Oligodendrocyte Progenitor Cell Proliferation and Fate after White Matter Stroke in Juvenile and Adult Mice.

The incidence of stroke in children is 2.4 per 100,000 person-years and results in long-term motor and cognitive disability. In ischemic stroke, white matter (WM) is frequently injured, but is relatively understudied compared to grey matter injury. Previous research suggests that the cellular response to WM ischemic injury is different at different ages. Little is known about whether WM repair mechanisms differ in children and adults. We utilized a model of focal ischemic WM injury to determine the oligodendrocyte (OL) response to focal WM ischemic injury in juvenile and adult mice. Methods: Juvenile (21-25 days of age) versus adult (2-3 months of age) mice underwent stereotaxic injection of the potent vasoconstrictor N5-(1-iminoethyhl)-L-ornithine (L-NIO) into the lateral corpus callosum (CC). Animals were sacrificed on postoperative day 3 (acute) or 21 (chronic). Cell birth-dating was performed acutely after WM stroke with 5-ethynyl-2-deoxyuridine (EdU) injected intraperitoneally. Immunohistochemistry was performed, as well as stereology, to measure injury volume. The acute oligodendrocyte progenitor cell (OPC) proliferation and the chronic OL cell fate were determined with immunohistochemistry. Compound action potentials were measured in the CC at acute and chronic time points. Results: Acutely WM injury volume was smaller in juveniles. There was significantly greater OPC proliferation in juvenile animals (acute) compared to adults, but newly born OLs did not survive and mature into myelinating cells at chronic time points. In addition, juveniles did not have improved histological or functional recovery when compared to adults. Protecting newly born OPCs is a potential therapeutic target in children with ischemic stroke.

Delayed inhibition of tonic inhibition enhances functional recovery following experimental ischemic stroke.

The current study focuses on the ability to improve cognitive function after stroke with interventions administered at delayed/chronic time points. In light of recent studies demonstrating delayed GABA antagonists improve motor function, we utilized electrophysiology, biochemistry and neurobehavioral methods to investigate the role of α5 GABAA receptors on hippocampal plasticity and functional recovery following ischemic stroke. Male C57Bl/6 mice were exposed to 45 min transient middle cerebral artery occlusion and analysis of synaptic and functional deficits performed 7 or 30 days after recovery. Our findings indicate that hippocampal long-term potentiation (LTP) is impaired 7 days after stroke and remain impaired for at least 30 days. We demonstrate that ex vivo administration of L655,708 reversed ischemia-induced plasticity deficits and importantly, in vivo administration at delayed time-points reversed stroke-induced memory deficits. Western blot analysis of hippocampal tissue reveals proteins responsible for GABA synthesis are upregulated (GAD65/67 and MAOB), increasing GABA in hippocampal interneurons 30 days after stroke. Thus, our data indicate that both synaptic plasticity and memory impairments observed after stroke are caused by excessive tonic GABA activity, making inhibition of specific GABA activity at delayed timepoints a potential therapeutic approach to improve functional recovery and reverse cognitive impairments after stroke.

Juvenile cerebral ischemia reveals age-dependent BDNF-TrkB signaling changes: Novel mechanism of recovery and therapeutic intervention.

Global ischemia in childhood often leads to poor neurologic outcomes, including learning and memory deficits. Using our novel model of childhood cardiac arrest/cardiopulmonary resuscitation (CA/CPR), we investigate the mechanism of ischemia-induced cognitive deficits and recovery. Memory is impaired seven days after juvenile CA/CPR and completely recovers by 30 days. Consistent with this remarkable recovery not observed in adults, hippocampal long-term potentiation (LTP) is impaired 7-14 days after CA/CPR, recovering by 30 days. This recovery is not due to the replacement of dead neurons (neurogenesis), but rather correlates with brain-derived neurotrophic factor (BDNF) expression, implicating BDNF as the molecular mechanism underlying impairment and recovery. Importantly, delayed activation of TrkB receptor signaling reverses CA/CPR-induced LTP deficits and memory impairments. These data provide two new insights (1) endogenous recovery of memory and LTP through development may contribute to improved neurological outcome in children compared to adults and (2) BDNF-enhancing drugs speed recovery from pediatric cardiac arrest during the critical school ages.

Endogenous Neuronal Replacement in the Juvenile Brain Following Cerebral Ischemia.

Replacement of dead neurons following ischemia, either via enhanced endogenous neurogenesis or stem cell therapy, has long been sought. Unfortunately, while various therapies that enhance neurogenesis or stem cell therapies have proven beneficial in animal models, they have all uniformly failed to truly replace dead neurons in the ischemic core to facilitate long-term recovery. Remarkably, we observe robust repopulation of medium-spiny neurons within the ischemic core of juvenile mice following experimental stroke. Despite extensive neuronal cell death in the injured striatum of both juveniles and adults at acute time points after ischemia (24 h and 7 d), mature newborn neurons replaced lost striatal neurons at 30 d post-ischemia. This neuronal repopulation was found only in juveniles, not adults, and importantly, was accompanied by enhanced post-ischemic behavioral recovery at 30 d. Ablation of neurogenesis using irradiation prevented neuronal replacement and functional recovery in MCAo-injured juvenile mice. In contrast, findings in adults were consistent with previous reports, that newborn neurons failed to mature and died, offering little therapeutic potential. These data provide support for neuronal replacement and consequent functional recovery following ischemic stroke and new targets in the development of novel therapies to treat stroke.

Endogenous Sex Steroids Dampen Neuroinflammation and Improve Outcome of Traumatic Brain Injury in Mice.

The role of biological sex in short-term and long-term outcome after traumatic brain injury (TBI) remains controversial. The observation that exogenous female sex steroids (progesterone and estrogen) reduce brain injury coupled with a small number of clinical studies showing smaller injury in women suggest that sex steroids may play a role in outcome from TBI. We used the controlled cortical impact (CCI) model of TBI in mice to test the hypothesis that after CCI, female mice would demonstrate less injury than male mice, related to the protective role of endogenous steroids. Indeed, adult females exhibit histological protection (3.7 ± 0.5 mm3) compared to adult male mice (6.8 ± 0.6 mm3), and females that lacked sex steroids (ovex) showed increased injury compared to intact females. Consistent with histology, sensorimotor deficits measured as reduced contralateral limb use were most pronounced in male mice (31.9 ± 6.9% reduced limb use) compared to a 12.7 ± 3.8% reduction in female mice. Ovex mice exhibited behavioral deficits similar to males (31.5 ± 3.9% reduced limb use). Ovex females demonstrated increased microglial activation relative to intact females in both the peri-injury cortex and the reticular thalamic nucleus. Ovex females also demonstrated increased astrogliosis in comparison to both females and males in the peri-injury cortex. These data indicate that female sex steroids reduce brain sensitivity to TBI and that reduced acute neuroinflammation may contribute to the relative protection observed in females.

Predicting Progression of Intracranial Arteriopathies in Childhood Stroke With Vessel Wall Imaging.

BACKGROUND AND PURPOSE: Childhood arterial ischemic stroke is frequently associated with an intracranial arteriopathy that often progresses in the first 3 to 6 months post stroke. We hypothesized that children with enhancing arteriopathies on vessel wall imaging (VWI) would have a higher risk of arteriopathy progression than those without enhancement. METHODS: Our institutional radiographic database was searched for cases of childhood stroke with VWI. Inclusion criteria consisted of age ranging from 1 month through 20 years, diagnosis of arterial ischemic stroke, available VWI, and follow-up magnetic resonance angiogram. Imaging was reviewed to systematically describe VWI findings, categorize arteriopathies, steroid therapy, and identify progressive arteriopathies using CACADE definitions. RESULTS: Sixteen cases of childhood stroke at Children's Hospital Colorado between January 1, 2010 and July 1, 2016 were reviewed. Strong vessel wall enhancement at presentation was associated with progressive arteriopathy in 83% of cases (10/12), when compared with 0% (0/4) without strong enhancement (P=0.008). CONCLUSIONS: Our case series demonstrates the potential benefit of VWI in children with stroke because it may identify patients who will have progressive arterial disease.

Autonomous CaMKII Activity as a Drug Target for Histological and Functional Neuroprotection after Resuscitation from Cardiac Arrest.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling, but its role in pathological glutamate signaling (excitotoxicity) remains less clear, with indications for both neuro-toxic and neuro-protective functions. Here, the role of CaMKII in ischemic injury is assessed utilizing our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). CaMKII inhibition (with tatCN21 or tatCN19o) at clinically relevant time points (30 min after resuscitation) greatly reduces neuronal injury. Importantly, CaMKII inhibition also works in combination with mild hypothermia, the current standard of care. The relevant drug target is specifically Ca2+-independent "autonomous" CaMKII activity generated by T286 autophosphorylation, as indicated by substantial reduction in injury in autonomy-incompetent T286A mutant mice. In addition to reducing cell death, tatCN19o also protects the surviving neurons from functional plasticity impairments and prevents behavioral learning deficits, even at extremely low doses (0.01 mg/kg), further highlighting the clinical potential of our findings.

Long-term depression in Purkinje neurons is persistently impaired following cardiac arrest and cardiopulmonary resuscitation in mice.

Cardiac arrest and cardiopulmonary resuscitation (CA/CPR) produce brain ischemia that results in cognitive and motor coordination impairments subsequent to injury of vulnerable populations of neurons, including cerebellar Purkinje neurons. To determine the effects of CA/CPR on plasticity in the cerebellum, we used whole cell recordings from Purkinje neurons to examine long-term depression (LTD) at parallel fiber (PF) synapses. Acute slices were prepared from adult male mice subjected to 8 min cardiac arrest at 1, 7, and 30 days after resuscitation. Concurrent stimulation of PF and climbing fibers (CFs) resulted in robust LTD of PF-evoked excitatory postsynaptic currents (EPSCs) in controls. LTD was absent in recordings obtained from mice subjected to CA/CPR, with no change in EPSC amplitude from baseline at any time point tested. AMPA and mGluR-mediated responses at the PF were not altered by CA/CPR. In contrast, CF-evoked NMDA currents were reduced following CA/CPR, which could account for the loss of LTD observed. A loss of GluN1 protein was observed following CA/CPR that was surprisingly not associated with changes in mRNA expression. These data demonstrate sustained impairments in synaptic plasticity in Purkinje neurons that survive the initial injury and which likely contribute to motor coordination impairments observed after CA/CPR.

The Clinical Significance of Collateral Ventilation.

Oxygen delivery and carbon dioxide removal being critical to cell survival, mammals have developed collateral vascular and ventilation systems to ensure tissue viability. Collateral ventilation, defined as ventilation of alveoli via pathways that bypass normal airways, is present in humans and many other species. The presence of collateral ventilation can be beneficial in certain disease states, whereas its relative absence can predispose to other diseases. These well defined anatomical pathways contribute little to ventilation in normal humans, but modulate ventilation perfusion imbalance in a variety of diseases, including obstructive diseases, such as asthma and emphysema. These pathways can be affected by pharmaceuticals and inhaled gas compositions. The middle lobe and lingula, constrained by their isolated, segmental anatomy, have reduced collateral ventilation, which predisposes them to disease. Recently, attempts to improve the quality of life of patients with emphysema, by performing nonsurgical lung volume reduction via use of endobronchial valves, have led to mixed results, because the role of collateral ventilation in the success or failure of the procedure was not initially appreciated. This review describes the anatomical pathways of collateral ventilation, their physiology and relationship to disease states, their modulatory effects on gas exchange, treatment considerations, and their effect on diagnostic procedures.

Neuropathophysiology of Brain Injury.

Every year in the United States, millions of individuals incur ischemic brain injury from stroke, cardiac arrest, or traumatic brain injury. These acquired brain injuries can lead to death or long-term neurologic and neuropsychological impairments. The mechanisms of ischemic and traumatic brain injury that lead to these deficiencies result from a complex interplay of interdependent molecular pathways, including excitotoxicity, acidotoxicity, ionic imbalance, oxidative stress, inflammation, and apoptosis. This article reviews several mechanisms of brain injury and discusses recent developments. Although much is known from animal models of injury, it has been difficult to translate these effects to humans.

Juvenile striatal white matter is resistant to ischemia-induced damage.

White matter injury following ischemic stroke is a major cause of functional disability. Injury to both myelinated axons and oligodendrocytes, the myelin producing cells in the central nervous system, occurs in experimental models of ischemic stroke. Age-related changes in white matter vulnerability to ischemia have been extensively studied and suggest that both the perinatal and the aged periods are times of increased white matter vulnerability. However, sensitivity of white matter following stroke in the juvenile brain has not been evaluated. Interestingly, the late pediatric period is an important developmental stage, as it is the time of maximal myelination. The current study demonstrates that neurons in late pediatric/juvenile striatum are vulnerable to ischemic damage, with neuronal injury being comparable in juvenile and adult mice following ischemia. By contrast, actively myelinating striatal oligodendrocytes in the juvenile brain are resistant to ischemia, whereas adult oligodendrocytes are quite sensitive. As a result, myelin sheaths are remarkably intact and axons survive well in the injured striatum of juvenile mice. In addition to relative resistance of juvenile white matter, other glial responses were very different in juvenile and adult mice following cerebral ischemia, including differences in astrogliosis, fibrosis, NG2-cell reactivity, and vascular integrity. Together, these responses lead to long-term preservation of brain parenchyma in juvenile mice, compared to severe tissue loss and scarring in adult mice. Overall, the current study suggests that equivalent ischemic insults may result in less functional deficit in children compared to adults and an environment more conducive to long-term recovery. GLIA 2016;64:1972-1986.

Sex-Related Differences in the Risk of Hospital-Acquired Sepsis and Pneumonia Post Acute Ischemic Stroke.

BACKGROUND AND OBJECTIVE: Infectious complications after ischemic stroke are frequent and lead to neurological deterioration, poor functional outcomes, and higher mortality. Local and systemic inflammatory responses to brain ischemia differ between males and females, but little is known about differences in poststroke susceptibility to infection by sex. The purpose of this study was to compare sex-related differences in the risk of hospital-acquired sepsis and pneumonia after acute ischemic stroke (AIS). MATERIALS AND METHODS: This is a retrospective, secondary analysis of the 2010-2011 California State Inpatient Database. Previously validated International Classification of Disease, Ninth Revision (ICD-9) codes were used to identify adult hospitalizations for AIS. The primary outcome was hospital-acquired sepsis or pneumonia, also identified using ICD-9 codes. Associations between sex and hospital-acquired sepsis or pneumonia were adjusted for baseline characteristics and comorbidities using multivariable logistic regression. RESULTS: There were 91,643 hospitalizations for AIS included in this analysis, of which 1027 had hospital-acquired sepsis and 1225 had hospital-acquired pneumonia. The in-hospital mortality without infection was 4.6%; the presence of hospital-acquired infections was associated with higher mortality for sepsis (32.7%) and pneumonia (21.9%). Female (versus male) sex was associated with lower adjusted odds of hospital-acquired sepsis (odds ratio [OR] .74, 95% confidence interval [CI] .65-.84) and pneumonia (OR .69, 95% CI .62-.78). This difference was similar across age strata. Among hospitalizations with either hospital-acquired sepsis or pneumonia, sex did not influence mortality. CONCLUSIONS: Female sex was associated with a lower risk of hospital-acquired sepsis and pneumonia after AIS. Further investigation is needed to determine the mechanisms underlying this clinical observation.

Arginase I release from activated neutrophils induces peripheral immunosuppression in a murine model of stroke.

Transient suppression of peripheral immunity is a major source of complication for patients suffering from ischemic stroke. The release of Arginase I (ArgI) from activated neutrophils has recently been associated with T-cell dysfunction in a number of pathologies. However, this pathway has not been previously explored in ischemic stroke. Using the murine model of transient middle cerebral artery occlusion, we explored effects of stroke on peripheral T-cell function and evaluated the role of neutrophils and ArgI. Stimulation of splenic T cells from post-stroke animals with anti-CD3/CD28 resulted in decreased proliferation and interferon-γ production when compared with sham-surgery controls. Flow cytometric analysis of intrasplenic leukocytes exposed the presence of a transient population of activated neutrophils that correlated quantitatively with elevated ArgI levels in culture media. In vitro activation of purified resting neutrophils from unmanipulated controls confirmed the capacity for murine neutrophils to release ArgI from preformed granules. We observed decreased expression of the L-arg-sensitive CD3ζ on T cells, consistent with decreased functional activity. Critically, L-arg supplementation restored the functional response of post-stroke T cells to mitogenic stimulation. Together, these data outline a novel mechanism of reversible, neutrophil-mediated peripheral immunosuppression related to ArgI release following ischemic stroke.

Animal models of stroke: translational potential at present and in 2050.

Translation from basic science bench research in ischemic stroke to bedside treatment of patients suffering ischemic stroke remains a difficult challenge. Despite literally hundreds of compounds and interventions that provide benefit in experimental models of cerebral ischemia, efficacy in humans remains to be demonstrated. The reasons for failure to translate the extensive positive basic science findings to successful clinical trials have been the focus of discussion for years. Some attribute the failure to flaws in clinical trial design, others question the predictive value of current animal models and some question the quality of preclinical data. It is likely that a combination of all these shortcomings have ultimately led to the failure. The purpose of this review is to analyze the commonly used animal models used in the field today, provide a framework for understanding the current state of basic science research in the ischemic stroke field and discuss a path forward.

Pro-inflammatory T-lymphocytes rapidly infiltrate into the brain and contribute to neuronal injury following cardiac arrest and cardiopulmonary resuscitation.

Although inflammatory mechanisms have been linked to neuronal injury following global cerebral ischemia, the presence of infiltrating peripheral immune cells remains understudied. We performed flow cytometry of single cell suspensions obtained from the brains of mice at varying time points after global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR) to characterize the influx of lymphocytes into the injured brain. We observed that CA/CPR caused a large influx of lymphocytes within 3h of resuscitation that was maintained for the 3day duration of our experiments. Using cell staining flow cytometry we observed that the large majority of infiltrating lymphocytes were CD4(+) T cells. Intracellular stains revealed a large proportion of pro-inflammatory T cells expressing either TNFα or INFγ. Importantly, the lack of functional T cells in TCRα knockout mice reduced neuronal injury following CA/CPR, implicating pro-inflammatory T cells in the progression of ischemic neuronal injury. Finally, we made the remarkable observation that the novel CD4(+)CD40(+) (Th40) population of pro-inflammatory T cells that are strongly associated with autoimmunity are present in large numbers in the injured brain. These data indicate that studies investigating the neuro-immune response after global cerebral ischemia should consider the role of infiltrating T cells in orchestrating the acute and sustained immune response.

Androgen and PARP-1 regulation of TRPM2 channels after ischemic injury.

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel was recently demonstrated to have a sexually dimorphic contribution to ischemic brain injury, with inhibition or knockdown of the channel protecting male brain preferentially. We tested the hypothesis that androgen signaling is required for this male-specific cell-death pathway. Additionally, we tested the hypothesis that differential activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is responsible for male-specific TRPM2 channel activation and neuronal injury. We observed that administration of the TRPM2 inhibitor clotrimazole (CTZ) 2 hours after onset of ischemia reduced infarct volume in male mice and that protection from ischemic damage by CTZ was abolished by removal of testicular androgens (castration; CAST) and rescued by androgen replacement. Male PARP-1 knockout mice had reduced ischemic damage compared with WT mice and inhibition of TRPM2 with CTZ failed to reduce infarct size. Lastly, we observed that ischemia increased PARP activity in the peri-infarct region of male mice to a greater extent than female mice and the difference was abolished in CAST male mice. Data presented in the current study indicate that TRPM2-mediated neuronal death in the male brain requires intact androgen signaling and PARP-1 activity.

Experimental pediatric arterial ischemic stroke model reveals sex-specific estrogen signaling.

BACKGROUND AND PURPOSE: Pediatric stroke, birth to 18 years, is a significant cause of long-term disability in the United States; however, there is currently little experimental data on the pathophysiology of childhood stroke owing to lack of animal models. We developed a novel mouse model of experimental childhood-onset arterial ischemic stroke to characterize the sex-specific response of the adolescent brain to cerebral ischemia and assess the neuroprotective effect of estrogen at this developmental stage. METHODS: Postnatal day 20 to 25 mice were subjected to 90 minutes experimental stroke via the intraluminal filament middle cerebral artery occlusion model and ischemic damage assessed 22 hours after reperfusion. Real-time quantitative real-time polymerase chain reaction was performed 22 hours after middle cerebral artery occlusion to determine the effects of ischemia and estrogen treatment on the proapoptotic gene Bax. RESULTS: Ischemic injury did not differ between male and female juvenile (postnatal day 20-25) mice after middle cerebral artery occlusion. However, estrogen reduced ischemic injury in female mice, whereas having no effect in juvenile males. No differences in estrogen receptor expression were observed on postnatal day between 20 males and females. In contrast, estrogen minimized the ischemia-induced increase in the proapoptotic gene Bax in female mice, whereas having no effect on Bax induction in the male brain. CONCLUSIONS: Focal ischemia has fundamentally different effects in the juvenile brain compared with the adult, as evidenced by the lack of sex difference in ischemic injury in the murine postnatal day 20 to 25 middle cerebral artery occlusion model and the sexually dimorphic response to estrogen neuroprotection.

Sex stratified neuronal cultures to study ischemic cell death pathways.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Normothermic cardiac arrest and cardiopulmonary resuscitation: a mouse model of ischemia-reperfusion injury.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality and which is most frequently caused by whole-body hypoperfusion. Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with difficulty. Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse. However, in recent years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI. This model reliably reproduces physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience with use of this model to assess a number of organ-specific outcomes in AKI.