Yvonne Couch

Background: Alterations in excitatory and inhibitory neurotransmission contribute to stroke pathology and recovery, yet longitudinal assessment of these changes in vivo remains limited. We investigated the feasibility of longitudinal edited proton magnetic resonance spectroscopy ( ¹ H-MRS) to quantify γ-aminobutyric acid (GABA) and glutamate following experimental focal cerebral ischaemia. Methods: Male rats underwent endothelin-1-induced striatal ischaemia or sham surgery. Longitudinal edited ¹ H-MRS was performed at baseline and 2-, 7- and 30-days post-stroke at 7-tesla with a MEGA-sLASER sequence. A voxel was positioned within the ipsilesional motor cortex adjacent to the lesion. Behavioural outcomes (grip strength, adhesive removal and CatWalk gait analysis) were assessed alongside spectroscopy, and immunohistochemistry for Iba1, GFAP and ICAM-1 was performed at 30 days. Results: Stroke induced a transient reduction in GABA+/tCr within the ipsilesional motor cortex, with significantly lower concentrations than sham animals at 7 days (p1 H-MRS is a feasible approach for monitoring dynamic changes in inhibitory neurochemistry following experimental stroke. The transient reduction in peri-lesional GABA, despite minimal behavioural impairment and an absence of persistent cortical inflammation, supports the use of edited spectroscopy as a translational tool to investigate neurochemical mechanisms underlying network dynamics in post-stroke recovery.

Mitochondrial Ca ²⁺ uptake governs metabolism and cell fate, yet how signals from other organelles shape this remains incompletely defined. Although lysosomes are relatively small Ca ²⁺ stores, their strategic positioning at organelle contact sites suggests they may amplify Ca ²⁺ transfer within nanodomains. Here, we show that activation of the lysosomal Two-pore channel 2 (TPC2) initiates rapid mitochondrial Ca ²⁺ uptake through an endoplasmic reticulum-dependent relay requiring IP₃ receptors and the mitochondrial calcium uniporter channel. The extent of mitochondrial Ca ²⁺ accumulation scales with TPC2 activity without affecting global Ca ²⁺ responses, identifying TPC2 as a specific amplifier of lysosome-mitochondria Ca ²⁺ exchange. Moderate TPC2 activation transiently enhances oxidative phosphorylation, whereas sustained enhancement increases susceptibility to Ca ²⁺ -induced mitochondrial permeability transition. In stroke models, hyperactivation of TPC2 exacerbates injury, while acute pharmacological inhibition at reperfusion confers neuroprotection, including in human iPSC-derived neurons. Thus, lysosomal Ca ²⁺ release acts as an upstream regulator of mitochondrial energetic resilience under stress.

Amid growing international efforts to reduce animal use in research, Couch explores the implications for neuroscience. She argues that moving away from in vivo models poses major scientific challenges, as whole-organism insights into brain circuitry and brain-body integration are difficult to replicate in vitro.

Extracellular vesicles (EVs) have long been understood to be important mediators of cell-to-cell communication and may lead to the molecular aftermath and exacerbation of brain injuries such as stroke. This study explored how the source of the EVs influenced their characteristics and the effect these differences had on naïve brain tissue. EVs were isolated from mice post-stroke in the acute or chronic stages of recovery in animals with and without reperfusion (transient and permanent middle cerebral artery occlusion) and from a model of systemic inflammation (i.p. lipopolysaccharide). The data show that neither stroke nor inflammation significantly increases EV numbers compared to sham or naïve animals. Post-stroke EVs exhibited a panel of different platelet and inflammatory markers when compared to EVs derived from a model of inflammation, reflecting differences between stroke and systemic immune activation. When injected into the brain, both stroke-derived and inflammation-derived EVs induced pro-inflammatory cytokine gene expression (IL-1β and CXCL1), suggesting a potential role in neuroinflammation. However, no clear group-level differences in microglial or astrocytic reactivity were detected at the level of regional histological assessment, despite consistent increases in ICAM-1 reactivity. The findings here underscore the complexity of EVs’ roles in pathophysiology and highlight the need for improved EV isolation methods. With further longitudinal studies, we may be able to more accurately determine how the context of the injury (reperfusion vs. no reperfusion vs. inflammation) might contribute to the EV populations and their function. Understanding more about EVs in different contexts will improve our ability to use EVs as biomarkers but also our capacity to interfere with EV biology as a novel therapeutic approach.

Changes in the peripheral metabolome, particularly in the blood, may provide biomarkers for assessing lesion severity and predicting outcomes after spinal cord injury (SCI). Using principal component analysis (PCA) and Orthogonal Partial Least Squares Discriminatory Analysis (OPLS-DA), we sought to discover how SCI severity and location acutely affect the nuclear magnetic resonance-acquired metabolome of the blood, spinal cord, and liver at 6 h post-SCI in mice. Unsupervised PCA of the spinal cord metabolome separated mild (30 kdyne) and severe (70 kdyne) contusion injury groups but did not distinguish between lesion level. However, OPLS-DA could discriminate thoracic level T2 from T9 lesions in both blood plasma (accuracy 86 ± 6%) and liver (accuracy 89 ± 5%) samples. These differences were dependent on alterations in energy metabolites (lactate and glucose), lipoproteins, and lipids. Lactate was the most discriminatory between mild and severe injury at T2, whereas overlapping valine/proline resonances were most discriminatory between injury severities at T9. Plasma lactate correlated with blood-spinal cord barrier breakdown and plasma glucose with microglial density. We propose that peripheral biofluid metabolites can serve as biomarkers of SCI severity and associated pathology at the lesion site; their predictive value is most accurate when the injury level is also considered.

Extracellular vesicles (EVs) have long been understood to be important mediators of cell-to-cell communication and may lead to the molecular aftermath and exacerbation of brain injuries such stroke. This study explored how the source of the EVs influenced their characteristics and the effect these differences had on naïve brain tissue. EVs were isolated from animals post-stroke in the acute or chronic stages of recovery in animals with and without reperfusion, and from a model of systemic inflammation (i.p. lipopolysaccharide). The data show that neither stroke nor inflammation significantly increase EV numbers compared to sham or naïve animals. Post-stroke EVs exhibited a panel of different platelet and inflammatory markers, when compared to EVs derived from a model of inflammation, reflecting differences between stroke and systemic immune activation. When injected into the brain, both stroke-derived and inflammation-derived EVs induced expression of pro-inflammatory cytokine gene expression, suggesting a potential role in neuroinflammation. However, there was a lack of distinct glial and astrocyte reactivity in response to any EVs, despite robust changes in ICAM reactivity. The findings here underscore the complexity of EV roles in pathophysiology and highlight the need for improved EV isolation methods. With further longitudinal studies we may be able to more accurately determine how the context of the injury (reperfusion vs no reperfusion vs inflammation) might contribute to the EV populations and their function. Understanding more about EVs in different contexts will improve our ability to use EVs as biomarkers, but also our capacity to interfere with EV biology as a novel therapeutic approach.

Ischaemic stroke treatment is limited to recanalizing the occluded vessel, while there is no approved adjunctive cerebroprotective therapy to protect either the neurons and parenchyma or the neurovascular unit. Pharmacological inhibition of mammalian target of rapamycin-1 (mTORC1) with rapamycin has shown promise in reducing infarct volume and improving functional outcomes. However, previous studies that investigated the effects of rapamycin on the vasculature and cerebral blood flow (CBF), administered rapamycin prior to or during stroke induction, thus limiting the potential for clinical translation. Therefore we investigated whether rapamycin maintains its cerebrovascular protective effect when administered immediately after recanalization following 90 minutes stroke in Wistar rats. We show, that rapamycin significantly improved post-recanalization cerebral blood flow (CBF), suggesting a beneficial neurovascular effect of rapamycin. Rats treated with rapamycin had smaller infarct volumes and improved functional outcomes compared to the control animals at three days post-stroke. The mechanisms of the overall positive effects seen in this study are likely due to rapamycin’s hyperacute effects on the neurovasculature, as shown with increased CBF during this phase. This paper shows that rapamycin treatment is a promising adjunct cerebroprotective therapy option for ischemic stroke.

Background: Inflammation has been found to be largely detrimental early in the acute phase of stroke but beneficial at more chronic stages. Fasting has been shown to reduce inflammation acutely. We aimed to determine whether post-ischemic fasting improves stroke outcomes through attenuated inflammation. Methods: After an endothelin-1 lesion was created in the striatum, animals were subjected to either normal feeding or water-only fasting for 24 hours. Results: It was found that at 24 hours, fasting reduced infarct volume and BBB breakdown and lowered both circulating and brain neutrophils. Conclusions: These findings suggest that fasting is a potentially beneficial non-pharmacological additive therapeutic option for cerebral ischemia, which might act by reducing inflammation in the acute disease stage.

Previously thought to be nothing more than cellular debris, extracellular vesicles (EVs) are now known to mediate physiological and pathological functions throughout the body. We now understand more about their capacity to transfer nucleic acids and proteins between distant organs, the interaction of their surface proteins with target cells, and the role of vesicle‐bound lipids in health and disease. To date, most observations have been made in reductionist cell culture systems, or as snapshots from patient cohorts. The heterogenous population of vesicles produced in vivo likely act in concert to mediate both beneficial and detrimental effects. EVs play crucial roles in both the pathogenesis of diseases, from cancer to neurodegenerative disease, as well as in the maintenance of system and organ homeostasis. This two‐part review draws on the expertise of researchers working in the field of EV biology and aims to cover the functional role of EVs in physiology and pathology. Part I will outline the role of EVs in normal physiology.

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change—size, number, and physicochemical composition—in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.