RESUMO
BACKGROUND: Respiratory dysfunction is a common complication of stroke, with an incidence of over 60%. Despite the high prevalence of stroke-induced respiratory dysfunction, how disordered breathing influences recovery and cognitive outcomes after ischemic stroke is unknown. We hypothesized that stroke induces chronic respiratory dysfunction, breathing instability, and apnea in mice, which would contribute to higher mortality and greater poststroke cognitive deficits. METHODS: Mice were subjected to a 60-minute transient middle cerebral artery occlusion or permanent distal middle cerebral artery occlusion. Whole body plethysmography was performed on C57BL/6 young (2-3 months) and aged (20 months) male and female mice. Animals were exposed to a variety of gas conditions to assess the contribution of peripheral and central chemoreceptors. A battery of cognitive tests was performed to examine behavioral function. RESULTS: Middle cerebral artery occlusion led to disordered breathing characterized by hypoventilation and apneas. Cognitive decline correlated with the severity of disordered breathing. Distal permanent middle cerebral artery occlusion, which produces a smaller cortical infarct, also produced breathing disorders and cognitive impairment but only in aged mice. CONCLUSIONS: Our data suggest that poststroke apnea is associated with cognitive decline and highlights the influence of aging on breathing disorders after stroke. Therefore, the treatment of respiratory instability may be a viable approach to improving cognitive outcomes after stroke.
Assuntos
Disfunção Cognitiva , Acidente Vascular Cerebral , Masculino , Feminino , Camundongos , Animais , Infarto da Artéria Cerebral Média/complicações , Apneia , Camundongos Endogâmicos C57BL , Disfunção Cognitiva/psicologiaRESUMO
Long-term disability after stroke is common but the mechanisms of post-stroke recovery remain unclear. Cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 contributes to functional recovery after ischemic stroke in mice. As Rac1 plays divergent roles in individual cell types after central neural system injury, we herein examined the specific role of neuronal Rac1 in post-stroke recovery and axonal regeneration. Young male mice were subjected to 60-min of middle cerebral artery occlusion (MCAO). Inducible deletion of neuronal Rac1 by daily intraperitoneal injection of tamoxifen (2 mg/40 g) into Thy1-creER/Rac1-floxed mice day 7-11 after MCAO worsened cognitive (assayed by novel object recognition test) and sensorimotor (assayed by adhesive removal and pellet reaching tests) recovery day 14-28 accompanied with the reduction of neurofilament-L (NFL) and myelin basic protein (MBP) and the elevation of glial fibrillary acidic protein (GFAP) in the peri-infarct zone assessed by immunostaining. Whereas the brain tissue loss was not altered assayed by cresyl violet staining. In another approach, delayed overexpression of neuronal Rac1 by injection of lentivirus encoding Rac1 with neuronal promotor into both the cortex and striatum (total 4 µl at 1 × 109 transducing units/mL) of stroke side in C57BL/6J mice day 7 promoted stroke outcome, NFL and MBP regrowth and alleviated GFAP invasion. Furthermore, neuronal Rac1 over-expression led to the activation of p21 activating kinases (PAK) 1, mitogen-activated protein kinase kinase (MEK) 1/2 and extracellular signal-regulated kinase (ERK) 1/2, and the elevation of brain-derived neurotrophic factor (BDNF) day 14 after stroke. Finally, we observed higher counts of neuronal Rac1 in the peri-infarct zone of subacute/old ischemic stroke subjects. This work identified a neuronal Rac1 signaling in improving functional recovery and axonal regeneration after stroke, suggesting a potential therapeutic target in the recovery stage of stroke.
Assuntos
Plasticidade Neuronal/fisiologia , Neuropeptídeos/metabolismo , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Axônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Aging is associated with dysfunction of the gut microbiota-immune-brain axis, a major regulatory axis in both brain health and in central nervous system (CNS) diseases. Antigen presenting cells (APCs) play a major role in sensing changes in the gut microbiota and regulation of innate and adaptive immune responses. APCs have also been implicated in various chronic inflammatory conditions, including age-related neurodegenerative diseases. The increase in chronic low-level inflammation seen with aging has also been linked to behavioral decline. Despite their acknowledged importance along the gut microbiota-immune-brain axis, there is limited evidence on how APCs change with aging. In this study, we examined age-related changes in myeloid APCs in the gut, spleen, and brain as well as changes in the gut microbiota and behavioral phenotype in mice ranging in age from 2 months up to 32 months of both sexes. Our data show that the number of peripherally-sourced myeloid APCs significantly increases with advanced aging in the brain. In addition, our data showed that age-related changes in APCs are subset-specific in the gut and sexually dimorphic in the spleen. Our work highlights the importance of studying myeloid APCs in an age-, tissue-, and sex-specific manner.
Assuntos
Doenças do Sistema Nervoso Central , Microbioma Gastrointestinal , Envelhecimento , Animais , Células Apresentadoras de Antígenos , Encéfalo , Feminino , Masculino , CamundongosRESUMO
Brain microvascular endothelial cells play an essential role in maintaining blood-brain barrier (BBB) integrity, and disruption of the BBB aggravates the ischemic injury. CaMKK (α and ß) is a major kinase activated by elevated intracellular calcium. Previously, we demonstrated that inhibition of CaMKK exacerbated outcomes, conversely, overexpression reduced brain injury after stroke in mice. Interestingly, CaMKK has been shown to activate a key endothelial protector, sirtuin 1 (SIRT1). We hypothesized that CaMKK protects brain endothelial cells via SIRT1 activation after stroke. In this study, Oxygen-Glucose Deprivation (OGD) was performed in human brain microvascular endothelial cells. Stroke was induced by middle cerebral artery occlusion (MCAO) in male mice. Knockdown of CaMKK ß using siRNA increased cell death following OGD. Inhibition of CaMKK ß by STO-609 significantly and selectively down-regulated levels of phosphorylated SIRT1 after OGD. Changes in the downstream targets of SIRT1 were observed following STO-609 treatment. The effect of STO-609 on cell viability after OGD was absent, when SIRT1 was concurrently inhibited. We also demonstrated that STO-609 increased endothelial expression of the pro-inflammatory proteins ICAM-1 and VCAM-1 and inhibition of CaMKK exacerbated OGD-induced leukocyte-endothelial adhesion. Finally, intracerebroventricular injection of STO-609 exacerbated endothelial apoptosis and reduced BBB integrity after 24-hr reperfusion following MCAO in vivo. Collectively, these results demonstrated that CaMKK inhibition reduced endothelial cell viability, exacerbated inflammatory responses and aggravated BBB impairment after ischemia. CaMKK activation may attenuate ischemic brain injury via protection of the microvascular system and a reduction in the infiltration of pro-inflammatory factors.
Assuntos
Barreira Hematoencefálica/enzimologia , Isquemia Encefálica/enzimologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Células Endoteliais/enzimologia , Acidente Vascular Cerebral/enzimologia , Animais , Benzimidazóis/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Morte Celular , Células Cultivadas , Humanos , Masculino , Camundongos Endogâmicos C57BL , Naftalimidas/farmacologia , Sirtuína 1/metabolismoRESUMO
Neonatal hypoxia-ischemia (HI) is a major cause of death and disability in neonates. HI leads to a dramatic rise in intracellular calcium levels, which was originally thought to be detrimental to the brain. However, it has been increasingly recognized that this calcium signaling may also play an important protective role after injury by triggering endogenous neuroprotective pathways. Calcium/calmodulin-dependent protein kinase kinase ß (CaMKK ß) is a major kinase activated by elevated levels of intracellular calcium. Here we evaluated the functional role of CaMKK ß in neonatal mice after HI in both acute and chronic survival experiments. Postnatal day ten wild-type (WT) and CaMKK ß knockout (KO) mouse male pups were subjected to unilateral carotid artery ligation, followed by 40 min of hypoxia (10% O2 in N2). STO-609, a CaMKK inhibitor, was administered intraperitoneally to WT mice at 5 minutes after HI. TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess infarct volume 24 h after HI. CaMKK ß KO mice had larger infarct volume than WT mice and STO-609 increased the infarct volume in WT mice after HI. In chronic survival experiments, WT mice treated with STO-609 showed increased tissue loss in the ipsilateral hemisphere three weeks after HI. Furthermore, when compared with vehicle-treated mice, they showed poorer functional recovery during the three week survival period, as measured by the wire hang test and corner test. Loss of blood-brain barrier proteins, a reduction in survival protein (Bcl-2), and an increase in pro-apoptotic protein Bax were also seen after HI with CaMKK ß inhibition. In conclusion, inhibition of CaMKK ß exacerbated neonatal hypoxia-ischemia injury in mice. Our data suggests that enhancing CaMKK signaling could be a potential target for the treatment of hypoxic-ischemic brain injury.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Hipóxia-Isquemia Encefálica/enzimologia , Hipóxia-Isquemia Encefálica/patologia , Animais , Animais Recém-Nascidos , Benzimidazóis/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Western Blotting , Morte Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naftalimidas/farmacologiaRESUMO
INTRODUCTION: Sex is increasingly recognized as an important factor in the epidemiology and outcome of many diseases. This also appears to hold for coronavirus disease 2019 (COVID-19). Evidence from China and Europe has suggested that mortality from COVID-19 infection is higher in men than women, but evidence from US populations is lacking. Utilizing data from a large healthcare provider, we determined if males, as compared to females have a higher likelihood of SARS-CoV-2 susceptibility, and if among the hospitalized COVID-19 patients, male sex is independently associated with COVID-19 severity and poor in-hospital outcomes. METHODS AND FINDINGS: Using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines, we conducted a cross-sectional analysis of data from a COVID-19 Surveillance and Outcomes Registry (CURATOR). Data were extracted from Electronic Medical Records (EMR). A total of 96,473 individuals tested for SARS-CoV-2 RNA in nasopharyngeal swab specimens via Polymerized Chain Reaction (PCR) tests were included. For hospital-based analyses, all patients admitted during the same time-period were included. Of the 96,473 patients tested, 14,992 (15.6%) tested positive, of whom 4,785 (31.9%) were hospitalized and 452 (9.5%) died. Among all patients tested, men were significantly older. The overall SARS-CoV-2 positivity among all tested individuals was 15.5%, and was higher in males as compared to females 17.0% vs. 14.6% [OR 1.20]. This sex difference held after adjusting for age, race, ethnicity, marital status, insurance type, median income, BMI, smoking and 17 comorbidities included in Charlson Comorbidity Index (CCI) [aOR 1.39]. A higher proportion of males (vs. females) experienced pulmonary (ARDS, hypoxic respiratory failure) and extra-pulmonary (acute renal injury) complications during their hospital course. After adjustment, length of stay (LOS), need for mechanical ventilation, and in-hospital mortality were significantly higher in males as compared to females. CONCLUSIONS: In this analysis of a large US cohort, males were more likely to test positive for COVID-19. In hospitalized patients, males were more likely to have complications, require ICU admission and mechanical ventilation, and had higher mortality than females, independent of age. Sex disparities in COVID-19 vulnerability are present, and emphasize the importance of examining sex-disaggregated data to improve our understanding of the biological processes involved to potentially tailor treatment and risk stratify patients.
Assuntos
COVID-19/epidemiologia , Cidades/epidemiologia , Índice de Gravidade de Doença , COVID-19/diagnóstico , Estudos Transversais , Suscetibilidade a Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Distribuição por Sexo , Estados Unidos/epidemiologiaRESUMO
Ischemic stroke is major cause of disability and mortality worldwide, and aging is strong risk factor for poor post-stroke outcome. Neutrophils traffic rapidly to the brain following ischemic stroke, and recent evidence has suggested that aging may alter neutrophil function after tissue injury. In this study, we hypothesize that aging enhances the pro-inflammatory function of neutrophils, directly contributing to the poorer outcomes seen in aging patients. We utilized demographic data and biological specimens from ischemic stroke patients and an experimental mouse model to determine the correlation between age, neutrophil function and stroke outcomes. In ischemic stroke patients, age was associated with increased mortality and morbidity and higher levels of neutrophil-activating cytokines. In mice, aged animals had higher stroke mortality and morbidity, higher levels of neutrophil-activating cytokines and enhanced generation of neutrophil reactive oxygen species compared to young mice. Finally, depletion of neutrophils via a specific monoclonal antibody after ischemic stroke led to long-term benefits in functional outcome in aged male and female animals, with no benefit observed in young. These results demonstrate that aging is associated with augmented neutrophil pathogenicity in ischemic stroke, and that neutrophil-targeted therapies may confer greater benefit in aged subjects.
Assuntos
Envelhecimento , Suscetibilidade a Doenças , Neutrófilos/imunologia , Neutrófilos/metabolismo , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologia , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/complicações , Isquemia Encefálica/etiologia , Citocinas/sangue , Modelos Animais de Doenças , Humanos , Contagem de Leucócitos , Camundongos , Morbidade , Mortalidade , Ativação de Neutrófilo/imunologia , Infiltração de Neutrófilos , Estudos Retrospectivos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , VirulênciaRESUMO
BACKGROUND AND PURPOSE: Long-term disability after stroke is common yet the mechanisms of post-stroke recovery are far from clear. It has been suggested that Ras-related C3 botulinum toxin substrate 1 (Rac1) contributes to functional recovery after ischemic stroke in mice. As Rac1 activation plays diverse roles in multiple cell types after central nervous system (CNS) injury, we herein examined the functional role of endothelial Rac1 in post-stroke recovery and angiogenesis. METHODS: Transient middle cerebral artery occlusion (MCAO) in mice and oxygen-glucose deprivation (OGD) in human brain endothelial cell line-5i (HEBC 5i) were performed to mimic ischemic stroke. Lentivirus vectors encoding Rac1 with GFP and endothelial promotor ENG were injected into the animal's brain after stroke to overexpress Rac1. After injection, stroke recovery was tested by multiple behavioral tests including novel object recognition, adhesive removal and single pellet reaching tests. Endothelial regeneration in the peri-infarct zone was detected by immunohistochemistry (IHC). In the vitro model, the effect of Rac1 and Pak1 inhibitors to cell proliferation and migration was examined by CCK-8 and wound healing assays after OGD. The cellular protein level of brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response element-binding protein (CREB), extracellular signal-regulated kinase (ERK) 1/2 and mitogen-activated protein kinase kinase (MEK) 1/2 were detected by western blots. RESULTS: Delayed overexpression of endothelial Rac1 after MCAO improved cognitive and sensorimotor recovery from day 14 to 21 after stroke, increased vascular density and the protein level of pericytes in the peri-infarct zone without altering tissue loss in mice. Consistently, inhibition of Rac1 prevented endothelial proliferation and migration after OGD. Pak1 inhibition exerted a similar effect on endothelial cells. However, co-incubation of Rac1 and Pak1 inhibitors with cells did not lead to additive effects when compared with either inhibitor alone. Moreover, individual inhibition of Rac1 or Pak1 suppressed OGD-induced activation of pro-regenerative molecules, including CREB, MEK1/2 and ERK1/2, as well as the production of BDNF in vitro. The level of these proteins did not further decrease if both Rac1 and Pak1 were simultaneously inhibited. CONCLUSIONS: We conclude that activation of endothelial Rac1 improves functional recovery and angiogenesis after stroke, and this process is mediated by Pak1 signaling. This study provides novel insight for Rac1 in the mechanism of long-term stroke recovery.
Assuntos
Neovascularização Fisiológica/fisiologia , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Aging and stroke alter the composition of the basement membrane and reduce the perivascular distribution of cerebrospinal fluid and solutes, which may contribute to poor functional recovery in elderly patients. Following stroke, TGF-ß induces astrocyte activation and subsequent glial scar development. This is dysregulated with aging and could lead to chronic, detrimental changes within the basement membrane. We hypothesized that TGF-ß induces basement membrane fibrosis after stroke, leading to impaired perivascular CSF distribution and poor functional recovery in aged animals. We found that CSF entered the aged brain along perivascular tracts; this process was reduced by experimental stroke and was rescued by TGF-ß receptor inhibition. Brain fibronectin levels increased with experimental stroke, which was reversed with inhibitor treatment. Exogenous TGF-ß stimulation increased fibronectin expression, both in vivo and in primary cultured astrocytes. Oxygen-glucose deprivation of cultured astrocytes induced multiple changes in genes related to astrocyte activation and extracellular matrix production. Finally, in stroke patients, we found that serum TGF-ß levels correlated with poorer functional outcomes, suggesting that serum levels may act as a biomarker for functional recovery. These results support a potential new treatment strategy to enhance recovery in elderly stroke patients.
Assuntos
Membrana Basal/patologia , Líquido Cefalorraquidiano/metabolismo , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Fator de Crescimento Transformador beta/farmacologia , Idoso , Animais , Benzamidas/farmacologia , Biomarcadores/sangue , Encéfalo/metabolismo , Feminino , Fibronectinas/metabolismo , Fibrose , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/sangueRESUMO
Neurite plasticity is a critical aspect of brain functional recovery after stroke. Emerging data suggest that Ras-related C3 botulinum toxin substrate 1 (Rac1) plays a central role in axonal regeneration in the injured brain, specifically by stimulating neuronal intrinsic growth and counteracting the growth inhibitory signaling that leads to growth cone collapse. Therefore, we investigated the functional role of Rac1 in axonal regeneration after stroke.Delayed treatment with a specific Rac1 inhibitor, NSC 23766, worsened functional recovery, which was assessed by the pellet reaching test from day 14 to day 28 after stroke. It additionally reduced axonal density in the peri-infarct zone, assessed 28 days after stroke, with no effect on brain cavity size or on the number of newly formed cells. Accordingly, Rac1 overexpression using lentivirus promoted axonal regeneration and functional recovery after stroke from day 14 to day 28. Rac1 inhibition led to inactivation of pro-regenerative molecules, including mitogen-activated protein kinase kinase (p-MEK)1/2, LIM domain kinase (LIMK)1, and extracellular signal-regulated kinase (p-ERK)1/2 at 14 days after stroke. Inhibition of Rac1 reduced axonal length and number in cultured primary mouse cortical neurons using microfluidic chambers after oxygen-glucose deprivation (OGD) without affecting cell viability. In contrast, inhibition of Rac1 increased levels of glial fibrillary acidic protein, an extrinsic inhibitory signal for axonal growth, after stroke in vivo and in primary astrocytes after OGD.In conclusion, Rac1 signaling enhances axonal regeneration and improve post-stroke functional recovery in experimental models of stroke.