James E Orfila

Increasing small conductance Ca2+‐activated potassium channel activity reverses ischemia‐induced impairment of long‐term potentiation

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca2+‐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.

Sirtuin-2 mediates male specific neuronal injury following experimental cardiac arrest through activation of TRPM2 ion channels.

OBJECTIVE Sirtuins (Sirt) are a class of deacetylase enzymes that play an important role in cell proliferation. Sirt2 activation produces O-acetylated-ADPribose (OAADPr) which can act as a ligand for transient receptor potential cation channel, M2 (TRPM2). We tested the hypothesis that Sirt2 is activated following global cerebral ischemia and contributes to neuronal injury through activation of TRPM2. METHODS Adult male and female mice (8-12 weeks old) C57Bl/6 and TRPM2 knock-out mice were subjected to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). The Sirt2 inhibitor AGK-2 was administered intravenously 30 min after resuscitation. Hippocampal CA1 injury was analyzed at 3 days after CA/CPR. Acute Sirt2 activity was analyzed at 3 and 24 h after CA/CPR. Long-term hippocampal function was assessed using slice electrophysiology 7 days after CA/CPR. RESULTS AGK-2 significantly reduced CA1 injury in WT but not TRPM2 knock-out males and had no effect on CA1 injury in females. Elevated Sirt2 activity was observed in hippocampal tissue from males at 24 h after cardiac arrest and was reduced by AGK-2. In contrast, Sirt2 activity in females was increased at 3 but not 24 h. Finally, we observed long-term benefit of AGK-2 on hippocampal function, with a protection of long-term potentiation at CA1 synapses at 7 and 30 days after ischemia. CONCLUSIONS In summary, we observed a male specific activation of Sirt2 that contributes to neuronal injury and functional deficits after ischemia specifically in males. These results are consistent with a role of Sirt2 in activating TRPM2 following global ischemia in a sex specific manner. These results support the growing body of literature showing that oxidative stress mechanisms predominate in males and converge on TRPM2 activation as a mediator of cell death.

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

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

Therapeutic hypothermia protects against ischemia-induced impairment of synaptic plasticity following juvenile cardiac arrest in sex-dependent manner

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 3days after resuscitation using hematoxylin and eosin (H&E) staining. Field excitatory post-synaptic potentials (fEPSPs) were recorded from acute hippocampal slices 7days after CA/CPR to determine LTP. Synaptic function was impaired 7days 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.

The role of T‐type calcium channels in the subiculum: to burst or not to burst?

Pharmacological, molecular and genetic data indicate a prominent role of low‐voltage‐activated T‐type calcium channels (T‐channels) in the firing activity of both pyramidal and inhibitory interneurons in the subiculum. Pharmacological inhibition of T‐channels switched burst firing with lower depolarizing stimuli to regular spiking, and fully abolished hyperpolarization‐induced burst firing. Our molecular studies showed that CaV3.1 is the most abundantly expressed isoform of T‐channels in the rat subiculum. Consistent with this finding, both regular‐spiking and burst firing patterns were profoundly depressed in the mouse with global deletion of CaV3.1 isoform of T‐channels. Selective inhibition of T‐channels and global deletion of CaV3.1 channels completely suppressed development of long‐term potentiation (LTP) in the CA1–subiculum, but not in the CA3–CA1 pathway.

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

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

529: SEX DIFFERENCES AFTER CONTROLLED CORTICAL IMPACT IN MICE

Crit Care Med 2015 • Volume 43 • Number 12 (Suppl.) agents or continuous analgesic IV infusion, neurodegenerative disease, quadriplegic patients. Data were collected between April and December in 2014. Patients were evaluated with transcranial Doppler sonography at 3 moments: at rest (M1), M2 during the pain pressure treshold (PPT), and 15 min after PPT (M3). We performed linear generalized model to evaluate differences between blood flow on middle cerebral artery in critical ill underwent pain stimulus Results: Sixty six paired assessment were performed, in 22 non-communicative critical. Mean age was 58.36 (SD ± 19,3) yr, mean APACHE score 24.05 (SD ±9,23), mean body mass index 30.8 kg/m2 (SD ±8); 59.1% were man, 90.9% were medical patient, the most frequent reason for admission at ICU was sepsis (59.1%), 59 % required mechanical ventilation and 95.5% had comorbidity, the most frequent was hypertension (68.2%) followed by diabetes mellitus (36.4%). The mean of blood flow on middle cerebral artery at rest was 46.88 cm/s (SD ± 15.73), 55.36 cm/s (SD ± 14.66) during PPT, and 47.59 cm/s (SD ± 15.16) 15 min after PPT. Generalized linear regression model showed that the flow in the moment M1 and M3 were different from M2 (p<0.001); M1 and M3 had no differ. Conclusions: This work shows that pain stimulus increased the cerebral blood flow. The increase in blood flow on middle cerebral artery reflects the hyperactivity of the neuromatrix of nociception in non-communicative critical ill.