Animal Stroke Research Articles

Determination of Vascular Reactivity of Middle Cerebral Arteries from Stroke and Spinal Cord Injury Animal Models Using Pressure Myography.

Stroke and other neurovascular derangements are main causes of global death. They, along with spinal cord injuries, are responsible for being the principal cause of disability due to neurological and cognitive problems. These problems then lead to a burden on scarce financial resources and societal care facilities as well as have a profound effect on patients' families. The mechanism of action in these debilitating diseases is complex and unclear. An important component of these problems arises from derangement of blood vessels, such as blockage due to clotting/embolism, endothelial dysfunction, and overreactivity to contractile agents, as well as alteration in endothelial permeability. Moreover, the cerebro-vasculature (large vessels and arterioles) is involved in regulating blood flow by facilitating auto-regulatory processes. Moreover, the anterior (middle cerebral artery and the surrounding region) and posterior (basilar artery and its immediate locality) regions of the brain play a significant role in triggering the pathological progression of ischemic stroke particularly due to inflammatory activity and oxidative stress. Interestingly, modifiable and non-modifiable cardiovascular risk factors are responsible for driving ischemic and hemorrhagic stroke and spinal cord injury. There are different stroke animal models to examine the pathophysiology of middle cerebral and basilar arteries. In this context, arterial myography offers an opportunity to determine the etiology of vascular dysfunction in these diseases. Herein, we describe the technique of pressure myography to examine the reactivity of cerebral vessels to contractile and vasodilator agents and a prelude to stroke and spinal cord injury.

Cryopreservation of Bone Marrow Mononuclear Cells Alters Their Viability and Subpopulation Composition but Not Their Treatment Effects in a Rodent Stroke Model.

The systemic administration of autologous bone marrow (BM) derived mononuclear cells (MNCs) is under investigation as a novel therapeutic modality for the treatment of ischemic stroke. Autologous applications raise the possibility that MNCs could potentially be stored as a banked source. There have been no studies that investigate the effects of cryopreservation of BM-MNCs on their functional abilities in stroke models. In the present study, C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAo) for 60 minutes and then divided into two treatment groups: fresh MNCs versus cryopreserved MNCs. BM-MNCs were collected at 22 hours after MCAo and were stored in liquid nitrogen for 12 months in cryopreserved MNCs group. BM-MNCs cellular viability, composition, and phenotype of the various subpopulations of mice BM-MNCs were evaluated by flow cytometry, and the behavioral recovery of stroke animals was tested with freshly harvested MNCs versus cryopreserved MNCs by corner test and ladder rung test. We found that long-term cryopreservation negatively impacts the cellular viability of bone marrow MNCs. Cryopreservation also alters the cellular composition of various subpopulations within the MNCs. However, despite the changes observed in cryopreserved cells, both fresh and frozen MNCs have similar beneficial effect on behavioral and histological outcomes.

Histone modification in activated microglia during ischemic stroke

Event Abstract Back to Event Histone modification in activated microglia during ischemic stroke Radhika Patnala1, Thiruma Arumugam2 and S. Thameem Dheen1* 1 National University of Singapore, Department of Anatomy, Yong Loo Lin School of Medicine, Singapore 2 National University of Singapore, Department of Physiology, Yong Loo Lin School of Medicine, Singapore Stroke pathogenesis is influenced by the microglia-mediated inflammatory response in the brain. While, healthy microglia are involved in surveillance and protection of the brain, a chronic activation of microglia, as seen in ischemic stroke, aggravates neuropathology and neurotoxicity. Hence, understanding the molecular and epigenetic mechanisms of microglial activation will aid in development of therapeutic strategies that mitigate microglia-mediated neurotoxicity in neuropathologies including ischemic stroke. In this study, we analysed the epigenetic regulation, in particular, the histone modification Histone 3-lysine 9-acetylation (H3K9ac), and its involvement in microglial activation in response to ischemia. H3K9ac has been found to be upregulated in microglia in the peri-infarct and infarct zones after ischemic stroke. The H3K9ac upregulation in microglia in response to 1 hr ischemia with 24hr reperfusion was observed in different regions of the brain. A similar upregulation was observed in lipopolysaccharide (LPS)-activated microglia in the adult rat brain, indicating that the H3K9ac upregulation is consistently associated with microglial activation. It is well established that Histone Deacetylases (HDAC) regulate H3K9ac. In this study, HDAC inhibition altered H3K9ac enrichment and transcription at pro-inflammatory (TNF-alpha, iNOS, STAT 1, IL6) and anti-inflammatory (STAT3 and IL-10) gene promoters in activated microglia in vitro. In the ischemic stroke animal model, HDAC inhibitors were found to downregulate TNF-alpha and iNOS expression in activated microglia, up-regulate IL10 expression and improve neuronal survival, indicating the neuroprotective ability of HDAC inhibitors via microglia. With the advent of HDAC inhibitors entering clinical trials, this study highlights the potential therapeutic value of HDAC inhibitors in treating microglia-mediated neuroinflammation and neurotoxicity in ischemic stroke. Keywords: Microglia, Stroke, Neuroinflammation, Epigenetic regulation, histone modification Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 6: Neuron-Glia Crosstalk in Healthy and Diseased Brain Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Patnala R, Arumugam T and Dheen S (2016). Histone modification in activated microglia during ischemic stroke. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00026 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. S. Thameem Dheen, National University of Singapore, Department of Anatomy, Yong Loo Lin School of Medicine, Singapore, Singapore, antstd@nus.edu.sg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Radhika Patnala Thiruma Arumugam S. Thameem Dheen Google Radhika Patnala Thiruma Arumugam S. Thameem Dheen Google Scholar Radhika Patnala Thiruma Arumugam S. Thameem Dheen PubMed Radhika Patnala Thiruma Arumugam S. Thameem Dheen Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Mitochondrial Impairment in Cerebrovascular Endothelial Cells is Involved in the Correlation between Body Temperature and Stroke Severity.

Stroke is the second leading cause of death worldwide. The prognostic influence of body temperature on acute stroke in patients has been recently reported; however, hypothermia has confounded experimental results in animal stroke models. This work aimed to investigate how body temperature could prognose stroke severity as well as reveal a possible mitochondrial mechanism in the association of body temperature and stroke severity. Lipopolysaccharide (LPS) compromises mitochondrial oxidative phosphorylation in cerebrovascular endothelial cells (CVECs) and worsens murine experimental stroke. In this study, we report that LPS (0.1 mg/kg) exacerbates stroke infarction and neurological deficits, in the mean time LPS causes temporary hypothermia in the hyperacute stage during 6 hours post-stroke. Lower body temperature is associated with worse infarction and higher neurological deficit score in the LPS-stroke study. However, warming of the LPS-stroke mice compromises animal survival. Furthermore, a high dose of LPS (2 mg/kg) worsens neurological deficits, but causes persistent severe hypothermia that conceals the LPS exacerbation of stroke infarction. Mitochondrial respiratory chain complex I inhibitor, rotenone, replicates the data profile of the LPS-stroke study. Moreover, we have confirmed that rotenone compromises mitochondrial oxidative phosphorylation in CVECs. Lastly, the pooled data analyses of a large sample size (n=353) demonstrate that stroke mice have lower body temperature compared to sham mice within 6 hours post-surgery; the body temperature is significantly correlated with stroke outcomes; linear regression shows that lower body temperature is significantly associated with higher neurological scores and larger infarct volume. We conclude that post-stroke body temperature predicts stroke severity and mitochondrial impairment in CVECs plays a pivotal role in this hypothermic response. These novel findings suggest that body temperature is prognostic for stroke severity in experimental stroke animal models and may have translational significance for clinical stroke patients - targeting endothelial mitochondria may be a clinically useful approach for stroke therapy.

Porphyromonas gingivalis infection modifies oral microcirculation and aortic vascular function in the stroke-prone spontaneously hypertensive rat (SHRSP)

The functional modulation of vascular endothelial cells associated with stroke and periodontal disease has not yet been clarified. The objective of this study is to analyze the vascular endothelial function of periodontitis and stroke animal models. We examined endothelial function and gingival blood flow in oral microcirculation in vivo and measured the isometric tension in vitro of the aorta in animal models for lifestyle-related diseases, such as periodontitis and stroke. Gingival reactive hyperemia (GRH) was measured using laser Doppler flowmetry. Wistar Kyoto rats (WKY) were used as control animals; Porphyromonas gingivalis (P. gingivalis) infected WKY (WKY + Pg) as the periodontitis model; stroke-prone spontaneously hypertensive rat (SHRSP) as the stroke model; and a final group consisting of P. gingivalis infected SHRSP (SHRSP + Pg). Furthermore, for each group, the relaxation of descending aortic ring preparations was measured using a force transducer. The GRH was estimated by maximum response (peak), time taken for the maximum response to fall to one half (T1/2), and increased total amount of blood flow (mass). The relative change in T1/2 and mass increased in SHRSP + Pg compared to WKY. However, mass significantly increased in WKY (758.59 ± 88.21 ml/min/100 g s to 1755.55 ± 226.10 ml/min/100 g s) and SHRSP (1214.87 ± 141.61 ml/min/100 g s to 2674.32 ± 675.48 ml/min/100 g s) after treatment with acetylcholine. In addition, T1/2 and mass significantly increased in WKY + Pg (624.18 ± 96.36 ml/min/100 g s to 2629.90 ± 612.01 ml/min/100 g s) and SHRSP + Pg (1116.36 ± 206.24 ml/min/100 g s to 1952.76 ± 217.39 ml/min/100 g s) after treatment with nitroglycerin. Furthermore, the endothelium-dependent relaxation of ring preparations, evoked by acetylcholine, was attenuated in SHRSP compared with WKY, but not in SHRSP + Pg. This attenuation effect in SHRSP could be prevented by superoxide dismutase pretreatment. Our results suggest altered endothelial function may occur in gingival tissue in animal models experiencing both periodontitis and stroke. Therefore, these results indicate the disruption of vascular function in oral microcirculation may be caused by the interaction between the oxidative stress induced by periodontitis and nitric oxide in periodontitis, similar to the interactions present in stroke cases.

Intravenous Bone Marrow Stem Cell Grafts Preferentially Migrate to Spleen and Abrogate Chronic Inflammation in Stroke.

Background and Purpose—Adult stem cell therapy is an experimental stroke treatment. Here, we assessed homing and anti-inflammatory effects of bone marrow stromal cells (hBMSCs) in chronic stroke.Methods—At 60 days post stroke, adult Sprague–Dawley rats received intravenous hBMSCs (4×106 labeled or nonlabeled cells) or vehicle (saline). A sham surgery group served as additional control. In vivo imaging was conducted between 1 hour and 11 days post transplantation, followed by histological examination.Results—Labeled hBMSCs migrated to spleen which emitted significantly higher fluorescent signal across all time points, especially during the first hour, and were modestly detected in the head region at the 12 hours and 11 days, compared with nonlabeled hBMSCs and vehicle-infused stroke animals, or sham (P<0.05). At 11 days post transplantation, ex vivo imaging confirmed preferential hBMSC migration to the spleen over the brain. Hematoxylin and eosin staining revealed significant 15% and 30% reductions in striatal infarct and peri-infarct area, and a trend of rescue against neuronal loss in the hippocampus. Unbiased stereology showed significant 75% and 60% decrements in major histocompatibility complex II–activated inflammatory cells in gray and white matter, and a 43% diminution in tumor necrosis factor-α cell density in the spleen of transplanted stroke animals compared with vehicle-infused stroke animals (P<0.05). Human antigen immunostaining revealed 0.03% hBMSCs survived in spleen and only 0.0007% in brain. MSC migration to spleen, but not brain, inversely correlated with reduced infarct, peri-infarct, and inflammation.Conclusions—hBMSC transplantation is therapeutic in chronic stroke possibly by abrogating the inflammation-plagued secondary cell death.

Time-Dependent Protection of CB2 Receptor Agonist in Stroke.

Recent studies have indicated that type 2 cannabinoid receptor (CB2R) agonists reduce neurodegeneration after brain injury through anti-inflammatory activity. The purpose of this study was to examine the time-dependent interaction of CB2R and inflammation in stroke brain. Adult male rats were subjected to right middle cerebral artery occlusion (MCAo). CB2R mRNA expression was significantly elevated >20 fold on day 2, peaked >40-fold on day 5, and normalized on day 10 post-stroke. Inflammatory markers IBA1 and TLR4 were significantly upregulated 15 fold until day 5 after MCAo. Because of the delayed upregulation of CB2R and IBA1, we next treated animals daily with CB2R agonist AM1241 or anti-inflammatory PPAR-γ agonist pioglitazone from 2 to 5 days after MCAo. Delayed treatment with pioglitazone significantly reduced abnormal neurological scores and body asymmetry as well as brain infarction in stroke animals. No behavioral improvement or reduction in brain infarction was found in animals receiving AM1241. Pioglitazone, but not AM1241, significantly reduced IBA1 expression in the stroke cortex, suggesting that delayed treatment with AM1241 failed to alter ischemia-mediated IBA-1 upregulation. In contrast, pretreatment with AM1241 significantly reduced brain infarction and neurological deficits. In conclusion, our data support a time-dependent neuroprotection of CB2 agonist in an animal model of stroke. Delayed post- treatment with PPAR-γ agonist induced behavioral recovery and microglial suppression; early treatment with CB2R agonist suppressed neurodegeneration in stroke animals.

Identification of proteins in hyperglycemia and stroke animal models

BackgroundStroke is a major cause of disability and death in adults. Diabetes mellitus is a metabolic disorder that strongly increases the risk of severe vascular diseases. This study compared changes in proteins of the cerebral cortex during ischemic brain injury between nondiabetic and diabetic animals. MethodsAdult male rats were injected with streptozotocin (40 mg/kg) via the intraperitoneal route to induce diabetes and underwent surgical middle cerebral artery occlusion (MCAO) 4 wk after streptozotocin treatment. Cerebral cortex tissues were collected 24 h after MCAO and cerebral cortex proteins were analyzed by two-dimensional gel electrophoresis and mass spectrometry. ResultsSeveral proteins were identified as differentially expressed between nondiabetic and diabetic animals. Among the identified proteins, we focused on the following metabolism-related enzymes: isocitrate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, adenosylhomocysteinase, pyruvate kinase, and glucose-6-phosphate isomerase (neuroleukin). Expression of these proteins was decreased in animals that underwent MCAO. Moreover, protein expression was reduced to a greater extent in diabetic animals than in nondiabetic animals. Reverse transcription–polymerase chain reaction analysis confirmed that the diabetic condition exacerbates the decrease in expression of metabolism-related proteins after MCAO. ConclusionsThese results suggest that the diabetic condition may exacerbate brain damage during focal cerebral ischemia through the downregulation of metabolism-related proteins.

Ischemic lesions localized to the medial prefrontal cortex produce selective deficits in measures of executive function in rats

Ischemic stroke is one of the leading causes of neurological disability worldwide, and it has been estimated that about one quarter of stroke survivors experience some measurable long-term cognitive impairments. Many higher order cognitive deficits occur because of damage to the prefrontal cortex (PFC), which is one of the main areas of the brain responsible for executive functioning in mammals. Currently, there are few animal models that examine the effects of stroke on executive function. In this study we used bilateral micro-injections (1μl) of the vasoconstricting peptide endothelin-1 (ET-1) into the medial PFC in male Sprague-Dawley rats (or vehicle control, N=17–18 per group) in order to model ischemic lesions in the medial PFC. The effects of these lesions on executive function were assessed using tests of set-shifting and temporal object recognition. ET-1 injections in the medial PFC resulted in replicable and specific lesions within the PFC with an average infarct volume of 16.63±2.71mm3. The ischemic lesions resulted in specific contextual set-shifting deficits within the maze, including an increased number of trials to criterion and a significant difference in learning curves. However, no deficits in temporal order memory processing were noted between sham and stroke animals. We conclude that ischemic lesions localized to the mPFC result in selective but not generalized deficits in executive function in rats.

V37. Interactions between primary and secondary motor areas for recovered hand functions after stroke

Objectives Goal oriented hand movements are the final product of complex interplay between multiple cortical regions of the frontoparietal cortex. Following stroke, recovery of function might be related to structural and functional modifications in the surviving brain networks. Strikingly, the ventral premotor cortex (PMv) plays a crucial role in the sensorimotor processing engaged in shaping finger movements and shares extensive reciprocal projections to the primary motor cortex (M1), making the PMv a promising structure involved in hand motor recovery after stroke. By instance, mapping studies in stroke animal models consistently showed that improvement in forelimb motor performance was associated with reorganization within the PMv. However, precise functional interactions between the PMv and the M1 in the process of hand motor recovery have not been investigated in stroke patients. Paired-pulse transcranial magnetic stimulation (pp-TMS) was used in order to evaluate PMv-M1 interactions in stroke patients and healthy controls. To further disentangle whether recovery-related changes are specific to PMv-M1 or apparent in all interactions between primary and secondary motor areas after stroke, connectivity between posterior parietal cortex (PPC) and M1 have been also assessed. Methods 22 patients (aged 62.3 ± 9.8 SD) with mild to severe unilateral hand motor deficit and 20 healthy controls (aged 59.7 ± 20.9 SD) participated in the present study. Within experiment-1, PMv-M1 interactions were investigated by using neuronavigated-ppTMS in both the affected and unaffected hemisphere, considering 6 interstimulus intervals (ISI; 2, 4, 6, 8, 10 and 15 ms) between conditioning and test pulse. In experiment-2, PPC-M1 interactions were investigated targeting two PPC regions: the anterior intraparietal sulcus (aIPS) and the caudal intraparietal sulcus (cIPS). Results Experiment-1: A consistent difference between patients and controls, with a group by ISI interaction, has been observed for both the affected (F = 14.9, p Conclusions The present pp-TMS approach enabled us to assess in vivo the specific contributions of individual brain areas to recovery of function after stroke. These findings demonstrate changes in interareal connectivity between the PMv and the M1 associated with recovered hand motor function. These data support a paramount role of the PMv within the process of functional reorganization and successful hand motor recovery, as suggested in previous animal work, contributing significantly to the understanding of the mechanisms underlying cortical reorganization after focal brain lesions.

MiR-335 Regulates Hif-1α to Reduce Cell Death in Both Mouse Cell Line and Rat Ischemic Models.

Hypoxia inducible factor-1α facilitates cellular adaptation to hypoxic conditions. Hence its tight regulation is crucial in hypoxia related diseases such as cerebral ischemia. Changes in hypoxia inducible factor-1α expression upon cerebral ischemia influence the expression of its downstream genes which eventually determines the extent of cellular damage. MicroRNAs are endogenous regulators of gene expression that have rapidly emerged as promising therapeutic targets in several diseases. In this study, we have identified miR-335 as a direct regulator of hypoxia inducible factor-1α and as a potential therapeutic target in cerebral ischemia. MiR-335 and hypoxia inducible factor-1α mRNA showed an inverse expression profile, both in vivo and in vitro ischemic conditions. Given the biphasic nature of hypoxia inducible factor-1α expression during cerebral ischemia, miR-335 mimic was found to reduce infarct volume in the early time (immediately after middle cerebral artery occlusion) of embolic stroke animal models while the miR-335 inhibitor appears to be beneficial at the late time of stroke (24 hrs after middle cerebral artery occlusion). Modulation of hypoxia inducible factor-1α expression by miR-335 also influenced the expression of crucial genes implicated in neurovascular permeability, cell death and maintenance of the blood brain barrier. These concerted effects, resulting in a reduction in infarct volume bring about a beneficial outcome in ischemic stroke.

Dexamethasone-Conjugated Polyamidoamine Dendrimer for Delivery of the Heme Oxygenase-1 Gene into the Ischemic Brain.

Heme oxygenase-1 (HO-1) has anti-apoptotic and anti-inflammatory effects. In this study, the HO-1 gene was delivered into the brain using dexamethasone-conjugated polyamidoamine generation 2 (PAMAM G2-Dexa) for the treatment of ischemic stroke. PAMAM G2-Dexa formed stable complexes with plasmid DNA (pDNA). The pDNA delivery efficiency of PAMAM G2-Dexa was higher than that of polyethylenimine (PEI25k, 25 kDa), dexamethasone-conjugated PEI (PEI-Dexa), and PAMAM G2 in Neuro2A cells. Therapeutic effect of PAMAM G2-Dexa/pHO-1 complexes was evaluated in a stroke animal model. PAMAM G2-Dexa delivered pHO-1 more efficiently into the ischemic brain than PEI25k and PEI-Dexa with higher therapeutic effect. Therefore, PAMAM G2-Dexa/pHO-1 complexes may be useful for ischemic stroke gene therapy.

Global proteomic analysis of brain tissues in transient ischemia brain damage in rats.

Ischemia-reperfusion injury resulting from arterial occlusion or hypotension in patients leads to tissue hypoxia with glucose deprivation, which causes endoplasmic reticulum (ER) stress and neuronal death. A proteomic approach was used to identify the differentially expressed proteins in the brain of rats following a global ischemic stroke. The mechanisms involved the action in apoptotic and ER stress pathways. Rats were treated with ischemia-reperfusion brain injuries by the bilateral occlusion of the common carotid artery. The cortical neuron proteins from the stroke animal model (SAM) and the control rats were separated using two-dimensional gel electrophoresis (2-DE) to purify and identify the protein profiles. Our results demonstrated that the SAM rats experienced brain cell death in the ischemic core. Fifteen proteins were expressed differentially between the SAM rats and control rats, which were assayed and validated in vivo and in vitro. Interestingly, the set of differentially expressed, down-regulated proteins included catechol O-methyltransferase (COMT) and cathepsin D (CATD), which are implicated in oxidative stress, inflammatory response and apoptosis. After an ischemic stroke, one protein spot, namely the calretinin (CALB2) protein, showed increased expression. It mediated the effects of SAM administration on the apoptotic and ER stress pathways. Our results demonstrate that the ischemic injury of neuronal cells increased cell cytoxicity and apoptosis, which were accompanied by sustained activation of the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways. Proteomic analysis suggested that the differential expression of CALB2 during a global ischemic stroke could be involved in the mechanisms of ER stress-induced neuronal cell apoptosis, which occurred via IRE1-alpha/TRAF2 complex formation, with activation of JNK1/2 and p38 MAPK. Based on these results, we also provide the molecular evidence supporting the ischemia-reperfusion-related neuronal injury.

Proper housing conditions in experimental stroke studies-special emphasis on environmental enrichment.

Environmental enrichment provides laboratory animals with novelty and extra space, allowing different forms of multisensory stimulation ranging from social grouping to enhanced motor activity. At the extreme end of the spectrum, one can have a super-enriched environment. Environmental enrichment is believed to result in improved cognitive and sensorimotor functions both in naïve rodents and in animals with brain lesions such as those occurring after a stroke. Robust behavioral effects in animals which have suffered a stroke are probably related not only to neuronal plasticity in the perilesional cortex but also in remote brain areas. There is emerging evidence to suggest that testing restorative therapies in an enriched environment can maximize treatment effects, e.g., the perilesional milieu seems to be more receptive to concomitant pharmacotherapy and/or cell therapy. This review provides an updated overview on the effect of an enriched environment in stroke animals from the practical points to be considered when planning experiments to the mechanisms explaining why combined therapies can contribute to behavioral improvement in a synergistic manner.

Forebrain neuronal specific ablation of p53 gene provides protection in a cortical ischemic stroke model

Cerebral ischemic injury involves death of multiple cell types at the ischemic sites. As a key regulator of cell death, the p53 gene has been implicated in the regulation of cell loss in stroke. Less focal damage is found in stroke animals pre-treated with a p53 inhibitor or in traditional p53 knockout (ko) mice. However, whether the p53 gene plays a direct role in regulating neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 ko mice, we utilized a neuronal specific conditional ko mouse line (CamcreTRP53loxP/loxP) to achieve forebrain neuronal specific deletion of p53 and examined the role of the p53 gene in ischemia-induced cell death in neurons. Expression of p53 after stroke is examined using the immunohistochemical method and the outcome of stroke is examined by analysis of infarction size and behavioral deficits caused by stroke. Our data showed that p53 expression is upregulated in the ischemic region in neuronal cells in wildtype (wt) mice but not in CamcreTRP53loxP/loxP ko mice. Deletion of the p53 gene in forebrain neurons results in a decreased infarction area in ko mice. Locomotor behavior, measured in automated activity chambers, showed that CamcreTRP53loxP/loxP ko mice have less locomotor deficits compared to wt mice after middle cerebral artery occlusion (MCAo). We conclude that manipulation of p53 expression in neurons may lead to unique therapeutic development in stroke.

Transplantation of human placenta-derived multipotent stem cells reduces ischemic brain injury in adult rats.

After the onset of stroke, a series of progressive and degenerative reactions, including inflammation, is activated, which leads to cell death. We recently reported that human placenta-derived multipotent stem cells (hPDMCs) process potent anti-inflammatory effects. In this study, we examined the protective effect of hPDMC transplants in a rodent model of stroke. Adult male Sprague-Dawley rats were anesthetized. hPDMCs labeled with a vital dye of fluorescing microparticles, DiI, or vehicle were transplanted into three cortical areas adjacent to the right middle cerebral artery (MCA). Five minutes after grafting, the right MCA was transiently occluded for 60 min. Stroke animals receiving hPDMCs showed a significant behavioral improvement and reduction in lesion volume examined by T2-weighted images 4 days poststroke. Brain tissues were collected 1 day later. Human-specific marker HuNu immunoreactivity and DiI fluorescence were found at the hPDMC graft sites, suggesting the survival of hPDMCs in host brain. Grafting of hPDMCs suppressed IBA1 immunoreactivity and deramification of IBA1(+) cells in the perilesioned area, suggesting activation of microglia was attenuated by the transplants. Taken together, our data indicate that hPDMC transplantation reduced cortical lesions and behavioral deficits in adult stroke rats, and these cells could serve as a unique anti-inflammatory reservoir for the treatment of ischemic brain injury.

Intranasal delivery of bone marrow mesenchymal stem cells improved neurovascular regeneration and rescued neuropsychiatric deficits after neonatal stroke in rats.

Neonatal stroke is a major cause of mortality and long-term morbidity in infants and children. Currently, very limited therapeutic strategies are available to protect the developing brain against ischemic damage and promote brain repairs for pediatric patients. Moreover, children who experienced neonatal stroke often have developmental social behavior problems. Cellular therapy using bone marrow mesenchymal stem cells (BMSCs) has emerged as a regenerative therapy after stroke. In the present investigation, neonatal stroke of postnatal day 7 (P7) rat pups was treated with noninvasive and brain-specific intranasal delivery of BMSCs at 6 h and 3 days after stroke (1 × 10(6)cells/animal). Prior to transplantation, BMSCs were subjected to hypoxic preconditioning to enhance their tolerance and regenerative properties. The effects on regenerative activities and stroke-induced sensorimotor and social behavioral deficits were specifically examined at P24 of juvenile age. The BMSC treatment significantly reduced infarct size and blood-brain barrier disruption, promoted angiogenesis, neurogenesis, neurovascular repair, and improved local cerebral blood flow in the ischemic cortex. BMSC-treated rats showed better sensorimotor and olfactory functional recovery than saline-treated animals, measured by the adhesive removal test and buried food finding test. In social behavioral tests, we observed functional and social behavioral deficits in P24 rats subjected to stroke at P7, while the BMSC treatment significantly improved the performance of stroke animals. Overall, intranasal BMSC transplantation after neonatal stroke shows neuroprotection and great potential as a regenerative therapy to enhance neurovascular regeneration and improve functional recovery observed at the juvenile stage of development.

Porphyromonas gingivalis-induced alveolar bone loss is accelerated in the stroke-prone spontaneously hypertensive rat

Porphyromonas gingivalis (P. gingivalis) is one of the prominent periodontal pathogens and is the most important bacteria involved in the onset and exacerbation of periodontitis. P. gingivalis is an anaerobic, Gram-negative coccobacillus that plays a role in the progression of periodontal disease by promoting alveolar bone resorption. The aim of the present study was to examine P. gingivalis-induced osteoclastic bone resorption in the stroke-prone spontaneously hypertensive rat (SHRSP), in which oxidative stress induced by reactive oxygen species (ROS) is increased.In the present study, we used animals orally challenged with P. gingivalis as a chronic inflammation model. Horizontal bone loss around the maxillary molars was assessed morphometrically. Animals were divided into four groups: (1) P. gingivalis-non-infected Wister Kyoto Rat (WKY), (2) orally challenged with P. gingivalis WKY (WKY+Pg), (3) P. gingivalis-non-infected SHRSP, and (4) orally challenged with P. gingivalis SHRSP (SHRSP+Pg).Alveolar bone resorption was significantly increased in the orally challenged with P. gingivalis groups, and was accelerated in the SHRSP group. Histological analysis revealed that the infiltration of inflammatory cells was absent in all groups. However, the infiltration of osteoclasts was observed in the SHRSP+Pg and SHRSP groups.We examined P. gingivalis-induced alveolar bone loss in both the SHRSP and WKY. The results obtained demonstrated that P. gingivalis-induced alveolar bone loss would be involved in hypertension and stroke animal model, such as SHRSP and/or periodontal disease.

Abstract W MP50: Pair Housing Reverses the Detrimental Effect of Social Isolation and Restores BDNF and MBP in Aged Mice after Stroke

Background: Age is an important non-modifiable risk factor for stroke. Stroke rates double every decade after age 55. Social isolation (SI) exacerbates behavioural deficits, slows functional recovery and worsens histological injury after stroke in young animals, primarily by increasing the inflammatory response. However, the inflammatory response differs in aging, and whether the detrimental effects of SI are seen in aged animals is unknown. We hypothesize that acute and chronic post stroke SI will worsen stroke pathology and recovery in aged mice and pair housing (PH) will reverse these effects. Methods: Eighteen-month-old male C57BL/6 mice were pair housed (PH) for two weeks prior to stroke and randomly assigned to various housing conditions immediately after stroke. Behavioral analysis was done weekly starting at day 7. Mice were sacrificed either at 72 hours or 4 weeks after 60-minute right MCAO or sham surgery (n=9-10/group). Results: Mice isolated after stroke (ST-ISO) mice had significantly greater hemispheric infarct volume and neurological deficit scores (p&lt;.05. n=13/group) than pair-housed (PH) stroke mice at 72 hours. SI mice that were isolated immediately after stroke showed significantly higher plasma IL-6 levels compared to PH sham (P&lt;.001, n=13/group ) or PH stroke mice (P&lt;.05) after 72 hour, but levels were similar by 4 weeks post-stroke (n=9-14/group). No change in tissue atrophy was seen after 4 weeks, however a significant interaction [F (1, 28) = 259.6, P&lt;0.001] between housing and stroke was found in the Novel Object Recognition Task (NORT) at day 14. PH led to increased expression of Brain-derived neurotrophic factor (BDNF) and myelin basic protein (MBP) by IHC and western blot (n=5/group for IHC and n= 4/ western blot). Conclusions: Social isolation immediately after stroke led to enhanced injury acutely. Despite similar infarcts at 4 weeks, SI mice had delayed behavioral recovery. Pair housing led to increased expression of BDNF and myelin protein expression. Therefore, the beneficial effects of pair housing may be related to BDNF and MBP expression and enhanced recovery after injury in aged animals.

Ultrasonic vocalization changes and FOXP2 expression after experimental stroke

Speech impairments affect one in four stroke survivors. However, animal models of post-ischemic vocalization deficits are limited. Male mice vocalize at ultrasonic frequencies when exposed to an estrous female mouse. In this study we assessed vocalization patterns and quantity in male mice after cerebral ischemia. FOXP2, a gene associated with verbal dyspraxia in humans, with known roles in neurogenesis and synaptic plasticity, was also examined after injury. Using a transient middle cerebral artery occlusion (MCAO) model, we assessed correlates of vocal impairment at several time-points after stroke. Further, to identify possible lateralization of vocalization deficits induced by left and right hemispheric strokes were compared. Significant differences in vocalization quantity were observed between stroke and sham animals that persisted for a month after injury. Injury to the left hemisphere reduced early vocalizations more profoundly than those to the right hemisphere. Nuclear expression of Foxp2 was elevated early after stroke (at 6h), but significantly decreased 24h after injury in both the nucleus and the cytoplasm. Neuronal Foxp2 expression increased in stroke mice compared to sham animals 4 weeks after injury. This study demonstrates that quantifiable deficits in ultrasonic vocalizations (USVs) are seen after stroke. USV may be a useful tool to assess chronic behavioral recovery in murine models of stroke.