Tissue Compliance and Intracranial Pressure Responses to Large Intracerebral Hemorrhage in Young and Aged Spontaneously Hypertensive Rats.

BACKGROUND: After a large intracerebral hemorrhage (ICH), the hematoma and swelling cause intracranial pressure (ICP) to increase, sometimes causing brain herniation and death. This is partly countered by widespread tissue compliance, an acute decrease in tissue volume distal to the stroke, at least in young healthy animals. Intracranial compensation dynamics seem to vary with age, but there is no data on old animals or those with hypertension, major factors influencing ICH risk and outcome. METHODS: We assessed hematoma volume, edema, ICP, and functional deficits in young and aged spontaneously hypertensive rats (SHRs) and young normotensive control strains after collagenase-induced ICH. Macroscopic and microscopic brain volume fractions, such as contralateral hemisphere volume, cortical thickness, and neuronal morphology, were assessed via histological and stereological techniques. RESULTS: Hematoma volume was 52% larger in young versus aged SHRs; surprisingly, aged SHRs still experienced proportionally worse outcomes following ICH, with 2× greater elevations in edema and ICP relative to bleed volume and 3× the degree of tissue compliance. Aged SHRs also experienced equivalent neurological deficits following ICH compared with their younger counterparts, despite the lack of significant age-related behavioral effects. Importantly, tissue compliance occurred across strains and age groups and was not impaired by hypertension or old age. CONCLUSIONS: Aged SHRs show considerable capacity for tissue compliance following ICH and seem to rely on such mechanisms more heavily in settings of elevated ICP. Therefore, the ICP compensation response to ICH mass effect varies across the lifespan according to risk factors such as chronic hypertension.

A systematic review and meta-analysis on the efficacy of glibenclamide in animal models of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating stroke with many mechanisms of injury. Edema worsens outcome and can lead to mortality after ICH. Glibenclamide (GLC), a sulfonylurea 1- transient receptor potential melastatin 4 (Sur1-Trpm4) channel blocker, has been shown to attenuate edema in ischemic stroke models, raising the possibility of benefit in ICH. This meta-analysis synthesizes current pre-clinical (rodent) literature regarding the efficacy of post-ICH GLC administration (vs. vehicle controls) on behaviour (i.e., neurological deficit, motor, and memory outcomes), edema, hematoma volume, and injury volume. Six studies (5 in rats and 1 in mice) were included in our meta-analysis (PROSPERO registration = CRD42021283614). GLC significantly improved behaviour (standardized mean difference (SMD) = -0.63, [-1.16, -0.09], n = 70-74) and reduced edema (SMD = -0.91, [-1.64, -0.18], n = 70), but did not affect hematoma volume (SMD = 0.0788, [-0.5631, 0.7207], n = 18-20), or injury volume (SMD = 0.2892, [-0.4950, 1.0734], n = 24). However, these results should be interpreted cautiously. Findings were conflicted with 2 negative and 4 positive reports, and Egger regressions indicated missing negative edema data (p = 0.0001), and possible missing negative behavioural data (p = 0.0766). Experimental quality assessed via the SYRCLE and CAMARADES checklists was concerning, as most studies demonstrated high risks of bias. Studies were generally low-powered (e.g., average n = 14.4 for behaviour), and future studies should employ sample sizes of 41 to detect our observed effect size in behaviour and 33 to detect our observed effect in edema. Overall, missing negative studies, low study quality, high risk of bias, and incomplete attention to key recommendations (e.g., investigating female, aged, and co-morbid animals) suggest that further high-powered confirmatory studies are needed before conclusive statements about GLC's efficacy in ICH can be made, and before further clinical trials are performed.

Colchicine pre-treatment and post-treatment does not worsen bleeding or functional outcome after collagenase-induced intracerebral hemorrhage.

Patients with intracerebral hemorrhage (ICH) are at increased risk for major ischemic cardiovascular and cerebrovascular events. However, the use of preventative antithrombotic therapy can increase the risk of ICH recurrence and worsen ICH-related outcomes. Colchicine, an anti-inflammatory agent, has the potential to mitigate inflammation-related atherothrombosis and reduce the risk of ischemic vascular events. Here we investigated the safety and efficacy of colchicine when used both before and acutely after ICH. We predicted that daily colchicine administration would not impact our safety measures but would reduce brain injury and improve functional outcomes associated with inflammation reduction. To test this, 0.05 mg/kg colchicine was given orally once daily to rats either before or after they were given a collagenase-induced striatal ICH. We assessed neurological impairments, intra-parenchymal bleeding, Perls positive cells, and brain injury to gauge the therapeutic impact of colchicine on brain injury. Colchicine did not significantly affect bleeding (average = 40.7 µL) at 48 hrs, lesion volume (average = 24.5 mm3) at 14 days, or functional outcome (median neurological deficit scale score at 2 days post-ICH = 4, i.e., modest deficits) from 1-14 days after ICH. Colchicine reduced the volume of Perls positive cells in the perihematomal zone, indicating a reduction in inflammation. Safety measures (body weight, food consumption, water consumption, hydration, body temperature, activity, and pain) were not affected by colchicine. Although colchicine did not confer neuroprotection or functional benefit, it was able to reduce perihematomal inflammation after ICH without increasing bleeding. Thus, our findings suggest that colchicine treatment is safe, unlikely to worsen bleeding, and is unlikely but may reduce secondary injury after an ICH if initiated early post ICH to reduce the risk of ischemic vascular events. These results are informative for the ongoing CoVasc-ICH phase II randomized trial (NCT05159219).

Glibenclamide does not improve outcome following severe collagenase-induced intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is a devastating insult with few effective treatments. Edema and raised intracranial pressure contribute to poor outcome after ICH. Glibenclamide blocks the sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) channel implicated in edema formation. While glibenclamide has been found to improve outcome and reduce mortality in animal models of severe ischemic stroke, in ICH the effects are less clear. In our previous study, we found no benefit after a moderate-sized bleed, while others have reported benefit. Here we tested the hypothesis that glibenclamide may only be effective in severe ICH, where edema is an important contributor to outcome. Glibenclamide (10 µg/kg loading dose, 200 ng/h continuous infusion) was administered 2 hours post-ICH induced by collagenase injection into the striatum of adult rats. A survival period of 24 hours was maintained for experiments 1-3, and 72 hours for experiment 4. Glibenclamide did not affect hematoma volume (~81 µL) or other safety endpoints (e.g., glucose levels), suggesting the drug is safe. However, glibenclamide did not lessen striatal edema (~83% brain water content), ionic dyshomeostasis (Na+, K+), or functional impairment (e.g., neurological deficits (median = 10 out of 14), etc.) at 24 hours. It also did not affect edema at 72 h (~86% brain water content), or overall mortality rates (25% and 29.4% overall in vehicle vs. glibenclamide-treated severe strokes). Furthermore, glibenclamide appears to worsen cytotoxic edema in the peri-hematoma region (cell bodies were 46% larger at 24 h, p = 0.0017), but no effect on cell volume or density was noted elsewhere. Overall, these findings refute our hypothesis, as glibenclamide produced no favorable effects following severe ICH.

An update to the Monro-Kellie doctrine to reflect tissue compliance after severe ischemic and hemorrhagic stroke.

High intracranial pressure (ICP) can impede cerebral blood flow resulting in secondary injury or death following severe stroke. Compensatory mechanisms include reduced cerebral blood and cerebrospinal fluid volumes, but these often fail to prevent raised ICP. Serendipitous observations in intracerebral hemorrhage (ICH) suggest that neurons far removed from a hematoma may shrink as an ICP compliance mechanism. Here, we sought to critically test this observation. We tracked the timing of distal tissue shrinkage (e.g. CA1) after collagenase-induced striatal ICH in rat; cell volume and density alterations (42% volume reduction, 34% density increase; p < 0.0001) were highest day one post-stroke, and rebounded over a week across brain regions. Similar effects were seen in the filament model of middle cerebral artery occlusion (22% volume reduction, 22% density increase; p ≤ 0.007), but not with the Vannucci-Rice model of hypoxic-ischemic encephalopathy (2.5% volume increase, 14% density increase; p ≥ 0.05). Concerningly, this 'tissue compliance' appears to cause sub-lethal damage, as revealed by electron microscopy after ICH. Our data challenge the long-held assumption that 'healthy' brain tissue outside the injured area maintains its volume. Given the magnitude of these effects, we posit that 'tissue compliance' is an important mechanism invoked after severe strokes.

Predicting stroke severity with a 3-min recording from the Muse portable EEG system for rapid diagnosis of stroke.

In this study, we demonstrated the use of low-cost portable electroencephalography (EEG) as a method for prehospital stroke diagnosis. We used a portable EEG system to record data from 25 participants, 16 had acute ischemic stroke events, and compared the results to age-matched controls that included stroke mimics. Delta/alpha ratio (DAR), (delta + theta)/(alpha + beta) ratio (DBATR) and pairwise-derived Brain Symmetry Index (pdBSI) were investigated, as well as head movement using the on-board accelerometer and gyroscope. We then used machine learning to distinguish between different subgroups. DAR and DBATR increased in ischemic stroke patients with increasing stroke severity (p = 0.0021, partial η2 = 0.293; p = 0.01, partial η2 = 0.234). Also, pdBSI decreased in low frequencies and increased in high frequencies in patients who had a stroke (p = 0.036, partial η2 = 0.177). Using classification trees, we were able to distinguish moderate to severe stroke patients and from minor stroke and controls, with a 63% sensitivity, 86% specificity and accuracy of 76%. There are significant differences in DAR, DBATR, and pdBSI between patients with ischemic stroke when compared to controls, and these effects scale with severity. We have shown the utility of a low-cost portable EEG system to aid in patient triage and diagnosis as an early detection tool.

The collagenase model of intracerebral hemorrhage in awake, freely moving animals: The effects of isoflurane.

Intracerebral hemorrhage (ICH) is a devastating stroke often modelled in rats. Isoflurane anesthetic, commonly used in preclinical research, affects general physiology (e.g., blood pressure) and electrophysiology (e.g., burst suppression) in many ways. These physiological changes may detract from the clinical relevance of the model. Here, we revised the standard collagenase model to produce an ICH in rats without anesthetic. Guide cannulas were implanted stereotaxically under anesthetic. After 3 days of recovery, collagenase was infused through an internal cannula into the striatum of animals randomly assigned to the non-anesthetized or isoflurane group. We assessed whether isoflurane affected hematoma volume, core temperature, movement activity, pain, blood pressure, and seizure activity. With a small ICH, there was a hematoma volume increased from 8.6 (±3.3, 95% confidence interval) µL in anesthetized rats to 13.2 (±3.1) µL in non-anesthetized rats (P = 0.008), but with a larger ICH, hematoma volumes were similar. Isoflurane decreased temperature by 1.3 °C (±0.16 °C, P < 0.001) for 2 h and caused a 35.1 (±1.7) mmHg group difference in blood pressure (P < 0.007) for 12 m. Blood glucose increased twofold after isoflurane procedures (P < 0.001). Pain, as assessed with the rat grimace scale, did not differ between groups. Seizure incidence rate (62.5%) in non-anesthetized ICH rats was similar to historic amounts (61.3%). In conclusion, isoflurane appears to have some significant and injury size-dependent effects on the collagenase model. Thus, when anesthetic effects are a known concern, the use of the standardized cannula infusion approach is scientifically and ethically acceptable.

Glibenclamide, a Sur1-Trpm4 antagonist, does not improve outcome after collagenase-induced intracerebral hemorrhage.

The sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) receptor is selectively expressed after intracerebral hemorrhage (ICH). This upregulation contributes to increases in intracellular sodium. Water follows sodium through aquaporin channels, leading to cytotoxic edema. Even after edema is thought to have resolved, ionic dyshomeostasis persists, as does blood-brain barrier (BBB) damage. Glibenclamide, a hypoglycemic agent that inhibits Sur1-Trpm4, has been shown to reduce BBB damage and edema following infusion of autologous blood into the brain (ICH) as well as after other brain injuries. In order to further assess efficacy, we used the collagenase ICH model in rats to test whether glibenclamide reduces edema, attenuates ion dyshomeostasis, improves BBB damage, and reduces lesion volume. We tested a widely-used glibenclamide dose shown effective in other studies (10 µg/kg loading dose followed by 200 ng/hr for up to 7 days). Early initiation of glibenclamide did not significantly impact edema (72 hours), BBB permeability (72 hours), or lesion volume after ICH (28 days). Recovery from neurological impairments was also not improved by glibenclamide. These results suggest that glibenclamide will not improve outcome in ICH. However, the treatment appeared to be safe as there was no effect on bleeding or other physiological variables.

Failure of bumetanide to improve outcome after intracerebral hemorrhage in rat.

After intracerebral hemorrhage (ICH), brain edema commonly occurs and can cause death. Along with edema, there are significant alterations in the concentrations of key ions such as sodium, potassium, and chloride, which are essential to brain function. NKCC1, a cation-chloride cotransporter, is upregulated after brain damage, such as traumatic injury and ischemic stroke. NKCC1 brings sodium and chloride into the cell, possibly worsening ion dyshomeostasis. Bumetanide, a specific NKCC1 antagonist, blocks the transport of chloride into cells, and thus should attenuate the increases in chloride, which should lessen brain edema and improve neuronal functioning post-ICH, as with other injuries. We used the collagenase model of ICH to test whether bumetanide treatment for three days (vs. vehicle) would improve outcome. We gave bumetanide beginning at two hours or seven days post-ICH and measured behavioural outcome, edema, and brain ion content after treatment. There was some evidence for a minor reduction in edema after early dosing, but this did not improve behaviour or lessen injury. Contrary to our hypothesis, bumetanide did not normalize ion concentrations after late dosing. Bumetanide did not improve behavioural outcome or affect lesion volume. After ICH, bumetanide is safe to use in rats but does not improve functional outcome in the majority of animals.

Acetazolamide Mitigates Intracranial Pressure Spikes Without Affecting Functional Outcome After Experimental Hemorrhagic Stroke.

Increased intracranial pressure (ICP) after stroke can lead to poor outcome and death. Novel treatments to combat ICP rises are needed. The carbonic anhydrase inhibitor acetazolamide diminishes cerebrospinal fluid (CSF) production, reduces ICP in healthy animals, and is beneficial for idiopathic intracranial hypertension patients. We tested whether acetazolamide mitigates ICP elevations by presumably decreasing CSF volume after collagenase-induced striatal hemorrhage in rats. We confirmed that acetazolamide did not adversely affect hematoma formation in this model or physiological variables, such as temperature. Then, we assessed the effects of acetazolamide on ICP. Lastly, we tested the effects of acetazolamide on behavioral and histological outcome. Acetazolamide reduced the magnitude and occurrence of short-timescale ICP spikes, assessed as disproportionate increases in ICP (sudden ICP increases > 10 mmHg), 1-min peak ICP, and the magnitude of spikes > 20 mmHg. However, mean ICP was unaffected. In addition, acetazolamide reduced ICP variability, reflecting improved intracranial compliance. Compliance measures were strongly correlated with high peak and mean ICP, whereas ipsilateral hemisphere water content was not correlated with ICP. Despite effects on ICP, acetazolamide did not improve behavioral function or affect lesion size. In summary, we show that intracerebral hemorrhage creates an impaired compliance state within the cranial space that can result in large, transient ICP spikes. Acetazolamide ameliorates intracranial compliance and mitigates ICP spikes, but does not improve functional outcome, at least for moderate-severity ICH in rats.

Prolonged Blood-Brain Barrier Injury Occurs After Experimental Intracerebral Hemorrhage and Is Not Acutely Associated with Additional Bleeding.

Intracerebral hemorrhage (ICH) causes blood-brain barrier (BBB) damage along with altered element levels in the brain. BBB permeability was quantified at 3, 7, and 14 days with Evans Blue dye after collagenase-induced ICH in rat. At peak permeability (day 3), a gadolinium (Gd)-based contrast agent was injected to further characterize BBB disruption, and X-ray fluorescence imaging (XFI) was used to map Gd, Fe, Cl, and other elements. XFI revealed that Ca, Cl, Gd, and Fe concentrations were significantly elevated, whereas K was significantly decreased. Therefore, using Gd-XFI, we co-determined BBB dysfunction with alterations in the metallome, including those that contribute to cell death and functional outcome. Warfarin was administered 3 days post-ICH to investigate whether additional or new bleeding occurs during peak BBB dysfunction, and hematoma volume was assessed on day 4. Warfarin administration prolonged bleeding time after a peripheral cut-induced bleed, but warfarin did not worsen hematoma volume. Accordingly, extensive BBB leakage occurred after ICH, but did not appear to affect total hematoma size.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. METHODS: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. RESULTS: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ). INTERPRETATION: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.