Alterations in Blood-Brain Barrier Integrity and Lateral Ventricle Differ in Rats Exposed to Space Radiation and Social Isolation.

The proposed Mars missions will expose astronauts to long durations of social isolation (SI) and space radiation (SR). These stressors have been shown to alter the brain's macrostructure and microenvironment, including the blood-brain barrier (BBB). Breakdown of the BBB is linked to impaired executive functions and physical deficits, including sensorimotor and neurocognitive impairments. However, the precise mechanisms mediating these effects remain unknown. Additionally, the synergistic effects of combined exposure to SI and SR on the structural integrity of the BBB and brain remain unknown. We assessed the BBB integrity and morphology in the brains of male rats exposed to ground-based analogs of SI and SR. The rats exposed to SR had enlarged lateral ventricles and increased BBB damage associated with a loss of astrocytes and an increased number of leaky vessels. Many deficits observed in SR-treated animals were attenuated by dual exposure to SI (DFS). SI alone did not show BBB damage but did show differences in astrocyte morphology compared to the Controls. Thus, determining how single and combined inflight stressors modulate CNS structural integrity is crucial to fully understand the multiple pathways that could impact astronaut performance and health, including the alterations to the CNS structures and cell viability observed in this study.

Evidence for a role of the basolateral amygdala in regulating regional metabolism in the stressed brain.

The brain regulates every physiological process in the body, including metabolism. Studies investigating brain metabolism have shown that stress can alter major metabolic processes, and that these processes can vary between regions. However, no study has investigated how metabolic pathways may be altered by stressor perception, or whether stress-responsive brain regions can also regulate metabolism. The basolateral amygdala (BLA), a region important for stress and fear, has reciprocal connections to regions responsible for metabolic regulation. In this study, we investigated how BLA influences regional metabolic profiles within the hippocampus (HPC) and medial prefrontal cortex (mPFC), regions involved in regulating the stress response and stress perception, using optogenetics in male C57BL/6 mice during footshock presentation in a yoked shuttlebox paradigm based on controllable (ES) and uncontrollable (IS) stress. RNA extracted from HPC and mPFC were loaded into NanoString® Mouse Neuroinflammation Panels, which also provides a broad view of metabolic processes, for compilation of gene expression profiles. Results showed differential regulation of carbohydrate and lipid metabolism, and insulin signaling gene expression pathways in HPC and mPFC following ES and IS, and that these differences were altered in response to optogenetic excitation or inhibition of the BLA. These findings demonstrate for the first time that individual brain regions can utilize metabolites in a way that are unique to their needs and function in response to a stressor, and that vary based on stressor controllability and influence by BLA.

Effects of social isolation and galactic cosmic radiation on fine motor skills and behavioral performance.

Future NASA missions will require astronauts to travel farther and spend longer durations in space than ever before. This will also expose astronauts to longer periods of several physical and psychological challenges, including exposure to space radiation (SR) and periods of social isolation (SI), which could have unknown negative effects on physical and mental health. Each also has the potential to negatively impact sleep which can reduce the ability to cope with stressful experiences and lead to sensorimotor, neurocognitive, and physical deficits. The effects of SI and SR on gross motor performance has been shown to vary, and depend on, individual differences in stress resilience and vulnerability based on our established animal model in which stress produces different effects on sleep. In this study, the impact that SI and SR, either alone or together, had on fine motor skill performance (bilateral tactile adhesive removal task (BTAR)) was assessed in male rats. We also examined emotional, exploratory, and other off-task behavioral responses during testing and assessed whether sensorimotor performance and emotion varied with individual differences in resilience and vulnerability. BTAR task performance was differentially impacted by SI and SR, and were further influenced by the stress resilience/vulnerability phenotype of the rats. These findings further demonstrate that identifying individual responses to stressors that can impact sensorimotor ability and behavior necessary to perform mission-related tasks will be of particular importance for astronauts and future missions. Should similar effects occur in humans, there may be considerable inter-individual variability in the impact that inflight stressors have on astronauts and their ability to perform mission-related tasks.

Femoral Fixation Strength as a Function of Bone Plug Length in Anterior Cruciate Ligament Reconstruction Utilizing Interference Screws.

PURPOSE: To determine femoral construct fixation strength as bone plug length decreases in anterior cruciate ligament reconstruction (ACLR). METHODS: Sixty fresh-frozen bone-patellar tendon-bone allografts were utilized and divided into 20-, 15-, and 10-mm length bone plug groups, subdivided further so that half utilized the patella side (P) for testing and half used the tibial side (T). Ten mm diameter recipient tunnels were created within the anatomic anterior cruciate ligament footprint of 60 cadaveric femurs. All bone plugs were 10 mm in diameter; grafts were fixed using a 7 × 23 mm metal interference screw. An Instron was used to determine the load to failure of each group. A one-way multivariate analysis of variance (MANOVA) was conducted to test the hypothesis that there would be one or more mean differences in fixation stability between 20- or 15-mm plug lengths (P or T) versus 10 mm T plug lengths when cross-compared, with no association between other P or T subgroups. RESULTS: The mean load to failure of the 20 mm plugs (20 P + T) was 457 ± 66N, 15 mm plugs (15 P + T) was 437 ± 74N, and 10 mm plugs (10 P + T) was 407 ± 107N. There was no significant difference between P + T groups: 20-versus 15-mm (p = 1.000), 15-versus 10-mm (p = 0.798), and 20-versus 10-mm (p = 0.200); P + T MANOVA (p = 0.291). Within groups, there was no significant difference between patella and tibial bone plug subgroups with a pullout force range between 469 ± 56N and 374 ± 116N and p-value ranging from p = 1.000 for longer bone plugs to p = 0.194 for shorter bone plugs; P versus T MANOVA (p = 0.113). CONCLUSION: In this human time zero cadaver model, there was no significant difference in construct failure between 20-,15-, and 10-mm bone plugs when fixed with an interference screw within the femoral tunnel, although fixation strength did trend down when from 20- to 15- to 10-mm bone plugs. CLINICAL RELEVANCE: There is a balance between optimal bone plug length on the femoral side for achieving adequate fixation as well as minimizing donor site morbidity and facilitating graft passage in ACLR. This study reveals utilizing shorter plugs with interference screw fixation is potentially acceptable on the femoral side if shorter plugs are harvested.

Controllable Stress Increases Rapid Eye Movement Sleep in Rats: Regulation by the Central Nucleus of the Amygdala.

BACKGROUND: Training with inescapable shock (IS; uncontrollable stressor) is followed by significant decreases in rapid eye movement sleep (REM). However, controllability is important in the effects of stress. We examined the effects of escapable shock (ES; controllable stressor) on sleep and whether the central nucleus of the amygdala (CNA) plays a role in regulating these effects. METHODS: Six Wistar rats implanted with a cannula located in CNA underwent two days of ES training (20 shock presentations; 0.5 mA; 5.0 s maximum duration; 1.0 min interstimulus interval). Five days later, they were re-exposed to the shock context. RESULTS: Following shock training, REM was significantly increased in both light and dark periods. Non-REM (NREM) and total sleep (TS) duration were decreased during the light period. Similar effects on REM and NREM were observed following re-exposure to the training context alone. Microinjections of saline into CNA immediately following ES also produced similar increases in REM, whereas microinjections of muscimol (MUS; GABAA (γ-aminobutyric acid) antagonist) subsequent to ES blocked the increases in REM. CONCLUSIONS: These data, along with previous work with ES and IS, demonstrate that stressor controllability is important in determining how stress impacts sleep. Moreover, the results of the microinjection study indicate that the effects of ES on REM are regulated through the CNA.

Stressor control and regional inflammatory responses in the brain: regulation by the basolateral amygdala.

Increasing evidence has connected the development of certain neuropsychiatric disorders, as well as neurodegenerative diseases, to stress-induced dysregulation of the immune system. We have shown that escapable (ES) and inescapable (IS) footshock stress, and memories associated with ES or IS, can differentially alter inflammatory-related gene expression in brain in a region dependent manner. We have also demonstrated that the basolateral amygdala (BLA) regulates stress- and fear memory-induced alterations in sleep, and that differential sleep and immune responses in the brain to ES and IS appear to be integrated during fear conditioning and then reproduced by fear memory recall. In this study, we investigated the role of BLA in influencing regional inflammatory responses within the hippocampus (HPC) and medial prefrontal cortex (mPFC) by optogenetically stimulating or inhibiting BLA in male C57BL/6 mice during footshock stress in our yoked shuttlebox paradigm based on ES and IS. Then, mice were immediately euthanized and RNA extracted from brain regions of interest and loaded into NanoString® Mouse Neuroinflammation Panels for compilation of gene expression profiles. Results showed differential regional effects in gene expression and activated pathways involved in inflammatory-related signaling following ES and IS, and these differences were altered depending on amygdalar excitation or inhibition. These findings demonstrate that the stress-induced immune response, or "parainflammation", is affected by stressor controllability and that BLA influences regional parainflammation to ES or IS in HPC and mPFC. The study illustrates how stress-induced parainflammation can be regulated at the neurocircuit level and suggests that this approach can be useful for uncovering circuit and immune interactions in mediating differential stress outcomes.

Sleep and Core Body Temperature Alterations Induced by Space Radiation in Rats.

Sleep problems in astronauts can arise from mission demands and stress and can impact both their health and ability to accomplish mission objectives. In addition to mission-related physical and psychological stressors, the long durations of the proposed Mars missions will expose astronauts to space radiation (SR), which has a significant impact on the brain and may also alter sleep and physiological functions. Therefore, in this study, we assessed sleep, EEG spectra, activity, and core body temperature (CBT) in rats exposed to SR and compared them to age-matched nonirradiated rats. Male outbred Wistar rats (8-9 months old at the time of the study) received SR (15 cGy GCRsim, n = 15) or served as age- and time-matched controls (CTRL, n = 15) without irradiation. At least 90 days after SR and 3 weeks prior to recording, all rats were implanted with telemetry transmitters for recording EEG, activity, and CBT. Sleep, EEG spectra (delta, 0.5-4 Hz; theta, 4-8 Hz; alpha, 8-12 Hz; sigma, 12-16 Hz; beta, 16-24 Hz), activity, and CBT were examined during light and dark periods and during waking and sleeping states. When compared to the CTRLs, SR produced significant reductions in the amounts of dark period total sleep time, total nonrapid eye movement sleep (NREM), and total rapid eye movement sleep (REM), with significant decreases in light and dark period NREM deltas and dark period REM thetas as well as increases in alpha and sigma in NREM and REM during either light or dark periods. The SR animals showed modest increases in some measures of activity. CBT was significantly reduced during waking and sleeping in the light period. These data demonstrate that SR alone can produce alterations to sleep and temperature control that could have consequences for astronauts and their ability to meet mission demands.

Differential Impact of Social Isolation and Space Radiation on Behavior and Motor Learning in Rats.

Future missions to Mars will expose astronauts to several physical and psychological challenges, including exposure to space radiation (SR) and periods of social isolation (SI). Each of these stressors, in addition to mission demands, can affect physical and mental health and potentially negatively impact sleep. The effects of inflight stressors may vary with duration and time course, may be additive or compounding, and may vary with individual differences in stress resilience and vulnerability. Determining how individual differences in resilient and vulnerable phenotypes respond to these mission-related stressors and their interactions with sleep will be crucial for understanding and mitigating factors that can impair performance and damage health. Here, we examined the single and compound effects of ground-based analogs of SI and SR on sensorimotor performance on the balance beam (BB) in rats. We also assessed emotional responses during testing on the BB and assessed whether sensorimotor performance and emotion varied with individual differences in stress resiliency using our established animal model in which stress produces different effects on sleep. Results showed differential motor performance and emotion in the BB task between SI and SR, and these varied based on resilient and vulnerable phenotypes. These findings demonstrate that identifying individual responses to stressors that can impact sensorimotor ability and behavior necessary to perform mission-related tasks will be of particular importance for astronauts and future missions. Should similar effects occur in humans, there may be considerable inter-individual variability in the impact that flight stressors have on the mental health of astronauts and their ability to perform mission-related tasks.

Modeling integrated stress, sleep, fear and neuroimmune responses: Relevance for understanding trauma and stress-related disorders.

Sleep and stress have complex interactions that are implicated in both physical diseases and psychiatric disorders. These interactions can be modulated by learning and memory, and involve additional interactions with the neuroimmune system. In this paper, we propose that stressful challenges induce integrated responses across multiple systems that can vary depending on situational variables in which the initial stress was experienced, and with the ability of the individual to cope with stress- and fear-inducing challenges. Differences in coping may involve differences in resilience and vulnerability and/or whether the stressful context allows adaptive learning and responses. We provide data demonstrating both common (corticosterone, SIH and fear behaviors) and distinguishing (sleep and neuroimmune) responses that are associated with an individual's ability to respond and relative resilience and vulnerability. We discuss neurocircuitry regulating integrated stress, sleep, neuroimmune and fear responses, and show that responses can be modulated at the neural level. Finally, we discuss factors that need to be considered in models of integrated stress responses and their relevance for understanding stress-related disorders in humans.

Increased medical student understanding of dementia through virtual embodiment.

Given the growing prevalence of Alzheimer's disease (AD), we assessed the impact of virtually embodying someone with progressive AD. This pilot explored students' understanding of individuals' needs with dementia, as well as, the efficacy of virtual reality (VR) as a curricular tool. Second-year medical students (n = 150) completed a pre-survey, Embodied Labs, Inc. Beatriz Lab VR module, and a post-survey. Most students knew someone with dementia (72%), were a family member of someone with dementia (52%) or had worked with a patient (61%) with dementia. Using paired survey questions, students reported significant increases in understanding how their lives would be affected by dementia (71% vs. 94%) and the needs of a person with dementia (64% vs. 95%) after VR. They reported increased understanding of being a caregiver of someone with dementia (24% vs. 81%) and the impact it can have on the entire family (64% vs. 97%). Overall students agreed this simulation made them think about their approach to clinical skills (94%) and should be utilized more in the curriculum (76%). This pilot study indicated that this VR experience can be used to advance understanding of a person's experiences with dementia and that integrating VR into the medical curricula should be considered.

Controllable and Uncontrollable Stress Differentially Impact Fear Conditioned Alterations in Sleep and Neuroimmune Signaling in Mice.

Stress induces neuroinflammation and disrupts sleep, which together can promote a number of stress-related disorders. Fear memories associated with stress can resurface and reproduce symptoms. Our previous studies have demonstrated sleep outcomes can be modified by stressor controllability following stress and fear memory recall. However, it is unknown how stressor controllability alters neuroinflammatory signaling and its association with sleep following fear memory recall. Mice were implanted with telemetry transmitters and experienced escapable or inescapable footshock and then were re-exposed to the shuttlebox context one week later. Gene expression was assessed with Nanostring® panels using RNA extracted from the basolateral amygdala and hippocampus. Freezing and temperature were examined as behavioral measures of fear. Increased sleep after escapable stress was associated with a down-regulation in neuro-inflammatory and neuro-degenerative related genes, while decreased sleep after inescapable stress was associated with an up-regulation in these genes. Behavioral measures of fear were virtually identical. Sleep and neuroimmune responses appear to be integrated during fear conditioning and reproduced by fear memory recall. The established roles of disrupted sleep and neuroinflammation in stress-related disorders indicate that these differences may serve as informative indices of how fear memory can lead to psychopathology.

Sleep Disturbance Alters Cocaine-Induced Locomotor Activity: Involvement of Striatal Neuroimmune and Dopamine Signaling.

Sleep disorders have high comorbidity with drug addiction and function as major risk factors for developing drug addiction. Recent studies have indicated that both sleep disturbance (SD) and abused drugs could activate microglia, and that increased neuroinflammation plays a critical role in the pathogenesis of both diseases. Whether microglia are involved in the contribution of chronic SDs to drug addiction has never been explored. In this study, we employed a mouse model of sleep fragmentation (SF) with cocaine treatment and examined their locomotor activities, as well as neuroinflammation levels and dopamine signaling in the striatum, to assess their interaction. We also included mice with, or without, SF that underwent cocaine withdrawal and challenge. Our results showed that SF significantly blunted cocaine-induced locomotor stimulation while having marginal effects on locomotor activity of mice with saline injections. Meanwhile, SF modulated the effects of cocaine on neuroimmune signaling in the striatum and in ex vivo isolated microglia. We did not observe differences in dopamine signaling in the striatum among treatment groups. In mice exposed to cocaine and later withdrawal, SF reduced locomotor sensitivity and also modulated neuroimmune and dopamine signaling in the striatum. Taken together, our results suggested that SF was capable of blunting cocaine-induced psychoactive effects through modulating neuroimmune and dopamine signaling. We hypothesize that SF could affect neuroimmune and dopamine signaling in the brain reward circuitry, which might mediate the linkage between sleep disorders and drug addiction.

Regulation of dark period sleep by the Amygdala: A microinjection and optogenetics study.

The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the influence of CNA inputs into reticularis pontis oralis (RPO), pedunculopontine tegmentum (PPT) and nucleus subcoeruleus (SubC) on dark period sleep. We compared these results to effects of microinjections into CNA of the GABAA agonist, muscimol (MUS, inhibition of cell bodies) and tetrodotoxin (TTX, inhibition of cell bodies and fibers of passage). For optogenetics, male Wistar rats received excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) opsins into CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre into RPO, PPT, or SubC. This enabled only CNA neurons synaptically connected to each region to express opsin. Optic cannulae for light delivery into CNA and electrodes for determining sleep were implanted. Sleep was recorded with and without blue or amber light stimulation of CNA. Separate rats received MUS or TTX into CNA prior to recording sleep. Optogenetic activation of CNA neurons projecting to RPO enhanced REM and did not alter non-REM (NREM) whereas activation of CNA neurons projecting to PPT or SubC did not significantly affect sleep. Inhibition of CNA neurons projecting to any region did not significantly alter sleep. TTX inactivation of CNA decreased REM and increased NREM whereas muscimol inactivation did not significantly alter sleep. Thus, the amygdala can regulate decreases and increases in REM, and RPO is important for CNA promotion of REM. Fibers passing through CNA, likely from the basolateral nucleus of the amygdala, also play a role in regulating sleep.

Sleep-Disturbance-Induced Microglial Activation Involves CRH-Mediated Galectin 3 and Autophagy Dysregulation.

Chronic sleep disturbances (CSDs) including insomnia, insufficient sleep time, and poor sleep quality are major public health concerns around the world, especially in developed countries. CSDs are major health risk factors linked to multiple neurodegenerative and neuropsychological diseases. It has been suggested that CSDs could activate microglia (Mg) leading to increased neuroinflammation levels, which ultimately lead to neuronal dysfunction. However, the detailed mechanisms underlying CSD-mediated microglial activation remain mostly unexplored. In this study, we used mice with three-weeks of sleep fragmentation (SF) to explore the underlying pathways responsible for Mg activation. Our results revealed that SF activates Mg in the hippocampus (HP) but not in the striatum and prefrontal cortex (PFc). SF increased the levels of corticotropin-releasing hormone (CRH) in the HP. In vitro mechanism studies revealed that CRH activation of Mg involves galectin 3 (Gal3) upregulation and autophagy dysregulation. CRH could disrupt lysosome membrane integrity resulting in lysosomal cathepsins leakage. CRHR2 blockage mitigated CRH-mediated effects on microglia in vitro. SF mice also show increased Gal3 levels and autophagy dysregulation in the HP compared to controls. Taken together, our results show that SF-mediated hippocampal Mg activation involves CRH mediated galectin 3 and autophagy dysregulation. These findings suggest that targeting the hippocampal CRH system might be a novel therapeutic approach to ameliorate CSD-mediated neuroinflammation and neurodegenerative diseases.

Differential Effect of Light and Dark Period Sleep Fragmentation on Composition of Gut Microbiome and Inflammation in Mice.

Bi-directional interactions amongst the gut microbiota, immune system, and brain function are thought to be critical mediators of health and disease. The role sleep plays in mediating these interactions is not known. We assessed the effects of sleep fragmentation (SF) on the microbiota-gut-brain axis. Male C57BL/6NCrl mice (4 to 5 per cage, fed standard lab chow) experienced SF via mechanical stimulation at 2 min intervals during the light (SF) and dark (DD, dark disturbances) periods. Home cage (HC) controls were undisturbed. After 10 days, fecal samples were collected at light onset, midday, light offset, and midnight. Samples were also collected after 10 days without SF. Subsequently, the mice were randomized across groups and allowed 20 additional days of recovery followed by 10 days of SF or DD. To assess effects on the microbiota, 16S rRNA sequencing was used, and mesenteric lymph nodes (MLNs) and cortex and medial prefrontal cortex were analyzed using cytokine arrays. SF and DD produced significant alterations in the microbiota compared to HC, and DD had greater impact than SF on some organisms. SF produced marked suppression in MLNs of chemokines that regulate inflammation (CCL3, CCL4 and their receptor CCR5) and maintain the immune mucosal barrier (Cxcl13) at the same time that cortical cytokines (IL-33) indicated neuroinflammation. DD effects on immune responses were similar to HC. These data suggest that SF alters the microbiome and suppresses mucosal immunity at the same time that mediators of brain inflammation are upregulated. The translational implications for potential application to clinical care are compelling.

Educational Case: Neisseria Meningitis.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, seehttp://journals.sagepub.com/doi/10.1177/2374289517715040.1.