Traumatic and non-traumatic brain injury results from severe disruptions in the cellular microenvironment leading to massive loss of neuronal populations and increased neuroinflammation. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity and free radical release contribute to neural tissue damage. NLRX1 is a member of the NLR family of pattern recognition receptors and is a potent negative regulator of several pathways that significantly modulate many of these events. Thus, we hypothesized that NLRX1 limits immune system signaling in the brain following trauma. To evaluate this hypothesis, we utilized Nlrx1−/− mice in a controlled cortical impact (CCI) injury murine model of traumatic brain injury (TBI). Here, we show that the Nlrx1−/− mice exhibited significantly larger brain lesions and increased motor deficits following CCI injury. Mechanistically, our data indicate that the NF-κB signaling cascade is significantly up-regulated in the Nlrx1−/− animals. This up-regulation is associated with increased microglia and macrophage populations in the cortical lesion. Utilizing a mouse neuroblastoma cell line (N2A), we also found that NLRX1 significantly reduced apoptosis under hypoxic conditions. In human patients, we identify 15 NLRs that are significantly dysregulated, including significant downregulation of NLRX1 in brain injury following aneurysm. We further demonstrate a concurrent increase in NF-κB signaling that is correlated with aneurysm severity in these human subjects. Together, our data extend the function of NLRX1 beyond its currently characterized role in host-pathogen defense and identify this highly novel NLR as a significant modulator of brain injury progression.

Traumatic brain injury (TBI) elicits the immediate production of proinflammatory cytokines which participate in regulating the immune response. While the mechanisms of adaptive immunity in secondary injury are well characterized, the role of the innate response is unclear. Recently, the NLR inflammasome has been shown to become activated following TBI, causing processing and release of interleukin-1β (IL-1β). The inflammasome is a multiprotein complex consisting of nucleotide-binding domain and leucine-rich repeat containing proteins (NLR), caspase-1, and apoptosis-associated speck-like protein (ASC). ASC is upregulated after TBI and is critical in coupling the proteins during complex formation resulting in IL-1β cleavage. To directly test whether inflammasome activation contributes to acute TBI-induced damage, we assessed IL-1β, IL-18, and IL-6 expression, contusion volume, hippocampal cell death, and motor behavior recovery in Nlrp1 −/−, Asc −/−, and wild type mice after moderate controlled cortical impact (CCI) injury. Although IL-1β expression is significantly attenuated in the cortex of Nlrp1 −/− and Asc −/− mice following CCI injury, no difference in motor recovery, cell death, or contusion volume is observed compared to wild type. These findings indicate that inflammasome activation does not significantly contribute to acute neural injury in the murine model of moderate CCI injury.

Key pointsr Intermittent reductions in respiratory neural activity, a characteristic of many ventilatory disorders, leads to inadequate ventilation and arterial hypoxia. Both intermittent reductions in respiratory neural activity and intermittent hypoxia trigger compensatory enhancements in inspiratory output when experienced separately, forms of plasticity called inactivity-induced inspiratory motor facilitation (iMF) and long-term facilitation (LTF), respectively.r Reductions in respiratory neural activity that lead to moderate, but not mild, arterial hypoxia occludes plasticity expression, indicating that concurrent induction of iMF and LTF impairs plasticity through cross-talk inhibition of their respective signalling pathways.r Moderate hypoxia undermines iMF by enhancing NR2B-containing NMDA receptor signalling, which can be rescued by exogenous retinoic acid, a molecule necessary for iMF.r These data suggest that in ventilatory disorders characterized by reduced inspiratory motor output, such as sleep apnoea, endogenous mechanisms of compensatory plasticity may be impaired, and that exogenously activating respiratory plasticity may be a novel strategy to improve breathing.Abstract Many forms of sleep apnoea are characterized by recurrent reductions in respiratory neural activity, which leads to inadequate ventilation and arterial hypoxia. Both recurrent reductions in respiratory neural activity and hypoxia activate mechanisms of compensatory plasticity that augment inspiratory output and lower the threshold for apnoea, inactivity-induced inspiratory motor facilitation (iMF) and long-term facilitation (LTF), respectively. However, despite frequent concurrence of reduced respiratory neural activity and hypoxia, mechanisms that induce and regulate iMF and LTF have only been studied separately. Here, we demonstrate that recurrent reductions in respiratory neural activity ('neural apnoea') accompanied by cessations in ventilation that result in moderate (but not mild) hypoxaemia do not elicit increased inspiratory output, suggesting that concurrent induction of iMF and LTF occludes plasticity. A key role for NMDA receptor activation in impairing plasticity following concurrent neural apnoea and hypoxia is indicated since recurrent hypoxic neural apnoeas triggered increased phrenic inspiratory Daryl Fields is currently a neurosurgery resident at the University of Pittsburgh. He completed his medical degree and research doctorate at the University of Wisconsin Madison under the mentorship of Dr Tracy Baker, PhD. Daryl's research is focused on creating readily translatable therapies for movement control disorders: (1) development of animal models to better understand deficits in movement control and (2) development of pharmacological therapies for improved motor rehabilitation. He has contributed to the development of several novel research directions that continue to progress the field of motor control/rehabilitation. 3952 D. P. Fields and others J Physiol 597.15 output in rats in which spinal NR2B-containing ...

Sleep apnea (SA) during pregnancy is detrimental to the health of the pregnancy and neonate, but little is known regarding long-lasting consequences of maternal SA during pregnancy on adult offspring. SA is characterized by repeated cessations in breathing during sleep, resulting in intermittent hypoxia (IH). We show that gestational IH (GIH) in rats reprograms the male fetal neuroimmune system toward enhanced inflammation in a region- and sex-specific manner, which persists into adulthood. Male GIH offspring also had deficits in the neural control of breathing, specifically in the ability to mount compensatory responses to central apnea, an effect that was rescued by a localized anti-inflammatory or microglial depletion. Female GIH offspring appeared unaffected. These results indicate that SA during pregnancy sex- and region-dependently skews offspring microglia toward a pro-inflammatory phenotype, which leads to long-lasting deficits in the capacity to elicit important forms of respiratory neuroplasticity in response to breathing instability. These studies contribute to the growing body of recent evidence indicating that SA during pregnancy may lead to sex-specific neurological deficits in offspring that persist into adulthood.

Mechanical trauma to the CNS results in the disruption of the cellular microenvironment leading to massive necrotic and apoptotic loss of neuronal and glia populations. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity and free radial release contribute to neural tissue damage. Members of the NLR family of pattern recognition receptors are essential mediators of the host immune response. Recently, our lab and others identified a novel sub-group of NLRs that function as negative regulators of inflammation. One of the members of this sub-group, NLRX1, is a potent regulator of interferon, NF-κB, ROS production and autophagy. Thus, we hypothesized that NLRX1 attenuates Traumatic Brain Injury (TBI) through the negative regulation of overzealous innate immune system signaling. To evaluate this hypothesis, we utilized Nlrx1−/− mice in a controlled cortical impact (CCI) injury model. The Nlrx1−/− mice exhibited significantly larger brain lesions and increased motor deficits following CCI injury. We also observed significant proliferation of microglia within the Nlrx1−/− lesions compared to wild type animals. Mechanistically, our data indicates that NLRX1 attenuates TBI progression through the microglia compartment via negative regulation of NF-κB signaling and IL-6 production. Together, our data extends the function of NLRX1 beyond its currently characterized role in host-pathogen defense and identifies this highly novel NLR as a significant modulator of TBI progression.

Sleep disordered breathing (SDB) during pregnancy is increasing in parallel with the obesity epidemic, yet the long‐term effect on the adult offspring are unknown. In our animal model of SDB during pregnancy, pregnant rats are exposed to chronic intermittent hypoxia (21/10.5% O2, 15 episodes/hr, 8 hrs/day) or intermittent normoxia from gestational days 10‐21. Preliminary data show that adult male (but not female) offspring exposed to gestational intermittent hypoxia (GIH) show deficits in respiratory control that manifest as increased spontaneous central apneas during presumptive sleep and impaired compensatory responses to recurrent reductions in respiratory neural activity, a form of plasticity known as inactivity‐induced inspiratory motor facilitation (iMF). Further, our data show that microglia isolated from male GIH offspring show primed inflammatory responses in respiratory control regions, and that microglial depletion or inhibition of inflammation in the region of the phrenic motor pool rescues deficits in respiratory control. Our preliminary data show that male GIH offspring suffer from gut dysbiosis. Most notably, they exhibit decreased levels of bacteria that produce butyrate, a short‐chain fatty acid (SCFA) necessary for the proper development of microglia. Because butyrate supplementation reduces microglial inflammation, we tested the hypothesis that adult butyrate supplementation restores the capacity to elicit iMF in adult male GIH offspring. Vehicle or tributyrin was delivered by oral gavage in 8 doses over 22 days (2g/kg). Phrenic inspiratory output was measured in urethane‐anesthetized, vagotomized, mechanically ventilated GIH offspring. As expected, recurrent neural apnea (5, ~1 min neural apnea episodes, separated by 5 minutes) elicited an increase in phrenic inspiratory output in GIH offspring treated with tributyrin compared to vehicle treated GIH offspring (78±30% and 15±2%, respectively, n = 3‐4, p = 0.057), indicating that butyrate supplementation rescues the capacity to elicit iMF in GIH offspring. These data indicate that gut dysbiosis may contribute to impaired capacity for recurrent reductions in respiratory neural activity to trigger compensatory forms of neuroplasticity.

Mammalian hibernation initiates dramatic changes in physiology that include large reductions in ventilation (V̇E), heart rate, oxygen consumption rate (V̇o2), and body temperature (Tb) upon entrance into torpor, effects that reverse upon arousal. Several physiologic processes must maintain function throughout a wide range of temperatures. Recent evidence indicates that neuronal synapses loosen and retract in regions of the brain that are silenced during a torpor bout, although little is understood yet about how these connections are regulated during hibernation. Microglia, brain resident immune cells, are potential facilitators of these essential neural homeostatic activities due to their critical roles in neuronal support and synaptic strengthening and pruning. Thus, we hypothesized that microglia are required for maintaining normal ventilatory neural control during hibernation. To test this, we treated 13‐lined ground squirrels with either vehicle or colony‐stimulating factor receptor‐1 (CSF1R) antagonist, PLX3397 (80mg/kg, po), a receptor whose activation is necessary for microglial survival in the adult nervous system, and measured V̇E, V̇o2, and Tb throughout a torpor bout. During entrance, PLX‐treated squirrels took ~1.5 hours longer to reach their minimum torpor temperature (Tb; 12°C) compared to control animals (Tb; 9.3°C) (ambient temperature; 5‐6°C). Similarly, arousal time in microglia‐depleted animals was as much as 1 hour longer relative to controls, despite starting at a higher minimum Tb. In both treatment groups, V̇o2 during entrance fell and spiked periodically. However, in PLX‐treated animals, these spikes were much greater and more frequent. Minimum V̇o2 in torpor was ~3 times greater in PLX‐treated animals compared to control animals. Upon arousal from torpor, V̇o2 remained elevated, longer, in PLX‐treated squirrels. Total V̇E tended to be elevated in PLX‐treated animals through entrance, steady state torpor, and arousal, but it was most striking during steady state torpor, where V̇E in PLX‐treated animals was 15‐fold higher than control animals. The net result in torpor was an elevated air convection requirement, thus microglia‐depleted animals hyperventilated throughout the torpor bout. Together, these results suggest that microglia play an important regulatory role in normal ventilatory and temperature control throughout torpor and influence the rate at which animals enter into and arouse from torpor. Mechanisms whereby microglia contribute to hibernation neurophysiology are currently under investigation.

Sleep disordered breathing (SDB) during pregnancy is increasing in parallel with the obesity epidemic, yet the long‐term effects on the adult offspring are unknown. In our animal model of SDB during pregnancy, pregnant rats are exposed to chronic intermittent hypoxia (8 hrs/day, 2 min 10.5% O2 separated by 2 min of 21% O2) or intermittent normoxia from gestational days 10–21. Preliminary data show that adult male (but not female) offspring exposed to gestational intermittent hypoxia (GIH) show deficits in respiratory control that manifest as increased spontaneous central apneas during presumptive sleep and impaired compensatory responses to recurrent reductions in respiratory neural activity, a form of plasticity known as inactivity‐induced inspiratory motor facilitation (iMF). Preliminary data also show that microglia isolated from male GIH offspring show primed inflammatory responses in respiratory control regions. Since neuroinflammation impairs respiratory plasticity, we tested the hypothesis that inhibiting NF‐kappaB and STAT3 inflammatory transcription factor activation restores the capacity to elicit iMF in GIH offspring. Phrenic inspiratory output was measured in urethane‐anesthetized, vagotomized, mechanically ventilated GIH or gestational intermittent normoxia (GNX) offspring. Vehicle or 2‐[(aminocarbonyl)amino]‐5 ‐(4‐fluorophenyl)‐3‐ thiophenecarboxamide (TPCA‐1) was delivered intrathecally over the phrenic motor pool ~1 hour before exposure to recurrent reductions in neural activity (5, ~1 min neural apnea episodes, separated by 5 min). As expected, recurrent neural apnea elicited a significant increase in phrenic inspiratory output in vehicle‐ and TPCA‐treated GNX offspring (54±17% and 62±12% baseline, n=6, 5, respectively, p<0.05) when compared to time controls not receiving activity deprivation (11±4% baseline, n=4). By contrast, recurrent neural apnea did not elicit iMF in vehicle‐treated GIH offspring (25±3%, n=7, p>0.05). However, in GIH offspring receiving intrathecal TPCA‐1, recurrent neural apnea induced a significant increase in phrenic inspiratory output (75+/−10% baseline, n=7, p<0.05), indicating that NF‐kappaB and STAT3 inflammatory transcription factor inhibition rescues the capacity to elicit iMF in GIH offspring. These data indicate that increased neuroinflammation in male GIH offspring impairs the capacity for recurrent reductions in respiratory neural activity to trigger compensatory forms of neuroplasticity. Support or Funding Information Supported by R01HL142752 (JJW and TLB)