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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 mm(3)) compared to adult male mice (6.8 ± 0.6 mm(3)), 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.

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.

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.

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

Introduction: Microglia are the first responders after stroke, mounting the immune response to CNS injury. While this response has been shown to be both neuroprotective and neurodegenerative following brain injury, it has been generally accepted that activation of microglia during the acute response to stroke is detrimental for neurogenesis and neuronal replacement. The vast majority of studies report that microglial activation reduces the survival of newborn neurons following stoke. However, our findings indicate that proinflammatory signaling is essential for survival of newborn neurons in juveniles. Remarkably, neurogenesis appears to require prolonged activation of microglia during the acute phase of cerebral ischemia. We utilized a juvenile stroke model to study the paracrine signaling triggered by ischemia in activated microglia and its consequence on newborn neuron survival. Methods: Electrocorticography (ECoG) was recorded from cortical surface electrodes and striatal depth electrodes implanted prior to middle cerebral artery occlusion (MCAO). Microglia were attenuated with Ibudilast (10 mg/kg), a glial cell activation inhibitor. Neurogenesis and inflammatory markers were examined with immunohistochemistry, and functional outcomes with neurobehavioral measures and ECoG recordings. Results and Conclusions: Acute inhibition of microglial activation after stroke resulted in reduced neurogenesis and long-lasting neurodegenerative effects, evidenced by increased astrogliosis, infiltrating macrophages, and glial scarring at 30d post-ischemia in the lateral striatum of MCAO+Ibudilast treated compared to MCAO+Vehicle treated juvenile mice. Behavioral tests and ECoG recordings revealed deficits in affected limb use, general motor functioning, and total ECoG power (μV 2 ) in both Ibudilast and vehicle treated MCAO-injured juveniles at 7d post-ischemia, but only MCAO+Vehicle treated juveniles returned to near sham-operated/baseline levels on neurobehavioral tasks and ECoG power in striatum and primary motor cortex at 30d. These surprising and striking findings provide evidence for a novel, supportive role for microglia in neuronal replacement and enhanced functional recovery following stroke in juveniles.

This chapter offers a set of stories, written as voices from the field that will be helpful to others who are building, or sustaining, team science in any venue. The first story centers on what the authors label as “team innovation,” that is, an activity that occurs in circumstances where innovation and surprising outcomes would not be possible without a team. The story also emphasizes how team members (or even leaders) may ask unschooled questions, which in an almost serendipitous way can lead to new and novel science. These examples arise from our experiences as National Institutes of Health (NIH)-funded stroke researchers who frequently use cell and animal models to investigate cellular and molecular mechanisms of ischemic brain injury. Next, we hypothesize that not only is team science greatly advantaged to be innovative and seek out novel science, this approach can lead to high productivity and effective competition in procuring funding and resources. Lastly, a final story emphasizes how strategic institutional actions and commitments can facilitate cross-disciplinary team science and assure that infrastructures are aligned to assure innovation.

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.

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.

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.

Introduction: Incidence of perinatal arterial ischemic stroke (PAIS) is surprisingly high, approaching that of the elderly. It has been postulated that the developing brain has a greater degree of plasticity and capacity for repair compared to the adult, thereby enhancing functional recovery. To address this issue, we utilized the recently developed intraluminal filament mouse model of ischemic stroke in postnatal day 10 mice. The aim of this study was to examine the cellular responses to ischemic stroke in the neonatal brain that contribute to the enhanced functional recovery. Method: Middle cerebral artery occlusion (45 minutes) was performed on adult and p10 mice using the intraluminal filament method. Immunohistochemistry was performed to identify oligodendrocyte precursors (PDGFRα/Olig2+), mature oligodendrocytes (CC1/Olig2+), vascular endothelial cells (Glut-1) and pericytes (CD13), and TUNEL to identify injured cells. Blood brain barrier integrity was examined by staining for fibrinogen. A protein array was conducted to examine extracellular protein levels. In situ hybridization was used to confirm source of IGF-1. Results: Oligodendrocytes were remarkably resistant to ischemic insult in the neonatal brain up to 7 days after injury; no change in oligodendrocyte precursor or mature oligodendrocyte cell count following neonatal stroke, compared to the significant loss of oligodendrocytes following adult stroke. In contrast, large numbers of TUNEL+ neurons were observed in both groups following stroke. In contrast to well-established vascular injury in adult stroke, no change in vascular integrity (Glut-1 positive vessel length/number or fibrinogen leakage) was detected following neonatal stroke. Unbiased analysis of extracellular protein levels in the adult and neonatal brain following stroke identified IGF-1 as a protein uniquely up-regulated following neonatal stroke. In situ hybridization revealed increased IGF-1 mRNA levels in oligodendrocytes following neonatal stroke. Conclusion: These results suggest that ischemia-resistant developing oligodendrocytes provide trophic support (IGF-1) to the vasculature following neonatal stroke.

Background: The aggressive management of atherosclerotic risk factors (ARF) has been accredited with the declining incidence of stroke in older adults. The goal of this study was to look at treatment patterns of ARF in young adults compared to older adults. Methods: A commercial claims database was used, containing enrollees between ages 15 to 45 for young adults and ≥ 55 for older adults from 2001 to 2014. Diagnosis for ARF including hypertension, diabetes and hyperlipidemia were established based on 2 outpatient or 1 inpatient claim. Hospitalization with the primary diagnosis of stroke were identified. Of patients with an ARF diagnosis, we looked for the presence of any medication claims including oral antihypertensive, antihyperlipidemics, and antidiabetics (including injectables) based on the Generic Product Identifier (GPI) codes. Results: Included in the analysis were 3,770,474 young adults with mean age 28 and 1,552,351 older adults with mean age 60. From 2001 to 2014, the overall prevalence of hypertension, diabetes and hyperlipidemia were 7.68%, 2.65%, and 7.85% respectively in young adults, compared to 47.8%, 18.0% and 46.6% respectively in older adults. Treatment rate of hypertension, diabetes and hyperlipidemia improved faster annually in older adults (1.9%, 1.7%, 1.8% respectively) than young adults (0.96 %, 0.99%, 0.61%, respectively, p Conclusion: Proportion of treatment for ARF in older adults improved at a faster rate than young adults. Overall treatment rates of diabetes was higher in young adults likely due to proportionally more cases of type 1 diabetes. Hyperlipidemia was treated at a much lower rate in young adults than older adults.

Introduction: Ischemic stroke is a common injury of the CNS, resulting in neuronal death and impaired brain function, likely contributing to long-term disability in adults. The lack of pharmacological interventions for patients with impairments in stroked-injured brain regions signals need for new strategies aimed at restoring function to the post-ischemic brain. Neurogenesis involves the generation of newborn neurons and occurs throughout life in the mammalian brain, making it an appealing target for potential interventions to enhance post-stroke recovery. Stroke-induced neurogenesis in adult mice has been reported to involve vigorous proliferation and migration of neural progenitors, but most cells die rapidly and fail to repopulate damaged areas. We tested the hypothesis that cortical neurogenesis is enhanced in the immature brain. Methods: We compared juvenile (P20-25) and adult (P60) mice following 45min transient middle cerebral artery occlusion (MCAo). Neurogenesis was examined with immunohistochemistry, including bromodeoxyuridine (BrdU) labeling and cell-type specific markers at 24hr, 7d, and 30d. Post-ischemic neurobehavioral outcomes were assessed at baseline, 7d, and 30d. Results and Conclusions: Despite extensive neuronal cell death in the injured cortex of both age groups at acute time points (24hr and 7d), a remarkable regenerative response was found at 30d, with substantially more mature newborn neurons found in juveniles than adults. Behavioral tests revealed reductions in affected limb use and general motor functioning in MCAo-injured adults and juveniles at 7d, with only the juveniles returning to near baseline levels on most tasks by 30d. When neurogenesis was arrested with ionizing radiation, 30d behavioral recovery was lost. Evidence of cortical neurogenesis is scarce; however, we discovered that newborn neurons in the developing brain not only survive, but post-stroke behavioral outcomes are only improved in non-irradiated juvenile mice. These results provide support for cortical neurogenesis, neuronal replacement, and enhanced functional recovery following stroke, which may have major biological significance for endogenous recovery and repair following CNS damage.

Background: Atherosclerotic risk factors (ARF) are known causes of stroke and acute myocardial infarction (AMI) in older adults. However, their role in young adult stroke incidence is largely unknown. Hypothesis: We hypothesized that if the trend in incidence of young adult strokes is driven by premature atherosclerosis then we would observe similar trends in young adult AMI. Methods: A 10% random sample of all enrollees, from a commercial claims database between 2001-2014, was used. Enrollees were divide into ages 15 to 45 for young adults and 55 to 75 for older adults. Individual claims for first ever stroke or AMI hospitalizations were identified using ICD-9 and ICD-10 codes. Annual incidence rate was adjusted for age in both groups. A simple linear regression model was used to plot trend lines for incidence. Results: Included in the analysis were 3,770,474 young adults with mean age 28 and 1,552,351 older adults with mean age 60. Of all young adults, there were 3,078 strokes and 3,408 AMIs compared to 38,707 strokes and 26,094 AMIs in older adults. Both older adult stroke and AMI incidence showed an annual decline with 36.8 and 21.9 less cases per 100,000 enrollees, respectively (R 2 = 0.85 and 0.81). Young adult stroke showed a slight increase in incidence overtime with overall, with 0.29 more cases a year per 100,000 enrollees (R 2 = 0.24). Meanwhile, young adult AMI incidence showed a slight decline overtime with 0.19 less cases a year per 100,000 enrollees (R 2 = 0.139). Though neither change was significantly different from 0, taken together their slopes are different from each other with p = 0.035. Conclusion: Despite the decreasing incidence of stroke and AMI in older adults, young strokes and AMI incidence remain largely unchanged. The slightly divergent trends between young adult stroke and AMI suggest that there may be more than atherosclerosis at play that is driving the increased incidence young adult strokes, which have been observed in other studies.

Introduction: Ischemic damage triggers glutamate excitotoxicity, resulting in neuronal cell death. Previous research has demonstrated that NMDA receptor activity triggers downstream calcium-dependent signaling pathways, specifically Ca2+/calmodulin-dependent protein kinase II (CaMKII). We hypothesize that Death Associated Protein Kinase I (DAPKI) and receptor interacting protein (RIP) are downstream of CaMKII, contributing to necroptosis. Focal and global ischemia studies have shown that inhibiting CaMKII is protective against hippocampal damage, but there is little known about cerebellar cell death mechanisms. The aim of this study is to examine the neuroprotective potential of inhibiting CaMKII in Purkinje cells using a global ischemia model. Methods: C57BL/6 male adult mice were subjected to 8 minutes of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Mice were randomized to tat-CN19o or control peptide (tat-SCR), administered 30 minutes after CA/CPR, and cerebellar injury analyzed 7 days after CA/CPR. Western blot analysis of cerebellar homogenates performed 6 hrs after CA/CPR. Phosphorylated and total CAMKII, DAPKI, and total RIP was quantified. Statistical analyses were performed using 1-WAY ANOVA, with P Results: Analysis of Purkinje cell death revealed injury at 7 days after CA/CPR that was prevented with tat-CN19o at doses of 0.1 and 1 mg/kg. We observed an increase in T286-P of CAMKII at 6 hours after CA/CPR, concurrent with increased DAPK1 activity (reduced phosphorylation) and increased RIP expression. Administration of tat-CN19o prevented CA/CPR-induced changes in DAPK1 and RIP, while having no effect on CAMKII. Conclusions: Autonomous activity of CAMKII is increased by ischemia in the cerebellum, which contributes to cell death. Neuroprotection with tat-CN19o occurred at doses 10-fold higher than that needed for protection in the hippocampus (Deng, Cell Reports, 2017). Importantly, we have identified signaling pathways downstream of CAMKII that may contribute to Purkinje cell death, DAPK1 and RIP. In summary, our findings indicate that inhibition of autonomous CaMKII activity is a promising therapeutic approach.

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.

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.

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

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.

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.

Understanding the anatomy and hemodynamic relationships of the cerebral vasculature is critical to an appreciation of the importance of these relationships in the control of the cerebrovasculature in normal and pathophysiological (disease) conditions. The cerebrovascular anatomy and the complexity of the arterial, venous, and capillary systems, and the blood–brain barrier and capillary permeability are briefly described. The chapter then provides an overview of cerebrovascular hemodynamics considering the basic physical principles of flow of fluids through tubes and blood vessels. Consideration is given to general hemodynamic principles of Poiseuille's law, perfusion pressure or driving pressure for flow, vascular resistance to flow, the influence of viscosity and blood vessel diameter/radius, blood volume, and autoregulation. These principles are then applied to the cerebral circulation, and cerebral pressure–volume and pressure–flow relationships are discussed.

Carbon dioxide (CO 2 ), oxygen (O 2 ), and nitric oxide (NO) profoundly affect the cerebrovasculature and are key players in the regulation of cerebral blood flow (CBF) and brain metabolism (i.e., cerebral metabolic rate of O 2 , CMRO 2 ). The brain is highly perfused and is extremely sensitive to any alteration in its blood supply. If the brain's circulation is completely obstructed, loss of consciousness occurs within seconds and irreversible pathological changes occur within minutes. Thus, the physiological mechanisms that regulate cerebral circulation are designed to ensure the constancy of CBF and CMRO 2 over a wide range of internal and external conditions. This may even occur at the expense of adequate blood flow to other organs. This chapter explores the physiological effects of CO 2 , O 2 , and NO on the cerebrovasculature and discusses their potential mechanisms of action. The effects of hyper- and hypocapnia, hypoxia, and hyperoxia, and the involvement of NO in cerebral ischemia are discussed.

Transient global cerebral ischemia due to cardiac arrest followed by resuscitation (CA/CPR) causes significant neurological damage in vulnerable neuron populations within the brain, such as hippocampal CA1 neurons. In recent years, we have implicated the transient receptor potential M2 (TRPM2) channel as a mediator of ischemic injury to neurons. We previously demonstrated that genetic and pharmacological strategies that reduce TRPM2 function preferentially protect male neurons in vitro and reduce infarct volume following experimental stroke. Due to the narrow therapeutic window for intervention following ischemic stroke, it is important to assess the role of TRPM2 in other models of cerebral ischemia. Therefore, this study utilized a modified mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR) to mimic more accurately the clinical condition by maintaining body and head temperatures near the physiological range throughout. Here we report that inhibition of TRPM2 activity with clotrimazole (CTZ) reduces hippocampal CA1 neuronal injury when administered 30 minutes after resuscitation from cardiac arrest. Consistent with our previous observations, neuroprotection was observed in male mice and no effect on injury was observed in the female. These findings provide further evidence for TRPM2 as a target for protection against cerebral ischemia in the male brain.

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.

Every year in the United States, millions of individuals incur ischemic brain injury from stroke, cardiac arrest, or traumatic brain injury (TBI). These forms of acquired brain injury can lead to death, or in many cases long-term neurologic and neuropsychological impairments. The mechanisms of ischemic and traumatic brain injuries that lead to these deficiencies result from a complex interplay of multiple interdependent molecular pathways that include excitotoxicity, acidotoxicity, ionic imbalance, oxidative stress, inflammation, and apoptosis. This article briefly reviews several of the traditional, well-known mechanisms of brain injury and then discusses more recent developments and newer mechanisms. Although much is known concerning mechanisms of injury and the manipulation of these mechanisms to result in protection of neurons and increased behavioral performance in animal models of injury, it has been difficult to translate these effects to humans. Attention is given to why this is so and newer outcome measures of injury are discussed.

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.

Female sex steroids, particularly estrogens, contribute to the sexually dimorphic response observed in cerebral ischemic outcome, with females being relatively protected compared to males. Using a mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR), we previously demonstrated that estrogen neuroprotection is mediated in part by the estrogen receptor β, with no involvement of estrogen receptor α. In this study we examined the neuroprotective effect of the novel estrogen receptor, G-protein coupled estrogen receptor 1 (GPER1/GPR30). Male mice administered the GPR30 agonist G1 exhibited significantly reduced neuronal injury in the hippocampal CA1 region and striatum. The magnitude of neuroprotection observed in G1 treated mice was indistinguishable from estrogen treated mice, implicating GPR30 in estrogen neuroprotection. Real-time quantitative RT-PCR indicates that G1 treatment increases expression of the neuroprotective ion channel, small conductance calcium-activated potassium channel 2. We conclude that GPR30 agonists show promise in reducing brain injury following global cerebral ischemia.

Background: Since genetically engineered mice are becoming more and more available, these animals become of high interest to study physiologic and pathophysiologic pathways of brain ischemia. The aim of this study was to examine body temperature (Tb), physical activity variation and neurohistopathology in mice exposed to normothermic and hyperthermic cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Methods: Male C57Bl/6 mice weighing 22 - 27 g were implanted intraperitoneally with a radio telemeter and subjected to 10 min cardiac arrest followed by cardiopulmonary resuscitation. Normothermia (37.5°C) or hyperthermia (39.0°C) was induced by controlling pericranial temperature during the arrest period. Results: Hyperthermia during the arrest resulted in a Tb decrease during early recovery to a nadir of 28°C ± 0.8°C (mean ± SE) and partially recovered to 34.4°C ± 1°C 36 hrs after CA/CPR. With normothermia during the arrest, Tb depression was less pronounced (nadir of 32.3°C ± 0.3°C) and recovered to physiologic levels within 24 hrs. Coupling of physical activity and body temperature was absent in all animals after CA/CPR. Neuronal injury in the caudoputamen was greater in the hyperthermia group. Conclusions: This study demonstrates that CA/CPR eliminates normal connectivity between body temperature and physical activity and induces long-lasting hypothermia, the depth of which is related to severity of brain injury. Long term temperature monitoring is required in survival murine experiments, if body temperature is a study variable.

Many theories have been advanced to explain the encephalopathy associated with chronic liver disease and with the less common acute form. A major factor contributing to hepatic encephalopathy is hyperammonemia resulting from portacaval shunting and/or liver damage. However, an increasing number of causes of hyperammonemic encephalopathy have been discovered that present with the same clinical and laboratory features found in acute liver failure, but without liver failure. Here, we critically review the physiology, pathology, and biochemistry of ammonia (i.e., NH3 plus NH4 +) and show how these elements interact to constitute a syndrome that clinicians refer to as hyperammonemic encephalopathy (i.e., acute liver failure, fulminant hepatic failure, chronic liver disease). Included will be a brief history of the status of ammonia and the centrality of the astrocyte in brain nitrogen metabolism. Ammonia is normally detoxified in the liver and extrahepatic tissues by conversion to urea and glutamine, respectively. In the brain, glutamine synthesis is largely confined to astrocytes, and it is generally accepted that in hyperammonemia excess glutamine compromises astrocyte morphology and function. Mechanisms postulated to account for this toxicity will be examined with emphasis on the osmotic effects of excess glutamine (the osmotic gliopathy theory). Because hyperammonemia causes osmotic stress and encephalopathy in patients with normal or abnormal liver function alike, the term “hyperammonemic encephalopathy” can be broadly applied to encephalopathy resulting from liver disease and from various other diseases that produce hyperammonemia. Finally, the possibility that a brain glutamine synthetase inhibitor may be of therapeutic benefit, especially in the acute form of liver disease, is discussed.

We evaluated long-term administration of estrogen after cardiac arrest and cardiopulmonary resuscitation (CA/CPR) on neurohistopathological and behavioral outcome. We also examined the effect of estrogen receptor (ER) stimulation using ER-alpha agonist propyl pyrazole triol (PPT) and ER-beta agonist diarylpropionitrile (DPN) on neuronal survival after CA/CPR to determine whether possible neuroprotective effects of estrogen are ER-mediated. Male C57Bl/6 mice underwent 10 mins of CA/CPR and 3-day survival. In protocol 1, intravenous injection of vehicle (NaCl 0.9%) and 0.5 or 2.5 microg 17beta-estradiol (E2 loading dose) was performed followed by subcutaneous implants containing vehicle (oil) or E2 (12.6 microg), according to a treatment group. In experimental protocol 2, mice were injected (intravenously) with the ER-alpha agonist PPT or ER-beta agonist DPN followed by Alzet pump implants (subcutaneously) containing PPT (200 microg) or DPN (800 microg). Long-term E2 administration reduced neuronal injury in the striatum after administration of either loading dose (41%+/-19%, 35%+/-26% of injured neurons), as compared with vehicle (68%+/-7%, P

Progress in regenerative medicine seems likely to produce new treatments for neurologic conditions that use human cells as therapeutic agents; at least one trial for such an intervention is already under way. The development of cell-based interventions for neurologic conditions (CBI-NCs) will likely include preclinical studies using animals as models for humans with conditions of interest. This paper explores predictive validity challenges and the proper role for animal models in developing CBI-NCs. In spite of limitations, animal models are and will remain an essential tool for gathering data in advance of first-in-human clinical trials. The goal of this paper is to provide a realistic lens for viewing the role of animal models in the context of CBI-NCs and to provide recommendations for moving forward through this challenging terrain. © 2009 ISCBFM All rights reserved.

While many pharmacological agents have been shown to protect the brain from cerebral ischemia in animal models, none have translated successfully to human patients. One potential clinical neuroprotective strategy in humans may involve increasing the brain’s tolerance to ischemia by pre-ischemic conditioning (preconditioning). There are many methods to induce tolerance via preconditioning such as: ischemia itself, pharmacological, hypoxia, endotoxin, and others. Inhalational anesthetic agents have also been shown to result in brain preconditioning. Mechanisms responsible for brain preconditioning are many, complex, and unclear and may involve Akt activation, ATP-sensitive potassium channels, and nitric oxide, amongst many others. Anesthetics, however, may play an important and unique role as preconditioning agents, particularly during the perioperative period.

Introduction: Following stroke, neurons are seriously damaged or die, impairing local brain function and contributing to long-term disability. Mounting evidence suggests that stroke recovery in children is enhanced compared to adults. Neurogenesis, a process involving the generation of functionally integrated neurons from progenitor cells, may play a role in enhanced plasticity and neuronal repair. Stroke-induced neurogenesis in adults involves massive proliferation and migration of newborn neurons, however these newborn neurons go on to die, never repopulating areas of damage. We tested the hypothesis that neurogenesis in the young brain effectively repopulates injured regions following ischemia. Methods: Stroke was induced in adult (2-3 mo, n=21) and pediatric (P20-25, n=21) mice by 45-min right middle cerebral artery occlusion (MCAo). Bromodeoxyuridine (BrdU) was injected on days 3 and 4, and mice sacrificed at 24 hr, 7 d or 30 d after recovery from MCAo. Immunohistochemistry was performed to assess cellular proliferation and neurogenesis. Results: The results revealed extensive neuronal cell death in the striatum of both pediatric and adult mice at 24 hr and 7 d after stroke. Remarkably, significant numbers of healthy, mature neurons (NeuN+) were observed in the striatum of pediatric mice at 30 d post-injury. Birth-dating with BrdU demonstrated robust, ischemia-induced proliferation of neural progenitor cells in both adult and pediatric brain. Consistent with previous reports, we observed very few mature NeuN+ neurons double labeled with BrdU in the injured adult brain. In contrast, significant numbers of BrdU+NeuN cells were observed in the pediatric brain 30 d after MCAo, indicative of mature neurons and most importantly, with COUP-TF1-interacting protein 2 (Ctip2) expression, a marker of medium spiny striatal neurons. Conclusion: Our results indicate that cerebral ischemia in pediatric mice increases neurogenesis and migration to sites of damage, and supports the possibility of true neuronal replacement in the pediatric brain. These findings have exciting implications for heightened restorative processes in the pediatric brain microenvironment.

Introduction: Recent data demonstrates that inhibition of TRPM2 channels protect the brain against ischemia, specifically in males. A major hindrance to our research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor developed in our laboratory. Methods: Fluorescent calcium imaging was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. tat-peptides were detected by immunohistochemistry using an anti-tat antibody. Adult (2-3 mo) and aged (18-20 mo) were subjected to 60 min middle cerebral artery occlusion (MCAo) and injected iv with tat-M2NX (20 mg/kg), control tat-SCR (20 mg/kg) or clotrimazole (CTZ) (30 mg/kg) either 20min prior or 3hr after reperfusion. Infarct size was assessed using TTC staining. Results: Successful TRPM2 inhibition in HEK293 cells was observed by decreased Ca2+ influx following H2O2 exposure. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR; 29.2±4.6% (n=8) vs. 43.4±3.2% (n=8; p Conclusions: These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a relatively wide therapeutic window. Furthermore, we took advantage of TRPM2-/- mice to confirm specificity of tat-M2NX protection.

Introduction: Ischemic stroke is the fourth leading cause of death in the United States and is increasingly being recognized as a disease that occurs in people of all ages, not just the elderly. Studies suggest that the immature developing brain may have a greater degree of plasticity compared to the adult, thereby enhancing functional recovery to a greater extent during development. Hypothesis: Pediatric mice exhibit greater recovery from hippocampal synaptic function following experimental stroke than adults. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared at, 7 or 30 days after recovery from middle cerebral artery occlusion (MCAO). A behavioral fear conditioning paradigm was done to evaluate contextual memory in both pediatric and adult mice 30 days after MCAO. α5 GABAA receptors and GABA transporter expression was evaluated by western blot analysis. Results: In adult mice following MCAO, hippocampal long-term potentiation (LTP), defined as an increase in synaptic strength, remained impaired for at least 30 days in the ipsilateral and contralateral, non-injured hemisphere. However, in pediatric mice following MCAO, LTP was only impaired in the ipsilateral side 7 days after MCAO and showed full recovery of synaptic function at 30 days. Behavioral data confirmed these data, showing that only adult mice displayed memory deficits 30 days after MCAO. L655,708 (100nM), an inverse agonist selective for α5 GABAA receptors, rescued LTP in acute slices in both pediatric and adult mice at all-time points tested. Western blot analysis failed to identify any changes in α5 GABAA receptors or GABA transporter (GAT1 or GAT3) levels after MCAO. Conclusion: The present study demonstrates that transient focal ischemia causes functional impairment in the hippocampus and that recovery of behavioral and synaptic function is more robust in the young brain. In addition, inhibition of tonic GABA activity rescues synaptic function, indicating that targeting of excessive GABA activity may provide a therapeutic approach to improve cognitive recovery after stroke.

Objective: Pediatric sudden cardiac arrest (CA) is an unfortunate and devastating condition, often leading to poor neurologic outcomes. However, little is known about the pathophysiology of pediatric CA. We have developed a unique pediatric cardiac arrest and cardiopulmonary (CA/CPR) mouse model to characterize the sex-specific response of the adolescent brain to global cerebral ischemia and assess the neuroprotective effect of estrogen at this developmental stage. Methods: C57BL/6 male and female mice (postnatal day 20 to 25 ) were subjected to 8 min of CA followed by CPR. Juvenile male and female mice were randomized to estradiol (E2, 100μg/kg, iv) or vehicle, administered 30 min after resuscitation. P38-MAPK inhibitor SB203580 (2 mg/kg) alone or in combination with E2 was given 30 min after resuscitation (iv) in females. Hippocampal neuronal damage was analyzed 3 days after CA/CPR by H&E staining. Statistical analyses were performed using ANOVA and t tests, with P Results: Analysis of histological damage 72 hrs after CA/CPR showed no difference between male (49.4 ± 10.2%, n=5) and female (62.8 ± 1.3%, n=5) pediatric mice. However, estrogen reduced neuronal injury from 62.8 ± 1.3% to 30.6 ± 5.6% (n=6, P Conclusions: Pre-pubertal male and female animals exhibit equvalent injury following global cerebral ischemia, in contrast to the extensive literature demonstrating relative protection in the adult female provided by estrogen. Interestingly, estradiol decreases neuronal damage in juvenile females, but not males following CA/CPR, indicating that there is sex difference in neuronal damage in the P20-25 mice after global cerebral ischemia. Moreover, this sex-specific estrogen neuroprotective signaling requires P38-MAPK pathway.

Pediatric cardiac arrest (CA) often leads to poor neurologic outcomes, including deficits in learning and memory. The only approved treatment for CA is therapeutic hypothermia, although its utility in the pediatric population remains unclear. This study analyzed the effect of mild therapeutic hypothermia after CA in juvenile mice on hippocampal neuronal injury and the cellular model of learning and memory, termed long-term potentiation (LTP). Juvenile mice were subjected to cardiac arrest and cardiopulmonary resuscitation (CA/CPR) followed by normothermia (37 °C) and hypothermia (30 °C, 32 °C). Histological injury of hippocampal CA1 neurons was performed 3 days after resuscitation using hematoxylin and eosin (H&E) staining. Field excitatory post-synaptic potentials (fEPSPs) were recorded from acute hippocampal slices 7 days after CA/CPR to determine LTP. Synaptic function was impaired 7 days after CA/CPR. Mice exposed to hypothermia showed equivalent neuroprotection, but exhibited sexually dimorphic protection against ischemia-induced impairment of LTP. Hypothermia (32 °C) protects synaptic plasticity more effectively in females, with males requiring a deeper level of hypothermia (30 °C) for equivalent protection. In conclusion, male and female juvenile mice exhibit equivalent neuronal injury following CA/CPR and hypothermia protects both males and females. We made the surprising finding that juvenile mice have a sexually dimorphic response to mild therapeutic hypothermia protection of synaptic function, where males may need a deeper level of hypothermia for equivalent synaptic protection.

OBJECTIVE Paraplegia from spinal cord ischemia is a devastating complication of thoracoabdominal aortic aneurysm repair. Perioperative hypoperfusion of the spinal cord is a critical determinant of residual neurologic deficits. We determined if functional and histologic outcome is dependent on systemic blood pressure in a rat model of spinal cord ischemia. DESIGN Randomized, controlled, prospective study. SETTING Research laboratory at a university teaching hospital. SUBJECTS Adult male Wistar rats. INTERVENTIONS Endotracheally intubated adult male Wistar rats (300-450 g) anesthetized with halothane underwent a thoracotomy and placement of a clip across the descending aorta for 27 mins. Mean proximal arterial blood pressure (MPABP) was monitored with a cannula placed in the left common carotid artery. Halothane was adjusted (1.25-1.5%) to maintain MPABP between 70 and 90 mm Hg (n = 20) or 140 and 150 mm Hg (n = 20). Shamoperated rats (n = 10) had a thoracotomy without aortic clamping at an MPABP of 70-90 mm Hg. Following 1, 24, 48, and 72 hrs of recovery from anesthesia, motor function of the hind paws was scored as follows: 0, no evidence of deficit; 1, toes flat under body when walking but with ataxia; 2, knuckle walks; 3, movements in hind limbs but unable to knuckle walk; 4, no movement, drags hind limbs. Body temperature was maintained between 37 and 38 degrees C throughout the experiment. MEASUREMENTS AND MAIN RESULTS All sham operated rats with MPABP 70-90 mm Hg recovered without neurologic deficits, whereas those that underwent aortic occlusion with MPABP between 70 and 90 mm Hg emerged from anesthesia with grade 3 and 4 deficits and remained in this condition without improvement at 72 hrs. Histopathology at 72 hrs demonstrated moderate to severe neuronal loss with involvement of dorsal, intermediate, and ventral horns. Only eight of 20 rats that underwent aortic occlusion with MPABP between 140 and 150 mm Hg had grade 1 and 2 deficits on emergence but had no neurologic deficit after 1 hr. Most of the surviving neurons in these animals appeared normal histologically, particularly motor neurons around the periphery of the ventral horn. CONCLUSIONS Systemic blood pressure is a critical determinant of outcome following spinal cord ischemia, and controlled peri-operative blood pressure augmentation may ameliorate neurologic deficits in patients who undergo thoracoabdominal vascular procedures and are at risk for spinal cord hypoperfusion.

Cardiac arrest is associated with high mortality and poor neurological outcome. We characterized functional and histological outcome in a novel mouse model of cardiac arrest and cardiopulmonary resuscitation (CPR) in order to study neuroprotective mechanisms. Cardiac arrest was induced in male C57Bl/6 and 129SVEV mice by i.v. injection of KCl. After 10 min cardiac standstill, CPR was initiated by administration of epinephrine, ventilation with 100% oxygen and chest compressions. Twenty-four hours before and 3 or 7 days after CPR, mice were subjected to behavioral testing using a passive avoidance task, locomotor activity in an open field, and spontaneous alternation in a T-maze. Hippocampal and caudoputamen injury was quantified 3 or 7 days after CPR. At both time points, caudoputamen injury was worse in 129SVEV mice. Post-ischemic mice of both strains showed a reduced number of correct choices in the T-maze up to 7 days after CPR, and were temporarily impaired in learning the passive avoidance task with a retention deficit on day 3 but not on day 7. Locomotor activity showed strain differences with C57Bl/6 mice being more active, but little ischemia-related effects. A dissociation between functional and histological outcome was found emphasizing the importance of combining both outcome measures for evaluation of neuroprotective strategies.

In the past decade, it has become possible to use the nuclear (proton, 1H) signal of the hydrogen atoms in water for noninvasive assessment of functional and physiological parameters with magnetic resonance imaging (MRI). Here we show that it is possible to produce pH-sensitive MRI contrast by exploiting the exchange between the hydrogen atoms of water and the amide hydrogen atoms of endogenous mobile cellular proteins and peptides. Although amide proton concentrations are in the millimolar range, we achieved a detection sensitivity of several percent on the water signal (molar concentration). The pH dependence of the signal was calibrated in situ, using phosphorus spectroscopy to determine pH, and proton exchange spectroscopy to measure the amide proton transfer rate. To show the potential of amide proton transfer (APT) contrast for detecting acute stroke, pH effects were noninvasively imaged in ischemic rat brain. This observation opens the possibility of using intrinsic pH contrast, as well as protein- and/or peptide-content contrast, as diagnostic tools in clinical imaging.

Poly (ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that facilitates DNA repair and may be important in neuronal cell death in a variety of diseases including cardiac arrest/cardiopulmonary resuscitation (CPR) and stroke. This chapter briefly reviews the characteristics of PARP, its activation, and its role in both focal (stroke) and global (cardiac arrest/CPR) cerebral ischemia. We demonstrate PARP deficiency to be effective in reducing neuronal reperfusion injury after stroke and cardiac arrest/CPR in mouse models. These data suggest a selective role of PARP-1 in glutamate excitotoxicity and that strategies of inhibiting PARP-1 may offer substantial neuroprotection in focal and global cerebral ischemia.

Increases in COX-2 enzymatic activity and prostaglandin production have been associated with neuronal injury in both acute and age-related degenerative neurological diseases. In this study, we tested the effects of increased COX-2 activity in a model of transient focal ischemia using a transgenic mouse model in which human COX-2 is constitutively expressed selectively in neurons of the striatum, cerebral cortex, and hippocampus. These COX-2 transgenic mice harbor elevated levels of PGE(2) that are 10-fold higher than nontransgenic levels. A significant increase in infarct volume was observed after middle cerebral artery occlusion with 4 days of reperfusion in COX-2 transgenic mice as compared with nontransgenic littermates. Pretreatment of nontransgenic mice with the selective COX-2 inhibitor SC58236 resulted in a significant reduction of infarct volume in nontransgenic mice, consistent with previous pharmacological studies. However, transgenic COX-2 mice treated with SC58236 did not show a significant reduction. This suggests that chronic increases in COX-2 expression and enzymatic activity, which can occur in aging and in pathological states characterized by oxidative stress and chronic inflammatory processes, can lead to downstream cellular changes that have a negative impact on neuronal survival in cerebrovascular disease.

BAK is a pro-apoptotic BCL-2 family protein that localizes to mitochondria. Here we evaluate the function of BAK in several mouse models of neuronal injury including neuronotropic Sindbis virus infection, Parkinson's disease, ischemia/stroke, and seizure. BAK promotes or inhibits neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. BAK protects neurons from excitotoxicity and virus infection in the hippocampus. As mice mature, BAK is converted from anti- to pro-death function in virus-infected spinal cord neurons. In addition to regulating cell death, BAK also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. BAK can alter neurotransmitter release in a direction consistent with its protective effects on neurons and mice. These findings suggest that BAK inhibits cell death by modifying neuronal excitability.

The in vivo signaling of ischemic neuroprotection provided by sigma-receptor ligands remains unclear. Catecholamines have been implicated in the propagation of ischemic neuronal injury, and previous in vitro studies suggest that sigma ligands modulate dopaminergic neurotransmission. In this study, we tested the hypothesis that the potent sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) attenuates the increase of extracellular dopamine in ischemic striatum. Under controlled physiological conditions, a microdialysis probe was implanted in right caudoputamen (CP) complex of adult male Wistar rats. Rats were subjected to 2 h of transient middle cerebral artery occlusion (MCAO) by the intraluminal suture technique. In a blinded, randomized fashion, rats were divided into five treatment groups: Group 1 (n = 8; saline-saline) continuous i.v. infusion of saline vehicle 30 min before MCAO followed by saline at reperfusion until the end of the experiment; Group 2 (n = 8; PPBP-PPBP) i.v. PPBP 30 min before MCAO followed by 1 micromol x kg(-1) x h(-1) of PPBP; Group 3 (n = 8; saline-PPBP) i.v. saline before MCAO followed by PPBP; Group 4 (n = 4) surgical shams (saline-saline); and Group 5 (n = 4) surgical shams (PPBP-PPBP). Infarction volume at 22 h of reperfusion in the CP complex (percentage of ipsilateral structure) was significantly attenuated in rats treated with PPBP-PPBP (27.3% +/- 9.1%) and saline-PPBP (27.8% +/- 12.7%) compared with saline-saline (59.3% +/- 7.3%) treatment. There was a three- to fourfold increase in dopamine concentrations in the microdialysates within 40 min of the onset of MCAO. Dopamine and its metabolites dihydroxy phenylacetic acid and homovallinic acid levels were similar among the three groups subjected to MCAO. Therefore, PPBP provides significant ischemic neuroprotection in the CP complex without altering the acute accumulation of dopamine in vivo during transient focal ischemia in the rat.

Neonatal hypoxia-ischemia (HI) can result in significant sensorimotor abnormalities, including movement and posture disorders. These neurological impairments are believed to result from basal ganglia (striatum) damage, but the exact cause of this injury is not known. One mechanism involved in brain injury after HI is the generation of reactive oxygen species, which damage cellular macromolecules. We tested the hypothesis that inactivation of plasma membrane enzyme Na,K-ATPase during striatal neurodegeneration after HI emerges with peroxynitrite attack on the enzyme. In vitro, reaction of peroxynitrite (100–500 μM) with purified Na,K-ATPase produced nitration of the α (catalytic) and β (transport) subunits, as quantified by immunoblots of the reaction products for nitrotyrosine. To evaluate for peroxynitrite damage to Na,K-ATPase in vivo, striatal plasma membrane fractions from 1-week-old piglets subjected to asphyxic cardiac arrest and recovery were also studied by immunoprecipitation. During the progression of striatal neurodegeneration and loss of enzyme function 3–24 h after arrest, nitration of the α3 (neuronal) isoform of Na,K-ATPase was not increased relative to sham control. Suprisingly, however, nitration of this α isoform occurs during normal brain development and peaks at 2 weeks of age. We conclude that Na,K-ATPase is a target of peroxynitrite, but that this mechanism is not responsible for enzyme inactivation after HI. Protein nitration may serve as marker of other normal, noninjurious cell processes in the developing brain.