Sex Differences in Poststroke Inflammation: a Focus on Microglia Across the Lifespan.

Stroke is one of the leading causes of death worldwide and currently only few therapeutic options are available. Stroke is a sexually dimorphic disease contributing to the difficulty in finding efficient treatments. Poststroke neuroinflammation is geared largely by brain microglia and infiltrating peripheral immune cells and largely contributes to sex differences in the outcome of stroke. Microglia, since very early in the development, are sexually divergent, imprinting specific sex-related features. The diversity in terms of microglial density, morphology, and transcriptomic and proteomic profiles between sexes remains in the adulthood and is likely to contribute to the observed sex-differences on the postischemic inflammation. The impact of sexual hormones is fundamental: changes in terms of risk and severity have been observed for females before and after menopause underlining the importance of altered circulating sexual hormones. Moreover, aging is a driving force for changes that interact with sex, shifting the inflammatory response in a sex-dependent manner. This review summarizes the present literature on sex differences in stroke-induced inflammatory responses, with the focus on different microglial responses along lifespan.

Microglial amyloid beta clearance is driven by PIEZO1 channels.

BACKGROUND: Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer's disease (AD), toxic amyloid beta (Aß) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aß. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia had been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. METHODS: Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aß pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. RESULTS: We show that PIEZO1 orchestrates Aß clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aß inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aß clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single-cell datasets. CONCLUSION: These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aß burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

Additive Behavioral Improvement after Combined Cell Therapy and Rehabilitation Despite Long-Term Microglia Presence in Stroke Rats.

Microglia are involved in the post-stroke immunomodulation of brain plasticity, repair, and reorganization. Here, we evaluated whether adipose-tissue-derived mesenchymal stem cells (ADMSCs) and/or rehabilitation improve behavioral recovery by modulating long-term perilesional inflammation and creating a recovery-permissive environment in a rat model of ischemic stroke. METHODS: A two-way mixed lymphocyte reaction was used to assess the immunomodulatory capacity of ADMSCs in vitro. Two or 7 days after permanent middle cerebral artery occlusion (pMCAO), rats were intravenously administered ADMSCs or vehicle and housed in a standard or enriched environment (EE). Behavioral performance was assessed with a cylinder test, then we performed stereological and ImageJ/Fiji quantifications of ionized calcium-binding adaptor molecule 1 (Iba1) cells and blood-brain barrier (BBB) leakage. RESULTS: Human ADMSCs were immunosuppressive in vitro. The cylinder test showed partial spontaneous behavioral recovery of pMCAO rats, which was further improved by combined ADMSCs and housing in EE on days 21 and 42 (p < 0.05). We detected an ischemia-induced increase in numbers, staining intensity, and branch length of Iba1+ microglia/macrophages as well as BBB leakage in the perilesional cortex. However, these were not different among pMCAO groups. CONCLUSION: Combined cell therapy and rehabilitation additively improved behavioral outcome despite long-term perilesional microglia presence in stroke rats.

Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray.

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.

Brain-derived neurotrophic factor alleviates the oxidative stress induced by oxygen and glucose deprivation in an ex vivo brain slice model.

Brain-derived neurotrophic factor (BDNF) is considered as a putative therapeutic agent against stroke. Since BDNF role on oxidative stress is uncertain, we have studied this role in a rat brain slice ischemia model, which allows BDNF reaching the neural parenchyma. Hippocampal and cerebral cortex slices were subjected to oxygen and glucose deprivation (OGD) and then returned to normoxic conditions (reperfusion-like, RL). OGD/RL increased a number of parameters mirroring oxidative stress in the hippocampus that were reduced by the BDNF presence. BDNF also reduced the OGD/RL-increased activity in a number of antioxidant enzymes in the hippocampus but no effects were observed in the cerebral cortex. In general, we conclude that alleviation of oxidative stress by BDNF in OGD/RL-exposed slices relies on decreasing cPLA2 activity, rather than modifying antioxidant enzyme activities. Moreover, a role for the oxidative stress in the differential ischemic vulnerability of cerebral cortex and hippocampus is also supported.

Post-ischemic salubrinal administration reduces necroptosis in a rat model of global cerebral ischemia.

Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Celecoxib Treatment Improves Neurologic Deficit and Reduces Selective Neuronal Loss and Glial Response in Rats after Transient Middle Cerebral Artery Occlusion.

Areas of selective neuronal loss (SNL) represent the first morphologic signs of damage in the penumbra region and are considered putative targets for ischemic stroke therapy. We performed a novel assessment of measuring the effects of the anti-inflammatory agent celecoxib by analyzing simultaneously the different neural populations (neurons, astrocytes, and microglia cells) in SNL and non-SNL areas. Rats were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and treated with celecoxib 1 and 24 hours after ischemia. Infarct volume measurements and triple immunostaining of neurons (neuronal nuclear antigen), microglia (ionized calcium-binding adaptor molecule 1), and astroglia were performed after 12 and 48 hours of reperfusion. Motor response was tested by standard behavioral assays at 3, 12, 24, and 48 hours. Confocal analysis revealed that the percentage of SNL areas, microglia densities, and glial activation increased at 48 hours of reperfusion. Celecoxib treatment improved the neurologic deficit, reduced the infarct volume by 50% after 48 hours of reperfusion, and resulted in a reduced percentage of SNL areas and microglia and astroglia reactivity after 48 hours of reperfusion. This study proves, for the first time, that celecoxib presents postischemic neuroprotective effects in a transient MCAO model, prevents or delays the presence of SNL areas, and reduces glial activation.

ciRS-7 and miR-7 regulate ischemia-induced neuronal death via glutamatergic signaling.

Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.

Celecoxib-Dependent Neuroprotection in a Rat Model of Transient Middle Cerebral Artery Occlusion (tMCAO) Involves Modifications in Unfolded Protein Response (UPR) and Proteasome.

Stroke is one of the main causes of death and disability worldwide. Ischemic stroke results in unfolded/misfolded protein accumulation in endoplasmic reticulum (ER), a condition known as ER stress. We hypothesized that previously reported neuroprotection of celecoxib, a selective inhibitor of cyclooxygenase-2, in transient middle cerebral artery occlusion (tMCAO) model, relies on the ER stress decrease. To probe this hypothesis, Sprague-Dawley rats were subjected to 1 h of tMCAO and treated with celecoxib or vehicle 1 and 24 h after ischemia. Protein and mRNA levels of the main hallmarks of ER stress, unfolded protein response (UPR) activation, UPR-induced cell death, and ubiquitin proteasome system (UPS) and autophagy, the main protein degradation pathways, were measured at 12 and 48 h of reperfusion. Celecoxib treatment decreased polyubiquitinated protein load and ER stress marker expression such as glucose-related protein 78 (GRP78), C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP), and caspase 12 after 48 h of reperfusion. Regarding the UPR activation, celecoxib promoted inositol-requiring enzyme 1 (IRE1) pathway instead of double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway. Furthermore, celecoxib treatment increased proteasome catalytic subunits transcript levels and decreased p62 protein levels, while the microtubule-associated protein 1 light chain 3 (LC3B) II/I ratio remained unchanged. Thus, the ability of celecoxib treatment on reducing the ER stress correlates with the enhancement of IRE1-UPR pathway and UPS degradation. These data support the ability of anti-inflammatory therapy in modulating ER stress and reveal the IRE1 pathway as a promising therapeutic target in stroke therapy.Graphical abstract.

Salubrinal and robenacoxib treatment after global cerebral ischemia. Exploring the interactions between ER stress and inflammation.

BACKGROUND: Blood reperfusion of the ischemic tissue after stroke promotes increases in the inflammatory response as well as accumulation of unfolded/misfolded proteins in the cell, leading to endoplasmic reticulum (ER) stress. Both Inflammation and ER stress are critical processes in the delayed death of the cells damaged after ischemia. The aim of this study is to check the putative synergic neuroprotective effect by combining anti-inflammatory and anti-ER stress agents after ischemia. METHODS: The study was performed on a two-vessel occlusion global cerebral ischemia model. Animals were treated with salubrinal one hour after ischemia and with robenacoxib at 8 h and 32 h after ischemia. Parameters related to the integrity of the blood-brain barrier (BBB), such as matrix metalloproteinase 9 and different cell adhesion molecules (CAMs), were analyzed by qPCR at 24 h and 48 h after ischemia. Microglia and cell components of the neurovascular unit, including neurons, endothelial cells and astrocytes, were analyzed by immunofluorescence after 48 h and seven days of reperfusion. RESULTS: Pharmacologic control of ER stress by salubrinal treatment after ischemia, revealed a neuroprotective effect over neurons that reduces the transcription of molecules involved in the impairment of the BBB. Robenacoxib treatment stepped neuronal demise forward, revealing a detrimental effect of this anti-inflammatory agent. Combined treatment with robenacoxib and salubrinal after ischemia prevented neuronal loss and changes in components of the neurovascular unit and microglia observed when animals were treated only with robenacoxib. CONCLUSION: Combined treatment with anti-ER stress and anti-inflammatory agents is able to provide enhanced neuroprotective effects reducing glial activation, which opens new avenues in therapies against stroke.