Cerebral edema and neurovascular dysfunction are reliable predictors of outcome after acute ischemic stroke (AIS), yet effective targeted therapies remain limited. We investigated the therapeutic potential of trifluoperazine (TFP), an FDA-approved antipsychotic and calmodulin inhibitor that modulates astrocytic aquaporin-4 expression. TFP administered after recanalization in a mouse model of AIS reduced infarct volume and improved early neurological recovery. Importantly, TFP restored neurovascular coupling and enhanced cerebral blood flow responses to spreading depolarizations, indicating improved cerebrovascular function. Mechanistic studies in acute brain slice preparations demonstrated that TFP attenuates cytotoxic tissue swelling, suppresses spreading depolarizations, reduces aquaporin-4 expression and preserves neuronal integrity under osmotic stress. These findings suggest that transient modulation of astrocytic cytotoxic edema and vascular reactivity represents a viable strategy to improve early stroke outcomes. Given its established clinical use, TFP emerges as a promising candidate for therapeutic repurposing in AIS.

BACKGROUND:Cerebral small vessel disease (cSVD) is a major cause of stroke and dementia, and is associated with increased blood-brain barrier permeability, neuroinflammation, and endothelial dysfunction. Endothelial Gαq/11 proteins are involved in vascular tone regulation and have been shown to affect capillary blood flow in the brain. Since factors downstream of activated Gαq/11 proteins, such as endothelial NO synthase (eNOS) activity, are discussed in cSVD, we asked whether the brain endothelial Gαq/11 signalling pathway might influence cSVD-related pathology. METHODS:Here, we generated mice carrying a brain endothelial-specific deletion of the Gαq/11 signalling and characterised these mice using different imaging and staining techniques, as well as behaviour tests measuring cognition in adult and aged mice. Immunoblots, electrophysiology, perfusion measurements, and in vitro experiments complemented those techniques. FINDINGS:The brain endothelial Gαq/11 signalling pathway preserves normal vascular reactivity, and its loss resembles mild endothelial dysfunction in the brain. While the vessel structure was maintained in adult mice, deletion of Gαq/11 signalling led to capillary rarefaction and blood-brain barrier disruption in aged mice. These effects were accompanied by disturbed VEGF signalling and an increase in senescence markers and oxidative stress in the vasculature, culminating in cognitive impairment with increased tau phosphorylation in the cortex and hippocampus and decreased myelination in the white matter. INTERPRETATION:These findings reflect the main hallmarks of cSVD and demonstrate a protective role of Gαq/11 in endothelial cells in ageing. Furthermore, our results show that the combination of cerebral endothelial dysfunction and ageing accelerates cognitive impairment. FUNDING:Research was supported by grants from the European Research Council, the Deutsche Forschungsgemeinschaft, the institutional priority program MI-VascAD of the University of Lübeck, and the Marie-Sklodowska-Curie European Union's Horizon 2020 research program.

Advanced age is associated with larger infarct volumes and poorer functional recovery after acute ischemic stroke (AIS). Carotid stenosis is also a common comorbidity in older individuals and often predicts subsequent AIS. However, no age-specific therapy is currently available to protect the aging brain from aggravated ischemic injury. Here, we investigated whether a senolytic approach could improve cerebrovascular status and reduce ischemic brain injury in a comorbid aging model of AIS. Unilateral common carotid artery occlusion was induced in young and aged rats and served as a diagnostic trigger for chronic senolytic therapy with dasatinib plus quercetin (D+Q). Two weeks later, the distal middle cerebral artery was occluded for 60 min. Compared with vehicle-treated animals, infarct size was measured, spreading depolarizations (SDs) were recorded electrophysiologically, cerebral blood flow (CBF) dynamics were monitored by laser speckle contrast imaging, and cerebrovascular senescent cell burden was assessed by immunocytochemistry. Cerebral angiogenesis, central and systemic inflammatory markers, and metabolic status were evaluated using protein arrays and blood glucose measurements. Aged rats developed larger infarcts than young controls, and this age-related increase was attenuated by D+Q treatment. D+Q reduced the higher frequency of SDs observed in the aged ischemic brain. Increased cerebrovascular senescence in aged animals was diminished by D+Q, accompanied by enhanced angiogenesis, although CBF responses to SDs and reperfusion were unchanged. In addition, D+Q modulated central and systemic inflammatory profiles and counteracted age-related metabolic impairment. Senolytic D+Q therapy administered after carotid artery occlusion confers multifaceted protection against subsequent AIS in the aged brain. By targeting fundamental aging mechanisms that exacerbate brain vulnerability to AIS, D+Q enhanced the resilience of the aging neurovascular niche. Long-term neurological outcomes were not assessed in the present study; therefore, the results provide a rationale for further investigation of senolytic strategies in age-related stroke vulnerability.

Cerebral ischemic stroke is a leading cause of mortality and morbidity worldwide, with systemic effects extending beyond brain injury to multiple organs, particularly the lungs. Clinical and experimental studies have demonstrated that altered immune function, post-stroke pneumonia, dysphagia and aspiration, pulmonary tissue inflammation, neurogenic pulmonary edema, mucus hypersecretion with impaired debris clearance, abnormal breathing patterns, respiratory muscle weakness, atelectasis and acute lung injury are all potential contributors to post-stroke pulmonary dysfunction development. These complications are associated with measurable changes in lung function, including altered inspiratory and expiratory pressures, impaired spirometry parameters, as well as altered respiratory mechanical parameters, including reduced lung compliance, changes in airway resistance, reduced functional residual capacity in both stroke survivors and animal models of global and focal cerebral ischemia. Mechanical ventilation, frequently required in severe stroke, poses additional short- and long-term challenges. This review integrates evidence from clinical and experimental studies to highlight the mechanisms underlying post-stroke pulmonary dysfunction and underscores the need for optimized ventilation strategies and longitudinal clinical studies to better understand long-term outcomes and reduce respiratory complications in stroke survivors.

Abstract Background Advanced age is associated with larger infarct volumes and poorer functional recovery after acute ischemic stroke (AIS). Carotid stenosis is also a common comorbidity in older individuals and often predicts subsequent AIS. However, no age-specific therapy is currently available to protect the aging brain from aggravated ischemic injury. Here, we investigated whether a senolytic approach could improve cerebrovascular status and reduce ischemic brain injury in a comorbid aging model of AIS. Methods Unilateral common carotid artery occlusion was induced in young and aged rats and served as a diagnostic trigger for chronic senolytic therapy with dasatinib plus quercetin (D+Q). Two weeks later, the distal middle cerebral artery was occluded for 60 min. Compared with untreated animals, infarct size was measured, spreading depolarizations (SDs) were recorded electrophysiologically, cerebral blood flow (CBF) dynamics were monitored by laser speckle contrast imaging, and cerebrovascular senescent cell burden was assessed by immunocytochemistry. Cerebral angiogenesis, central and systemic inflammatory markers, and metabolic status were evaluated using protein arrays and blood glucose measurements. Results Aged rats developed larger infarcts than young controls, and this age-related increase was attenuated by D+Q treatment. D+Q reduced the higher frequency of SDs observed in the aged ischemic brain. Increased cerebrovascular senescence in aged animals was diminished by D+Q, accompanied by enhanced angiogenesis, although CBF responses to SDs and reperfusion were unchanged. In addition, D+Q modulated central and systemic inflammatory profiles and counteracted age-related metabolic impairment. Conclusions Senolytic D+Q therapy administered after carotid artery occlusion confers multifaceted protection against subsequent AIS in the aged brain. By targeting fundamental aging mechanisms that exacerbate brain vulnerability to AIS, D+Q enhances the resilience of the aging neurovascular niche. These results identify senolytic therapy as a promising preventive personalized approach to mitigate the disproportionate impact of AIS in older individuals and warrant further investigation.

Major cerebral artery occlusion causes acute ischemic stroke and triggers systemic responses that extend beyond the brain, including lung injury. Although arterial recanalization limits neuronal damage, reperfusion may also modulate peripheral organ dysfunction. In this study, we compared the pulmonary mechanical, structural, and inflammatory consequences of permanent versus transient focal cerebral ischemia. Young male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion for 3 days (n = 10), transient occlusion for 60 min followed by 3 days of reperfusion (n = 9), or SHAM operation (n = 10). Lung injury was assessed by measuring respiratory mechanics by forced oscillation at positive end-expiratory pressures of 0, 3, and 6 cmH2O, histology, wet-to-dry ratio, bronchoalveolar lavage fluid (BALF), and serum cytokine profiling. Permanent ischemia increased respiratory tissue damping and elastance, accompanied by pronounced alveolar septal thickening, fibrin deposition, pulmonary edema, hemoconcentration, and systemic inflammatory alterations. Conversely, transient ischemia showed similar respiratory mechanical changes or lung injury, evidenced by histological and inflammatory responses, with tissue mechanical parameters remaining close to sham values. Several inflammatory and tissue remodeling markers were differentially regulated in serum and BALF, indicating compartment-specific lung responses. Moreover, the extent of cerebral ischemia correlated with deterioration of lung tissue mechanics. These findings demonstrate that persistent cerebral ischemia promotes lung injury triggered by adverse secondary alveolar inflammatory and structural changes, whereas transient cerebral ischemia is associated with milder pulmonary tissue damage. These findings highlight that the temporal dynamics of cerebral ischemia are an important determinant of stroke-associated lung injury.NEW & NOTEWORTHY By comparing permanent and transient focal cerebral ischemia, this study reveals that the duration of stroke determines secondary pulmonary outcomes. Permanent ischemia induced pronounced lung tissue injury, inflammation, edema, and impaired tissue mechanics, whereas transient ischemia with reperfusion largely preserved lung structure and function. These findings identify the temporal dynamics of cerebral ischemia as an important modulator of brain-lung interactions and suggest that limiting ischemic exposure may attenuate stroke-associated pulmonary complications.

Abstract Background and aims Spreading depolarizations (SDs) exacerbate neuronal injury during acute ischemic stroke (AIS). The sigma-1 receptor (S1R) agonist dimethyltryptamine (DMT) reduces cellular damage, inhibits SDs, and enhances neuronal survival in rodent models of AIS. Although DMT primarily acts on S1R, it also binds to aminergic receptors. Therefore, we aimed to determine the S1R-mediated neuroprotective effects of DMT. Methods 350-μm-thick brain slices were prepared from wild-type C57BL/6 (WT) and S1R receptor knockout (S1R-KO) mice (n=12). To model acute ischemic stroke (AIS), a medium of reduced glucose content was applied, and SDs were induced by hypoxia. SDs were recorded using intrinsic optical signal (white-light reflectance) imaging and local field potential recordings. Brain slices were incubated in solutions containing DMT (20 μM) or its vehicle. Neuronal viability was assessed using NeuN immunohistochemistry. Results DMT reduced the cortical area affected by SDs in both WT and S1R-KO animals (53.3±21.6% vs. 65.7±13.8% and 42.1±11.2% vs. 61.0±12.7%; WT+DMT vs. WT and S1R-KO+DMT vs. S1R-KO). Furthermore, DMT reduced the area under the curve of SDs (299.9±148.0 vs. 543.3±270.5 mV·s, S1R-KO+DMT vs. S1R-KO) and their propagation velocity (2.6±1.0 vs. 3.8±1.8 mm/min) in S1R KO animals only. NeuN-positive cell number tended to increase following DMT treatment. Contrary to our expectations, these results demonstrate that DMT is more effective in S1R-KO than in WT animals, suggesting that its neuroprotective effects are mediated by aminergic receptors in addition to S1R activation. These findings support the potential adjuvant use of DMT in the treatment of AIS. Conflict of interest Funding: EU H2020-HCEMM (No. 739593), NKFIH (No. K146725), The Hungarian Brain Research Program 3.0. to E.F. and Á.M., National Research, Development and Innovation Office of Hungary (PD137565) to N.S.

Background: Cytotoxic brain edema, marked by astrocyte swelling, develops early after acute ischemic stroke (AIS). While the ischemic core is irreversibly injured, the penumbra remains vulnerable to spreading depolarization (SD), which propagates through viable tissue and exacerbates lesion progression. Swollen astrocytes fail to maintain extracellular ion and glutamate homeostasis, thus facilitating SD initiation and promoting excitotoxicity. Using an acute brain slice model of stroke-related cytotoxic edema, we tested whether inhibiting astrocyte swelling limits SD and glutamate accumulation. Methods: Coronal brain slices (350 µm) from Wistar rats (n=28) were prepared. Cytotoxic edema was achieved by hypo-osmotic stress (NaCl 130→60 mM; HM60), SD was elicited by transient hypoxia (2.5 min). The spatio-temporal characteristics of SD were detected by local field potential and intrinsic optical signal recordings. Extracellular glutamate levels were measured by enzymatic biosensors. To counteract astrocyte swelling Bumetanide (Bum; 1 mM), TGN-020 (100 µM), and DCPIB (20 µM) were applied to inhibit sodium potassium chloride cotransporter 1 (NKCC1), aquaporin-4 (AQP4) and volume regulated anion channels (VRAC). Results: In HM60 the SD area affected nearly the entire cortex (82.5±11.5%) which was reduced by DCPIB and TGN-020+Bum treatments (45.1±11.6 and 44.8±13.2%). Surprisingly, despite limiting spatial spread, both treatments increased SD propagation velocity compared to HM60 (5.8±1.5 vs. 5.1±1.5 vs. 3.7±1.2 mm/min; DCPIB vs.TGN-020+Bum vs. HM60). SDs in HM60 were accompanied by a glutamate peak of 61.7±14.2 µM, which remained elevated during recovery (22±10 µM). DCPIB and TGN-020+Bum reduced the glutamate peak (25.4±6.6 and 21.7±7 µM), the post-SD glutamate accumulation (9.9±4.1 and 8.7±2.8 µM) and shortened the SD plateau (68±44 vs. 111.5±52.3 vs. 176.7±57.6 s; DCPIB vs.TGN-020+Bum vs. HM60), thereby reducing the overall duration of SD. Conclusion: Our results indicate that astrocyte swelling enhances SD propagation and glutamate excitotoxicity in a stroke-related cytotoxic edema model. Pharmacological inhibition of astrocyte swelling may help limit secondary injury and slow lesion progression during the acute phase of ischemic stroke.

IntroductionCerebral edema is a hallmark of lesion progression after acute ischemic stroke (AIS) and a major contributor to the evolution of spreading depolarizations (SDs). SDs trigger extracellular glutamate accumulation and excitotoxic injury, yet the mechanisms linking edema formation, glutamate dysregulation, and SD dynamics remain incompletely understood. Here, we investigated how inhibiting glial swelling and volume-regulated glutamate release, or blocking neuronal ionotropic glutamate receptors alters SD features under hypo-osmotic stress in vitro.MethodsAcute 350-µm-thick brain slices were prepared from male Wistar rats (n = 24). Edema was induced using hypoosmotic medium (130→60 mM NaCl), and SD was triggered by hypoxia. SD evolution and extracellular glutamate levels were monitored using local field potential recordings, intrinsic optical signal imaging, and enzyme-based glutamate biosensors. Astrocyte swelling was reduced by blocking AQP4+NKCC1 (TGN-020 + bumetanide) and VRAC channels (DCPIB), while neuronal NMDA and AMPA/kainate receptors were antagonized with MK-801 + CNQX.ResultsInhibition of AQP4, NKCC1, or VRAC channels restricted the cortical area invaded by SD, shortened SD duration, and reduced extracellular glutamate accumulation. In contrast, blockade of NMDA or AMPA/kainate receptors markedly decreased SD propagation and glutamate buildup. Both astrocytic and neuronal interventions disrupted typical SD initiation patterns, producing atypical, multifocal SD events.DiscussionThese findings demonstrate that astrocyte volume regulation and neuronal ionotropic glutamate receptors jointly shape SD characteristics under osmotic stress, identifying astrocytic water/ion homeostasis and glutamatergic signaling as potential therapeutic targets to limit excitotoxic injury in acute cerebrovascular disease.

Cognitive impairment is a major medical problem in the aging population. The risk of developing cognitive impairment is higher in several systemic conditions like hypertension, diabetes, and obesity[1][1]–[3][2]. While a vascular contribution to cognitive impairment in these pathologies is well established, the underlying mechanisms are not fully understood[4][3]. Endothelial dysfunction, which frequently accompanies the abovementioned conditions and increases with age[5][4]–[7][5], might be a key causal and mechanistic factor in cognitive deficits associated with systemic conditions. In this study, we demonstrate that the inducible deletion of the Gαq/11 signaling pathway in brain endothelial cells leads to an impaired reactivity of the brain vasculature to vasodilating stimuli such as neuronal activity, representing an isolated cerebral endothelial dysfunction. These mice develop mild cognitive impairment with aging that could be explained by increased tau phosphorylation, decreased myelination, and capillary rarefaction, suggesting that endothelial cell-driven processes protect cognition in aging and providing a mechanistic explanation for how endothelial dysfunction can lead to cognitive impairment in cerebral small vessel disease. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-5 [5]: #ref-7

BACKGROUND AND PURPOSE:Nimodipine, an L-type voltage-gated calcium channel blocker, is an approved cerebral vasorelaxant. We hypothesized that nimodipine attenuates the pro-inflammatory shift in microglial phenotypes. Here, we analysed the effects of nimodipine on morphological and functional microglial phenotypes as well as their transcriptomic profile. EXPERIMENTAL APPROACH:Live brain slice preparations from C57BL/6 mice and primary microglia cultures from neonatal Sprague Dawley rats were used. Microglia were activated either by ischemia or lipopolysaccharide (LPS), and preparations were treated with nimodipine (5-10-20 μM). Microglial morphological phenotypes, phagocytic activity, Iba1 expression and TNF-α levels were evaluated. Total RNA was isolated from monocultures and processed for next generation RNA sequencing. KEY RESULTS:LPS resulted in a pro-inflammatory microglial phenotype, affecting the expression of cytokines, the complement system and phagocytosis-related genes. LPS increased the transcription of ionotropic purinergic and TRP channels but decreased the expression of voltage- and ligand-gated calcium channels, down-regulated the expression of Ryr and IP3 receptors and increased transcription of the SERCA calcium pump. Nimodipine suppressed the amoeboid morphological transformation and phagocytosis and altered the expression of 110 genes in the opposite direction to LPS activation, of which at least 20 were associated with the microglial immune response, seven with cell adhesion and two with autophagy regulation. CONCLUSION AND IMPLICATIONS:The effect of nimodipine goes beyond cerebral vasorelaxation. Nimodipine attenuates microglial activation by modulating Ca2+-dependent gene expression involved in intracellular signalling cascades to drive microglial immune responses. Consideration should be given to expanding the use of nimodipine beyond vasorelaxation.

This research demonstrates that hydrophobically modified chitosan (Chit) with adequate pH sensitivity can deliver drugs to ischemic brain tissues (pH 6.5-6.0). In this study, Chit was modified using hydrophobic hexyl and dodecyl aldehydes in reductive amination reactions. DSC and TG analyses showed that increasing the aldehydic side chain length reduced Chit's water content from 78.5 % to 47.1 %. Consequently, the cut-off pH for nanoparticle (NP) formation was reduced from 7.45 (initial Chit) to 6.74 (hexyl aldehyde modified Chit, C6-chit) and 2.49 (dodecyl aldehyde modified Chit, C12-chit). To target drug release in ischemic regions, the C6-chit polymer was selected for NP development. Different amounts of Pluronic F127 (PF) were incorporated within these NPs to achieve controlled and sustained drug release. The NPs successfully encapsulated two hydrophobic drugs, Nimodipine (NIMO) and HC-067047 (HC). Notably, this study is the first to achieve HC encapsulation within any polymeric particles. The encapsulated drugs exhibited reduced crystallinity compared to bare forms, enhancing both water solubility and stability in aqueous medium. The pH-responsive drug release profiles highlighted the potential of releasing drugs in ischemic tissues rather than normal ones. Additionally, increasing the PF content resulted in prolonged drug release at pH 6, thereby extending the therapeutic effect.

Intracellular sigma-1 receptors (σ1 receptors) have a versatile function through the regulation of lipid rafts, neuroreceptors and ion channels, and can influence signal transduction and neuronal plasticity. Since decreased activity of σ1 receptors is a common pathological feature in the early stages of many neurological diseases, σ1 receptor agonists may represent a promising therapeutic tool for the treatment of these disorders. In this study, we aimed to comprehensively investigate the potential protective effects of the novel synthetic σ1 receptor agonist (S)-L1 against endothelial endoplasmic reticulum (ER) stress and cerebral ischemia. In binding affinity experiments, we showed that (S)-L1 has a high affinity and selectivity for σ1 receptor with virtually no affinity for any of the other receptors tested. Next, (S)-L1 exerted protection against endoplasmic reticulum stress in human brain endothelial cells, consistent with the localization of σ1 receptors in endothelial cells. Furthermore, (S)-L1 penetration was demonstrated across the cell culture model of the blood-brain barrier, providing a rationale for neuronal action in addition to endothelial protection. Finally, (S)-L1 inhibited spreading depolarization, suppressed apoptosis and rescued astrocytes in a rat model of cerebral ischemia. Based on our results, (S)-L1 exerts a protective effect on both brain endothelial cells and neural tissue. Moreover, since these experiments revealed no affinity for serotonergic receptors, the compound holds promise as an adjuvant therapy for the treatment of cerebrovascular disease without potential psychedelic side effects.

Understanding the relationship between the degree of neurological deficit and lesion volume is key to predicting outcomes in patients with acute ischemic stroke (AIS). Over the past 40 years, AIS research has relied on a perceived linear relationship between lesion volumes and neurological deficit. Here, we found that these variables do not show a relationship in a mouse model of AIS. Acute ischemic stroke was induced by transient (60 minutes) intraluminal microfilament occlusion of the middle cerebral artery in 15 male isoflurane (0.8%-1%)-anesthetized mice. Acute ischemic stroke-induced sensorimotor deficits were assessed daily for 72 hours using the Garcia Neuroscore Scale (GNS). Lesion size was estimated 72 hours after AIS using a rodent MRI system. Lesion sizes ranged from 17 to 130 mm3. In 3/15 mice (atypical cases: lesion <30 mm3 and GNS <11), small infarcts (14.6 ± 6.2 vs 51.7 ± 19.9 mm3, atypical vs typical) were associated with low GNS values at 72 hours (9 ± 2 vs 11 ± 2 pts; atypical vs typical). Consequently, we found no relationship between lesion size and GNS in this AIS model (R = 0.058). These results suggest that lesion size is not a reliable predictor of neurological outcome in AIS models.

Spreading depolarization (SD) is assumed to be the pathophysiological correlate of migraine aura, leading to spreading depression of activity and a long-lasting vasoconstriction known as spreading oligemia. Furthermore, cerebrovascular reactivity is reversibly impaired after SD. Here, we explored the progressive restoration of impaired neurovascular coupling to somatosensory activation during spreading oligemia. Also, we evaluated whether nimodipine treatment accelerated the recovery of impaired neurovascular coupling after SD. Male, 4-9-month-old C57BL/6 mice (n = 11) were anesthetized with isoflurane (1%-1.5%), and SD was triggered with KCl through a burr hole made at the caudal parietal bone. EEG and cerebral blood flow (CBF) were recorded minimally invasively with a silver ball electrode and transcranial laser-Doppler flowmetry, rostral to SD elicitation. The L-type voltage-gated Ca2+ channel blocker nimodipine was administered i.p. (10 mg/kg). Whisker stimulation-related evoked potentials (EVPs) and functional hyperemia were assessed under isoflurane (0.1%)-medetomidine (0.1 mg/kg i.p.) anesthesia before, and repeatedly after SD, at 15-min intervals for 75 minutes. Nimodipine accelerated the recovery of CBF from spreading oligemia (time to full recovery, 52 +/- 13 vs. 70 +/- 8 min, nimodipine vs. control) and exhibited a tendency to shorten the duration of the SD-related EGG depression duration. The amplitudes of EVP and functional hyperemia were markedly reduced after SD, and progressively recovered over an hour post-SD. Nimodipine exerted no impact on EVP amplitude but consistently increased the absolute level of functional hyperemia from 20 min post-CSD (93 +/- 11% vs. 66 +/- 13%, nimodipine vs. control). A linear, positive correlation between EVP and functional hyperemia amplitude was skewed by nimodipine. In conclusion, nimodipine facilitated CBF restoration from spreading oligemia and the recovery of functional hyperemia post-SD, which were linked to a tendency of an accelerated return of spontaneous neural activity after SD. The use of nimodipine in migraine prophylaxis is suggested to be re-visited.

Microglia are the resident macrophages in the central nervous system, accounting for 10-15% of the cell mass in the brain. Next to their physiological role in development, monitoring neuronal function and the maintenance of homeostasis, microglia are crucial in the brain’s immune defense. Brain injury and chronic neurological disorders are associated with neuroinflammation, in which microglia activation is a central element. Microglia acquire a wide spectrum of activation states in the diseased or injured brain, some of which are neurotoxic. The investigation of microglia (patho)physiology and therapeutic interventions targeting neuroinflammation is a substantial challenge. In addition to in vivo approaches, the application of in vitro model systems has gained significant ground and is essential to complement in vivo work. Primary microglia cultures have proved to be a useful tool. Microglia cultures have offered the opportunity to explore the mechanistic, molecular elements of microglia activation, the microglia secretome, and the efficacy of therapeutic treatments against neuroinflammation. As all model systems, primary microglia cultures have distinct strengths and limitations to be weighed when experiments are designed and when data are interpreted. Here, we set out to provide a succinct overview of the advantages and pitfalls of the use of microglia cultures, which instructs the refinement and further development of this technique to remain useful in the toolbox of microglia researchers. Since there is no conclusive therapy to combat neurotoxicity linked to neuroinflammation in acute brain injury or neurodegenerative disorders, these research tools remain essential to explore therapeutic opportunities.

Brain pH is precisely regulated, and pH transients associated with activity are rapidly restored under physiological conditions. During ischemia, the brain's ability to buffer pH changes is rapidly depleted. Tissue oxygen deprivation causes a shift from aerobic to anaerobic metabolism and the accumulation of lactic acid and protons. Although the degree of tissue acidosis resulting from ischemia depends on the severity of the ischemia, spreading depolarization (SD) events emerge as central elements to determining ischemic tissue acidosis. A marked decrease in tissue pH during cerebral ischemia may exacerbate neuronal injury, which has become known as acidotoxicity, in analogy to excitotoxicity. The cellular pathways underlying acidotoxicity have recently been described in increasing detail. The molecular structure of acid or base carriers and acidosis-activated ion channels, the precise (dys)homeostatic conditions under which they are activated, and their possible role in severe ischemia have been addressed. The expanded understanding of acidotoxic mechanisms now provides an opportunity to reevaluate the contexts that lead to acidotoxic injury. Here, we review the specific cellular pathways of acidotoxicity and demonstrate that SD plays a central role in activating the molecular machinery leading to acid-induced damage. We propose that SD is a key contributor to acidotoxic injury in cerebral ischemia.