Animal Stroke Research Articles

Nose-to-brain delivery of stem cells in stroke: the role of extracellular vesicles.

Stem cell transplantation offers a promising therapy that can be administered days, weeks, or months after a stroke. We recognize 2 major mitigating factors that remain unresolved in cell therapy for stroke, notably: (1) well-defined donor stem cells and (2) mechanism of action. To this end, we advance the use of ProtheraCytes, a population of non-adherent CD34+ cells derived from human peripheral blood and umbilical cord blood, which have been processed under good manufacturing practice, with testing completed in a phase 2 clinical trial in post-acute myocardial infarction (NCT02669810). We also reveal a novel mechanism whereby ProtheraCytes secrete growth factors and extracellular vesicles (EVs) that are associated with angiogenesis and vasculogenesis. Our recent data revealed that intranasal transplantation of ProtheraCytes at 3 days after experimentally induced stroke in adult rats reduced stroke-induced behavioral deficits and histological damage up to 28 days post-stroke. Moreover, we detected upregulation of human CD63+ EVs in the ischemic brains of stroke animals that were transplanted with ProtheraCytes, which correlated with increased levels of DCX-labeled neurogenesis and VEGFR1-associated angiogenesis and vasculogenesis, as well as reduced Iba1-marked inflammation. Altogether, these findings overcome key laboratory-to-clinic translational hurdles, namely the identification of well-characterized, clinical grade ProtheraCytes and the elucidation of a potential CD63+ EV-mediated regenerative mechanism of action. We envision that additional translational studies will guide the development of clinical trials for intranasal ProtheraCytes allografts in stroke patients, with CD63 serving as a critical biomarker.

Hematopoietic Endothelial Progenitor cells enhance motor function and cortical motor map integrity following cerebral ischemia.

Hematopoietic stem cells (HSC) are recruited to ischemic areas in the brain and contribute to improved functional outcome in animals. However, little is known regarding the mechanisms of improvement following HSC administration post cerebral ischemia. To better understand how HSC effect post-stroke improvement, we examined the effect of HSC in ameliorating motor impairment and cortical dysfunction following cerebral ischemia. Baseline motor performance of male adult rats was established on validated motor tests. Animals were assigned to one of three experimental cohorts: control, stroke, stroke + HSC. One, three and five weeks following a unilateral stroke all animals were tested on motor skills after which intracortical microstimulation was used to derive maps of forelimb movement representations within the motor cortex ipsilateral to the ischemic injury. Stroke + HSC animals significantly outperformed stroke animals on single pellet reaching at weeks 3 and 5 (28±3% and 33±3% versus 11±4% and 17±3%, respectively, p < 0.05 at both time points). Control animals scored 44±1% and 47±1%, respectively. Sunflower seed opening task was significantly improved in the stroke + HSC cohort versus the stroke cohort at week five-post stroke (79±4 and 48±5, respectively, p < 0.05). Furthermore, Stroke + HSC animals had significantly larger forelimb motor maps than animals in the stroke cohort. Overall infarct size did not significantly differ between the two stroked cohorts. These data suggest that post stroke treatment of HSC enhances the functional integrity of residual cortical tissue, which in turn supports improved behavioral outcome, despite no observed reduction in infarct size.

Impaired Peripheral Vascular Function Following Ischemic Stroke in Mice: Potential Insights into Blood Pressure Variations in the Post-Stroke Patient.

High systolic blood pressure and increased blood pressure variability after the onset of ischemic stroke are associated with poor clinical outcomes. One of the key determinants of blood pressure is arteriolar size, determined by vascular smooth muscle tone and vasodilatory and vasoconstrictor substances that are released by the endothelium. The aim of this study is to outline alterations in vasomotor function in isolated peripheral arteries following ischemic stroke. The reactivity of thoracic aortic segments from male C57BL/6 mice to dilators and constrictors was quantified using wire myography. Acetylcholine-induced endothelium-dependent vasodilation was impaired after ischemic stroke (LogIC50 Sham = -7.499, LogIC50 Stroke = -7.350, p = 0.0132, n = 19, 31 respectively). The vasodilatory responses to SNP were identical in the isolated aortas in the sham and stroke groups. Phenylephrine-induced vasoconstriction was impaired in the aortas isolated from the stroke animals in comparison to their sham treatment counterparts (Sham LogEC50= -6.652 vs. Stroke LogEC50 = -6.475, p < 0.001). Our study demonstrates that 24 h post-ischemic stroke, peripheral vascular responses are impaired in remote arteries. The aortas from the stroke animals exhibited reduced vasoconstrictor and endothelium-dependent vasodilator responses, while the endothelium-independent vasodilatory responses were preserved. Since both the vasodilatory and vasoconstrictor responses of peripheral arteries are impaired following ischemic stroke, our findings might explain increased blood pressure variability following ischemic stroke.

Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke.

Ischemic stroke patients are more prone to developing another cardiovascular event. This study aims to examine potential biological predispositions to cardiovascular recurrence in patients with ischemic stroke. Human and preclinical studies. Quantitative proteomic analysis, animal stroke, atherosclerosis models and circulating endothelial cells (CECs) were employed to examine candidate biomarkers derived from an ischemic stroke cohort in Singapore. Proteomic analysis of pooled microvesicles of "Event" (n = 24) and without "Event" (n = 24) samples identified NOTCH3 as a candidate marker; plasma NOTCH3 were shown to be elevated in "Event" patients compared to those without "Events" and age-matched controls. In a validation cohort comprising 431 prospectively recruited ischemic stroke patients (mean age 59.1 years; median follow-up 3.5 years), men with plasma NOTCH3 (>1600pg/ml) harbored increased risk of cardiovascular recurrence (adjusted hazards ratio 2.29, 95% CI 1.10-4.77); no significant association was observed in women. Chronic renal failure, peripheral artery disease and NT-pro-brain natriuretic peptide were significant predictors of plasma NOTCH3 in men without ischemic stroke (adjusted r2=0.43). Following middle cerebral artery occlusion, NOTCH3 expression in mouse sera increased and peaked at 24 hrs, persisting thereafter for at least 72 hours. In Apoe-/- atherosclerotic mice, NOTCH3 stained the endothelium of defective arterial lining and atherosclerotic plaques. Analysis of CECs isolated from stroke patients revealed increased gene expression of NOTCH3, further supporting endothelial damage underpinning NOTCH3-mediated atherosclerosis. Findings from this study suggests that NOTCH3 could be important in cardiovascular recurrence following an ischemic stroke.

Emerging Roles of Exosomes in Stroke Therapy.

Stroke is the number one cause of morbidity in the United States and number two cause of death worldwide. There is a critical unmet medical need for more effective treatments of ischemic stroke, and this need is increasing with the shift in demographics to an older population. Recently, several studies have reported the therapeutic potential of stem cell-derived exosomes as new candidates for cell-free treatment in stoke. This review focuses on the use of stem cell-derived exosomes as a potential treatment tool for stroke patients. Therapy using exosomes can have a clear clinical advantage over stem cell transplantation in terms of safety, cost, and convenience, as well as reducing bench-to-bed latency due to fewer regulatory milestones. In this review article, we focus on (1) the therapeutic potential of exosomes in stroke treatment, (2) the optimization process of upstream and downstream production, and (3) preclinical application in a stroke animal model. Finally, we discuss the limitations and challenges faced by exosome therapy in future clinical applications.

Stem cell grafts enhance endogenous extracellular vesicle expression in the stroke brain

Endogenous brain repair occurs following an ischemic stroke but is transient, thus unable to fully mount a neuroprotective response against the evolving secondary cell death. Finding a treatment strategy that may render robust and long-lasting therapeutic effects stands as a clinically relevant therapy for stroke. Extracellular vesicles appear to be upregulated after stroke, which may represent a candidate target for neuroprotection. In this study, we probed whether transplanted stem cells could enhance the expression of extracellular vesicles to afford stable tissue remodeling in the ischemic stroke brain. Aged rats were initially exposed to the established ischemic stroke model of middle cerebral artery occlusion then received intravenous delivery of either bone marrow-derived mesenchymal stem cell transplantation or vehicle. A year later, the animals were assayed for brain damage, inflammation, and extracellular vesicle expression. Our findings revealed that while core infarction was not reduced, the stroke animals transplanted with stem cells displayed a significant reduction in peri-infarct cell loss that coincided with downregulated Iba1-labeled inflammatory cells and upregulated CD63-positive extracellular vesicles that appeared to be co-localized with GFAP-positive astrocytes. Interestingly, grafted stem cells were not detected at one year post-transplantation period, suggesting that the extracellular vesicles likely originated within the host brain. That long-lasting functional benefits persisted in the absence of surviving transplanted stem cells, but with upregulation of endogenous extracellular vesicles, advances the concept that transplantation of stem cells acutely after stroke propels host extracellular vesicles to the ischemic brain, altogether promoting chronic brain remodeling.

Prophylactic zinc and therapeutic selenium administration in adult rats prevents long-term cognitive and behavioral sequelae by a transient ischemic attack

The transient hypoxic-ischemic attack, also known as a minor stroke, can result in long-term neurological issues such as memory loss, depression, and anxiety due to an increase in nitrosative stress. The individual or combined administration of chronic prophylactic zinc and therapeutic selenium is known to reduce nitrosative stress in the first seven days post-reperfusion and, due to an antioxidant effect, prevent cell death. Besides, zinc or selenium, individually administered, also causes antidepressant and anxiolytic effects. Therefore, this work evaluated whether combining zinc and selenium could prevent stroke-elicited cognition and behavior deficits after 30 days post-reperfusion. Accordingly, we assessed the expression of growth factors at 7 days post-reperfusion, a four-time course of memory (from 7 to 28 days post-learning test), and cell proliferation, depression, and anxiety-like behavior at 30 days post-reperfusion. Male Wistar rats with a weight between 190 and 240 g) were treated with chronic prophylactic zinc administration with a concentration of 0.2 mg/kg for 15 days before common carotid artery occlusion (10 min) and then with therapeutic selenium (6 μg/kg) for 7 days post-reperfusion. Compared with individual administrations, the administration combined of prophylactic zinc and therapeutic selenium decreased astrogliosis, increased growth factor expression, and improved cell proliferation and survival in two regions, the hippocampus, and cerebral cortex. These effects prevented memory loss, depression, and anxiety-like behaviors. In conclusion, these results demonstrate that the prophylactic zinc administration combined with therapeutic selenium can reduce the long-term sequelae caused by the transient ischemic attack.Significance statement.A minor stroke caused by a transient ischemic attack can result in psychomotor sequelae that affect not only the living conditions of patients and their families but also the economy. The incidence of these micro-events among young people has increased in the world. Nonetheless, there is no deep understanding of how this population group responds to regular treatments (Ekker and et al., 2018) [1]. On the basis that zinc and selenium have antioxidant, anti-inflammatory, and regenerative properties in stroke animal models, our work explored whether the chronic combined administration of prophylactic zinc and therapeutic selenium could prevent neurological sequelae in the long term in a stroke rat model of unilateral common carotid artery occlusion (CCAO) by 10-min. Our results showed that this combined treatment provided a long-term neuroprotective effect by decreasing astrogliosis, memory loss, anxiety, and depression-like behavior.

Retinoic acid attenuates ischemic injury-induced activation of glial cells and inflammatory factors in a rat stroke model.

Stroke is a leading cause of death and long-term disability which can cause oxidative damage and inflammation of the neuronal cells. Retinoic acid is an active metabolite of vitamin A that has various beneficial effects including antioxidant and anti-inflammatory effects. In this study, we investigated whether retinoic acid modulates oxidative stress and inflammatory factors in a stroke animal model. A middle cerebral artery occlusion (MCAO) was performed on adult male rats to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected into the peritoneal cavity for four days before MCAO surgery. The neurobehavioral tests were carried out 24 h after MCAO and cerebral cortex tissues were collected. The cortical damage was assessed by hematoxylin-eosin staining and reactive oxygen species assay. In addition, Western blot and immunohistochemical staining were performed to investigate the activation of glial cells and inflammatory cytokines in MCAO animals. Ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) were used as markers of microglial and astrocyte activation, respectively. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were used as representative pro-inflammatory cytokines. Results showed that MCAO damage caused neurobehavioral defects and histopathological changes in the ischemic region and increased oxidative stress. Retinoic acid treatment reduced these changes caused by MCAO damage. We detected increases in Iba-1 and GFAP in MCAO animals treated with vehicle. However, retinoic acid alleviated increases in Iba-1 and GFAP caused by MCAO damage. Moreover, MCAO increased levels of nuclear factor-κB and pro-inflammatory cytokines, including TNF-α and IL-1β. Retinoic acid alleviated the expression of these inflammatory proteins. These findings elucidate that retinoic acid regulates microglia and astrocyte activation and modulates pro-inflammatory cytokines. Therefore, this study suggests that retinoic acid exhibits strong antioxidant and anti-inflammatory properties by reducing oxidative stress, inhibiting neuroglia cell activation, and preventing the increase of pro-inflammatory cytokines in a cerebral ischemia.

Matrilin-3 supports neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation

In the brain, the role of matrilin-3, an extracellular matrix component in cartilage, is unknown. Here, we identify that matrilin-3 decreased in reactive astrocytes but was unchanged in neurons after ischemic stroke in animals. Importantly, it declined in serum of patients with acute ischemic stroke. Genetic or pharmacological inhibition or supplementation of matrilin-3 aggravates or reduces brain injury, astrocytic cell death, and glial scar, respectively, but has no direct effect on neuronal cell death. RNA sequencing demonstrates that Matn3-/- mice display an increased inflammatory response profile in the ischemic brain, including the nuclear factor κB (NF-κB) signaling pathway. Both endogenous and exogenous matrilin-3 reduce inflammatory mediators. Mechanistically, extracellular matrilin-3 enters astrocytes via caveolin-1-mediated endocytosis. Cytoplasmic matrilin-3 translocates into the nucleus by binding to NF-κB p65, suppressing inflammatory cytokine transcription. Extracellular matrilin-3 binds to BMP-2, blocking the BMP-2/Smads pathway. Thus, matrilin-3 is required for astrocytes to exert neuroprotection, at least partially, by suppressing astrocyte-mediated neuroinflammation.

Green synthesis, chemical characterization, and protective potentials of silver nanoparticles on the development of cerebrovascular diseases in rat cerebral ischemia reperfusion injury

Thrombolysis or mechanical recanalization are two methods that can be used to restore blood flow after an ischemic stroke. However, it is important to note that in certain situations, reperfusion can actually exacerbate the initial damage caused by ischemia, leading to a condition known as “cerebral reperfusion injury.” This injury involves several pathological processes, including the infiltration of leukocytes, activation of platelets and complement, postischemic hyperperfusion, and disruption of the blood–brain barrier. The main objective of research conducted globally is to create a novel therapeutic medication for stroke. In light of the nano revolution, it is imperative to merge nanoscience and medicine. Knowledge acquired from nature has paved the path for the development of biogenic technologies, facilitating the production of cutting-edge nanomaterials. We have recently conducted a study to evaluate the neuroprotective properties of a blend of Mellisa officinalis leaf extract and green-mediated silver nanoparticles on the development of cerebrovascular diseases in rat cerebral ischemia reperfusion injury. The silver nanoparticles were thoroughly examined using UV–Vis, FE-SEM, and EDX analyses to ensure comprehensive characterization. The Ag NPs were observed to have various polymorphic shapes and were found to be aggregated. Also, the particles average size was measured by ImageJ software and it is about 29.15 nm. The UV–Vis measurements showed that the spectra displayed the highest absorption at around 432 nm. The stroke occurred as a result of the occlusion of the middle cerebral artery for 1.5 h, followed by reperfusion for 2 days. Before the ischemia reperfusion, the rats received an intravenous injection of AgNPs at doses of 40, 80 and 160 µg/kg once daily for a consecutive period of 3 days. The evaluation of the cell damage index involved assessing various factors such as immunoglobulin G (IgG) extravasation into the cerebral parenchyma, lactate dehydrogenase efficacy, serum MDA level, infarct volume, and learning and memory function. The results of the study showed that pretreatment with AgNPs had a protective effect on stroke in animals. Moreover, AgNPs effectively alleviated the memory and learning impairments caused by stroke. Additionally, AgNPs significantly reduced infarct volume, IgG extravasation, serum MDA level, and lactate dehydrogenase efficacy.

Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Intracranial pressure is elevated at 24 h post‐stroke in mice

AbstractBackgroundIt has long been assumed that post‐stroke intracranial pressure (ICP) elevation occurs because of large infarct and edema volumes. However, we have repeatedly shown ICP elevation at 24 h post‐stroke in the presence of little to no edema in rats. Biological processes are often conserved across species and types of injury. Therefore, we aimed to determine if an ICP rise occurs at 24 h post‐stroke in the presence of small infarct and edema volumes in mice.MethodsMice were randomized by random number table to either photothrombotic stroke or sham surgery (n = 15). Epidural ICP was recorded using a fiber optic catheter at 1 h post‐stroke (baseline) and between 23 and 24 h post‐stroke.ResultsICP was significantly higher at 24 h compared to baseline in stroke animals (n = 6; 10.71 ± 6.45 mmHg vs. 3.74 ± 2.20 mmHg, respectively; p = 0.03). ICP at 24 h was also significantly higher in stroke mice compared to sham (n = 6; 3.45 ± 1.43 mmHg; p = 0.02). There was no change in ICP in sham mice (p = 0.9). Edema volumes in stroke animals were small (0.04 ± 0.04 mm3) and unlikely to have caused significant ICP elevation.ConclusionThis study provides evidence of an edema‐independent ICP elevation following small ischemic stroke in mice. The occurrence of this rise supports our findings in other species and suggests it is caused by a previously undescribed mechanism.

Abstract WP321: Novel Therapeutic Potential of Intestinal Organoids Secretomes for Cerebrovascular Stroke

Background: Our recent studies show that transplants of intestine epithelial stem cells (IESCs) promote acute and long term recovery after stroke in middle-aged rats. To improve the translational potential of this therapy, we investigated whether injections of the IESC conditioned media (CM) would be similarly effective in improving acute and chronic stroke outcomes. Methods: Conditioned media was collected intestinal organoids culture from young animals and processed for proteomic and tropic factors analysis. CM efficacy was tested in middle-aged male and female rats assigned to the following groups: Control (sham), stroke+vehicle injection, or stroke+IESC-CM treatment. Middle cerebral artery occlusion (MCAo) was induced by Endothelin-1 (vasoconstrictor). Conditioned media was injected ip (CMip) 4h/24h/48h after stroke. In the acute phase, infarct volume, sensorimotor function and serum levels of the gut permeability markers were measured 4d post stroke. Long term (4 weeks after stroke) depressive-like behavior was assessed by the Social Interaction test, and cognitive performance by the Novel object Recognition test and Barnes maze test. Results: Proteomic analysis of CM showed elevated levels of small peptide growth factors ad large matrix molecules. IESC-CMip treatment significantly improved acute stroke outcomes and reduced gut permeability in middle-aged hosts of either sex. In long term study, vehicle-treated MCAo animals showed depressive-like behaviors compared to their pre-stroke behaviors. In contrast, stroke animals that received IESC secretome were no different from their pre stroke performance and were similar sham (no-stroke) animals. In cognition assesments. MCAo+vehicle animals showed poor ability to discriminate the novel object and displayed impaired learning and recall on the Barnes maze. MCAo animals that received IESC secretome did not show significant improved during the Barnes learning trials, but retrieval (memory recall) performance was improved and significantly different from the vehicle group. Conclusion: Overall, these data show that IESC secretome derived from a healthy young animal can significantly improve acute stroke outcomes and specific aspects of cognitive performance.

Interferon Beta Modulation of Brain Endothelial Cell Activation in Ischemic Stroke

Background and Hypothesis:Every year, more than 690,000 people in the United States suffer an ischemic stroke. Many survivors are left with long-term disability. While the initial insult to the brain is caused by hypoxia resulting from cerebral artery occlusion, a secondary insult is caused by peripheral immune cell infiltration across the blood brain barrier (BBB) and subsequent cytotoxic insult. Previous studies have demonstrated that interferon beta (IFNβ) limits peripheral immune cell infiltration across the BBB and reduces brain infarction volume. We hypothesize that during ischemic stroke IFNβ suppresses brain endothelial cells (bECs) activation to reduce their expression of adhesion molecules as one of the mechanisms by which it decreases peripheral immune cell infiltration across the BBB.&#x0D; Experimental Design:In this project, bEnd.3 cells, a cell line of bECs, were activated by TNF-α, a pro-inflammatory cytokine. Tissue plasminogen activator (tPA), an FDA-approved thrombolytic for ischemic stroke, was included in the study. bEnd.3 cells were treated with IFNβ at 1.5 hours prior TNF-α or TNF-α + tPA stimulation to evaluate its modulation of adhesion cell expression. The adhesion molecule expression was determined by flow cytometry. Results were further confirmed by in vivo studies in which stroke animals were subjected to tPA treatment in the presence or absence of IFNβ.&#x0D; Results:Our results showed that TNF-α induced ICAM-1, VCAM-1, E-selectin, and P-selectin expression. Importantly, we found IFNβ suppressed the expression of aforementioned adhesion molecules in bEnd.3 cells treated with TNF-α or TNF-α+tPA. Our in vivo results demonstrated that IFNβ treatment reduced ICAM-1 and E-selectin, but not VCAM-1 or P-selectin expression in the ischemic brain.&#x0D; Conclusion and Potential Impact:Our study demonstrates that IFNβ modulates bEC expression of adhesion molecules in vitro and in vivo of ischemic stroke, suggesting IFNβ, an FDA-approved drug for Multiple Sclerosis, shows potential to improve ischemic stroke outcomes.

Peptide-mediated targeted delivery of SOX9 nanoparticles into astrocytes ameliorates ischemic brain injury.

Astrocytes are highly activated following brain injuries, and their activation influences neuronal survival. Additionally, SOX9 expression is known to increase in reactive astrocytes. However, the role of SOX9 in activated astrocytes following ischemic brain damage has not been clearly elucidated yet. Therefore, in the present study, we investigated the role of SOX9 in reactive astrocytes using a poly-lactic-co-glycolic acid (PLGA) nanoparticle plasmid delivery system in a photothrombotic stroke animal model. We designed PLGA nanoparticles to exclusively enhance SOX9 gene expression in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Our observations indicate that PLGA nanoparticles encapsulated with GFAP:SOX9:tdTOM reduce ischemia-induced neurological deficits and infarct volume through the prostaglandin D2 pathway. Thus, the astrocyte-targeting PLGA nanoparticle plasmid delivery system provides a potential opportunity for stroke treatment. Since the only effective treatment currently available is reinstating the blood supply, cell-specific gene therapy using PLGA nanoparticles will open a new therapeutic paradigm for brain injury patients in the future.

Ultrasound biomicroscopy in the quantification of brain perfusion parameters of a rat stroke model: Analysis of contrast agent bolus kinetic dynamics

BackgroundIschemic stroke represents a significant global health concern, necessitating thorough investigations and the utilization of stroke animal models to explore novel treatment modalities and diagnostic imaging techniques. New methodUltrasound biomicroscopy (BMU), operating at a center frequency of 21 MHz, along with ultrasound contrast agents (UCAs), was used to quantify microcirculation cerebral blood flow in a rat model of ischemic stroke. The microcirculation parameters were derived from time intensity curve (TIC) plots obtained based on UCA-bolus kinetics. ResultsSemiquantitative perfusion-related parameters were assessed. The TIC curves showed differences in amplitude when compared intra-animal between the left and right sides, and three situations were observed: normal perfusion, hypoperfusion, and nonperfusion. ROC analysis of delays between the left and right time intensity peak (TIP) for regions of interest (ROIs) in the control and stroke-hypoperfusion groups revealed an optimal cutpoint of 0.39 s to indicate when hypoperfusion is occurring in rats, with a sensitivity of 93.33 % and a specificity of 80 %. Comparison with existing method(s)Ultrasound perfusion imaging through the temporal bone window has been clinically applied to stroke patients using a UCA bolus for TIC analysis. TIC parameters were correlated with MRI- and CT-based measurements. ConclusionsThis investigation quantified cerebral blood flow in a rat model of ischemic stroke by measuring microcirculation parameters. The study demonstrated the efficacy of this approach as a valuable tool for conducting preclinical studies.

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.

Discovery of the Next Generation of Non-peptidomimetic Neurolysin Activators with HighBlood-BrainBarrier Permeability: a Pharmacokinetics Study in Healthy and Stroke Animals.

There is growing interest in seeking pharmacological activation of neurolysin (Nln) for stroke treatment. Discovery of central nervous system drugs remains challenging due to the protection of the blood-brain barrier (BBB). The previously reported peptidomimetic Nln activators display unsatisfactory BBB penetration. Herein, we investigate the next generation of non-peptidomimetic Nln activators with high BBB permeability. A BBB-mimicking model was used to evaluate their in vitro BBB permeability. Protein binding, metabolic stability, and efflux assays were performed to determine their unbound fraction, half-lives in plasma and brains, and dependence of BBB transporter P-glycoprotein (P-gp). The in vivo pharmacokinetic profiles were elucidated in healthy and stroke mice. Compounds KS52 and KS73 out of this generation exhibit improved peptidase activity and BBB permeability compared to the endogenous activator and previous peptidomimetic activators. They show reasonable plasma and brain protein binding, improved metabolic stability, and independence of P-gp-mediated efflux. In healthy animals, they rapidly distribute into brains and reach peak levels of 18.69% and 12.10% injected dose (ID)/ml at 10min. After 4h, their total brain concentrations remain 7.78 and 12.34 times higher than their A50(minimal concentration required for enhancing 50% peptidase activity). Moreover, the ipsilateral hemispheres of stroke animals show comparable uptake to the corresponding contralateral hemispheres and healthy brains. This study provides essential details about the pharmacokinetic properties of a new generation of potent non-peptidomimetic Nln activators with high BBB permeability and warrants the future development of these agents as potential neuroprotective pharmaceutics for stroke treatment.

Chlorogenic acid alleviates the reduction of 14-3-3 protein in the cerebral cortex of stroke animal model

Chlorogenic acid alleviates the reduction of 14-3-3 protein in the cerebral cortex of stroke animal model

Dexmedetomidine compared to low-dose ketamine better protected not only the brain but also the lungs in acute ischemic stroke

BackgroundDexmedetomidine (DEX) and low-dose ketamine (KET) present neuroprotective effects in acute ischemic stroke (AIS); however, to date, no studies have evaluated which has better protective effects not only on the brain but also lungs in AIS. MethodsAIS-induced Wistar rats (390 ± 30 g) were randomized after 24-h, receiving dexmedetomidine (STROKE-DEX, n = 10) or low-dose S(+)-ketamine (STROKE-KET, n = 10). After 1-h protective ventilation, perilesional brain tissue and lungs were removed for histologic and molecular biology analysis. STROKE animals (n = 5), receiving sodium thiopental but not ventilated, had brain and lungs removed for molecular biology analysis. Effects of DEX and KET mean plasma concentrations on alveolar macrophages, neutrophils, and lung endothelial cells, extracted primarily 24-h after AIS, were evaluated. ResultsIn perilesional brain tissue, apoptosis did not differ between groups. In STROKE-DEX, compared to STROKE-KET, tumor necrosis factor (TNF)-α and vascular cell adhesion molecule-1 (VCAM-1) expressions were reduced, but no changes in nuclear factor erythroid 2-related factor-2 (Nrf2) and super oxide dismutase (SOD)-1 were observed. In lungs, TNF-α and VCAM-1 were reduced, whereas Nrf2 and SOD-1 were increased in STROKE-DEX. In alveolar macrophages, TNF-α and inducible nitric oxide synthase (M1 macrophage phenotype) were lower and arginase and transforming growth factor-β (M2 macrophage phenotype) higher in STROKE-DEX. In lung neutrophils, CXC chemokine receptors (CXCR2 and CXCR4) were higher in STROKE-DEX. In lung endothelial cells, E-selectin and VCAM-1 were lower in STROKE-DEX. ConclusionsIn the current AIS model, dexmedetomidine compared to low-dose ketamine reduced inflammation and endothelial cell damage in both brain and lung, suggesting greater protection.