Rhoa/ROCK, mTOR and Secretome-Based Treatments for Ischemic Stroke: New Perspectives.

Ischemic stroke triggers a complex cascade of cellular and molecular events leading to neuronal damage and tissue injury. This review explores the potential therapeutic avenues targeting cellular signaling pathways implicated in stroke pathophysiology. Specifically, it focuses on the articles that highlight the roles of RhoA/ROCK and mTOR signaling pathways in ischemic brain injury and their therapeutic implications. The RhoA/ROCK pathway modulates various cellular processes, including cytoskeletal dynamics and inflammation, while mTOR signaling regulates cell growth, proliferation, and autophagy. Preclinical studies have demonstrated the neuroprotective effects of targeting these pathways in stroke models, offering insights into potential treatment strategies. However, challenges such as off-target effects and the need for tissue-specific targeting remain. Furthermore, emerging evidence suggests the therapeutic potential of MSC secretome in stroke treatment, highlighting the importance of exploring alternative approaches. Future research directions include elucidating the precise mechanisms of action, optimizing treatment protocols, and translating preclinical findings into clinical practice for improved stroke outcomes.

Pharmacological and stem cell therapy of stroke in animal models: Do they accurately reflect the response of humans?

Cerebrovascular diseases are the second leading cause of death worldwide. Despite significant research investment, the only available therapeutic options are mechanical thrombectomy and tissue plasminogen activator thrombolysis. None of the more than a thousand drugs tested on animal models have proven successful in human clinical trials. Several factors contribute to this poor translation of data from stroke-related animal models to human stroke patients. Firstly, our understanding of the molecular and cellular processes involved in recovering from an ischemic stroke is severely limited. Secondly, although the risk of stroke is particularly high among older patients with comorbidities, most drugs are tested on young, healthy animals in controlled laboratory conditions. Furthermore, in animal models, the tracking of post-stroke recovery typically spans only 3 to 28 days, with occasional extensions to 60 days, whereas human stroke recovery is a more extended and complex process. Thirdly, young animal models often exhibit a considerably higher rate of spontaneous recovery compared to humans following a stroke. Fourth, only a very limited number of animals are utilized for each condition, including control groups. Another contributing factor to the much smaller beneficial effects in humans is that positive outcomes from numerous animal studies are more readily accepted than results reported in human trials that do not show a clear benefit to the patient. Useful recommendations for conducting experiments in animal models, with increased chances of translatability to humans, have been issued by both the STEPS investigative team and the STAIR committee. However, largely, due to economic factors, these recommendations are largely ignored. Furthermore, one might attribute the overall failures in predicting and subsequently developing effective acute stroke therapies beyond thrombolysis to potential design deficiencies in clinical trials.

Oxidative Stress and Antioxidant Defense Mechanisms in Acute Ischemic Stroke Patients with Concurrent COVID-19 Infection.

Stroke remains a debilitating cerebrovascular condition associated with oxidative stress, while COVID-19 has emerged as a global health crisis with multifaceted systemic implications. This study investigates the hypothesis that patients experiencing acute ischemic stroke alongside COVID-19 exhibit elevated oxidative stress markers and altered antioxidant defense mechanisms compared to those with acute ischemic stroke. We conducted a single-center prospective cross-sectional study to investigate oxidative stress balance through oxidative damage markers: TBARS (thiobarbituric acid reactive substances level) and PCARB (protein carbonyls); antioxidant defense mechanisms: TAC (total antioxidant capacity), GPx (glutathione peroxidase), GSH (reduced glutathione), CAT (catalase), and SOD (superoxide dismutase); as well as inflammatory response markers: NLR (neutrophil-to-lymphocyte ratio), CRP (C-reactive protein), and ESR (erythrocyte sedimentation rate). Statistical analyses and correlation models were employed to elucidate potential associations and predictive factors. Our results revealed increased oxidative stress, predominantly indicated by elevated levels of TBARS in individuals experiencing ischemic stroke alongside a concurrent COVID-19 infection (p < 0.0001). The Stroke-COVID group displayed notably elevated levels of GSH (p = 0.0139 *), GPx (p < 0.0001 ****), SOD (p = 0.0363 *), and CAT (p = 0.0237 *) activities. Multivariate analysis found a significant association for TBARS (p < 0.0001 ****), PCARB (p = 0.0259 *), and GPx activity (p < 0.0001 ****), together with NLR (p = 0.0220 *) and CRP (p = 0.0008 ***). Notably, the interplay between stroke and COVID-19 infection appears to amplify oxidative damage, potentially contributing to exacerbated neurological deficits and poorer outcomes. This study highlights the intricate relationship between oxidative stress, inflammation, and concurrent health conditions. Understanding these interactions may open avenues for novel therapeutic strategies aimed at ameliorating oxidative damage in patients with acute ischemic stroke and COVID-19, ultimately improving their prognosis and quality of life.

Clinical Ageing.

Ageing is generally characterised by the declining ability to respond to stress, increasing homeostatic imbalance, and increased risk of ageing-associated diseases . Mechanistically, the lifelong accumulation of a wide range of molecular and cellular impairments leads to organismal senescence. The aging population poses a severe medical concern due to the burden it places on healthcare systems and the general public as well as the prevalence of diseases and impairments associated with old age. In this chapter, we discuss organ failure during ageing as well as ageing of the hypothalamic-pituitary-adrenal axis and drugs that can regulate it. A much-debated subject is about ageing and regeneration. With age, there is a gradual decline in the regenerative properties of most tissues. The goal of regenerative medicine is to restore cells, tissues, and structures that are lost or damaged after disease, injury, or ageing. The question arises as to whether this is due to the intrinsic ageing of stem cells or, rather, to the impairment of stem-cell function in the aged tissue environment. The risk of having a stroke event doubles each decade after the age of 55. Therefore, it is of great interest to develop neurorestorative therapies for stroke which occurs mostly in elderly people. Initial enthusiasm for stimulating restorative processes in the ischaemic brain with cell-based therapies has meanwhile converted into a more balanced view, recognising impediments related to survival, migration, differentiation, and integration of therapeutic cells in the hostile aged brain environment. Therefore, a current lack of understanding of the fate of transplanted cells means that the safety of cell therapy in stroke patients is still unproven. Another issue associated with ischaemic stroke is that patients at risk for these sequels of stroke are not duly diagnosed and treated due to the lack of reliable biomarkers. However, recently neurovascular unit-derived exosomes in response to Stroke and released into serum are new plasma genetic and proteomic biomarkers associated with ischaemic stroke. The second valid option, which is also more economical, is to invest in prevention.

Predictive Value of Pulmonary Involvement in Stroke Patients Co-Infected with COVID-19.

In response to the intricate clinical challenges posed by the intersection of COVID-19 and acute ischemic stroke, the Neuropsychiatry Hospital of Craiova, Romania, initiated a comprehensive study. This research aims to unravel the impacts of pulmonary complications on ischemic stroke outcomes, comparing patients with concurrent SARS-CoV-2 infection to those without. The study integrates pulmonary assessments, acknowledging the significant role respiratory involvement plays in the progression and prognosis of stroke patients during the pandemic. By systematically examining individuals with both acute ischemic stroke and COVID-19, the study seeks to shed light on the complex interplay between cerebral and pulmonary health. The findings are expected to enhance patient care by informing clinical decisions and leading to more effective management approaches for stroke patients in the COVID-19 era.

Mesenchymal stromal cell-derived small extracellular vesicles promote neurological recovery and brain remodeling after distal middle cerebral artery occlusion in aged rats.

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) promote neurological recovery after middle cerebral artery occlusion (MCAO) in young rodents. Ischemic stroke mainly affects aged humans. MSC-sEV effects on stroke recovery in aged rodents had not been assessed. In a head-to-head comparison, we exposed young (4-5 months) and aged (19-20 months) male Sprague-Dawley rats to permanent distal MCAO. At 24 h, 3 and 7 days post-stroke, vehicle or MSC-sEVs (2 × 106 or 2 × 107 MSC equivalents/kg) were intravenously administered. Neurological deficits, ischemic injury, brain inflammatory responses, post-ischemic angiogenesis, and endogenous neurogenesis were evaluated over 28 days. Post-MCAO, aged vehicle-treated rats exhibited more severe motor-coordination deficits evaluated by rotating pole and cylinder tests and larger brain infarcts than young vehicle-treated rats. Although infarct volume was not influenced by MSC-sEVs, sEVs at both doses effectively reduced motor-coordination deficits in young and aged rats. Brain macrophage infiltrates in periinfarct tissue, which were evaluated as marker of a recovery-aversive inflammatory environment, were significantly stronger in aged than young vehicle-treated rats. sEVs reduced brain macrophage infiltrates in aged, but not young rats. The tolerogenic shift in immune balance paved the way for structural brain tissue remodeling. Hence, sEVs at both doses increased periinfarct angiogenesis evaluated by CD31/BrdU immunohistochemistry in young and aged rats, and low-dose sEVs increased neurogenesis in the subventricular zone examined by DCX/BrdU immunohistochemistry. Our study provides robust evidence that MSC-sEVs promote functional neurological recovery and brain tissue remodeling in aged rats post-stroke. This study encourages further proof-of-concept studies in clinic-relevant stroke settings.

Dietary habits, lifestyle factors and neurodegenerative diseases.

Worldwide stroke is increasing in parallel with modernization, changes in lifestyle, and the growing elderly population. Our review is focused on the link between diet, as part of 'modern lifestyle', and health in the context of genetic predisposition of individuals to 'unhealthy' metabolic pathway activity. It is concluded that lifestyle including high sugar diets, alcohol and tobacco addiction or high fat diets as well as ageing, brain injury, oxidative stress and neuroinflammation, negatively influence the onset, severity and duration of neurodegenerative diseases. Fortunately, there are several healthy dietary components such as polyunsaturated fatty acids and the anti-oxidants curcumin, resveratrol, blueberry polyphenols, sulphoraphane, salvionic acid as well as caloric restriction and physical activity, which may counteract ageing and associated neurodegenerative diseases via increased autophagy or increased neurogenesis in the adult brain.

Intracortical Administration of the Complement C3 Receptor Antagonist Trifluoroacetate Modulates Microglia Reaction after Brain Injury.

Worldwide, millions of individuals suffer an ischemic stroke each year, causing major disability, especially in the elderly, where stroke is the number one cause of disability. However, to date, no effective therapy exists that targets the functional recovery after stroke. After necrosis, neuroinflammation is a common feature of the acute stroke and a major obstacle to tissue restoration. In the lesioned area, the dying neurons release chemotactic signals, such as fractalkine/CX3CL1, which evoke "eat-me" signals that are recognized by microglia expressing complement C3a receptor (C3aR), resulting in phagocytosis of the dying but still viable neurons, known as secondary phagocytosis. Using a mouse model of stroke and two-photon microscopy, we aimed to attenuate poststroke phagocytosis of the dying but still viable neurons by using SB 290157, an antagonist of C3aR. We found that intracortical administration of SB 290157 reduced the number of inflammatory microglial cells expressing ED1 and Iba1 antigens at the lesion site. We could show, in vivo, that two days after a needle-induced cortical lesion there were less microglial cells present around the injury site, displaying less high-order branches and an increase in the lower order ones, suggesting an attenuated phagocytic phenotype in treated animals as compared with controls. We conclude that the C3aR antagonist, SB 290157, may be used in the future to limit the neuronal death by limiting secondary phagocytosis after stroke.

Recent Advances in Mono- and Combined Stem Cell Therapies of Stroke in Animal Models and Humans.

Following the failure of acute neuroprotection therapies, major efforts are currently made worldwide to promote neurological recovery and brain plasticity in the subacute and post-acute phases of stroke. Currently, there is hope that stroke recovery might be promoted by cell-based therapies. The field of stem cell therapy for cerebral ischemia has made significant progress in the last five years. A variety of stem cells have been tested in animal models and humans including adipose stem cells, human umbilical cord blood-derived mesenchymal stem cells, human amnion epithelial cells, human placenta amniotic membrane-derived mesenchymal stem cells, adult human pluripotent-like olfactory stem cells, human bone marrow endothelial progenitor cells, electrically-stimulated human neuronal progenitor cells, or induced pluripotent stem cells (iPSCs) of human origin. Combination therapies in animal models include a mix of two or more therapeutic factors consisting of bone marrow stromal cells, exercise and thyroid hormones, endothelial progenitor cells overexpressing the chemokine CXCL12. Mechanisms underlying the beneficial effects of transplanted cells include the "bystander" effects, paracrine mechanisms, or extracellular vesicles-mediated restorative effects. Mitochondria transfer also appears to be a powerful strategy for regenerative processes. Studies in humans are currently limited to a small number of studies using autologous stem cells mainly aimed to assess tolerability and side-effects of human stem cells in the clinic.

Vasculature Remodeling in a Rat Model of Cerebral Ischemia. The Fate of the BrdU-Labeled Cells Prior to Stroke.

Despite the clinical significance of post-stroke angiogenesis, a detailed phenotypic analysis of pre-stroke vascular remodeling and post-stroke angiogenesis had not yet been done in a model of focal ischemia. In this study, using BrdU-labeling of proliferating cells and immunofluorescence of pre- and post-stroke rats, we found that, (i) BrdU administered before stroke was incorporated preferentially into the nuclei of endothelial cells lining the lumen of existing blood vessels and newly born neurons in the dentate gyrus but not in the subventricular zone or proliferating microglia, (ii) BrdU injection prior to stroke led to the patchy distribution of the newly incorporated endothelial cells into existing blood vessels of the adult rat brain, (iii) BrdU injection prior to stroke specifically labeled neuronal precursors cells in a region of soft tissue beyond the inhibitory scar, which seems to be permissive to regenerative events, (iv) BrdU injection after stroke led to labeling of endothelial cells crossing or detaching from the disintegrating blood vessels and their incorporation into new blood vessels in the stroke region, scar tissue and the region beyond, (v) BrdU injection after stroke led to specific incorporation of BrdU-positive nuclei into the "pinwheel" architecture of the ventricular epithelium, (vi) blood vessels in remote areas relative to the infarct core and in the contralateral non-lesioned cortex, showed co-labeled BrdU/RECA+ endothelial cells shortly after the BrdU injection, which strongly suggests a bone marrow origin of the endothelial cells. In the damaged cortex, a BrdU/prolyl 4-hydroxylase beta double labeling in the close proximity to collagen IV-labeled basement membrane, suggests that, in addition to bone marrow derived endothelial cells, the disintegrating vascular wall itself could also be a source of proliferating endothelial cells, (vii) By day 28 after stroke, new blood vessels were observed in the perilesional area and the scar tissue region, which is generally considered to be resistant to regenerative events. Finally, (viii) vigorous angiogenesis was also detected in a region of soft tissue, also called "islet of regeneration," located next to the inhibitory scar. Conclusion: BrdU administered prior to, and after stroke, allows to investigate brain vasculature remodeling in the adult brain.

Present Status and Future Challenges of New Therapeutic Targets in Preclinical Models of Stroke in Aged Animals with/without Comorbidities.

The aging process, comorbidities, and age-associated diseases are closely dependent on each other. Cerebral ischemia impacts a wide range of systems in an age-dependent manner. However, the aging process has many facets which are influenced by the genetic background and epigenetic or environmental factors, which can explain why some people age differently than others. Therefore, there is an urgent need to identify age-related changes in body functions or structures that increase the risk for stroke and which are associated with a poor outcome. Multimodal imaging, electrophysiology, cell biology, proteomics, and transcriptomics, offer a useful approach to link structural and functional changes in the aging brain, with or without comorbidities, to post-stroke rehabilitation. This can help us to improve our knowledge about senescence firstly, and in this context, aids in elucidating the pathophysiology of age-related diseases that allows us to develop therapeutic strategies or prevent diseases. These processes, including potential therapeutical interventions, need to be studied first in relevant preclinical models using aged animals, with and without comorbidities. Therefore, preclinical research on ischemic stroke should consider age as the most important risk factor for cerebral ischemia. Furthermore, the identification of effective therapeutic strategies, corroborated with successful translational studies, will have a dramatic impact on the lives of millions of people with cerebrovascular diseases.

Cellular and Molecular Mechanisms Underlying Non-Pharmaceutical Ischemic Stroke Therapy in Aged Subjects.

The incidence of ischemic stroke in humans increases exponentially above 70 years both in men and women. Comorbidities like diabetes, arterial hypertension or co-morbidity factors such as hypercholesterolemia, obesity and body fat distribution as well as fat-rich diet and physical inactivity are common in elderly persons and are associated with higher risk of stroke, increased mortality and disability. Obesity could represent a state of chronic inflammation that can be prevented to some extent by non-pharmaceutical interventions such as calorie restriction and hypothermia. Indeed, recent results suggest that H2S-induced hypothermia in aged, overweight rats could have a higher probability of success in treating stroke as compared to other monotherapies, by reducing post-stroke brain inflammation. Likewise, it was recently reported that weight reduction prior to stroke, in aged, overweight rats induced by caloric restriction, led to an early re-gain of weight and a significant improvement in recovery of complex sensorimotor skills, cutaneous sensitivity, or spatial memory. CONCLUSION: animal models of stroke done in young animals ignore age-associated comorbidities and may explain, at least in part, the unsuccessful bench-to-bedside translation of neuroprotective strategies for ischemic stroke in aged subjects.

Molecular mechanisms underlying neurodevelopmental disorders, ADHD and autism.

Neurodevelopmental disorders such as attention deficit hyperactivity disorder and autism represent a significant economic burden, which justify vigorous research to uncover its genetics and developmental clinics for a diagnostic workup. The urgency of addressing attention deficit hyperactivity disorder comorbidities is seen in the chilling fact that attention deficit hyperactivity disorder (ADHD), mood disorders, substance use disorders and obesity each increase the risk for mortality. However, data about comorbidity is mainly descriptive, with mechanistic studies limited to genetic epidemiological studies that document shared genetic risk factors among these conditions. Autism and intellectual disability affects 1.5 to 2% of the population in Western countries with many individuals displaying social-emotional agnosia and having difficulty in forming attachments and relationships. Underlying mechanisms include: (i) dysfunctions of neuronal miRNAs; (ii) deletions in the chromosome 21, subtelomeric deletions, duplications and a maternally inherited duplication of the chromosomal region 15q11-q13; (iii) microdeletions in on the long (q) arm of the chromosome in a region designated q21.1 increases the risk of delayed development, intellectual disability, physical abnormalities, and neurological and psychiatric problems associated with autism, schizophrenia, and epilepsy and weak muscle tone (hypotonia); (iv) interstitial duplications encompassing 16p13.11.