Alok S. Shah

Primary Blast Traumatic Brain Injury in the Rat: Relating Diffusion Tensor Imaging and Behavior

The incidence of traumatic brain injury (TBI) among military personnel is at its highest point in U.S. history. Experimental animal models of blast have provided a wealth of insight into blast injury. The mechanisms of neurotrauma caused by blast, however, are still under debate. Specifically, it is unclear whether the blast shockwave in the absence of head motion is sufficient to induce brain trauma. In this study, the consequences of blast injury were investigated in a rat model of primary blast TBI. Animals were exposed to blast shockwaves with peak reflected overpressures of either 100 or 450 kPa (39 and 110 kPa incident pressure, respectively) and subsequently underwent a battery of behavioral tests. Diffusion tensor imaging (DTI), a promising method to detect blast injury in humans, was performed on fixed brains to detect and visualize the spatial dependence of blast injury. Blast TBI caused significant deficits in memory function as evidenced by the Morris Water Maze, but limited emotional deficits as evidenced by the Open Field Test and Elevated Plus Maze. Fractional anisotropy, a metric derived from DTI, revealed significant brain abnormalities in blast-exposed animals. A significant relationship between memory deficits and brain microstructure was evident in the hippocampus, consistent with its role in memory function. The results provide fundamental insight into the neurological consequences of blast TBI, including the evolution of injury during the sub-acute phase and the spatially dependent pattern of injury. The relationship between memory dysfunction and microstructural brain abnormalities may provide insight into the persistent cognitive difficulties experienced by soldiers exposed to blast neurotrauma and may be important to guide therapeutic and rehabilitative efforts.

Behavioral Outcomes Differ between Rotational Acceleration and Blast Mechanisms of Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) can result from a number of mechanisms, including blunt impact, head rotational acceleration, exposure to blast, and penetration of projectiles. Mechanism is likely to influence the type, severity, and chronicity of outcomes. The objective of this study was to determine differences in the severity and time course of behavioral outcomes following blast and rotational mTBI. The Medical College of Wisconsin (MCW) Rotational Injury model and a shock tube model of primary blast injury were used to induce mTBI in rats and behavioral assessments were conducted within the first week, as well as 30 and 60 days following injury. Acute recovery time demonstrated similar increases over protocol-matched shams, indicating acute injury severity equivalence between the two mechanisms. Post-injury behavior in the elevated plus maze demonstrated differing trends, with rotationally injured rats acutely demonstrating greater activity, whereas blast-injured rats had decreased activity that developed at chronic time points. Similarly, blast-injured rats demonstrated trends associated with cognitive deficits that were not apparent following rotational injuries. These findings demonstrate that rotational and blast injury result in behavioral changes with different qualitative and temporal manifestations. Whereas rotational injury was characterized by a rapidly emerging phenotype consistent with behavioral disinhibition, blast injury was associated with emotional and cognitive differences that were not evident acutely, but developed later, with an anxiety-like phenotype still present in injured animals at our most chronic measurements.

Voluntary alcohol intake following blast exposure in a rat model of mild traumatic brain injury

Alcoholism is a frequent comorbidity following mild traumatic brain injury (mTBI), even in patients without a previous history of alcohol dependence. Despite this correlational relationship, the extent to which the neurological effects of mTBI contribute to the development of alcoholism is unknown. In this study, we used a rodent blast exposure model to investigate the relationship between mTBI and voluntary alcohol drinking in alcohol naïve rats. We have previously demonstrated in Sprague Dawley rats that blast exposure leads to microstructural abnormalities in the medial prefrontal cortex (mPFC) and other brain regions that progress from four to thirty days. The mPFC is a brain region implicated in alcoholism and drug addiction, although the impact of mTBI on drug reward and addiction using controlled models remains largely unexplored. Alcohol naïve Sprague Dawley rats were subjected to a blast model of mTBI (or sham conditions) and then tested in several common measures of voluntary alcohol intake. In a seven-week intermittent two-bottle choice alcohol drinking test, sham and blast exposed rats had comparable levels of alcohol intake. In a short access test session at the conclusion of the two-bottle test, blast rats fell into a bimodal distribution, and among high intake rats, blast treated animals had significantly elevated intake compared to shams. We found no effect of blast when rats were tested for an alcohol deprivation effect or compulsive drinking in a quinine adulteration test. Throughout the experiment, alcohol drinking was modest in both groups, consistent with other studies using Sprague Dawley rats. In conclusion, blast exposure had a minimal impact on overall alcohol intake in Sprague Dawley rats, although intake was increased in a subpopulation of blast animals in a short access session following intermittent access exposure.

Effects of Blast Overpressure on Neurons and Glial Cells in Rat Organotypic Hippocampal Slice Cultures

Due to recent involvement in military conflicts, and an increase in the use of explosives, there has been an escalation in the incidence of blast-induced traumatic brain injury (bTBI) among US military personnel. Having a better understanding of the cellular and molecular cascade of events in bTBI is prerequisite for the development of an effective therapy that currently is unavailable. The present study utilized organotypic hippocampal slice cultures (OHCs) exposed to blast overpressures of 150 kPa (low) and 280 kPa (high) as an in vitro bTBI model. Using this model, we further characterized the cellular effects of the blast injury. Blast-evoked cell death was visualized by a propidium iodide (PI) uptake assay as early as 2 h post-injury. Quantification of PI staining in the cornu Ammonis 1 and 3 (CA1 and CA3) and the dentate gyrus regions of the hippocampus at 2, 24, 48, and 72 h following blast exposure revealed significant time dependent effects. OHCs exposed to 150 kPa demonstrated a slow increase in cell death plateauing between 24 and 48 h, while OHCs from the high-blast group exhibited a rapid increase in cell death already at 2 h, peaking at ~24 h post-injury. Measurements of lactate dehydrogenase release into the culture medium also revealed a significant increase in cell lysis in both low- and high-blast groups compared to sham controls. OHCs were fixed at 72 h post-injury and immunostained for markers against neurons, astrocytes, and microglia. Labeling OHCs with PI, neuronal, and glial markers revealed that the blast-evoked extensive neuronal death and to a lesser extent loss of glial cells. Furthermore, our data demonstrated activation of astrocytes and microglial cells in low- and high-blasted OHCs, which reached a statistically significant difference in the high-blast group. These data confirmed that our in vitro bTBI model is a useful tool for studying cellular and molecular changes after blast exposure.

Rate-dependent fracture characteristics of lumbar vertebral bodies

Experimental testing incorporating lumbar columns and isolated components is essential to advance the understanding of injury tolerance and for the development of safety enhancements. This study incorporated a whole column axial acceleration model and an isolated vertebral body model to quantify compression rates during realistic loading and compressive tolerance of vertebrae. Eight lumbar columns and 53 vertebral bodies from 23 PMHS were used. Three-factor ANOVA was used to determine significant differences (p<0.05) in physiologic and failure biomechanics based on compression rate, spinal level, and gender. Results demonstrated a significant increase in ultimate force (i.e., fracture) from lower to higher compression rates. Ultimate stress also increased with compression rate. Displacement and strain to failure were consistent at both compression rates. Differences in ultimate mechanics between vertebral bodies obtained from males and females demonstrated non-significant trends, with female vertebral bodies having lower ultimate force that would be associated with decreased injury tolerance. This was likely a result of smaller vertebrae in that population. Combined with existing literature, results presented in this manuscript contribute to the understanding of lumbar spine tolerance during axial loading events that occur in both military and civilian environments with regard to effects of compression rate and gender.

Methodology to Study Attenuation of a Blast Wave Through the Cranium

The purpose of the study was to quantify attenuation of open field shockwaves passing through the PMHS (Post Mortem Human Subject) cranium. A better understanding of the relationship between shockwave characteristics external to the cranium and insults experienced by the brain is essential for understanding injury mechanisms, validation of finite element models, and development of military safety devices for soldiers in the field. These relationships are being developed using experimental PMHS techniques. Our existing shock tube produced open field shockwaves by increasing input pressure behind a Mylar membrane using compressed nitrogen until the membrane burst. Increasing membrane thickness resulted in greater bursting pressure and peak shockwave pressure. Peak pressure decreased predictably with greater distance from the shock tube outlet. Input pressures between 1.6 and 3.2 MPa resulted in peak shockwave pressures between 45 kPa and 90 kPa measured between 40 and 60 cm from the shock tube exit. The experimental protocol consisted of obtaining a PMHS head, filling the voided cranium with Sylgard gel, and securing the head in front of the shock tube using a Hybrid III dummy neck. Pressure transducers were mounted on the external cranium surface on the ipsilateral side and on the internal cranium surface on the ipsilateral and contralateral sides. Because the specimen was tested in multiple orientations, the ipsilateral side referred to the frontal or temporal sides. Transducers were mounted prior to adding the Sylgard gel. Data from all tests indicated shockwave rise times less than 10 μs external to the skull and internal to the skull on the ipsilateral side. Therefore, the sampling rate was 10 MHz using a digital oscilloscope. Shockwave characteristics were quantified including peak overpressure, peak underpressure, and duration of positive phase. The results show peak overpressure attenuations between 14 and 26% from the external ipsilateral transducer to the contralateral transducers in frontal and lateral orientation. In addition, there was a 93–96% reduction in the rate of onset between those transducers. Each characteristic may affect injury type/severity. This setup can be used to understand injury mechanisms for blast-induced mTBI, to quantify effects of interventions (e.g., helmets) on attenuation of open field blast waves, and for validation of finite element models.Copyright

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

Prediction of Post-Concussive Behavioral Changes in a Rodent Model Based on Head Rotational Acceleration Characteristics.

Quantifying injury tolerance for concussion is complicated by variability in the type, severity, and time course of post-injury physiological and behavioral changes. The current study outlined acute and chronic changes in behavioral metrics following rotational acceleration-induced concussion in rats. The Medical College of Wisconsin (MCW) rotational injury model independently controlled magnitude and duration of the rotational acceleration pulse. Increasing rotational acceleration magnitude produced longer recovery times, which were used in this study and our prior work as an assessment of acute injury severity. However, longer duration rotational accelerations produced changes in emotionality as measured using the elevated plus maze. Cognitive deficits were for the most part not apparent in the Morris water maze assessment, possibly due to the lower severity of rotational acceleration pulses incorporated in this study. Changes in emotionality evolved between acute and chronic assessments, in some cases increasing in severity and in others reversing polarity. These findings highlight the complexity of quantifying injury tolerance for concussion and demonstrate a need to incorporate rotational acceleration magnitude and duration in proposed injury tolerance metrics. Rotational velocity on its own was not a strong predictor of the magnitude or type of acute behavioral changes following concussion, although its combination with rotational acceleration magnitude using multivariate analysis was the strongest predictor for acute recovery time and some chronic emotional-type behavioral changes.

Effects of Mild Blast Traumatic Brain Injury on Cognitive- and Addiction-Related Behaviors

Traumatic brain injury (TBI) commonly results in cognitive and psychiatric problems. Cognitive impairments occur in approximately 30% of patients suffering from mild TBI (mTBI), and correlational evidence from clinical studies indicates that substance abuse may be increased following mTBI. However, understanding the lasting cognitive and psychiatric problems stemming from mTBI is difficult in clinical settings where pre-injury assessment may not be possible or accurate. Therefore, we used a previously characterized blast model of mTBI (bTBI) to examine cognitive- and addiction-related outcomes. We previously demonstrated that this model leads to bilateral damage of the medial prefrontal cortex (mPFC), a region critical for cognitive function and addiction. Rats were exposed to bTBI and tested in operant learning tasks several weeks after injury. bTBI rats made more errors during acquisition of a cue discrimination task compared to sham treated rats. Surprisingly, we observed no differences between groups in set shifting and delayed matching to sample, tasks known to require the mPFC. Separate rats performed cocaine self-administration. No group differences were found in intake or extinction, and only subtle differences were observed in drug-primed reinstatement 3–4 months after injury. These findings indicate that bTBI impairs acquisition of a visual discrimination task and that bTBI does not significantly increase the ability of cocaine exposure to trigger drug seeking.

Comparison of Head Impact Exposure Between Concussed Football Athletes and Matched Controls: Evidence for a Possible Second Mechanism of Sport-Related Concussion

Studies of football athletes have implicated repetitive head impact exposure in the onset of cognitive and brain structural changes, even in the absence of diagnosed concussion. Those studies imply accumulating damage from successive head impacts reduces tolerance and increases risk for concussion. Support for this premise is that biomechanics of head impacts resulting in concussion are often not remarkable when compared to impacts sustained by athletes without diagnosed concussion. Accordingly, this analysis quantified repetitive head impact exposure in a cohort of 50 concussed NCAA Division I FBS college football athletes compared to controls that were matched for team and position group. The analysis quantified the number of head impacts and risk weighted exposure both on the day of injury and for the season to the date of injury. 43% of concussed athletes had the most severe head impact exposure on the day of injury compared to their matched control group and 46% of concussed athletes had the most severe head impact exposure for the season to the date of injury compared to their matched control group. When accounting for date of injury or season to date of injury, 72% of all concussed athletes had the most or second most severe head impact exposure compared to their matched control group. These trends associating cumulative head impact exposure with concussion onset were stronger for athletes that participated in a greater number of contact activities. For example, 77% of athletes that participated in ten or more days of contact activities had greater head impact exposure than their matched control group. This unique analysis provided further evidence for the role of repetitive head impact exposure as a predisposing factor for the onset of concussion. The clinical implication of these findings supports contemporary trends of limiting head impact exposure for college football athletes during practice activities in an effort to also reduce risk of concussive injury.