Coleen M. Atkins

Protection in animal models of brain and spinal cord injury with mild to moderate hypothermia

For the past 20 years, various laboratories throughout the world have shown that mild to moderate levels of hypothermia lead to neuroprotection and improved functional outcome in various models of brain and spinal cord injury (SCI). Although the potential neuroprotective effects of profound hypothermia during and following central nervous system (CNS) injury have long been recognized, more recent studies have described clinically feasible strategies for protecting the brain and spinal cord using hypothermia following a variety of CNS insults. In some cases, only a one or two degree decrease in brain or core temperature can be effective in protecting the CNS from injury. Alternatively, raising brain temperature only a couple of degrees above normothermia levels worsens outcome in a variety of injury models. Based on these data, resurgence has occurred in the potential use of therapeutic hypothermia in experimental and clinical settings. The study of therapeutic hypothermia is now an international area of investigation with scientists and clinicians from every part of the world contributing to this important, promising therapeutic intervention. This paper reviews the experimental data obtained in animal models of brain and SCI demonstrating the benefits of mild to moderate hypothermia. These studies have provided critical data for the translation of this therapy to the clinical arena. The mechanisms underlying the beneficial effects of mild hypothermia are also summarized.

Modulation of the cAMP signaling pathway after traumatic brain injury

Traumatic brain injury (TBI) results in both focal and diffuse brain pathologies that are exacerbated by the inflammatory response and progress from hours to days after the initial injury. Using a clinically relevant model of TBI, the parasagittal fluid-percussion brain injury (FPI) model, we found injury-induced impairments in the cyclic AMP (cAMP) signaling pathway. Levels of cAMP were depressed in the ipsilateral parietal cortex and hippocampus, as well as activation of its downstream target, protein kinase A, from 15 min to 48 h after moderate FPI. To determine if preventing hydrolysis of cAMP by administration of a phosphodiesterase (PDE) IV inhibitor would improve outcome after TBI, we treated animals intraperitoneally with rolipram (0.3 or 3.0 mg/kg) 30 min prior to TBI, and then once per day for 3 days. Rolipram treatment restored cAMP to sham levels and significantly reduced cortical contusion volume and improved neuronal cell survival in the parietal cortex and CA3 region of the hippocampus. Traumatic axonal injury, characterized by beta-amyloid precursor protein deposits in the external capsule, was also significantly reduced in rolipram-treated animals. Furthermore, levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), were significantly decreased with rolipram treatment. These results demonstrate that the cAMP-PKA signaling cascade is downregulated after TBI, and that treatment with a PDE IV inhibitor improves histopathological outcome and decreases inflammation after TBI.

Bidirectional Regulation of Cytoplasmic Polyadenylation Element-Binding Protein Phosphorylation by Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Phosphatase 1 during Hippocampal Long-Term Potentiation

Induction of hippocampal long-term potentiation (LTP) requires activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), whereas maintenance of LTP additionally requires protein synthesis. We recently reported that CaMKII stimulates protein synthesis in depolarized hippocampal neurons through phosphorylation of the mRNA translation factor cytoplasmic polyadenylation element-binding protein (CPEB), and this phosphorylation is rapidly reversed by protein phosphatase 1 (PP1). Protein synthesis-dependent late-phase LTP (L-LTP) in the hippocampus requires calcium influx through the NMDA-type glutamate receptor (NMDA-R) to activate CaMKII as well as concomitant inhibition of PP1 mediated by protein kinase A. Therefore, we investigated the regulation of CPEB phosphorylation during L-LTP. Pharmacological stimulation of the NMDA-R in hippocampal slices to produce chemical long-term depression induced a brief dephosphorylation of CPEB. Modest LTP induction (once at 100 Hz), which induces a protein synthesis-independent early-phase LTP (E-LTP), resulted in a transient phosphorylation of CPEB. However, stronger stimulation (four times at 100 Hz), known to induce protein synthesis-dependent L-LTP, elicited a prolonged phosphorylation of CPEB. Furthermore, CPEB phosphorylation correlated with phosphorylation of PP1 inhibitor dopamine- and cAMP-regulated phosphoprotein, a known substrate for protein kinase A. These results evoke the hypothesis that bidirectional regulation of CPEB phosphorylation by CaMKII and protein phosphatases may serve as a mechanism to convert E-LTP into protein synthesis-dependent L-LTP by stimulating protein synthesis and thereby stabilizing synaptic enhancement.

Alterations in Mammalian Target of Rapamycin Signaling Pathways after Traumatic Brain Injury

In response to traumatic brain injury (TBI), neurons initiate neuroplastic processes through the activation of intracellular signaling pathways. However, the molecular mechanisms underlying neuroplasticity after TBI are poorly understood. To study this, we utilized the fluid-percussion brain injury (FPI) model to investigate alterations in the mammalian target of rapamycin (mTOR) signaling pathways in response to TBI. Mammalian target of rapamycin stimulates mRNA translation through phosphorylation of eukaryotic initiation factor 4E binding protein-1 (4E-BP1), p70 ribosomal S6 kinase (p70S6K), and ribosomal protein S6 (rpS6). These pathways coordinate cell growth and neuroplasticity via dendritic protein synthesis. Rats received sham surgery or moderate parasagittal FPI on the right side of the parietal cortex, followed by 15 mins, 30 mins, 4 h, 24 h, or 72 h of recovery. Using Western blot analysis, we found that mTOR, p70S6K, rpS6, and 4E-BP1 phosphorylation levels were significantly increased in the ipsilateral parietal cortex and hippocampus from 30 mins to 24 h after TBI, whereas total protein levels were unchanged. Using confocal microscopy to localize these changes, we found that rpS6 phosphorylation was increased in the parietal cortex and all subregions of the hippocampus. In accordance with these results, eIF4E, a key, rate-limiting mRNA translation factor, was also phosphorylated by mitogen-activated protein kinase-interacting kinase 1 (Mnk1) 15 mins after TBI. Together, these results suggest that changes in mRNA translation may be one mechanism that neurons use to respond to trauma and may contribute to the neuroplastic changes observed after TBI.

Post-traumatic seizure susceptibility is attenuated by hypothermia therapy

Traumatic brain injury (TBI) is a major risk factor for the subsequent development of epilepsy. Currently, chronic seizures after brain injury are often poorly controlled by available antiepileptic drugs. Hypothermia treatment, a modest reduction in brain temperature, reduces inflammation, activates pro‐survival signaling pathways, and improves cognitive outcome after TBI. Given the well‐known effect of therapeutic hypothermia to ameliorate pathological changes in the brain after TBI, we hypothesized that hypothermia therapy may attenuate the development of post‐traumatic epilepsy and some of the pathomechanisms that underlie seizure formation. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid‐percussion brain injury, and were then maintained at normothermic or moderate hypothermic temperatures for 4 h. At 12 weeks after recovery, seizure susceptibility was assessed by challenging the animals with pentylenetetrazole, a GABAA receptor antagonist. Pentylenetetrazole elicited a significant increase in seizure frequency in TBI normothermic animals as compared with sham surgery animals and this was significantly reduced in TBI hypothermic animals. Early hypothermia treatment did not rescue chronic dentate hilar neuronal loss nor did it improve loss of doublecortin‐labeled cells in the dentate gyrus post‐seizures. However, mossy fiber sprouting was significantly attenuated by hypothermia therapy. These findings demonstrate that reductions in seizure susceptibility after TBI are improved with post‐traumatic hypothermia and provide a new therapeutic avenue for the treatment of post‐traumatic epilepsy.

Activation of calcium/calmodulin-dependent protein kinases after traumatic brain injury

A prominent cognitive impairment after traumatic brain injury (TBI) is hippocampal-dependent memory loss. Although the histopathologic changes in the brain are well documented after TBI, the underlying biochemical mechanisms that contribute to memory loss have yet to be thoroughly delineated. Thus, we determined if calcium/calmodulin-dependent protein kinases (CaMKs), known to be necessary for the formation of hippocampal-dependent memories, are regulated after TBI. Sprague—Dawley rats underwent moderate parasagittal fluid-percussion brain injury on the right side of the parietal cortex. The ipsilateral hippocampus and parietal cortex were Western blotted for phosphorylated, activated α-calcium/calmodulin-dependent protein kinase II (α-CaMKII), CaMKIV, and CaMKI. α-Calcium/calmodulin-dependent protein kinase II was activated in membrane subcellular fractions from the hippocampus and parietal cortex 30 mins after TBI. CaMKI and CaMKIV were activated in a more delayed manner, increasing in phosphorylation 1 h after TBI. The increase in activated α-CaMKII in membrane fractions was accompanied by a decrease in cytosolic total α-CaMKII, suggesting redistribution to the membrane. Using confocal microscopy, we observed that α-CaMKII was activated within hippocampal neurons of the dentate gyrus, CA3, and CA1 regions. Two downstream substrates of α-CaMKII, the AMPA-type glutamate receptor GluR1, and cytoplasmic polyadenylation element-binding protein, concomitantly increased in phosphorylation in the hippocampus and cortex 1 h after TBI. These results demonstrate that several of the biochemical cascades that subserve memory formation are activated unselectively in neurons after TBI. As memory formation requires activation of CaMKII signaling pathways at specific neuronal synapses, unselective activation of CaMKII signaling in all synapses may disrupt the machinery for memory formation, resulting in memory loss after TBI.

Hypothermia treatment potentiates ERK1/2 activation after traumatic brain injury

Traumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal‐dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid‐percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal‐regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element‐binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen‐activated protein kinase‐interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI.

STAT3 signaling after traumatic brain injury

J. Neurochem. (2012) 120, 710–720.

Phosphodiesterase Inhibition Rescues Chronic Cognitive Deficits Induced by Traumatic Brain Injury

Traumatic brain injury (TBI) modulates several cell signaling pathways in the hippocampus critical for memory formation. Previous studies have found that the cAMP-protein kinase A signaling pathway is downregulated after TBI and that treatment with a phosphodiesterase (PDE) 4 inhibitor rolipram rescues the decrease in cAMP. In the present study, we examined the effect of rolipram on TBI-induced cognitive impairments. At 2 weeks after moderate fluid-percussion brain injury or sham surgery, adult male Sprague Dawley rats received vehicle or rolipram (0.03 mg/kg) 30 min before water maze acquisition or cue and contextual fear conditioning. TBI animals treated with rolipram showed a significant improvement in water maze acquisition and retention of both cue and contextual fear conditioning compared with vehicle-treated TBI animals. Cue and contextual fear conditioning significantly increased phosphorylated CREB levels in the hippocampus of sham animals, but not in TBI animals. This deficit in CREB activation during learning was rescued in TBI animals treated with rolipram. Hippocampal long-term potentiation was reduced in TBI animals, and this was also rescued with rolipram treatment. These results indicate that the PDE4 inhibitor rolipram rescues cognitive impairments after TBI, and this may be mediated through increased CREB activation during learning.

Proinflammatory cytokine regulation of cyclic AMP-phosphodiesterase 4 signaling in microglia in vitro and following CNS injury

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β on cyclic AMP‐phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF‐α or IL‐1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL‐1β but remained suppressed with TNF‐α through 24 h. Cyclic AMP was also reduced in TNF‐α‐stimulated primary microglia, albeit to a lesser extent. Accompanying TNF‐α‐induced cyclic AMP reductions, but not IL‐1β, was increased cyclic AMP‐PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF‐α; IL‐1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF‐α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF‐α but not IL‐1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX‐42+ microglia; PDE4B was absent in OX‐42+ cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up‐regulation from 24 h to 1 wk post‐SCI, the peak of microglia activation. These studies show that TNF‐α and IL‐1β differentially affect cyclic AMP‐PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases.