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JOURNAL OF NEUROTRAUMA Volume 22, Number 2, 2005 © Mary Ann Liebert, Inc. Pp. 252–265

Spatial and Temporal Characteristics of Neurodegeneration after Controlled Cortical Impact in Mice: More than a Focal Brain Injury EDWARD D. HALL, PATRICK G. SULLIVAN, TONYA R. GIBSON, KRISSI M. PAVEL, BRIAN M. THOMPSON, and STEPHEN W. SCHEFF

ABSTRACT The present study examined the neuropathology of the lateral controlled cortical impact (CCI) traumatic brain injury (TBI) model in mice utilizing the de Olmos silver staining method that selectively identifies degenerating neurons and their processes. The time course of ipsilateral and contralateral neurodegeneration was assessed at 6, 24, 48, 72, and 168 h after a severe (1.0 mm, 3.5 M/sec) injury in young adult CF-1 mice. At 6 hrs, neurodegeneration was apparent in all layers of the ipsilateral cortex at the epicenter of the injury. A low level of degeneration was also detected within the outer molecular layer of the underlying hippocampal dentate gyrus and to the mossy fiber projections in the CA3 pyramidal subregions. A time-dependent increase in cortical and hippocampal neurodegeneration was observed between 6 and 72 hrs post-injury. At 24 h, neurodegeneration was apparent in the CA1 and CA3 pyramidal and dentate gyral granule neurons and in the dorsolateral portions of the thalamus. Image analysis disclosed that the overall volume of ipsilateral silver staining was maximal at 48 h. In the case of the hippocampus, staining was generalized at 48 and 72 h, indicative of damage to all of the major afferent pathways: perforant path, mossy fibers and Schaffer collaterals as well as the efferent CA1 pyramidal axons. The hippocampal neurodegeneration was preceded by a significant increase in the levels of calpain-mediated breakdown products of the cytoskeletal protein -spectrin that began at 6 h, and persisted out to 72 h post-injury. Damage to the corpus callosal fibers was observed as early as 24 h. An anterior to posterior examination of neurodegeneration showed that the cortical damage included the visual cortex. At 168 h (7 days), neurodegeneration in the ipsilateral cortex and hippocampus had largely abated except for ongoing staining in the cortical areas surrounding the contusion lesion and in hippocampal mossy fiber projections. Callosal and thalamic neurodegeneration was also very intense. This more complete neuropathological examination of the CCI model shows that the associated damage is much more widespread than previously appreciated. The extent of ipsilateral and contralateral neurodegeneration provides a more complete anatomical correlate for the cognitive and motor dysfunction seen in this paradigm and suggests that visual disturbances are also likely to be involved in the post-CCI neurological deficits Key words: calpain; controlled cortical impact; de Olmos silver staining; neurodegeneration; -spectrin Spinal Cord and Brain Injury Research Center, University of Kentucky Chandler Medical Center, Lexington, Kentucky.

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POST-TBI NEURODEGENERATION

INTRODUCTION

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(CCI) model of traumatic brain injury (TBI), first described by Lighthall (Lighthall, 1988; Lighthall et al., 1990) and Dixon, Hayes and colleagues (Dixon et al., 1991), has become one of the most widely employed model in experimental TBI studies. It was initially developed as a midline model in ferrets (Lighthall, 1988) and rats (Dixon et al., 1991), but was subsequently altered to be a lateral injury model in rats (McIntosh et al., 1989). The lateral CCI model has since been adapted for use in mice (Smith et al., 1995; Raghupathi et al., 1998; Hannay et al., 1999; Sullivan et al., 1999a,b). Initial neuropathological characterization of the midline CCI model in the ferret reported extensive axonal injury in subcortical white matter, internal capsule, thalamic relay nuclei, midbrain, pons and medulla in addition to a cortical contusion at the CCI epicenter (Lighthall et al., 1990). The extent of the damage is proportional to the depth of cortical deformation. Subsequent lateral CCI studies in rats revealed rather diffuse damage in the ipsilateral hippocampus (Newcomb et al., 1997), and loss of CA3 and CA1 pyramidal neurons in particular that are also related to the depth of the cortical impact (Goodman et al., 1994; Baldwin et al., 1997; Scheff et al., 1997). The hippocampal injury probably explains deficits in spatial memory function in Morris water maze tests (Hamm et al., 1992, 1993a,b; Goodman et al., 1994; Scheff et al., 1997). Motor dysfunction is also seen and probably is the result of cortical and thalamic damage (Hamm et al., 1992; Goodman et al., 1994; Hannay et al., 1999). Secondary insults such as hypoxia exacerbate the neuropathological and functional deficits associated with CCI (Clark et al., 1997). Despite the fact that the lateral CCI-TBI model has been used extensively for many years, and the involvement of cortical, hippocampal, and thalamic damage has been noted, no systematic neuropathological characterization had been carried out that defines in detail the spatial and temporal characteristics of post-traumatic damage. Recently, a 1, 4, 7, and 28 day time course of cellular pathology has been reported in rats subjected to moderate lateral CCI (Chen et al., 2003). In that study, conventional cresyl violet and neurofilament staining was employed to show neuronal damage. The present study was conducted in the context of the mouse severe lateral CCI paradigm. To allow for a highly sensitive and quantitative characterization of post-traumatic neuronal degeneration, the de Olmos aminocupric silver staining method was employed followed by quantitation of the volume and regional distribution of damage over the first seven days after injury. This method, which very selecHE CONTROLLED CORTICAL IMPACT

tively stains degenerating neurons and neuronal processes (de Olmos et al., 1994; Switzer, 2000), was previously used to characterize the time course of post-traumatic neurodegeneration in a mouse diffuse TBI model (Kupina et al., 2003; Hall et al., 2004) and to study the ability of certain neuroprotective agents to reduce brain damage (Kupina et al., 2001; Kupina et al., 2002). The results demonstrate that the lateral CCI injury is associated with widespread neurodegeneration in cortex, hippocampus, thalamus in the ipsilateral hemisphere. The spatial extent and intensity peaks at 48–72 h, and contralateral cortical and hippocampal neurodegeneration is also observed. Hippocampal neurodegeneration is preceded by a significant increase in the level of the calpain-mediated -spectrin breakdown products which began as early as 6 h and extended out to 72 h post-injury implying a mechanistic role of calpain in the secondary injury process.

MATERIALS AND METHODS Subjects All studies employed young adult male CF-1 mice (Charles River, Portage, MI) weighing 29–31 g. A total of 73 mice were used. All procedures have been approved by the University of Kentucky Institutional Animal Care and Use Committee.

Mouse Model of Focal (Lateral Controlled Cortical Impact) Traumatic Brain Injury Mice were anesthetized with 0.175 mg/kg tribromoethanol i.p. (Avertin, Aldrich), and placed in a stereotaxic frame (David Kopf, Tujunga, CA). The head was positioned in the horizontal plane with the nose bar set at zero. Following a midline incision exposing the skull, a 4-mm craniotomy made lateral to the sagittal suture, and centered between bregma and lambda. The skull cap at the craniotomy was carefully removed without damaging the underlying dura, and the exposed cortex was injured using a pneumatically controlled impactor device as previously described (Sullivan et al., 1999a,b). The impactor tip diameter was 3 mm, the impact velocity was 3.5 m/sec, and the depth of cortical deformation was set at 1.0 mm. After injury, Surgiseal (Johnson & Johnson, Arlington, TX) was laid on the dura, and the skull cap replaced. The skin was sutured and the animals placed in a Hova-Bator incubator (model 1583; Randall Burkey Co., Boerne, TX), which was set at 37°C until consciousness (return of righting reflex and mobility) was regained (approximately 1 h) in order to prevent hypothermia. Injured mice were allowed to survive from 6 h to 7 days depending upon experimental group assign-

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HALL ET AL. ment. A sham group was included that had a craniotomy, but no injury and was killed at 24 h after surgery.

Histochemistry of Neuronal Degeneration by Silver Staining and Image Analysis

sections analyzed (420 m) and  % a (s) is the sum of percent area of silver staining in all sections examined (13 for each brain).

Western Blot Analysis of -Spectrin Degradation

Neurodegeneration in injured brains was examined by the de Olmos aminocupric silver histochemical technique as previously described (Grafe and Leonard, 1980; Yamamoto et al., 1986; de Olmos et al., 1994; Switzer, 2000; Kupina et al., 2003). At the intended time of sacrifice, the mice were deeply anesthetized with sodium pentobarbital (200 mg/kg I.P.) and transcardially perfused with 0.9% sodium chloride followed by a fixative solution containing 4% paraformaldehyde. Following decapitation, the heads were stored in fixative containing 15% sucrose for 24 h, after which the brains were removed, placed in fresh fixative and shipped (4°C) for histological processing (Neuroscience Associates, Inc., Knoxville, TN). The 25 brains used in this study were embedded into a single block of gelatin (MultiBlock® technology, Neuroscience Associates). Frozen coronal sections, 35 m, were silver stained for neuronal degeneration and counter-stained with Neutral Red. Thirteen sections, 420 m apart taken between 1.1 mm anterior and 4.4 mm posterior to Bregma were used for analysis of silver staining. Brain sections were photographed on an Olympus Provis A70 microscope at using an Olympus Magnafire digital camera. The imported images were analyzed with the Image Pro program (MediaCybernetics; San Diego, CA). Using a pre-programmed method of contrast-enhancement and densitometric thresholding, the area of silver staining and the overall area in the ipsilateral or the contralateral hemisphere was measured for each of 13 brain sections (35 M between each, 420 M total distance). After setting the threshold for the sections on one slide, it was not changed for analysis of the subsequent slides. This was possible since all of the 25 brains used in the current study were embedded in a single gelatin block and all sections taken from the block (i.e., separate slides) were silver stained at the same time, and then counter-stained with neutral red. This served to minimize staining variability between each brain in a given section through the block or across the different sections such that the densitometric thresholding did not need to be readjusted for analyzing each brain section. The percentage area of silver staining for each brain section was calculated by dividing the area of silver staining in each section by the area of the total hemispheric section and multiplying by 100. The volume of silver staining in the hemisphere as a percentage of the overall hemispheric volume was estimated by the by the equation % V  t   %a(s), where % V is percent silver stain volume, t  the distance between

At the time of killing (24-h sham, 6, 24, 48, 72, and 168 h post-injury), mice (n  8 per group) were deeply anesthetized with pentobarbital (200 mg/kg I.P.), the ipsilateral hippocampus was dissected on a chilled stage and immediately transferred to a centrifuge tube containing 800 l ice-cold triton lysis buffer [1 % triton, 20 mM TrisHCL, 150 mM NaCl, 5 mM EGTA, 10 mM EDTA, 10% glycerol and protease inhibitor cocktail (Roche)] and briefly sonicated. Following dismembranation, brain tissues were centrifuged at 14,000 rpm for 30 min at 4°C, protein concentrations are determined in the cleared lysates by a modified Lowry assay (Bio-Rad). Each sample was normalized to contain 1 mg/mL of protein, and 15 g of each was run on SDS/PAGE [3–8% (w/v) acrylamide, Bio-Rad Criterion XT precast gel] with a Tris/acetate running buffer system and then transferred to nitrocellulose membranes using a semi-dry electrotransferring unit (Bio-Rad) at 20 mA for 15 min. The blots were probed with an anti--spectrin antibody (monoclonal, Affinity, UK) followed by a goat anti-mouseHRP antibody (Jackson ImmunoResearch) and developed in a linear range using a standard ECL kit (Amersham Biosciences). Densitometric analysis of Western blots was done using a color scanner (HP psc 950) and the Scion Image software version 4.02, to quantify the content of the 145 and 150 kD -spectrin breakdown products (SBDP 145 and SBDP 150).

Statistical Analysis Injury-induced increases in the volumes and average densities of silver staining and the levels of -spectrin degradation were compared to sham, (non-injured) animals using a one-way ANOVA followed by StudentNewman-Keuls (SNK) post-hoc testing to determine the significance of changes between sham and individual timepoints. A p value of 0.05 compared to sham was considered significant.

RESULTS Time Course of Neurodegeneration Figure 1 displays the time course of silver staining (i.e., neurodegeneration) in the ipsilateral and contralateral hemispheres between 6 h and 7 days after severe CCITBI. For the ipsilateral hemisphere, there was a significant main effect for time post-injury (F  29.5, df  5,

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FIG. 1. Post-traumatic time course of neurodegeneration as revealed by the de Olmos silver staining technique in brains of male mice subjected to severe (1.0 mm) CCI TBI. Graph shows the volume of silver staining as a percent of hemispheric volume determined by image analysis. The increased post-injury silver staining was compared to the low level (2%) seen in sham (craniotomy, but no injury), and found to be significantly increased at 24, 48, and 72 h and 7 days (one-way ANOVA followed by post-hoc t-test; *p  0.05 vs. sham). Insets show typical examples of neurodegeneration at coronal plane 2.3 mm posterior to Bregma (CCI epicenter) in the ipsilateral and contralateral hemispheres at each time point. Values  mean  standard error; numbers of animals at each time point are given in parentheses. The brains were counter-stained with neutral red. Bar  1 mm.

FIG. 2. High-power view of post-traumatic neurodegeneration in ipsilateral hemisphere (same sections as in Fig. 1) at CCI epicenter at various post-traumatic time points. CA1, hippocampal CA1 region; CA3, hippocampal CA3 region; DG, dentate gyrus; SOr, stratum oriens; SRa, stratum radiatum; Mol, molecular layer of the dentate gyrus; PoDG, polymorphic layer of dentate gyrus; SLu, stratum lucidum. Bar  1 mm.

HALL ET AL. p  0.0001). Post-hoc SNK testing revealed that the volume of silver staining, as a percent of overall volume of the ipsilateral hemisphere, was increased significantly compared to sham, non-injured animals at 24, 48, and 72 h and at 168 h (7 days) after injury. The greatest volume of damage was observed at 48 h. Changes in the volume of staining were also analyzed in the contralateral hemisphere. Despite the fact that this hemisphere did not receive the mechanical force of the injury, there was a significant main effect for time postinjury (F  13.3, df  5, p  0.0001). Silver staining was observed in discrete regions of the contralateral cortex and hippocampus and was significantly higher than that seen in sham, non-injured animals at 48, 72, and 168 h. Since the volume of silver staining was expressed as percentage of the ipsilateral or contralateral hemispheres, we also looked at possible changes in the overall hemispheric volume since changes in the overall volume could affect the silver staining measurement. However, for the ipsilateral hemisphere, no significant change in volume was seen across post-injury time points (F  1.9, df  5, p  0.15). Thus, the lack of change in overall hemispheric volume could not have impacted the measurement of the volume of staining as a percentage of hemispheric volume. It should be mentioned that although the sham group was killed and analyzed at 24 h post-surgery, the identical staining volumes for the ipsilateral hemisphere, which had experienced a craniotomy, and the contralateral hemisphere, which did not, argues against the possibility that craniotomy alone produced a discernable injury response that might have been revealed later. Moreover, this conclusion is supported by the fact that staining in the ipsilateral hemisphere of the 24-h injured group was significantly different from the sham (24 h post-craniotomy) group. It is important to note that for all brains in the study, all the sections in each of the coronal planes were stained together on the same slide thus preventing the possibility of staining variations. Figures 2 and 3 provide a higher power view of the time course and pattern of silver staining in the ipsilateral (Fig. 2) and contralateral (Fig. 3) hemispheres. 6 h post-injury. At 6 h after CCI, silver staining was observed in all layers of the ipsilateral cortex immediately below the site of impact as shown in Figures 1 and 2. A modest amount of staining was also detected within the molecular layer of the underlying hippocampal dentate gyrus. This is indicative of damage to the perforant pathway axons that innervate the dentate granule cells, and to the mossy fiber projections from the granule cells to the CA3 pyramidal neurons. A very low level of staining was also apparent in the stratum lucidum of the con-

tralateral hippocampus as a result of the loss of commissural fiber projections from the ipsilateral CA3. 24 h post-injury. By 24 h post-injury, a cavitation lesion had developed that was surrounded by more intense silver staining than that seen at 6 h. The ipsilateral hippocampus and dentate gyrus displayed staining in all subfields. Staining was particularly evident in the CA3 region and in the medial portion of the CA1 subfield. In all three areas, staining of the neuronal perikarya and processes was apparent as seen more clearly in the higher power view of the ipsilateral CA3 subfield shown in Figure 2. The intensity of the staining in the molecular layer of the dentate gyrus, first seen at 6 h, increased dramatically by 24 h. This layer carries the perforant pathway axons that originate in the entorhinal cortex. Increased silver staining also became apparent at 24 h in the dorsolateral portions of the ipsilateral thalamus. 48 and 72 h post-injury. As noted above, image analysis showed that the overall areas and volume of ipsilateral silver staining was the greatest at 48 h (Figs. 1 and 2). In the case of the hippocampus, staining was widespread at 48 and 72 h, indicative of damage to all major afferent pathways: perforant path, mossy fibers and Schaffer collaterals as well as efferent CA1 pyramidal axons. The area of dorsolateral thalamic silver staining was increased at 48 and 72 h. Figure 3 shows increased staining in the contralateral hippocampus as a consequence of degeneration of the ipsilateral CA3 commissural fiber projections to the contralateral dentate gyrus and CA1 regions. Damage to the corpus callosal fibers was also observed as early as 24 h. Its intensity increased over the ensuing time points and was paralleled by staining in the contralateral cortex representing the degeneration of inter-hemispheric projections. An anterior to posterior examination of neurodegeneration as pictured in Figure 4 shows that the cortical damage included the ipsilateral and contralateral visual cortex. 168 h (7 days) post-injury. At 168 h following CCI, neurodegeneration in the ipsilateral cortex and hippocampus had largely abated except for residual staining in the deeper cortical layers and in the cortical areas surrounding the contusion lesion and in hippocampal mossy fiber projections. The cortex at the epicenter of the CCI injury was atrophied, and scar tissue was apparent on the surface. Callosal and thalamic neurodegeneration was still very intense (Figs. 1 and 2). In the contralateral cortex, intense silver staining was prominent in the deeper cortical layers and in the hippocampal CA1 stratum radiatum.

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FIG. 3. High-power view of post-traumatic neurodegeneration in contralateral hemisphere (same sections as in Fig. 1) at CCI epicenter at various post-traumatic time points.

FIG. 4. Anterior  posterior distribution of neurodegeneration in an injured brain at 48 h post-injury. Injury epicenter was at Bregma-2.3 mm. Brains were counter-stained with neutral red. Bar  1 mm.

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Time Course of Neurodegeneration in the Hippocampus

Time Course of Calpain-Mediated Cytoskeletal Degradation in the Hippocampus

Figures 5, 6, and 7 provide high power views of the hippocampal formation that show the time course of degeneration in the CA1, CA3, and the dentate gyral subfields, respectively. In CA1 (Fig. 5), limited silver staining is apparent at 6 h. However, by 24 h, degeneration of CA1 pyramidal neurons is seen in the medial portion of the subfield along with staining of the apical (stratum oriens) and basal (stratum radiatum) dendrites. The fact that these are CA1 dendrites is apparent from the fact that the pattern of staining tends to run perpendicular to the line of the CA1 neurons. At 48 h, the loss of the CA1 pyramidal somata seems to have been completed, but intense staining is apparent in the stratum oriens and stratum radiatum. The character of the latter changed at 24–48 h. Staining in the stratum radiatum had a small granular appearance. The most likely explanation is that this represents the degeneration of CA3 Schaffer collaterals coupled with some residual degenerative debris from the CA1 basal dendrites. The less intense, but persistent staining in the stratum oriens is most likely degeneration of CA1 axons, and degeneration of CA1 apical dendrites. At 48–72 h, this pattern of staining continues, but the intensity appears reduced. In CA3 (Fig. 6), scattered neuronal degeneration is seen, particularly in the more dorsal portion near the border with CA2. Light staining is also seen at 6 h in the stratum lucidum which contains apical dendrites and mossy fiber projections originating from ipsilateral dentate gyrus granule cells. Since this is coincident with the CA3 neuronal staining, it probably also represents dendritic degeneration. At 24 h, the CA3 neuronal degeneration has become more generalized and probably includes the CA3 pyramidal cell bodies and their apical and basal dendrites along with loss of fiber inputs to the apical dendritic field. By 48 h, the intensity of the staining appears to be reduced and 72 h is essentially over, following the same pattern as that seen CA1 (Fig. 5). However, at 72 h, there is very dense staining in the stratum lucidum, representing massive degeneration of mossy fiber terminals. In dentate gyrus (Fig. 7), scattered staining is seen within the neuronal layer at 6 hrs post-injury. The position of these cells suggests that they are granule cells (Amaral, 1995). Degeneration of the dendrites of these neurons is apparent in the perpendicular striated staining seen in the molecular and polymorphic layers. By 24 h, the loss of granule cells continues as does staining in the molecular and polymorphic layers. However, the character of the staining is more granular. At 48 and 72 h, loss of the granule cells continues to occur in contrast to the cessation of CA1 (Fig. 5) and CA3 (Fig. 6) somatic degeneration (i.e., staining).

Figure 8 shows the time course of changes in the levels of the breakdown products of the cytoskeletal protein -spectrin which has been demonstrated to be produced by the proteolytic action of calpain (Wang, 2000). An overall difference was seen across groups by one-way ANOVA for both the SBDP 145 (F  57.3, df  7, p  0.0001) and SBDP 150 (F  30.0, df  7, p  0.0001). Post-hoc testing showed a significant increase in the calpain-specific SBDP145 and the calpain/caspase 3–generated SBDP 150 compared to sham, non-injured brains as early as 6 h post-injury. This increase continued out to 72 h. However, by 168 h post-injury, the earlier increase was followed by a significant decrease in both SBDP 145 and SBDP 150 levels compared to sham. As shown in the example immunoblot in Figure 8, there was no apparent increase in the caspase 3–specific (Wang, 2000) 120-kD SBDP.

DISCUSSION Previous reports have described various neuropathological aspects of the midline (Lighthall, 1988; Lighthall et al., 1990) and lateral (Lighthall, 1988; McIntosh et al., 1989; Lighthall et al., 1990; Goodman et al., 1994; Baldwin et al., 1997; Newcomb et al., 1997; Chen et al., 2003; Grady et al., 2003) versions of the CCI-TBI model. The present results, obtained with quantitative image analysis of tissue stained with the de Olmos aminocupric silver staining method (de Olmos et al., 1994; Switzer, 2000), provide a much clearer and more complete definition of the time course and extent of post-traumatic neurodegeneration in the lateral CCI paradigm. The lateral CCI model, which is generally referred to as a focal contusion injury model, causes extensive neurodegeneration not only in the cortex but also throughout all areas of the ipsilateral hippocampal formation, the dorsolateral thalamic nuclei, the dorsal portion of the striatum and the corpus callosum. Although the epicenter of the lateral CCI injury is located at approximately bregma-2.3 mm (area where only the dorsal hippocampus is seen), intense silver staining can be observed as far posterior as the visual cortex (bregma-4.4 mm). Moreover, silver staining was found as far forward as bregma  1.1 mm. Along with the ipsilateral injury and neurodegeneration, contralateral silver staining is also observed. This appears to represent anterograde degeneration of axons originating in the ipsilateral hemisphere that project into the contralateral hemisphere via the corpus callosum or commissural pathway as well as retrograde degeneration of

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FIG. 5. High-power view of the time course of neurodegeneration in the hippocampal CA1 subfield. SOr, stratum oriens; SRa, stratum radiatum. Bar  0.2 mm.

FIG. 6. High-power view of the time course of neurodegeneration in the hippocampal CA3 subfield. SLu, stratum lucidum. Bar  0.2 mm.

FIG. 7. High-power view of the time course of neurodegeneration in the dentate gyral subfield. Mol, molecular layer; PoDG, polymorphic layer. Bar  0.2 mm.

HALL ET AL.

FIG. 8. Time course of the post-traumatic increase in -spectrin breakdown products in ipsilateral hippocampus after CCI. SBDP 145 is entirely attributable to calpain whereas SBDP 150 is produced by calpain and perhaps in part by caspase 3. Values  mean  standard error; n  8 mice at each time point. Inset shows typical Western blot illustrating the increase in both SBDP 150 and 145 which occurred as early as 6 h, which was maximal from 24–72 h. At 168 h, the levels were reduced below the level seen in the sham, non-injured group. In contrast to the changes in the SBDP 150 and 145, the caspase 3–specific 120kD band did not appear to change after injury. *p  0.05 vs. sham, non-injured group.

axons whose cell bodies are in the cortex and hippocampus of the contralateral hemisphere. At 6 h post-injury, the gross architecture of the brain at the impact site is largely intact (i.e., no post-traumatic cavitation), and silver staining in the cortical layers, although clearly visible, is relatively light. Likewise, staining in the underlying hippocampus is largely confined to CA1, CA3 and granule cells of the dentate gyrus, and their dendritic fields. In contrast, at 6–24 h, a large cavity develops that is surrounded by intense staining in the adjacent cortex and staining appears throughout the hippocampus. Staining is also present in the ipsilateral dorsolateral thalamus. By 48 h, the area and volume of silver staining reaches its maximum, and extends laterally to include the auditory cortex, anteriorly to include the dorsal striatum and posteriorly to take in much of the visual cortex. Over the next 24 h (i.e.,

72-h timepoint), the volume of staining in the ipsilateral hemisphere remains stable. By 7 days, the volume of staining in the cortex and hippocampus has waned. As a result of the intense cortical neurodegeneration that has taken place, the cortical thickness at the injury site is noticeably reduced. In contrast, degeneration in the corpus callosum, the stratum lucidum of CA3 and the dorsolateral thalamus is still intense at 7 days. This time course and pattern of neural damage is consistent with recently published work from another laboratory using the CCI model in rats (Chen et al., 2003). However, that study did not look at 6, 24, or 48 h timepoints, which our results show are perhaps the most interesting in regards to the evolution of post-traumatic neurodegeneration. Similarities in the pattern of degeneration suggests that our results in mice may be generalizable to rats.

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POST-TBI NEURODEGENERATION The initial neurodegeneration at 6 and 24 h primarily effects the cortical and hippocampal cell bodies and their dendrites in CA1, CA3 and dentate gyrus. However, it is apparent that a substantial amount of axonal degeneration is taking place during the first 24 hrs as well. Although some of this is probably Wallerian (anterograde) degeneration of the axonal projections of the degenerating CA1, CA3, and dentate granule cells, it is likely that a good deal is due to diffuse axonal injury. This finding was described by the earliest CCI investigators (Lighthall et al., 1990) who noted greater axonal injury compared to that previously reported in the fluid percussion model (Povlishock et al., 1983). The early investigators suggested that the CCI model was likely to be quite useful for studying the biomechanics and pathophysiology of traumatic axonal injury. In contrast, the idea that all of the axonal degeneration is simply anterograde degeneration of the axons of degenerating CA1, CA3, and dentate granule neurons is not tenable since the magnitude of the neuronal loss which was mainly seen at 24 h is in fact not sufficient to explain the substantial staining in the axon-rich hippocampal strata. Although this is certainly a component of the axonal degeneration, the magnitude of the axonal degeneration suggests that most of it is due to traumatic secondary axonal injury processes. Despite the fact that there is some silver staining of CA1, CA3, and dentrate granule cell perikarya at 6 and 24 h, it is apparent from the neutral red staining in the each of these regions at the later time points (i.e., that there is relatively little loss of the cell bodies compared to the intense degree of axonal staining). Most importantly, the originators of the model concluded that while some immediate mechanical disruption of cell bodies, dendrites and stretching and shearing of axons occurs as part of the primary mechanical injury after CCI, most of the damage is due to a progressive secondary process (Lighthall et al., 1990). This view is consistent with the present data showing that the greatest axonal degeneration in the hippocampus, corpus callosum and the dorsolateral thalamus does not occur until 48–72 h post-injury. Silver staining associated with primary mechanical disruption of axons would have been expected to appear much earlier. Perhaps most convincing regarding the likelihood that much of the neurodegeneration seen in the CCI model is secondary in nature is the fact that various pharmacological approaches have been shown to reduce brain damage in this model (Scheff and Sullivan, 1999; Sullivan et al., 1999b, 2000a,b,c; Albensi et al., 2000; Stover et al., 2000, 2003; Whalen et al., 2000; Kim et al., 2001; Kline et al., 2001; Rao et al., 2001; Aoyama et al., 2002; Kline et al., 2002; McCullers et al., 2002; Cherian et al., 2003; Dempsey and Raghavendra Rao, 2003; Dixon et al., 2003; Faden et al.,

2003; Mahmood et al., 2003). Additionally, the extent of post-traumatic damage is alterable by genetic manipulations (Sullivan et al., 1999a; Igarashi et al., 2001; Longhi et al., 2001). Virtually all of these studies have focused their neuroprotective assessments on the volume of the cortical contusion, the volume of spared tissue and/or preservation of neurons in the hippocampal CA1, CA3, or dentate gyral subfields. In contrast, the fact that much of the injury involves axonal damage suggests that a quantification of changes in axonal injury would be an important focus of attention in future neuroprotection studies, and that agents which target the secondary injury mechanisms responsible for diffuse axonal injury would be appropriate to evaluate this model. Substantial morphometric evidence has been obtained supporting a critical role of cytoskeletal degradation in post-TBI axonal injury. Specifically, the loss of neurofilament proteins has been linked to the pathophysiology of secondary diffuse axonal injury (Ross et al., 1994; Pettus and Povlishock, 1996; Saatman et al., 1998; Buki et al., 1999, 2000; McCracken et al., 1999; Shreiber et al., 1999; Maxwell et al., 2003; Saatman et al., 2003). A principal mechanism of post-traumatic neuronal injury involves the activation of the Ca2-activated neutral proteases known as calpains (Bartus, 1997; Kampfl et al., 1997; McIntosh, 1997; McCracken et al., 1999; Kupina et al., 2003; Ringger et al., 2004). When activated, calpains are known to degrade cytoskeletal proteins as well as receptor proteins, signal transduction enzymes and transcription factors (Yuen and Wang, 1996; Kampfl et al., 1997). In the case of cytoskeleton proteins, -spectrin (a 280-kDa protein that provides structural support to membranes) can be cleaved by calpain at tyrosine 1176 to yield a 150-kDa spectrin breakdown product (SBDP150) or at glycine 1230 to yield a 145-kDa fragment (SBDP145) (Harris and Morrow, 1988). Calpains have also been shown to be involved in the dissolution of neurofilament (NF) proteins [triplet member proteins H (200 kDa), M (160 kDa), and L (68 kDa)], which make up the bulk of axonal volume and are responsible for stabilizing and maintaining neuronal morphology. It is believed that the collapse and compaction of neurofilaments following injury is due, in part, to NF side arms being a primary target for cleavage by M calpain, leading to impaired axoplasmic transport (Pettus and Povlishock, 1996). On the other hand, it should also be noted that TBI-induced cytoskeletal proteolysis can be mediated by caspase-3 activation (Rink et al., 1995; Yakovlev et al., 1997; Pike et al., 1998), which is reflected in an additional 150-kD  spectrin (distinct from the calpain-mediated 150-kD fragment) and a caspase-3–specific 120-kD fragment (Wang, 2000). However, previous work has shown, in the context of the mouse diffuse TBI model, that most of the cytoskeletal

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HALL ET AL. damage (i.e., -spectrin degradation) is due to calpain (Kupina et al., 2002b, 2003). The present results showing a substantial increase in the SBDP 145 and 150 fragments without much, if any, increase in SBDP120, likewise points to a major role of calpain in the CCI model, and a lesser role of caspase 3. The fact that this increase, and its peak, precedes the onset and peak of neurodegeneration, strongly suggests that calpain-mediated proteolysis is a key contributor to the post-traumatic damage. This view is in line with the work of others that has demonstrated that compounds which inhibit calpain, either directly (Saatman et al., 1996; Posmantur et al., 1997; Kupina et al., 2001) or indirectly (Kupina et al., 2002), are neuroprotective in TBI models. Furthermore, in the case of the neuroimmunophilin V-10,367 (Kupina et al., 2002), that compound attenuates both -spectrin degradation and silver staining in mice after diffuse TBI, which strongly supports a linkage between post-traumatic cytoskeletal damage and neuronal/axonal degeneration. Previous immunohistochemical studies from Hayes and colleagues (Newcomb et al., 1997) have provided a time course of the occurrence of -spectrin breakdown products in rats subjected to lateral CCI. Their results show that staining for SBDPs occurs in the ipsilateral cortex within the first 3 h after injury and that the staining is specifically associated with neuronal cell bodies and dendrites. The appearance of SBDPs in cortical neurons coincided with histochemical evidence of neuropathology. However, SBDP staining is also observed in hippocampal neurons that preceded neuronal death. By 24 h, the SBDP staining includes axons. This suggests that the origin of the significant calpain-mediated increase in hippocampal SBDP 145 and 150 levels at the 6 hr time point in our results is mainly coming from neuronal cell bodies and dendrites whereas at later time points, the continuing elevation of SBDP levels is more a reflection of the substantial axonal injury. The decrease in SDBP 145 and 150 levels at 168 h, below that seen in the sham mice, is almost certainly due to an overall decrease in cytoskeletal protein levels reflective of extensive post-traumatic neurodegeneration. The time course of the spectrin loss and neurodegeneration implies that in the CCI model, the critical window for preventing calpain-mediated secondary brain damage is the first 6 h. In contrast, our previous experiments with the diffuse TBI model, which were also conducted in male CF-1 mice, showed that post-traumatic increase in hippocampal SBDP levels was not significant until 72 h postinjury (Kupina et al., 2003). Similarly, the peak in the volume of silver staining in the diffuse model occurred at 72 h (Hall et al., 2004), whereas we presently find in the CCI model that the zenith in neurodegeneration occurs at 48 h. Thus, the therapeutic window for pharmacologically inhibiting calpain-mediated secondary injury may differ by

as much as 48 hrs between the diffuse and CCI models. This difference in the time course and therapeutic window may also be relevant to humans who sustain a characteristically diffuse versus a more focal contusion injury. If so, then anti-calpain treatments may need to be instituted much more quickly in the focal injury context. The histological pattern of neurodegeneration seen in the CCI-TBI model suggests that several behavioral tests are needed to fully assess the neurological impact of the post-traumatic damage. The cortical and thalamic degeneration would be apparent in several tests of motor function (Hamm et al., 1992; Goodman et al., 1994; Hannay et al., 1999). The extensive hippocampal damage is undoubtedly the basis for the defects in spatial memory function observed in studies using the Morris water maze paradigms for short term spatial memory acquisition and retention (Scheff et al., 1997). However, the extension of post-CCI neurodegeneration into the ipsilateral visual cortex, suggests that visual impairments could play a role in the water maze spatial memory deficits. In summary, the current study employing the de Olmos silver staining method has provided a more complete neuropathological analysis of the CCI-TBI model. Mice subjected to a severe level of injury displayed neurodegeneration in the ipsilateral cortex, hippocampus, dorsolateral thalamus, dorsal striatum and the corpus callosum. Contralateral neurodegeneration is due to anterograde and retrograde degeneration of fibers carried in the callosum and commissural pathway. Although degeneration of cortical and hippocampal CA1, CA3, and dentate gyral neuronal somata was observed, much of the neurodegeneration seems to be related to diffuse axonal injury. The greatest neurodegeneration, seen at 48–72 h, was preceded by an increase in calpain-mediated cytoskeletal degradation as early as 6 hrs post-injury. The characteristics of the damage provide an anatomical explanation for the spatial memory and motor deficits associated with the CCI model and show that the assessment of these may be complicated by visual deficits. A comparison of the time course of cytoskeletal degradation and neurodegeneration with previous work in a mouse diffuse TBI model (Kupina et al., 2003; Hall et al., 2004) leads to the conclusion that the temporal characteristics of the two types of injury may differ significantly which in turn indicates that the therapeutic window for pharmacological neuroprotection may be shorter after focal injuries compared to diffuse TBI.

ACKNOWLEDGMENTS This work was supported by funding from 1R01 NS046566 and from a grant from the Kentucky Spinal Cord and Head Injury Research Trust. We gratefully acknowl-

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POST-TBI NEURODEGENERATION edge the excellent contribution of Dr. Robert Switzer and Neuroscience Associates (Knoxville, TN), who carried out the de Olmos silver staining employed in this study.

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