Rebecca J. Hood

Intracranial hypertension is a major modifiable pathway of secondary brain injury after acute neurological insults. Osmotherapy is an early cornerstone of tiered intracranial pressure management. Hypertonic saline and mannitol reliably reduce intracranial pressure through osmotic reduction in brain water, with additional haemodynamic and rheological effects. However, neither agent has shown consistent improvement in survival or long-term neurological recovery. Current practice is therefore guided by physiological rather than patient-centred outcomes. This review summarises the physiological basis, clinical evidence, and practical limitations of conventional osmotherapy, and explores investigational chloride-free osmotherapies including sodium lactate, sodium bicarbonate, sodium ascorbate, and sodium acetate. Chloride-free therapies aim to preserve sodium-driven osmotic efficacy while reducing chloride exposure or targeting metabolic and oxidative pathways implicated in secondary brain injury. At present, evidence is limited to small physiological trials, retrospective cohort studies, and mechanistic human studies. Future progress requires large collaborative trials that combine pragmatic treatment comparisons with multimodal neuromonitoring, biochemical safety endpoints and patient-centred outcomes.

Physiological factors, such as cerebral blood flow, blood pressure, respiratory rate, and body temperature are critical determinants of ischemic stroke pathophysiology, infarct development, and overall outcomes, and vice versa. However, despite long-standing guidelines recommending rigorous monitoring of key physiological factors, this aspect is not always incorporated into experimental designs and is generally underreported. Further, even when collected it is not always clear whether the data are integrated into the interpretation of results, potentially affecting translation. This review outlines the importance of monitoring, reporting and interpreting key physiological parameters in experimental stroke models, including the cardiovascular and respiratory systems, metabolism, temperature, circadian rhythm and intracranial pressure. Our aim is to provide researchers with an understanding of (1) different aspects of physiology that can affect experimental outcomes; (2) why and when it is important to monitor animal physiology in the context of ischemic stroke; (3) potential options for monitoring equipment and procedures; and (4) why accurate and transparent reporting and deliberate incorporation of physiological data into interpretation will enhance experimental rigor and strengthen future translational outcomes.

The ontological categorization of the cellular elements of the brain was proposed over a century ago by Santiago Ramón y Cajal (neurons, astroglia) and Pío del Río-Hortega (oligodendroglia, microglia). It combines histochemical observations of morphology with allied inferences about the specialized functions and origins (ectoderm or mesoderm) of each cellular element. This ontology shapes modern neuroscience, with the main non-neuronal cells—astroglia, oligodendroglia and microglia—viewed as having distinct primary roles relating respectively to the metabolic support, myelination and immunoprotection of neurons, the information signaling cells. Yet contemporary techniques, ranging from electrophysiology to single-cell transcriptomics and ultrahigh resolution spectroscopy, are revealing intersecting molecular profiles and functional capacities of these cell groups, for example metabolic support, neuroimmune and signaling functions in oligodendroglia. Here we identify discrepancies in current glial paradigms, from empirical, evolutionary and pragmatic perspectives. We suggest a subset of small, iron-rich glial cells, usually with few processes, often viewed as oligodendroglia with myelin-related primary functions, instead have iron-related primary functions that are central to all aspects of brain activity. We call these ‘ferriglia’. We discuss implications for pathogenesis across the spectrum of neuropsychiatric and neurological disorders, including neurodegenerative conditions such as Alzheimer’s disease and other less common cognitive, movement and neurobehavioral disorders, stroke and cerebrovascular disease, glioblastoma and other brain cancers and neuroimmune conditions. We also briefly address the question of where ferriglia may reside within existing glial compartments and lineages, implications for the ontological classification of other glial cells, and research challenges that must be overcome going forward.

Clopidogrel has been the most commonly used therapy for preventing secondary cardiovascular events since 1997 by inhibiting the purinergic receptor P2Y, G-protein coupled, 12 protein receptor (P2RY12). P2RY12 is critical for microglia function in the brain, where it facilitates repair processes following injury. Under normal conditions, the blood-brain barrier (BBB) prevents peripheral drugs like clopidogrel from entering the brain. However, stroke-induced BBB disruption may allow clopidogrel to interfere with neural recovery by impairing microglia activity. Recently, we demonstrated that clopidogrel worsened cognitive outcomes in young mice after stroke. In this study, we examined the effects of clopidogrel on aged mice, focusing on survival, body weight, neurovascular changes, immune response, and amyloid beta accumulation. Aged male mice underwent photothrombotic stroke (or sham surgery) and received daily clopidogrel or control treatment for 14 days. On day 15, brain tissue was analyzed. Clopidogrel treatment significantly reduced survival and body weight, decreased vessel density, increased vascular permeability, altered microglia activity, and increased amyloid beta levels in the peri-infarct region. Notably, some of these effects were not observed in young mice. These results suggest that BBB disruption in stroke mice enables clopidogrel to enter the central nervous system, where it impairs microglia-mediated restoration of BBB integrity and promotes amyloid accumulation, factors that may contribute to worsened cognitive recovery. This study raises the possibility that clopidogrel may similarly cross the BBB in older stroke patients, impacting microglial function, and emphasizes the need for further research into its mechanisms of action.

Digitized imaging of the spatial distribution of a targeted metal over a given area converts a continuous spectrum of data into a set of discrete pixels of intensities that ideally correlate strongly with the areal density map. This should be independent of the metal imaging modality, allowing semiquantitative analysis. In practice, correlation strength may vary between data sets obtained by different modalities. One enduring problem for all modalities is selecting a cutoff threshold intensity appropriately distinguishing the true signal from background noise. This may entail subjective choices between “conservative” thresholds prioritizing specificity over sensitivity (i.e., true positives despite possible false negatives) and “discovery-driven” thresholds prioritizing identification of putative effects for subsequent validation (i.e., true negatives despite possible false positives). Computerized data processing may help make this more objective by comparing outcomes for the set of all possible threshold values, termed cumulative threshold analysis. We address pitfalls in performing valid cumulative threshold analysis using ImageJ/Fiji for relative quantification of brain iron in mice with normal or genetically elevated iron, assessed by classical histochemistry or synchrotron X-ray fluorescence. In addition to pitfalls in choosing settings for generating analysis histograms, these include data loss with conversion between bit depths and selecting both appropriate X-axis directionality and image display range minima and maxima for analyzing multiple images. If these factors are handled appropriately, cumulative thresholding provides a powerful approach for more objective analysis of biometal imaging. This has important applications for metal imaging in research and clinical settings.

UK Biobank studies show Parkinson’s disease risk is almost doubled in men homozygous for the homeostatic iron regulator gene HFE p.C282Y polymorphism, associated with the common genetic iron disorder hemochromatosis. Whether this relationship is causal or spurious is unknown. We previously reported a novel Hfe ^-/- xTfr2 ^mut mouse model of hemochromatosis with elevated brain iron (∼1.5-1.8x). We now show these mice have reduced substantia nigra tyrosine hydroxylase expression at 3 months and 9 months age, sometimes exhibit severe hindlimb clasping by 7-8 months, have impaired rotarod and balance beam performance at 9 months and are untestable on the pole test. These parkinsonian features place the model at the forefront of genetic mouse models of PD, which generally do not show both TH loss and motor impairment. This confirms hemochromatosis-related mutations can cause parkinsonian features, substantiating causality of epidemiological relationships. Despite total brain iron elevation, neuronal iron remains low in Hfe ^-/- xTfr2 ^mut mice, consistent with hemochromatosis-related mutations disrupting the normal, iron-responsive regulation of the neuronal iron exporter ferroportin by hepcidin. Parkinsonian features may reflect reduced mitochondrial respiratory complex (MRC) activity due to functional neuronal iron depletion. This may be exacerbated by indiscriminate chelation and could instead respond to drugs targeting the hepcidin-ferroportin axis or MRC activity. This new model of chronic parkinsonism that increases with age provides unprecedented insights into the complex relationships of brain iron regulation and movement impairment. Since parkinsonism of diverse etiologies can exhibit iron dysregulation, the model may facilitate pre-clinical to end-stage studies relevant to both sporadic and genetic PD.

The ontological categorization of the cellular elements of the brain was proposed over a century ago by Santiago Ramón y Cajal (neurons, astroglia) and Pío del Río-Hortega (oligodendroglia, microglia). It combines histochemical observations of morphology with allied inferences about the specialized functions and origins (ectoderm or mesoderm) of each cellular element. This ontology shapes modern neuroscience, with the main non-neuronal cells – astroglia, oligodendroglia and microglia – viewed as having distinct primary roles relating to metabolic support, myelination and protection of neurons, the information signaling cells. Yet contemporary techniques, ranging from electrophysiology to single-cell transcriptomics and ultrahigh resolution spectroscopy, are revealing intersecting molecular profiles and functional capacities of these cell groups, for example metabolic support, neuroimmune and signaling functions in oligodendroglia. Here we identify discrepancies in current glial paradigms from empirical, evolutionary and pragmatic perspectives. We suggest that a subset of cells often viewed as oligodendroglia instead have iron-related primary functions that are central to all aspects of brain activity, rather than myelin-related primary functions. We discuss implications for pathogenesis across the spectrum of neuropsychiatric and neurological disorders, including neurodegenerative conditions such as Alzheimer’s disease and other less common cognitive, movement and neurobehavioral disorders, stroke and cerebrovascular disease, glioblastoma and other brain cancers and neuroimmune conditions. We also briefly address the question of where these cells reside within existing glial compartments and lineages, implications for the ontological classification of other glial cells and research limitations that must be overcome going forward.

In patients with ischaemic stroke, retrograde perfusion of the penumbra by the leptomeningeal collateral vessels (LMCs) is a strong predictor of clinical outcome, thus raising the possibility that enhancing LMC flow could offer a novel therapeutic approach. Here, using computational modelling we show that LMCs experience elevated fluid shear stress that is significantly higher than that in other blood vessels during ischaemic stroke in animals and humans. We take advantage of this to selectively enhance flow in LMCs using shear-activated nanoparticle aggregates carrying the vasodilator nitroglycerin (NG-NPAs) that specifically release drug in regions of vessels with high shear stress (≥100 dyne/cm ² ). The NG-NPAs significantly increased LMC-mediated penumbral perfusion, decreased infarct volume, and reduced neurological deficit without altering systemic blood pressure in a rat ischaemic stroke model. The NG-NPAs also did not cause known common side effects of systemic nitrate administration, such as systemic hypotension, cerebral vascular steal, cortical vein dilation, or intracranial pressure elevation. Systemic administration of free NG at the maximal tolerated dose, which was ten times higher than the dose of NG used in the NG-NPAs, did not enhance LMC perfusion and dropped blood pressure. Thus, packaging NG within shear-activated NPAs can potentially enable this widely available vasodilator to become a highly effective therapeutic for ischaemic stroke.

Stroke is the leading cause of acquired disability. The development of acute ischemic stroke treatments, such as mechanical thrombectomy and tissue plasminogen activator, has resulted in more patients surviving the initial insult. However, long‐term complications, such as post‐stroke cognitive impairment (PSCI) and dementia (PSD), are at an all‐time high. Notably, 80% of stroke survivors suffer from cognitive impairment, and a history of stroke doubles a patient's lifetime risk of developing dementia. A combination of greater life expectancy, an increase in the number of strokes in young individuals, and improved survival have inherently increased the number of years patients are living post‐stroke, highlighting the critical need to understand the long‐term effects of stroke, including how pathological changes in the brain might give rise to functional and behavioral changes in stroke survivors. Even with this increased risk of PSCI and PSD in stroke survivors, understanding of how the stroke itself develops into these conditions remains incomplete. Recently, secondary neurodegeneration (SND) following stroke has been linked with PSCI and PSD. SND is the degeneration of brain regions outside the original stroke site. Degeneration in these sites is thought to arise due to functional diaschisis with the infarct core; however, observation of SND pathology in multiple regions without direct connectivity to the stroke infarct suggests that the degeneration in these regions is likely more complex. Moreover, pathological hallmarks of dementia, such as a deposition of neurodegenerative proteins and iron, cell death, inflammation and blood–brain barrier alterations, have all been found in regions such as the thalamus, hippocampus, basal ganglia, amygdala and prefrontal cortex following stroke. Hence, in this review, we present the current understanding of PSCI and PSD in the context of SND and outline how remote anatomical and molecular changes may drive the development of these conditions.

We have previously demonstrated that a cortical stroke causes persistent impairment of hippocampal‐dependent cognitive tasks concomitant with secondary neurodegenerative processes such as amyloid‐β accumulation in the hippocampus, a region remote from the primary infarct. Interestingly, there is emerging evidence suggesting that deposition of amyloid‐β around cerebral vessels may lead to cerebrovascular structural changes, neurovascular dysfunction, and disruption of blood–brain barrier integrity. However, there is limited knowledge about the temporal changes of hippocampal cerebrovasculature after cortical stroke. In the current study, we aimed to characterise the spatiotemporal cerebrovascular changes after cortical stroke. This was done using the photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Cerebrovascular morphology as well as the co‐localisation of amyloid‐β with vasculature and blood–brain barrier integrity were assessed in the cortex and hippocampal regions at 7, 28 and 84 days post‐stroke. Our findings showed transient cerebrovascular remodelling in the peri‐infarct area up to 28 days post‐stroke. Importantly, the cerebrovascular changes were extended beyond the peri‐infarct region to the ipsilateral hippocampus and were sustained out to 84 days post‐stroke. When investigating vessel diameter, we showed a decrease at 84 days in the peri‐infarct and CA1 regions that were exacerbated in vessels with amyloid‐β deposition. Lastly, we showed sustained vascular leakage in the peri‐infarct and ipsilateral hippocampus, indicative of a compromised blood–brain‐barrier. Our findings indicate that hippocampal vasculature may represent an important therapeutic target to mitigate the progression of post‐stroke cognitive impairment.