also investigated. TBN showed significant activity in both the transient MCAO (t-MCAO) and permanent MCAO (p-MCAO) stroke models in the rat. The therapeutic time window is 8 hr in the t-MCAO model. TBN readily crossed the blood-brain barrier and in vitro had strong activity in neutralizing ·OH, O(-)(2)·, and ONOO(-) and significantly decreased intracellular Ca(2+) concentration. TBN is a promising new treatment forischemic stroke, with multiple mechanisms of action. It blocks Ca(2+) overload and neutralizes ·OH, O(-)(2)·, and ONOO(-).

Abstract 3842: Angiographic Assessment Of Leptomeningeal Collateral Perfusion Parameters Following Experimental Transient Middle Cerebral Artery Occlusion

Objectives: This work aims to develop and validate an angiographically based quantitative assessment of leptomeningeal collateral perfusion (QLCP) in experimental reversible middle cerebral artery occlusion (MCAO). Methods: Pial collaterals were assessed during MCAO using an angiographically based transient MCAO model in eight mongrel dogs (20-30 kg). Angiographic images were analyzed using a custom-made MATLAB program which measured contrast density over time. Using bivariate linear fit analysis relative cerebral blood volume (rCBV), relative transit time (rTT) and relative cerebral blood flow (rCBF) derived from regions of interest (ROI) from the normal and abnormal hemispheres were extracted and compared to one hour post reperfusion MRI based infarct volume calculations and leptomeningeal collateral scoring using previously published methods. Results: QLCP was reproducibly assessed but variably predictive of infarct volume on one hour post reperfusion mean diffusivity maps using rCBV (p<0.0001; r2=0.937), rTT (p = 0.05, r2 = 0.494), and rCBF (p=0.0024, r2=0.807). Leptomeningeal collateral scoring variably correlated with rCBV (p<0.0001, r2 = 0.948), rTT (p=0.0285, r2= 0.578) and rCBF (p0.0021, r2= 0.817). Conclusion: QLCP was validated in an experimental MCAO model based on correlation with a leptomeningeal collateral scoring system. QLCP assessment of rCBV is a better predictor for infarct volume than rTT or rCBF in a transient MCAO model. It is noteworthy that an angiographically based assessment of rCBV, rTT and rCBF differs from CT and MRI based assessments. In particular , the time frame used and the relative density of the vasculature on the derived color maps differ ( figure 1). figure 1: QLCP derived cerebral blood volume map. The boxes indicate regions of interest for analysis.

MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor, ameliorates ischemic brain damage in a non-human primate model

Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) in response to DNA damage is considered to play a crucial role in the development of post-ischemic neuronal injury, such as ischemic stroke. The present study was undertaken to clarify the beneficial effects of MP-124, a novel PARP-1 inhibitor, on neurological deficits and cerebral infarcts following middle cerebral artery occlusion (MCAO) in the monkey. The effects of MP-124 on cerebral infarcts and neurological deficits in monkeys were investigated in permanent MCAO (pMCAO) and transient MCAO (tMCAO) models. In a dose-dependency study, the neurological deficits and cerebral infarct volume were assessed at 28 h after pMCAO. MP-124 significantly reduced the total infarct volume, including that in the cortex/white matter and striatum, at doses of 0.3, 1 and 3 mg/kg/h by 22, 54 and 64%, respectively. In addition, MP-124 at all doses significantly reduced the overall neurological deficits. Such ameliorative effects of MP-124 were observed in female as well as male monkeys. In the therapeutic time window (TTW) study, the neurological deficits and cerebral infarct volume were assessed at several time points after pMCAO or tMCAO. Treatment with MP-124 at 3 and 6 h after MCAO significantly ameliorated not only the neurological deficits but also the infarct volume. MP-124 is thought to exhibit neuroprotective effects with a broad TTW regardless of sex in MCAO models. Such findings suggest that MP-124 may be beneficial for the treatment of acute ischemic stroke.

Neuroprotective effects of a novel water-soluble poly(ADP-ribose) polymerase-1 inhibitor, MP-124, in in vitro and in vivo models of cerebral ischemia

Cerebral ischemia induces excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), leading to neuronal cell death and the development of post-ischemic dysfunction. Blockade of PARP-related signals during cerebral ischemia has become a focus of interest as a new therapeutic approach for acute stroke treatment. The purpose of the present study was to examine the pharmacological profiles of MP-124, a novel water-soluble PARP-1 inhibitor, and its neuroprotective effects on ischemic injury in vitro and in vivo. MP-124 demonstrated competitive inhibition of the PARP-1 activity of human recombinant PARP-1 enzyme (Ki = 16.5 nmol/L). In P388D 1 cells, MP-124 inhibited the LDH leakage induced by H 2O 2 in a concentration-dependent manner. (IC 50 = 20.8 nmol/L). In rat primary cortical neurons, MP-124 also inhibited the NAD depletion and polymerized ADP-ribose formation induced by H 2O 2 exposure. Moreover, we investigated the neuroprotective effects of MP-124 in rat permanent and transient stroke models. In the rat permanent middle cerebral artery occlusion (MCAO) model, MP-124 was administered intravenously for 24 h from 5 min after the onset of MCAO. MP-124 (1, 3 and 10 mg/kg/h) significantly inhibited the cerebral infarction in a dose-dependent manner (18, 42 and 48%). In rat transient MCAO model, MP-124 was administered intravenously from 30 min after the onset of MCAO. MP-124 (3 and 10 mg/kg/h) significantly reduced the infarct volume (53% and 50%). The present findings suggest that MP-124 acts as a potent neuroprotective agent in focal ischemia and its actions can be attributed to a reduction in NAD depletion and PAR formation.

Characterization of Gadolinium-Based Dynamic Susceptibility Contrast Perfusion Measurements in Permanent and Transient MCAO Models with Volumetric Based Validation by CASL

Perfusion imaging is crucial in imaging of ischemic stroke to determine 'tissue at risk' for infarction. In this study we compared the volumetric quantification of the perfusion deficit in two rat middle-cerebral-artery occlusion (MCAO) models using two gadolinium-based contrast agents (P1152 (Guerbet) and Magnevist (Bayer-Schering, Pittsburgh, PA, USA)) as compared with our well established continuous arterial spin labeling (CASL) perfusion imaging technique. Animals underwent either permanent MCAO or transient MCAO with 80-min reperfusion. Imaging was performed at four different time points after MCAO. A region-of-interest (ROI) analysis of the subregions of the ischemic zone (core, penumbra, transient reversal (TR), and sustained reversal (SR)) using P1152 showed significant reduction in blood flow in the core and TR subregions relative to the penumbral and SR subregions while occluded. After reperfusion, a significant increase in blood flow was recorded at all time points after reperfusion in all regions except TR. From the ROI analysis the threshold for the penumbra was determined to be -62+/-11% and this value was subsequently used for quantification of the volumetric deficit. The ischemic volume as defined by dynamic susceptibility contrast (DSC), was only statistically different from the CASL-derived ischemic volume when using Magnevist at post-reperfusion time points.

Embryonic neural stem cells transplanted in middle cerebral artery occlusion model of rats demonstrated potent therapeutic effects, compared to adult neural stem cells

Cell therapy using stem cells is awaited by stroke patients with impaired movement and cognitive functions, although intravenous alteplase-administration ameliorated outcomes of patients receiving the therapy within 3 h of onset. In this study, we explored the therapeutic effects of neural progenitor cells (NPC) upon middle cerebral artery occlusion (MCAO) model of rats with exploration of the differences between adult and embryonic NPCs in therapeutic effects. GFP-labeled adult or embryonic NPCs were transplanted for transient MCAO model of rats at 1h after reperfusion. Rats were examined behaviorally using limb placement test, rotarod test and cylinder test with neuroradiological assessment using magnetic resonance imaging (MRI). Consequently after euthanasia, rats were immunohistochemically investigated to explore graft survival and immune reaction. MRI of rats receiving NPCs revealed significant reduction of infarct volumes, compared to vehicle-treated rats with corresponding behavioral amelioration. The transplanted cells were surviving in rats receiving NPCs, although the number of embryonic NPCs was significantly higher than that of adult NPCs. Iba-1-positive inflammatory cells of rats receiving adult NPCs were prominent, compared to those receiving embryonic NPCs, which might be a rationale for the differences between rats receiving adult and embryonic NPCs in the number of surviving NPCs. On the contraries, adult NPCs surely demonstrated therapeutic effects with a few surviving cells, thus indicating that the therapeutic effects might be due to trophic/growth factor-secretion from transplanted NPCs, rather than replacement of damaged host neurons. Therapeutic effects of NPCs for MCAO model of rats were clarified in this study. Transplantation of NPCs will be a hopeful strategy for stroke patients, although further studies are required for the patient safety and underlying mechanisms.