Failure of Indomethacin and Radiation to Prevent Blast-induced Heterotopic Ossification in a Sprague-Dawley Rat Model.

BACKGROUND: Although use of nonsteroidal antiinflammatory drugs and low-dose irradiation has demonstrated efficacy in preventing heterotopic ossification (HO) after THA and surgical treatment of acetabular fractures, these modalities have not been assessed after traumatic blast amputations where HO is a common complication that can arise in the residual limb. QUESTIONS/PURPOSES: The purpose of this study was to investigate the effectiveness of indomethacin and irradiation in preventing HO induced by high-energy blast trauma in a rat model. METHODS: Thirty-six Sprague-Dawley rats underwent hind limb blast amputation with a submerged explosive under water followed by irrigation and primary wound closure. One group (n = 12) received oral indomethacin for 10 days starting on postoperative Day 1. Another group (n = 12) received a single dose of 8 Gy irradiation to the residual limb on postoperative Day 3. A control group (n = 12) did not receive either. Wound healing and clinical course were monitored in all animals until euthanasia at 24 weeks. Serial radiographs were taken immediately postoperatively, at 10 days, and every 4 weeks thereafter to monitor the time course of ectopic bone formation until euthanasia. Five independent graders evaluated the 24-week radiographs to quantitatively assess severity and qualitatively assess the pattern of HO using a modified Potter scale from 0 to 3. Assessment of grading reproducibility yielded a Fleiss statistic of 0.41 and 0.37 for severity and type, respectively. By extrapolation from human clinical trials, a minimum clinically important difference in HO severity was empirically determined to be two full grades or progression of absolute grade to the most severe. RESULTS: We found no differences in mean HO severity scores among the three study groups (indomethacin 0.90 ± 0.46 [95% confidence interval {CI}, 0.60-1.19]; radiation 1.34 ± 0.59 [95% CI, 0.95-1.74]; control 0.95 ± 0.55 [95% CI, 0.60-1.30]; p = 0.100). For qualitative HO type scores, the radiation group had a higher HO type than both indomethacin and controls, but indomethacin was no different than controls (indomethacin 1.08 ± 0.66 [95% CI, 0.67-1.50]; radiation 1.89 ± 0.76 [95% CI, 1.38-2.40]; control 1.10 ± 0.62 [95% CI, 0.70-1.50]; p = 0.013). The lower bound of the 95% CI on mean severity in the indomethacin group and the upper bound of the radiation group barely spanned a full grade and involved only numeric grades < 2, suggesting that even if a small difference in severity could be detected, it would be less than our a priori-defined minimum clinically important difference and any differences that might be present are unlikely to be clinically meaningful. CONCLUSIONS: This work unexpectedly demonstrated that, compared with controls, indomethacin and irradiation provide no effective prophylaxis against HO in the residual limb after high-energy blast amputation in a rat model. Such an observation is contrary to the civilian experience and may be potentially explained by either a different pathogenesis for blast-induced HO or a stimulus that overwhelms conventional regimens used to prevent HO in the civilian population. CLINICAL RELEVANCE: HO in the residual limb after high-energy traumatic blast amputation will likely require novel approaches for prevention and management.

Rat Model of Brain Injury to Occupants of Vehicles Targeted by Land Mines: Mitigation by Elastomeric Frame Designs.

Many victims of blast traumatic brain injury (TBI) are occupants of vehicles targeted by land mines. A rat model of under-vehicle blast TBI was used to test the hypothesis that the ensuing neuropathology and altered behavior are mitigated by vehicle frame designs that dramatically reduce blast-induced acceleration (G force). Male rats were restrained on an aluminum platform that was accelerated vertically at up to 2850g, in response to detonation of an explosive positioned under a second platform in contact with the top via different structures. The presence of elastomeric, polyurea-coated aluminum cylinders between the platforms reduced acceleration by 80% to 550g compared with 2350g with uncoated cylinders. Moreover, 67% of rats exposed to 2850g, and 20% of those exposed to 2350g died immediately after blast, whereas all rats subjected to 550g blast survived. Assays for working memory (Y maze) and anxiety (Plus maze) were conducted for up to 28 days. Rats were euthanized at 24 h or 29 days, and their brains were used for histopathology and neurochemical measurements. Rats exposed to 2350g blasts exhibited increased cleaved caspase-3 immunoreactive neurons in the hippocampus. There was also increased vascular immunoglobulin (Ig)G effusion and F4/80 immunopositive macrophages/microglia. Blast exposure reduced hippocampal levels of synaptic proteins Bassoon and Homer-1, which were associated with impaired performance in the Y maze and the Plus maze tests. These changes observed after 2350g blasts were reduced or eliminated with the use of polyurea-coated cylinders. Such advances in vehicle designs should aid in the development of the next generation of blast-resistant vehicles.

Pulsatile Lavage of Musculoskeletal Wounds Causes Muscle Necrosis and Dystrophic Calcification in a Rat Model.

BACKGROUND: Adequate irrigation of open musculoskeletal injuries is considered the standard of care to decrease bacterial load and other contaminants. While the benefit of debris removal compared with the risk of further seeding by high-pressure lavage has been studied, the effects of irrigation on muscle have been infrequently reported. Our aim in the present study was to assess relative damage to muscle by pulsatile lavage compared with bulb-syringe irrigation. METHODS: In an animal model of heterotopic ossification, 24 Sprague-Dawley rats underwent hindlimb blast amputation via detonation of a submerged explosive, with subsequent through-the-knee surgical amputation proximal to the zone of injury. All wounds were irrigated and underwent primary closure. In 12 of the animals, pulsatile lavage (20 psi [138 kPa]) was used as the irrigation method, and in the other 12 animals, bulb-syringe irrigation was performed. A third group of 6 rats did not undergo the blast procedure but instead underwent surgical incision into the left thigh muscle followed by pulsatile lavage. Serial radiographs of the animals were made to monitor the formation of soft-tissue radiopaque lesions until euthanasia at 6 months. Image-guided muscle biopsies were performed at 8 weeks and 6 months (at euthanasia) on representative animals from each group. Histological analysis was performed with hematoxylin and eosin, alizarin red, and von Kossa staining on interval biopsy and postmortem specimens. RESULTS: All animals managed with pulsatile lavage, with or without blast injury, developed soft-tissue radiopaque lesions, whereas no animal that had bulb-syringe irrigation developed these lesions (p = 0.001). Five of the 12 animals that underwent blast amputation with pulsatile lavage experienced wound complications, whereas no animal in the other 2 groups experienced wound complications (p = 0.014). Radiopaque lesions appeared approximately 10 days postoperatively, increased in density until approximately 16 weeks, then demonstrated signs of variable regression. Histological analysis of interval biopsy and postmortem specimens demonstrated tissue damage with inflammatory cells, cell death, and dystrophic calcification. CONCLUSIONS: Pulsatile lavage of musculoskeletal wounds can cause irreversible insult to tissue, resulting in myonecrosis and dystrophic calcification. CLINICAL RELEVANCE: The benefits and offsetting harm of pulsatile lavage (20 psi) should be considered before its routine use in the management of musculoskeletal wounds.

Aeromedical evacuation-relevant hypobaria worsens axonal and neurologic injury in rats after underbody blast-induced hyperacceleration.

BACKGROUND: Occupants of military vehicles targeted by explosive devices often suffer from traumatic brain injury (TBI) and are typically transported by the aeromedical evacuation (AE) system to a military medical center within a few days. This study tested the hypothesis that exposure of rats to AE-relevant hypobaria worsens cerebral axonal injury and neurologic impairment caused by underbody blasts. METHODS: Anesthetized adult male rats were secured within cylinders attached to a metal plate, simulating the hull of an armored vehicle. An explosive located under the plate was detonated, resulting in a peak vertical acceleration force on the plate and occupant rats of 100G. Rats remained under normobaria or were exposed to hypobaria equal to 8,000 feet in an altitude chamber for 6 hours, starting at 6 hours to 6 days after blast. At 7 days, rats were tested for vestibulomotor function using the balance beam walking task and euthanized by perfusion. The brains were then analyzed for axonal fiber injury. RESULTS: The number of internal capsule silver-stained axonal fibers was greater in animals exposed to 100G blast than in shams. Animals exposed to hypobaria starting at 6 hours to 6 days after blast exhibited more silver-stained fibers than those not exposed to hypobaria. Rats exposed to 100% oxygen (O2) during hypobaria at 24 hours postblast displayed greater silver staining and more balance beam foot-faults, in comparison with rats exposed to hypobaria under 21% O2. CONCLUSION: Exposure of rats to blast-induced acceleration of 100G increases cerebral axonal injury, which is significantly exacerbated by exposure to hypobaria as early as 6 hours and as late as 6 days postblast. Rats exposed to underbody blasts and then to hypobaria under 100% O2 exhibit increased axonal damage and impaired motor function compared to those subjected to blast and hypobaria under 21% O2. These findings raise concern about the effects of AE-related hypobaria on TBI victims, the timing of AE after TBI, and whether these effects can be mitigated by supplemental oxygen.

Central Nervous System Changes Induced by Underbody Blast-Induced Hyperacceleration: An in Vivo Diffusion Tensor Imaging and Magnetic Resonance Spectroscopy Study.

Blast-related traumatic brain injury (bTBI) resulting from improvised explosive devices is the hallmark injury of recent wars, and affects many returning veterans who experienced either direct or indirect exposure. Many of these veterans have long-term neurocognitive symptoms. However, there is very little evidence to show whether blast-induced acceleration alone, in the absence of secondary impacts, can cause mild TBI. In this study, we examine the effect of under-vehicle blast-induced hyperacceleration (uBIH) of ∼1700 g on the biochemical and microstrucutral changes in the brain using diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS). Two groups of adult male Sprague-Dawley (SD) rats were subjected to a sham procedure and uBIH, respectively. Axonal and neurochemical alterations were assessed using in vivo DTI and MRS at 2 h, 24 h, and 7 days after uBIH. Significant reduction in mean diffusivity, axial diffusivity, and radial diffusivity were observed in the hippocampus, thalamus, internal capsule, and corpus callosum as early as 2 h, and sustained up to 7 days post-uBIH. Total creatine (Cr) and glutamine (Gln) were reduced in the internal capsule at 24 h post-uBIH. The reductions in DTI parameters, Cr and Gln in vivo suggest potential activation of astrocytes and diffuse axonal injury following a single underbody blast, confirming previous histology reports.

Neuropathology and neurobehavioral alterations in a rat model of traumatic brain injury to occupants of vehicles targeted by underbody blasts.

Many victims of blast-induced traumatic brain injury are occupants of military vehicles targeted by land mines. Recently improved vehicle designs protect these individuals against blast overpressure, leaving acceleration as the main force potentially responsible for brain injury. We recently developed a unique rat model of under-vehicle blast-induced hyperacceleration where exposure to acceleration as low as 50G force results in histopathological evidence of diffuse axonal injury and astrocyte activation, with no evidence of neuronal cell death. This study investigated the effects of much higher blast-induced accelerations (1200 to 2800G) on neuronal cell death, neuro-inflammation, behavioral deficits and mortality. Adult male rats were subjected to this range of accelerations, in the absence of exposure to blast overpressure, and evaluated over 28days for working memory (Y maze) and anxiety (elevated plus maze). In addition, brains obtained from rats at one and seven days post-injury were used for neuropathology and neurochemical assays. Sixty seven percent of rats died soon after being subjected to blasts resulting in 2800G acceleration. All rats exposed to 2400G acceleration survived and exhibited transient deficits in working memory and long-term anxiety like behaviors, while those exposed to 1200 acceleration G force only demonstrated increased anxiety. Behavioral deficits were associated with acute microglia/macrophage activation, increased hippocampal neuronal death, and reduced levels of tight junction- and synapse- associated proteins. Taken together, these results suggest that exposure of rats to high underbody blast-induced G forces results in neurologic injury accompanied by neuronal apoptosis, neuroinflammation and evidence for neurosynaptic alterations.

Does Blast Medium Affect Heterotopic Ossification in a Blast-amputation Model?

BACKGROUND: Heterotopic ossification (HO) develops after nearly 2/3 of traumatic blast amputations in the contemporary battlefield. This phenomenon has potentially devastating consequences for servicemen and women and its pathophysiology warrants further investigation using a previously developed animal blast model. QUESTIONS/PURPOSES: We asked, what is the (1) severity (quantity) and (2) type (location) of HO bone formation after a hindlimb blast amputation with two distinct blast media. We hypothesized that a more "war-relevant" blast medium could be a more accurate model and potentially intensify the development of HO. METHODS: Using a Sprague-Dawley rat model, the pathophysiology of ectopic bone formation in a traumatic hindlimb blast amputation was evaluated. Twenty-four animals underwent blast amputations and closure based on a previously established experimental model. Half the amputations were subjected to blasted sand and the other 1/2 to blasted water. Serial orthogonal radiography was performed on each animal until euthanasia at 24 weeks to track the development of HO. Heterotopic bone severity and type were assessed by three independent graders at each time using a novel grading scale to assess quantity and quality of HO. RESULTS: All animals had radiographic evidence of HO develop. No differences were observed in ectopic bone development between sand and water blasting regarding severity or type at any time. Animals that received water and sand blasting had moderate HO develop at 24 weeks (median, 2.0 and 2.5 weeks, respectively; range, 1-3 weeks; difference of medians, 0.5; p=0.67). At the time of euthanasia, 10 animals that were water blasted had Type 3 HO compared with 11 in the sand-blasted group (p=1.00). CONCLUSIONS: Our study showed a clear development of HO after hindlimb blast amputation in a Sprague-Dawley rat model; however, no difference was observed in HO development based on the type of blast media. This suggests it is the blast mechanism that induces ectopic bone development, regardless of the blasted medium. The grading scale we developed for our animal-model study provided a reliable means of assessing HO severity and type. CLINICAL RELEVANCE: We anticipate that future investigations will elucidate similarities between service members' wartime extremity injuries and the animal model used in our study, and with focused future research this model may have beneficial therapeutic implications as the pathophysiology of HO development is further understood.

Rat model of brain injury caused by under-vehicle blast-induced hyperacceleration.

BACKGROUND: More than 300,000 US war fighters in Operations Iraqi and Enduring Freedom have sustained some form of traumatic brain injury (TBI), caused primarily by exposure to blasts. Many victims are occupants in vehicles that are targets of improvised explosive devices. These underbody blasts expose the occupants to vertical acceleration that can range from several to more than 1,000 G; however, it is unknown if blast-induced acceleration alone, in the absence of exposure to blast waves and in the absence of secondary impacts, can cause even mild TBI. METHODS: We approached this knowledge gap using rats secured to a metal platform that is accelerated vertically at either 20 G or 50 G in response to detonation of a small explosive (pentaerythritol tetranitrate) located at precise underbody standoff distances. All rats survived the blasts and were perfusion fixed for brain histology at 4 hours to 30 days later. RESULTS: Robust silver staining indicative of axonal injury was apparent throughout the internal capsule, corpus callosum, and cerebellum within 24 hours after blast exposure and was sustained for at least 7 days. Astrocyte activation, as measured morphologically with brains immunostained for glial fibrillary acidic protein, was also apparent early after the blast and persisted for at least 30 days. CONCLUSION: Exposure of rats to underbody blast-induced accelerations at either 20 G or 50 G results in histopathologic evidence of diffuse axonal injury and astrocyte activation but no significant neuronal death. The significance of these results is that they demonstrate that blast-induced vertical acceleration alone, in the absence of exposure to significant blast pressures, causes mild TBI. This unique animal model of TBI caused by underbody blasts may therefore be useful in understanding the pathophysiology of blast-induced mild TBI and for testing medical and engineering-based approaches toward mitigation.