Middle Cerebral Artery Occlusion Procedure Research Articles

The novel FAAH inhibitor, MCH1, reduces the infarction area in the motor cortex-related region but does not affect the sensorimotor function or memory and spatial learning in rats exposed to transient middle cerebral artery occlusion

Macamides, amides of fatty acids first isolated from maca (Lepidium meyenii) are potentially responsible for the reduction of ischemic injury in the stroke animal model followed by maca extract administration. This deduction comes from its ability to inhibit the fatty acid amide hydrolase activity, an enzyme related to the endocannabinoid anandamide hydrolysis. However, no study about the effects of isolated macamides on in-vivo models has been published yet. Our objective was to evaluate the effect of a 10-day 30 mg/kg i.p. MCH1 administration, the macamide with the higher FAAH inhibition capability, on the neurological recovery and brain infarction area of Sprague-Dawley rats exposed to the transient middle cerebral artery occlusion (MCAO) model. Our results showed that the group receiving MCH1 for 10 days did not improve Garcia’s neurological score compared to receiving the vehicle only. Likewise, the MCH1 group did not improve their sensorimotor dysfunction as indicated by the latency to detect and remove the tape from the contralateral forepaw in the adhesive removal test, and a similar number of errors with the contralateral forepaw in the foot fault test compared to the vehicle group at the 10th day. Evaluation of the spatial memory and learning using the Barnes test showed longer latency to reach the escape box in the Vehicle and MCH1 groups compared to the control group (no MCAO) only in the retrieval test, while no effect of MCAO procedure or MCH1 administration was observed in the reversal learning test. Despite the lack of behavioral effect of MCH1, analysis of the infarcted areas in the brain using the 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining method in the seven consecutive coronal sections revealed that the infarcted area in the first (bregma + 4.2 mm) and fifth (bregma −3.8 mm) coronal sections of the MCAO + MCH1 group remained similar to the Control group. These results provide evidence that MCH1 can limit damage from ischemic stroke, although it is not reflected in neurological or sensorimotor behavior and spatial learning and memory.

Active fraction of Polyrhachis vicina (Roger) alleviated cerebral ischemia/reperfusion injury by targeting SIRT3-mediated mitophagy and angiogenesis

BackgroundDamaged mitophagy and impaired angiogenesis involve in the pathogenic development of ischemic stroke. Active fraction of Polyrhachis vicina (Roger) (AFPR) showed great potential on neurological disease with it's remarkable anti-inflammatory and anti-oxidative effects. PurposeThis study designed to clarify the correlation between Pink1/Parkin-mediated mitophagy and angiogenesis after stroke, and to elucidate the role of SIRT3 in regulating mitophagy and angiogenesis, and to address the mechanism of AFPR on promoting mitophagy and angiogenesis in microvessels endothelium of ischemic brain. Study designA cerebral ischemia/reperfusion (CIR) rat model was developed by middle cerebral artery occlusion procedure. bEnd.3 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic CIR process. Neurological function, mitophagy and angiogenesis related indicators were measured. SIRT3 siRNA and 3-MA were used to verify the interaction between SIRT3-mediated mitophagy and angiogenesis. MethodsCIR rats were orally treated with AFPR (8 and 4 g raw drug /kg) and Nimodipine (10.8 mg/kg) for 12 days to mimic the recovery phase post-stroke. The neurological function assessment, TTC staining, HE staining, TUNEL staining and Nissl staining were performed to assess neuroprotective effects of AFPR against CIR. Then CD31-labeled microvessel density in brain was visualized and quantified by immunofluorescence staining. Mitochondrial ultrastructure was assessed by transmission electron microscope scanning. Expressions of relative proteins,e.g. SIRT3, Pink1, Parkin, LC3-II, p62, VEGFA, involving in mitophagy and angiogenesis, were detected by Western blotting analysis. In vitro, bEnd.3 cells were cultured with AFPR or in combination of autophagy inhibitor 3-MA during the reoxygenation. Then cell viability, and LDH releasing were measured. Angiogenic indicators,such as migration and tube formation activity, VEGFA level were determined. To assess effects of AFPR on mitophagy, mitophagy-related proteins were detected, as well as the autophagosome engulfment and lysosome degradation of mitochondria. To address the role of SIRT3, deacetylation activity of SIRT3 was validated by detecting acetylated FOXO3A level with co-immunoprecipitation (Co-IP) assay. Pre-treatment of siRNA or combination use of 3-MA were used to verify the detailed mechanism. ResultsAFPR remarkably reduced neurological scores and infarct size, alleviated neuron apoptosis in cortex, and increased Nissl density in hippocampus of CIR rats. In addition, AFPR significantly promoted angiogenesis by increasing microvessels density and VEGFA expressions, increased SIRT3 expression, and activated Pink1/Parkin mediated mitophagy. In bEnd.3 cells, the combination use of 3-MA and AFPR further demonstrated that AFPR might promote angiogenesis after OGD/R injury through activating Pink1/Parkin mediated mitophagy. Co-IP assay suggested AFPR reduced acetylated FOXO3A level. This might be correlated with an elevation of SIRT3 expression and it's deacetylation activity. SIRT3 siRNA pretreatment significantly abolished the activation of mitophagy through Pink1/Parkin axis, eventually inhibited angiogenesis. ConclusionAFPR promoted angiogenesis through activating mitophagy after cerebral ischemia reperfusion, which might partially involved in the amelioration of SIRT3-mediated regulation on Pink1/Parkin axis. Our study will shed new light on the role of SIRT3 in ischemic brain, especially in regulating mitophagy and angiogenesis after stroke.

Feature-based Quality Assessment of Middle Cerebral Artery Occlusion Using 18F-Fluorodeoxyglucose Positron Emission Tomography.

In animal experiments, ischemic stroke is usually induced through middle cerebral artery occlusion (MCAO), and quality assessment of this procedure is crucial. However, an accurate assessment method based on 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is still lacking. The difficulty lies in the inconsistent preprocessing pipeline, biased intensity normalization, or unclear spatiotemporal uptake of FDG. Here, we propose an image feature-based protocol to assess the quality of the procedure using a 3D scale-invariant feature transform and support vector machine. This feature-based protocol provides a convenient, accurate, and reliable tool to assess the quality of the MCAO procedure in FDG PET studies. Compared with existing approaches, the proposed protocol is fully quantitative, objective, automatic, and bypasses the intensity normalization step. An online interface was constructed to check images and obtain assessment results.

SRC3 Promotes the Protective Effects of Bone Marrow Mesenchymal Stem Cell Transplantation on Cerebral Ischemia in a Mouse Model.

Mesenchymal stem cells (MSCs) derived from the bone marrow (BM) are reported to protect against ischemic brain injury. This study aimed to investigate whether the steroid receptor cofactor 3 (SRC3) was involved in MSC-induced neuroprotection. BM-MSCs were isolated from wild-type (WT) and SRC3 knockout (SRC3-/-) mice and transplanted into mice with middle cerebral artery occlusion (MCAO). The MSC identification and differentiation were determined by flow cytometry and Alizarin Red S staining after osteogenic and adipogenic stimulations. The effects of MSCs on brain injury were assessed by brain water content, modified neurological severity score (mNSS), Morris water maze test, and open field test. Finally, the effects of MSCs on MCAO-induced oxidative stress were assessed by measuring the levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) and mRNA levels of SOD1, SOD2, and CAT. We found that SRC3 deficiency did not impact the MSC identification or osteogenic and adipogenic differentiation. MSC-SRC3-/- transplantation in mice that underwent the MCAO procedure exhibited diminished effects on suppression of brain edema, neurological deficits, cognitive disruption, locomotor impairment, and anxiety compared to comparable levels of MSC-WT. Finally, MSC-WT transplantation inhibited MCAO-induced oxidative stress, and the effects were significantly attenuated in MCAO mice transplanted with MSC-SRC3-/-. MSCs suppressed the MCAO-induced upregulation of MDA activity and the inhibition of SOD, GSH, SOD1, SOD2, and CAT levels, and SRC3-deficient MSCs showed significantly reduced effects. Our results indicate that SRC3 plays an important role in mediating the neuroprotective effects of MSCs in mice that experienced ischemic stroke.

NLRP3 Inflammasome: A Potential Target in Isoflurane Pretreatment Alleviates Stroke-Induced Retinal Injury in Diabetes.

Ischemic stroke remains a devastating disease which is the leading cause of death worldwide. Visual impairment after stroke is a common complication which may lead to vision loss, greatly impacting life quality of patients. While ischemic stroke is traditionally characterized by a blockage of blood flow to the brain, this may coincide with reduced blood flow to the eye, resulting in retinal ischemia and leading to visual impairment. Diabetes increases the risk of ischemic stroke and induces diabetic retinopathy; the latter may be more sensitive to the ischemic retinal injury. In diabetic status, the underlying mechanism in stroke-induced retinal injury has not been fully clarified. The NLR pyrin domain containing 3 (NLRP3) inflammasome is an important activator of inflammation, which may play a critical role in catalyzing and forming certain pro-inflammatory cytokines in both cerebral and retinal ischemia. Isoflurane has been demonstrated to inhibit the activation of the NLRP3 inflammasome and show neuroprotective effects. In this study, we established a diabetic mouse model and performed the middle cerebral artery occlusion procedure to induce ischemic stroke. Our results revealed that cerebral ischemia-induced retinal injury in the diabetic model. Isoflurane pretreatment alleviated the cerebral and retinal injury after ischemic stroke. Of note, isoflurane pretreatment inhibited the NLRP3 inflammasome activation in the retina, indicating that isoflurane pretreatment may provide substantial retinal protection in stroke-induced retinal injury in diabetes.

A low-cost mouse cage warming system provides improved intra-ischemic and post-ischemic body temperature control – Application for reducing variability in experimental stroke studies

BackgroundBrain temperature is a strong determinant of ischemic stroke injury. For this reason, tight management of brain or body temperature (Tcore) in experimental rodent stroke models is recommended to improve the rigor and reproducibility of outcomes. However, methods for managing Tcore during and after stroke vary widely in approach and effectiveness. New MethodWe developed a low-cost warm ambient air cage (WAAC) system to provide improved temperature control during the intra-ischemic and post-ischemic recovery periods. The system is incorporated into standard holding cages for maintaining Tcore during the intra-ischemic period as well as for several hours into the recovery period. Results and Comparison with Existing MethodsWe compared the WAAC system with a commonly used heat support method, consisting of a cage on a heating pad. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) stroke model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra- ischemic period (60 min) and post-ischemic period (3 h). Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54 % lower in the WAAC system group. ConclusionsMice and other small rodents are highly vulnerable to heat loss during and after the MCAo procedure. The WAAC system provides more precise and controlled Tcore maintenance compared with frequently used induction heating methods in mice undergoing the MCAo stroke model. The improved temperature control should enhance experimental rigor and reduce the number of experimental animals needed.

Xanthoangelol alleviates cerebral ischemia reperfusion injury in rats.

Ischemia/reperfusion (I/R) injury accounts to be a prime cause of neurological deficit following stroke. This study aimed to explore the neuro-protective effects of Xanthoangelol (XAG) on I/R-induced injury in both in vivo and in vitro models. Our data demonstrated that XAG can shrink infarct size and brain edema in middle cerebral artery occlusion (MCAO) model. In addition, XAG was capable of alleviating the neurological deficit in rats that have undergone MCAO procedure. Meanwhile, antiapoptotic activities of XAG against I/R-induced neuronal injury were evidenced and further illustrated that XAG elicits antiapoptotic activities by suppressing excessive oxidative stress via nuclear factor erythroid-2-related factor 2 activation. Overall, our study revealed that XAG displayed the potential to be utilized as a neuroprotective agent against I/R-induced neurological injury.

Technical report: Simple method of animal stroke model of luminal occlusion of middle cerebral artery in Indonesia.

Background:Although there are many experimental studies describing the suture method of middle cerebral artery occlusion (MCAO) in rats, this is still a new procedure in Indonesia and the techniques for applying this stroke model in animal research are not well known. There has been a perception in Indonesian scientific community that the technique would be difficult and require advanced equipment. The aim of this study is to demonstrate that it is possible to perform the technique with minimal resources using simple method and basic surgical loupe equipment.Methods:A total of 30 male Wistar rats, aged 6 months, weighing 250 g–400 g Wistar rats, were obtained from the Bandung Biofarma Pasteur. Preliminary trials were performed to gain an understanding of the detailed anatomy of the animals and to master the techniques. An ×8 loupe magnification was used for all surgical steps in this study except taking of surgical operation photos. The procedures applied simple methods, using two loop temporary knots instead of any animal vascular clips.Results:After an extensive training period, two of the 30 rats died within 4 weeks after the procedure. The effects of MCAO were confirmed clinically and by hematoxylin-eosin staining pathology slides.Conclusion:With simple methods, this MCAO procedure could be implemented in developing countries such as Indonesia without the requirement for advanced equipment.

Involvement of d-amino acid oxidase in cerebral ischaemia induced by transient occlusion of the middle cerebral artery in mice.

d-Amino acid oxidase (DAAO) is a flavine adenine dinucleotide-containing flavoenzyme and specifically catalyses oxidative deamination of d-amino acids. This study aimed to explore the association between increased cerebral DAAO expression or enzymic activity and the development of cerebral ischaemia. A mouse model of transient (90 min) middle cerebral artery occlusion (MCAO) was established, and western blotting, enzymic activity assay, and fluorescent immunostaining techniques were used. The expression and enzymic activity of DAAO increased over time in the cortical peri-infarct area of the mice subjected to transient MCAO. The DAAO was specifically expressed in astrocytes, and its double immunostaining with the astrocytic intracellular marker, glial fibrillary acidic protein, in the cortical peri-infarct area was up-regulated following ischaemic insult, with peak increase on Day 5 after MCAO. Single intravenous injection of the specific and potent DAAO inhibitor Compound SUN reduced the cerebral DAAO enzymic activity and attenuated neuronal infarction and neurobehavioural deficits with optimal improvement apparent immediately after the MCAO procedure. The neuroprotective effect was dose dependent, with ED50 values of 3.9-4.5mg·kg-1 . Intracerebroventricular injection of the DAAO gene silencer siRNA/DAAO significantly reduced cerebral DAAO expression and attenuated MCAO-induced neuronal infarction and behavioural deficits. Our results, for the first time, demonstrated that increased cerebral astrocytic DAAO expression and enzymic activity were causally associated with the development of neuronal destruction following ischaemic insults, suggesting that targeting cerebral DAAO could be a potential approach for treatment of neurological conditions following cerebral ischaemia.

Simultaneous knockdown of TLR2 and TLR4 after focal cerebral ischemia facilitates the post‐stroke neurological recovery in rats

Ischemic stroke is the primary reason for serious long‐term disability. Our recent studies demonstrated that the suppression of toll‐like receptors (TLRs) 2 and 4 alone or in combination attenuated the post‐ischemic inflammation and brain damage. The purpose of this study is to investigate the effect of the simultaneous knockdown of both TLR2 and TLR4 on post‐stroke neurological recovery in young adult male rats. Transient focal cerebral ischemia was induced to male Sprague‐Dawley rats by middle cerebral artery occlusion (MCAO) procedure. A silicone rubber‐coated monofilament suture was introduced into the internal carotid artery via the external and common carotid arteries and pushed until it reaches the origin of the middle cerebral artery followed by removal of the monofilament after 2 hours to initiate reperfusion. Appropriate cohorts of rats were treated with a nanoparticle formulations of TLR2shRNA (T2sh) and TLR4shRNA (T4sh) expressing plasmids (1mg/kg each of T2sh and T4sh) or scrambled sequence inserted vector (SVsh; vehicle) expressing plasmids (2 mg/kg) intravenously via tail vein immediately after reperfusion. The rats from both the cohorts were subjected to various standard neurological tests such as modified neurological severity scores (mNSS), modified adhesive removal (sticky‐tape), beam walking and accelerating rotarod performance before MCAO procedure and at days 1, 3, 5, 7, and 14 after reperfusion. The sensory and motor functions of vehicle treated stroke‐induced rats were markedly impaired when tested at 1d reperfusion followed by a gradual improvement over 14 days. Prevention of the post‐ischemic induction of TLR2 and TLR4 attenuated the degree of neurological impairment and facilitated the magnitude of functional recovery. Therefore, we conclude that the post‐stroke induction of TLR2 and TLR4 in rat ischemic brain contributes to neurological deficits.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MMP‐12 knockout mouse: Is it a relevant animal model to study the role of MMP‐12 in post‐stroke brain damage?

We recently reported for the first time the detrimental role of matrix metalloproteinase (MMP)‐12 on post‐stroke brain damage. We hypothesize that the post‐stroke MMPs dysregulation is similar across species and the genetic deletion of MMP‐12 does not affect the post‐stroke expression of other MMPs. We tested our hypothesis by determining the pre‐ and post‐ischemic expression profile of MMPs in MMP‐12 knockout and respective wild type mice. Focal cerebral ischemia was induced in MMP‐12 knockout and wild type mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischemia, reperfusion was initiated by removing the monofilament. One‐day after reperfusion, ischemic brain tissues from various groups of mice were collected for real‐time PCR analysis and PCR analysis followed by agarose gel electrophoresis. Although the post‐stroke regulation of several MMPs was altered in wild type mice, huge upregulation of MMP‐12 (~763 fold over sham) was noticed in mice similar to rats as reported earlier by our group. Unlike in rats, MMP‐8 was prominently upregulated (~280 fold over sham) in ischemic mice brains. As expected, MMP‐12 expression is absent in the ischemic brains of MMP‐12 knockout mice. Except the expression of MMP‐9, the expression of several other MMPs such as MMP‐1a, −2, −8, −10, −15, −16, −17, −21 and −28 was altered in the ischemic brain of MMP‐12 knockout mice as compared to wild type mice under similar experimental conditions. In addition, the expression of various MMPs in MMP‐12 knockout mice under non‐ischemic conditions was altered as compared to the wild type mice that did not subject to ischemia. Based on our results, we conclude that the mice with genetic deletion of MMP‐12 are constitutive in nature that warrants the attention of their use in elucidating the role of MMP‐12 in post‐stroke brain damage.Support or Funding InformationThis work was supported in part by the OSF HealthCare Foundation and OSF Illinois Neurological Institute, Peoria, IL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract TP106: Sex Differences After Mesenchymal Stem Cell Treatment on Post-Stroke Neurological Outcome

Introduction: A major problem exists in stroke management because of the sex differences in frequency and severity of disease and in treatment outcomes. Our recent studies demonstrated the potential of human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) in attenuating the post-stroke brain damage. Methods: Sprague-Dawley rats of both sexes were obtained and randomly assigned to various groups. Rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure by inserting a silicone rubber-coated monofilament suture followed by fourteen days of reperfusion. The body weight of rats on the day of the MCAO procedure and the size of the monofilament for male and female rats were standardized to obtain the same depth of post-stroke infarction. Previously standardized effective dose (0.25 million cells/animal) of HUCB-MSCs were intravenously injected via tail vein 24 hours post-MCAO to the designated groups of rats. Rats were subjected to neurological evaluation at various reperfusion time points by modified neurological severity scores ( mNSS), modified adhesive removal ( sticky-tape) beam walking and accelerating Rotarod performance tests. Results: The fourteen-day post-stroke survival period in males and females is approximately 20% and 70%, respectively. Females are more resistant to stroke-induced mortality. Although the difference in post-stroke percent survival is prominent in both sexes, there was no change in the body weight gain of both sexes. It was evident from the mNSS and sticky-tape tests that the magnitude of natural neurological recovery is the same in both sexes. However, the improvement in coordination and integration of motor function in females is better than males as evidenced by other tests. Stem cell treatment prevented mortality in males without any change in the treatment-induced body weight gain. Further, stem cell treatment improved neurological recovery in males, and the coordination and integration of motor function in females. Conclusions: Sex differences exist in post-stroke mortality and neurological function. Stem cell treatment improved neurological recovery and there was no sex difference in the magnitude of stem cell treatment improved neurological function

PH-sensitive amide proton transfer effect dominates the magnetization transfer asymmetry contrast during acute ischemia-quantification of multipool contribution to in vivo CEST MRI.

To determine the origins of in vivo magnetization transfer asymmetry contrast during acute ischemic stroke, particularly in the diffusion lesion, perfusion lesion, and their mismatch using a middle cerebral artery occlusion rat model of acute stroke. Adult male Wistar rats underwent multiparametric MRI of diffusion, perfusion, T1 , and amide proton transfer (APT) imaging at 4.7 T following a middle cerebral artery occlusion procedure. A multipool Lorentzian model, including the nuclear Overhauser effect, magnetization transfer, direct water saturation, amine and amide chemical exchange saturation transfer effects, was applied for Z-spectrum fitting to determine the sources of in vivo magnetization transfer asymmetry following acute stroke. We showed that changes in amine chemical exchange saturation transfer (2 ppm) and APT (3.5 ppm) effects, particularly the APT MRI effect, dominate the commonly used magnetization transfer asymmetry analysis and hence confer pH sensitivity to APT imaging of acute stroke. Also, the nuclear Overhauser effect and magnetization transfer show small changes that counteracted each other, contributing less than 0.3% to magnetization transfer asymmetry at 3.5 ppm. Moreover, we showed that diffusion lesion had worsened acidosis from perfusion/diffusion lesion mismatch (P < 0.05). The study complements recent in vivo quantitative chemical exchange saturation transfer work to shed light on the sensitivity and specificity of endogenous APT MRI to tissue acidosis. Magn Reson Med 79:1602-1608, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

상백피(桑白皮) 메탄올 추출물 전처치가 일과성 허혈에 의한 생쥐의 뇌 손상에 미치는 영향

Objectives : Mori Radicis Cortex (MRC), the root epidermis of Morus alba L., has been traditionally used to treat lung-related diseases in Korean Medicine. The common of MRC is Mulberry bark Morus bark, and it's pharmaceutical properties and taste are known as sweet and cold, and it promotes urination and reduce edema by reducing heat from the lungs and soothe asthma. In the present study, anti-apoptotic mechanism of MRC in middle cerebral artery occlusion (MCAO) model in mice. Methods : Two-hundred grams of MRC was extracted with methanol at room temperature for 5 days, and this was repeated one time. After filtration, the methanol was removed using vacuum evaporator, then stored at <TEX>$-20^{\circ}C$</TEX> until use. C57BL/6 male mice were housed in an environment with controlled humidity, temperature, and light cycle. In order to determine beneficial effects of MRC on ischemia induced brain damage, infarct volume, neurological deficit scores, activities of several apoptosis-related proteins such as caspase-8, -9, Bcl-xL in MCAO-induced brains of mice were analyzed. Mice in MRC-treated groups were orally administered 30, 100, or 300 mg/kg of body weight for three consecutive days before commencing the MCAO procedure. Results : Pre-treatment of MRC significantly reduced infarct volume in MCAO subjected mice applied with 300 mg/kg of MRC methanol extract, and MRC effectively inhibited Bcl-xL reduction and caspase-9 activation caused by MCAO-induced brain damage. Conclusions : MRC showed neuro-protective effects by regulating apoptosis-related protein signals, and it can be a potential candidate for the therapy of ischemia-induced brain damage.

Exercício físico promove neuroproteção estrutural e funcional em ratos com isquemia cerebral

Objetive. The present study investigated the engine performance and of volume of cerebral infarction in sedentary rats or who practiced physical exercise before and after cerebral ischemia, promoted by transitory middle cerebral artery occlusion (MCAO). Method. Forty-eight rats were randomized into two groups, one subjected to MCAO and the other on the absence of ischemia. Both groups were subdivid­ed into AI (n=6): animals that did exercise prior to MCAO; DI (n=6): animals that did exercise after MCAO; ADI (n=6): animals that did exercise before and after MCAO and SI (n=6): animals that did not exercise. Physical training was performed on the mat for 6 weeks (30min/day), 5 days a week. Results. The results showed weight loss in animals of group SI after the MCAO procedure (81 days) and at the end of the experiment the of ADI group (p&lt;0.0001). The animals in groups AI and ADI showed lower frequency of errors in the test misstep and parallel bars in 76 days. Conclusion. At the end of the experiment the ADI group showed better motor performance in func­tional tests and reduced volume of cerebral infarction.

Nafamostat mesilate attenuates transient focal ischemia/reperfusion-induced brain injury via the inhibition of endoplasmic reticulum stress

Nafamostat mesilate (NM), a serine protease inhibitor, has a broad range of clinical applications that include use as an anticoagulant during hemodialysis in cerebral hemorrhage patients, as a hemoperfusion anticoagulant for patients with intravascular coagulation, hemorrhagic lesions, and hemorrhagic tendencies, and for the improvement of acute pancreatitis. However, the effects of NM on acute cerebral ischemia have yet to be investigated. Thus, the present study utilized a rat model in which transient middle cerebral artery occlusion (MCAO) was used to induce ischemic injury to investigate the effects of NM on infarct volume and histological and biological changes. NM (1mg/kg) was intravenously administered prior to and after the MCAO procedure. Compared to control rats, the administration of NM significantly decreased infarct size and the extent of brain edema after the induction of focal ischemia via MCAO. Additionally, NM treatment attenuated MCAO-induced neuronal degeneration and activation of microglia and astrocytes. NM treatment also inhibited the MCAO-induced expression levels of glucose-regulated protein 78 (GRP78), CATT/EBP homologous protein (CHOP), and p-eukaryotic initiation factor 2α (eIF2α), which are endoplasmic reticulum (ER) stress markers, in the cerebral cortex. The present findings demonstrate that NM exerts neuroprotective effects in the brain following focal ischemia via, at least in part, the inhibition of ER stress.

Abstract 168: Intra-arterial Stem Cell Treatment Reduces Ischemic Brain Injury In Reproductively Senescent Female Rats

Background: Cell therapy is emerging as a promising treatment for stroke. We recently we demonstrated the efficacy of intra-arterial delivery of MSCs (IA MSCs) at 24h after a reversible middle cerebral artery occlusion (MCAo). Our study also identified a maximum tolerated dose of MSCs that could be delivered IA without compromising middle cerebral artery flow. The IA approach avoids first-pass trapping of MSCs in the lungs and liver as seen with intravenous delivery. Intra-arterial treatment is also minimally invasive and widely available in clinical practice and thus appealing for clinical translation. Since majority of ischemic strokes in women occur after onset of menopause it is crucial that we test the efficacy of IA MSCs in reproductively senescent females consistent with STAIR recommendations. We aimed to validate the efficacy of IA MSCs in reproductively senescent female rats. Methods: Retired breeder female (9-11 months; 280-350 g) Sprague-Dawley rats showing estrous acyclicity were exposed to MCAo (90 min). A day later, rats were treated with IA MSCs (1x10^5 cells) or phosphate-buffered saline (PBS). MSCs or PBS treated rats were sacrificed at 28-30 days for infarct volume measurement using histology. To test motor function, the rotarod test was performed. Rats were trained for 3 consecutive days for the rotarod test before undergoing the MCAo procedure. The mean duration (in seconds) on the device was recorded from 3 rotarod measurements 1 day before surgery. The rats were tested at 1, 7, 15 and 28-30 days after MCAo. Results: We observed significantly lower mean infarct volume in the MSC-treated group (12 ± 3 mm3; n=6) compared to the PBS-treated group (29 ± 7 mm3; Mean ± SEM; n=4, p&lt;0.05). Treatment at 1 day after MCAO with MSCs significantly improved functional recovery, as evidenced by improved rotarod test results and neurological scores at 7, 15 and 30 days (P&lt;0.05) compared with the PBS-treated group. Conclusions: Intra-arterial stem cell treatment reduces ischemic brain injury and improves functional outcomes in reproductively senescent female rats. Validating the efficacy of IA MSC treatment using a reproductively senescent animal stroke model suggests high potential for future clinical translation in a population at high risk for stroke.

Interleukin-33 ameliorates ischemic brain injury in experimental stroke through promoting Th2 response and suppressing Th17 response

Ischemic stroke causes brain injury with activation of an inflammatory response that can contribute to clinical impairment. As a novel cytokine of the interleukin-1 (IL-1) family, IL-33 has been suggested to be involved in regulating pathophysiology and inflammatory responses in the central nervous system (CNS). However, the role and underlying mechanisms of IL-33 in ischemic stroke remain poorly understood. Here, adult male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) for stroke induction. The MCAO procedure resulted in the enhanced Th1 and Th17 immune responses from 6h after transient cerebral ischemia/reperfusion even up to day 3. Meanwhile, the protein and mRNA level of IL-33 expression was significantly decreased at 6h and 72h, but not at 24h after MCAO. Moreover, recombinant mouse IL-33 administration substantially attenuated ischemic brain damage and neurological deficit at 24h and 72h, but not at 6h after MCAO. Interestingly, the reduced CNS inflammation in IL-33-treated MCAO mice may be at least partly due to an induced immuno-shift of Th cells from Th1 to Th2 response and suppressing Th17 immune response. These findings demonstrate that IL-33 can play a protective role after MCAO and may be a new target for therapy of ischemic stroke.

Development and characterization of a Yucatan miniature biomedical pig permanent middle cerebral artery occlusion stroke model

BackgroundEfforts to develop stroke treatments have met with limited success despite an intense need to produce novel treatments. The failed translation of many of these therapies in clinical trials has lead to a close examination of the therapeutic development process. One of the major factors believed to be limiting effective screening of these treatments is the absence of an animal model more predictive of human responses to treatments. The pig may potentially fill this gap with a gyrencephalic brain that is larger in size with a more similar gray-white matter composition to humans than traditional stroke animal models. In this study we develop and characterize a novel pig middle cerebral artery occlusion (MCAO) ischemic stroke model.MethodsEleven male pigs underwent MCAO surgery with the first 4 landrace pigs utilized to optimize stroke procedure and 7 additional Yucatan stroked pigs studied over a 90 day period. MRI analysis was done at 24 hrs and 90 days and included T2w, T2w FLAIR, T1w FLAIR and DWI sequences and associated ADC maps. Pigs were sacrificed at 90 days and underwent gross and microscopic histological evaluation. Significance in quantitative changes was determined by two-way analysis of variance and post-hoc Tukey’s Pair-Wise comparisons.ResultsMRI analysis of animals that underwent MCAO surgery at 24 hrs had hyperintense regions in T2w and DWI images with corresponding ADC maps having hypointense regions indicating cytotoxic edema consistent with an ischemic stroke. At 90 days, region of interest analysis of T1 FLAIR and ADC maps had an average lesion size of 59.17 cc, a loss of 8% brain matter. Histological examination of pig brains showed atrophy and loss of tissue, consistent with MRI, as well as glial scar formation and macrophage infiltration.ConclusionsThe MCAO procedure led to significant and consistent strokes with high survivability. These results suggest that the pig model is potentially a robust system for the study of stroke pathophysiology and potential diagnostics and therapeutics.

Decreased expression of neuronal Per-Arnt-Sim domain protein 4 gene in the hippocampus of a post-stroke depression rat model

Neuronal Per-Arnt-Sim domain protein 4 (NPAS4) is important in regulating transcription and function in the limbic system and in brain development. Post-stroke depression (PSD) is a common complication following a stroke. Furthermore, organic damage as a result of a stroke affects the restoration of nerve function and indicates that hippocampal neural activity may be associated with PSD. A PSD rat model was established via a middle cerebral artery occlusion procedure, which was combined with isolation and chronic unexpected mild stress, and was used to investigate the expression of the NPAS4 gene in the hippocampus. The neurological deficit and behavior were evaluated and NPAS4 mRNA expression was measured by semi-quantitative reverse transcription-polymerase chain reaction; furthermore, the association with cognitive impairment was analyzed. The PSD rats displayed neuropsychopathic disorders and the NPAS4 mRNA expression levels in the hippocampus were significantly lower in the depression and PSD groups compared with the control group. Therefore, the present study identified that NPAS4 expression was decreased in the hippocampus of PSD rats.