Histological comparison of repeated mild weight drop and lateral fluid percussion injury models of traumatic brain injury (TBI) in female and male rats.

ABSTRACT: In preclinical traumatic brain injury (TBI) research, the animal model should be selected based on the research question and outcome measures of interest. Direct side by side comparisons of different injury models are essential for informing such decisions. Here, we used immunohistochemistry to compare the outcomes from two common models of TBI, lateral fluid percussion (LFP) and repeated mild weight drop (rmWD) in adult female and male Wistar rats. Specifically, we measured the effects of LFP and rmWD on markers of cerebrovascular and tight junction disruption, neuroinflammation, mature neurons, and perineuronal nets in the cortical site of injury, cortex adjacent to injury, dentate gyrus, and the CA2/3 area of the hippocampus. Animals were randomized into LFP or rmWD groups. On day 1, the LFP group received a craniotomy and on day 4, injury (or sham procedure; randomly assigned). The rmWD animals underwent either injury or isoflurane-only (randomly assigned) on each of those 4 days. Seven days after injury, brains were harvested for analysis. Overall, our observations revealed that the most significant disruptions were evident in response to LFP, followed by craniotomy-only, while rmWD animals showed the least residual changes compared to isoflurane-only controls, supporting consideration of rmWD as a mild injury. LFP led to longer-lasting disruptions, perhaps more representative of moderate TBI. We also report that craniotomy and LFP produced greater disruptions in females relative to males. These findings will assist the field in the selection of animal models based on target severity of post-injury outcomes, and support the inclusion of both sexes and appropriate control groups.

Histological comparison of repeated mild weight drop and lateral fluid percussion injury models of traumatic brain injury (TBI) in female and male rats.

Traumatic brain injury (TBI) heterogeneity has led to the development of several preclinical models, each modeling a distinct subset of outcomes. Selection of an injury model should be guided by the research question and the specific outcome measures of interest. Consequently, there is a need for conducting direct comparisons of different TBI models. Here, we used immunohistochemistry to directly compare the outcomes from two common models, lateral fluid percussion (LFP) and repeat mild weight drop (rmWD), on neuropathology in adult female and male Wistar rats. Specifically, we used immunohistochemistry to measure the effects of LFP and rmWD on cerebrovascular and tight junction disruption, inflammatory markers, mature neurons and perineuronal nets in the cortical site of injury, cortex adjacent to injury, dentate gyrus, and the CA2/3 area of the hippocampus. Animals were randomized into either LFP or rmWD groups. The LFP group received a craniotomy prior to LFP (or corresponding sham procedure) three days later, while rmWD animals underwent either weight drop or sham (isoflurane only) on each of those four days. After a recovery period of 7 days, animals were euthanized, and brains were harvested for analysis of RECA-1, claudin-5, GFAP, Iba-1, CD-68, NeuN, and wisteria floribunda lectin. Overall, our observations revealed that the most significant disruptions were evident in response to LFP, followed by craniotomy-only, while rmWD animals showed the least residual changes compared to isoflurane-only controls. These findings support consideration of rmWD as a mild, transient injury. LFP leads to longer-lasting disruptions that are more closely associated with a moderate TBI. We further show that both craniotomy and LFP produced greater disruptions in females relative to males at 7 days post-injury. These findings support the inclusion of a time-matched experimentally-naïve or anesthesia-only control group in preclinical TBI research to enhance the validity of data interpretation and conclusions.

Neuropathological Outcomes of Traumatic Brain Injury and Alcohol Use in Males and Females: Studies Using Pre-Clinical Rodent and Clinical Human Specimens.

Traumatic brain injury (TBI) and alcohol misuse are inextricably linked and can increase the risk for development of neurodegenerative diseases, particularly in military veterans and contact sport athletes. Proteinopathy (defects in protein degradation) is considered an underlying factor in neurodegenerative diseases. Whether it contributes to TBI/alcohol-mediated neurodegeneration is unexplored, however. Our recent studies have identified ISGylation, a conjugated form of ISG15 (Interferon-Stimulated Gene 15) and inducer of proteinopathy, as a potential mechanistic link underlying TBI-mediated neurodegeneration and proteinopathy in veterans. In the current study, a rat model of combined TBI and alcohol use was utilized to investigate the same relationship. Here, we report sustained induction of Interferon ß (IFNß), changes in TAR DNA Binding 43 (TDP-43) ISGylation levels, TDP-43 proteinopathy (C-terminal fragmentation [CTF]), and neurodegeneration in the ventral horns of the lumbar spinal cords (LSCs) and/or motor cortices (MCs) of female rats post-TBI in a time-dependent manner. In males, these findings mostly remained non-significant, although moderate alcohol use appears to decrease neurodegeneration in males (but not females) post-TBI. We, however, do not claim that moderate alcohol consumption is beneficial for preventing TBI-mediated neurodegeneration. We have previously demonstrated that ISGylation is increased in the LSCs of veterans with TBI/ALS (amyotrophic lateral sclerosis). Here, we show increased ISGylation of TDP-43 in the LSCs of TBI/ALS-afflicted female veterans compared with male veterans. Knowing that ISGylation induces proteinopathy, we suggest targeting ISGylation may prevent proteinopathy-mediated neurodegeneration post-TBI, particularly in women; however, causal studies are required to confirm this claim.

Conditioned place avoidance is associated with a distinct hippocampal phenotype, partly preserved pattern separation, and reduced reactive oxygen species production after stress.

Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.