Post Injury Research Articles

Mitigating Critical Peripheral Nerve Deficit Therapy with Reactive Oxygen Species/Ca2+-Responsive Dynamic Hydrogel-Mediated mRNA Delivery.

Critical peripheral nerve deficiencies present as one of the most formidable conundrums in the realm of clinical medicine, frequently culminating in structural degradation and derangement of the neuromuscular apparatus. Engineered extracellular vesicles (EVs) exhibit the potential to ameliorate nerve impairments. However, the advent of Wallerian degeneration (WD), an inexorable phenomenon that ensues post peripheral nerve injury, serves as an insurmountable impediment to the direct therapeutic efficacy of EVs. In this investigation, we have fashioned a dynamic network for the conveyance of PTEN-induced kinase 1 (PINK1) mRNA (E-EV-P@HPCEP) using an adaptive hydrogel with reactive oxygen species (ROS)/Ca2+ responsive ability as the vehicle, bearing dual-targeted, engineered EVs. This intricate system is to precisely deliver PINK1 to senescent Schwann cells (SCs) while concurrently orchestrating a transformation in the inflammatory-senescent milieu following injury, thereby stymying the progression of WD in peripheral nerve fibers through the stimulation of autophagy within the mitochondria of the injured cells and the maintenance of mitochondrial mass equilibrium. WD, conventionally regarded as an inexorable process, E-EV-P@HPCEP achieved functionalized EV targeting, orchestrating a dual-response dynamic release mechanism via boronate ester bonds and calcium chelation, effectuating an enhancement in the inflammatory-senescent microenvironment, which expedites the therapeutic management of nerve deficiencies and augments the overall reparative outcome.

#2027 Activation of histone deacetylase-1 in fibroblasts and pericytes induces renal interstitial fibrosis after ischemia-reperfusion injury

Abstract Background and Aims Following an acute kidney ischemic event the chromatin remodelling enzyme, histone deacetylase-1 (HDAC1), is activated in many cell types of the kidney including fibroblasts and pericytes. During kidney injury, the fibroblasts/pericytes differentiate into the myofibroblasts and produce extracellular matrix that can lead to a permanent decline in kidney function. Pharmacological inhibition of HDACs can attenuate ischemia-reperfusion-injury (IRI) mediated interstitial fibrosis. The hypothesis of this study was that fibroblast/pericyte HDAC1 activation promotes myofibroblast differentiation and interstitial fibrosis. Method Tamoxifen inducible, fibroblast/pericyte HDAC1 knockout (KO) mice (HDAC1Fl/Fl; Col1a2-CreEr) and littermate controls (HDAC1Fl/Fl) were used. Male and female mice were given tamoxifen by ip at 8-10 weeks of age and IRI or sham surgery completed after a 2-week tamoxifen washout period. A mild, 18 min, bilateral, warm IRI model was used, and samples collected over 4 weeks. Glomerular filtration rate (GFR) was assessed using transdermal FITC-Sinistrin clearance. In vitro experiments with kidney fibroblast cells (NRK49F) overexpressing empty vector or HDAC1 were used for RNA-sequencing to determine the HDAC1-dependent myofibroblast transcriptome. Results We confirmed that HDAC1 was knockdown in myofibroblast cells from KO mice with co-localization of HDAC1 and PDGFRβ or α-smooth muscle actin in the kidneys of IRI mice. Twenty-four hours post-surgery, all IRI mice exhibited 2-3 times increase in serum creatinine and a ∼50% reduction in GFR compared to sham mice (control IRI = 513 ± 129 μl /min/100 g body mass, control sham = 1010 ± 218, n = 4-5, p = 0.02; KO IRI = 529 ± 116, KO sham = 1187 ± 59 μl/min/100 g body mass, n = 4-5, p = 0.004). Subsequent GFR measurements over the next 4 weeks showed a gradual recovery of renal function of IRI mice starting at week 2 and fully restored to sham values by week 4. However, the male control IRI mice had significant focal interstitial fibrosis 2- and 4-weeks post injury (4-week data: sham = 0.3 ± 0.09 % of cortical area, IRI = 1.2 ± 0.2%, n = 6/group, p = 0.0011) but this was attenuated in the KO male IRI mice (sham = 0.4 ± 0.07% n = 4, IRI = 0.5 ± 0.1%, n = 7, p = 0.93). The female mice, regardless of genotype, had very little kidney damage or interstitial fibrosis at 4 weeks. Transcriptomes of NRK49F cells overexpressing HDAC1 had an upregulation of 55 genes related to pro-inflammatory cytokines and tumour necrosis factor signalling and downregulation of 367 genes related to cell junctions, cytoskeleton proteins, and cell adhesion compared to the empty vector control cells. Fibroblast proliferation rates were not statistically different among the groups, suggesting HDAC1 does not affect myofibroblast proliferation but rather pro-inflammatory gene expression. Conclusion In male mice, fibroblast/pericyte activation of HDAC1 leads to the production of pro-inflammatory cytokines that we predict will lead to immune cell infiltration and crosstalk that leads to the excessive production of interstitial fibrosis following IRI.

Virtual reality as a method of cognitive training of processing speed, working memory, and sustained attention in persons with acquired brain injury: a protocol for a randomized controlled trial.

Acquired brain injury (ABI) often leads to persisting somatic, cognitive, and social impairments. Cognitive impairments of processing speed, sustained attention, and working memory are frequently reported and may negatively affect activities of daily living and quality of life. Rehabilitation efforts aiming to retrain these cognitive functions have often consisted of computerized training programs. However, few studies have demonstrated effects that transfer beyond the trained tasks. There is a growing optimism regarding the potential usefulness of virtual reality (VR) in cognitive rehabilitation. The research literature is sparse, and existing studies are characterized by considerable methodological weaknesses. There is also a lack of knowledge about the acceptance and tolerability of VR as an intervention method for people with ABI. The present study aims to investigate whether playing a commercially available VR game is effective in training cognitive functions after ABI and to explore if the possible effects transfer into everyday functioning. One hundred participants (18-65years), with a verified ABI, impairments of processing speed/attention, and/or working memory, and a minimum of 12months post injury will be recruited. Participants with severe aphasia, apraxia, visual neglect, epilepsy, and severe mental illness will be excluded. Participants will be randomized into two parallel groups: (1) an intervention group playing a commercial VR game taxing processing speed, working memory, and sustained attention; (2) an active control group receiving psychoeducation regarding compensatory strategies, and general cognitive training tasks such as crossword puzzles or sudoku. The intervention period is 5weeks. The VR group will be asked to train at home for 30min 5days per week. Each participant will be assessed at baseline with neuropsychological tests and questionnaires, after the end of the intervention (5weeks), and 16weeks after baseline. After the end of the intervention period, focus group interviews will be conducted with 10 of the participants in the intervention group, in order to investigate acceptance and tolerability of VR as a training method. This study will contribute to improve understanding of how VR is tolerated and experienced by the ABI population. If proven effective, the study can contribute to new rehabilitation methods that persons with ABI can utilize in a home setting, after the post-acute rehabilitation has ended.

Exploring the challenges of avoiding collisions with virtual pedestrians using a dual-task paradigm in individuals with chronic moderate to severe traumatic brain injury.

Individuals with a moderate-to-severe traumatic brain injury (m/sTBI), despite experiencing good locomotor recovery six months post-injury, face challenges in adapting their locomotion to the environment. They also present with altered cognitive functions, which may impact dual-task walking abilities. Whether they present collision avoidance strategies with moving pedestrians that are altered under dual-task conditions, however, remains unclear. This study aimed to compare between individuals with m/sTBI and age-matched control individuals: (1), the locomotor and cognitive costs associated with the concurrent performance of circumventing approaching virtual pedestrians (VRPs) while attending to an auditory-based cognitive task and; (2) gaze behaviour associated with the VRP circumvention task in single and dual-task conditions. Twelve individuals with m/sTBI (age = 43.3 ± 9.5 yrs; >6 mo. post injury) and 12 healthy controls (CTLs) (age = 41.8 ± 8.3 yrs) were assessed while walking in a virtual subway station viewed in a head-mounted display. They performed a collision avoidance task with VRPs, as well as auditory-based cognitive tasks (pitch discrimination and auditory Stroop), both under single and dual-task conditions. Dual-task cost (DTC) for onset distance of trajectory deviation, minimum distance from the VRP, maximum lateral deviation, walking speed, gaze fixations and cognitive task accuracy were contrasted between groups using generalized estimating equations. In contrast to CTLs who showed locomotor DTCs only, individuals with m/sTBI displayed both locomotor and cognitive DTCs. While both groups walked slower under dual-task conditions, only individuals with m/sTBI failed to modify their onset distance of trajectory deviation and maintained smaller minimum distances and smaller maximum lateral deviation compared to single-task walking. Both groups showed shorter gaze fixations on the approaching VRP under dual-task conditions, but this reduction was less pronounced in the individuals with m/sTBI. A reduction in cognitive task accuracy under dual-task conditions was found in the m/sTBI group only. Individuals with m/sTBI present altered locomotor and gaze behaviours, as well as altered cognitive performances, when executing a collision avoidance task involving moving pedestrians in dual-task conditions. Potential mechanisms explaining those alterations are discussed. Present findings highlight the compromised complex walking abilities in individuals with m/sTBI who otherwise present a good locomotor recovery.

Sex hormone supplementation improves breathing and restores respiratory neuroplasticity following C2 hemisection in rats.

In addition to loss of sensory and motor function below the level of the lesion, traumatic spinal cord injury (SCI) may reduce circulating steroid hormones that are necessary for maintaining normal physiological function for extended time periods. For men, who comprise nearly 80% of new SCI cases each year, testosterone is the most abundant circulating sex steroid. SCI often results in significantly reduced testosterone production and may result in chronic low testosterone levels. Testosterone plays a role in respiratory function and the expression of respiratory neuroplasticity. When testosterone levels are low, young adult male rats are unable to express phrenic long-term facilitation (pLTF), an inducible form of respiratory neuroplasticity invoked by acute, intermittent hypoxia (AIH). However, testosterone replacement can restore this respiratory neuroplasticity. Complicating the interpretation of this finding is that testosterone may exert its influence in three possible ways: 1) directly through androgen receptor (AR) activation, 2) through conversion to dihydrotestosterone (DHT) by way of the enzyme 5α-reductase, or 3) through conversion to 17β-estradiol (E2) by way of the enzyme aromatase. DHT signals via AR activation similar to testosterone, but with higher affinity, while E2 activates local estrogen receptors. Evidence to date supports the idea that exogenous testosterone supplementation exerts its influence through estrogen receptor signaling under conditions of low circulating testosterone. Here we explored both recovery of breathing function (measured with whole body barometric plethysmography) and the expression of AIH-induced pLTF in male rats following C2-hemisection SCI. One week post injury, rats were supplemented with either E2 or DHT for 7days. We hypothesized that E2 would enhance ventilation and reveal pLTF following AIH in SCI rats. To our surprise, though E2 did beneficially impact overall breathing recovery following C2-hemisection, both E2 supplementation and DHT restored the expression of AIH-induced pLTF 2weeks post-SCI.

Exosomes from hypoxic ADSCs ameliorate neuronal damage post spinal cord injury through circ-Wdfy3 delivery and inhibition of ferroptosis

BackgroundExosomes generated from adipose-derived mesenchymal stem cells (Exos), and in particular hypoxia-pretreated ADSCs (HExos), possess therapeutic properties that promote spinal cord repair following spinal cord injury (SCI). Nevertheless, the regulatory mechanisms through which HExos exert their effects remain unclear. MethodsHere, next-generation sequencing (NGS) was utilized to examine abnormal circRNA expression comparing HExos to Exos. Bioinformatics analysis and RNA pulldown assays together with luciferase reporter assays were applied to determine interactions among miRNAs, mRNAs and circRNAs. ELISA and immunofluorescence staining were used to examine inflammatory cytokine levels, apoptosis and ROS deposition in LPS-treated HT-22 cells, respectively. The therapeutic effects of Exos and HExos on a mouse model of SCI were analyzed by immunohistochemistry and immunofluorescence staining. ResultsOur findings confirmed that HExos have more significant therapeutic influences on decreasing ROS and inflammatory cytokine levels post-SCI than Exos. NGS revealed that circ-Wdfy3 expression levels were significantly higher in HExos than Exos. Downregulation of circ-Wdfy3 led to a decrease in HExo-induced therapeutic effects on spinal cord repair post-SCI, indicating that circ-Wdfy3 has a critical role in the regulation of HExo-mediated protection against SCI. Our bioinformatics, RNA pulldown and luciferase reporter data demonstrated that GPX4 and miR-423-3p were downstream targets of circ-Wdfy3. GPX4 downregulation or miR-423-3p overexpression reversed the protective effects of circ-Wdfy3 on LPS-treated HT-22 cells. Furthermore, overexpression of circ-Wdfy3 led to an in increase in the Exo-induced therapeutic effects on spinal cord repair post-SCI through the inhibition of ferroptosis. Conclusionscirc-WDfy3-overexpressing Exos promote spinal cord repair post-SCI through mediation of ferroptosis via the miR-138-5p/GPX4 pathway.

NGAL in the Development of Acute Kidney Injury in a Murine Model of Remote Ischaemic Preconditioning and Liver Ischaemia Reperfusion.

Acute kidney injury (AKI) is common following liver transplantation and is associated with liver ischeamia reperfusion (IR) injury. The purpose of this study was to use a mouse model of liver IR injury and AKI to study the role of Neutrophil Gelatinase Associated Lipocalin (NGAL), a biomarker of AKI, in liver IR injury and AKI. We demonstrate an adapted, reproducible model of liver IR injury and AKI in which remote ischemic preconditioning (RIPC) by repeated episodes of hindleg ischemia prior to liver IR reduced the severity of the IR injury. In this model, serum NGAL at 2 h post reperfusion correlated with AKI development early following IR injury. This early rise in serum NGAL was associated with hepatic but not renal upregulation of NGAL mRNA, suggesting NGAL production in the liver but not the kidney in the early phase post liver IR injury.

Non-TGFβ profibrotic signaling in ulcerative colitis after in vivo experimental intestinal injury in humans.

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high-resolution endoscopic imaging, histological assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 patients with UC in remission and 20 control subjects. Human ER had a 48-h lag before induction of regenerative epithelial cells [wound-associated epithelial (WAE) and transit amplifying (TA) cells] along with the increase of fibroblast-derived stem cell growth factor gremlin 1 mRNA (GREM1). However, UC deconvolution data showed rapid induction of inflammatory fibroblasts and upregulation of major structural ECM collagen mRNAs along with tissue inhibitor of metalloproteinase 1 (TIMP1), suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor β (TGFβ) mRNA, whereas the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC, suggesting that human postinjury responses could be TGFβ-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end-organ failure, i.e., intestinal damage.NEW & NOTEWORTHY The study reveals the regulatory dynamics of epithelial regeneration and extracellular matrix remodeling after experimental injury of the human colon in vivo and shows that human intestinal regeneration is different from data obtained from animals. A lag phase in epithelial restitution is associated with induction of stromal cell-derived epithelial growth factors. Postinjury regeneration is transforming growth factor β-independent, and we find a profibrotic response in patients with ulcerative colitis despite being in remission.

Migrasomes from adipose derived stem cells enrich CXCL12 to recruit stem cells via CXCR4/RhoA for a positive feedback loop mediating soft tissue regeneration

BackgroundAdipose-derived stem cells (ASCs) represent the most advantageous choice for soft tissue regeneration. Studies proved the recruitment of ASCs post tissue injury was mediated by chemokine CXCL12, but the mechanism by which CXCL12 is generated after tissue injury remains unclear. Migrasomes are newly discovered membrane-bound organelles that could deliver CXCL12 spatially and temporally in vivo. In this study, we sought to investigate whether migrasomes participate ASC-mediated tissue regeneration.MethodsDiscrepant and asymmetrical soft tissue regeneration mice model were established, in which HE staining, immunofluorescent staining, western blot and qPCR were conducted to confirm the role of CXCL12 and migrasomes in ASC-mediated tissue regeneration. Characterization of ASC-derived migrasomes were carried out by confocal microscopy, scanning electron microscopy, transmission electron microscopy as well as western blot analysis. The function and mechanism of migrasomes were further testified by assisting tissue regeneration with isolated migrasomes in vivo and by in vitro transwell combined with co-culture system.ResultsHere, we show for the first time that migrasomes participate in soft tissue regeneration. ASCs generate migrasomes enriched with CXCL12 to mediate tissue regeneration. Migrasomes from ASCs could promote stem cells migration by activating CXCR4/RhoA signaling in vivo and in vitro. Chemoattracted ASCs facilitate regeneration, as demonstrated by the upregulation of an adipogenesis-associated protein. This positive feed-back-loop creates a favorable microenvironment for soft tissue regeneration. Thus, migrasomes represent a new therapeutic target for ASC-mediated tissue regeneration.ConclusionsOur findings reveal a previously unknown function of ASCs in mediating tissue regeneration by generating migrasomes. The ASC-derived migrasomes can restore tissue regeneration by recruiting stem cells, which highlighting the potential application of ASC-derived migrasomes in regenerative medicine.

Abnormal Static Sagittal Cervical Curvatures following Motor Vehicle Collisions: A Retrospective Case Series of 41 Patients before and after a Crash Exposure.

Three spine clinic records were reviewed over a 2-year period, looking for patients where both an initial lateral cervical X-ray and an examination were performed prior to the patient being exposed to a MVC; afterwards, an additional exam and radiographic analysis were obtained. A total of 41 patients met the inclusion criteria. Examination records of pain intensity on numerical pain rating scores (NPRS) and neck disability index (NDI), if available, were analyzed. The CLRs were digitized and modeled in the sagittal plane using curve fitting and the least squares error approach. Radiographic variables included total cervical curve (ARA C2-C7), Chamberlain's line to horizontal (skull flexion), horizontal translation of C2 relative to C7, segmental translations (retrolisthesis and anterolisthesis), and circular modelling radii. There were 15 males and 26 females with an age range of 8-65 years. Most participants were drivers (28) involved in rear-end impacts (30). The pre-injury NPRS was 2.7 while the post injury was 5.0; p < 0.001. The NDI was available on 24/41 (58.5%) patients and increased after the MVC from 15.7% to 32.8%, p < 0.001. An altered cervical curvature was identified following exposure to MVC, characterized by an increase in the mean radius of curvature (265.5 vs. 555.5, p < 0.001) and an approximate 8° reduction of lordosis from C2-C7; p < 0.001. The mid-cervical spine (C3-C5) showed the greatest curve reduction with an averaged localized mild kyphosis at these levels. Four participants (10%) developed segmental translations that were just below the threshold of instability, segmental translations < 3.5 mm. The post-exposure MVC cervical curvature was characterized by an increase in radius of curvature, an approximate 8° reduction in C2-C7 lordosis, a mild kyphosis of the mid-cervical spine, and a slight increase in anterior translation of C2-C7 sagittal balance. The modelling result indicates that the post-MVC cervical sagittal alignment approximates a second-order buckling alignment, indicating a significant alteration in curve geometry. Future biomechanics experiments and clinical investigations are needed to confirm these findings.

1,25-Dihydroxyvitamin D3 reduces early mortality post severe burn injury via alleviating endotoxemia, oxidative stress and inflammation

Severe burn patients frequently suffer from 1,25-Dihydroxyvitamin D3 (1,25-[OH]2-D3) deficiency. In this study, we investigated the effect of 1,25-[OH]2-D3 on early mortality post severe burn and potential underlying mechanisms. Our results indicate that 1,25-[OH]2-D3 significantly reduced early mortality in mice post severe burn injury. A decrease in serum lipopolysaccharide levels and an increase in serum superoxide dismutase activity were found after administration of 1,25-[OH]2-D3. Furthermore, 1,25-[OH]2-D3 demonstrated protective effects on both intestinal and lung histology and ameliorated lung inflammation. Its anti-inflammatory effect was further confirmed in airway epithelial cells. In conclusion, our study provides evidence that 1,25-[OH]2-D3 has a significant impact on the reduction of early mortality post severe burn injury, possibly through its ability to alleviate endotoxemia, oxidative stress, and inflammation. Our findings highlight the potential of 1,25-[OH]2-D3 to protect the intestinal mucosal barrier in the early stage following major burn injury and opens up new avenues for clinical application of 1,25-[OH]2-D3 in burn patients.

Lived Experiences of Sexuality and Sexual Functioning in Males with SCI: A Mixed-Methods Study.

Sexual dysfunction is highly prevalent in males with spinal cord injury (SCI) and has been recognized to be a key recovery priority. This cross-sectional, mixed-methods study aimed to investigate the major themes linked to sexual functioning in males with chronic (>1 year) SCI. Twenty male participants with SCI, aged 25 to 59 years, completed validated questionnaires exploring sexual function/satisfaction and health-related quality of life and a semi-structured interview with an experienced sexual medicine physician. Sex hormone concentrations and metabolic biomarkers, along with body composition and habitual physical activity levels, were assessed. Interview recordings were transcribed and thematic analysis performed using combined COM-B (Capability, Opportunity, Motivation, and Behavior) and biopsychosocial models to identify and organize major contributors and barriers to sexual functioning. Metabolic and hormonal biomarkers largely fell within normal physiological ranges despite reduced sexual functioning reported in our cohort (19/20 participants reported some degree of erectile dysfunction). Qualitative analysis of interview transcripts revealed 24 themes. Adaptability was important for improving sexual satisfaction. Attraction and attentiveness to sex and partners remained stable over time, while the desire for intimacy increased post injury. Sexual social norms, and comparisons to the able-bodied population, provided challenges for sexual activity and partnership. Environmental concerns regarding access to sexual health resources and accessible physical spaces during intimacy were relevant. Mood disorders and general life stressors negatively impacted sexual desire, while physical activity encouraged sexual activity. By considering a holistic view of sexuality in males with SCI, we identified key contributors and barriers to sexual functioning for the cohort studied.

Modeling Mild Traumatic Brain Injury In-Vitro Using PHuman Choroid Plexus Endothelial Cells

Over 2.5 million Traumatic Brain Injuries (TBIs) occur each year due to falls, motor vehicle accidents, assaults, and sports (Brain Trauma Foundation, 2023). Short- and long-term consequences of TBI can include headache, cognitive impairment, seizures, hypoxia, and ischemia. Human TBI studies are diffcult due to the confounding variables of type and mechanism of injury, severity of injury, and length of time to present to emergency departments. While animal models exist, it is our goal to develop an in-vitro model of TBI using primary human brain cell cultures to examine cellular responses in real time. While the blood-brain barrier has been studied extensively post-TBI due to its role in ischemia and hypoxia, there is a dearth of research on the blood-CSF-barrier (BCSFB), the choroid plexus, and its response post-TBI. After culturing primary human choroid plexus endothelial cells (ScienCell Cat #1300) on a flexible membrane, we subjected them to mechanical strain using the FlexCell tension system and collected supernatant at 3-, 6-, 12-, 24-, 48-, 72-, and 96-hours post injury. The FlexCell tension bioreactor system uses positive and negative air pressure to apply mechanical elongation strain mimicking mild shearing injury. Using R&amp;D cytokine arrays (R&amp;D Systems, Cat# ARY005B), we measured 36 different cytokines, chemokines and acute phase proteins including C5a, GM-CSF, ICAM-1, IL-1, IL-6, TNFα, IFNγ, CCL5/RANTES, etc. We hypothesized that we would see a pro-inflammatory response (IL-1β, IL-6, TNFα) within 24 hours after one or two 12% mechanical strain injuries compared to the control of no strain, as seen in the literature. However, there were not significant increases in cytokine signaling until 24-48 hours post-injury, and in regulatory cytokines, such as CCL2, MIP1α/MIP1β, IL-2, IL-12, &amp; IL-17, that lasted as long as 96-hours. This effect was more pronounced in the two strain verses one strain model and control, as expected. Interestingly, anti-inflammatory cytokine IL-10 was significantly increased in the one strain model, but not two strain model, 48 hours post-insult. As the choroid plexus is a niche for T cell stimulation in the central nervous system (Strominger et al., 2018), these results indicate a complex immunological response that seems to favor T-Cell response over monocytes/macrophages. These experiments were completed with the lowest mechanical strain of the FlexCell system and future experiments are warranted to determine if a stronger mechanical strain is needed to produce the pro-inflammatory response seen in the literature in mild to severe models of TBI. This project was funded by a Senior Research Grant from the Indiana Academy of Science and from a Faculty Research Development Grant from Marian University College of Osteopathic Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuromuscular Junction Remodeling: the Role of Sub-Synaptic Nuclei, Terminal Schwann Cells, and Trophic Signaling Following Neuromusculoskeletal Injuries

Peripheral nerve crush injury is a well-established model of neuromuscular junction (NMJ) denervation and subsequent re-innervation. Functionally, the muscle tissue follows a similar pattern as neural recovery, with immediate loss of force production that steadily improves in parallel with rates of re-innervation. On the other hand, traumatic injury to the muscle tissue itself, specifically volumetric muscle loss (VML), results in an irrecoverable loss of muscle function. Recent work has indicated significant impairments to the NMJ following VML that appear chronic in nature, alongside functional recovery. Thus, the goal of this study was to compare the effects of nerve and muscle injury on NMJ remodeling, with the hypothesis that VML will result in chronic alterations to the NMJ, including a higher degree of cellular localization to the NMJ. Even numbers of adult male and female mice were used with three experimental groups: injury naive, nerve crush, and VML-injury; and three terminal timepoints: 3-, 48-, and 112-days post injury. Data were analyzed by three-way ANOVA with Tukey’s HSD post-hoc. Confirming the assumed recoverability of the two injury models, we found in vivo maximal torque was fully restored following nerve crush injury (Tukey’s HSD post hoc p=0.730), but remained at a significant deficit following VML (~41% lower than injury naïve; Tukey’s HSD post hoc p&lt;0.001). Compared to nerve crush and injury naïve, we found VML results in aberrantly high trophic signaling and numbers of supporting cells. Protein expression of neuregulin-1 and connective tissue growth factor were both highest in the VML group (~1.2-fold and 12-fold increase compared to injury naïve, respectively; Main effect of group, p&lt;0.001). While nerve crush injury elicited a transient increase in terminal Schwann cell numbers (~38% higher than injury naïve at 48dpi; Tukey’s HSD post hoc p&lt;0.001), VML resulted in a chronic increase (~53% and 27% higher than injury naïve at 48 and 112dpi, respectively; Tukey’s HSD post hoc p&lt;0.001). Moreover, nerve crush injury did not alter the number of sub-synaptic nuclei per NMJ, while VML resulted in significant increases at both 48- and 112-days post injury (~27% and 24% higher than injury naïve, respectively; Tukey’s HSD post hoc p=0.002). In some cases, sex differences were detected, including higher rates of innervation in females than males (Main effect of sex p=0.003), particularly following VML-injury (Tukey’s HSD post hoc p=0.004). The findings herein bear a striking resemblance to NMJs found in dystrophic muscle, which are thought to be the result of continuous cycles of muscle fiber degeneration and regeneration. Thus, the NMJ changes observed following VML may be indicative of a larger issue, such as chronic muscle fiber remodeling and pervasive fibrotic accumulation. R01-AR078903 (JAC &amp; SMG); K02-AG081488 (SMG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Closed-loop Epidural Stimulation Elicits Respiratory Plasticity in a Rat Model of Acute Spinal Cord Injury

Epidural stimulation restores motor control in animal models and humans after spinal cord injury (SCI) with the most notable example being locomotion; however, it has not yet been clinically translated to breathing. Further, neuroplasticity elicited by epidural stimulation is largely unexplored. We showed in a rat C2 hemisection (C2HS) model of SCI that one bout of closed-loop epidural stimulation (CLES) restores some ipsilesional (ipsi) diaphragm EMG acutely post paralysis, and that in some rats, activity briefly lasts post CLES (&lt;15 seconds) (Mickle et al., 2023). Here we explore if this brief plasticity increases in magnitude and duration via multiple bouts of CLES, respiratory challenge, or altered sensory input. Adult Sprague Dawley (n=5) rats were urethane anesthetized, implanted with diaphragm EMG recording electrodes and dorsal C4 stimulating electrodes and given a C2HS plus &gt;15 minute recovery period to stabilize and confirm hemidiaphragm paralysis. Rats were delivered two &lt;5 minute periods of CLES during which current amplitude increased by 25 uA steps every 30 breaths until breathing instability. Afferent input was removed via bilateral cervical dorsal rhizotomy (CDR) prior to CLES (n = 2) or between the first two bouts (n = 3). During the first two bouts of CLES, rats were administered 50% O2 via a cycle triggered ventilator which delivers breaths in-phase with contralesional (contra) EMG. For the 3rd bout, rats were challenged with room air and removal of ventilatory support. CLES increased contra EMG output in 5/5 and restored ipsi EMG in 4/5 rats during all bouts. There were no differences in PCO2 prior to each bout (mmHg +/- SEM: 44 +/- 6, 43 +/- 4, 41 +/- 4) though PO2 was much lower under room air (mmHg +/- SEM 172 +/- 24. 165 +/- 31, 73 +/- 5). Contra peak amplitude was elevated in the 30 seconds post-CLES vs baseline for the 1st bout of CLES (77 vs 93 mV, SE of diff. 3, p = 0.009) with no difference in rats with or without CDR (20 vs 23% increase from baseline, SE of diff. 10), and no difference in the 1st and 2nd bouts (93 vs 90 mV, SE of diff. 5). While contra peak EMG did not change between bouts, there was a trend towards an increase in length of residual ipsi activity post bout 2 vs 1 (223 vs 54 sec, SE of diff. 170). Removing the ventilator increased contra peak (77 vs 132 mV, SE of diff. 16, p = 0.025) and unveiled ipsi activity in 3/5 rats. CLES during challenge trended towards increased ipsi response during CLES (15 vs 61 V cumulative sum, SE of diff. 8) and length of residual ispi activity (223 vs 1819 sec, SE of diff. 1031). In 3/5 rats, the residual ipsi activity post challenge CLES bout continued until the end of the experimental period, in one case lasting &gt;20 min. Finally, across all CLES bouts, the magnitude of ipsi activity in the 30 seconds post CLES was correlated with the magnitude of response during CLES (r2 0.34, p = 0.05) indicating conditions that increase response may be best for neuroplasticity. Work is ongoing to clarify the roles of multiple bouts, cervical afferents, challenge, and time post injury in eliciting the enduring activity seen in some rats, but this work represents the first evidence that CLES can elicit respiratory neuroplasticity on a timescale and magnitude which may be relevant for functional benefit. T32HL134621 (AM), R01HL153102 (ED), SPARC OT2OD023854 (ED), Craig H. Neilsen Pilot Grant (ED). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cerebrovascular Dysfunction in a Mouse Model of Marfan Syndrome: Increased Vulnerability to Neuropathology &amp; Mild Traumatic Brain Injury

Marfan syndrome (MFS) is a connective tissue disorder (CTD) associated with mutations in fibrillin-1 ( Fbn1), leading to chronic systemic increases in transforming growth factor-β (TGF-β) availability and signaling that could lead to aortic wall weakening and aneurysm/rupture. TGF-β has been implicated in cerebrovascular dysfunction, loss of blood-brain barrier (BBB) integrity, age-related neuroinflammation and neurological deficits. Improvements in MFS treatment and life expectancy have uncovered a risk for systemic vascular dysfunction including cerebrovascular complications such as stroke, and cerebral aneurysm. Therefore, we hypothesized that Fbn1 mutation ( Fbn1C1041G/+) is associated with systemic vascular dysfunction, BBB permeability, neuroinflammation, neurobehavioral alterations, and impaired glutamate homeostasis, leaving the brain more vulnerable to mild traumatic brain injury (mTBI). Our experimental groups included: 6M-old male and female MFS ( Fbn1C1041G/+) and C57BL/6 (WT) control, and middle-aged 12M WT mice (N=3-10/group, P &lt; 0.05). In vivo ultrasound imaging analyzed by two blinded investigators demonstrated increased aortic root diameters and stiffness in MFS mice compared to 6M and 12M WT mice, where MFS male diameters were affected more than females. Vascular dysfunction measured by blood flow velocity (BFV) extended to the left pulmonary artery where 6M MFS and 12M WT mice were reduced compared to 6M WT mice. This reduction continued through decreased posterior cerebral artery BFV in MFS males, where MFS females were comparable to 6 and 12M WT females. In 6M MFS male mice, Evans blue extravasation and Immunoglobulin G staining demonstrated increased BBB permeability in the hippocampus to large and small molecules, respectively, as well as to small molecules in the lateral hypothalamus as compared to 6M WT and like 12M WT. In adjacent sections, iba-1-stained cells had morphologies indicative of reactive microglia. In vivo electrochemical recordings in the hippocampus showed a trend towards lowered peak glutamate amplitudes and elevated baseline glutamate concentrations in MFS and 12M WT mice demonstrating disrupted glutamate homeostasis. Furthermore, exogenous TGF-β applied in the lateral hippocampus of 6M WT males revealed an evoked glutamate response that may support this elevated baseline in MFS mice. Higher neurobehavioral severity scale (NSS) scores in 6M MFS mice suggested neurobehavioral alterations that were more like 12M WT. Midline fluid percussion was used to induce mTBI, where 6M MFS male mice and both female groups required a 15% lower pressure to induce mTBI righting reflex times (5-10 minutes) compared to 6M WT male mice. Mice were evaluated at 1-day post injury, where BBB permeability to large and small molecules and microglia activation were increased, glutamate clearance was slowed, and NSS scores were increased compared to 6M WT and were like 6M MFS and 12M WT mice. These novel findings signify MFS as a condition of systemic vascular dysfunction and pre-mature cerebrovascular aging phenotype with more vulnerability to brain injury. Funding: NIH-R36AG083385, Valley Research Partnership-P1A-5012, NIH-R15HL145646, NIH-R01NS100793, Midwestern Graduate Funds, Phoenix Children’s Hospital Leadership Circle, Graduate and Professional Council Research and Projects Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aloe emodin promotes mucosal healing by modifying the differentiation fate of enteroendocrine cells via regulating cellular free fatty acid sensitivity

The proper differentiation and reorganization of the intestinal epithelial cell population is critical to mucosal regeneration post injury. Label retaining cells (LRCs) expressing SRY-box transcription factor 9 (SOX9) promote epithelial repair by replenishing LGR5+ intestinal stem cells (ISCs). While, LRCs are also considered precursor cells for enteroendocrine cells (EECs) which exacerbate mucosal damage in inflammatory bowel disease (IBD). The factors that determine LRC-EEC differentiation and the effect of intervening in LRC-EEC differentiation on IBD remain unclear. In this study, we investigated the effects of a natural anthraquinone called aloe emodin (derived from the Chinese herb rhubarb) on mucosal healing in IBD models. Our findings demonstrated that aloe emodin effectively interfered with the differentiation to EECs and preserved a higher number of SOX9+ LRCs, thereby promoting mucosal healing. Furthermore, we discovered that aloe emodin acted as an antagonist of free fatty acid receptors (FFAR1), suppressing the FFAR1-mediated Gβγ/serine/threonine-protein kinase (AKT) pathway and promoting the translocation of forkhead box protein O1 (FOXO1) into the nucleus, ultimately resulting in the intervention of differentiation fate. These findings reveal the effect of free fatty acid accessibility on EEC differentiation and introduce a strategy for promoting mucosal healing in IBD by regulating the FFAR1/AKT/FOXO1 signaling pathway.

Nanosponge for Iron Chelation and Efflux: A Ferroptosis-Inhibiting Approach for Myocardial Infarction Therapy.

Myocardial infarction (MI), a consequence of coronary artery occlusion, triggers the degradation of ferritin, resulting in elevated levels of free iron in the heart and thereby inducing ferroptosis. Targeting myocardial ferroptosis through the chelation of excess iron has therapeutic potential for MI treatment. However, iron chelation in post ischemic injury areas using conventional iron-specific chelators is hindered by ineffective myocardial intracellular chelation, rapid clearance, and high systemic toxicity. A chitosan-desferrioxamine nanosponge (CDNS) is designed by co-crosslinking chitosan and deferoxamine through noncovalent gelation to address these challenges. This architecture facilitates direct iron chelation regardless of deferoxamine (DFO) release due to its sponge-like porous hydrogel structure. Upon cellular internalization, CDNS can effectively chelate cellular iron and facilitate the efflux of captured iron, thereby inhibiting ferroptosis and associated oxidative stress and lipid peroxidation. In MI mouse models, myocardial injection of CDNS promotes sustainable retention and the suppression of ferroptosis in the infarcted heart. This intervention improves cardiac function and alleviates adverse cardiac remodeling post-MI, leading to decreased oxidative stress and the promotion of angiogenesis due to ferroptosis inhibition by CDNS in the infarcted heart. This study reveals a nanosponge-based nanomedicine targeting myocardial ferroptosis with efficient iron chelation and efflux, offering a promising MI treatment.

Elucidating a genomic signature associated with behavioral and executive function after moderate to severe pediatric TBI: a systems biology informed approach

Introduction: There is significant unexplained variability in behavioral and executive functioning after pediatric traumatic brain injury (TBI). Prior research indicates that there are likely genetic contributions; however, current research is limited. The purpose of this study is to use a systems biology informed approach to characterize the genomic signature related to behavioral and executive functioning ∼12 months after moderate through severe TBI in children.Methods: Participants were from two prospective cohorts of children with severe TBI (Cohort #1) and moderate-severe TBI and an orthopedic injury (OI) group (Cohort #2). Participants included 196 children (n = 72 and n = 124 total from each respective cohort), ranging in age between 0–17 years at the time of injury. In total, 86 children had severe TBI, 49 had moderate TBI, and 61 had an OI. Global behavioral functioning assessed via the Child Behavior Checklist and executive function assessed via the Behavioral Rating Inventory of Executive Function at ∼ 12 months post injury served as outcomes. To test for a genomic signature, we compared the number of nominally significant (p &amp;lt; 0.05) polymorphisms associated with the outcomes in our systems biology identified genes to a set 10,000 permutations using control genes (e.g., not implicated by systems biology). We used the ToppFun application from Toppgene Suite to identify enriched biologic processes likely to be associated with behavioral and executive function outcomes.Results: At 12 months post injury, injury type (TBI vs OI) by polymorphism interaction was significantly enriched in systems biology selected genes for behavioral and executive function outcomes, suggesting these genes form a genomic signature. Effect sizes of the associations from our genes of interest ranged from .2–.5 for the top 5% of variants. Systems biology analysis of the variants associated with the top 5% effect sizes indicated enrichment in several specific biologic processes and systems.Discussion: Findings indicate that a genomic signature may explain heterogeneity of behavioral and executive outcomes after moderate and severe TBI. This work provides the foundation for constructing genomic signatures and integrating systems biology and genetic information into future recovery, prognostic, and treatment algorithms.

Ex vivo method for rapid quantification of post traumatic brain injury lesion volumes using ultrasound

BackgroundStudies of traumatic brain injury often involve the quantification of the lesion volume as a major outcome measure. The determination of lesion volume typically employs the cutting and mounting of brain tissue, and the calculation of the cross-sectional area of the lesion within each section of brain after histological staining. This is a time consuming and laborious task often requiring many weeks to determine the lesion volume for an individual brain. MethodsIn this report we present a method for determining the lesion volume within the brain following traumatic brain injury that involves the use of ultrasound imaging. With this process the lesion volume can be determined within a time period of 90 min per brain rather than weeks and months. Moreover, we have developed a pipeline that will combine the cross-sectional ultrasound images of the brain with the Allen Mouse Brain Atlas to provide the precise anatomical structures that are affected by traumatic injury to the brain. The anatomical detail was lastly paired with behavioral data showing neurological deficits correlated with specific areas of brain injury. ResultsThe accuracy and precision of this method was shown to be highly consistent with the traditional histological approach. Additionally, the mapping process and behavioral data show that neurological recovery from 1 to 3 weeks post injury is not correlated with gross anatomical recovery of the TBI lesion in our TBI model. ConclusionTogether these approaches will enhance the pipeline for processing brain tissue in experimental conditions where the lesion volume is an important outcome parameter and provide more high resolution information about the identity of the damaged regions of the brain.