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    Molecular modeling and binding function of the RGS12 PDZ domain variant associated with familial bipolar disorder
    (2024-03-21) Agogo-Mawuli, Percy; Bosch, Dustin; Oestreich, Emily; Siderovski, David
    Purpose – Bipolar disorder (BD) is a highly heritable neuropsychiatric disease characterized by recurrent episodes of depression and mania. BD is considered one of the most vexing health disorders to medicate appropriately, yet affects ~2% of U.S. adults and is the most costly mental health condition for American health insurers nationwide [1]. In reporting the whole-exome sequencing of 81 individuals from 27 multiply-affected BD families, Forstner et al. [2] discovered a single nucleotide polymorphism co-segregating with BD in the gene encoding “Regulator of G protein Signaling” (RGS) type 12, predicted to cause a missense change (arginine-59 to glutamine) in the sequence of the encoded protein’s PDZ domain. RGS proteins are negative regulators of neurotransmitter signaling via G protein-coupled receptors (GPCRs) [3], specifically serving to accelerate signaling shutoff by increasing the GTP hydrolysis rate of GPCR-associated G-alpha subunits [4]. In prior mouse genetics studies, the Siderovski lab has demonstrated that RGS12 acts to regulate kappa opioid receptor signaling and extracellular dopamine levels in the basal ganglia of the brain, and that RGS12 is able to bind to various protein targets in neurons, such as the kinase MEK2 via its PDZ domain. To address whether the R59Q amino-acid variation in the RGS12 PDZ domain associated with BD affects the function of the RGS12 protein, molecular modeling and biochemical binding experiments were performed. Methods – Schrodinger’s software suite of molecular modeling and dynamics tools was employed to create structural models of the R59Q variant of RGS12’s PDZ domain, starting from the published NMR-derived structure of the domain with wildtype sequence (i.e., Protein Data Bank record id 2KV8). Desmond molecular dynamics software was used to relax both structural models and then assess their overall stability over 50 nanoseconds of elapsed simulation. Models of the wildtype and R59Q variant PDZ domain bound to short polypeptides from the C-termini of candidate RGS12-interacting proteins were similarly created and molecular dynamics simulations performed. Both PDZ domains were expressed as glutathione-S-transferase (GST) fusion proteins by E. coli cultures, purified by fast protein liquid chromatography (FPLC), and then tested for their binding to biotinylated polypeptides from the C-termini of candidate RGS12-interacting proteins using surface plasmon resonance (SPR). Results and Conclusion – The position of the missense change within RGS12 was not predicted to directly engage the polypeptide binding-site of the PDZ domain; however, when tested using SPR, the fusion protein bearing the R59Q-substituted PDZ domain exhibited less binding affinity for target polypeptide partners than the wildtype sequence. These initial in silico and in vitro findings suggest that the R59Q variant associated with BD may lead to reduced RGS12 protein function in vivo. References: [1] PMID: 35816713; DOI: 10.7326/AITC202207190 [2] PMID: 32066727; PMCID: PMC7026119; DOI: 10.1038/s41398-020-0732-y [3] PMID: 21737532; PMCID: PMC3141876; DOI: 10.1124/pr.110.003038 [4] PMID: 20351284; PMCID: PMC2872438; DOI: 10.1073/pnas.0912934107
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    Early psychosis structural abnormalities in the midbrain correlate with positive and negative symptoms
    (2024-03-21) Zhou, Zicong; Jones, Kylie; Ivleva, Elena; Colon-Perez, Luis
    Early psychosis (EP) is a critical period in developing psychotic disorders. During the EP period, timely intervention strategies can effectively mitigate symptoms of psychotic disorders. A primary target in current therapeutics for psychotic disorders is the dopaminergic system; however, current therapeutics are often inadequate in treating the advanced stages of psychotic disorders. Currently, there are hypotheses that the advent of psychotic disorders are alterations in the brain’s structure and functional connectivity leading to aberrant network organization. Given the prominence of the midbrain in synthesizing dopamine and being a hub in the circuitry of dopaminergic function in this study, we will quantify the structural properties of the midbrain using a publicly available dataset of EP subjects. The Human Connectome Project (HCP) is a global effort to determine human brain connectivity objectively. Specifically, there is a subset where the focus is on subjects within five years of their first psychotic episode, which is the focus of this study. In this project, we employ various computational tools (including FSL, ANTs, packages of Python and RStudio, etc.) to acquire relevant measures of brain structure in EP. Specifically, we performed gross anatomical analysis of brain volumetrics, regional microstructural analysis, and correlation analysis between brain and behavior indices. First, Deformation-based Morphometry (DBM) is a test performed on T1 MRI scans to determine brain volumes. DBM measures shape movements to align individuals with a registration template using the Jacobian determinant (JD), whereas a standard VBM focuses on voxel intensity differences between individuals and the template. In our results, DBM revealed mild changes around the midbrain between EP and healthy controls. Second, we analyzed Diffusion Tensor Imaging (DTI) data by obtaining indices such as Fractional Anisotropy (FA), Axial Diffusivity (AD), and Mean Diffusivity (MD). A midbrain mask was created based on Freesurfer atlas ROI labels, allowing a seed-based analysis centered around the midbrain. Group differences were estimated using the Welch two-group t-tests on ROI means of JD, FA, AD, MD, and midbrain volumes. Interestingly, group differences in JD and midbrain volumes were insignificant, but differences were more pronounced for FA, AD, and MD. Third, we employed Tract-Based Spatial Statistics (TBSS) to determine microstructural changes in white matter tracts. TBSS successfully captured structural variabilities within the midbrain, aligning with our study’s expectations, in addition to moderate changes in other main white matter tracts, such as corticospinal tract and cingulum, suggesting an initiation of altered brain connectivity emanating from alterations in the midbrain or a putative reorganization of dopaminergic circuitry. Fourth, correlations between these quantities in the EP group and behavioral scores (i.e., PANSS and CAINS tests) were explored. It is found that midbrain volume noticeably correlates with the Cognitive score of PANSS and JD, strongly correlates with AD and MD; FA correlates with the Negative score of PANSS, and MD correlates with the Positive score of PANSS. Overall, these findings contribute to understanding midbrain involvement in early psychosis and underscore the interest for further investigation in this research path.
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    Unilateral to Bilateral Progressive Sciatic Neuropathy After Radiotherapy: A Case Report
    (2024-03-21) Mathew, Ezek; Drown, Mariah; Abarquez, Angela; Shivnani, Anand; Ortega, Sterling; Dickerman, Rob
    Background: Radiation therapy is often an adjunct treatment for prostate cancer. However, this procedure is not without risks; as the lumbosacral plexus is not routinely contoured during radiotherapy treatment plans, this raises potential for unintended consequences. As this case, especially this particular presentation, is an extremely rare occurrence, we will examine relevant literature and discuss the challenging diagnosis. Case Presentation: In this report, we detail the case of a 66-year-old male patient who suffered from unilateral sciatic neuropathy. Unfortunately, this unilateral neuropathy became bilateral, and was deemed idiopathic at the time, causing the patient severe distress. However, further workup which consisted of examination of patient history, scrutinizing imaging, and electromyography (EMG), painted a different picture. The onset of the patient’s complaints appeared to be initiated by adaptive radiotherapy, which the patient underwent during his treatment regimen for prostate cancer. Conclusions: As radiation-induced lumbosacral plexopathy (RILSP) may present in a delayed fashion after treatment, diagnosis could become difficult. While radiculopathy was the differential diagnosis which initially led to neurosurgical consultation, the patient’s presentation did not align with this diagnosis. Further workup, especially strategic usage of EMG, allowed for discernment of a neuropathic condition, versus a mechanically induced radiculopathy. While RILSP appears to be an underreported phenomenon subsequent to pelvic radiation, there exists only one other case of such neuropathy after prostate radiotherapy. Knowledge of this case will enable clinicians to modify their workup and avoid spine surgery in cases where it may cause harm.
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    Impact of sex and hypoxia on brain region-specific expression of androgen receptor AR45 and G protein Gαq in young adult rats
    (2024-03-21) Wilson, Elizabeth; Bradshaw, Jessica; Mabry, Steve; Shrestha, Pawan; Gardner, Jennifer; Cunningham, Rebecca
    Purpose: Sex differences in oxidative stress-associated cognitive decline are influenced by sex hormone levels. However, little is known regarding the expression of hormone receptors in brain regions associated with cognitive function. Notably, oxidative stress-associated neuronal cell death is exacerbated through testosterone signaling via membrane-associated androgen receptor AR45 and G protein Gαq. The objective of this study was to elucidate the expression of AR45 and Gαq in brain regions associated with cognitive function. Additionally, we investigated whether chronic intermittent hypoxia (CIH), an oxidative stressor with sex-specific effects, would modulate AR45 and Gαq expression. Methods: Adult male and female Sprague-Dawley rats were exposed to CIH or normoxia for 14 days. We quantified AR45 and Gαq protein expression in various cognition-associated brain regions [dorsal hippocampal CA1, CA3, DG, and entorhinal cortex (ETC)] via western blotting. For comparisons, AR45 and Gαq protein expression were also assessed in brain regions outside the hippocampal-ETC circuit [thalamus (TH) and striatum (STR)]. Results: The highest AR45 levels were expressed in the CA1 while the lowest expression was observed in the STR. The highest Gαq levels were expressed in the DG and ETC while the lowest expression was observed in the TH. We observed no effect of sex on AR45 or Gαq expression regardless of brain region assessed. Similarly, there was no effect of CIH on AR45 expression in any of the brain regions examined. However, CIH exposure increased Gαq expression only in the CA3 regardless of sex. Conclusions: Our findings reveal enrichment of AR45 and Gαq protein expression within the hippocampal-ETC circuit, which is vulnerable to oxidative stress and neurodegeneration during cognitive decline. Moreover, our data suggest the CA3 is the most vulnerable region to CIH-mediated oxidative stress. Overall, these findings were observed in both sexes, indicating that there are no observed sex differences in AR45 and Gαq expression or their modulation by CIH.
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    Cocaine Administration but Not Morphine Leads to Increases in Impulsivity as Measured in a Delay Discounting Task
    (2024-03-21) Keitzer, Jeri; Anchondo, Olivia; Brunetti, Kaylee; Shuchi, Samia; Colon-Perez, Luis
    Cocaine Administration but Not Morphine Leads to Increases in Impulsivity as Measured in a Delay Discounting Task Jeri Keitzer, Olivia Anchondo, Kaylee Brunetti, Samia Shuchi, and Luis Colon-Perez Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, 76107 Purpose: Inhibitory and cognitive control are mental process susceptible to repeated exposure to drugs of abuse. Specifically, impulsivity refers to the tendency to act prematurely without regard of the future. Substance use disorders patients (SUD) show impaired impulsivity; however, it is unclear whether psychostimulant use precedes impulsive behavior or vice versa. The delay discounting task is an assessment of impulsive choice used in behavioral neuroscience research. In this task, subjects are given a choice between an immediate small reward or a delayed large reward in which the time of delay increases across trials. Subjects that discount the delayed reward earlier are determined to act with more impulsivity. In this current study, we looked at impulsivity as it relates to performance in a delay discounting task before and after IP injection drug administration of either morphine or cocaine. We hypothesized that the subjects that were administered drugs would devalue the delayed reward earlier within the delay discounting task than control subjects. Methods: For this study, we used Sprague Dawley rats split into 4 different groups: cocaine, morphine, saline, and naïve. All subjects were food restricted to increase motivation to obtain the reward food pellets. Rats began training on the delay discounting task after a training phase in which the rats were introduced to the behavioral chambers and exposed to the levers within the chambers in which they were trained to press to receive pellets. The delay discounting task consisted of a daily session in which the subject went through 5 phases (a total of 60 total trials) choosing between two levers to receive pellets. Each phase is characterized by two forced trials and ten choice trials. In the forced choice trials, the animal has only one option: either the immediate small reward or delayed choice reward. At each phase the delay increases from no delay to 4s, 8s, 16s, and 32s. During choice trials, rats choose between the immediate and delayed levers. Once a decision has been made, both levers retract until the next trial. First, baseline data of each subject’s impulsive choice was acquired, then the subjects underwent IP injection of either cocaine, morphine, or saline. A naïve group did not receive any injections. Rats were injected with drugs according to their weight (5 mg/kg) once per day for seven days, then they completed the delay discounting task again under experimental conditions. Results: The cocaine drug group showed a significant decrease in delayed lever presses compared to all other groups during the delay discounting task following drug administration. No other significant differences were seen across any of the other groups. Conclusion: The results of the study suggest that passive administration of cocaine in rats may lead to an increase in impulsivity but not morphine.
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    Hyperbaric Oxygen as Potential Treatment for Chemotherapy-Related Cognitive Impairments
    (2024-03-21) Trinh, Oanh; Mensah-Kane, Paapa; Shi, Helen; Sumien, Nathalie
    Purpose: “Chemobrain”, characterized by impaired attention, learning and memory retention, is a prevalent condition affecting approximately 75% of patients undergoing cancer treatments. Chemotherapy-related cognitive impairment (CRCI), a subtype of “chemobrain”, can persist for up to 20 years post-treatment, significantly diminishing the daily quality of life for survivors and caretakers. Very few interventions are available to alleviate the effects of chemotherapy on the brain. One potential treatment is hyperbaric oxygen therapy (HBOT), which has shown neuroprotective effects in conditions such as Alzheimer’s disease, traumatic brain injury, and stroke. The current study investigated the effects of HBOT on cognitive impairments induced by commonly used chemotherapeutic agents, and studied the underlying mechanisms with a focus on cellular senescence. Methods: Four-month-old male and female C57BL/6 mice were injected (i.p.) with saline or chemotherapeutic cocktails (Methotrexate (37.5 mg/kg) + 5-Fluorouracil (50 mg/kg)) once a week for three weeks. Simultaneously, half of the mice underwent daily HBOT session (2.4 ATM for 90 min). Morris water maze was used to measure spatial learning and memory. Hippocampus was evaluated for markers of cellular senescence using western blot analyses. Results: Chemotherapy exposure impaired spatial learning and memory, which was attenuated by HBOT in male mice only. The exposure was also associated with increased levels of p16INK4a, ɣH2AX, and b-galactosidase (cellular senescence markers), and increased levels of cleaved-Lamin B1 (a surrogate marker of caspase-6 activity relating to apoptosis) in male hippocampus. HBOT seemed to reduce the effects of chemotherapy on ɣH2AX, b-galactosidase, and cleaved-Lamin B1, but not on p16INK4a in males. Conclusion: HBOT reduced cognitive impairments associated with chemotherapy exposure. Interestingly, females were not impaired by the chosen chemotherapeutic cocktail. Cellular senescence and apoptosis may play a role in the beneficial effects of HBOT.
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    Sex-dependent effects of chronic intermittent hypoxia: Implication for obstructive sleep apnea
    (2024-03-21) Mabry, Steve; Bradshaw, Jessica; Gardner, Jennifer; Wilson, Elizabeth; Cunningham, Rebecca
    Background: Obstructive sleep apnea (OSA) is a highly prevalent sleeping disorder in the USA with known sex differences in prevalence and severity. Men have a higher incidence and experience greater severity of OSA than women. However, recent reports indicate the incidence of OSA in women, particularly mild cases of OSA, may be under-reported and left untreated. OSA is characterized by elevated oxidative stress and inflammation, mechanisms that involve mitochondrial function. This study addressed the role of 1) sex and 2) mitochondrial oxidative stress in OSA induced circulatory oxidative stress and inflammatory cytokines. Methods: Adult Sprague-Dawley male and female rats were implanted (s.c.) with an osmotic pump containing either MitoTEMPOL (mitochondrial oxidative stress inhibitor; MT) or saline vehicle and then exposed to a model of OSA, chronic intermittent hypoxia (CIH), or normoxic room-air for 14 days. The CIH protocol consisted of 10 CIH cycles/hour/8 hrs/day, in which each CIH cycle was composed of 3 minutes of normoxia at 21% O2 and 3 minutes of hypoxia at 10% O2. This protocol replicates an apnea-hypopnea index (AHI) of 10, which is consistent with mild OSA in humans. At the conclusion of the CIH protocol, rats were sacrificed and plasma was collected to quantify markers of oxidative stress (Advanced Oxidized Protein Products, AOPP) and inflammation (pro-inflammatory IL-6, anti-inflammatory IL-10, IL-6/IL-10 ratio). To determine statistical significance, ANOVA followed by Tukey’s post-hoc test was used. Significance level was set a p<0.05. Results: We found circulating oxidative stress was dependent on CIH and sex. Sex differences were observed in control normoxic rats, in which females had higher oxidative stress than males. Interestingly, the impact of CIH on oxidative stress was dependent on sex, wherein CIH decreased oxidative stress in females but increased oxidative stress in males. Inhibiting mitochondria-associated oxidative stress reduced oxidative stress in vehicle females, but only blocked the effect of CIH-induced oxidative stress in males. In contrast to oxidative stress, CIH increased the level of IL-6 only in females. Further, CIH overall induced a pro-inflammatory state as measured by an elevated IL6/IL10 ratio in females. The inflammatory effects of CIH in females were blocked by inhibiting mitochondrial-associated oxidative stress, despite no effect on circulating oxidative stress in CIH. Neither CIH nor MT impacted inflammatory markers in males. Discussion: These results indicate CIH-induced mechanisms underlying oxidative stress and inflammation are dependent on sex. Specifically, males experience a mitochondria-associated oxidative stress phenotype and females experience a mitochondria-associated inflammatory phenotype. These findings indicate that the OSA phenotype is sex-dependent, which may be related to the under-reported OSA incidence in women compared to men. Further, these data indicate that women may be at unique risk from OSA, particularly when AHIs are mild. Interestingly, inhibition of mitochondrial oxidative stress may be a potential drug target for both men and women with OSA.
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    Impact of nicotine's autonomic effect on nicotine's discriminative stimulus
    (2024-03-21) Kusi-Boadum, Nana Kofi; Forster, Michael
    Purpose: As the leading cause of preventable death, tobacco smoking has inspired years of research into the mechanisms of addiction and pharmacological targets for smoking cessation. Nicotine, an active psychostimulant in tobacco, activates the brain's reward system and drives the addiction to smoking via dopaminergic neurons. Although some studies have suggested that the autonomic effects of psychostimulants may independently serve as cues for the release of dopamine in the brain, it has not been demonstrated behaviorally. A previous locomotor activity study in our lab showed that hexamethonium, a brain-impermeable nicotinic receptor antagonist blocks the locomotor stimulant effects of nicotine in mice. To further assess the impact of the autonomic effects of nicotine on behavior, we conducted a nicotine discrimination study. In this study, we subtracted nicotine’s autonomic effects from the overall subjective effect by using hexamethonium. Method: Using a two-lever drug discrimination operant chamber, six male Sprague-Dawley rats were trained to discriminate 0.1 mg/kg nicotine tartrate from saline by lever pressing. Subcutaneous injections of nicotine or saline for training occurred five minutes prior to the start of the training session. On test days, rats received hexamethonium (1, 2.5, 5, 10, 25, or 50 mg/kg) intraperitoneally 25 minutes prior to subcutaneous administration of nicotine at the training dose (0.1 mg/kg). Percentage of drug lever responses and response rate were recorded to measure antagonism and analyzed using repeated measures ANOVA. Results: Within the dose range of 10-50 mg/kg, hexamethonium partially antagonized nicotine’s discriminative stimulus effect by reducing the percentage nicotine-lever response to 42% of nicotine’s maximum discriminative stimulus effect. Conclusion: Although full antagonism was not observed, the partial antagonism of nicotine’s discriminative stimulus by a solely peripherally-acting antagonist shows that the autonomic effects of nicotine are a component of the overall subjective effect of nicotine that influences behavior and can be explored as a target for smoking cessation. More direct assays of reward, such as the self-administration assay will be required to affirm the autonomic contributions to the neurobiology of nicotine addiction.
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    Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain
    (2024-03-21) Ampofo, Hannah; Berry, Raymond; Spann, Claire; Liu, Ran; Yang, Shaohua
    Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain Hannah Ampofo, Raymond Berry, Claire Spann, Ran Liu, Shaohua Yang Pharmacology and Neuroscience Department, School of Biomedical Sciences, University of North Texas Health Science Center, Texas, United States. Purpose-To establish Expansion microscopy (ExM) for super-resolution imaging of the rodent brain. Background- ExM is a remarkable imaging technology that enables nanoscale resolution in three-dimensional (3-D) imaging of preserved cells and tissues. ExM, which was invented in 2015, physically expands specimens using a hydrogel, allowing high-resolution imaging to be done with conventional diffraction-limited microscopes. The basic idea is to attach anchors to biomolecules or labels chemically and link them to a hydrogel that is uniformly distributed throughout the material. This polymerization technique separates biomolecules while maintaining their spatial organization by enabling isotropic expansion. The procedure is similar to sketching an outline on an inflating object and blowing it up: the ink particles will move apart, but their relative organization remains the same. Traditional optical imaging is unable to resolve nanoscale structures with dimensions smaller than 200–300 nm due to the fundamental physical limitations imposed by diffraction. ExM offers faster imaging speeds as compared to super-resolution methods, and enhanced antibody efficiency due to the decrowding effect generated by expanding biomolecules. The original ExM could resolve the specimen at 70 nm, however, new variants such as iterative ExM, 10X ExM microscopy, and nine-fold microscopy can resolve down to 15 to 30 nm, comparable to super-resolution microscopes. Method- The Paper-MAP version of expansion microscopy, a modified MAP method that allows immunostaining and expansion within two days was employed. The procedure involved staining floating mouse brain sections and incubating with a Paper-MAP cocktail (consisting of TEMED and sodium acrylate) and ammonium persulfate solution. The hydrogel matrix was created in situ through the crosslinking of sodium acrylate and bisacrylamide, forming a dense polyelectrolyte hydrogel. A denaturing solution was used to mechanically homogenize the sample and then expanded using deionized water. The pre and post-expanded sections were imaged using a Zeiss LSM 510 confocal microscope. Results- Following the addition of the monomer solution, the expansion procedure produced a 2 fold increase in size. This was followed by an evident 4 to 5 fold increase after the expansion was completed. We compared the pre-expansion image to the post-expansion image and observed intricate and detailed structures with significantly enhanced resolution that were previously indistinguishable in the pre-expansion section using confocal microscope. Conclusion- Expansion microscopy is a versatile and accessible imaging technique that resulted in significant improvements in imaging the microscopic configuration of the mouse brain. Its broad application offers a powerful tool for biological research in diverse organisms.
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    Experimental ischemic stroke induces secondary white matter degeneration and long-term cognitive impairment
    (2024-03-21) Berry, Raymond; Liu, Ran; Winters, Ali; Spann, Clair; Ampofo, Hannah; Colon-Perez, Luis; Sumien, Nathalie; Yang, Shao-Hua
    Clinical investigations have detected extensive white matter degeneration in individuals affected by ischemic stroke. Nonetheless, current stroke research has primarily concentrated on the infarct and periinfarct penumbra regions. The exploration of white matter degeneration's role after ischemic stroke and its contribution to post-stroke cognitive impairment and dementia (PSCID) has been limited in experimental models. Understanding the impact of white matter degeneration on PSCID in these models could offer valuable insights into potential therapeutic targets and interventions for alleviating cognitive decline following ischemic stroke. In this study, we analyzed the progression of locomotor and cognitive function up to 4 months after inducing ischemic stroke by middle cerebral artery occlusion in young adult rats. Despite evident ongoing locomotor recovery, long-term cognitive and affective impairment persisted after ischemic stroke, as indicated by Morris water maze, elevated plus maze, and open field performance. At 4-month after stroke, multimodal MRI was conducted to assess white matter degeneration. T2-weighted MRI (T2WI) unveiled bilateral cerebroventricular enlargement after ischemic stroke. Fluid Attenuated Inversion Recovery MRI (FLAIR) revealed white matter hyperintensities in the corpus callosum and fornix across bilateral hemispheres. A positive association between the volume of white matter hyperintensities and total cerebroventricular volume was noted in stroke rats. Further evidence of bilateral white matter degeneration was indicated by the reduction of fractional anisotropy (FA) and quantitative anisotropy (QA) in diffusion-weighted MRI (DWI) analysis. FA measures water diffusion directionality; reduced FA implies decreased white matter tract coherence. QA, linked to diffusion directionality, indicates microstructural white matter changes with decreased QA. Reduced FA and QA in DWI MRI suggest brain microstructural integrity changes, involving myelin sheath disruption, axonal damage, or overall white matter deterioration. Additionally, microglia and astrocyte activation were identified in the bilateral corpus callosum after stroke. This inflammatory response indicates the involvement of glial cells in the post-stroke environment, suggesting a complex interplay between structural alterations and neuroinflammatory processes that may contribute to the observed changes in white matter integrity. Understanding these multifaceted mechanisms is crucial for developing targeted interventions aimed at promoting recovery and minimizing long-term neurological consequences following ischemic stroke. The importance of these results is underscored by their potential connection to neurological or neurodegenerative conditions, given that white matter degeneration is commonly noted in diverse neurological disorders, including Alzheimer's disease, multiple sclerosis, and other related conditions. Our study suggests that experimental ischemic stroke induced by MCAO in young rats replicates long-term cognitive impairment and pervasive white matter degeneration observed in ischemic stroke patients. This model provides an invaluable tool for unraveling the mechanisms underlying post-stroke secondary white matter degeneration and its contribution to PSCID. Researchers and clinicians use these metrics to understand and monitor the progression of neurological diseases, potentially aiding in early diagnosis and treatment planning. This research may pave the way for a more comprehensive understanding of the mechanisms underlying post-stroke cognitive impairment and dementia, ultimately leading to improved strategies for patient care and rehabilitation.
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    Neuroinflammation and gut diversity effects due to opioid self-administration
    (2024-03-21) Brunetti, Kaylee; Shuchi, Samia; Colon-Perez, Luis
    Purpose: Opioids are a great resource to treat pain in humans, but prolonged use can lead users to physical dependence and opioid use disorders (OUD). The use of opioids increases the risk of developing OUDs in humans via activation ofμ-opioid receptors. OUD is extensively studied for its effects in the central nervous system including the brain; however, Gut-Brain Axis (GBA) research will potentially expand our understanding of addiction and provide a new paradigm for developing new substance use disorder (SUD) therapeutics. GBA in animal models of OUDs can elucidate the complex interactions between the brain and gut that lead to pathological drug seeking and consumption and their relation to GBA components (i.e., bacterial populations, gut peptides, and gut signaling). In this study, we will share the determining temporal hallmarks of gut alterations in rats self-administering morphine for 15 days and relate it to neuroinflammatory features in the brain. One of our hypotheses is that abuse of drugs, such as morphine, starts by inducing inflammation of the brain and gut taxonomic changes similar to those observed in human opioid users. Methods: In this project, we used Sprague Dawley (SD) rats, and both the experimental and control groups were surgically implanted with intravenous (IV) catheters. The control group was exposed to the self-administration box, but only received sucrose pellets instead of morphine to avoid differences in behavior due to instrument learning or exposure to self-administration chambers. The experimental group was trained to self-administer morphine dosed to their body weight of 0.4 mg/kg for 14 days. The experimental timeline was (1) baseline: 7 days after catheter implantation, (2) acute: 24 hours after the second day of self-administration, and (3) chronic: 24 hours after completion of the 12 days of self-administration. Fecal samples were acquired at the three-time points and analyzed with 16s DNA sequencing to determine the relative abundance of microbial species at the time points. After the last day, we collected the brains from all animals and prepared tissue FFPE for immunohistochemistry and spatial transcriptomics analysis. Concurrently we acquired MRI diffusion-weighted scans in a 7.0 T preclinical scanner. Rats were restrained under sedation (isoflurane 5% induction, 2% maintenance) reducing the stressor of noise and restriction while scanning. This project will help us identify whether neuroinflammation markers occur due to large doses of morphine repetitively in rodents. Results: Preliminary analysis points out that chronic and voluntary administration of morphine leads to a neuroinflammatory response in the habenula possibly detected with diffusion MRI. Our preliminary16s DNA sequencing analysis has shown some key microbiome differences in our experimental drug group versus our control pellet group. Conclusion: As we continue analyzing our data, we hope to provide more insight on early effects of chronic drug use on gut-brain axis.
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    Three Pediatric cases of Bow hunter's Syndrome
    (2024-03-21) Falana, Zainab; Schenk, Allyson; Herring, Rachelle
    Background: Bow hunter’s syndrome is a rare condition in which the vertebral artery is compressed during head rotation, potentially leading to recurrent vessel injury, dissection, and stroke. If gone unrecognized and left untreated, this syndrome can result in recurrent strokes. This case series describes three pediatric males diagnosed with bow hunter’s syndrome subsequently treated with cervical spinal fusion. Case information: Three previously healthy male patients (median age: 8) with posterior circulation strokes were treated at a single institution. Demographics, stroke score, and treatments are summarized in Table 1. Two patients experienced recurrent strokes while the third underwent endovascular thrombectomy for an acute basilar artery occlusion. Acute medical treatment otherwise included anticoagulation and neck stabilization with cervical collar due to clinical suspicion of bow hunter's syndrome. The diagnosis was subsequently confirmed via dynamic conventional angiography performed with neck rotation. All three patients received an occiput to C3 vertebra fusion. Follow up imaging performed 3 months post fusion showed no evidence of additional strokes. Conclusions: Bow hunter’s syndrome is a rare cause of posterior circulation strokes amongst children and should be included in the differential diagnosis of likely patients given the high likelihood of stroke recurrence. The diagnostic gold standard is dynamic conventional angiography to confirm vertebral artery compression. Cervical spine stabilization to limit neck rotation and anticoagulation may help reduce the risk of stroke recurrence until neurosurgical intervention can be pursued. Further studies are necessary to follow the long term outcomes of patients treated with cervical spinal fusion and develop a standard of care for bow hunter’s syndrome.
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    Tractography as a method for mapping brain connectivity
    (2024-03-21) Spann, Claire; Yang, Shaohua; Liu, Ran; Berry, Raymond; Ampofo, Hannah; Colon-Perez, Luis
    Purpose. Mapping the brain and its complex connectivity has proved a challenging feat for neuroscience, though with the development of diffusion tensor imaging and tractography, we are one step closer to understanding brain anatomical connections. This method utilizes diffusion-weighted magnetic resonance imaging, which takes advantage of the Brownian motion of water molecules, to produce a diffusion tensor. In the white matter of the brain, diffusion varies in direction due to cellular membranes and myelin, and the diffusion tensor measures this anisotropic diffusivity to indicate possible tissue orientation. The generalized q-sampling imaging tractography method, developed by Frank Yeh in 2010, uses the diffusion tensor to approximate the course of white matter tracts and can be used to determine the exact location and termination of white matter bundles to assess connectivity between and within different brain regions. Despite limitations that decrease the accuracy of white matter tracking, tractography remains the only method to visualize white matter trajectories in vivo and non-invasively. Though commonly used for human diffusion-weighted images, here we verify tractography as a method to visualize and measure white matter trajectories in the rat brain. Methods. A male 3-month Sprague Dawley rat was used to acquire DWI images that were analyzed using DSI Studio. The DWI was superimposed with the corresponding T2W image and regions of interest (ROIs) were drawn in the corpus callosum and were applied via the built-in Waxholm Space rat atlas. Fiber tracking was seeded from the ROI, and fractional anisotropy, quantitative anisotropy, isotropy, mean diffusivity, axial diffusivity, and radial diffusivity was calculated at each ROI by DSI software. Results. Tractography of the corpus callosum was easily visualized using both drawn and atlas-applied ROIs. Fiber tractography from both ROIs included fibers from the internal and external capsules to ensure the integrity of all corpus callosum fibers. Diffusion metrics were not drastically different between the two seeding methods. Conclusion. This study presents tractography as a tool for visualizing white matter tracts and quantifying different diffusion metrics. Using both hand-drawn regions and regions from the rat atlas, white matter tracts in diseased brains can be compared to controls to measure several aspects of pathology, such as edema, axonal integrity, and axonal density. One application includes the imaging and quantification of both acute and chronic stroke, which exhibit different pathologies that can be visualized and measured with diffusion metrics, allowing for more precise targets of therapy. The use of tractography in adjunct with other established methods can improve the understanding of disease and assist in the development of better treatment.
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    In-Vitro Assessment of Cortical Repair Induced by Branched-chain Amino Acid Treatment
    (2024-03-21) Mathew, Ezek; Jones, Nathan; Dickerman, Rob; Ortega, Sterling
    Purpose: Traumatic Brain Injury (TBI) refers to a constellation of pathologies resulting from mechanical damage to cortical tissue. The neurological sequala of such injuries can be devastating, and definitive treatment does not exist at this time. Branched-chain Amino Acid (BCAA) treatment has demonstrated neuroprotective effects in clinical literature and in various animal models of TBI. However, there is a lack of in-vitro literature referencing the repair capacity of BCAA administration after neuronal injury, particularly in the context of TBI. To fill this gap in knowledge, a scratch assay was repurposed for use in cortical culture, to assess the repair capacity of BCAA treatment. Methods: Mouse-derived Mixed Cortical Culture (MCC) cells were extracted and seeded in 24 well plates. A scratch assay was performed, where a vertical scratch was drawn across each well in a reproducible manner with a 200 uL pipette tip. This procedure is meant to recapitulate aspects of mechanical damage induced by TBI on cortical tissue. Subsequently, images were taken immediately post-injury (0 hour time point), at 24 hour, and at 48 hour time points post-scratch to quantify the area of scratch unfilled by cells. Various dose concentrations of BCAA were tested in comparison to control (media only) and vehicle control (water). Test conditions included the customary BCAA ratio, which is a 2:1:1 mix of leucine, isoleucine, and valine; additionally, a 1:1:1 ratio of leucine, isoleucine, and valine was also tested. Results: At 48 hours post-scratch, significant differences were found in open wound area when comparing the media only control to 10 uM (p < 0.01), 30 uM (p < 0.01), 300 uM (p < 0.001), and 1000 uM (p < 0.01) of the 2:1:1 BCAA dose. Significant differences were also found in the wound area when comparing the water vehicle control to 10 uM (p < 0.05), 30 uM (p < 0.01), 300 uM (p < 0.01), and 1000 uM (p < 0.01) of the 2:1:1 BCAA dose. No significant differences were found in the open wound area when comparing the controls and BCAA doses, at the 24 hour time point. Of note, no significant differences were found between control and treatment with the 1:1:1 ratio of leucine, isoleucine, and valine at any time point. Conclusion: BCAA treatment at the 2:1:1 ratio was seen to accelerate injury recovery at various dose concentrations, as quantified by open wound area after scratch injury was induced. This cell culture model demonstrates the importance of BCAA ratios. While this aligns with animal models and clinical literature, this is the first in-vitro assessment of BCAA repair capacity, in the context of cortical culture. Future studies will be undertaken to further elucidate the constituents of the repair mechanism.
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    Changes in Cerebral Mitochondrial Function in Postpartum Dams Exposed to a Low-resource Environment during Weaning
    (2024-03-21) Jones, Kylie; Smith, Savanna; Smith, Jonna; Castillo, Angie; Powell, Madison; Cunningham, Mark
    Background: Postpartum depression is a serious mental-health condition, affecting over 14% of all mothers in the U.S. Poverty, a lack of educational and economic resources, is a major determinant of adult mental health. In the U.S., poverty impacts 11% of adults, with a greater incidence in women of childbearing age (roughly 16%) and postpartum women (roughly 14%). While pregnancy and poverty separately increase risks of developing depression, few studies have combined these conditions to determine their role in the development of depression. Further, no studies have explored mechanisms contributing to depression following pregnancy and poverty. However, cerebral mitochondrial dysfunction (C-mtDys) may be one mechanism. The aim of this study is to examine C-mtDys in postpartum (PP) dams exposed to the limited-bedding-nesting (LBN) model in rats, which reduces their nesting material to simulate a low-resource environment. We hypothesize that PP dams in the LBN model will exhibit C-mtDys and elevated oxidative stress. Methods. Pregnant Sprague-Dawley rats gave birth naturally and were divided randomly into LBN (n =2) or control (n = 2) groups. LBN dams were exposed to the LBN model from PD 2 through PD 9. To validate the success of the LBN model, entropy scores, which are a measure of behavior unpredictability, were recorded for each dam. At 17 weeks PP, equivalent to 8 years PP in humans, brains were collected and used to isolate mitochondria through differential centrifugation. Mitochondrial function was evaluated via respiration (mtRes) using respiratory states. Oxidative stress in the whole brain was examined through H2O2 and total antioxidant capacity biochemical assays. Results. LBN PP dams displayed higher entropy scores (1.10 ± 0.04 v. 0.79 ± 0.04, p < 0.05), as expected based on prior literature, serving as validation of the model. At 17 weeks, PP LBN dams had reduced mtRes in all states, including the basal state (176.09 ± 5.90 v. 360.70 ± 7.73 pmol/s/mg, ns), State 2 (1044.72 ± 14.00 v. 1703.00 ± 18.10 pmol/s/mg, ns), State 3 (3843.27 ± 31.86 v. 6705.72 ± 37.54 pmol/s/mg, ns) and State 4 (776.83 ± 13.65 v. 1533.54 ± 16.46 pmol/s/mg, ns). PP LBN dams exhibited reduced total antioxidant capacity by roughly 20% (54.10 ± 1.24 v. 63.04 ± 0.99 mM Trolox) and elevated H2O2 by roughly 50% (2.12 ± 0.52 v. 1.35 ± 0.28 nM/mg, ns). Summary. In summary, preliminary data shows C-mtDys via reduced mtRes in PP dams exposed to an impoverished environment during weaning. Furthermore, this study suggests that decreased mtRes could contribute to increased oxidative stress in the brain. Elevated oxidative stress may cause damage at the cellular and circuitry levels in the brain that could facilitate the development of depression later in life. Future studies will further examine C-mtDys, oxidative stress, and depressive behaviors in PP dams exposed to LBN. This study is significant because it identifies C-mtDys as a possible mechanism causing depression after exposure to both pregnancy and poverty.
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    Deep Brain Stimulation in Pediatric Status Dystonicus: A Case Series Investigation on Rapid Activation & ICU Stays
    (2024-03-21) Schneider, Glen; Marks, Warren
    Purpose: Status dystonicus (SD) is a life-threatening movement disorder characterized by a sudden and severe worsening of generalized dystonia, often complicated by rhabdomyolysis with associated metabolic and respiratory consequences, including death. Deep brain stimulation (DBS) is a potential treatment modality for pharmaco-resistant SD (PRSD) which, despite growing interest in its use, requires further research into ideal patient selection and optimum timing for device activation. Timely deployment of this therapy can lead to shorter ICU stays and better outcomes for patients by reducing the need for additional invasive procedures and escalating pharmaceutical therapy. Methods: This is a retrospective case series of 6 consecutive pediatric SD patients who were admitted to the PICU at Cook Children’s Medical Center between October 2010 and March 2022. Following their admission, these patients were treated with DBS and were all subsequently discharged from the ICU. We examined ICU time between patients who underwent rapid activation of DBS systems and those who delayed the start of this intervention. Results: Patients with acquired forms of dystonia demonstrated a mean ICU stay of 58 following DBS activation, and patients with genetic dystonias had a mean stay of 18 days after the procedure. Rapid deployment of DBS systems and two stage procedures were associated with more positive patient outcomes, Conclusions: SD is a condition with a wide range of clinical presentations and responsiveness to treatment. This unpredictability and variation in disease outcomes emphasizes the need for further controlled studies to clarify optimal timing and patient selection for DBS. Ethical and parental concerns regarding the recruitment of children into clinical trials remains a major limiting factor to this goal, making meaningful clinical reports and data sharing between care centers essential in advancing the collective understanding of this condition and in optimizing patient care.