Behavioral and histological inflammatory analysis of a single, mild traumatic brain injury and repeated subconcussive brain injury using a rodent model.

Subconcussive (SC) impacts have become a growing concern within the neuroscience community regarding the immediate and long-lasting effects of sports-related injuries. While a single low-level impact, i.e., a subconcussion, may not cause cerebral perturbations, it has been increasingly recognized that repeated SC exposure can induce deleterious effects. Therefore, determining the lower limits of systematic perturbation resulting from multiple SC impacts is of critical importance in expanding our understanding of cerebral vulnerability and recovery. Currently, there is a lack of correlation between a mild traumatic brain injury (mTBI) and repeated SC impacts with respect to injury biomechanics. Moreover, the cumulative threshold for repetitive low-level impacts is currently undefined. Thus, this research was designed to determine the pathophysiological differences between a single impact of an mTBI and repeated SC impacts with a subdivided cumulative kinetic energy of the single mTBI impact. In order to address this gap in knowledge, the present investigation employed a surgery-free, closed-head, weight drop injury device capable of producing repeatable, head impacts within a rat model. General locomotion and anxiety-like behavior were assessed using an Open Field Test and motor coordination dysfunction was measured using the rotarod assay. Neuroinflammation was measured using immunohistochemical assessment of astrogliosis (GFAP) and microgliosis (Iba-1) within the hippocampus. Additionally, immunohistochemical assessment of neuronal loss (NeuN) was measured within the hippocampus. To investigate the tolerance and the persistence of cerebral vulnerability following a single mTBI and repeated subconcussive impacts, measurement outcomes were assessed over two-time points (3- and 7-days) post final impact. Although injury groups were not statistically different from their associated sham groups with respect to behavioral outcomes; on average, RSC injury rats displayed a significant increase in anxious-like behavior after 7-days of recovery compared to the single mTBI group. From an inflammatory perspective, both mTBI and RSC injury groups led to extensive microgliosis in the gray matter following 3-days post-impact. Overall, this work’s findings do not provide evidence in support of the notion that repeated subconcussive impacts do result in behavioral disturbances and neuroinflammation, that do not manifest following a single mTBI of the same energy input

An experimental protocol for mimicking pathomechanisms of traumatic brain injury in mice

Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI

The cellular senescence response and neuroinflammation in juvenile mice following controlled cortical impact and repetitive mild traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of disability and increases the risk of developing neurodegenerative diseases. The mechanisms linking TBI to neurodegeneration remain to be defined. It has been proposed that the induction of cellular senescence after injury could amplify neuroinflammation and induce long-term tissue changes. The induction of a senescence response post-injury in the immature brain has yet to be characterised. We carried out two types of brain injury in juvenile CD1 mice: invasive TBI using controlled cortical impact (CCI) and repetitive mild TBI (rmTBI) using weight drop injury. The analysis of senescence-related signals showed an increase in γH2AX-53BP1 nuclear foci, p53, p19ARF, and p16INK4a expression in the CCI group, 5 days post-injury (dpi). At 35 days, the difference was no longer statistically significant. Gene expression showed the activation of different senescence pathways in the ipsilateral and contralateral hemispheres in the injured mice. CCI-injured mice showed a neuroinflammatory early phase after injury (increased Iba1 and GFAP expression), which persisted for GFAP. After CCI, there was an increase at 5 days in p16INK4, whereas in rmTBI, a significant increase was seen at 35 dpi. Both injuries caused a decrease in p21 at 35 dpi. In rmTBI, other markers showed no significant change. The PCR array data predicted the activation of pathways connected to senescence after rmTBI. These results indicate the induction of a complex cellular senescence and glial reaction in the immature mouse brain, with clear differences between an invasive brain injury and a repetitive mild injury

The Effects of Repeat Concussive Injury on Hippocampal Neurogenesis in Rats

Abstract A concussion is one of the most common types of traumatic brain injury. Repeat concussion has been associated with an increased likelihood of developing neurodegenerative diseases, such as Parkinson\u27s and Alzheimer\u27s Disease, and chronic traumatic encephalopathy. Although treatments are available to tolerate the symptoms, one potential treatment to address the pathology is to use adult neurogenesis to promote and recruit cells to injury sites for repair. Neurogenesis is the study of new neurons from neural stem cells (NSCs) present in adult humans at the hippocampal dentate gyrus. This study explored whether repeat concussion induces the proliferative capabilities of NSCs using BrdU and NSCs\u27 commitment in becoming neurons using anti-DCX+ in both male and female rats. Long-Evans adult rats were divided into treatment (sham and repeat concussion) and sex (male and female) groups (n=5-6/group). The injury was performed once every 48 hrs for three days using a clinically relevant model of closed head injury (Jamnia et al., 2017). A novel object recognition test was also used to assess working memory and compare it to changes in the BrdU+ and DCX+ populations. BrdU was injected three times every 2hr on day 45, and then animals were euthanized 24hrs later. Unbiased stereology was used to manually count cells expressing BrdU, DCX or both at randomly selected sites on the dentate gyrus. The current study has shown no significant results due to the chronic effects of repeat concussions on cell proliferation for neurogenesis (p= 0.6501), nor on neuronal commitment (p= 0.3944). Normal estrus-cycling female rats’ BrdU (p= 0.7777) and DCX (p= 0.6743) populations did not change compared to male rats. However, the injured female rats did show a trend for a greater amount of DCX+ expression than the injured male population. The injured females reported the strongest correlation (R=0.702) between the novel object test and the DCX+ population, but it was not statistically significant. The current study did not show any significant effects of repeat concussion on the neurogenic response in both males and females. However, given a limited sample size, and trends present in this study, future studies should be conducted to continue this line of inquiry. This would potentially lead to treating the pathology of a concussion with future implications of treating other brain pathologies, such as neurodegenerative diseases

Efficacy of N-acetyl cysteine in traumatic brain injury

In this study, using two different injury models in two different species, we found that early post-injury treatment with NAcetyl Cysteine (NAC) reversed the behavioral deficits associated with the TBI. These data suggest generalization of a protocol similar to our recent clinical trial with NAC in blast-induced mTBI in a battlefield setting [1], to mild concussion from blunt trauma. This study used both weight drop in mice and fluid percussion injury in rats. These were chosen to simulate either mild or moderate traumatic brain injury (TBI). For mice, we used novel object recognition and the Y maze. For rats, we used the Morris water maze. NAC was administered beginning 30-60 minutes after injury. Behavioral deficits due to injury in both species were significantly reversed by NAC treatment. We thus conclude NAC produces significant behavioral recovery after injury. Future preclinical studies are needed to define the mechanism of action, perhaps leading to more effective therapies in man

Maturation-Dependent Response of the Piglet Brain to Scaled Cortical Impact

Object. The goal of this study was to investigate the relationship between maturational stage and the brain\u27s response to mechanical trauma in a gyrencephalic model of focal brain injury. Age-dependent differences in injury response might explain certain unique clinical syndromes seen in infants and young children and would determine whether specific therapies might be particularly effective or even counterproductive at different ages. Methods. To deliver proportionally identical injury inputs to animals of different ages, the authors have developed a piglet model of focal contusion injury by using specific volumes of rapid cortical displacement that are precisely scaled to changes in size and dimensions of the growing brain. Using this model, the histological response to a scaled focal cortical impact was compared at 7 days after injury in piglets that were 5 days, 1 month, and 4 months of age at the time of trauma. Despite comparable injury inputs and stable physiological parameters, the percentage of hemisphere injured differed significantly among ages, with the youngest animals sustaining the smallest lesions (0.8%, 8.4%, and 21.5%, for 5-day-, 1-month-, and 4-month-old animals, respectively, p = 0.0018). Conclusions. These results demonstrate that, for this particular focal injury type and severity, vulnerability to mechanical trauma increases progressively during maturation. Because of its developmental and morphological similarity to the human brain, the piglet brain provides distinct advantages in modeling age-specific responses to mechanical trauma. Differences in pathways leading to cell death or repair may be relevant to designing therapies appropriate for patients of different ages

The immune response to bacterial pneumonia following traumatic brain injury

Thesis (Ph.D.)--Boston UniversityTraumatic brain injury (TBI) is an important clinical problem affecting 1.7 million Americans annually. TBI affects peripheral organs beyond the nervous system with particularly profound effects on the lung, predisposing TBI patients to develop respiratory dysfunction due to bacterial pneumonia. Previous clinical and basic science studies have suggested TBI induces an immune depressed state rendering TBI patients more susceptible to pneumonia. As no mechanism has been proven as a cause for TBI-induced immune modulation we created an animal model of TBI and bacterial pneumonia to investigate the effects of TBI on pulmonary immune function. Our model revealed that instead of increasing susceptibility to bacterial pneumonia, TBI results in a more robust neutrophil recruitment in the lung that allows for faster bacterial clearance and increased survival after bacterial challenge. This response is paradoxically accomplished with significant decreases in pro-inflammatory cytokine production. An important neural mediator of pulmonary inflammation is substance P which acts through the neurokinin-1 receptor (NK-1R) to recruit neutrophils to the lung and increase pulmonary vascular permeability. Treatment with an NK-1R antagonist abolished the increased bacterial killing and recruitment in TBI mice but treatment of sham injury animals with an NK-1R agonist increased their lung neutrophil recruitment and bacterial killing. These findings point to an important role of substance P after TBI and in the immune response to pneumonia. We complemented the findings in our animal model with patient data from the National Trauma Database comparing the incidence of pneumonia among TBI and non-neurotrauma patients. After matching patients by demographics, vital signs, hospital, and importantly injury severity score, we found TBI patients had a decreased incidence of pneumonia. This finding is contrary to the findings of previously published studies that did not account for the confounding factor of injury severity. Our studies offer a new perspective on immune function after TBI and possibly a new therapeutic approach to pneumonia in TBI and non-TBI patients alike

Neural circuit mechanisms of post–traumatic epilepsy

Traumatic brain injury (TBI) greatly increases the risk for a number of mental health problems and is one of the most common causes of medically intractable epilepsy in humans. Several models of TBI have been developed to investigate the relationship between trauma, seizures, and epilepsy-related changes in neural circuit function. These studies have shown that the brain initiates immediate neuronal and glial responses following an injury, usually leading to significant cell loss in areas of the injured brain. Over time, long-term changes in the organization of neural circuits, particularly in neocortex and hippocampus, lead to an imbalance between excitatory and inhibitory neurotransmission and increased risk for spontaneous seizures. These include alterations to inhibitory interneurons and formation of new, excessive recurrent excitatory synaptic connectivity. Here, we review in vivo models of TBI as well as key cellular mechanisms of synaptic reorganization associated with post-traumatic epilepsy (PTE). The potential role of inflammation and increased blood–brain barrier permeability in the pathophysiology of PTE is also discussed. A better understanding of mechanisms that promote the generation of epileptic activity versus those that promote compensatory brain repair and functional recovery should aid development of successful new therapies for PTE

Anti-Inflammatory Effect of Centella asiatica (L.) Extract by Decreasing TNF-α Serum Levels in Rat Model of Traumatic Brain Injury

Centella asiatica (L.) has many active ingredients with many important roles, including as antioxidant, anti-inflamation and neuroprotectant. Centella asiatica (L.) can reduce inflammatory reactions by inhibiting the activity of TNF-α. Thus, Centella asiatica (L.) is a potential alternative therapy for traumatic brain injury by reducing inflammation via TNF-α expression modulation. This study aimed to determine the effect of Centella asiatica (L.) on serum TNF-α levels in rat model of traumatic brain injury. This study was conducted during the period of July 3-17, 2020 at the LPPT Unit IV, Gajah Mada University. This was a true experimental with post-test only control group study on 35 male wistar rats as  the experimental animals. The rats were divided into 5 groups: P1, P2, and traumatic brain injury groups that received Centella asiatica (L.) treatement at 150, 300, and 600mg/kgBW/d doses, respectively. Blood samples were collected after the experimental animals were terminated to assess serum TNF-α levels. Mean TNF-α levels were 60,980±4,057, 76,931±0,698, P3=75,889±0,948, P4=75,868±1,163, and 74,508±1,126 for P1, P2, P3, P4, and P5, respectively. The Kruskal Wallis test results showed a statistically different between groups (p = 0.005). This study shows that Centella asiatica (L.) can decrease serum TNF-α level in rat model of traumatic brain injury

Effects of Traumatic Brain Injury on the Intestinal Tract and Gut Microbiome

Traumatic brain injury (TBI) initiates not only complex neurovascular and glial changes within the brain but also pathophysiological responses that extend beyond the central nervous system. The peripheral response to TBI has become an intensive area of research, as these systemic perturbations can induce dysfunction in multiple organ systems. As there are no approved therapeutics for TBI, it is imperative that we investigate the peripheral response to TBI to identify targets for future intervention. Of particular interest is the gastrointestinal (GI) system. Even in the absence of polytrauma, brain-injured individuals are at increased risk of suffering from GI-related morbidity and mortality. Symptoms such as intestinal dysmotility, inflammation, ulceration, and fecal incontinence can drastically diminish quality of life. The GI tract is inhabited by trillions of microbes that have been implicated as modulators of many neurological disorders. Clinical and preclinical studies implicate gut dysbiosis, a pathological imbalance in the normally symbiotic microbiota, as both a consequence of TBI as well as a contributing factor to brain damage.However, our understanding of this interplay is still limited. While relatively little is known about the effects of TBI on the structure and function of the GI tract, prior studies report that experimental TBI induces intestinal barrier dysfunction and morphological changes. To confirm these findings, male C57BL/6J mice underwent a sham control or a controlled cortical impact (CCI) procedure to induce a contusive brain injury, and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d post-injury. An acute, transient increase in permeability was observed at 4 h after CCI. Histological analyses of the ileum and colon at multiple time points from 4 h to 4 wks revealed no overt morphological changes, suggesting that CCI induced a short-lived physiologic dysfunction without major structural alterations to the GI tract. As the microbiome is a modulator of GI physiology, we performed 16s gene sequencing on fecal samples collected prior to and over the first month after CCI or sham injury. Microbial community diversity was assessed using common metrics of alpha and beta diversity. Alpha diversity was lower in the CCI injury group and beta diversity differed among groups, although these effects were not observed in all metrics. Subsequent differential abundance analysis revealed that the phylum Verrucomicrobiota was increased in CCI mice at 1, 2, and 3 d post-injury when compared to sham mice. Subsequent qPCR identified the Verrucomicrobiota species as Akkermansia muciniphila, an obligate anaerobe that resides in and helps regulate the intestinal mucus layer and barrier. To determine whether TBI promotes changes to the GI tract favorable for the proliferation of A. muciniphila, mucus-producing goblet cells and the level of GI hypoxia were evaluated. Goblet cell density in the medial colon was significantly increased at 1 d, while colon hypoxia was significantly increased at 3 d. Taken together, these studies show that CCI induces transient intestinal barrier dysfunction followed by increased goblet cell density and hypoxia in the colon with a concomitant increase in A. muciniphila that may suggest a compensatory response to systemic stress after TBI