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Cathepsin B Relocalization in Late Membrane Disrupted Neurons Following Diffuse Brain Injury in Rats.

ABSTRACT: Traumatic brain injury (TBI) has consequences that last for years following injury. While TBI can precipitate a variety of diffuse pathologies, the mechanisms involved in injury-induced neuronal membrane disruption remain elusive. The lysosomal cysteine protease, Cathepsin B (Cath B), and specifically its redistribution into the cytosol has been implicated in cell death. Little is known about Cath B or neuronal membrane disruption chronically following diffuse TBI. Therefore, the current study evaluated Cath B and diffuse neuronal membrane disruption over a more chronic post-injury window (6 h-4 w). We evaluated Cath B in adult male Sprague-Dawley rats following central fluid percussion injury (CFPI). Expression of Cath B, as well as Cath B-associated pro (Bak and AIF) and anti-apoptotic (Bcl-xl) proteins, were assessed using western blot analysis. Cath B activity was also assessed. Localization of Cath B was evaluated in the membrane disrupted and non-disrupted population following CFPI using immunohistochemistry paired with quantitative image analysis and ultrastructural verification. There was no difference in expression or activity of Cath B or any of the associated proteins between sham and CFPI at any time post-injury. Immunohistological studies, however, showed a sub-cellular re-localization of Cath B at 2 w and 4 w post-injury in the membrane disrupted neuronal population as compared to the time-point matched non-disrupted neurons. Both membrane disruption and Cath B relocalization appear linked to neuronal atrophy. These observations are indicative of a late secondary pathology that represents an opportunity for therapeutic treatment of these neurons following diffuse TBI. Summary Statement Lysosomal cathepsin B relocalizes to the cytosol in neurons with disrupted plasmalemmal membranes weeks following diffuse brain injury. Both the membrane disrupted and cathepsin B relocalized neuronal subpopulations displayed smaller soma and nucleus size compared to non-pathological neurons, indicating atrophy.

SUBMITTER: Hernandez ML

PROVIDER: S-EPMC9069603 | biostudies-literature | 2022 Jan-Dec

REPOSITORIES: biostudies-literature

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Cathepsin B Relocalization in Late Membrane Disrupted Neurons Following Diffuse Brain Injury in Rats.

Hernandez Martina L ML Marone Michael M Gorse Karen M KM Lafrenaye Audrey D AD

ASN neuro 20220101

Traumatic brain injury (TBI) has consequences that last for years following injury. While TBI can precipitate a variety of diffuse pathologies, the mechanisms involved in injury-induced neuronal membrane disruption remain elusive. The lysosomal cysteine protease, Cathepsin B (Cath B), and specifically its redistribution into the cytosol has been implicated in cell death. Little is known about Cath B or neuronal membrane disruption chronically following diffuse TBI. Therefore, the current study e ...[more]

PMID: 35503242

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Project description:The repercussions of traumatic brain injury (TBI) endure years following the initial insult and involve chronic impairments/disabilities. Studies indicate that these morbidities stem from diffuse pathologies, however, knowledge regarding TBI-mediated diffuse pathologies, and in particular, diffuse neuronal membrane disruption, is limited. Membrane disruption has been shown to occur acutely following injury, primarily within neurons, however, the progression of TBI-induced membrane disruption remains undefined. Therefore, the current study investigated this pathology over a longer temporal profile from 6 h to 4 w following diffuse TBI induced using the central fluid percussion injury (CFPI) model in rats. To visualize membrane disruption, animals received an intracerebroventricular infusion of tagged cell-impermeable dextran 2 h prior to experimental endpoints at 6 h, 1 d, 3 d, 1 w, 2 w, or 4 w post-CFPI. The percentage of total neurons demonstrating dextran uptake, indicative of membrane disruption, was quantified within the lateral neocortex layers V and VI from 6 h to 4 w post-injury. We found that membrane disruption displayed a biphasic pattern, where nearly half of the neurons were membrane disrupted sub-acutely, from 6 h to 3 d post-TBI. At 1 w the membrane disrupted population was dramatically reduced to levels indistinguishable from sham controls. However, by 2 and 4 w following CFPI, approximately half of the neurons analyzed displayed membrane disruption. Moreover, our data revealed that a subset of these late membrane disrupted neurons were NeuN negative (NeuN-). Correlative western blot analyses, however, revealed no difference in NeuN protein expression in the lateral neocortex at any time following injury. Furthermore, the NeuN- membrane disrupted neurons did not co-label with traditional markers of astrocytes, microglia, oligodendrocytes, or NG2 cells. Immunohistochemistry against NeuN, paired with a hematoxylin and eosin counter-stain, was performed to quantify the possibility of overall NeuN+ neuronal loss following CFPI. A NeuN- population was observed consistently in both sham and injured animals regardless of time post-injury. These data suggest that there is a consistent subpopulation of NeuN- neurons within the lateral neocortex regardless of injury and that these NeuN- neurons are potentially more vulnerable to late membrane disruption. Better understanding of membrane disruption could provide insight into the mechanisms of diffuse pathology and lead to the discovery of novel treatments for TBI.

Project description:BackgroundDiffuse traumatic brain injury (TBI) is known to lead to microstructural changes within both white and grey matter detected in vivo with diffusion tensor imaging (DTI). Numerous studies have shown alterations in fractional anisotropy (FA) and mean diffusivity (MD) within prominent white matter tracts, but few have linked these to changes within the grey matter with confirmation via histological assessment. This is especially important as alterations in the grey matter may be predictive of long-term functional deficits.MethodsA total of 33 male Sprague Dawley rats underwent severe closed-head TBI. Eight animals underwent tensor-based morphometry (TBM) and DTI at baseline (pre-TBI), 24 hours (24 h), 7, 14, and 30 days post-TBI. Immunohistochemical analysis for the detection of ionised calcium-binding adaptor molecule 1 (IBA1) to assess microglia number and percentage of activated cells, β-amyloid precursor protein (APP) as a marker of axonal injury, and myelin basic protein (MBP) to investigate myelination was performed at each time-point.ResultsDTI showed significant alterations in FA and RD in numerous white matter tracts including the corpus callosum, internal and external capsule, and optic tract and in the grey-matter in the cortex, thalamus, and hippocampus, with the most significant effects observed at 14 D post-TBI. TBM confirmed volumetric changes within the hippocampus and thalamus. Changes in DTI were in line with significant axonal injury noted at 24 h post-injury via immunohistochemical analysis of APP, with widespread microglial activation seen within prominent white matter tracts and the grey matter, which persisted to 30 D within the hippocampus and thalamus. Microstructural alterations in MBP+ve fibres were also noted within the hippocampus and thalamus, as well as the cortex.ConclusionThis study confirms the widespread effects of diffuse TBI on white matter tracts which could be detected via DTI and extends these findings to key grey matter regions, with a comprehensive investigation of the whole brain. In particular, the hippocampus and thalamus appear to be vulnerable to ongoing pathology post-TBI, with DTI able to detect these alterations supporting the clinical utility in evaluating these regions post-TBI.

Project description:Traumatic brain injury (TBI) is a global health problem affecting millions of individuals annually, potentially resulting in persistent neuropathology, chronic neurological deficits, and death. However, TBI not only affects neural tissue, but also affects the peripheral immune system's homeostasis and physiology. TBI disrupts the balanced signaling between the brain and the peripheral organs, resulting in immunodysregulation and increasing infection susceptibility. Indeed, secondary infections following TBI worsen neurological outcomes and are a major source of mortality and morbidity. Despite the compelling link between the damaged brain and peripheral immune functionality, little is known about how injury severity affects the peripheral immune system in closed-head diffuse TBI, the most common clinical presentation including all concussions. Therefore, we characterized peripheral blood mononuclear cells (PBMCs) and plasma changes over time and across injury severity using an established large-animal TBI model of closed-head, non-impact diffuse rotational acceleration in pigs. Across all timepoints and injury levels, we did not detect any changes to plasma cytokine concentrations. However, changes to the PBMCs were detectable and much more robust. We observed the concentration and physiology of circulating PBMCs changed in an injury severity-dependent manner, with most cellular changes occurring within the first 10 days following a high rotational velocity injury. Here, we report changes in the concentrations of myeloid and T cells, changes in PBMC composition, and changes in phagocytic clearance over time. Together, these data suggest that following a diffuse brain injury in a clinically relevant large-animal TBI model, the immune system exhibits perturbations that are detectable into the subacute timeframe. These findings invite future investigations into therapeutic interventions targeting peripheral immunity and the potential for peripheral blood cellular characterization as a diagnostic tool.

Project description:Traumatic brain injury (TBI) is a common phenomenon, accounting for significant cost and adverse health effects. While there is information about focal pathologies following TBI, knowledge of more diffuse processes is lacking, particularly regarding how analgesics affect this pathology. As buprenorphine is the most commonly used analgesic in experimental TBI models, this study investigated the acute effects of the opioid analgesic buprenorphine (Bup-SR-Lab) on diffuse neuronal/glial pathology, neuroinflammation, cell damage, and systemic physiology. We utilized a model of central fluid percussion injury (CFPI) in adult male rats treated with a single subcutaneous bolus of Bup-SR-Lab or saline 15 min post-injury. Microscopic assessments were performed at 1 day post-injury. Cell impermeable dextran was infused intraventricularly prior to sacrifice to assess neuronal membrane disruption. Axonal injury was assessed by investigating labeling of the anterogradely transported amyloid precursor protein. Neuroinflammation was assessed by analyzing Iba-1 + microglial and GFAP + astrocyte histological/morphological features as well as cytokine levels in both regions of interest (ROIs). Myelin pathology was assessed by evaluating the expression of myelin basic protein (MBP) and the propensity of MBP + myelin debris. Acute physiologic data showed no difference between groups except for reduction in weight loss following cFPI in Bup treated animals compared to saline. There were no discernable differences in axonal injury or membrane disruption between treatment groups. Cytokine levels were consistent between Bup and saline treated animals, however, microglia and astrocytes revealed region specific histological changes at 1d following Bup treatment. Myelin integrity and overall MBP expression showed no differences between Bup and saline treated animals, but there were significant regional differences in MBP expression between the cortex and thalamus. These data suggest effects of Bup treatment on weight following CFPI and potential regional specificity of Bup-associated microglial and astrocyte alterations, but very little change in other acute pathology at 1-day post-injury. Overall, this preliminary study indicates that use of Bup-SR-Lab in preclinical work does have effects on acute glial pathology, however, longer term studies will be needed to assess potential effects of Bup treatment on more chronic pathological progressions.

Dataset Information

Cathepsin B Relocalization in Late Membrane Disrupted Neurons Following Diffuse Brain Injury in Rats.

Publications

Cathepsin B Relocalization in Late Membrane Disrupted Neurons Following Diffuse Brain Injury in Rats.

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