Postmortem Histological Analysis Research Articles

Quantitative micro-CT-derived biomarkers elucidate age-related lung fibrosis in elder mice

BackgroundIdiopathic Pulmonary Fibrosis (IPF), prevalently affecting individuals over 60 years of age, has been mainly studied in young mouse models. The limited efficacy of current treatments underscores the need for animal models that better mimic an aged patient population. We addressed this by inducing pulmonary fibrosis in aged mice, using longitudinal micro-CT imaging as primary readout, with special attention to animal welfare.MethodsA double bleomycin dose was administered to 18–24 months-old male C57Bl/6j mice to induce pulmonary fibrosis. Bleomycin dosage was reduced to as low as 75% compared to that commonly administered to young (8–12 weeks-old) mice, resulting in long-term lung fibrosis without mortality, complying with animal welfare guidelines. After fibrosis induction, animals received Nintedanib once-daily for two weeks and longitudinally monitored by micro-CT, which provided structural and functional biomarkers, followed by post-mortem histological analysis as terminal endpoint.ResultsCompared to young mice, aged animals displayed increased volume, reduced tissue density and function, and marked inflammation. This increased vulnerability imposed a bleomycin dosage reduction to the lowest tested level (2.5 µg/mouse), inducing a milder, yet persistent, fibrosis, while preserving animal welfare. Nintedanib treatment reduced fibrotic lesions and improved pulmonary function.ConclusionsOur data identify a downsized bleomycin treatment that allows to achieve the best trade-off between fibrosis induction and animal welfare, a requirement for antifibrotic drug testing in aged lungs. Nintedanib displayed significant efficacy in this lower-severity disease model, suggesting potential patient stratification strategies. Lung pathology was quantitatively assessed by micro-CT, pointing to the value of longitudinal endpoints in clinical trials.

Bioreactor-produced iPSCs-derived dopaminergic neuron-containing neural microtissues innervate and normalize rotational bias in a dose-dependent manner in a Parkinson rat model

A breadth of preclinical studies now support the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, most cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such a format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with cryopreserved single-cell DA progenitors. Here, we used a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabling the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods, including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner using a Parkinsonian rat model. The survival of the cells was confirmed by post-mortem histological analysis, characterised by the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson's disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks after transplantation using cryopreserved 3D format.

PET imaging of synaptic vesicle glycoprotein 2 subtype A for neurological recovery in ischemic stroke.

[18F]SynVesT-1 is a novel radiopharmaceutical for assessing synaptic density in vivo. This study aims to investigate the potential of [18F]SynVesT-1 positron emission tomography (PET) in evaluating neurological recovery in the rat model of ischemic stroke, and to compare its performance with [18F]FDG PET. Sprague-Dawley rats were subjected to photothrombotic cerebral infarction, and safinamide was administered intraperitoneally from day 3 to day 14 post-stroke to alleviate neurological deficits. Cylinder test and forelimb placing test were performed to assess the neurological function. MRI, [18F]SynVesT-1 PET/CT and [18F]FDG PET/CT imaging were used to evaluate infarct volume, synaptic density, and cerebral glucose metabolism pre- and post-treatment. [18F]SynVesT-1 and [18F]FDG PET images were compared using Statistical Parametric Mapping (SPM) and region of interest (ROI)-based analysis. Post-mortem histological analysis was performed to validate PET images. Safinamide treatment improved behavioral outcomes in stroke-damaged rats. Both [18F]SynVesT-1 and [18F]FDG PET detected stroke-induced injury, with the injured region being significantly larger in [18F]FDG PET than in [18F]SynVesT-1 PET. Compared with the saline group, radiotracer uptake in the injured area significantly increased in [18F]SynVesT-1 PET after safinamide treatment, whereas no notable change was observed in [18F]FDG PET. Additionally, [18F]SynVesT-1 PET imaging showed a better correlation with neurological function recovery than [18F]FDG PET. Post-mortem analysis revealed increased neuronal numbers, synaptic density, and synaptic neuroplasticity, as well as decreased glia activation in the stroke-injured area after treatment. [18F]SynVesT-1 PET effectively quantified spatiotemporal dynamics of synaptic density in the rat model of stroke, and showed different capabilities in detecting stroke injury and neurological recovery compared with [18F]FDG PET. The utilization of [18F]SynVesT-1 PET holds promise as a potential non-invasive biomarker for evaluating ischemic stroke in conjunction with [18F]FDG PET.

Abstract Mo132: Pouring Salt in the Wound: Sex-specific Cardiometabolic Responses to Increased Dietary Fat, Sugar, and Salt.

Introduction: The general population is becoming more obese, and diabetes rates continue to rise, with overnutrition and consumption of processed foods likely contributing factors. Many people consume diets high in fat, sugar, and salt; however, this triad of factors is rarely examined in unison in studies. In addition, studies that examined diets high in fat and sugar in the context of heart failure with preserved ejection fraction and cardiometabolic outcomes have often performed short dietary interventions, with many studies performed in only male mice, and dietary salt content is often not considered or manipulated. Study Goal: This study aimed to assess the long-term impact of high-fat, high-sucrose diets with differing levels of salt (NaCl) on systemic and cardiovascular physiology in male and female mice. Methods and Results: 12-week-old C57BL/6J male and female mice ( n = 4/group) were fed one of the following diets for 25 weeks: normal chow (NC), low-fat diet (LFD; 10 kcal%), high-fat high sucrose (HFHS; 60 kcal%) with 0.33% NaCl, HFHS with 1% NaCl (HFHS1%), and HFHS with 8% NaCl (HFHS8%). Mice were subjected to basal tail blood metabolite assessment and weighed weekly. In week 24, mice were subjected to glucose tolerance testing, dual-energy X-ray absorptiometry followed by postmortem gravimetric, blood metabolite, histological, and biochemical analyses at 25 weeks. We found that irrespective of sex, body weight was significantly greater in mice receiving HFHS ( p <0.001) and HFHS1% ( p <0.001) versus the other diets. By 25 weeks, male mice were significantly larger than female mice with all diets except HFHS1% ( p <0.05), which correlated with increased percent body fat ( p <0.01), impaired glucose tolerance ( p <0.05) and increased ketone levels ( p <0.05) in female mice subjected to HFHS1% versus males on the same diet. There was also increased heart weight and atrial weights in female mice subjected to HFHS and HFHS1% versus the other females, which was not observed in male mice. Conclusions: Although previous short-term dietary studies have shown that males have altered cardiometabolic responses more than females, our findings together show that long-term dietary interventions with HFHS diets with salt may adversely impact female systemic and cardiovascular physiology.

Abstract 2017: Interleukin-10 Hydrogel-Based Therapy For Abdominal Aortic Aneurysm In Rodent Model

Abdominal aortic aneurysms (AAA) are diagnosed in over 200,000 patients per year in the United States. Endovascular AAA repair offers a strategy for those unable to undergo open operation; however there still exists a treatment gap for those whose anatomy is not suitable for current stent graft options. In response to this need for novel therapeutics to prevent aneurysm proliferation, the objective of this work was to expand AAA treatment options via anti-inflammatory hydrogel application with a handheld bioprinting device. Induction of AAA in 8-week-old, male, Sprague-Dawley rats was achieved via intraluminal infusion of porcine elastase and topical application of calcium chloride to the infrarenal aorta. Following a 4-week incubation period, rats were treated with IL-10 loaded hydrogel or blank control hydrogel. The IL-10 loaded hydrogel was placed concentrically around the aorta from the lowest renal artery to the aortic bifurcation. Rats were sacrificed 4 weeks post-treatment. Histologic analysis included hematoxylin and eosin, picrosirius red, and Verhoeff-Van Gieson for inflammatory cell infiltration, collagen and elastin content, respectively. Three-dimensional aneurysm reconstruction and analysis was performed using SkyScan1076 micro-CT at baseline, post-induction/pre-treatment and sacrifice. Immunohistochemistry (IHC) was used to examine the presence of macrophage infiltration (ED1/CD68) as a marker of inflammation. Three-dimensional reconstruction demonstrated appropriate aneurysm formation in both groups and successful aneurysm mitigation in the treatment group only. Post-mortem histologic analysis showed irregularity of the aortic intima, fibrosis, abnormal collagen organization and dramatically decreased elastin concentration in control rodents while treated rats demonstrated preservation of aortic architecture. IHC demonstrated a significant increase in macrophage infiltration in the control group when compared to treated. Taken together, the results demonstrate that peri-aortic application of an IL-10 loaded hydrogel mitigates AAA expansion in a rat model. This novel approach may be a potential strategy for treating patients with AAA whose anatomy is unsuitable for endovascular intervention.

Prenatal Programming of Monocyte Chemotactic Protein-1 Signaling in Autism Susceptibility.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that involves functional and structural defects in selective central nervous system (CNS) regions, harming the individual capability to process and respond to external stimuli, including impaired verbal and non-verbal communications. Etiological causes of ASD have not been fully clarified; however, prenatal activation of the innate immune system by external stimuli might infiltrate peripheral immune cells into the fetal CNS and activate cytokine secretion by microglia and astrocytes. For instance, genomic and postmortem histological analysis has identified proinflammatory gene signatures, microglia-related expressed genes, and neuroinflammatory markers in the brain during ASD diagnosis. Active neuroinflammation might also occur during the developmental stage, promoting the establishment of a defective brain connectome and increasing susceptibility to ASD after birth. While still under investigation, we tested the hypothesis whether the monocyte chemoattractant protein-1 (MCP-1) signaling is prenatally programmed to favor peripheral immune cell infiltration and activate microglia into the fetal CNS, setting susceptibility to autism-like behavior. In this review, we will comprehensively provide the current understanding of the prenatal activation of MCP-1 signaling by external stimuli during the developmental stage as a new selective node to promote neuroinflammation, brain structural alterations, and behavioral defects associated to ASD diagnosis.

Cardiac Fibrosis and Innervation State in Uncorrected and Corrected Transposition of the Great Arteries: A Postmortem Histological Analysis and Systematic Review

In the transposition of the great arteries (TGA), alterations in hemodynamics and oxygen saturation could result in fibrotic remodeling, but histological studies are scarce. We aimed to investigate fibrosis and innervation state in the full spectrum of TGA and correlate findings to clinical literature. Twenty-two human postmortem TGA hearts, including TGA without surgical correction (n = 8), after Mustard/Senning (n = 6), and arterial switch operation (ASO, n = 8), were studied. In newborn uncorrected TGA specimens (1 day-1.5 months), significantly more interstitial fibrosis (8.6% ± 3.0) was observed compared to control hearts (5.4% ± 0.8, p = 0.016). After the Mustard/Senning procedure, the amount of interstitial fibrosis was significantly higher (19.8% ± 5.1, p = 0.002), remarkably more in the subpulmonary left ventricle (LV) than in the systemic right ventricle (RV). In TGA-ASO, an increased amount of fibrosis was found in one adult specimen. The amount of innervation was diminished from 3 days after ASO (0.034% ± 0.017) compared to uncorrected TGA (0.082% ± 0.026, p = 0.036). In conclusion, in these selected postmortem TGA specimens, diffuse interstitial fibrosis was already present in newborn hearts, suggesting that altered oxygen saturations may already impact myocardial structure in the fetal phase. TGA-Mustard/Senning specimens showed diffuse myocardial fibrosis in the systemic RV and, remarkably, in the LV. Post-ASO, decreased uptake of nerve staining was observed, implicating (partial) myocardial denervation after ASO.

Downregulation of striatal CaV1.3 inhibits the escalation of levodopa-induced dyskinesia in male and female parkinsonian rats of advanced age

In the past 25 years, the prevalence of Parkinson's disease (PD) has nearly doubled. Age remains the primary risk factor for PD and as the global aging population increases this trend is predicted to continue. Even when treated with levodopa, the gold standard dopamine (DA) replacement therapy, individuals with PD frequently develop therapeutic side effects. Levodopa-induced dyskinesia (LID), a common side effect of long-term levodopa use, represents a significant unmet clinical need in the treatment of PD. Previously, in young adult (3-month-old) male parkinsonian rats, we demonstrated that the silencing of CaV1.3 (Cacan1d) L-type voltage-gated calcium channels via striatal delivery of rAAV-CaV1.3-shRNA provides uniform protection against the induction of LID, and significant reduction of established severe LID. With the goal of more closely replicating a clinical demographic, the current study examined the effects of CaV1.3-targeted gene therapy on LID escalation in male and female parkinsonian rats of advanced age (18-month-old at study completion). We tested the hypothesis that silencing aberrant CaV1.3 channel activity in the parkinsonian striatum would prevent moderate to severe dyskinesia with levodopa dose escalation. To test this hypothesis, 15-month-old male and female F344 rats were rendered unilaterally parkinsonian and primed with low-dose (3–4 mg/kg) levodopa. Following the establishment of stable, mild dyskinesias, rats received an intrastriatal injection of either the Cacna1d-specific rAAV-CaV1.3-shRNA vector (CAV-shRNA), or the scramble control rAAV-SCR-shRNA vector (SCR-shRNA). Daily (M-Fr) low-dose levodopa was maintained for 4 weeks during the vector transduction and gene silencing window followed by escalation to 6 mg/kg, then to 12 mg/kg levodopa. SCR-shRNA-shRNA rats showed stable LID expression with low-dose levodopa and the predicted escalation of LID severity with increased levodopa doses. Conversely, complex behavioral responses were observed in aged rats receiving CAV-shRNA, with approximately half of the male and female subjects—therapeutic ‘Responders’—demonstrating protection against LID escalation, while the remaining half—therapeutic ‘Non-Responders’—showed LID escalation similar to SCR-shRNA rats. Post-mortem histological analyses revealed individual variability in the detection of Cacna1d regulation in the DA-depleted striatum of aged rats. However, taken together, male and female therapeutic ‘Responder’ rats receiving CAV-shRNA had significantly less striatal Cacna1d in their vector-injected striatum relative to contralateral striatum than those with SCR-shRNA. The current data suggest that mRNA-level silencing of striatal CaV1.3 channels maintains potency in a clinically relevant in vivo scenario by preventing dose-dependent dyskinesia escalation in rats of advanced age. As compared to the uniform response previously reported in young male rats, there was notable variability between individual aged rats, particularly females, in the current study. Future investigations are needed to derive the sex-specific and age-related mechanisms which underlie variable responses to gene therapy and to elucidate factors which determine the therapeutic efficacy of treatment for PD.

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy

Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.

Abstract TP231: Low Hemoglobin Causes Cerebral Hypoxia Resulting In Changes In Blood-brain Barrier Function In Mice

Introduction: Low hemoglobin is associated with stroke and poor cerebrovascular outcomes. It is unclear whether hemoglobin concentrations directly impact cerebrovascular health. Thus, we sought to investigate the role of hemoglobin concentration on cerebral hypoxia and blood brain barrier (BBB) function in murine models of anemia. Hypothesis: Low hemoglobin will cause relevant cerebral hypoxic changes and impair BBB function. Methods: Two different models of anemia and their respective controls were generated from 30 8-week-old, female C57/BL6 mice. The separate cohorts generated included: 1) an acute anemia model cohort via anti-TER119 injection (red blood cell hemolysis) compared to IgG control injected mice; 2) chronic anemia model cohort via iron-deficient chow compared to iron replete diet control. Hemoglobin concentrations were assessed using modified Drabkin assays. Mice were euthanized by intracardiac perfusion with saline and fixative and the brain was dissected and processed for histological analysis of vascular permeability (IgG), microglia number and activation (Iba1 and CD68), response to hypoxia (HIF1α) and endothelial markers of BBB function (GLUT1). Statistical analyses were performed using two-tailed Student's t-tests. Results: Hemoglobin concentrations were lower in our separate anemia model mice compared to their respective controls (acute anemia: 5.5 ± 4.02 vs 13.1 ± 1.69, p=0.007; chronic anemia: 7.9 ± 1.05 vs 11.9 ± 2.31, p=0.0006). Postmortem histological analyses revealed stronger microglial activation (p<0.005) and higher microglial expression of hypoxia-response protein HIF1α (p<0.05) in both acute and chronic anemia models compared to their respective controls. Additionally, anemic mice had increased vascular permeability to serum IgG and decreased expression of the endothelial-specific glucose transporter GLUT1 (p<0.05). Conclusions: Anemic mice appear to display stronger microglial activation, greater HIF1α expression, and BBB dysfunction compared to non-anemic mice. Future studies will be needed to further clarify cellular and molecular mechanisms driving relationships between anemia, hypoxia, BBB dysfunction and clinical outcomes in cerebrovascular disease.

Identification of vacuolar autophagic aggregates in the skeletal muscles of inbred C57BL/6NCrl mice.

A comprehensive pathological analysis of inbred strains is essential to define strain-specific spontaneous lesions and to understand whether a specific phenotype results from experimental intervention or reflects a naturally occurring disease. This study aimed to report and describe a novel condition affecting the skeletal muscles of an inbred C57BL/6NCrl mouse colony characterised by large sarcoplasmic vacuoles in the muscle fibres of male mice in the subsarcolemmal spaces and the intermyofibrillary network. There was no muscle weakness, loss of ambulation or cardiac/respiratory involvement. Post-mortem evaluation and histological analysis excluded the presence of pathological accumulations or lesions in other tissues and organs. Changes were seen in fibre size, with many hypotrophic and some slightly hypertrophic fibres. Histological, immunohistochemical and molecular analyses of the vacuolar content revealed dysregulation of the autophagy machinery while ruling out a morphologically similar condition marked by the accumulation of tubular aggregates.

Histopathological Spectrum of Cardiac Lesions in Sudden Cardiac DeathAn Autopsy Study

Introduction: Sudden Cardiac Death (SCD) is a serious health concern, and the incidence of SCD is rising globally. A number of causes can result in SCD in apparently healthy individuals and in people with undiagnosed cardiac disease. The study was done to evaluate the probable cause of death by observing various histomorphological changes in cardiac autopsies. Aim: To establish the cause of SCD and study the histopathology, age and sex distribution, frequency, and location of different types of cardiac lesions. Materials and Methods: The study was a descriptive, crosssectional, observational study carried out in the Department of Pathology and Forensic Medicine and Toxicology (FMT) at PRM Medical College and Hospital, Baripada, Odisha, over a period of three years. Gross and microscopic findings on Haematoxylin & Eosin (H&E)-stained cardiac sections were studied. The final diagnosis was made on the basis of clinical, autopsy, and histopathological findings. The study was compared with other relevant studies. The data was analysed using Microsoft Excel 2019 software. Results: A total of 164 cases of SCD were included in this study. The maximum number of deaths occurred in the age group of 51-60 years (42 cases). The male to female ratio was 2.6:1, indicating an overall male preponderance. Out of 164 autopsied hearts, 85 cases of Ischemic Heart Disease (IHD), including new, old, and mixed lesions were found, followed by Hypertrophic Cardiomyopathy (HCM) in 25 cases, multiple lesions in 12 cases, dilated cardiomyopathy in four cases, tubercular pericarditis with myocarditis in one case, infective endocarditis in three cases, atherosclerosis in one case, coronary insufficiency in one case, cardiac myxoma in one case. Conclusion: Many factors can lead to SCD in apparently healthy individuals or in people with cardiac disease. In the present study, the most common cause contributing to SCD was IHD. The cause of SCD can be identified by a thorough post-mortem examination and histological analysis.

219 ISOLATED CORONARY OSTIAL STENOSIS DUE TO RECURRENT AORTIC WALL THROMBOSIS

Abstract Background Coronary ostial lesions are a rare entity and isolated coronary ostial stenosis is even less frequent. Diffuse coronary artery atherosclerosis is the main cause for the non-isolated stenosis, based on old post-mortem series and histological analysis of tissue removed during atherectomies. Case presentation A 49-year-old woman suffering from mild non-familial hypercholesterolemia reported to her family physician repeated episodes of chest pain related to exercise. During a cardiological outpatient evaluation, 12 lead electrocardiogram (ECG) and echocardiography turned out normal. During a stress test she had recurrent chest pain coupled with ECG ischemic changes (diffuse ST segment elevation). She was immediately referred to the emergency department where the ECG was back to normal and the troponin dosage within normal range. Coronary angiography was performed: non-selective cannulation revealed some irregularities of the aortic wall profile at the level of the right coronary ostium, with inability to visualize the right coronary artery. The selective cannulation of the left main coronary artery (LMCA), left anterior descending (LAD) and left circumflex branches showed a critical ostial stenosis at the origin of the LMCA without any other relevant lumen restriction, in a left-dominant pattern of coronary circulation. A suspicion of LAD dissection was raised when a laminar flux was observed at its origin. At the end of the procedure, the patient suddenly collapsed. The recorded rhythm was initially bradycardia, evolving to episodes of ventricular tachycardia for which multiple shocks were applied. Despite the resuscitation efforts, the patient never recovered. Autopsy was performed at the local hospital. At gross analysis, a non-uniform pale-tan thickening at the level of the sino-tubular junction was evident with involvement of both the right and left coronary ostia. Histological examination allowed to ascribe the thickening to recurrent thrombosis, without any underlying inflammatory process at the level of the aortic wall and with only minimal atherosclerotic plaques. No coronary artery dissection nor significant stenosis was present in all the coronary tree. Regarding the ventricular myocardium, one focus of replacement-type fibrosis was identified at subendocardial level in the infero-septal wall coupled with a small spot of loose connective tissue rich in hemosiderin-laden macrophages in the same region, consistent with an area of subacute ischemic damage. Conclusion Several series reported a higher frequency of isolated coronary ostial stenosis in women versus men. In fact, female sex along with hypertriglyceridemia have been recognized as independent risk factors for ostial lesions. The case herein reported highlights the diagnostic relevance of the autopsy investigation together with clinicopathological correlation, particularly in cases where the definite diagnosis can't be achieved in vivo.

Early Joint Use Following Elbow Dislocation Limits Range-of-Motion Loss and Tissue Pathology in Posttraumatic Joint Contracture.

Simple elbow dislocation occurs at an incidence of 2.9 to 5.21 dislocations per 100,000 person-years, with as many as 62% of these patients experiencing long-term elbow joint contracture, stiffness, and/or pain. Poor outcomes and the need for secondary surgical intervention can often be prevented nonoperatively with early or immediate active mobilization and physical therapy. However, immobilization or limited mobilization may be necessary following trauma, and it is unknown how different periods of immobilization affect pathological changes in elbow joint tissue and how these changes relate to range of motion (ROM). The purpose of this study was to investigate the effects of varying the initiation of free mobilization on elbow ROM and histological features in an animal model of elbow posttraumatic joint contracture. Traumatic elbow dislocation was surgically induced unilaterally in rats. Injured forelimbs were immobilized in bandages for 3, 7, 14, or 21 days; free mobilization was then allowed until 42 days after injury. Post-mortem joint ROM testing and histological analysis were performed. One-way analysis of variance was used to compare ROM data between control and injured groups, and Pearson correlations were performed between ROM parameters and histological outcomes. Longer immobilization periods resulted in greater ROM reductions. The anterior and posterior capsule showed increases in cellularity, fibroblasts, adhesions, fibrosis, and thickness, whereas the measured outcomes in cartilage were mostly unaffected. All measured histological characteristics of the capsule were negatively correlated with ROM, indicating that higher degrees of pathology corresponded with less ROM. Longer immobilization periods resulted in greater ROM reductions, which correlated with worse histological outcomes in the capsule in an animal model of posttraumatic elbow contracture. The subtle differences in the timing of ROM and capsule tissue changes revealed in the present study provide new insight into the distinct timelines of biomechanical changes as well as regional tissue pathology. This study showed that beginning active mobilization 3 days after injury minimized posttraumatic joint contracture, thereby supporting an immediate-motion clinical treatment strategy (when possible). Furthermore, uninjured but pathologically altered periarticular tissues near the injury location may contribute to more severe contracture during longer immobilization periods as the disease state progresses.

Abstract 10128: Recombinant Human Placental Growth Factor-2 Treatment in Porcine Ischemic Cardiomyopathy

Background: Placental growth factor (PlGF)-2 induces neovascularization and improves myocardial function in atherosclerotic rodent myocardial ischemia models without stimulating plaque angiogenesis. This promising therapeutic profile of PlGF-2 warrants a double blind, randomized, and placebo-controlled feasibility and efficacy study in a large animal model, representative of human disease. Methods: We induced myocardial infarction (MI) in domestic pigs using a 75 min balloon occlusion of the mid-LAD followed by reperfusion. After 4 w, we randomized pigs with marked LV dysfunction (LVEF<40%) to continuous IV infusion of 5, 15, 45 μg/kg/day rhPlGF-2 or PBS (CON) for 2 w using osmotic pumps. We evaluated treatment effect 8 w after MI using comprehensive MRI analysis of LV function and structure and performed postmortem histological analyses of LV vascularization and fibrotic remodeling. Results: In the pilot study (3 pigs/dose), PIGF plasma concentrations showed stable and sustained dose-dependent increases for 28 days without organ-specific side effects or systemic toxicity. Myocardial Ischemia/reperfusion induced LV dysfunction at 4 w (infarct size, 16±4 % of LV mass, n=42; LVEF, 34±4 %, range 19%-40%). In the dose escalation study using 5 (n=10), 15 or 45 μg/kg/day PlGF (n=11 per group), global LV function, and regional myocardial blood flow at rest or during stress, and infarct size did not differ between CON (n=10) and PlGF (Figure 1). Capillary densities and extent of LV interstitial fibrosis in infarct core and border zone were comparable between CON and PlGF. Conclusions: In this blinded randomized controlled trial, rhPIGF-2 did not improve global cardiac function in a porcine model of chronic ischemic cardiomyopathy, and failed to recapitulate therapeutic neovascularization reported in rodents. Our data emphasize the critical need for robust double blind, placebo controlled trials in representative large animal models before clinical translation.

Molecular Imaging of Ultrasound-Mediated Blood-Brain Barrier Disruption in a Mouse Orthotopic Glioblastoma Model.

Glioblastoma (GBM) is an aggressive and malignant primary brain tumor. The blood-brain barrier (BBB) limits the therapeutic options available to tackle this incurable tumor. Transient disruption of the BBB by focused ultrasound (FUS) is a promising and safe approach to increase the brain and tumor concentration of drugs administered systemically. Non-invasive, sensitive, and reliable imaging approaches are required to better understand the impact of FUS on the BBB and brain microenvironment. In this study, nuclear imaging (SPECT/CT and PET/CT) was used to quantify neuroinflammation 48 h post-FUS and estimate the influence of FUS on BBB opening and tumor growth in vivo. BBB disruptions were performed on healthy and GBM-bearing mice (U-87 MG xenograft orthotopic model). The BBB recovery kinetics were followed and quantified by [99mTc]Tc-DTPA SPECT/CT imaging at 0.5 h, 3 h and 24 h post-FUS. The absence of neuroinflammation was confirmed by [18F]FDG PET/CT imaging 48 h post-FUS. The presence of the tumor and its growth were evaluated by [68Ga]Ga-RGD2 PET/CT imaging and post-mortem histological analysis, showing that tumor growth was not influenced by FUS. In conclusion, molecular imaging can be used to evaluate the time frame for systemic treatment combined with transient BBB opening and to test its efficacy over time.

Neurodegeneration in multiple sclerosis.

Axonal loss in multiple sclerosis (MS) is a key component of disease progression and permanent neurologic disability. MS is a heterogeneous demyelinating and neurodegenerative disease of the central nervous system (CNS) with varying presentation, disease courses, and prognosis. Immunomodulatory therapies reduce the frequency and severity of inflammatory demyelinating events that are a hallmark of MS, but there is minimal therapy to treat progressive disease and there is no cure. Data from patients with MS, post-mortem histological analysis, and animal models of demyelinating disease have elucidated patterns of MS pathogenesis and underlying mechanisms of neurodegeneration. MRI and molecular biomarkers have been proposed to identify predictors of neurodegeneration and risk factors for disease progression. Early signs of axonal dysfunction have come to light including impaired mitochondrial trafficking, structural axonal changes, and synaptic alterations. With sustained inflammation as well as impaired remyelination, axons succumb to degeneration contributing to CNS atrophy and worsening of disease. These studies highlight the role of chronic demyelination in the CNS in perpetuating axonal loss, and the difficulty in promoting remyelination and repair amidst persistent inflammatory insult. Regenerative and neuroprotective strategies are essential to overcome this barrier, with early intervention being critical to rescue axonal integrity and function. The clinical and basic research studies discussed in this review have set the stage for identifying key propagators of neurodegeneration in MS, leading the way for neuroprotective therapeutic development. This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology Neurological Diseases > Molecular and Cellular Physiology.

No Reduction in the 226-Hz Probe Tone Acoustic Reflex Amplitude Following Severe Inner Hair Cell Loss in Chinchillas.

The relationship between the middle ear acoustic reflex (AR) and inner hair cell (IHC) loss is currently unknown. Given that IHC are believed to convey nearly all acoustic information to the central auditory nervous system, it has been assumed that loss of IHC would significantly impact the AR. To evaluate this relationship, we assessed the presence and amplitude of the AR in chinchillas before and after treatment with carboplatin, an anticancer drug that reliably and selectively destroys IHC in this species. Baseline measures of hearing sensitivity, including auditory brainstem response (ABR) thresholds and distortion product otoacoustic emissions (DPOAE), were assessed and then re-evaluated following carboplatin treatment. Post-carboplatin ABR thresholds and DPOAE were found to be unchanged or slightly elevated; results were consistent with published reports. Our main hypothesis was that loss of IHC would abolish the reflex or significantly reduce its amplitude. Contrary to our hypothesis, the ipsilateral 226-Hz AR continued to be reliably elicited following carboplatin treatment. Post-mortem histological analysis confirmed significant IHC loss (65-85 %), but no measurable loss of outer hair cells (OHCs). Given that loss of IHC alone does not significantly reduce the 226-Hz AR, our results suggest that few IHC are needed to maintain the 226-Hz AR response. These results suggest additional studies are needed to better understand the role of IHC in the reflex arc, present opportunities to further study the reflex pathway, and could change how we use the clinical AR as a potential diagnostic tool for IHC dysfunction, including those related to IHC synaptopathy.

Accumulation of pathological Ataxin‐7 in the medulla leads to hypoglossal (XII) motor unit pathology

SCA7 is an autosomal dominant neurological disorder caused by a deleterious CAG repeat expansion in the coding region of the ataxin‐7 gene on chromosome 3. Patients with infantile SCA‐7 have the largest repeat expansion characterized by progressive loss of coordination, dysarthria, dysphagia, retinal degeneration and respiratory distress. These infants succumb to death due to severe hypotonia, aspiration pneumonia and respiratory failure. Our objective is to comprehensively analyze the underlying mechanisms that result in upper airway pathology which exacerbates respiratory insufficiency in SCA7. To do this, we will use an infantile SCA7 mouse model: SCA7266Q Knock‐in (KI) mouse. SCA7266Q KI mice exhibit progressive respiratory dysfunction along with irregular breathing and prolonged apneic events with a significant loss of hypoglossal (XII) motoneurons in the medulla. The XII motoneurons innervate the tongue to maintain the shape, stiffness and position of the tongue and regulate the upper airway patency. Therefore, we hypothesize that the expression of pathological ataxin‐7 in the medulla causes XII motor unit pathology leading to upper airway pathology. To confirm if the reduced number of motoneurons resulted in hypoglossal nerve pathology, we analyzed the efferent nerve output of the XII nerve of 9‐week‐old WT control and SCA72666Q KI mice during eupnea and hypercapnia (7% CO2). Later we performed post‐mortem histological analysis of the XII respiratory centers in the medulla and XII nerves. Specifically, we used immunohistochemistry to identify the cells that accumulate ataxin‐7 and we performed TUNEL assay to evaluate the extent of neurodegeneration in the XII motor pool. We analyzed the demyelination of the nerves by light microscopy and electron microscopy. To identify the cellular mechanisms involved in the XII pathology, we performed transcriptome analysis by bulk RNA sequencing of the medulla and confirmed the findings with qPCR.ResultsXII nerve efferent output is blunted at baseline and hypercapnic challenge in SCA7266QKI mice. There is increased TUNEL+ cells in the XII motor pool with intranuclear accumulation of ataxin‐7 in the astrocytes. Further, demyelination is present in the XII nerves in SCA7266QKI mice. RNA sequencing data of the medulla supports these findings and reveals diminished expression of the transcripts regulating the myelination pathway such as Plp1. There is also a significant decrease in activation of astrocytes with attenuated expression of neurotransmitter transporter such as Slc6a9 and Slc710 that are critical for breathing. In conclusion, intranuclear accumulation of ataxin‐7 in SCA7266QKI mice modulates transcription of astrocytes and influences different signaling pathways that result in neurodegeneration, demyelination, and decreased efferent XII nerve output. These findings are clinically relevant and emphasize the importance of neuropathology that results in dysphagia, aspiration pneumonia and respiratory failure in infantile SCA7.

Impact of Limb Phenotype on Tongue Denervation Atrophy, Dysphagia Penetrance, and Survival Time in a Mouse Model of ALS.

Current treatments for dysphagia in ALS do not target the underlying tongue weakness and denervation atrophy that is prevalent in spinal and bulbar ALS cases. To address this clinical gap, we studied the low copy number SOD1-G93A (LCN-SOD1) mouse model of ALS to quantify the impact of limb phenotype on tongue denervation atrophy, dysphagia penetrance, and survival time in preparation for future treatment-based studies. Two male LCN-SOD1 breeders and 125 offspring were followed for limb phenotype inheritance, of which 52 (30 LCN-SOD1 and 22 wild-type/WT, both sexes) underwent characterization of dysphagia penetrance (via videofluoroscopic swallow study; VFSS) and survival time at disease end-stage (15-20% body weight loss). From these, 16 mice (8/genotype) underwent postmortem histological analysis of the genioglossus for evidence of denervation atrophy. Results revealed that both breeders displayed a mixed (hindlimb and forelimb) ALS phenotype and sired equal proportions of hindlimb vs. mixed phenotype offspring. Dysphagia penetrance was complete for mixed (100%) versus incomplete for hindlimb (64%) phenotype mice; yet survival times were similar. Regardless of limb phenotype, LCN-SOD1 mice had significantly smaller genioglossus myofibers and more centralized myonuclei compared to WT mice (p < 0.05). These biomarkers of denervation atrophy were significantly correlated with VFSS metrics (lick and swallow rates, p < 0.05) but not survival time. In conclusion, both LCN-SOD1 phenotypes had significant tongue denervation atrophy, even hindlimb phenotype mice without dysphagia. This finding recapitulates human ALS, providing robust rationale for using this preclinical model to explore targeted treatments for tongue denervation atrophy and ensuing dysphagia.