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Background The formation and degradation of an intracerebral hemorrhage causes protracted cell death, and an extended window for intervention. Experimental studies find that rehabilitation mitigates late cell death, with accelerated hematoma clearance as a potential mechanism. Objective We assessed whether early, intense, enriched rehabilitation (ER, environmental enrichment and massed skills training) enhances functional benefit, reduces brain injury, and augments hematoma clearance. Methods In experiment 1, rats (n = 56) were randomized to intervention in the light (-L) or dark phase (-D) of their housing cycle, then to 10 days of ER or control (CON) treatment after collagenase-induced striatal intracerebral hemorrhage (ICH). ER rats were treated from 5 to 14 days after ICH. Behavior and residual hematoma volume was assessed on day 14. In experiment 2, rats (n = 72) were randomized to ER-D10, ER-D20, or CON-D. ER rats completed 10 or 20 days of training in the dark. Rats were euthanized on day 60 for histology. In both experiments, behavioral assessment was completed pre-ICH, pre-ER (day 4 post-ICH), and post-ER (experiment 1: days 13-14; experiment 2: days 16-17 and 30-31). Results Reaching intensity was high but similar between ER-D10 and ER-L10. Unlike previous work, rehabilitation did not alter skilled reaching or hematoma resolution. Varying ER duration also did not affect reaching success or lesion volume. Conclusions In contrast to others, and under these conditions, our findings show that striatal ICH was generally unresponsive to rehabilitation. This highlights the difficulty of replicating and extending published work, perhaps owing to small inter-study differences.

Background Intracerebral hemorrhage remains the deadliest form of stroke worldwide, inducing neuronal death through a wide variety of pathways. Therapeutic hypothermia is a robust and well-studied neuroprotectant widely used across a variety of specialties. Aims This review summarizes results from preclinical and clinical studies to highlight the overall effectiveness of therapeutic hypothermia to improve long-term intracerebral hemorrhage outcomes while also elucidating optimal protocol regimens to maximize therapeutic effect. Summary of review A systematic review was conducted across three databases to identify trials investigating the use of therapeutic hypothermia to treat intracerebral hemorrhage. A random-effects meta-analysis was conducted on preclinical studies, looking at neurobehavioral outcomes, blood brain barrier breakdown, cerebral edema, hematoma volume, and tissue loss. Several mixed-methods meta-regression models were also performed to adjust for variance and variations in hypothermia induction procedures. Twwenty-one preclinical studies and five human studies were identified. The meta-analysis of preclinical studies demonstrated a significant benefit in behavioral scores (ES = −0.43, p = 0.02), cerebral edema (ES = 1.32, p = 0.0001), and blood brain barrier (ES = 2.73, p ≤ 0.00001). Therapeutic hypothermia was not found to significantly affect hematoma expansion (ES = −0.24, p = 0.12) or tissue loss (ES = 0.06, p = 0.68). Clinical study outcome reporting was heterogeneous; however, there was recurring evidence of therapeutic hypothermia-induced edema reduction. Conclusions The combined preclinical evidence demonstrates that therapeutic hypothermia reduced multiple cell death mechanisms initiated by intracerebral hemorrhage; yet, there is no definitive evidence in clinical studies. The cooling strategies employed in both preclinical and clinical studies were highly diverse, and focused refinement of cooling protocols should be developed in future preclinical studies. The current data for therapeutic hypothermia in intracerebral hemorrhage remains questionable despite the highly promising indications in preclinical studies. Definitive randomized controlled studies are still required to answer this therapeutic question.

Therapeutic hypothermia (TH) has applications dating back millennia. In modern history, however, TH saw its importation into medical practice where investigations have demonstrated that TH is efficacious in ischemic insults, notably cardiac arrest and hypoxic-ischemic encephalopathy. As well, studies have been undertaken to investigate whether TH can provide benefit in focal stroke (i.e., focal ischemia and intracerebral hemorrhage). However, clinical studies have encountered various challenges with induction and maintenance of post-stroke TH. Most clinical studies have attempted to use body-wide cooling protocols, commonly hindered by side effects that can worsen post-stroke outcomes. Some of the complications and difficulties with systemic TH can be circumvented by using local hypothermia (LH) methods. Additional advantages include the potential for lower target temperatures to be achieved, and faster TH induction rates with LH. This systematic review summarizes the body of clinical and preclinical LH focal stroke studies, and raises key points to consider for future LH research. We conclude with an overview of LH neuroprotective mechanisms and a comparison of LH mechanisms with those observed with systemic TH. Overall, whereas many LH studies have been conducted preclinically in the context of focal ischemia, insufficient work has been done in intracerebral hemorrhage. Furthermore, key translational studies have yet to be done in either stroke subtype (e.g., varied models and time-to-treat, studies considering aged animals or animals with comorbidities). Few clinical LH investigations have been performed and the optimal LH parameters to achieve neuroprotection are unknown.

Rising intracranial pressure (ICP) aggravates secondary injury and heightens risk of death following intracerebral hemorrhage (ICH). Long-recognized compensatory mechanisms that lower ICP include reduced cerebrospinal fluid and venous blood volumes. Recently, we identified another compensatory mechanism in severe stroke, a decrease in cerebral parenchymal volume via widespread reductions in cell volume and extracellular space (tissue compliance). Here, we examined how age affects tissue compliance and ICP dynamics after severe ICH in rats (collagenase model). A planned comparison to historical young animal data revealed that aged SHAMs (no stroke) had significant cerebral atrophy (9% reduction, p ≤ 0.05), ventricular enlargement (9% increase, p ≤ 0.05), and smaller CA1 neuron volumes (21%, p ≤ 0.05). After ICH in aged animals, contralateral striatal neuron density and CA1 astrocyte density significantly increased (12% for neurons, 7% for astrocytes, p ≤ 0.05 vs. aged SHAMs). Unlike young animals, other regions in aged animals did not display significantly reduced cell soma volume despite a few trends. Nonetheless, overall contralateral hemisphere volume was 10% smaller in aged ICH animals compared to aged SHAMs (p ≤ 0.05). This age-dependent pattern of tissue compliance is not due to absent ICH-associated mass effect (83.2 mm3 avg. bleed volume) as aged ICH animals had significantly elevated mean and peak ICP (p ≤ 0.01), occurrence of ICP spiking events, as well as bilateral evidence of edema (e.g., 3% in injured brain, p ≤ 0.05 vs. aged SHAMs). Therefore, intracranial compliance reserve changes with age; after ICH, these and other age-related changes may cause greater fluctuation from baseline, increasing the chance of adverse outcomes like mortality.

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 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.

Localized brain hypothermia (HYPO) can be achieved by infusing cold saline into the carotid artery of animals and patients. Studies suggest that HYPO improves behavioral and histological outcomes in focal ischemia models. Given that ischemic stroke and intracerebral hemorrhage (ICH) share pathophysiological overlap, we tested whether cold saline infusion is safe and neuroprotective when given during collagenase-induced ICH. Eighty-five adult male Sprague-Dawley rats were used. Experiment 1 investigated brain and body temperature changes associated with a cold saline infusion paradigm that was scaled from patients according to brain weight and blood volume (3 mL/20-minute infusion). Experiment 2 determined whether HYPO aggravated bleeding volume. Experiment 3 investigated if cerebral edema or elemental concentrations were altered by HYPO. We also collected core body temperature and activity data through telemetry. Experiment 4 investigated whether behavioral outcomes (e.g., skilled reaching) and tissue loss were influenced by HYPO. Our HYPO protocol decreased the ipsilateral striatal temperature by ∼0.20°C (p 0.05 for all). Brain tissue loss was not different between groups on day 28 post-ICH (p = 0.90). Although cold saline infusion appears to be safe in the acute post-ICH period, there was no evidence that this therapy improved outcome. However, our treatment protocol was relatively mild and additional interventions might help improve efficacy. Finally, our findings may also speak to the safety of this cooling approach in focal ischemia where hemorrhagic transformation is a risk; future studies on this issue are needed.

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.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease with many known structural and functional changes in the central nervous system. A well-recognized, but poorly understood, complication of MS is chronic pain. Little is known regarding the influence of sex on the development and maintenance of MS-related pain. This is important to consider, as MS is a predominantly female disease. Using the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we demonstrate sex differences in measures of spinal cord inflammation and plasticity that accompany tactile hypersensitivity. Although we observed substantial inflammatory activity in both sexes, only male EAE mice exhibit robust staining of axonal injury markers and increased dendritic arborisation in morphology of deep dorsal horn neurons. We propose that tactile hypersensitivity in female EAE mice may be more immune-driven, whereas pain in male mice with EAE may rely more heavily on neurodegenerative and plasticity-related mechanisms. Morphological and inflammatory differences in the spinal cord associated with pain early in EAE progression supports the idea of differentially regulated pain pathways between the sexes. Results from this study may indicate future sex-specific targets that are worth investigating for their functional role in pain circuitry.

One major aim of preclinical intracerebral hemorrhage (ICH) research is to develop and test potential neuroprotectants. Published guidelines for experimental design and reporting stress the importance of clearly and completely reporting results and methodological details to ensure reproducibility and maximize information availability. The current review has two objectives: first, to characterize current ICH neuroprotection research and, second, to analyze aspects of translational design in preclinical ICH studies. Translational design is the adoption and reporting of experimental design characteristics that are thought to be clinically relevant and critical to reproducibility in animal studies (e.g., conducting and reporting experiments according to the STAIR and ARRIVE guidelines, respectively). Given that ICH has no current neuroprotective treatments and an ongoing reproducibility crisis in preclinical research, translational design should be considered by investigators. We conducted a systematic review of ICH research from 2015 to 2019 using the PubMed database. Our search returned 281 published manuscripts studying putative neuroprotectants in animal models. Contemporary ICH research predominantly uses young, healthy male rodents. The collagenase model is the most commonly used. Reporting of group sizes, blinding, and randomization are almost unanimous, but group size calculations, mortality and exclusion criteria, and animal model characteristics are infrequently reported. Overall, current ICH neuroprotection research somewhat aligns with experimental design and reporting guidelines. However, there are areas for improvement. Because failure to consider translational design is associated with inflation of effect sizes (and possibly hindered reproducibility), we suggest that researchers, editors, and publishers collaboratively consider enhanced adherence to published guidelines.

Author(s): Karuppagounder, Saravanan S; Alin, Lauren; Chen, Yingxin; Brand, David; Bourassa, Megan W; Dietrich, Kristen; Wilkinson, Cassandra M; Nadeau, Colby A; Kumar, Amit; Perry, Steve; Pinto, John T; Darley-Usmar, Victor; Sanchez, Stephanie; Milne, Ginger L; Pratico, Domenico; Holman, Theodore R; Carmichael, S Thomas; Coppola, Giovanni; Colbourne, Frederick; Ratan, Rajiv R | Abstract: ObjectivesN-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.MethodsHemin 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.ResultsNAC 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 ).InterpretationNAC 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.

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.

Coordination chemistry enables a variety of vital functions in biological systems; however, characterising the chemical form of metal ions in cells and tissue is notoriously difficult. One technique that is gaining substantial momentum in this research area is X-ray absorption near-edge structure (XANES) spectroscopy. The XANES spectrum can be a rich source of information with respect to the coordination environment of metal ions. Further, XANES spectroscopy is compatible with microscopy mapping protocols as the spectra are recorded across a relatively narrow range of data points (typically 50–100). Although the potential of XANES spectroscopy to study metal ion coordination chemistry has long been known, data collection speed has only relatively recently reached the state in which maps can be collected with a full spectrum per pixel. The realisation of this capability now places XANES spectroscopic mapping among a suite of other spectroscopic imaging techniques, such as Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy, which are available to characterise biochemical composition, in situ within cells and tissue. Herein, we report a proof-of-concept application of XANES spectroscopic mapping to begin exploration of Fe speciation in brain tissue, which demonstrates the potential of this method for the biomedical sciences, and identifies important areas for consideration with respect to future protocol developments.

Background: Raised intracranial pressure (ICP) following severe strokes can impede blood flow to the brain, causing injury or death. There are compensatory mechanisms regulating ICP, but they are often inadequate. Interestingly, our recent observations in intracerebral hemorrhage (ICH) suggest that a volume reduction in remaining brain may serve a compensatory role after large strokes. We observed neurons well outside the hematoma reduce their volume and intercellular space 7 days after the bleed. Here, we tested the hypotheses that this volume reduction would occur sooner in both ischemic and hemorrhagic insults. Methods: Rats received either an ICH via stereotaxic infusion of collagenase or a sham procedure. Euthanasia and perfusion fixation occurred either 1 (N=12), 3 (N=12), or 7 (N=12) days following the ICH. Following histological processing, cellular volume, density, and cortical thickness were assessed with stereological techniques in representative brain regions such as hippocampus, striatum, and primary somatosensory cortex (S1). In a separate experiment, rats received either a middle cerebral artery occlusion (MCAO) via intraluminal suture occlusion (N=8), or a sham procedure (N=7). Rats were euthanized and perfusion fixed 1 day following the MCAO, with brains processed and assessed as described above. Results: After ICH, the decrease in neuronal volume (up to 60%) compared to shams had the largest effect one day following the insult in hippocampal layers CA1 and CA3 (p Discussion: Our data challenges the assumption that ‘healthy’ brain tissue outside the injured area maintains its volume after stroke. Given the magnitude of cell volume reductions, we posit that this is an important intracranial compliance mechanism invoked after severe strokes.

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.

Intracerebral hemorrhage (ICH) is a devastating stroke with high morbidity and mortality. Post-ICH seizures are a common complication, potentially increasing brain injury and the risk of delayed epilepsy. Mild therapeutic hypothermia (HYPO, ∼33°C) is neuroprotective against several brain insults, such as ischemia, while also mitigating seizure incidence and severity in some instances. Therefore, we tested whether brain-selective HYPO reduced electrographic seizure activity after a collagenase-induced striatal ICH in rats. Animals were injected unilaterally with 0.14 U of bacterial collagenase, implanted with a unilateral brain cooling device, and a probe to bilaterally record electroencephalographic (EEG) activity. Cooling began 6 hours after collagenase infusion and was maintained for 48 hours, followed by rewarming over 6 hours. Our protocol did not affect EEG activity in naïve rats, nor did it increase bleeding after ICH (∼50 μL). Epileptiform activity commonly occurred in untreated ICH rats (∼60% of animals), but HYPO did not affect the incidence, timing, total duration of seizures, or the peaks occurring during epileptiform activity. However, longer average duration was detected on the ipsilateral side to stroke in the HYPO group (p 0.05). Cooling did not affect neurological deficits (e.g., circling), measured 7 and 14 days after ICH, or lesion volume (∼35 mm(3)). In addition, there was no relationship among endpoints (e.g., seizures and lesion size). In summary, HYPO failed to reduce seizure activity after ICH, which fits with our separate findings that cooling does not mitigate thrombin and iron-mediated secondary injury mechanisms thought to cause seizures after ICH. Additional research is needed to identify better HYPO protocols and the use of cotreatments to maximize the benefit of HYPO to ICH patients.

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.

Oxygen (O2) is a crucial element for physiological functioning in mammals. In particular, brain function is critically dependent on a minimum amount of circulating blood levels of O2 and both immediate and lasting neural dysfunction can result following anoxic or hypoxic episodes. Although the effects of deficiencies in O2 levels on the brain have been reasonably well studied, less is known about the influence of elevated levels of O2 (hyperoxia) in inspired gas under atmospheric pressure. This is of importance due to its typical use in surgical anesthesia, in the treatment of stroke and traumatic brain injury, and even in its recreational or alternative therapeutic use. Using local field potential (EEG) recordings in spontaneously breathing urethane-anesthetized and naturally sleeping rats, we characterized the influence of different levels of O2 in inspired gases on brain states. While rats were under urethane anesthesia, administration of 100% O2 elicited a significant and reversible increase in time spent in the deactivated (i.e., slow-wave) state, with concomitant decreases in both heartbeat and respiration rates. Increasing the concentration of carbon dioxide (to 5%) in inspired gas produced the opposite result on EEG states, mainly a decrease in the time spent in the deactivated state. Consistent with this, decreasing concentrations of O2 (to 15%) in inspired gases decreased time spent in the deactivated state. Further confirmation of the hyperoxic effect was found in naturally sleeping animals where it similarly increased time spent in slow-wave (nonrapid eye movement) states. Thus alterations of O2 in inspired air appear to directly affect forebrain EEG states, which has implications for brain function, as well as for the regulation of brain states and levels of forebrain arousal during sleep in both normal and pathological conditions. NEW & NOTEWORTHY We show that alterations of oxygen concentration in inspired air biases forebrain EEG state. Hyperoxia increases the prevalence of slow-wave states. Hypoxia and hypercapnia appear to do the opposite. This suggests that oxidative metabolism is an important stimulant for brain state.

Studies treating intracerebral hemorrhage (ICH) with therapeutic hypothermia (TH) have shown inconsistent benefits. We hypothesized that TH’s anti-inflammatory effects may be responsible as inflammatory cells are essential for removing degrading erythrocytes. Here, we subjected rats to a collagenase-induced striatal ICH followed by whole-body TH (∼33℃ for 11–72 h) or normothermia. We used X-ray fluorescence imaging to spatially quantify total and peri-hematoma iron three days post-injury. At three and seven days, we measured non-heme iron levels. Finally, hematoma volume was quantified on one, three, and seven days. In the injured hemisphere, total iron levels were elevated ( p

An intracerebral hemorrhage (ICH) is a devastating stroke that results in high mortality and significant disability in survivors. Unfortunately, the underlying mechanisms of this injury are not yet fully understood. After the primary (mechanical) trauma, secondary degenerative events contribute to ongoing cell death in the peri-hematoma region. Oxidative stress is thought to be a key reason for this delayed injury, which is likely due to free-Fe-catalyzed free radical reactions. Unfortunately, this is difficult to prove with conventional biochemical assays that fail to differentiate between alterations that occur within the hematoma and peri-hematoma zone. This is a critical limitation, as the hematoma contains tissue severely damaged by the initial hemorrhage and is unsalvageable, whereas the peri-hematoma region is less damaged but at risk from secondary degenerative events. Such events include oxidative stress mediated by free Fe presumed to originate from hemoglobin breakdown. Therefore, minimizing the damage caused by oxidative stress following hemoglobin breakdown and Fe release is a major therapeutic target. However, the extent to which free Fe contributes to the pathogenesis of ICH remains unknown. This investigation used a novel imaging approach that employed resonance Raman spectroscopic mapping of hemoglobin, X-ray fluorescence microscopic mapping of total Fe, and Fourier transform infrared spectroscopic imaging of aggregated protein following ICH in rats. This multimodal spectroscopic approach was used to accurately define the hematoma/peri-hematoma boundary and quantify the Fe concentration and the relative aggregated protein content, as a marker of oxidative stress, within each region. The results revealed total Fe is substantially increased in the hematoma (0.90 μg cm(-2)), and a subtle but significant increase in Fe that is not in the chemical form of hemoglobin is present within the peri-hematoma zone (0.32 μg cm(-2)) within 1 day of ICH, relative to sham animals (0.22 μg cm(-2)). Levels of aggregated protein were significantly increased within both the hematoma (integrated band area 0.10 AU) and peri-hematoma zone (integrated band area 0.10 AU) relative to sham animals (integrated band area 0.056 AU), but no significant difference in aggregated protein content was observed between the hematoma and peri-hematoma zone. This result suggests that the chemical form of Fe and its ability to generate free radicals is likely to be a more critical predictor of tissue damage than the total Fe content of the tissue. Furthermore, this article describes a novel approach to colocalize nonheme Fe and aggregated protein in the peri-hematoma zone following ICH, a significant methodological advancement for the field.

Animal studies testing whether therapeutic hypothermia is neuroprotective after intracerebral hemorrhage (ICH) have been inconclusive. In rodents, ICH is often produced in the striatum by infusing collagenase, which causes prolonged hemorrhaging from multiple vessels. Our previous data shows that this bleeding (hematoma) is worsened by systemic hypothermia given soon after collagenase infusion. In this study we hypothesized that localized brain hypothermia would also aggravate bleeding in this model (0.2 U of collagenase in 1.2 μL of saline). We also evaluated cooling after intrastriatal thrombin infusion (1 U in 30 μL of saline)-a simplified model of ICH thought to cause bleeding. Focal hypothermia was achieved by flushing cold water through an implanted cooling device attached to the skull underneath the temporalis muscle of adult rats. Previous work and data at this time shows this method cools the striatum to ∼33°C, whereas the body remains normothermic. In comparison to normothermic groups, cooling significantly worsened bleeding when instituted at 6 hours (∼94 vs. 42 μL, p=0.018) and 12 hours (79 vs. 61 μL, p=0.042) post-ICH (24-hour survival), but not after a 24-hour delay (36-hour survival). Rats were cooled until euthanasia when hematoma size was determined by a hemoglobin-based spectrophotometry assay. Cooling did not influence cerebral blood volume after just saline or thrombin infusion. The latter is explained by the fact that thrombin did not cause bleeding beyond that caused by saline infusion. In summary, local hypothermia significantly aggravates bleeding many hours after collagenase infusion suggesting that bleeding may have confounded earlier studies with hypothermia. Furthermore, these findings serve as a cautionary note on using cooling even many hours after cerebral bleeding.

Brain injury, such as from stroke and trauma, can be complicated by elevated intracranial pressure (ICP). Although raised ICP can be a significant determinant of morbidity and mortality, clinical studies often report widely varying ICP measurements depending on location of measurement and technique used. For the same reasons, reported ICP measurements also vary widely in animal models. The need for anesthesia or tethered connections with some methods of ICP measurement in animals may introduce additional confounds. Moreover, these methods are not well suited for prolonged, continuous measurement. Here, we describe an approach to continually measure ICP in awake, freely moving rats for several days. This technique uses a commercially available, wireless pressure sensor mounted on the head to measure ICP from the epidural space via a fluid-filled catheter. We have demonstrated that this approach reliably detects elevations in ICP that last for several days after ischemic and hemorrhagic strokes in rat.

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

Therapeutic hypothermia (TH) mitigates neuronal injury in models of ischemic stroke. Although this therapy is meant for injured tissue, most protocols cool the whole body, including the contralesional hemisphere. Neuroplasticity responses within this hemisphere can affect functional outcome. Thus, cooling the contralesional hemisphere serves no clear neuroprotective function and may instead be detrimental. In this study, we cooled the contralesional hemisphere to determine whether this harms behavioral recovery after cortical injury in rats. All rats were trained on skilled reaching and walking tasks. Rats then received a motor cortex insult contralateral to their dominant paw after which they were randomly assigned to focal contralesional TH (∼33°C) for 1-48, 1-97, or 48-96 hours postinjury, or to a normothermic control group. Contralesional cooling did not impact lesion volume (p = 0.371) and had minimal impact on neurological outcome of the impaired limb. However, rats cooled early were significantly less likely to shift paw preference to the unimpaired paw (p ≤ 0.043), suggesting that cooling reduced learned nonuse. In a second experiment, we tested whether cooling impaired learning of the skilled reaching task in naive rats. Localized TH applied to the hemisphere contralateral or ipsilateral to the preferred paw did not impair learning (p ≥ 0.677) or dendritic branching/length in the motor cortex (p ≥ 0.105). In conclusion, localized TH did not impair learning or plasticity in the absence of neural injury, but contralesional TH may reduce unwanted shifts in limb preference after stroke.

Brain injury, such as from stroke and trauma, can be complicated by elevated intracranial pressure (ICP). Although raised ICP can be a significant determinant of morbidity and mortality, clinical studies often report widely varying ICP measurements depending on location of measurement and technique used. For the same reasons, reported ICP measurements also vary widely in animal models. The need for anesthesia or tethered connections with some methods of ICP measurement in animals may introduce additional confounds. Moreover, these methods are not well suited for prolonged, continuous measurement. Here, we describe an approach to continually measure ICP in awake, freely moving rats for several days. This technique uses a commercially available, wireless pressure sensor mounted on the head to measure ICP from the epidural space via a fluid-filled catheter. We have demonstrated that this approach reliably detects elevations in ICP that last for several days after ischemic and hemorrhagic strokes in rat.

Seizures are a frequent complication of brain injury, including intracerebral hemorrhage (ICH), where seizures occur in about a third of patients. Rodents are used to study pathophysiology and neuroprotective therapies after ICH, but there have been no studies assessing the occurrence of seizures in these models. Thus, we compared seizure incidence and characteristics after infusing collagenase (0.14 U), which degrades blood vessels, and autologous blood (100 μL) into the striatum of rats. Saline was infused in others as a negative control, whereas iron, a by-product of degrading erythrocytes, served as a positive control. Ipsilateral and contralateral electroencephalographic (EEG) activity was continuously monitored with telemetry probes for a week after the stroke. There were no electrographic abnormalities during baseline recordings. As expected, saline did not elicit any epileptiform activity whereas iron caused seizure activity. Seizures occurred in 66 % of the collagenase group between 10 and 36 h, their duration ranged from 5 to 90 s, and these events were mostly observed bilaterally. No such activity occurred after blood infusion despite comparable lesion sizes of 32.5 and 40.9 mm(3) in the collagenase and blood models, respectively (p = 0.222). Therefore, seizures are a common acute occurrence in the collagenase but not whole blood models of striatal ICH (p = 0.028, for incidence). These findings have potential implications for ICH studies such as for understanding model differences, helping select which model to use, and determining how seizures may affect or be affected by treatments applied after stroke.

In some patients, intracerebral hemorrhage (ICH) causes life-threatening elevations in intracranial pressure (ICP) arising from mass effect of the hematoma and edema. Accordingly, edema is a common endpoint to gauge treatment efficacy in rodent ICH models. Despite widespread reliance on edema, its relationship with ICP and cerebral perfusion pressure (CPP) is unknown. Blood pressure (BP) and ICP were measured by telemetry devices in rats after collagenase (“severe” — 0.3 U, and “moderate” — 0.15 U doses) or blood infusion (100 μL) into striatum (vs. saline infused shams). We compared epidural and intraparenchymal ICP readings (collagenase), evaluated CPP (collagenase), and compared models. Moderate (9.46 mm Hg ± 4.72 SD, 3 day average) and severe collagenase ICHs (10.79 ± 3.50) significantly increased ICP versus shams (4.02 ± 2.09), whereas blood infusion did not (5.37 ± 0.55). The two monitoring locations gave similar readings after severe collagenase ICH. Increased ICP reduced CPP by ~ 7.5 mm Hg for days after the larger collagenase infusion. CPP averaged from 103–112 mm Hg in shams. Edema occurred in all ICH models and predicted ICP. However, ICP and CPP were only modestly changed even after severe ICH and edema. Thus, small changes in edema typically reported in the literature, which often use smaller bleeds than presently used, likely minimally affects ICP and CPP. Further research into the face validity of these models, endpoints, and their ability to evaluate therapeutics is needed.

Selective brain cooling is a promising advent for reducing final infarct volume and improving outcomes in ischemic stroke victims. Despite the robust body of evidence from animal studies, evidence supporting the use of selective hypothermia in stroke patients is lacking. A recent study provided promising results on the safety and possible efficacy of selective brain hypothermia via intraarterial infusion of cooled saline. Better understanding of the patients’ population that may attain benefit from this approach will be informative. Details of infarct progression using perfusion imaging will also help understand the mechanism of effect of selective hypothermia to inform future trials.

Iron chelators, such as the intracellular ferrous chelator 2,2'-bipyridine, are a potential means of ameliorating iron-induced injury after intracerebral hemorrhage (ICH). We evaluated bipyridine against the collagenase and whole-blood ICH models and a simplified model of iron-induced damage involving a striatal injection of FeCl2 in adult rats. First, we assessed whether bipyridine (25 mg/kg beginning 12 h post-ICH and every 12 h for 3 days) would attenuate non-heme iron levels in the brain and lessen behavioral impairments (neurological deficit scale, corner turn test, and horizontal ladder) 7 days after collagenase-induced ICH. Second, we evaluated bipyridine (20 mg/kg beginning 6 h post-ICH and then every 24 h) on edema 3 days after collagenase infusion. Body temperature was continually recorded in a subset of these rats beginning 24 h prior to ICH until euthanasia. Third, bipyridine was administered (as per experiment 2) after whole-blood infusion to examine tissue loss, neuronal degeneration, and behavioral impairments at 7 days post-stroke, as well as body temperature for 3 days post-stroke. Finally, we evaluated whether bipyridine (25 mg/kg given 2 h prior to surgery and then every 12 h for 3 days) lessens tissue loss, neuronal death, and behavioral deficits after striatal FeCl2 injection. Bipyridine caused a significant hypothermic effect (maximum drop to 34.6 °C for 2-5 h after each injection) in both ICH models; however, in all experiments bipyridine-treated rats were indistinguishable from vehicle controls on all other measures (e.g., tissue loss, behavioral impairments, etc.). These results do not support the use of bipyridine against ICH.

Background and Purpose— We assessed the elemental and biochemical effects of rehabilitation after intracerebral hemorrhage, with emphasis on iron-mediated oxidative stress, using a novel multimodal biospectroscopic imaging approach. Methods— Collagenase-induced striatal hemorrhage was produced in rats that were randomized to enriched rehabilitation or control intervention starting on day 7. Animals were euthanized on day 14 or 21, a period of ongoing cell death. We used biospectroscopic imaging techniques to precisely determine elemental and molecular changes on day 14. Hemoglobin content was assessed with resonance Raman spectroscopy. X-ray fluorescence imaging mapped iron, chlorine, potassium, calcium, and zinc. Protein aggregation, a marker of oxidative stress, and the distribution of other macromolecules were assessed with Fourier transform infrared imaging. A second study estimated hematoma volume with a spectrophotometric assay at 21 days. Results— In the first experiment, rehabilitation reduced hematoma hemoglobin content ( P =0.004) and the amount of peri-hematoma iron ( P P =0.004). Lipid content in the peri-hematoma zone was increased by rehabilitation ( P =0.016). Rehabilitation reduced the size of calcium deposits ( P =0.040) and attenuated persistent dyshomeostasis of Cl − ( P + ( P =0.060). The second study confirmed that rehabilitation decreased hematoma volume ( P =0.024). Conclusions— Rehabilitation accelerated clearance of toxic blood components and decreased chronic oxidative stress. As well, rehabilitation attenuated persistent ion dyshomeostasis. These novel effects may underlie rehabilitation-induced neuroprotection and improved recovery of function. Pharmacotherapies targeting these mechanisms may further improve outcome.

Brief forebrain ischemia in rodents induces selective and delayed neuronal death, particularly of hippocampal CA1 pyramidal neurons. Neuronal death is preceded by down-regulation specific to CA1 of GluR2, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that limits Ca 2+ influx. This alteration is hypothesized to cause neurodegeneration by permitting a lethal influx of Ca 2+ and/or Zn 2+ through newly formed GluR2-lacking AMPA receptors. Two days of mild hypothermia induced 1 h after ischemia potently and lastingly protects against ischemic injury. We examined molecular mechanisms underlying hypothermia-induced neuroprotection. We report that hypothermia rescues most hippocampal CA1 neurons from ischemia-induced cell death and attenuates ischemia-induced down-regulation of mRNA encoding the AMPA receptor subunit GluR2. Ischemia induced a marked down-regulation of GluR2 mRNA and a small down-regulation of GluR1 mRNA in CA1 at 2 days, as assessed by quantitative in situ hybridization. The ischemia-induced changes in gene expression were cell-specific in that GluR2 was not significantly altered in CA3 or dentate gyrus. After ischemia treated by hypothermia GluR2 mRNA expression was modestly reduced at 2 days and exhibited complete recovery to control levels at 7 days. Hypothermia prevented ischemia induced changes in GluR1 mRNA expression. These findings suggest that intervention at the level of transcriptional regulation of the GluR2 gene may be a mechanism by which prolonged postischemic cooling rescues CA1 neurons otherwise “destined to die.”

Extensive animal research facilitated the clinical translation of therapeutic hypothermia for cardiac arrest in adults and hypoxic-ischemic injury in infants. Similarly, clinical interest in hypothermia for other brain injuries, such as stroke, has been greatly supported by positive findings in preclinical work. The reliability, validity, and utility of animal models, among many research practices (blinding, randomization, etc.), are key to successful clinical translation. Here, we review methods used to induce and maintain hypothermia in animal models. These include physical and pharmacological methods. We emphasize the advantages and limitations of each approach, and the importance of using clinically relevant cooling protocols and appropriate monitoring and reporting approaches. Moreover, we performed a literature survey of ischemic stroke studies published in 2015 to highlight the continuing risk of temperature confounds in neuroprotection studies. For example, many still do not accurately monitor and report temperature during surgery (23.5%), even though almost half of these studies (46.0%) use pharmaceutical agents that likely influence temperature. We hope this review stimulates awareness and discussion of the importance of temperature in neuroprotective studies.

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier–permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.

Hypothermia is an effective neuroprotectant for cardiac arrest and perinatal ischemic injury. Hypothermia also improves outcome after traumatic brain injury and stroke. Although the ideal treatment parameters (duration, delay, and depth) are not fully delineated, prolonged cooling is usually more effective than shorter periods. There is the concern that extended cooling may be hazardous to brain plasticity and cause damage. In order to evaluate this possibility, we assessed the effects of 3 days of systemic hypothermia (32°C) in rats subjected to a sham stroke surgery. There were no detrimental behavioral effects or signs of brain damage. As even longer cooling may be needed in some patients, we cooled (∼32°C) the right hemisphere of rats for 3 or 21 days. Physiological variables, functional outcome, and measures of cell injury were examined. Focal brain cooling for 21 days modestly decreased heart rate, blood pressure, and core temperature. However, focal hypothermia did not affect subsequent behavior (e.g., spontaneous limb usage), cell morphology (e.g., dendritic arborization, ultrastructure), or cause cell death. In conclusion, prolonged mild hypothermia did not harm the brain of normal animals. Further research is now needed to evaluate whether such treatments affect plasticity after brain injury.

Hyperthermia accelerates and increases ischemic brain damage. Owing to overlapping mechanisms of injury, many assume that hyperthermia also worsens outcome after intracerebral hemorrhage (ICH). However, clinical data do not conclusively prove this, and there is only one animal study examining the impact of hyperthermia. In that study (MacLellan and Colbourne, 2005), several hyperthermia protocols were administered after collagenase-induced ICH in rats; none worsened injury. While the collagenase model is widely used, it differs in important ways from another common model - injecting autologous blood directly into the brain. Thus, we evaluated the impact of immediate hyperthermia (HYP, 39 °C for 3 hr) after a 100-µL infusion of blood into the striatum of rats. This treatment, which markedly increases ischemic damage, was compared to control rats kept normothermic (NOR, 37 °C). Three separate experiments were done to measure: 1) edema at 24 hr, 2) edema at 72 hr, and 3) behavioral impairment and lesion size out to 1 month post-ICH. The HYP treatment did not significantly affect edema at 24 hr, but surprisingly, it modestly reduced edema at 72 hr and partly improved behavioral outcome. However, there were no lasting effects of HYP on behavior (e.g., skilled reaching) or the volume of tissue lost (NOR: 14.0 mm(3) vs. HYP: 14.5 mm(3)). In summary, our findings do not support the common belief that hyperthermia worsens outcome after ICH. Additional research is needed to determine whether more severe or prolonged heating or fever and its cause (e.g., infection) affect morbidity and mortality after ICH.

Co-existing protein-energy malnutrition (PEM), characterized by deficits in both protein and energy status, impairs functional outcome following global ischemia and has been associated with increased reactive gliosis. Since temperature is a key determinant of brain damage following an ischemic insult, the objective was to investigate whether alterations in post-ischemic temperature regulation contribute to PEM-induced reactive gliosis following ischemia. Male Sprague-Dawley rats (190-280 g) were assigned to either control diet (18% protein) or PEM induced by feeding a low protein diet (2% protein) for 7 days prior to either global ischemia or sham surgery. There was a rapid disruption in thermoregulatory function in rats fed the low protein diet as assessed by continuous recording of core temperature with bio-electrical sensor transmitters. Both daily temperature fluctuation and mean temperature increased within the first 24 hours, and these remained significantly elevated throughout the 7 day pre-ischemic period (p < 0.027). In the immediate post-surgical period, PEM decreased body temperature to a greater extent than that in well-nourished controls (p = 0.003). The increase in daily temperature fluctuation caused by PEM persisted throughout the 7 day post-surgical period (p < 0.001), and this interacted with the effects of global ischemia on days 8 (p = 0.018) and 11 (p = 0.021). The astrocytic and microglial responses induced at 7 days after global ischemia were not influenced by PEM, but this preliminary analysis needs to be confirmed with a more reliable global ischemia model. In conclusion, exposure to a low protein diet rapidly impairs the ability to maintain thermoregulatory homeostasis, and the resultant PEM also diminishes the ability to thermoregulate in response to a challenge. Since temperature regulation is a key determinant of brain injury following ischemia, these findings suggest that the pathophysiology of brain injury could be altered in stroke victims with coexisting PEM.

Rehabilitation, consisting of enriched environment and skilled reach training, improves recovery after intracerebral hemorrhage (ICH) in rats. We tested whether rehabilitation influences dendritic morphology (Golgi-Cox staining-experiment 1) or cell proliferation (immunohistochemistry-experiment 2). In the latter experiment, BrdU was given from 14 to 18 days post stroke, and cells were labeled for BrdU along with NeuN, Iba1 or GFAP. One week after a striatal ICH, via collagenase infusion, the rats were given rehabilitation for 2 weeks or control treatment (group housing in standard cages). Behavioral outcome (e.g., skilled reaching, walking) was assessed at multiple times. Rats were euthanized at 5 (experiment 2) or 6 (experiment 1) weeks post-ICH. As expected, rehabilitation significantly improved skilled reaching and walking ability. There was also a concomitant increase in dendritic length in peri-hematoma striatum and ipsilateral cortex as well as in the contralateral striatum. Lesion volume did not differ between groups, nor did cell proliferation. There was no evidence of neurogenesis, but there was increased Iba1 and GFAP labeling in the injured hemisphere. Thus, rehabilitation likely improves outcome after ICH though a plasticity response (e.g., increased dendritic growth) that does not involve neurogenesis.