The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

<p>Abstract</p> <p>Background</p> <p>Small-conductance Ca²⁺activated K⁺channels are expressed in the CNS, where <it>KCNN2</it>/SK2/KCa2.2 and <it>KCNN3</it>/SK3/KCa2.3 help shape the electrical activity of some neurons. The SK3 channel is considered a potential therapeutic target for diseases and disorders involving neuron hyper-excitability but little is known about its expression and roles in non-neuronal cells in either the healthy or damaged CNS. The purpose of this study was to examine expression of <it>KCNN3</it>/SK3 in CNS microglia <it>in vivo </it>and <it>in vitro</it>, and to use an established <it>in vitro </it>model to determine if this channel contributes to the neurotoxic capacity of activated microglia.</p> <p>Methods</p> <p><it>KCNN3 </it>mRNA (real-time RT-PCR) and SK3 immunoreactivity were examined in rat microglia. Lipopolysaccharide was then used to activate microglia (monitored by iNOS, nitric oxide, activation of NF-κB and p38 MAPK) and transform them to a neurotoxic state. Microglia-mediated neuron damage (TUNEL, activated caspase 3) and nitrotyrosine levels were quantified using a two-chamber system that allowed microglia to be treated with channel blockers, washed and then added to neuron/astrocyte cultures. Contributions of SK3 to these processes were discriminated using a subtractive pharmacological approach with apamin and tamapin. ANOVA and post-hoc tests were used to assess the statistical significance of differences between treatment groups. SK3 immunoreactivity was then compared in the normal and damaged adult rat striatum, by injecting collagenase (a hemorrhagic stroke) or endothelin-1 (a transient ischemic stroke).</p> <p>Results</p> <p><it>KCNN3 </it>mRNA was prevalent in cultured microglia and increased after lipopolysaccharide-induced activation; SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons. SK3 immunoreactivity was prevalent in cultured microglia and throughout the adult rat striatum (except white matter tracts). After strokes, SK3 was highly expressed in activated microglia/macrophages within the lesions, but reduced in other cells.</p> <p>Conclusions</p> <p>SK3 is expressed in microglia in both the healthy and damaged adult striatum, and mechanistic <it>in vitro </it>studies show it contributes to transformation of microglia to an activated neurotoxic phenotype. Thus, SK3 might be a therapeutic target for reducing inflammation-mediated acute CNS damage. Moreover, its roles in microglia must be considered when targeting this channel for CNS diseases, disorders and reducing neuron hyper-excitability.</p

Medulloblastoma has a global impact on health related quality of life: Findings from an international cohort.

BackgroundUnderstanding the global impact of medulloblastoma on health related quality of life (HRQL) is critical to characterizing the broad impact of this disease and realizing the benefits of modern treatments. We evaluated HRQL in an international cohort of pediatric medulloblastoma patients.MethodsSeventy-six patients were selected from 10 sites across North America, Europe, and Asia, who participated in the Medulloblastoma Advanced Genomics International Consortium (MAGIC). The Health Utilities Index (HUI) was administered to patients and/or parents at each site. Responses were used to determine overall HRQL and attributes (ie specific subdomains). The impact of various demographic and medical variables on HRQL was considered-including molecular subgroup.ResultsThe majority of patients reported having moderate or severe overall burden of morbidity for both the HUI2 and HUI3 (HUI2&nbsp;=&nbsp;60%; HUI3&nbsp;=&nbsp;72.1%) when proxy-assessed. Self-care in the HUI2 was rated as higher (ie better outcome) for patients from Western versus Eastern sites, P&nbsp;=&nbsp;.02. Patients with nonmetastatic status had higher values (ie better outcomes) for the HUI3 hearing, HUI3 pain, and HUI2 pain, all P&nbsp;&lt;&nbsp;.05. Patients treated with a gross total resection also&nbsp;had better outcomes for the HUI3 hearing (P&nbsp;=&nbsp;.04). However, those who underwent a gross total resection reported&nbsp;having worse outcomes on the HUI3 vision (P&nbsp;=&nbsp;.02). No differences in HRQL were evident as a function of subgroup.ConclusionsBy examining an international sample of survivors, we characterized the worldwide impact of medulloblastoma. This is a critical first step in developing global standards for evaluating long-term outcomes

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

Background: Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. Methods: In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. Results: Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. Conclusion: Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice

Age-Related Comparisons of Evolution of the Inflammatory Response After Intracerebral Hemorrhage in Rats

In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response within the brain characterized by the infiltration of peripheral neutrophils and macrophages and the activation of brain-resident microglia and astrocytes. Despite the strong correlation of aging and ICH incidence, and increasing information about cellular responses, little is known about the temporal- and age-related molecular responses of the brain after ICH. Here, we monitored a panel of 27 genes at 6 h and 1, 3, and 7 days after ICH was induced by injecting collagenase into the striatum of young adult and aged rats. Several molecules (CR3, TLR2, TLR4, IL-1β, TNFα, iNOS, IL-6) were selected to reflect the classical activation of innate immune cells (macrophages, microglia) and the potential to exacerbate inflammation and damage brain cells. Most of the others are associated with the resolution of innate inflammation, alternative pathways of macrophage/microglial activation, and the repair phase after acute injury (TGFβ, IL-1ra, IL-1r2, IL-4, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22). In young animals, the up-regulation of 26 in 27 genes (not IL-4) was detected within the first week. Differences in timing or levels between young and aged animals were detected for 18 of 27 genes examined (TLR2, GFAP, IL-1β, IL-1ra, IL-1r2, iNOS, IL-6, TGFβ, MMP9, MMP12, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22), with a generally less pronounced or delayed inflammatory response in the aged animals. Importantly, within this complex response to experimental ICH, the induction of pro-inflammatory, potentially harmful mediators often coincided with resolving and beneficial molecules

Medulloblastoma has a global impact on health related quality of life: Findings from an international cohort

Background: Understanding the global impact of medulloblastoma on health related quality of life (HRQL) is critical to characterizing the broad impact of this disease and realizing the benefits of modern treatments. We evaluated HRQL in an international cohort of pediatric medulloblastoma patients. Methods: Seventy-six patients were selected from 10 sites across North America, Europe, and Asia, who participated in the Medulloblastoma Advanced Genomics International Consortium (MAGIC). The Health Utilities Index (HUI) was administered to patients and/or parents at each site. Responses were used to determine overall HRQL and attributes (ie specific subdomains). The impact of various demographic and medical variables on HRQL was considered—including molecular subgroup. Results: The majority of patients reported having moderate or severe overall burden of morbidity for both the HUI2 and HUI3 (HUI2 = 60%; HUI3 = 72.1%) when proxy-assessed. Self-care in the HUI2 was rated as higher (ie better outcome) for patients from Western versus Eastern sites, P =.02. Patients with nonmetastatic status had higher values (ie better outcomes) for the HUI3 hearing, HUI3 pain, and HUI2 pain, all P <.05. Patients treated with a gross total resection also had better outcomes for the HUI3 hearin

Neuroinflammation after intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is a particularly severe type of stroke for which no specific treatment has been established yet. Although preclinical models of ICH have substantial methodological limitations, important insight into the pathophysiology has been gained. Mounting evidence suggests an important contribution of inflammatory mechanisms to brain damage and potential repair. Neuroinflammation evoked by intracerebral blood involves the activation of resident microglia, the infiltration of systemic immune cells and the production of cytokines, chemokines, extracellular proteases and reactive oxygen species (ROS). Previous studies focused on innate immunity including microglia, monocytes and granulocytes. More recently, the role of adaptive immune cells has received increasing attention. Little is currently known about the interactions among different immune cell populations in the setting of ICH. Nevertheless, immunomodulatory strategies are already being explored in ICH. To improve the chances of translation from preclinical models to patients, a better characterization of the neuroinflammation in patients is desirable

White Matter Microstructure and Emotional Functioning in Children Treated for Posterior Fossa Tumours

Children treated for brain tumours that arise in the posterior fossa (PF) experience lifelong cognitive and emotional difficulties, and exhibit white matter (WM) damage. This thesis combined diffusion tensor imaging (DTI), eye-tracking and standardized measures of cognitive and emotional functioning in children treated for PF tumours and typically developing children, to: 1) characterize outcomes related to decreases in treatment intensity, 2) objectively evaluate emotional functioning, and 3) examine the associations between WM microstructure and emotional functioning. It was found that treatment with the lowest intensity craniospinal irradiation protocol spared WM in the temporal lobe of children treated for malignant PF tumours. In addition, patients treated on lower intensity protocols had largely preserved cognitive, social and affective functioning. However, novel eye-tracking tasks designed to evaluate emotional functioning uncovered some remaining deficits; children treated for PF tumours had difficulty recognizing facial emotions despite attending to the faces, and difficulty regulating their initial attention away from emotional faces. Notably, this eye-tracking measure of emotion regulation was associated with emotional control in daily life. The current work also revealed that relations between WM microstructure and emotional functioning can diverge in the injured and uninjured brain; WM predicted facial emotion recognition in typically developing children only, whereas WM was associated with emotion regulation in patients only. In a field dominated by findings that characterize negative sequelae, this work highlights the possibility of favourable outcomes for PF tumour patients who are eligible for treatment with lower intensity protocols. Despite these positive outcomes, subtle emotional functioning deficits not captured by standardized questionnaires persist. To better understand how emotional processes are altered in children treated for PF tumours, novel objective measures are required. This thesis detailed one such behavioural marker to evaluate emotion regulation, using eye-tracking technology, and characterized an oculomotor response that may warrant interventional follow-up.Ph.D

White Matter Damage and Inflammation in Rat Models of Ischemic and Hemorrhagic Stroke

Cerebral ischemia and intracerebral hemorrhage (ICH) are both characterized by a prolonged inflammatory response and secondary injury phase, yet the spatial/temporal relationships between inflammation and white matter (WM) damage were largely unknown. Thus, I quantified the development of WM damage and inflammation over 7 days after ischemia, and 14 days after ICH. Following ischemia, myelin and axons were progressively damaged, and myelin damage coincided with neutrophil infiltration. Activated microglia/macrophages increased dramatically in the lesion core and edge, and selectively infiltrated damaged WM tracts while surrounding undamaged ones. To investigate the involvement of neutrophils in WM damage and inflammation after ICH, rats were rendered neutropenic before performing ICH. Neutrophil depletion reduced peri-hematomal axonal damage, BBB breakdown, and MMP-9 production at early times, and lessened microglia/macrophage and astrocyte responses at later times. Activated microglia/macrophages infiltrated peri-hematomal WM tracts, correlating with myelin fragmentation and axonal loss, and this was reduced with neutrophil depletion.MAS

Neuropsychological Outcome following Cranio-spinal Radiation in Medulloblastoma Patients: A Longitudinal Analysis of Predictors

Medulloblastoma is the most common malignant central nervous system (CNS) tumor in childhood. The cranio-spinal radiation (CSR) required to treat this disease results in long-term cognitive and neurologic impairments. Medulloblastoma was recently categorized into four genetic subgroups (WNT, SHH, Group 3, and Group 4). This study examined neuropsychological and intellectual functioning in 91 medulloblastoma patients (41 Group 4; 20 Group 3; 18 SHH; 12 WNT) following treatment, and examined the impact of several medical, treatment and demographic factors on functioning over time. Longitudinal growth curve analyses revealed hydrocephalus most clearly predisposes to poor neuropsychological functioning. Results also indicate medulloblastoma subgroups have heterogeneous intellectual outcomes following treatment. All subgroups experience intellectual declines following treatment; however, comparing between subgroups revealed Group 4 performs most poorly, and Group 3 has the best overall intellectual outcome. Lastly, qualitative analyses suggest treatment with a larger CSR dose may contribute to poor intellectual functioning.MAS