Controlled cortical impact (CCI), using a pneumatically driven impactor to produce traumatic brain injury, has been characterized previously in both the ferret and in the rat. In the present study, we applied this technique to establish and characterize the CCI model of brain injury in another species, the mouse, evaluating cognitive and histopathologic outcome. In anesthetized (sodium pentobarbital, 65 mg/kg) male C57BL mice, we performed sham treatment (no injury, n = 12) or CCI injury (n = 12) at a velocity of 5.7-6.2 m/sec and depth of 1 mm, using a 3-mm diameter rounded-tip impounder, positioned over the left parietotemporal cortex (parasagittal). At this level of injury, we observed highly significant deficits in memory retention of a Morris water maze task 2 days following injury (p < 0.001). Postmortem histopathologic analysis performed at 48 h following injury revealed substantial cortical tissue loss in the region of impact and selective hippocampal neuronal cell loss in the CA2, CA3, and CA3c regions, using Nissl staining. Analysis of degenerating neurons using modified Gallyas silver staining techniques demonstrated consistent ipsilateral injury of neurons in the cortex adjacent to the impact site and in the dentate gyrus of the ipsilateral hippocampus. Bilateral degeneration was observed at the gray matter-white matter interface along the corpus callosum. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed extensive reactive gliosis appearing diffusely through the bilateral cortices, hippocampi, and thalami at 48 h postinjury. Breakdown of the blood-brain barrier was demonstrated with antimouse IgG immunohistochemistry, revealing extravasation of endogenous IgG throughout the ipsilateral cortex, hippocampus, and thalamus. These results suggest that this new model of parasagittal CCI in the mouse mimics a number of well-established sequelae observed in previously characterized brain injury models using other rodent species. This mouse model may be a particularly useful experimental tool for comparing behavioral and histopathologic characteristics of traumatic brain injury in wild-type and genetically altered mice.

Matrix metalloproteinases (MMPs) belong to a class of extracellular proteinases responsible for maintaining and remodeling the extracellular matrix. In addition to multiple functions in normal physiology, abnormal MMP expression and activity may also participate in the pathophysiology of cerebral disease. Here, we show that MMP-9 (gelatinase B; EC.3.4.24.35) contributes to the pathophysiology of traumatic brain injury. After controlled cortical impact in mice, MMP-9 was increased in traumatized brain. Total MMP-9 levels at 24 hr were significantly increased as measured by a substrate cleavage assay. Zymograms showed that MMP-9 was elevated as early as 3 hr after traumatic brain injury, reaching a maximum at approximately 24 hr. Increased MMP-9 levels persisted for up to 1 week. Western blot analysis indicated increased profiles of MMP-9 expression that corresponded with the zymographic data. Knock-out mice deficient in MMP-9 gene expression were compared with wild-type littermates in terms of morphological and motor outcomes after trauma. Motor outcomes were measured at 1, 2, and 7 d after traumatic brain injury by the use of a rotarod device. MMP-9 knock-out mice had less motor deficits than wild-type mice. At 7 d, traumatic brain lesion volumes on Nissl-stained histological sections were significantly smaller in MMP-9 knock-out mice. These data demonstrate that MMP-9 contributes to the pathophysiology of traumatic brain injury and suggest that interruption of the MMP proteolytic cascade may be a possible therapeutic approach for preventing the secondary progression of damage after brain trauma.

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified.

Blast-induced traumatic brain injury (TBI) is the signature insult in combat casualty care. Survival with neurological damage from otherwise lethal blast exposures has become possible with body armor use. We characterized the neuropathologic alterations produced by a single blast exposure in rats using a helium-driven shock tube to generate a nominal exposure of 35 pounds per square inch (PSI) (positive phase duration ∼ 4 msec). Using an IACUC-approved protocol, isoflurane-anesthetized rats were placed in a steel wedge (to shield the body) 7 feet inside the end of the tube. The left side faced the blast wave (with head-only exposure); the wedge apex focused a Mach stem onto the rat's head. The insult produced ∼ 25% mortality (due to impact apnea). Surviving and sham rats were perfusion-fixed at 24 h, 72 h, or 2 weeks post-blast. Neuropathologic evaluations were performed utilizing hematoxylin and eosin, amino cupric silver, and a variety of immunohistochemical stains for amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), ED1, and rat IgG. Multifocal axonal degeneration, as evidenced by staining with amino cupric silver, was present in all blast-exposed rats at all time points. Deep cerebellar and brainstem white matter tracts were most heavily stained with amino cupric silver, with the morphologic staining patterns suggesting a process of diffuse axonal injury. Silver-stained sections revealed mild multifocal neuronal death at 24 h and 72 h. GFAP, ED1, and Iba1 staining were not prominently increased, although small numbers of reactive microglia were seen within areas of neuronal death. Increased blood-brain barrier permeability (as measured by IgG staining) was seen at 24 h and primarily affected the contralateral cortex. Axonal injury was the most prominent feature during the initial 2 weeks following blast exposure, although degeneration of other neuronal processes was also present. Strikingly, silver staining revealed otherwise undetected abnormalities, and therefore represents a recommended outcome measure in future studies of blast TBI.

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OBJECTIVES: Traumatic brain injury (TBI) triggers multiple cell death pathways, possibly including ferroptosis -a recently described cell death pathway that results from accumulation of 15-lipoxygenase (15-LOX)-mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine (PE) containing arachidonic (AA) or adrenic (AdA) acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo TBI, and whether inhibition of 15-LOX provided neuroprotection. DESIGN:Cell culture study and randomized, controlled animal study.

The therapeutic benefits of dopamine (DA) agonists after traumatic brain injury (TBI) imply a role for DA systems in mediating functional deficits post-TBI. We investigated how experimental TBI affects striatal dopamine systems using fast scan cyclic voltammetry (FSCV), western blot, and d-amphetamine-induced rotational behavior. Adult male Sprague-Dawley rats were injured by a controlled cortical impact (CCI) delivered unilaterally to the parietal cortex, or were naïve controls. Amphetamine-induced rotational behavior was assessed 10 days post-CCI. Fourteen days post-CCI, animals were anesthetized and underwent FSCV with bilateral striatal carbon fiber microelectrode placement and stimulating electrode placement in the medial forebrain bundle (MFB). Evoked DA overflow was assessed in the striatum as the MFB was electrically stimulated at 60 Hz for 10 s. In 23% of injured animals, but no naïve animals, rotation was observed with amphetamine administration. Compared with naïves, striatal evoked DA overflow was lower for injured animals in the striatum ipsilateral to injury (p < 0.05). Injured animals exhibited a decrease in V max (52% of naïve, p < 0.05) for DA clearance in the hemisphere ipsilateral to injury compared with naïves. Dopamine transporter (DAT) expression was proportionally decreased in the striatum ipsilateral to injury compared with naïve animals (60% of naïve, p < 0.05), despite no injury-related changes in vesicular monoamine transporter or D 2 receptor expression (DRD 2 ) in this region. Collectively, these data appear to confirm that the clinical efficacy of dopamine agonists in the treatment of TBI may be related to disruptions in the activity of subcortical dopamine systems.