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.

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.

Traumatic brain injury (TBI) affects millions of people each year. Previous studies using the central fluid percussion injury (CFPI) model in adult male rats indicated that elevated intracranial pressure (ICP) was associated with long-term effects, including neuronal cell loss and increased sensory sensitivity post-injury and secondary ICP elevation, which were not seen following injury alone. Investigations also indicated that cathepsin B (Cath B), a lysosomal cysteine protease, may play a role in the pathological progression of neuronal membrane disruption; however, the specific impact of Cath B inhibition following CFPI remained unknown. Thus, the focus of this study was to evaluate the effects of Cath B inhibition via the intracerebroventricular infusion of the Cath B inhibitor to the CA-074 methyl ester (CA-074Me) 2w following injury with or without secondary ICP elevation. This was accomplished using adult male rats continuously infused with CA-074Me or 10% DMSO as a vehicle control for 2w following either sham injury, CFPI only, or CFPI with subsequent ICP elevation to 20 mmHg. We assessed Cath B activity and evaluated the protein levels of Cath B and Cath B-binding partners AIF, Bcl-XL, and Bak. We also conducted histological analyses of the total cell counts to assess for cell loss, membrane disruption, and Cath B localization. Finally, we investigated somatosensory changes with the whisker nuisance task. Overall, this study demonstrated that Cath B is not a direct driver of membrane disruption; however, the administration of CA-074Me alters Cath B localization and reduces hypersensitivity, emphasizing Cath B as an important component in late secondary pathologies.

Traumatic brain injury (TBI) remains a significant global public health epidemic with adverse health and cost implications. Due to its complex, heterogeneous nature and wide-ranging impacts, definitive TBI treatments remain elusive. As such, continued laboratory research using animal models is warranted. In accordance with guidelines set forth for the humane treatment of research animals, TBI animal models are often administered analgesics for pain management. The choice of drug, timing, dose, and formulation of analgesic can vary depending on the study’s unique needs and can potentially and unintentionally influence experimental results. In TBI studies utilizing rats as animal models, buprenorphine is a common analgesic administered. In addition to pain management in such studies, investigators must also monitor the research animals post-operatively and make the decision for humane euthanasia before intended experimental survival timepoint if the animals are assessed to be excessively suffering. This study investigated the differences in adult, male Sprague Dawley rats used for various TBI studies that reached weight-loss-induced humane endpoints following a single administration of buprenorphine slow-release LAB (bup-SR-LAB) or buprenorphine slow-release HCl (bup-SR-HCl). Our findings indicate that TBI-induced rats receiving bup-SR-LAB in conjunction with a secondary surgical insult such as artificial intracranial pressure elevation and/or osmotic pump implantation reach a weight-loss-induced humane euthanasia endpoint more often compared to sham-injured rats. When stratifying into the same groups, we did not find this pattern to hold true for rats administered bup-SR-HCl. Overall, this study contributes to the limited body of literature addressing different analgesic formulations’ effects on laboratory animals.

Traumatic brain injury (TBI) affects millions of people each year. Previous studies using the central fluid percussion injury (CFPI) model in adult male rats indicated that elevated intracranial pressure (ICP) was associated with long-term effects, including neuronal cell loss and increased sensory sensitivity post-injury and secondary ICP elevation, that were not seen following injury alone. Investigations also indicated that Cathepsin B (Cath B), a lysosomal cysteine protease, may play a role in the pathological progression of neuronal membrane disruption; however, the specific impact of Cath B inhibition following CFPI remains unknown. Thus, the focus of this study was to evaluate the effects of Cath B inhibition via intracerebroventricular infusion of CA-074Me 2w following injury with or without secondary elevation of ICP. This was accomplished using adult male rats continuously infused with CA-074Me or 10% DMSO as a vehicle control for 2w following either sham injury, a CFPI only, or a CFPI with subsequent ICP elevation to 20mmHg. We assessed Cath B activity in the lateral neocortices and liver, evaluated protein levels of Cath B and Cath B binding partners AIF, Bcl-XL, and Bak. We also conducted histological analyses of total cell counts to assess for cell loss, membrane disruption, and Cath B localization. Finally, we investigated somatosensory changes with the whisker nuisance task. Overall, this study demonstrated that Cath B is not a direct driver of membrane disruption, however, administration of CA-074Me alters Cath B localization and reduced hypersensitivity, emphasizing Cath B being an important component in late secondary pathologies.

Neural Regen Res, Official publication of Publishing House of Neural Regeneration Research China Association of Rehabilitation Medicine,China