Traumatic Brain Injury in the Immature Mouse Brain: Characterization of Regional Vulnerability

Experimental Neurology 176, 105–116 (2002) doi:10.1006/exnr.2002.7941

Traumatic Brain Injury in the Immature Mouse Brain: Characterization of Regional Vulnerability Winnie Tong,* Takuji Igarashi,* Donna M. Ferriero,† ,‡ and Linda J. Noble* ,1 *Department of Neurological Surgery, †Department of Neurology, and ‡Department of Pediatrics, University of California, San Francisco, California 94143 Received November 30, 2001; accepted April 11, 2002

INTRODUCTION We characterized the regional and temporal patterns of neuronal injury and axonal degeneration after controlled cortical impact of moderate severity in mice at postnatal day 21. Animals were euthanized at 1, 3, or 7 days after injury or sham operation. The brains were removed and prepared for immunolocalization of neurons and microglia/macrophages or subjected to Fluoro-Jade and silver stains, indicators of irreversible neuronal cell injury and axonal degeneration. There was significant neuronal loss in both the ipsi- and the contralateral cortices, ipsilateral hippocampus, and ipsilateral thalamus by 7 days post injury compared to sham-operated animals. Activated microglia/macrophages were most prominent in regions of neuronal loss including the ipsilateral cortex, hippocampus, and thalamus. Neuronal injury, as evidenced by Fluoro-Jade labeling, was not apparent in sham-operated animals. In injured animals, labeling was identified in the ipsilateral cortex and hippocampus at 1 and 3 days post injury. Silver- and FluoroJade-labeled degenerating axons were observed in the ipsilateral subcortical white matter by 1 day post injury, in the ipsilateral external capsule, caudate putamen, and contralateral subcortical white matter by 3 days post injury, and in the internal capsule, pyramidal tracts, and cerebellar peduncles by 7 days post injury. Our findings demonstrate that controlled cortical impact in the developing brain generates neuronal loss in both the ipsilateral and the contralateral cortex, a temporally distinct pattern of subcortical neuronal injury/death, and widespread white matter damage. These observations serve as an important baseline for studying human brain injury and optimizing therapies for the brain-injured child. © 2002 Elsevier Science (USA)

Key Words: traumatic brain injury; controlled cortical impact; immature brain; delayed neuronal damage; Fluoro-Jade; axonal degeneration.

1 To whom correspondence should be addressed. Fax: (415) 4765634. E-mail: [email protected].

Traumatic brain injury is a major cause of morbidity and mortality in children (21, 24, 37, 39) but the precise pathomechanical responses to traumatic injury in the developing brain have not been thoroughly described. It was originally believed that the developing brain, which has the capacity for substantial plasticity, exhibits remarkable recovery after injury. This belief has been challenged, however, by reports of long-term neurological (22, 37), cognitive (11, 36, 38, 39, 41), behavioral (23), and psychosocial (33, 49) deficits and impaired motor function (20, 30, 34) in children who have sustained moderate or severe head injuries. The precise pathomechanical responses to traumatic brain injury in the developing brain are not well understood. The few experimental models that address such injuries in the brain have been developed primarily in immature rats and to a lesser extent in neonatal pigs. These models include devices that produce cortical ablation (35) or a scaled cortical impact (18), induce impact to the intact and freely movable cranium (2, 27), deliver an insult to the exposed dura (3), and rapidly inject physiological saline into a closed cranium (fluid percussion) (4, 47). In this study, we describe the characterization of a model of controlled cortical impact (CCI) in the developing murine brain. We selected this injury model for several reasons. The clinical relevance of this model is evident from studies of CCI in adult animals which have demonstrated both contusions and subdural hematomas, features that are also observed in the traumatically brain-injured child (26, 28, 29, 56). The CCI model allows easy manipulation and accurate quantification of biomechanical forces, resulting in the generation of a reproducible injury (7). Finally, the development of the CCI model in the immature mouse is advantageous with regard to the existence of normative data for a wide range of physiological and behavioral variables in adult mice and the ability to study genetically altered animals and distinct inbred strains.

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0014-4886/02 $35.00 2002 Elsevier Science (USA) All rights reserved.

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In this study, we evaluated traumatic brain injury in mice at postnatal day 21. This age is roughly equivalent to the 2-year-old child (48, 55). We have selected this time point because of the particular susceptibility of this age group to traumatic brain injury as reported in clinical studies (5, 28, 29, 36, 41) and in an experimental model of hypoxic-ischemic brain damage (55). Furthermore, the first 3 postnatal weeks in the rodent correspond to a critical period of neural growth in the human during which the foundation for the cytoarchitecture of the mature brain is established (10, 15). MATERIALS AND METHODS

Surgical Preparations Male C57BL/6 mice at postnatal day 21, weighing from 6.0 to 9.3 g, were evaluated in this study. Within each litter, animals were randomized to either a CCI insult or a sham surgery. Each animal was initially anesthetized with 2,2,2-tribromoethanol (12.5 mg/kg, intraperitoneal). The head was positioned in a stereotaxic frame, and the scalp and soft tissues covering the dorsum of the skull were reflected laterally from a midsagittal incision. Body temperature was maintained at 37°C by resting the animal on a warming pad. A burr hole, 4 mm in diameter, was drilled over the left parietal cortex, approximately 2 mm from sagittal suture, 1 mm from lambda, and 1 mm from bregma suture. The dura was left intact at this opening. Depth of anesthesia was continuously monitored throughout the surgery, and anesthetic was supplemented as needed. These procedures were approved by the UCSF Committee on Animal Research and are consistent with NIH designated guidelines. Controlled Cortical Injury The impact device consisted of a pneumatic piston with a 3-mm-diameter impactor tip. The impactor tip was angled at 5° to the vertical and was aligned to the center of the craniotomy. Injury was produced at a velocity of 4.0 to 4.6 m/s and an impactor depth of 0.5 mm. Animals whose dura was torn after impact were excluded from the study. The scalp incision was then sutured closed. The animals recovered from the anesthesia while resting on a warming pad at 37°C. Sham controls underwent anesthesia, scalp incision, drilling of a burr hole, and closure. Animals were euthanized at 1 (n ⫽ 3), 3 (n ⫽ 3), or 7 (n ⫽ 10) days after head injury or at 1 (n ⫽ 1), 3 (n ⫽ 1), or 7 (n ⫽ 7) days after sham surgery. Each animal was anesthetized and transcardially perfused with 50 ml of 4% paraformaldehyde in 0.05 M phosphate-buffered saline (PBS). The brain was removed and placed in fixative at 4°C either for 4 h (immunocytochemistry or Fluoro-Jade labeling) or 5 days (silver staining). Serial coronal sections (40 –50

␮m thick), centered over the cortical impact site and the cerebellum, were cut with a vibratome. Immunocytochemistry Selected brain sections at each time point were immunostained with a monoclonal antibody to vertebrate neuronal nuclei (NeuN; Chemicon, Temecula, CA), a marker of neurons, and with a polyclonal antibody to complement type 3 receptor (CD11b; Serotec, UK), a marker of microglia/macrophages. Degenerating neuronal somata and their processes were detected with Fluoro-Jade B (Histochem, Jefferson, AK) at 1 and 3 days. Patterns of axonal degeneration were analyzed at 7 days after injury in silver-stained sections. Free-floating sections were rinsed in 0.05 M PBS followed by incubation in 1% H 20 2 for 10 min to quench endogenous peroxidase activity. Sections were preincubated in either Mouse-On-Mouse blocking reagent (M.O.M. Kit; Vector, Burlingame, CA) for 1 h (NeuN) or in 2% rabbit serum/0.2% Triton X-100/0.1% bovine serum albumin (RS/TX/BSA) for 5 min followed by 10% RS/TX/BSA for 20 min (CD11b). Sections were rinsed in PBS 3⫻ for 5 min each rinse between each step. Following this, sections were incubated in either M.O.M. diluent for 5 min followed by NeuN (1:1000 in M.O.M. diluent) for 30 min or in CD11b (1:1000 in 2% RS/TX/BSA) overnight. Sections were then placed in either biotinylated M.O.M. anti-mouse IgG (1:250 in M.O.M. diluent) for 10 min (NeuN) or in biotinylated anti-rat IgG (1:200 in 2% RS/TX/BSA) for 1 h. They were next treated with either Vectastain Elite ABC Reagent (Vector Laboratories, Burlingame, CA) for 5 min (NeuN) or with Vectastain ABC Regent (Vector) for 30 min (CD11b). Visualization was achieved using 0.05% 3,3-diaminobenzidine tetrachloride (Life Technologies, Gaithersburg, MD) as the chromogen in the presence of 0.02% H 2O 2. Sections were subsequently mounted on Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA), dehydrated in graded alcohols, cleared in HemoDe (Fisher), and coverslipped. Immunocytochemical controls consisted of adjacent sections of brain, processed as described in the preceding paragraph but in the absence of primary antibody. Histochemistry Sections were stained with Fluoro-Jade as originally described by Schmued and Hopkins (52, 53) or the FD NeuroSilver Kit (FD Neuro Technologies, Baltimore, MD). Fluoro-Jade staining was conducted on sections mounted on Superfrost/Plus slides and dried at room temperature for 2 days. These sections were incubated in each of the following solutions for the time indicated: 100% alcohol, 3 min; 70% alcohol, 1 min; distilled water (dH 2O), 1 min; 0.06% potassium permanganate, 15 min; dH 2O, 1 min; 0.001% Fluoro-Jade B in 0.09% acetic acid, 30 min; dH 20, 3⫻ 1 min. Stained sections

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FIG. 1. Fluoro-Jade labeling at 1 day post injury in the ipsilateral (A, C, E) versus the contralateral (B, D, F) cortex. Prominent Fluoro-Jade-positive neural profiles, indicative of damaged neurons and degenerating processes, are observed in the ipsilateral (A) relative to the contralateral (B) cortex. Fluoro-Jade-labeled processes and a labeled neuronal cell body are shown in the ipsilateral hippocampal CA2/CA3 region (arrow, C), whereas the contralateral hippocampus (D) is unlabeled. Robust labeling of neuronal cell bodies and processes are detected in the ipsilateral hilus and polymorphic and granular layer of the dentate gyrus (E) as compared to the contralateral side (F). Scale bar, 5 ␮m.

were dried either at room temperature for 2 days or at 37°C for 20 min, cleared in HemoDe, and coverslipped. Sections were evaluated for Fluoro-Jade fluorescence using a Nikon Optiphot microscope equipped with an epifluorescence attachment and a fluorescein filter (HQ FITC 4100 306; Nikon, Tokyo, Japan).

Quantitative Analysis The number of NeuN-labeled neurons was quantified within the subfields of the cortex adjacent to the impact site, the ipsilateral latero-dorsal thalamus, and the pyramidal cell layer of ipsilateral hippocampal

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FIG. 2. Fluoro-Jade labeling at 3 days post injury. Intensely labeled neurons and processes are observed in the penumbra immediately adjacent to the impact site (A). Fluoro-Jade-positive neuronal cell bodies and processes are scattered throughout the ipsilateral hippocampus including the CA2/CA3 pyramidal cell layer (B). Fluoro-Jade-labeled neuronal cell bodies and processes are detected in the granular and polymorphic layers of the dentate gyrus and the hilar portion of CA3 (C). Labeled neuronal cell bodies and processes are dispersed throughout the ipsilateral thalamus (D). Scale bar, 5 ␮m.

CA2/CA3 at 7 days after CCI. The number of labeled neurons was also counted in matched areas of the contralateral side and in sham-operated brains. Neuronal counts were performed on three adjacent sections using a magnification of ⫻400 over a microscopic field of 0.0189 mm 2. The numbers of neurons within each region of the injured and sham-operated brain were compared using unpaired, two-tailed t tests. Statistical significance was defined at P ⬍ 0.05. RESULTS

General Observations Animals that underwent traumatic brain injury experienced apneic episodes ranging from 2 to 7 immediately after the insult and typically exhibited a subdural hematoma. Although the dura remained intact in each animal, the cortex sustained gross tissue loss in the form of cavitation at the impact site by 1 day after injury.

Temporal Pattern of Cell Injury Fluoro-Jade labeling was restricted to the microvasculature and the choroid plexus in the sham-operated animals at all time points. No evidence of neuronal or glial labeling was observed in these animals. In general the temporal pattern of cell injury was reproducible across animals at any given time point. By 1 day after traumatic brain injury, the penumbral region, bordering the impact site, exhibited numerous Fluoro-Jade-positive neuronal bodies and processes (Fig. 1). In contrast, no Fluoro-Jade labeling was detected in the contralateral cortex. Fluoro-Jade-labeled cells were most consistently noted in processes of the ipsilateral stratum radiatum of hippocampal CA2/CA3 and in the granular and polymorphic layer of the ipsilateral dentate gyrus (Fig. 1). Fluoro-Jade labeling was not detected in the thalami. Robust Fluoro-Jade labeling of cell bodies and processes was observed in the ipsilateral cortical penumbra at 3 days post injury (Fig. 2). In addition, the adjacent cortical mantle exhibited labeled neurons.

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FIG. 3. NeuN immunostaining at 7 days post injury. There is overt loss of neurons in the ipsilateral cortex (B) as compared to the contralateral cortex (A). There is also pronounced loss of neurons in CA2/CA3 of the ipsilateral hippocampus (D) as compared to the contralateral hippocampus (C). Scale bars (A–D), 500 ␮m.

The hippocampus appeared to be more extensively injured at 3 days post injury as demonstrated by more widespread Fluoro-Jade-labeled structures particularly in the ipsilateral pyramidal cell layer and stratum radiatum of CA2/CA3 and the granular and polymorphic layer of the dentate gyrus (Fig. 2). Fluoro-Jade labeling was noted in neurons and processes of the lateral posterior thalamus and the dorsal lateral and ventral lateral geniculate nuclei (Fig. 2). Regional Neuronal Loss, Silver Staining, and Microglial Activation at 7 Days Post Injury In the sham-operated brain, there was no evidence of neuronal cell loss, based upon sections immunolabeled with NeuN. In addition, neuronal labeling was not detected when the primary antibody was omitted from the incubations in sections from either injured or sham-operated animals. In the injured brain, there was an overt loss of neurons within the penumbral region, bordering the impact site, relative to the corresponding region in the sham-operated animal (Figs. 3, 4). Neuronal loss was also noted in the pyramidal cell layer of CA2/CA3 of the

ipsilateral side and in thalamic nuclei (medial and lateral ventroposterior, posterior, and reticular thalmic nuclei) that receive afferents from the parietal cortex. Furthermore, there appeared to be cell loss in the lateral geniculate, lateral dorsal, and lateral posterior nuclei, all of which receive afferents from the adjacent occipital or frontal cortices. No silver staining was observed in the brains of sham controls (Fig. 5). In contrast, in injured brains robust silver staining (Fig. 5) and prominent microglial/macrophage activation (Fig. 6), occurred in the ipsilateral cortex, hippocampus, and thalamus, regions that likewise were labeled with Fluoro-Jade. The extent of neuronal loss at 7 days after injury was quantified within the subfields of the superficial and deep layers of the cortex, hippocampus, and laterodorsal thalamus (Fig. 7). There was significant neuronal loss in the ipsilateral superficial and deep cortex, pyramidal cell layer of the hippocampal CA2/CA3, and thalamus after CCI as compared to matched regions of the ipsilateral hemisphere of sham-operated controls (Fig. 7). Interestingly, the contralateral superficial and deep layers of the cortex of injured animals also

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FIG. 4. NeuN immunostaining at 7 days post injury. The ipsilateral cortex, adjacent to the impact site, appears devoid of neurons (A) relative to that in the sham-operated animal (B). There is a widespread loss of neuronal cell bodies and processes in the ipsilateral hippocampal CA2/CA3 pyramidal cell layer (C) relative to sham (D). Dystrophic cells with shrunken and darkly stained nuclei are noted in the thalamus of injured animals (E) and contrast the typical appearance of neurons in the sham (F). Scale bar, 10 ␮m.

showed significant neuronal loss relative to those of sham controls (Fig. 8). Temporal Pattern of White Matter Injury White matter damage, as evidenced with both silver and Fluoro-Jade labeling of neural processes, was apparent as early as 1 day post injury and became more widespread over time. Degenerating processes were first limited to the ipsilateral subcortical white matter at 1 day post injury (Figs. 9A and

9B) but extended into the external capsule, caudate putamen, and the contralateral subcortical white matter by 3 days (Figs. 9C and 9D). This Fluoro-Jade pattern correlated with the pattern of silver deposits in the internal and external capsules at day 7 post injury (Fig. 10). Similarly, the pyramidal tracts and cerebellar peduncles first showed robust Fluoro-Jade staining at day 3. This pattern was consistent with intense silver deposits in these regions by 7 days post injury (Fig. 10).

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FIG. 5. Silver staining at 7 days post injury. Intense silver deposits are detected in the ipsilateral cortex (A) relative to sham (B). Labeling in the hippocampal CA2/CA3 region is more subtle and involves stratum oriens, the pyramidal cell layer, stratum lucidum, and stratum ramidal (C), in comparison to the sham (D). Robust staining is detected in the injured thalamus (E) relative to the sham (F). Scale bar, 10 ␮m.

DISCUSSION

This is the first study to describe and characterize a model of CCI in the developing mouse brain. There has been an increasing effort to develop experimental models of traumatic brain injury in the neonatal animal to better define how the immature brain responds to injury (2– 4, 18, 27, 35, 47). Head trauma is the leading cause of disability in children. Cognitive and motor deficits are not limited to severe injury but can also

accompany mild to moderate injury in this young population. Given these clinical observations, there is a substantial need to understand the vulnerability of the developing brain to trauma and to identify the most optimal therapies. The present study demonstrates that CCI in the mouse at postnatal day 21 results in a temporal pattern of neuronal loss that is regionally specific. Neuronal injury, as demonstrated by Fluoro-Jade labeling, was apparent in the ipsilateral cortical penumbra, hip-

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FIG. 6. Immunolabeling of microglia/macrophages (CD11b) 7 days post injury. Activated microglia/macrophages are observed in the cortical region adjacent to the impact site and in the ipsilateral hippocampal CA2/CA3 pyramidal cell layer, polymorphic and granular layer of the dentate gyrus, and thalamus (arrows). Scale bar, 10 ␮m.

pocampal CA2/CA3 regions, and polymorphic and granular layers of the dentate gyrus within 1 day post injury. In contrast, cell injury in the thalamus was delayed in onset. This regionally and temporally specific pattern of neuronal injury is consistent with reported mechanisms of primary and secondary pathogenesis after traumatic brain injury in the adult brain. Early neuronal injury in the cortex and hippocampus has been attributed to direct mechanical damage, excitotoxicity, and oxidative insults that are initiated rapidly after traumatic brain injury. The tissue, comprising the cortical mantle, is subjected to shearing and tearing forces that together generate prominent intraparenchymal hemorrhage. Both the cortex and the hippocampus are likewise exposed to oxidative stress/injury and excitotoxicity. Trauma-induced production of reactive oxygen

FIG. 7. Quantification of NeuN-labeled neurons in ipsilateral injured and sham-operated brains at 7 days post injury. There is a statistically significant neuronal loss in the ipsilateral superficial cortex (SCx), deep cortex (DCx), hippocampal CA2/CA3 region (CA2/ 3), and thalamus (Thal) in the injured animals relative to corresponding regions in the sham controls. Values are the means ⫾ SE. *P ⬍ 0.005.

FIG. 8. Quantification of NeuN-labeled neurons in the contralateral injured and sham-operated brains at 7 days post injury. There is a statistically significant neuronal loss in the contralateral superficial cortex (SCx) and deep cortex (DCx) in the injured animals relative to shams. Values are the means ⫾ SE. *P ⬍ 0.002.

species such as oxygen free radicals can cause peroxidative destruction of the cell membrane and oxidation of cellular proteins and nucleic acids. Our findings of delayed neuronal injury in the thalamus are consistent with those reported in the adult brain after traumatic brain injury (19) and the neonatal brain after hypoxia–ischemia (44). Studies have shown that programmed cell death pathways are involved in the delayed and progressive degenerative response to brain trauma with massive apoptotic cell loss occurring in the thalamus at 1 week post injury and continuing for up to 1 month post injury (13). The finding of apoptotic cell death in areas removed from the injury site is in keeping with reported findings of neuronal cell death after interruption of neuronal connectivity and trophic factor removal (40). Given the profound loss of neurons within the cortex in our model, it is quite possible that delayed neuronal loss in the thalamus is secondary to target deprivation. The loss of connectivity is further supported by our finding of fiber tract degeneration in these areas. This damage to interconnecting white matter pathways has been associated with delayed neurodegeneration in brain regions remote from primary sites of injury in the neonatal ischemia model also (45). We demonstrated that CCI produces a focal cortical injury and a pattern of early selective vulnerability in the pyramidal cell layer of hippocampal CA2/CA3 and the granular and polymorphic layer of the dentate gyrus, a finding consistent with other reports on experimental and clinical traumatic brain injury (1, 16, 19, 31, 54). Dunn-Meynell and Levin (19) demonstrated selective damage in the hippocampal CA3 region in adult rodents after head trauma. Dietrich et al. (16) reported the presence of dark shrunken neurons, consistent with dying cells, selectively in the hippocampal CA3/CA4 regions and the dentate hilus 1 h after brain injury in a fluid percussion model of injury in the adult rat. Findings of hippocampal damage in the form of

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FIG. 9. Regional characterization of white matter degeneration, as seen with Fluoro-Jade labeling at 1 (A, B) and 3 (C, D) days post injury in the ipsilateral hemisphere (A, C) of an injured animal relative to the contralateral side (B, D). Mildly labeled processes are visualized in the ipsilateral subcortical white matter at 1 day post injury (A), whereas no label is detected in a matching region of the contralateral cortex (B). Fluoro-Jade-labeled processes are diffusely scattered throughout the ipsilateral subcortical white matter and external capsule at 3 days post injury (C). A modest but apparent labeling is also noted in the contralateral subcortical white matter at this time point (D). Scale bar, 10 ␮m.

acidophilic neurons have also been observed in CA2/ CA3 regions at 24 h after a similar brain injury (14). Bramlett et al. (9) reported selective damage to hippocampal CA3, dentate gyrus, fissure, and hilus at 8 weeks after lateral fluid percussion injury in adult rats. Together, these studies demonstrate a specific pattern of regional neuronal vulnerability after traumatic injury to the adult brain that are similar to our findings in the developing brain. A key question is whether the developing brain exhibits a degree of vulnerability different from that of the adult brain after traumatic brain injury. There is evidence for age-related differences in vasoreactivity (4, 6, 27), apoptotic cell death (8), preservation of function (47), and the generation of and response to oxidants between the immature and the mature nervous system. With regard to the latter, overexpression of the antioxidant superoxide dismutase-1 has been shown to result in marked neuroprotection after focal and global ischemic insults and after traumatic brain injury in the adult rodent brain (12, 42). These findings, however, contrast those observed after injury to the immature

rodent brain. Superoxide dismutase-1 overexpression in the neonatal animal exacerbates hypoxic–ischemic brain injury (17). This pronounced vulnerability is likely due to the imbalanced overproduction of H 2O 2 from O 2•⫺ and a lack of compensatory protective mechanisms (25). Similar differences in response to oxidants may likewise occur in the developing brain subjected to traumatic brain injury. In the present study diffuse injury was evidenced by significant neuronal loss in both the ipsilateral and the contralateral cortices and widespread white matter damage. Our data suggest that the contralateral cortex, although injured to a lesser degree than the ipsilateral side, nonetheless sustained significant damage. The cause of the neuronal loss in the contralateral cortex may be due to a contra-coup contusive effect and/or delayed neuronal degeneration following the prior loss of synaptic connectivity with the injured cortex. White matter damage in injured animals was detected as early as 1 day post injury in the ipsilateral subcortical white matter with subsequent delayed involvement of the ipsilateral internal and external cap-

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FIG. 10. Regional characterization of white matter degeneration at 7 days post injury. Prominent silver staining (A) and Fluoro-Jade labeling (B) are apparent in the pyramidal tracts in adjacent sections. Silver deposits are diffusely scattered throughout the white matter fiber tracts of the internal and external capsules, extending into several thalamic nuclei (C). An adjacent Fluoro-Jade-labeled section likewise reveals white matter degeneration in similar regions (D). Scale bar, 10 ␮m.

sules, contralateral subcortical white matter, and bilateral pyramidal tracts. Degeneration of the subcortical white matter could be due to loss of cortico-cortical innervation following loss of neurons in the ipsilateral cortex. It is also likely that widespread neuronal injury and degeneration of white matter are in part a consequence of calcium activation of calpain and the subsequent degradation of the membrane-associated cytoskeletal protein spectrin during the posttraumatic period (32, 43, 46, 50). It has, for example, been demonstrated that calpain-mediated proteolysis of ␣-spectrin is not limited to the site of contusion injury but rather occurs in more distal sites (43). The clinical significance of calpain-mediated proteolysis is emphasized in a study by Saatman et al, (51) who demonstrated that a selective calpain antagonist significantly attenuated posttraumatic motor and cognitive deficits following lateral fluid percussion brain injury in rats (51). The present study has successfully utilized developing mice as an experimental species to study controlled cortical head injury. Our data emphasize the profound effect that CCI has on both gray and white matter in the developing brain. The neuropathologic findings of widespread injury to the white matter and progressive

neuronal damage to the cortex, hippocampus, and thalamus provide an essential foundation for better understanding of the biologic basis for posttraumatic cognitive deficits and motor dysfunction that persist in the brain-injured child (11, 20, 22, 30, 34, 36 –39, 41). Further studies to identify cellular mechanisms of injury and the underlying motor and cognitive deficits that occur after CCI to the developing brain are underway. ACKNOWLEDGMENTS This research was supported by NS14543, the Lucille Packard Foundation for Children’s Health Initiative, and the University of California Neurotrauma Research Initiative.

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