Melissa D. Laird

Opposing roles for reactive astrocytes following traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. Current medical therapies exhibit limited efficacy in reducing neurological injury and the prognosis for patients remains poor. While most research is focused on the direct protection of neuronal cells, non-neuronal cells, such as astrocytes, may exert an active role in the pathogenesis of TBI. Astrocytes, the predominant cell type in the human brain, are traditionally associated with providing only structural support within the CNS. However, recent work suggests astrocytes may regulate brain homeostasis and limit brain injury. In contrast, reactive astrocytes may also contribute to increased neuroinflammation, the development of cerebral edema, and elevated intracranial pressure, suggesting possible roles in exacerbating secondary brain injury following neurotrauma. The multiple, opposing roles for astrocytes following neurotrauma may have important implications for the design of directed therapeutics to limit neurological injury. As such, a primary focus of this review is to summarize the emerging evidence suggesting reactive astrocytes influence the response of the brain to TBI.

Critical Role of NADPH Oxidase in Neuronal Oxidative Damage and Microglia Activation following Traumatic Brain Injury

Background Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS) following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O2 −), and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. Methodology/Principal Findings The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24–96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O2 − induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimers disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. Conclusions/Significance As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

Curcumin attenuates cerebral edema following traumatic brain injury in mice: a possible role for aquaporin-4?

J. Neurochem. (2010) 113, 637–648.

HIGH MOBILITY GROUP BOX PROTEIN-1 PROMOTES CEREBRAL EDEMA AFTER TRAUMATIC BRAIN INJURY VIA ACTIVATION OF TOLL-LIKE RECEPTOR 4

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Cerebral edema, a life‐threatening medical complication, contributes to elevated intracranial pressure (ICP) and a poor clinical prognosis after TBI. Unfortunately, treatment options to reduce post‐traumatic edema remain suboptimal, due in part, to a dearth of viable therapeutic targets. Herein, we tested the hypothesis that cerebral innate immune responses contribute to edema development after TBI. Our results demonstrate that high‐mobility group box protein 1 (HMGB1) was released from necrotic neurons via a NR2B‐mediated mechanism. HMGB1 was clinically associated with elevated ICP in patients and functionally promoted cerebral edema after TBI in mice. The detrimental effects of HMGB1 were mediated, at least in part, via activation of microglial toll‐like receptor 4 (TLR4) and the subsequent expression of the astrocytic water channel, aquaporin‐4 (AQP4). Genetic or pharmacological (VGX‐1027) TLR4 inhibition attenuated the neuroinflammatory response and limited post‐traumatic edema with a delayed, clinically implementable therapeutic window. Human and rodent tissue culture studies further defined the cellular mechanisms demonstrating neuronal HMGB1 initiates the microglial release of interleukin‐6 (IL‐6) in a TLR4 dependent mechanism. In turn, microglial IL‐6 increased the astrocytic expression of AQP4. Taken together, these data implicate microglia as key mediators of post‐traumatic brain edema and suggest HMGB1‐TLR4 signaling promotes neurovascular dysfunction after TBI. GLIA 2013;62:26–38

Hemin-induced necroptosis involves glutathione depletion in mouse astrocytes

Intracerebral hemorrhage (ICH) is a devastating neurological injury associated with significant mortality. Astrocytic inflammation may contribute to the pathogenesis of ICH, although the underlying cellular mechanisms remain unclear. In this study, the hemoglobin oxidation by-product, hemin, concentration dependently induced necroptotic cell death in cortical astrocytes within 5 h of treatment. Hemin-induced cell death was preceded by increased inflammatory gene expression (COX-2, IL-1beta, TNF-alpha, iNOS). Inhibition of the NF-kappaB transcription factor reversed inflammatory gene expression and attenuated cell death after hemin treatment, suggesting a possible role for inflammatory mediators in astrocytic injury. Superoxide production paralleled the increase in iNOS expression, and inhibition of either iNOS (aminoguanidine or iminopiperdine) or superoxide (apocynin) significantly reduced cell death. Similarly, reduced formation of peroxynitrite, the damaging product of nitric oxide and superoxide, significantly reduced hemin injury. Hemin-induced peroxidative injury was associated with a rapid depletion of intracellular glutathione (GSH), culminating in lipid peroxidation and cell death, effects that were reduced by cotreatment with exogenous GSH, N-acetyl-L-cysteine, or the glutathione peroxidase mimetic ebselen. Together, these studies suggest a novel role for GSH depletion in necroptotic astrocyte injury after a hemorrhagic injury and indicate that therapeutic targeting of GSH may exert a beneficial effect after ICH.

Curcumin Attenuates Vascular Inflammation and Cerebral Vasospasm After Subarachnoid Hemorrhage in Mice

Cerebral vasospasm is a major cause of death and disability after subarachnoid hemorrhage (SAH); however, clinical therapies to limit the development of cerebral vasospasm are lacking. Although the causative factors underlying the development of cerebral vasospasm are poorly understood, oxidative stress contributes to disease progression. In the present study, curcumin (150 or 300 mg/kg) protected against the development of cerebral vasospasm and limited secondary cerebral infarction after SAH in mice. The protective effect of curcumin was associated with a significant attenuation of inflammatory gene expression and lipid peroxidation within the cerebral cortex and the middle cerebral artery. Despite the ability of curcumin to limit the development of cerebral vasospasm and secondary infarction, behavioral outcome was not improved, indicating a dissociation between cerebral vasospasm and neurologic outcome. Together, these data indicate a novel role for curcumin as a possible adjunct therapy after SAH, both to prevent the development of cerebral vasospasm and to reduce oxidative brain injury after secondary infarction.

Delayed reduction in hippocampal postsynaptic density protein-95 expression temporally correlates with cognitive dysfunction following controlled cortical impact in mice

OBJECT Traumatic brain injury (TBI) induces significant neurological damage, including deficits in learning and memory, which contribute to a poor clinical prognosis. Treatment options to limit cognitive decline and promote neurological recovery are lacking, in part due to a poor understanding of the secondary or delayed processes that contribute to brain injury. In the present study, the authors characterized the temporal and spatial changes in the expression of postsynaptic density protein-95 (PSD-95), a key scaffolding protein implicated in excitatory synaptic signaling, after controlled cortical impacts in mice. Neurological injury, as assessed by the open-field activity test and the novel object recognition test, was compared with changes in PSD-95 expression. METHODS Adult male CD-1 mice were subjected to controlled cortical impacts to simulate moderate TBI in humans. The spatial and temporal expression of PSD-95 was analyzed in the cerebral cortex and hippocampus at various time points following injury and sham operations. Neurological assessments were performed to compare changes in PSD-95 with cognitive deficits. RESULTS A significant decrease in PSD-95 expression was observed in the ipsilateral hippocampus beginning on Day 7 postinjury. The loss of PSD-95 corresponded with a concomitant reduction in immunoreactivity for NeuN (neuronal nuclei), a neuron-specific marker. Aside from the contused cortex, a significant loss of PSD-95 immunoreactivity was not observed in the cerebral cortex. The delayed loss of hippocampal PSD-95 directly correlated with the onset of behavioral deficits, suggesting a possible causative role for PSD-95 in behavioral abnormalities following head trauma. CONCLUSIONS A delayed loss of hippocampal synapses was observed following head trauma in mice. These data may suggest a cellular mechanism to explain the delayed learning and memory deficits in humans after TBI and provide a potential framework for further testing to implicate PSD-95 as a clinically relevant therapeutic target.

Repeated-bout exercise in the heat in young athletes: physiological strain and perceptual responses

A short recovery period between same-day competitions is common practice in organized youth sports. We hypothesized that young athletes will experience an increase in physiological strain and perceptual discomfort during a second identical exercise bout in the heat, with 1 h (21 degrees C) between bouts, even with ample hydration. Twenty-four athletes (6 boys and 6 girls: 12-13 yr old, 47.7 +/- 8.3 kg; 6 boys and 6 girls: 16-17 yr old, 61.0 +/- 8.6 kg) completed two 80-min intermittent exercise bouts (treadmill 60%, cycle 40% peak oxygen uptake) in the heat (33 degrees C, 48.9 +/- 6.1% relative humidity). Sweat loss during each bout was similar within each age group (12-13 yr old: bout 1, 943.6 +/- 237.1 ml; bout 2, 955.5 +/- 250.3 ml; 16-17 yr old: bout 1, 1,382.2 +/- 480.7 ml; bout 2, 1,373.1 +/- 472.2 ml). Area under the curve (AUC) was not statistically different (P > 0.05) between bouts for core body temperature (12-13 yr old: bout 1 peak, 38.6 +/- 0.4 degrees C; bout 2, 38.4 +/- 0.2 degrees C; 16-17 yr old: bout 1 peak, 38.8 +/- 0.7 degrees C; bout 2, 38.7 +/- 0.6 degrees C), physiological strain index (12-13 yr old: bout 1 peak, 7.9 +/- 0.9; bout 2, 7.5 +/- 0.7; 16-17 yr old: bout 1 peak, 8.1 +/- 1.5; bout 2, 7.9 +/- 1.4), or thermal sensation for any age/sex subgroup or for all subjects combined. However, rating of perceived exertion AUC and peak were higher (P = 0.0090 and 0.0004, respectively) during bout 2 in the older age group. Notably, four subjects experienced consistently higher responses throughout bout 2. With these healthy, fit, young athletes, 1 h of complete rest, cool down, and rehydration following 80 min of strenuous exercise in the heat was generally effective in eliminating any apparent carryover effects that would have resulted in greater thermal and cardiovascular strain during a subsequent identical exercise bout.

INHIBITION OF NFκB REDUCES CELLULAR VIABILITY IN GH3 PITUITARY ADENOMA CELLS

OBJECTIVE Adenomas of the pituitary gland are among the most common types of tumors of the adult brain. Although adenomas are histologically benign, they may be associated with significant morbidity and mortality, mostly because of their invasive growth pattern and hormone hypersecretion. Current medical therapies are suppressive, acting at a receptor level. Thus, there is a need to identify novel cellular and molecular targets for pituitary tumors. We investigated the possible role of the NFkappaB transcription factor in pituitary tumor cell growth. METHODS The effect of NFkappaB pathway inhibition on cellular viability was studied in the GH3 pituitary adenoma cell line, a well-characterized rat cell line that secretes growth hormone and prolactin. Cells were treated with mechanistically diverse pharmacological NFkappaB pathway inhibitors or with molecular inhibitors that were overexpressed in tumor cells before the assessment of cellular viability. NFkappaB activity was also assessed in GH3 cells using deoxyribonucleic acid binding assays. RESULTS GH3 cells exhibited constitutive NFkappaB activity, which contributed to increased cellular proliferation. Treatment with wedelolactone, an IkappaB kinase inhibitor, or overexpression of an IkappaB super-repressor reduced cell viability, further implicating NFkappaB in pituitary tumor cell growth. Pharmacological or molecular inhibition of Akt similarly reduced GH3 viability and NFkappaB binding, suggesting that constitutive activation of NFkappaB may be, at least in part, mediated by Akt. CONCLUSION Directed targeting of the Akt and NFkappaB signaling pathways may be a useful adjunct in the clinical management of pituitary tumors. Further elucidation of this pathway may yield novel information regarding the behavior of pituitary tumors in humans.

Elucidating novel mechanisms of brain injury following subarachnoid hemorrhage: an emerging role for neuroproteomics

Subarachnoid hemorrhage (SAH) is a devastating neurological injury associated with significant patient morbidity and death. Since the first demonstration of cerebral vasospasm nearly 60 years ago, the preponderance of research has focused on strategies to limit arterial narrowing and delayed cerebral ischemia following SAH. However, recent clinical and preclinical data indicate a functional dissociation between cerebral vasospasm and neurological outcome, signaling the need for a paradigm shift in the study of brain injury following SAH. Early brain injury may contribute to poor outcome and early death following SAH. However, elucidation of the complex cellular mechanisms underlying early brain injury remains a major challenge. The advent of modern neuroproteomics has rapidly advanced scientific discovery by allowing proteome-wide screening in an objective, nonbiased manner, providing novel mechanisms of brain physiology and injury. In the context of neurosurgery, proteomic analysis of patient-derived CSF will permit the identification of biomarkers and/or novel drug targets that may not be intuitively linked with any particular disease. In the present report, the authors discuss the utility of neuroproteomics with a focus on the roles for this technology in understanding SAH. The authors also provide data from our laboratory that identifies high-mobility group box protein-1 as a potential biomarker of neurological outcome following SAH in humans.