Repeated mild traumatic brain injury causes sex-specific increases in cell proliferation and inflammation in juvenile rats.

Childhood represents a period of significant growth and maturation for the brain, and is also associated with a heightened risk for mild traumatic brain injuries (mTBI). There is also concern that repeated-mTBI (r-mTBI) may have a long-term impact on developmental trajectories. Using an awake closed head injury (ACHI) model, that uses rapid head acceleration to induce a mTBI, we investigated the acute effects of repeated-mTBI (r-mTBI) on neurological function and cellular proliferation in juvenile male and female Long-Evans rats. We found that r-mTBI did not lead to cumulative neurological deficits with the model. R-mTBI animals exhibited an increase in BrdU + (bromodeoxyuridine positive) cells in the dentate gyrus (DG), and that this increase was more robust in male animals. This increase was not sustained, and cell proliferation returning to normal by PID3. A greater increase in BrdU + cells was observed in the dorsal DG in both male and female r-mTBI animals at PID1. Using Ki-67 expression as an endogenous marker of cellular proliferation, a robust proliferative response following r-mTBI was observed in male animals at PID1 that persisted until PID3, and was not constrained to the DG alone. Triple labeling experiments (Iba1+, GFAP+, Brdu+) revealed that a high proportion of these proliferating cells were microglia/macrophages, indicating there was a heightened inflammatory response. Overall, these findings suggest that rapid head acceleration with the ACHI model produces an mTBI, but that the acute neurological deficits do not increase in severity with repeated administration. R-mTBI transiently increases cellular proliferation in the hippocampus, particularly in male animals, and the pattern of cell proliferation suggests that this represents a neuroinflammatory response that is focused around the mid-brain rather than peripheral cortical regions. These results add to growing literature indicating sex differences in proliferative and inflammatory responses between females and males. Targeting proliferation as a therapeutic avenue may help reduce the short term impact of r-mTBI, but there may be sex-specific considerations.

Effects of Ethanol on Synaptic Plasticity and NMDA Currents in the Juvenile Rat Dentate Gyrus.

BACKGROUND AND OBJECTIVES: We examined how acute ethanol (EtOH) exposure affects long term depression (LTD) in the dentate gyrus (DG) of the hippocampus in juvenile rats. EtOH is thought to directly modulate n-methyl-D-aspartate receptor (NMDAr) currents, which are believed important for LTD induction. LTD in turn is believed to play an important developmental role in the hippocampus by facilitating synaptic pruning. METHODS: Hippocampal slices (350µm) were obtained at post-natal day (PND) 14, 21, or 28. Field EPSPs (excitatory post-synaptic potential) or whole-cell EPSCs (excitatory post-synaptic conductance) were recorded from the DG (dentate gyrus) in response to medial perforant path activation. Low-frequency stimulation (LFS; 900 pulses; 120 s pulse) was used to induce LTD. RESULTS: Whole-cell recordings indicated that EtOH exposure at 50mM did not significantly impact ensemble NMDAr EPSCs in slices obtained from animals in the PND14 or 21 groups, but it reliably produced a modest inhibition in the PND28 group. Increasing the concentration to 100 mM resulted in a modest inhibition of NMDAr EPSCs in all three groups. LTD induction and maintenance was equivalent in magnitude in all three age groups in control conditions, however, and surprisingly, NMDA antagonist AP5 only reliably blocked LTD in the PND21 and 28 age groups. The application of 50 mM EtOH attenuated LTD in all three age groups, however increasing the concentration to 100 mM did not reliably inhibit LTD. CONCLUSIONS: These results indicate that the effect of EtOH on NMDAr-EPSCs recorded from DGCs is both age and concentration dependent in juveniles. Low concentrations of EtOH can attenuate, but did not block LTD in the DG. The effects of EtOH on LTD do not align well with it's effects on NNMDA receptors.

An Engineered Infected Epidermis Model for In Vitro Study of the Skin's Pro-Inflammatory Response.

Wound infection is a major clinical challenge that can significantly delay the healing process, can create pain, and requires prolonged hospital stays. Pre-clinical research to evaluate new drugs normally involves animals. However, ethical concerns, cost, and the challenges associated with interspecies variation remain major obstacles. Tissue engineering enables the development of in vitro human skin models for drug testing. However, existing engineered skin models are representative of healthy human skin and its normal functions. This paper presents a functional infected epidermis model that consists of a multilayer epidermis structure formed at an air-liquid interface on a hydrogel matrix and a three-dimensionally (3D) printed vascular-like network. The function of the engineered epidermis is evaluated by the expression of the terminal differentiation marker, filaggrin, and the barrier function of the epidermis model using the electrical resistance and permeability across the epidermal layer. The results showed that the multilayer structure enhances the electrical resistance by 40% and decreased the drug permeation by 16.9% in the epidermis model compared to the monolayer cell culture on gelatin. We infect the model with Escherichia coli to study the inflammatory response of keratinocytes by measuring the expression level of pro-inflammatory cytokines (interleukin 1 beta and tumor necrosis factor alpha). After 24 h of exposure to Escherichia coli, the level of IL-1ß and TNF-α in control samples were 125 ± 78 and 920 ± 187 pg/mL respectively, while in infected samples, they were 1429 ± 101 and 2155.5 ± 279 pg/mL respectively. However, in ciprofloxacin-treated samples the levels of IL-1ß and TNF-α without significant difference with respect to the control reached to 246 ± 87 and 1141.5 ± 97 pg/mL respectively. The robust fabrication procedure and functionality of this model suggest that the model has great potential for modeling wound infections and drug testing.

A Rapid Neurological Assessment Protocol for Repeated Mild Traumatic Brain Injury in Awake Rats.

Preclinical models for mild traumatic brain injury (mTBI) need to recapitulate several essential clinical features associated with mTBI, including a lack of significant neuropathology and the onset of neurocognitive symptoms normally associated with mTBI. Here we show how to establish a protocol for reliably and repeatedly inducing a mild awake closed head injury (ACHI) in rats, with no mortality or clinical indications of persistent pain. Moreover, we implement a new rapid neurological assessment protocol (NAP) that can be completely conducted within 1 min of each impact. This ACHI model will help to rectify the paucity of data on how repeated mTBI (r-mTBI) impacts the juvenile brain, an area of significant concern in clinical populations where there is evidence that behavioral sequelae following injury can be more persistent in juveniles. In addition, the ACHI model can help determine if r-mTBI early in life can predispose the brain to exhibiting greater neuropathology (i.e., chronic traumatic encephalopathy) later in life and can facilitate the identification of critical periods of vulnerability to r-mTBI across the lifespan. This article describes the protocol for administering an awake closed head mTBI (i.e., ACHI) to rats, as well as how to perform a rapid NAP following each ACHI. Methods for administering the ACHI to individual subjects repeatedly are described, as are the methods and scoring system for the NAP. The goal of this article is to provide a standardized set of procedures allowing the ACHI and NAP protocols to be used reliably by different laboratories. © 2019 by John Wiley & Sons, Inc.

Repeated mild traumatic brain injury can cause acute neurologic impairment without overt structural damage in juvenile rats.

Repeated concussion is becoming increasingly recognized as a serious public health concern around the world. Moreover, there is a greater awareness amongst health professionals of the potential for repeated pediatric concussions to detrimentally alter the structure and function of the developing brain. To better study this issue, we developed an awake closed head injury (ACHI) model that enabled repeated concussions to be performed reliably and reproducibly in juvenile rats. A neurological assessment protocol (NAP) score was generated immediately after each ACHI to help quantify the cumulative effects of repeated injury on level of consciousness, and basic motor and reflexive capacity. Here we show that we can produce a repeated ACHI (4 impacts in two days) in both male and female juvenile rats without significant mortality or pain. We show that both single and repeated injuries produce acute neurological deficits resembling clinical concussion symptoms that can be quantified using the NAP score. Behavioural analyses indicate repeated ACHI acutely impaired spatial memory in the Barnes maze, and an interesting sex effect was revealed as memory impairment correlated moderately with poorer NAP score performance in a subset of females. These cognitive impairments occurred in the absence of motor impairments on the Rotarod, or emotional changes in the open field and elevated plus mazes. Cresyl violet histology and structural magnetic resonance imaging (MRI) indicated that repeated ACHI did not produce significant structural damage. MRI also confirmed there was no volumetric loss in the cortex, hippocampus, or corpus callosum of animals at 1 or 7 days post-ACHI. Together these data indicate that the ACHI model can provide a reliable, high throughput means to study the effects of concussions in juvenile rats.

Diffusion MRI abnormalities in adolescent rats given repeated mild traumatic brain injury.

OBJECTIVE: Mild traumatic brain injury (mTBI) is a serious health concern in the adolescent population. Repeated mTBI may result in more pronounced deficits, and has been associated with long-term neurological consequences including neurodegeneration. As such, there is a critical need for the development of objective mTBI biomarkers to help guide medical management. Diffusion-weighted imaging (DWI) is an advanced magnetic resonance imaging (MRI) technique that may detect brain abnormalities after mTBI. Diffusion tensor imaging (DTI) is the most commonly applied DWI method, and initial studies have reported DTI changes in mTBI patients. Furthermore, new DWI methods (e.g., track-weighted imaging; TWI) are being developed that may also be sensitive to mTBIs, but remain to be comprehensively studied. METHODS: This study utilized the Awake Closed Head Injury (ACHI) model of mTBI to investigate changes in DTI and TWI following repeated mTBI in adolescent male and female rats. A total of four ACHI impacts, two/day over two consecutive days, were delivered beginning on postnatal day 25. At 1 day and 7 days postinjury, rats were euthanized and brains were collected for DWI analyses. RESULTS: Rats given repeated mTBI displayed changes in fractional anisotropy and radial diffusivity (i.e., DTI measures), as well as track density (i.e., TWI). INTERPRETATION: These findings are consistent with initial DTI findings in mTBI patients, suggest that TWI may complement DTI, support the utility of DWI measures as biomarkers in mTBI, and further validate the ACHI rat model of mTBI.