p17/C18-ceramide-mediated mitophagy is an endogenous neuroprotective response in preclinical and clinical brain injury.

Repeat concussions (or repetitive mild traumatic brain injury [rmTBI]) are complex pathological processes consisting of a primary insult and long-term secondary complications and are also a prerequisite for chronic traumatic encephalopathy (CTE). Recent evidence implies a significant role of autophagy-mediated dysfunctional mitochondrial clearance, mitophagy, in the cascade of secondary deleterious events resulting from TBI. C18-ceramide, a bioactive sphingolipid produced in response to cell stress and damage, and its synthesizing enzyme (CerS1) are precursors to selective stress-mediated mitophagy. A transporter, p17, mediates the trafficking of CerS1, induces C18-ceramide synthesis in the mitochondrial membrane, and acts as an elimination signal in cell survival. Whether p17-mediated mitophagy occurs in the brain and plays a causal role in mitochondrial quality control in secondary disease development after rmTBI are unknown. Using a novel repetitive less-than-mild TBI (rlmTBI) injury paradigm, ablation of mitochondrial p17/C18-ceramide trafficking in p17 knockout (KO) mice results in a loss of C18-ceramide-induced mitophagy, which contributes to susceptibility and recovery from long-term secondary complications associated with rlmTBI. Using a ceramide analog with lipid-selenium conjugate drug, LCL768 restored mitophagy and reduced long-term secondary complications, improving cognitive deficits in rlmTBI-induced p17KO mice. We obtained a significant reduction of p17 expression and a considerable decrease of CerS1 and C18-ceramide levels in cortical mitochondria of CTE human brains compared with age-matched control brains. These data demonstrated that p17/C18-ceramide trafficking is an endogenous neuroprotective mitochondrial stress response following rlmTBI, thus suggesting a novel prospective strategy to interrupt the CTE consequences of concussive TBI.

Angiotensin II Type 2 Receptor Agonism Alleviates Progressive Post-stroke Cognitive Impairment in Aged Spontaneously Hypertensive Rats.

Hypertension and aging are leading risk factors for stroke and vascular contributions to cognitive impairment and dementia (VCID). Most animal models fail to capture the complex interplay between these pathophysiological processes. In the current study, we examined the development of cognitive impairment in 18-month-old spontaneously hypertensive rats (SHR) before and following ischemic stroke. Sixty SHRs were housed for 18 months with cognitive assessments every 6 months and post-surgery. MRI scans were performed at baseline and throughout the study. On day 3 post-stroke, rats were randomized to receive either angiotensin II type 2 receptor (AT2R) agonist Compound 21 (C21) or plain water for 8 weeks. SHRs demonstrated a progressive cognitive decline and significant MRI abnormalities before stroke. Perioperative mortality within 72 h of stroke was low. Stroke resulted in significant acute brain swelling, chronic brain atrophy, and sustained sensorimotor and behavioral deficits. There was no evidence of anhedonia at week 8. C21 enhanced sensorimotor recovery and ischemic lesion resolution at week 8. SHRs represent a clinically relevant animal model to study aging and stroke-associated VCID. This study underscores the importance of translational disease modeling and provides evidence that modulation of the AT2R signaling via C21 may be a useful therapeutic option to improve sensorimotor and cognitive outcomes even in aged animals.

Stimulation of Angiotensin II Type 2 Receptor Modulates Pro-Inflammatory Response in Microglia and Macrophages: Therapeutic Implications for the Treatment of Stroke.

BACKGROUND: Sustained microglial activation contributes to the development of post-stroke cognitive impairment (PSCI). Compound 21 (C21), an angiotensin II type 2 receptor agonist, has shown some neurovascular protection after stroke. This study aimed to investigate the direct anti-inflammatory effects of C21 on macrophages, as well as brain innate immune cells. METHODS: Murine microglial cell line (C8-B4) and RAW 264.7 macrophages were exposed to lipopolysaccharide (LPS) and co-treated with C21. Pro-inflammatory mediators were assessed via RT-qPCR and ELISA. Cellular reactive oxygen species (ROS) were evaluated via CellROXGreen staining, and nitrate production was assessed using Griess assay. RESULTS: C21 suppressed LPS-induced inflammation and ROS generation in both cells. In microglia, C21 blunted LPS-induced mRNA expression of IL-1ß, IL-12b, COX-1, iNOS, and IL-6. A similar pattern was observed in macrophages, where C21 suppressed LPS-induced IL-1ß, TNF-α, and CXCL1 expression. These anti-inflammatory effects in microglia and macrophages were associated with increased neuroprotective gene expression, including GDNF and BDNF, in a dose-dependent manner. CONCLUSIONS: Our findings suggest a protective effect of C21 against the inflammatory response, in both macrophages and microglia, via suppression of the release of pro-inflammatory cytokines/chemokines and the generation of ROS while stimulating the production of neurotrophic factors.

Acute Ischemic Stroke by Middle Cerebral Artery Occlusion in Rat Models of Diabetes: Importance of Pre-op and Post-op Care, Severity of Hyperglycemia, and Sex.

Diabetes mellitus (DM) is associated with poor stroke outcomes, including high mortality and disability rates. Ischemic injury modeling large artery stroke in diabetic animals also results in high mortality and poor acute and long-term outcomes. In this chapter, we describe middle cerebral artery occlusion (MCAO) in a high-fat diet/low-dose streptozotocin (STZ) model of diabetes including details on pre-op and post-op care that improve survival rate for successful completion of the studies.

Cerebral microvascular matrix metalloproteinase-3 (MMP3) contributes to vascular injury after stroke in female diabetic rats.

Diabetes exacerbates hemorrhagic transformation (HT) after stroke and worsens clinical outcomes. Female patients with diabetes are at a greater risk of stroke and worsened recovery. We have shown that activation of matrix metalloprotease 3 (MMP3) in hyperglycemic settings mediates HT in male rats. In light of our recent findings that diabetic female rats develop greater HT, the current study was designed to test the hypotheses that: 1) cerebral microvascular MMP3 activation contributes to poor functional outcomes and increased hemorrhagic transformations (HT) after ischemic stroke, and 2) MMP3 inhibition can improve functional outcomes in female diabetic rats. Female control and diabetic Wistar rats were subjected to 60 min of middle cerebral artery occlusion (MCAO). One cohort of diabetic animals received a single dose of MMP3 inhibitor (UK356618; 15 mg/kg; iv) or vehicle after reperfusion. Neurobehavioral outcomes, brain infarct size, edema, HT, and MMPs were measured in brain tissue. Diabetic rats had significant neurological deficits on Day 3 after stroke. MMP3 expression and enzyme activity were significantly increased in both micro and macro vessels of diabetic animals. MMP3 inhibition improved functional outcomes and reduced brain edema and HT scores. In conclusion, cerebral endothelial MMP3 activation to vascular injury in female diabetic rats. Our findings identify MMP3 as a potential therapeutic target in diabetic stroke.

Deferoxamine prevents poststroke memory impairment in female diabetic rats: potential links to hemorrhagic transformation and ferroptosis.

Diabetes increases the risk of poststroke cognitive impairment (PSCI). Greater hemorrhagic transformation (HT) after stroke is associated with vasoregression and cognitive decline in male diabetic rats. Iron chelator deferoxamine (DFX) prevents vasoregression and improves outcomes. Although diabetic female rats develop greater HT, its impact on poststroke cerebrovascularization and cognitive outcomes remained unknown. We hypothesized that diabetes mediates pathological neovascularization, and DFX attenuates poststroke cerebrovascular remodeling and improves neurological outcomes in female diabetic rats. Female control and diabetic animals were treated with DFX or vehicle for 7 days after stroke. Vascular indices, microglial activation, and blood-brain barrier (BBB) integrity were evaluated on day 14. Results from diabetic female rats were partially compared with our previously published findings in male counterparts. Hemin-induced programmed cell death was studied in male and female brain microvascular endothelial cell lines (BMVEC). There was no vasoregression after stroke in either control or diabetic female animals. DFX prevented diabetes-mediated gliovascular remodeling and compromised BBB integrity while improving memory function in diabetes. Comparisons of female and male rats indicated sex differences in cognitive and vascular outcomes. Hemin mediated ferroptosis in both male and female BMVECs. DFX improved survival but had differential effects on ferroptosis signaling in female and male cells. These results suggest that stroke and associated HT do not affect cerebrovascularization in diabetic female rats, but iron chelation may provide a novel therapeutic strategy in the prevention of poststroke memory impairment in females with diabetes via the preservation of gliovascular integrity and improvement of endothelial cell survival.NEW & NOTEWORTHY The current study shows for the first time that diabetes does not promote aberrant cerebrovascularization in female rats. This contrasts with what we reported in male animals in various diabetes models. Deferoxamine preserved recognition memory function in diabetic female animals after stroke. The effect(s) of stroke and deferoxamine on cerebrovascular density and microglial activation also appear(s) to be different in female diabetic rats. Lastly, deferoxamine exerts detrimental effects on animals and BMVECs under control conditions.

Endothelin A receptors contribute to senescence of brain microvascular endothelial cells.

Cellular senescence plays a pivotal role in the aging and progression of neurodegenerative diseases, including vascular cognitive impairment and dementia (VCID). In postmortem brains from individuals with VCID, endothelin-1 (ET-1) levels closely correlate with blood barrier breakdown and cerebral hypoperfusion. Brain microvascular endothelial cells (BMVECs), previously thought to have exclusively endothelin B receptors, also possess endothelin A (ETA) receptors; however, the functional significance of this receptor in BMVECs is not known. We hypothesize that ETA receptors mediate BMVEC senescence. Serum-starved human BMVECs (HBEC5i) were incubated with ET-1 (1 µmol/L) in the presence/absence of ETA receptor antagonist BQ-123 (20 µmol/L). Cells were collected for Western blot and quantitative real-time PCR analyses. Treatment of ET-1 increased protein expression of ETA receptor, while it was prevented by the ETA receptor antagonist. ET-1 increased p21, p16, p53, LIF1 and cyclin D1 protein levels, and ß-galactosidase accumulation, which were prevented in the presence of ETA blockade. While there was no change in tight junction proteins, ET-1 decreased adherent junction protein vascular endothelial cadherin (VE-cadherin) levels. In conclusion, ET-1 upregulates ETA receptors in BMVECs in an autocrine manner and triggers the activation of senescence. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of endothelial senescence in VCID.

Magnetic resonance imaging reveals microemboli-mediated pathological changes in brain microstructure in diabetic rats: relevance to vascular cognitive impairment/dementia.

Diabetes doubles the risk of vascular cognitive impairment, but the underlying reasons remain unclear. In the present study, we determined the temporal and spatial changes in the brain structure after microemboli (ME) injection using diffusion MRI (dMRI). Control and diabetic rats received cholesterol crystal ME (40-70 µm) injections. Cognitive tests were followed up to 16 weeks, while dMRI scans were performed at baseline and 12 weeks post-ME. The novel object recognition test had a lower d2 recognition index along with a decrease in spontaneous alternations in the Y maze test in diabetic rats with ME. dMRI showed that ME injection caused infarction in two diabetic animals (n=5) but none in controls (n=6). In diabetes, radial diffusivity (DR) was increased while fractional anisotropy (FA) was decreased in the cortex, indicating loss of tissue integrity and edema. In the dorsal hippocampus, mean diffusivity (MD), axial diffusivity (DA), and DR were significantly increased, indicating loss of axons and myelin damage. Histological analyses confirmed more tissue damage and microglial activation in diabetic rats with ME. These results suggest that ME injury and associated cerebrovascular dysfunction are greater in diabetes, which may cause cognitive deficits. Strategies to improve vascular function can be a preventive and therapeutic approach for vascular cognitive impairment.

Vascular contributions to cognitive impairment/dementia in diabetes: role of endothelial cells and pericytes.

Vascular contributions to cognitive impairment/dementia (VCID) are a leading cause of dementia, a known neurodegenerative disorder characterized by progressive cognitive decline. Although diabetes increases the risks of stroke and the development of cerebrovascular disease, the cellular and vascular mechanisms that lead to VCID in diabetes are yet to be determined. A growing body of research has identified that cerebrovascular cells within the neurovascular complex display an array of cellular responses that impact their survival and reparative properties, which plays a significant role in VCID development. Specifically, endothelial cells and pericytes are the primary cell types that have gained much attention in dementia-related studies due to their molecular and phenotypic heterogeneity. In this review, we will discuss the various morphological subclasses of endothelial cells and pericytes as well as their relative distribution throughout the cerebrovasculature. Furthermore, the use of diabetic and stroke animal models in preclinical studies has provided more insight into the impact of sex differences on cerebral vascularization in progressive VCID. Understanding how cellular responses and sex differences contribute to endothelial cell and pericyte survival and function will set the stage for the development of potential preventive therapies for dementia-related disorders in diabetes.

p38 MAPK Is a Major Regulator of Amyloid Beta-Induced IL-6 Expression in Human Microglia.

The accumulation of amyloid beta (Aß) plaques in the brain is a hallmark of Alzheimer's disease (AD) pathology. Microglial activation-mediated neuroinflammation has been implicated in the pathogenesis of AD and the expression levels of interleukin-6 (IL-6) were increased in the brains of AD patients. However, the mechanisms by which IL-6 expression is regulated in human microglia are incompletely understood. Here, we show that Aß1-40 oligomers (Aß40) dose-dependently stimulate IL-6 expression in HMC3 human microglial cells. Treatment with Aß40 promotes the transcription of IL-6 and tumor necrosis factor α (TNFα) mRNAs in both HMC3 and THP-1 cells. Mechanistic studies reveal that Aß40-induced increase of IL-6 secretion is associated with the activation of p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK by BIRB 796 or SB202190 abrogates Aß40-induced increase of IL-6 production. Through analyzing brain specimens, we found that the immunoreactivity for IL-6 and phosphorylated (the activated form) p38 MAPK was markedly higher in microglia of AD patients than in age-matched control subjects. Moreover, our studies identified the co-localization of IL-6 with phosphorylated p38 MAPK in microglia in the cortices of AD patients. Taken together, these results indicate that p38 MAPK is a major regulator of Aß-induced IL-6 production in human microglia, which suggests that targeting p38 MAPK may represent a new approach to ameliorate Aß accumulation-induced neuroinflammation in AD.

Contralesional angiotensin type 2 receptor activation contributes to recovery in experimental stroke.

We and others have previously shown that angiotensin II receptor type 2 receptor (AT2R) is upregulated in the contralesional hemisphere after stroke in normoglycemic Wistar rats. In this study, we examined the expression of AT2R in type 2 diabetic Goto-Kakizaki (GK) rats and control Wistars after stroke. We also tested the contribution of the contralesional AT2R in recovery after stroke through a specific knockdown of the AT2R in this hemisphere only. Two experiments were conducted. In the first experiment, GK rats were subjected to middle cerebral artery occlusion (MCAO) and treated with the angiotensin II receptor type 1 receptor (AT1R) blocker candesartan or saline at reperfusion. Stroke outcomes, as well as AT2R expression, were examined and compared to control Wistars at 24 h. In the second experiment, localized AT2R knockdown was achieved through intrastriatal injection of short hairpin RNA (shRNA) lentiviral particles or non-targeting control into the left-brain hemisphere of Wistar rats. After 14 days, rats were subjected to right MCAO and treated with the AT2R agonist, Compound 21 (C21), or saline for 7 days. Behavioral outcomes were assessed for up to 10 days. In the first experiment, stroke reduced the expression of AT2R in GK rats. Candesartan treatment failed to improve the neurobehavioral outcomes, preserve vascular integrity or reduce oxidative/nitrative stress or apoptotic markers at 24 h post stroke in these animals. In the second experiment, contralesional AT2R knockdown reduced the C21-mediated functional recovery after stroke. In conclusion, contralesional AT2R upregulation after stroke is blunted in diabetic rats which show reduced sensitivity to post-stroke candesartan treatment. Contralesional AT2R could be involved in C21-mediated functional recovery after stroke.

Cerebral Microvascular Senescence and Inflammation in Diabetes.

Stress-induced premature senescence can contribute to the accelerated metabolic aging process in diabetes. Progressive accumulation of senescent cells in the brain, especially those displaying the harmful inflammatory senescence-associated secretory phenotype (SASP), may lead to cognitive impairment linked with metabolic disturbances. In this context, the senescence within the neurovascular unit (NVU) should be studied as much as in the neurons as emerging evidence shows that neurogliovascular communication is critical for brain health. It is also known that cerebrovascular dysfunction and decreased cerebral blood flow (CBF) precede the occurrence of neuronal pathologies and overt cognitive impairment. Various studies have shown that endothelial cells, the major component of the NVU, acquire a senescent phenotype via various molecular mediators and pathways upon exposure to high glucose and other conditions mimicking metabolic disturbances. In addition, senescence in the other cells that are part of the NVU, like pericytes and vascular smooth cells, was also triggered upon exposure to diabetic conditions. The senescence within the NVU may compromise functional and trophic coupling among glial, vascular, and neuronal cells and the resulting SASP may contribute to the chronic neurovascular inflammation observed in Alzheimer's Disease and Related Dementias (ADRD). The link between diabetes-mediated cerebral microvascular dysfunction, NVU senescence, inflammation, and cognitive impairment must be widely studied to design therapeutic strategies.

Porphyromonas gingivalis infection upregulates the endothelin (ET) system in brain microvascular endothelial cells.

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction and brain ET-1 levels were shown to be related to Alzheimer's disease and related dementias (ADRD) progression. ET-1 also contributes to neuroinflammation, especially in infections of the central nervous system. Recent studies causally linked chronic periodontal infection with an opportunistic anaerobic bacterium Porphyromonas gingivalis (Coykendall et al.) Shah & Collins to AD development. Thus, the goal of the study was to determine the impact of P. gingivalis infection on the ET system and cell senescence in brain microvascular endothelial cells. Cells were infected with a multiplicity of infection 50 P. gingivalis with and without extracellular ATP-induced oxidative stress for 24 h. Cell lysates were collected for analysis of endothelin A receptor (ETA)/endothelin B receptor (ETB) receptor as well as senescence markers. ET-1 levels in cell culture media were measured with enzyme-linked immunosorbent assay. P. gingivalis infection increased ET-1 (pg/mL) secretion, as well as the ETA receptor expression, whereas decreased lamin A/C expression compared to control. Tight junction protein claudin-5 was also decreased under these conditions. ETA or ETB receptor blockade during infection did not affect ET-1 secretion or the expression of cell senescence markers. Current findings suggest that P. gingivalis infection may compromise endothelial integrity and activate the ET system.

Impact of diabetes and ischemic stroke on the cerebrovasculature: A female perspective.

There is a very complex interaction between the brain and the cerebral vasculature to meet the metabolic demands of the brain for proper function. Preservation of vascular networks and cerebrovascular function ultimately plays a key role in this intricate communication within the brain in health and disease. Experimental evidence showed that diabetes not only affects the architecture of cerebral blood arteries causing adverse remodeling, pathological neovascularization, and vasoregression, but also alters cerebrovascular function resulting in compromised myogenic reactivity and endothelial dysfunction. Coupled with the disruption of blood brain barrier (BBB) integrity, changes in blood flow and microbleeds into the brain can rapidly occur. When an ischemic insult is superimposed on this pathology, not only is the neurovascular injury greater, but repair mechanisms fail, resulting in greater physical and cognitive deficits. While clinically it is known that women suffer disproportionately from diabetes as well as ischemic stroke and post-stroke cognitive impairment, the cerebrovascular architecture, patho/physiology, as well as cerebrovascular contributions to stroke recovery in female and diabetic animal models are inadequately studied and highlighted in this review.

Stimulation of angiotensin II receptor 2 preserves cognitive function and is associated with an enhanced cerebral vascular density after stroke.

Angiotensin signaling is known to be sexually dimorphic. Although it is a well-studied target for intervention in stroke and cognitive impairment, female studies are rare. With females suffering a disproportionately greater negative impact of stroke and dementia vs. males, effective interventions are of utmost urgency. The aim of the current study was to determine the impact of activation of the angiotensin II type 2 receptor (AT2R) with the agonist compound 21 (C21) on the development of post-stroke cognitive impairment, after experimental ischemic stroke. Ovariectomized (OVX) spontaneously hypertensive rats (SHRs) were subjected to 1 h of middle cerebral artery occlusion (MCAO). At 24 h, rats with a significant neurologic deficit were randomized to receive either saline or C21 (0.03 mg/kg/day) intraperitoneally (IP) for 5 days, then orally (0.12 mg/kg/day) for a total of 6 weeks. Cognitive function, brain structure by MRI and vascular architecture by microCT angiography were measured. C21 preserved cognitive function, specifically spatial memory, and improved vascular density in the ischemic hemisphere at 6 weeks, reflecting both arteriogenesis and angiogenesis. In conclusion, C21 prevented cognitive impairment after stroke, likely through a mechanism involving vascular protection and restoration.

Delayed Administration of Angiotensin Receptor (AT2R) Agonist C21 Improves Survival and Preserves Sensorimotor Outcomes in Female Diabetic Rats Post-Stroke through Modulation of Microglial Activation.

About 70% of stroke victims present with comorbid diseases such as diabetes and hypertension. The integration of comorbidities in pre-clinical experimental design is important in understanding the mechanisms involved in the development of stroke injury and recovery. We recently showed that administration of compound C21, an angiotensin II type 2 receptor agonist, at day 3 post-stroke improved sensorimotor outcomes by lowering neuroinflammation in diabetic male animals. In the current study, we hypothesized that a delayed administration of C21 would also lower chronic inflammation post-stroke in diabetic female animals. Young female diabetic rats were subjected to 1 h of middle cerebral artery occlusion (MCAO). Three days post-stroke, rats were administered C21 or vehicle in drinking water at a dose of 0.12 mg/kg/day for 4 weeks. The impact of C21 on microglial polarization was analyzed by flow cytometry in vivo and in vitro. Compound 21 treatment improved fine motor skills after MCAO through modulation of the microglia/macrophage inflammatory properties. In addition, C21 increased M2 polarization and reduced the M1:M2 ratio in vitro. In conclusion, delayed administration of C21 downregulates post-stroke inflammation in female diabetic animals. C21 may be a useful therapeutic option to lower neuro-inflammation and improve the post-stroke recovery in diabetes.

Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis.

It is a clinically well-established fact that patients with diabetes have very poor stroke outcomes. Yet, the underlying mechanisms remain largely unknown. Our previous studies showed that male diabetic animals show greater hemorrhagic transformation (HT), profound loss of cerebral vasculature in the recovery period, and poor sensorimotor and cognitive outcomes after ischemic stroke. This study aimed to determine the impact of iron chelation with deferoxamine (DFX) on (1) cerebral vascularization patterns and (2) functional outcomes after stroke in control and diabetic rats. After 8 weeks of type 2 diabetes induced by a combination of high-fat diet and low-dose streptozotocin, male control and diabetic animals were subjected to thromboembolic middle cerebral artery occlusion (MCAO) and randomized to vehicle, DFX, or tPA/DFX and followed for 14 days with behavioral tests. Vascular indices (vascular volume and surface area), neurovascular remodeling (AQP4 polarity), and microglia activation were measured. Brain microvascular endothelial cells (BMVEC) from control and diabetic animals were evaluated for the impact of DFX on ferroptotic cell death. DFX treatment prevented vasoregression and microglia activation while improving AQP4 polarity as well as blood-brain barrier permeability by day 14 in diabetic rats. These pathological changes were associated with improvement of functional outcomes. In control rats, DFX did not have an effect. Iron increased markers of ferroptosis and lipid reactive oxygen species (ROS) to a greater extent in BMVECs from diabetic animals, and this was prevented by DFX. These results strongly suggest that (1) HT impacts post-stroke vascularization patterns and recovery responses in diabetes, (2) treatment of bleeding with iron chelation has differential effects on outcomes in comorbid disease conditions, and (3) iron chelation and possibly inhibition of ferroptosis may provide a novel disease-modifying therapeutic strategy in the prevention of post-stroke cognitive impairment in diabetes.

Progress and challenges in preclinical stroke recovery research.

Significant innovations in the management of acute ischemic stroke have led to an increased incidence in the long-term complications of stroke. Therefore, there is an urgent need for improvements in and refinement of rehabilitation interventions that can lead to functional and neuropsychological recovery. The goal of this review is to summarize the current progress and challenges involved with preclinical stroke recovery research. Moving forward, stroke recovery research should be placing an increased emphasis on the incorporation of comorbid diseases and biological variables in preclinical models in order to overcome translational roadblocks to establishing successful clinical rehabilitation interventions.