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.

P-glycoprotein Expression Is Upregulated in a Pre-Clinical Model of Traumatic Brain Injury.

Athletes participating in contact sports are at risk for sustaining repeat mild traumatic brain injury (rmTBI). Unfortunately, no pharmacological treatment to lessen the pathophysiology of brain injury has received U.S. Food and Drug Administration (FDA) approval. One hurdle to overcome for potential candidate agents to reach effective therapeutic concentrations in the brain is the blood-brain barrier (BBB). Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp), line the luminal membrane of the brain capillary endothelium facing the vascular space. Although these transporters serve to protect the central nervous system (CNS) from damage by effluxing neurotoxicants before they can reach the brain, they may also limit the accumulation of therapeutic drugs in the brain parenchyma. Thus, increased Pgp expression following brain injury may result in reduced brain availability of therapeutic agents. We therefore questioned if repeat concussive injury increases Pgp expression in the brain. To answer this question, we used a rodent model of repeat mild closed head injury (rmCHI) and examined the messenger RNA (mRN) and protein expression of both isoforms of rodent Pgp (Abcb1a and Abcb1b). Compared with sham-operated controls (n = 5), the mRNA levels of both Abcb1a and Abcb1b were found to be increased in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury. Using a validated antibody, we show increased immunolabeling for Pgp in the dorsal cortex at day 5 and in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury compared with sham controls (n = 6). Taken together, these results suggest that increased expression of Pgp after rmCHI may reduce the brain accumulation of therapeutic drugs that are Pgp substrates. It is plausible that including a Pgp inhibitor with a candidate therapeutic agent may be an effective approach to treat the pathophysiology of rmCHI.

Acute damage to the blood­brain barrier and perineuronal net integrity in a clinically-relevant rat model of traumatic brain injury.

Closed-head, frontal impacts in which the brain undergoes both lateral and rotational acceleration comprise the majority of human traumatic brain injury (TBI). Here, we utilize a clinically relevant model to examine the effects of a single concussion on aspects of brain integrity: the blood-brain barrier, the perineuronal nets (PNNs), and diffuse axonal injury. Adult, male Sprague-Dawley rats received either a frontal, closed-head concussive TBI, or no injury and were evaluated at 1- or 7-day post-injury. Using immunolabeling for albumin, we observed a significant increase in the permeability of the blood-brain barrier at 1-, but not 7-day post-injury. Breakdown of the PNN, as measured by the binding of wisteria floribunda, was observed at 1-day post-injury in the dorsal, lateral, and ventral cortices. This difference was resolved at 7-day. Finally, axonal injury was identified at both 1- and 7-day post-injury. This preclinical model of closed-head, frontal TBI presents a useful tool with which to understand better the acute pathophysiology of a single, frontal TBI.

Traumatic brain injury and hippocampal neurogenesis: Functional implications.

In the adult brain, self-renewing radial-glia like (RGL) progenitor cells have been shown to reside in the subventricular zone and the subgranular zone of the hippocampus. A large body of evidence shows that experiences such as learning, enriched environment and stress can alter proliferation and differentiation of RGL progenitor cells. The progenitor cells present in the subgranular zone of the hippocampus divide to give rise to newborn neurons that migrate to the dentate gyrus where they differentiate into adult granule neurons. These newborn neurons have been found to have a unique role in certain types of hippocampus-dependent learning and memory, including goal-directed behaviors that require pattern separation. Experimental traumatic brain injury (TBI) in rodents has been shown to alter hippocampal neurogenesis, including triggering the acute loss of newborn neurons, as well as progenitor cell hyper-proliferation. In this review, we discuss the role of hippocampal neurogenesis in learning and memory. Furthermore, we review evidence for the molecular mechanisms that contribute to newborn neuron loss, as well as increased progenitor cell proliferation after TBI. Finally, we discuss strategies aimed at enhancing neurogenesis after TBI and their possible therapeutic benefits.

Sarm1 loss reduces axonal damage and improves cognitive outcome after repetitive mild closed head injury.

One of the consistent pathologies associated with both clinical and experimental traumatic brain injury is axonal injury, especially following mild traumatic brain injury (or concussive injury). Several lines of experimental evidence have demonstrated a role for NAD+ metabolism in axonal degeneration. One of the enzymes that metabolizes NAD+ in axons is Sarm1 (Sterile Alpha and TIR Motif Containing 1), and its activity is thought to play a key role in axonal degeneration. Using a Sarm1 knock-out mouse, we examined if loss of Sarm1 offers axonal injury protection and improves cognitive outcome after repeated mild closed head injury (rmCHI). Our results indicate that rmCHI caused white matter damage that can be observed in the corpus callosum, cingulum bundle, alveus of the hippocampus, and fimbria of the fornix of wild-type mice. These pathological changes were markedly reduced in injured Sarm1-/- mice. Interestingly, the activation of astrocytes and microglia was also attenuated in the areas with white matter damage, suggesting reduced inflammation. Associated with these improved pathological outcomes, injured Sarm1-/- mice performed significantly better in both motor and cognitive tasks. Taken together, our results suggest that strategies aimed at inhibiting Sarm1 and/or restoring NAD+ levels in injured axons may have therapeutic utility.