Traumatic brain injury (TBI) is a leading cause of death and disability in children, especially those under five, with younger children more vulnerable to persistent cognitive and neuropsychological effects due to disrupted brain development. Paediatric brains are biomechanically more susceptible to diffuse axonal injury due to anatomical differences, with axonal injury observed in up to 80

By 2050, one-fifth of the world is expected to be over 60, and the prevalence of age-related neurological conditions is predicted to increase dramatically. Aged animals are currently underutilised in neurological research, leading to a gap in knowledge about the contribution of biological age to the pathophysiology of age-related neurological conditions. Additionally, it is unclear whether age-related changes differ across species used in preclinical models, and how these differences may compare to the aged human brain. Understanding these points is critical for successful translation of findings from preclinical studies to the human context. The current study presents a cross-species characterisation of microglia, the key regulator of the brain’s immune response, during ageing. Microglial number, proliferation and morphology were assessed in archival tissue from Sprague Dawley rats (males; 3 to 18 months old) and Merino sheep (males and females; 1 to 6 years old), with these two species selected for their relevance to preclinical modelling of neurological disease. Increased numbers of proliferating microglia were observed in the cortex, hippocampus and portions of the striatum in both species. This proliferation declined at the oldest timepoint assessed (i.e. 18 months old) in rats, a pattern not seen in the sheep. Total microglial number was largely unchanged with age in the rat brain; however, in sheep, the number of microglia decreased significantly in the dentate gyrus in older animals. Notably, microglia in 18-month-old rats were larger in all regions, but changes in branching were observed exclusively the striatum. Similarly, in sheep, morphological changes were localised to the striatum, with increased cell and soma size in the caudate nucleus, and increased cell size and process length in the putamen. These changes suggest a shift away from homeostasis in the cortex and hippocampus and towards a semi-ramified morphology in the striatum in late middle adulthood that is largely conserved across these two species. Nevertheless, the age of the oldest animals here equates to only 60 years old in humans, rather than reflecting an aged human population. Thus, future work is needed to understand how species-specific differences continue to evolve in older age.

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.

Concussion-related symptoms, such as impaired balance, slower processing speed, attention deficits, memory dysfunction, and irritability, are thought to result from diffuse axonal injury (DAI), characterized by selective damage to white matter axons. Axons subjected to this mechanical stretch injury exhibit diverse pathological changes, including disruption of axonal transport, neurofilament compaction and degradation, myelin sheath disruption, and loss of sodium channels required for action potential generation and propagation. These distinct forms of axonal pathology may evolve differentially over time and preferentially localize to specific white matter tracts. In this study, we employed the clinically relevant ferret model of concussion using the closed head impact model of engineered rotational acceleration (CHIMERA). 55 male ferrets were randomly allocated to sham or injury groups and then to either 24 h, 72 h, or 14d survival time points. We confirmed that axonal transport disruption and neurofilament pathology represent independent processes, with minimal colocalization but a shared peak of around 72 h following injury. Furthermore, we observed a persistent loss of ankyrin-G, a critical anchoring protein for sodium channels at the node of Ranvier, up to 14d postinjury, suggesting that the resultant impairment in axonal transmission may underlie many concussion symptoms. Indeed, injured ferrets displayed significant deficits in balance, working memory, spatial memory, and recognition memory. These findings demonstrate that the CHIMERA model in ferrets recapitulates key axonal pathologies and their associated clinical manifestations following concussion. This model offers a valuable platform for investigating the temporal evolution of axonal injury and developing targeted therapeutic interventions to mitigate concussion-related deficits.

Early stroke detection and treatment are critical for improving patient outcomes. Optical brain pulse monitoring (OBPM) uses red and infrared light to capture brain pulse waveforms reflecting arteriole-to-venous pressure levels driving microvascular blood flow. This study assessed OBPMs potential to detect middle cerebral artery occlusion (MCAo) and reperfusion in a clinically relevant sheep model. Stroke was induced in 11 Merino wethers via 4-hour occlusion of the right MCA, followed by 6 hours of reperfusion. OBPM recordings were taken at baseline, MCAo, early and late reperfusion. The OBPM brain pulse waveform classes were classified based on the presence of arterial or central venous circulation wave features. Magnetic resonance imaging assessed infarct volume at 2 hours post-reperfusion. Invasive brain tissue oxygen and intracranial pressures were also monitored. The OBPM brain pulse waveform classes changed during MCAo and reperfusion (p <0.0001). MCAo was associated brain pulses with venous circulation features (p = 0.0007). Reperfusion was associated with the return of arterial circulation features (p = 0.001). Early reperfusion was also associated with an increase in the brain pulse amplitude (p < 0.05) and the respiratory wave amplitude (p < 0.05). OBPM may aid in early stroke detection and reperfusion assessment following intervention. ### Competing Interest Statement B.D. is the founder and Chief Scientific Officer of Cyban, Pty Ltd and reports grants and personal fees from Cyban, during the conduct of the study; In addition, B.D. has patents US9717446B2 and WO2008134813A1 issued to Cyban. E.J.T., S.A.G., S.P., S.W.C, and J.H. are paid employees of Cyban. J.M.S, was a paid employee at the time of conducting the research. H.P., T.K. and M.P.M are founders and shareholders of Camoxis Pharmaceuticals Ltd. The remaining authors report no conflict of interest.

Background: Early detection and intervention of cerebral ischemia is critical to improving patient outcomes. Optical brain pulse monitoring (OBPM) is a unique technique that offers non-invasive, continuous monitoring of brain hemodynamics via brain pulse waveform visualization. This method has the potential to identify ischemic changes and facilitate expeditious intervention. Here, we assessed OBPM signal changes during middle cerebral artery occlusion (MCAo) and following reperfusion in a clinically relevant sheep model to enable pre-clinical waveform characterization. Methods: Eleven Merino wethers underwent MCAo for 4 hours followed by reperfusion for 6 hours. Brain tissue oxygen monitoring (PbtO 2 ; n=11) and OBPM (n=11) were performed in the MCAo hemisphere overlying the MCA territory, while intracranial pressure (ICP; n=11) and OBPM (n=6) were performed in the contralateral MCA territory. OBPM measurements were taken at baseline, MCAo, and following reperfusion. Magnetic resonance imaging (MRI) was used to measure infarct volume at 1 hour post reperfusion. Brain pulse waveforms were grouped into 5 classes by 3 blinded observers. Interrater agreement and frequency distributions were analyzed using Fleiss k and Fischer’s exact test, respectively. Generalized estimating equations (GEE) identified neuromonitoring changes (OBPM, StO 2 %, PbtO 2 , ICP) at each time point, adjusted with the Benjamini-Hochberg method. Results: Observers were in moderate agreement (k=0.433; p< 0.0001). Consensus classification of the OBP waveforms showed significant differences in the distribution of classes in the ipsilesional hemisphere over time ( p =0.02), but not the contralesional hemisphere ( p >0.99). One waveform class was observed only during MCAo. Reductions in brain pulse amplitude were found during MCAo when infarct volume was controlled for ( p <0.05 for all time points). Slow wave activity was observed in the OBPM optical signal and was associated with changes in ICP and PbtO 2 . OBPM StO 2 % showed significant decreases in both ipsi- and contra-lesional hemispheres ( p <0.001). A significant reduction in PbtO 2 ( p < 0.001) was observed during MCAo. Conclusion: MCAo was associated with distinct changes in the OBPM brain pulse waveform class and reductions in StO 2 % in a sheep model. These findings demonstrate that the OBPM captures cerebrovascular signatures of MCAo identifiable by human observers or by a drop in StO 2 %, providing an initial preclinical characterization of waveforms.

Introduction: Ischemia reperfusion injury (IRI) is a paradoxical and deleterious consequence of current interventions for acute ischemic stroke (AIS). Rapid restoration of oxygen to brain tissue upon reperfusion initiates mitochondrial reverse electron transport (RET) and production of reactive oxygen species (ROS), which exacerbate cell death. A pivotal role of the citric acid cycle intermediate succinate has been identified in driving RET post-reperfusion, whereby succinate accumulated during ischemia is rapidly reoxidized following reperfusion leading to a burst of ROS. Disodium malonate (DSM), a competitive inhibitor of succinate dehydrogenase, has been shown to attenuate RET ROS production following reperfusion and reduce infarct volume in rodent models. Here, we sought to evaluate the effect of DSM on infarct evolution post-reperfusion in a clinically-relevant sheep model of AIS for enhanced clinical translation. Methods: Male Merino sheep (N=13, 24-36 months, 62±6 kgs) underwent right pterional craniotomy and middle cerebral artery occlusion (MCAo) via aneurysm clip application for 4 hrs followed by reperfusion. Animals were pre-operatively randomized into vehicle (0.9% saline, N=5), medium dose DSM (0.5 mmole/min; N=4) and high dose DSM (1.0 mmole/min; N=4). Treatment was administered via right common carotid catheter at a rate of 15 mL/min for 10 min, starting 5 min prior to reperfusion. MCAo and reperfusion were confirmed on digital subtraction angiography (DSA). One hour following reperfusion, animals underwent magnetic resonance imaging (MRI) with a follow-up MRI performed 6 hours later. Infarct volume was calculated on diffusion weighted imaging (DWI) at each time-point to assess ischemic evolution. Results: All animals displayed evidence of MCAo and successful reperfusion following aneurysm clip removal (Figure 1). Infarct volume between groups was comparable at 1 hr post reperfusion (P>0.05), however, by 6 hrs infarct expansion was attenuated in animals receiving DSM compared with vehicles (P=0.0037). This was apparent in both the medium (P=0.006) and high (P=0.011) DSM groups. Conclusions: Intraarterial DSM administration reduces infarct expansion following reperfusion in a sheep model of MCAo. Evaluation of treatment efficacy in a larger cohort of animals is essential to address stroke therapeutic and industry roundtable (STAIR) guidelines and provide evidence to progress DSM to clinical trial for the treatment of IRI in AIS.

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.

Following ischemic stroke, substance P (SP)-mediated neurogenic inflammation is associated with profound blood-brain barrier (BBB) dysfunction, cerebral edema, and elevated intracranial pressure (ICP). SP elicits its effects by binding the neurokinin 1 tachykinin receptor (NK1-R), with administration of an NK1-R antagonist shown to ameliorate BBB dysfunction and cerebral edema in rodent and permanent ovine stroke models. Given the importance of reperfusion in clinical stroke, this study examined the efficacy of NK1-R antagonist treatment in reducing cerebral edema and ICP in an ovine model of transient middle cerebral artery occlusion (tMCAo). Anesthetized sheep ( n = 24) were subject to 2-hours tMCAo and randomized ( n = 6/group) to receive early NK1-R treatment (days 1–3 post-stroke), delayed NK1-R treatment (day 5 post-stroke), or saline vehicle. At 6-days post-stroke animals were re-anaesthetized and ICP measured, followed by MRI to evaluate infarction, edema and BBB dysfunction. Following both early and delayed NK1-R antagonist administration, ICP was significantly reduced on day 6 compared to vehicle animals (p < 0.05), accompanied by a reduction in cerebral edema, midline shift and BBB dysfunction (p < 0.05). This study demonstrates that NK1-R antagonist treatment is an effective novel therapy for cerebral edema and elevated ICP following stroke in an ovine model, warranting future clinical evaluation.

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 colocalization 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 was 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.

Background Assessment of functional impairment following ischaemic stroke is essential to determine outcome and efficacy of intervention in both clinical patients and pre-clinical models. Although paradigms are well described for rodents, comparable methods for large animals, such as sheep, remain limited. This study aimed to develop methods to assess function in an ovine model of ischaemic stroke using composite neurological scoring and gait kinematics from motion capture. Methods Merino sheep (n = 26) were anaesthetised and subjected to 2 hours middle cerebral artery occlusion. Animals underwent functional assessment at baseline (8-, 5-, and 1-day pre-stroke), and 3 days post-stroke. Neurological scoring was carried out to determine changes in neurological status. Ten infrared cameras measured the trajectories of 42 retro-reflective markers for calculation of gait kinematics. Magnetic resonance imaging (MRI) was performed at 3 days post-stroke to determine infarct volume. Intraclass Correlation Coefficients (ICC's) were used to assess the repeatability of neurological scoring and gait kinematics across baseline trials. The average of all baselines was used to compare changes in neurological scoring and kinematics at 3 days post-stroke. A principal component analysis (PCA) was performed to determine the relationship between neurological score, gait kinematics, and infarct volume post-stroke. Results Neurological scoring was moderately repeatable across baseline trials (ICC > 0.50) and detected marked impairment post-stroke (p < 0.05). Baseline gait measures showed moderate to good repeatability for the majority of assessed variables (ICC > 0.50). Following stroke, kinematic measures indicative of stroke deficit were detected including an increase in stance and stride duration (p < 0.05). MRI demonstrated infarction involving the cortex and/or thalamus (median 2.7 cm3, IQR 1.4 to 11.9). PCA produced two components, although association between variables was inconclusive. Conclusion This study developed repeatable methods to assess function in sheep using composite scoring and gait kinematics, allowing for the evaluation of deficit 3 days post-stroke. Despite utility of each method independently, there was poor association observed between gait kinematics, composite scoring, and infarct volume on PCA. This suggests that each of these measures has discreet utility for the assessment of stroke deficit, and that multimodal approaches are necessary to comprehensively characterise functional impairment.

Diffuse axonal injury (DAI) is a significant feature of traumatic brain injury (TBI) across all injury severities and is driven by the primary mechanical insult and secondary biochemical injury phases. Axons comprise an outer cell membrane, the axolemma which is anchored to the cytoskeletal network with spectrin tetramers and actin rings. Neurofilaments act as space-filling structural polymers that surround the central core of microtubules, which facilitate axonal transport. TBI has differential effects on these cytoskeletal components, with axons in the same white matter tract showing a range of different cytoskeletal and axolemma alterations with different patterns of temporal evolution. These require different antibodies for detection in post-mortem tissue. Here, a comprehensive discussion of the evolution of axonal injury within different cytoskeletal elements is provided, alongside the most appropriate methods of detection and their temporal profiles. Accumulation of amyloid precursor protein (APP) as a result of disruption of axonal transport due to microtubule failure remains the most sensitive marker of axonal injury, both acutely and chronically. However, a subset of injured axons demonstrate different pathology, which cannot be detected via APP immunoreactivity, including degradation of spectrin and alterations in neurofilaments. Furthermore, recent work has highlighted the node of Ranvier and the axon initial segment as particularly vulnerable sites to axonal injury, with loss of sodium channels persisting beyond the acute phase post-injury in axons without APP pathology. Given the heterogenous response of axons to TBI, further characterization is required in the chronic phase to understand how axonal injury evolves temporally, which may help inform pharmacological interventions.

Neuroinflammation is considered to be a significant component in a range of neuropathologies. Unfortunately, whilst its role is well recognised, the options for therapeutic intervention are limited. As such, there is a need to identify novel targets in order to increase treatment options. Given its role as both a neurotransmitter and an immune modulator, substance P (SP) and its NK1 receptor (NK1R) have been widely studied as a potential therapeutic target. There is evidence that NK1R antagonists may exert beneficial effects in a range of conditions, including traumatic brain injury and stroke. Blocking the NK1R has been shown to reduce blood–brain barrier dysfunction, reduce cerebral oedema, and reduce the levels of pro-inflammatory cytokines. These actions are associated with improved survival and functional outcomes. The NK1R has also been shown to be involved in the inflammatory reaction to CNS infection, and hence antagonists may have some benefit in reducing infection-driven inflammation. However, the NK1R may also play a role in the host immune response to infection, and so here, the potential beneficial and detrimental effects need to be carefully balanced. The purpose of this review is to provide a summary of evidence for the involvement of the NK1R in acute CNS inflammation, particularly in the context of traumatic brain injury and stroke.

Damage to the axonal white matter tracts within the brain is a key cause of neurological impairment and longterm disability following traumatic brain injury (TBI). Understanding how axonal injury develops following TBI requires gyrencephalic models that undergo shear strain and tissue deformation similar to the clinical situation and investigation of the effects of post-injury insults like hypoxia. The aim of this study was to determine the effect of post-traumatic hypoxia on axonal injury and inflammation in a sheep model of TBI. Fourteen male Merino sheep were allocated to receive a single TBI via a modified humane captive bolt animal stunner, or sham surgery, followed by either a 15 min period of hypoxia or maintenance of normoxia. Head kinematics were measured in injured animals. Brains were assessed for axonal damage, microglia and astrocyte accumulation and inflammatory cytokine expression at 4 hrs following injury. Early axonal injury was characterised by calpain activation, with significantly increased SNTF immunoreactivity, a proteolytic fragment of alpha-II spectrin, but not with impaired axonal transport, as measured by amyloid precursor protein (APP) immunoreactivity. Early axonal injury was associated with an increase in GFAP levels within the CSF, but not with increases in IBA1 or GFAP+ve cells, nor in levels of TNF & alpha;, IL1 & beta; or IL6 within the cerebrospinal fluid or white matter. No additive effect of post-injury hypoxia was noted on axonal injury or inflammation. This study provides further support that axonal injury post-TBI is driven by different pathophysiological mechanisms, and detection requires specific markers targeting multiple injury mechanisms. Treatment may also need to be tailored for injury severity and timing post-injury to target the correct injury pathway.