Time course of cellular pathology after controlled cortical impact injury

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Experimental Neurology 182 (2003) 87–102

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Time course of cellular pathology after controlled cortical impact injury S. Chen,a,c J.D. Pickard,a,b and N.G. Harrisa,b,* a

Academic Neurosurgery, Centre for Brain Repair, University of Cambridge, Cambridge, UK b Wolfson Brain Imaging Centre, Addenbrooke’s Hospital, Cambridge, UK c Department of Physical Medicine & Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan Received 24 April 2002; revised 19 September 2002; accepted 1 October 2002

Abstract Several different models of brain trauma are currently used and each simulates different aspects of the clinical condition and to varying degrees of accuracy. While numerous studies have characterized the cellular pathology after weight-drop or fluid percussion injury, detailed information on the histopathology that evolves after the controlled cortical impact model is incomplete. We have determined the spatiotemporal pathologies of neuronal, axonal, vascular, and macro- and microglial elements at 1, 4, 7, and 28 days after moderate controlled cortical impact injury. Neuronal injury identified by pyknotic perikarya and disrupted neurofilament-stained axonal profiles were evident by 1 day in ipsilateral cortex and hippocampus and at later times in the thalamus. glial fibrillary acidic protein-reactive astrocytes were more widespread, reaching a maximum immunointensity at 4 days across the ipsilateral hemisphere but declining to control levels thereafter. Microglia/macrophage-OX42 staining was initially restricted to the contusion site and then later to the thalamus, consistent with the pattern of neuronal injury. Increases in nestin immunoreactivity—a postulated marker of neural progenitor cells, and in NG2 proteoglycan—a marker of oligodendrocyte precursor cells, were detected by 1 day, reaching maximal immunointensity at 4 –7 days after injury. Mean density and diameter of cortical microvessels was significantly reduced and increased respectively but only at the initial time points, suggesting that some degree of vascular remodeling takes place after injury. We discuss these results in light of recent evidence that suggests there may be some degree of endogenous repair after central nervous system injury. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Controlled cortical impact; Trauma; Animal model; Neurofilament; Gliosis; Microglia; Macrophage; Oligodendrocyte precursor; Microvessel; Histopathology

Introduction The design of effective therapies for the treatment of traumatic brain injury (TBI) victims is the ultimate goal of experimental TBI research, but a thorough understanding of the evolving pathophysiology is required to establish the most suitable targets for such treatments. A number of experimental models have been developed over the last few decades that simulate different aspects of the clinical condition and to varying degrees of accuracy (Gennavelli, 1994). Two of the most widely employed models are fluid percussion and controlled cortical impact (CCI). While the * Corresponding author. Academic Neurosurgery, Centre for Brain Repair, University of Cambridge, Forvie Site, Box 85, Robinson Way, Cambridge CB2 2PY, UK. Fax: ⫹44-1223-331174. E-mail address: [email protected] (N.G. Harris).

advantage of the fluid percussion model resides primarily in its simplicity and its ability to produce significant disturbances in the brain, including axonal injury and intraparenchymal hemorrhages, it allows little control over injury parameters and midline injury often causes brainstem injury and death (Lighthall et al., 1989). We have used the CCI model, which has previously been shown to produce a more precise injury as indicated by experiments titrating impact velocity and depth with injury severity (Dixon et al., 1991). Although numerous studies have characterized the temporospatial pattern of cellular response to brain injury in terms of neuronal necrosis (Cortez et al., 1989; DunnMeynell and Levin, 1997; Goss et al., 1998; Hicks et al., 1996; Sutton et al., 1993) axonal damage (Leonard et al., 1997; Okonkwo et al., 1998; Posmantur et al., 2000; Stone et al., 2001; Yaghmai and Povlishock, 1992), microglia

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activation/macrophage infiltration (Aihara et al., 1995; Mathew et al., 1994; Soares et al., 1995) and astrocyte reaction (Baldwin and Scheff, 1996; Dietrich et al., 1999; Hill-Felbag et al., 1999; Raghavendra et al., 2000), the majority of these studies are confined to either fluid percussion or weight-drop injury models. Studies on the alternative CCI model are largely confined to investigating neuronal injury (Dunn-Meynell and Levin, 1997; Newcomb et al., 1999; Posmantur et al., 2000), while details on other cell pathology are more limited (Baldwin and Schoff, 1996; Raghavendra et al., 2000). Therefore, a systematic, quantitative survey of the major cellular events that occur following CCI is required, not only to provide further clues as to how the process of injury and intrinsic repair mechanisms progress, but also to establish a firm baseline from which other interventional studies may proceed in this model. Cortical apoptotic cellular reactions peak at 1 day after contusion injury (Newcomb et al., 1999) with necrosis occurring for only slightly longer (Dunn-Meynell and Levin, 1997) so that the therapeutic window for pharmacological intervention on neuronal targets may well be limited. Although in the thalamus, the appearance of degenerating neurons is delayed after fluid percussion injury (Conti et al., 1998) or traumatic lesioning (Hermann et al., 2000), this is presumably a result of retrograde degeneration from damaged cortical targets and it is questionable whether they are salvageable (Watanabe et al., 1997). The astrocytic reaction is a more prolonged event that is characterized by a metabolic and morphological response culminating in the formation of a dense glial scar (Baldwin and Scheff, 1996; Dietrich et al., 1999; Holmin et al., 1997; Mathewson and Berry, 1985; Novenburg, 1994; Sahin et al., 1999; Takamiya et al., 1988). The glial scar has been suggested to impede the reestablishment of axonal connections (Fawcett and Asher, 1999) so that it may present a therapeutic target from which to improve the functional outcome after brain injury. We have contributed to the knowledge of glial scar formation after CCI by extending existing information on the cortex (Baldwin and Scheff, 1996) to quantitatively map the glial fibrillary acidic protein (GFAP) response throughout the whole brain. Loss of hippocampal astrocytes after fluid percussion injury is not fully compensated for by astrocytic proliferation (Hill-Felberg et al., 1999) so that a return to normal numbers over subsequent weeks may well result from proliferation of existing precursor cells. We have mapped the injury response of the intermediate filament nestin, a marker of developmentally immature cells (Lendahl et al., 1990) that has previously been shown to be upregulated after brain injury (Duggal et al., 1997; Holmin et al., 1997; Li and Chopp, 1999; Sahin et al., 1999; Schwab et al., 2001). The number of oligodendrocyte precursor cells (OPCs) are increased in response to brain injury (Levine, 1994; Ong and Levine, 1999; Tanaka et al., 2001), but their exact role is uncertain; they have been implicated in glial scar formation (Fawcett and Asher, 1999) synaptic plasticity (Ong and

Levine, 1999), as well as in remyelination (Levine et al., 2001). In this study we have quantified the OPC response to contusion injury using an NG2-proteoglycan antibody to clarify their role in the pathogenesis of brain trauma. Microglia activation occurs at a very early stage in response to brain injury (Aihara et al., 1995; Carbonell and Grady, 1999; Raghavendra et al., 2000; Soares et al., 1995) and although they have numerous function, not all of which are fully characterized, a role in repair and regeneration has been ascribed (Kreutzberg, 1996; Prewitt et al., 1997). We have investigated the traumatic response of microglia/macrophage activation and recruitment and considered the response in relation to axonal injury and microvascular pathology. While some of the cellular responses that occur after CCI have been presented separately before, the findings we report here are an attempt to describe the evolution of a number of different cellular pathologies that occur after injury and the spatiotemporal relationship between them. We present especially novel findings on the glial response, a hitherto underreported pathology after brain trauma, given the potential importance of glial cells as a source of neuronal precursors (Seri et al., 2001) and as a therapeutic target to enable axon regeneration (Fawcett and Asher, 1999).

Material and methods Surgical procedures Following CCI or sham operation, rats were processed for histology at 1, 4, 7, and 28 days (n ⫽ 6 and 3/time point, respectively) together with three additional rats that were used as controls. All animals were treated in accordance with the Animals Scientific Procedure Act, 1986, and within the guidelines of the local animal ethics committee. Male Sprague-Dawley rats (200 –300 g in body weight) were anesthetized with 3% isoflurane vaporized in O2 flowing at 0.8 L/min and then maintained with 2% isoflurane during surgery. The head was fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and after retracting the scalp, a dental drill-trephine was used to make a 5-mm craniotomy over the left parietal cortex, 0.5 mm posterior to the coronal suture and 3 mm lateral to the sagittal suture. Considerable care was taken to avoid injury to the underlying dura, which was continuously bathed in sterile physiological saline during the procedure. Body temperature was monitored throughout the surgery by a rectal probe and maintained at 37.0 ⫾ 0.5°C by using a heated pad (Harvard Apparatus Ltd. Edenbridge, Kent, UK). Anesthesia was reduced to 1–1.5% isofluorane in N2O/O2 (0.8/0.4 L/min) prior to injury, which was produced 10 min later by using a pneumatic piston with a rounded metal tip, 2.5 mm in diameter and angled at 22.5° to the vertical so that the tip was perpendicular with the brain surface at the center of the craniotomy. A velocity of 4 m/s and a deformation depth below the dura of 2 mm was used.

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Immediately after CCI, the bone flap was replaced and sealed with dental acrylic cement (Bracon Ltd., Etchingham, Sussex, UK) and the scalp was sutured closed. Rats were placed in a heated cage to maintain body temperature while recovering from anesthesia and soluble paracetamol (1 mg/ml; Cox Pharmaceuticals, Barnstaple, Devon, UK) was administered in the drinking water for 3 days postoperatively. Sham-operated rats received a craniotomy as before but no CCI; the impact tip was placed lightly on the dura before sealing the wound. Tissue preparation and histology Following terminal anesthesia (Euthatal, 2 ml/kg; Roche Meriaux, UK), the decending aorta was clamped and the brain was immediately fixed by transcardial perfusion, initially flushing with phosphate-buffered saline for 1 min followed by 4% paraformaldehyde for 3 min. All solutions were maintained at pH 7.4 and 4°C. Brains were removed and postfixed in 4% paraformaldehyde overnight and transferred to phosphate-buffered saline containing 30% sucrose and 0.1% sodium azide (Sigma-Aldrich, Poole, Dorset, UK) for cryoprotection. Coronal sections were cut at 50 ␮m using a sledge microtome from the level of the olfactory bulbs to the visual cortex and assigned to one of 12 groups that was processed for either immunohistochemistry or cresyl violet staining. The distance between similarly stained sections within each group was thus 600 ␮m and there were approximately 18 sections for each group. The following primary antibodies and dilutions were used: (1) mouse monoclonal anti-neurofilament, nonphosphorylated medium and heavy chain proteins (SMI-312, Sternberger Monoclonals, Libreville, Maryland, USA; 1:25,000), (2) rabbit polyclonal anti-GFAP (Dako, Cambridge, UK; 1:50,000), (3) mouse monoclonals anti-nestin (Rat 401, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA; 1:400), (4) mouse monoclonal anti-OX42 (Serotec, Oxford, UK; 1:400), (5) mouse monoclonals anti-NG2 (produced by J. Levine, State University of New York at Stony Brook, NY, USA; 1:4), and (6) mouse monoclonal anti-rat endothelial cell antigen-1 (RECA-1, Serotec, Oxford, UK; 1:50) Immunohistochemistry For each immunostaining protocol, one series of sections from each brain was stained as free-floating sections simultaneously, to reduce the variability in staining intensity. Sections from control brains were incubated in the same solution but without a primary antibody to ensure that immunostaining was specific. All sections were quenched in a solution of 10% methanol and 10% hydrogen peroxide in distilled water for 5 min before washing three times in Trizma (Sigma-Aldrich, UK)-buffered saline (TBS). Sections were then blocked for 60 minutes in TXTBS (TBS containing 0.2% Triton X-100; Sigma) with 3% normal goat

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serum (NGS; Dako, Cambridge, UK) and then incubated overnight at room temperature in the relevant primary antibody in TXTBS containing 1% NGS. After washing three times in TBS, sections were left in the appropriate biotinylated secondary antibody (Dako biotinylated anti-rabbit IgG and Vector biotinylated rat-adsorbed ant-mouse IgG; Burlingame, VT, USA) at a concentration of 1:200 in TBS with 1% NGS for 3 h followed by three washes in TBS. The primary antibody was visualized with the diaminobenzidine reaction by using the streptavidin-biotinylated horseradish peroxidase complex kit (Dako, Cambridge, UK) in 1% NGS in TBS for 2 h followed by three washes in TBS and two washes in Trizma nonsaline (TNS). Sections were developed with diaminobenzidine in TNS containing 0.03% hydrogen peroxide and excess stain was removed by washing in TNS three times. Finally, sections were mounted on gel-coated slides, dehydrated, and coverslipped with DPX mounting medium. Morphometric analysis Sections were digitized as grayscale images after background subtraction to correct for errors in optical density due to the light source and camera optics (NIH Image, Bethesda, MD, USA). Images were captured at a single sitting for each immunostained group of sections to further reduce the variability in the measurement of immunointensity. Contusion volume Contusion area was calculated from all images of cresyl violet-stained sections that contained contused brain and a volume measurement was computed by summation of areas multiplied by the interslice distance (600 ␮m). The criteria used for inclusion of pixels into the contusion area were set to those pixels with a grayscale value of less than 2 SD below the region-of-interest (ROI) mean intensity in a similar, but unaffected region in the posterior section of the same brain at ⫺5.80 mm from Bregma (Fig. 1A). Glial immunohistochemistry The intensity of GFAP, nestin, OX42, and NG2 immunostaining was quantitatively assessed by making optical density ROI measurements directly from the section image corresponding to ⫺2.80 mm from Bregma (Fig. 1B) within the following brain regions: medial and lateral cortex over laminae 3 and 4 (298 ␮m2 ROI), corpus callosum (144 ␮m2), thalamus (1620 ␮m2), and over the whole hippocampus. All ROI values were normalized to the corresponding white or gray matter value in a remote, unaffected, contralateral ROI in a posterior brain section at ⫺5.80 mm Bregma (Fig. 1A) using the formula:

冋

册

ipsilateral intensity ⫺ unaffected remote area intensity unaffected remote area intensity

⫻ 100 The hippocampal OX42 and NG2 staining intensity was

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Lillie for correction) and Levene median tests were used to describe the distribution of the data and to determine the equality of the variances, respectively. A one-way analysis of variance or Kruskal-Wallis test was used to test for an overall difference between the groups and a post hoc (Dunnett test) was used to determine individual group differences. There was no significant difference between control and sham group optical density measurements within each brain region, at each time point, and for each immunostained dataset, so that these groups were pooled and compared to the injury groups at each time point. Difference between the means was assessed at the probability level of P ⬍ 0.05, 0.01, and 0.001.

Results Gross observations

Fig. 1. Brain atlas coronal brain section of (A) a remote brain region unaffected by the injury at ⫺5.80 mm from Bregma and (B) a core contused region (hatched area) at ⫺2.80 mm from Bregma. Region-ofinterest optical density measurements were determined in (1) corpus callosum, (2) whole hippocampus/CA3 region, (3, 3a, 3b) cortical grey matter, and (4) dorsolateral thalamus at both coronal levels (A and B) and used to either compute the criteria for inclusion of pixels into the contusion area on cresyl violet-stained sections or to obtain normalized optical density measurements of glial fibrillary acidic protein, nestin, OX42, or NG2 immunoreactivity of injured brain (see text for details).

highly heterogenous so that additional mean intensity measurements were obtained from the CA3 obtained from the CA3 region. Vascular morphology Vascular morphology was quantified on a RECA-1stained section at ⫺2.80 mm from Bregma by using a light microscopic interfaced to an image-analysis system (Olympus computer-assisted stereological toolbox-grid system; Sikelborg, Denmark). Measurements were made in three regions placed along the tangential orientation of the cortex, i.e., at the lesion margin, at a distance of 500 ␮m, and within the midline ipsilateral cortex. Within each region, vessels intersecting with a series of nine equally spaced points superimposed on the three randomly positioned field-of-views (FOV) were counted and assessed for mean diameter. Vessel number was expressed per 400-␮m2 FOV and mean diameter was determined for vessels both greater than and less than 10 ␮m per 200-␮m2 FOV.

There were no deaths following injury or sham operation and all rats recovered spontaneous locomotion within 2 to 3 h after injury. The dura mater was torn as a result of the impact in most rats and there was immediate swelling of the cortex with some hemorrhaging below the injury site in all rats. Inspection of the sham-control brain sections revealed minor, superficial pathology when compared to control brains, including occasional distortion in the superficial cortical layers and limited reactive gliosis at the surgery site. No axonal pathology was noted in sham-control animals. The volume of the cortical contusion estimated from cresyl violet-stained sections was 43.3 ⫾ 2.10 mm3 at 1 day and this increased with time post injury so that it was 54.6 ⫾ 4.15 mm3 at 28 days (Fig. 2). The volume of the insult was highly reproducible at the initial stages with only a 12% and 12.5% coefficient of variation at 1 and 4 days, respectively, and this increased to 31% and 19%, at 7 and 28 days, respectively. Neuronal damage One day after injury, cresyl violet-stained sections demonstrated gross reductions in staining intensity within the ipsilateral cortex, hippocampus, and thalamus and this was

Statistical analysis Data are expressed as group mean ⫾ standard error of the mean (SEM). For all data the Kolmogorov-Smirnov (with

Fig. 2. Time course plot of mean contusion volume obtained by the summation of hypointense areas on cresyl violet-stained sections. (Values are mean ⫾ SEM.)

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Fig. 3. Regional characterization of neuronal injury in cresyl violet-stained sections. (A) The low power image demonstrates that an obvious cavitation occurs immediately below the impact site by 1 day post injury, indicated by an asterisk (*) together with hypointense regions in the cortex, hippocampal CA3 region, and in the thalamus (boxed ROIs). (B) High power view from the centre of the ROIs in (A) show that a reduction in the number of large, neuronal cell bodies at 1 day underlie the low power hypointensity in the cortex and hippocampal regions. Thalamic neurons appear either faintly stained or shrunken and darkly stained at 1 day. By 4 and 7 days there is an increase in the number of small glial-like cell bodies in the cortex and hippocampus that subsides by 28 days but remains evident in the thalamus. Scale bar is 50 ␮m. ROI, region-of-interest.

most prominent around the contusion site within the cortex (Fig. 3A). Surrounding this was an area of partial damage identified by less densely stained neurons with irregular cytoplasm together with shrunken and darkly stained neurons (Fig. 3B). Within the ipsilateral hippocampus, small zones of hypointensity were also found in the CA3 and dentate gyrus regions at 1 day (Fig. 3B), although the CA1 region remained largely unaffected (not shown). More mild injury was observed in the ipsilateral dorsal thalamus at 1 day, indicated by less severe reductions in staining intensity and fewer shrunken neurons (Fig. 3B). No obvious pathology was detected in the contralateral hemisphere. By 4 days, the zone of partial damage observed at 1 day had decreased at the lesion margin and there was an extensive loss of large, presumably neuronal cells around the

contusion edge together with an increase in the number of smaller, glial cells/leukocytes in all regions (Fig. 3B). By 7 and 28 days, the contusion cavity was larger than at previous time points and the impact site was bordered by a band of tightly packed cells that were GFAP positive, indicating scar tissue. At 28 days, the cortex bordering the contusion appeared normal while neuronal and glial cell loss was evident in the hippocampus. Gliosis was still marked at this time in the thalamus and this was particularly pronounced in the dorsal thalamic edge (Fig. 3B). Axonal injury All brain sections from control and sham-operated rats demonstrated only light immunostaining (Fig. 4). At 1 day

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Fig. 4. Neurofilament immunoreactivity in the ipsilateral corpus callosum within region-of-interest 1, as shown in Fig. 1B, in a sham control and in injured rats at different times after trauma. Large numbers of immunoreactive profiles are observed 1 day after injury together with a number of swollen, bulbous axons (arrow). Fewer reactive profiles are observed after 4 days, although there are a marked number of disconnected axonal bulbs (arrowhead) at 4 and 7 days. By 28 days there is no significant difference in immunoreactivity. Scale bar is 50 ␮m.

post injury, brain sections were more intensely stained compared to control and two types of axonal pathology were observed; multiple varicosities spread along large lengths of intact axon or a single terminal retraction bulb along a continous or discontinous axon (Fig. 4). This axonal pathology was primarily found in areas close to the impact site

such as the contusion margin, the subcortical white matter immediately below the injury site, the ipsilateral corpus callosum, the internal and external capsule, the hippocampal CA3 region, and the dorsal regions of the striatum and thalamus. No terminal axonal bulbs were observed in the contralateral hemisphere, although occasional axonal swell-

Fig. 5. Low power light micrographs of (A) glial fibrillary acidic protein (GFAP) and (B) nestin immunoreactivity in the ipsilateral hemisphere in sham-control and injured rats at different times after trauma. Generalized plots of the maximal extent of increased immunoreactivity are presented for each protein (far right). Increased GFAP immunoreactivity was present throughout the whole ipsilateral cortex at 1–7 days and this extended into the dorsolateral thalamus, although it was most intense in pericontusioned areas. Increased nestin immunoreactivity was confined to the cortex, corpus callosum, and hippocampus; none was present in the thalamus. (C) High power view of nestin-stained reactive astrocytes in the pericontusional area showing gross hypertrophy and thick radiating processes at 4 and 7 days. Scale bar is 50 ␮m.

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ings were seen in the contralateral cortex and subcortical white matter. By 4 and 7 days after injury, the SMI-312 immunroeactivity had decreased and the majority of the terminal axon bulbs were disconnected from the proximal axonal segments (Fig. 4). Varicose swellings were still observed but less frequently than at 1 day, although the regional extent of axonal damage was similar. By 28 days, the SMI-312 immunoreactivity had declined to control levels and no axonal bulbs were detected (Fig. 4). GFAP immunoreactivity A mild elevation of GFAP immunoreactivity above control brain levels was observed as early as 1 day in the injured ipsilateral cortex, corpus callosum, and hippocampus, and this was significant at 4 to 7 days but similar to control levels by 28 days (Figs. 5 and 6A). The peak of the immunoreactive response was marked by the distribution of GFAP-positive astrocytes with hypertrophic soma and thick processes throughout the entire ipsilateral cortex, and especially around the contusion site (Fig. 5). Hippocampal GFAP expression was also intense and widespread at this time, involving all hippocampal subfields as well as the dentate gyrus. In the thalamus, however, the increase in GFAP immunoreactivity was similar to that observed in the cortex at 4 and 7 days but this remained significantly elevated even at 28 days (Fig. 6A). The temporal pattern of white matter GFAP immunoreactivity was similar to the cortex, i.e., significantly increased above control levels at 4 and 7 days but similar to control at 28 days. Nestin immunoreactivity In normal and sham-operated brains, the vasculature was lightly stained and nestin-positive cells were found along the ependyma and choroid plexus. At 1 day after injury, nestin-positive cells with morphological characteristics of reactive astrocytes; i.e., hypertrophic soma with thick radiating processes were observed around the cortical contusion, ipsilateral corpus callosum, and hippocampus (Fig. 5). Both the amplitude and regional extent of nestin immunoreactivity increased with time so that by 4 and 7 days it encompassed the whole ipsilateral cortex and all hippocampal subfields (Figs. 5, 6B), although this returned to control levels in all regions by 28 days. Similar to the GFAP response, nestin expression was strictly ipsilateral in the cortical gray matter and hippocampus, while nestin-positive cells with an elongated morphology were observed in the contralateral corpus callosum and external capsule at 4 days (not shown). However in the thalamus, no nestin immunoreactivity was detected at any time after injury despite a significant GFAP response (Figs. 5, 6A,B). It appears therefore that the temporal and spatial profiles of GFAP and nestin response are similar in the cortex, hippocampus, and white matter structures but different in the thalamus.

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OX42 immunoreactivity In normal and sham-operated brains, OX42-positive microglia with highly ramified processes were distributed uniformly throughout the white and gray matter. At 1 day after injury OX42 immunoreactivity was increased above control levels around the cortical contusion margin and this was maximal at 4 days but not significantly different from control levels at 28 days (Fig. 7A). High-power magnification of the contusion margin revealed the presence of reactive microglia, plump cell bodies with stout processes and macrophage-like round cells (Fig. 7B). In contrast to GFAP and nestin expression, which extended throughout the whole ipsilateral cortex, cortical OX42 expression was restricted to regions adjacent to the cortical contusion at all time points (Fig. 7A). In the corpus callosum, the temporal pattern of OX42 expression was similar to that observed in the cortex and similar to cortical GFAP and nestin expression (Fig. 6A and B). A mild increase in immunoreactivity was also observed in the contralateral side at 4 and 7 days compared to the ipsilateral side, similar to GFAP and nestin (not shown). The time course of OX42 expression in the hippocampus was similar to the cortex and the areas most affected were all layers of the CA3 subfield as well as the dentate gyrus and these areas corresponded to regions of neuronal injury on cresyl violet-stained sections. However, in the thalamus, increased OX42 immunoreactivity persisted and increased at each time point, in contrast to all other brain regions and all other histochemical protocols used. NG2 immunoreactivity In normal and sham-operated brains, regularly spaced, NG2-positive cells with irregularly shaped cell bodies and long thin, highly stellate branches were seen throughout the cortex, hippocampus, and white matter tracts, and these differed morphologically from OX42-positive microglia and GFAP-positive astrocytes. There was a distinct temporally related change in NG2-positive cell morphology, from an enlarged cell body with swollen processes at 1 day, to cells with fewer and thicker processes with dense surface deposits of anti-NG2 product at 4 –7 days, and finally a multipolar appearance similar to that in normal brain at 28 days (Fig. 8A). Similar to the OX42 response, increased cortical NG2 immunoreactivity was restricted to the cortical contusion margin at all ages and the amplitude of this expression was maximal at 4 days and decreased thereafter (Fig. 8B). Although NG2 immunoreactivity was increased in the ipsilateral corpus callosum at 1– 4 days, unlike the cortex, the amplitude was much smaller when compared to GFAP, nestin, and OX42 immunoreactivity in the same region. However, similar to other histochemical methods, a mild increase in immunoreactivity was observed in the contralateral corpus callosum at 1– 4 days that gradually declined at later time points (not shown). Increased hippocampal NG2 immunoreactivity was observed in the CA3

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Fig. 6. Time course and regional characterization of (A) glial fibrillary acidic protein (GFAP) and (B) nestin immunoreactivity quantified by optical density (O. D.) measurements normalized to a remote, unaffected brain region in sham-injured (E), injured (F), and control rats (Œ). In the cortex, corpus callosum, and hippocampus, both GFAP and nestin expression is significantly increased on or after 1 day and this returns to control levels by 28 days. Thalamic expression of GFAP is also significantly increased while nestin remains at control levels throughout. ***, **, and * represent a significant difference compared to the pooled sham-control group at the probability levels of P ⬍ 0.001, 0.01, and 0.05, respectively.

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Fig. 7. Regional characterization of OX42 immunoreactivity. (A) Low power light micrographs of the ipsilateral hemisphere of a sham-control rat and rats at different times after trauma showing the accumulation of microglial/macrophages in pericontused cortex by 4 days and the more gradual accumulation in the dorsolateral thalamus. (B) High power view of the pericontused cortex showing reactive microglia with stellate morphologies (arrow) and macrophagelike round cells (arrow head). (C) Time course of OX42 immunoreactivity in different regions quantified by optical density measurements and normalized to a remote, unaffected brain region in sham-injured (E), injured (F), and control rats (Œ). Immunoreactivity peaked at 4 days post trauma in cortex, corpus callosum, and hippocampus but remained elevated throughout in the thalamus. ***, **, and * represent a significant difference compared to the pooled sham-control group at the probability levels of P ⬍ 0.001, 0.01, and 0.05, respectively. Scale bar is 50 ␮m.

subfield and dentate gyrus at 4 –7 days and this was spatially consistent with areas of neuronal injury. The temporal profile of NG2 immunoreactivity in the thalamus was similar to the hippocampus.

above control values in all regions and this was significant for pericontused cortex (P ⬍ 0.01). At later time points mean diameter returned to control values.

Vascular response

Discussion

The density of RECA-1-positive blood vessels at the contusion margin was significantly decreased from shamcontrol levels at 1–7 days post injury (P ⬍ 0.01) and this returned to control values by 28 days (Figs. 9A and B). At a distance of 500 ␮m from the lesion margin, there was a mild but nonsignificant reduction in the number of RECA1-positive blood vessels at 1 day and this returned to control values by 4 days. Further from the contusion core within the ipsilateral midline cortex there was no change in vessel density at any time after injury. The mean diameter of all vessels and capillaries in all regions examined was unaffected at 1 day post injury, but at 4 days it was increased

We have shown that cortical contusion injury results in a highly reproducible lesion size that results in consistent, unilateral injury to cortical gray and white matter, hippocampus, and thalamus. Damage to the cortex and hippocampus was characterized by early neuronal loss together with a transient reactive astrocytosis, microglial/macrophage response, and an increase in both nestin-positive and NG2-positive cells. The pattern of damage to dorsal regions of the thalamus was markedly different with less severe reductions in neuronal staining, a persistent astrocyte and microglial/macrophage response, a transient increase in NG-positive cells, and an absence of any increase in nestin-positive cells.

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Fig. 9. (A) RECA-1-immunostained cortical microvessels at the contusion margin at different times after injury compared to sham control. (B) The density of all blood vessels, regardless of size, was significantly reduced from sham-control levels in the pericontused cortical margin at 1, 4, and 7 days post injury. Vessel density in cortical regions at 500 ␮m from the contusion margin and in the midline was unaffected. (C) The mean diameter of all blood vessels and (D) vessels less than 10 ␮m was increased in all three regions in sham control rats and this was significant at 4 days post-injury in the peri-contused cortical margin. Key: 1, 4, 7, 28, C ⫽ days post injury compared to sham control, respectively, also indicated by progressively increasing gray scale bars; ***, **, and * represent a significant difference compared to the pooled sham-control group at the probability levels of P ⬍ 0.001, 0.01, and 0.05, respectively.

Neuronal injury The pattern of reduced staining intensity obtained in this study was similar to that in other studies on the same model, albeit with some variations in the injury protocol (Colicos and Dash, 1996; Colicos et al., 1996; Dunn-Meynell and Levin, 1999; Eayrs, 1954; Kaya et al., 1999; Newcomb et al., 1999; Sutton et al., 1993). We did not attempt to quan-

tify neuronal damage since Nissl stain is not a good marker for this type of analysis. However, loss of cresyl violet staining is spatially coincident with heat-shock protein and acid fuchsin staining, more robust markers of cell injury after CCI (Dunn-Meynell and Levin, 1997). Since acute changes in these markers are consistent with cell loss at more chronic time points (Sutton et al., 1993), a qualitative loss of Nissl staining in this study can be broadly interpreted

Fig. 8. (A) High power views showing increased NG2 immunoreactivity in the pericontused cortex at different times post truama compared to sham control. (B) Time course of NG2 immunoreactivity in different regions quantified by optical density measurements in sham-injured (E), injured (F), and control rats (Œ). The greatest increase occurred around the cortical margin in the first week after injury together with some upregulation in hippocampal and thalamic regions. ***, **, and * represent a significant difference compared to the pooled sham-control group at the probability levels of P ⬍ 0.001, 0.01, and 0.05, respectively. Scale bar is 30 ␮m.

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as injured or dying neurons. It is difficult to determine whether the observed increase in contusion volume is due to cell loss or to neuronal atrophy; a distinction between them in future studies may be important since the latter may be amenable to trophic intervention. The spatial pattern of axonal injury— unilateral and centered around the contusion site and adjacent structures— confirms other work on the CCI model (Dunn-Meynell and Levin, 1997; Posmantur et al., 2000; Yaghmai and Povlishock, 1992). The time course of evolving axonal pathology in this study, from swollen axons to eventual terminal axon bulbs, is consistent with work on numerous trauma models (Maxwell et al., 1997). The mechanisms responsible for the swellings are complex, not least because recent work demonstrates neurofilament compaction and impaired axonal transport, pathologies previously thought to occur at the same site which may be independent phenomena (Stone et al., 2001). Astrocytosis The general temporal pattern of the astrocytic response was broadly similar to another qualitative study on the CCI model (Dunn-Meynell and Levin, 1997), in other brain trauma models (Carbonell and Grady, 1999; Dietrich et al., 1997; Hill et al., 1996; Hill-Felberg et al., 1999; Holmin et al., 1997; Raghavendra et al., 2000) stab wound (Mathewson and Berry, 1985), and cerebral infarction (Duggal et al., 1997). While many functions have been attributable to astrocytes such as ion homeostasis and neuromodulation (Aragul et al., 2001), when transformed to the activated cell type, other than a major involvement in glial scar formation, their role is far from clear; astrocytes are the major source of nerve growth factor after injury and in certain conditions they are permissive to axon growth, implying a possible role in neuronal plasticity (Goss et al., 1998; Ridet et al., 1997) but there is considerable evidence that they are also inhibitory to axon outgrowth (Fawcett and Asher, 1999). We observed a peak in cortical GFAP immunodensity at 4 days post trauma while a similar study on the CCI model found a peak increase in the density of GFAP-positive astrocytes at 10 days in the cortical contused region (Baldwin and Scheff, 1996), making it difficult to interpret our results as an increase in the number of reactive astrocytes. A likely reason for this discrepancy is that the optical density measurements that we used to quantify the response did not enable us to distinguish between an upregulation of filament protein per cell and an increase in cell number. The difference may also relate to the severity of the injury since, in the current study, a moderate to severe insult was used, compared to a relatively mild trauma in previous work (Baldwin and Scheff, 1996). In this study the pattern of GFAP staining was more widespread compared to the other pathologies investigated, and this is more consistent with another study that demonstrated an extensive pattern of neuronal activation after contusion injury, indicated by FOS staining

(Dunn-Meynell and Levin, 1997). As commented on by those authors, the mechanisms responsible for the GFAP response may differ spatially; around the contusion core the robust response may be stimulated by injured or degenerating neurons, compared with a more mild, remote response that may be linked with neuronal activation. Nestin-positive cells The time course and regional differences in amplitude of nestin immunoreactivity was similar to GFAP, OX42 and NG2 in the cortex, corpus callosum, and the hippocampus but not the thalamus. The intermediate filament protein nestin is a marker of developmentally immature cells (Lendahl et al., 1990) and adult vascular and ependymal cells (Duggal et al., 1997; Li and Chopp, 1999). It has previously been shown to be upregulated after CCI injury in cortex, hippocampus, and, unlike in the present study, it was also present in the thalamus (Sahin et al., 1999). This discrepancy may be related to injury severity because a bilateral craniectomy CCI protocol was used compared to a unilateral protocol in the present study. Furthermore, nestin was confined to cortex and hippocampal structures after moderate weight-drop injury (Holmin et al., 1997), while it was present in all damaged regions after transient ischemia (Li and Chopp, 1999). Nestin has been localized to at least astrocytes and microglia after brain injury (Holmin et al., 1997; Sahin et al., 1999) and more recent work on a transient ischemia model has shown it to be also present in oligodendrocytes and in neurons on the outer border zone of the ischemic core (Li and Chopp, 1999). These observations, together with data during CNS embryogensis that show a transient nestin expression in neuroepithelial stem cells prior to differentiation along a determined cell path (Lendahl et al., 1990), imply an attempt by the brain to repair the injury. A number of observations support the speculation that the widespread nestin immunoreactivity observed after injury in the present study is the result of transformation of endogenous stem cells to a differentiated cell type as part of an injury-induced repair mechanism. For example, nestin-positive cells have been observed in the ependyma of normal animals both in this study and by others (Duggal et al., 1997; Li and Chopp, 1999), and this region is considered to be neurogenic (Morshead and Vander, 2001); nestin-positive cells appear to migrate from the subependymal zone to the lesion site after weight-drop injury (Holmin et al., 1997); nestin expression coincides with proliferating (Bromodeoxyridine [BrdU]-labeled) cells after contusion injury in the mouse (Kernie et al., 2001) and an increase in BrdU-labeled cells occurred in the dentate gyrus after contusion injury in the rat (Newcomb et al., 1999)—a region of nestin stained cells in the present study. If nestin expression is considered to be a marker of endogenous neurogenesis, then the absence of any newly formed cells in the thalamus in the current work may be due to its location furthest from the neurogenic zones. A greater

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response of nestin-positive cells is presumed to occur after a more severe, direct injury to the thalamus and this might account for its presence in other models of severe trauma (Li and Chopp, 1999; Sahin et al., 1999) Although the fate of neural precursor cells after injury has been shown to be largely astrocytic (Holmin et al., 1997; Li and Chopp, 1999), proliferating cells coexpressing neuronal markers have been observed in the hippocampus after contusion injury (Dash et al., 2001; Kernie et al., 2001). Furthermore, reactive astrocytes can revert to a developmental immature state after injury as indicated by reexpression of proteins such as vimentin (Baldwin and Scheff, 1996; Hill et al., 1996) and nestin (Holmin et al., 1997; Lin et al., 1995), and, in the hippocampus at least, these cells can give rise to new neurons (Seri et al., 2001). Although the experimental design used in the current study does not enable us to distinguish between nestin upregulation in differentiated astrocytes and recruitment of neural precursor cells, based on the evidence above it is likely that the nestin response observed in our study represents part of a compensatory mechanism to repair injury. However, caution must be used when evaluating nestin expression because it is also upregulated in response to depolarization (Holmin et al., 2001) so that its appearance after injury may also result from either direct trauma or from spreading depression that occurs acutely post injury (Rogatsky et al., 1996). Oligodendrocyte precursor cells OPCs, a fourth major glial element of the mature brain (Levine et al., 2001), are transiently increased in number after CNS injury (Levine, 1994; Ong and Levine, 1999; Tanaka et al., 2001) or demyelination (Di Bello et al., 1999). We utilized the antibody to the cell surface molecule NG chondroitin sulfate proteoglycan to label OPCs and have extended the previous observations by describing the spatial-temporal response of this cell population to TBI. The results reported here indicated that NG2-positive OPCs responded rapidly to TBI and localize only to those regions of brain that show neuronal damage or loss. Although the rapid increase in NG2 immunoreactivity after TBI is comparable to that observed after stab wound (Levine, 1994), OPCs were not confined to the primary injury site but were also found in remote brain regions, such as the hippocampus and the thalamus, where neuronal injury develops over a more prolonged time course. While the exact reasons for this observation are unclear, the high extracellular glutamate and calcium concentrations and axonal damage present after trauma are factors that have been implicated in stimulation of either endogenous OPCs or migration from distant regions (Levine et al., 2001) and references therein). OPCs have been shown to develop in vitro into either astrocytes or oligodendrocytes (Wolswijk and Noble, 1989) and, although this has not yet been confirmed in vivo, OPCs have been shown to rapidly repopulate a lesioned area in a de-

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myelination model (Keirstead et al., 1998), suggesting a role in remyelination and brain repair after injury. The OPC membrane surface molecule NG2 has been shown to be inhibitory to axon growth (Dou and Levine, 1994), and a synaptic regulatory function has been proposed (Ong and Levine, 1999) so that the widespread accumulation of these cells after TBI may also represent a role in plasticity. Microgliosis The temporal response of microglia and macrophages in this study is in broad agreement with previous work that described the acute time course and localization of ED1and OX42-immunopositive cells following fluid percussion injury (Aihara et al., 1995; Soares et al., 1995). Our results demonstrate that the pattern of enhanced OX42 staining, which labels both microglia and macrophages regardless of whether they originate from the brain or systemically, was correlated anatomically with the distribution of neuronal damage and this was most conspicuously in the cortical contusion margin, CA3, dentate gyrus, and dorsal-lateral thalamus. Since acute leukocyte recruitment is spatially distinct from regions of neuronal injury (Soares et al., 1995), then the cellular response we observe may be mostly a response of endogenous rather than blood-borne cells to neuronal injury. We observed increasing OX42 immunoreactivity in the thalamus at all times post injury in accordance with another study on the CCI model (Raghavendra et al., 2000). The acute appearance of these cells in this region occurs when neurons become activated and show signs of early injury in this model (Dunn-Meynell and Levin, 1997), reflecting the idea that neuronal injury results in the transformation of microglia into phagocytic cells for the purpose of neuronal debris removal as well as activation of neighbouring, nonphagocytotic microglia (Kreutzberg, 1996). The prolonged upregulation of the thalamic OX42 response is most likely due to progressive degeneration of thalamic neurons and this has been shown to occur by both anterograde and retrograde degeneration after cortical aspiration injury (Sorensen et al., 1996). Thalamic neuronal death is largely apoptotic after cryogenic injury to the cortex (Hermann et al., 2000), but this does not appear to be the case within the first 2 weeks following contusion injury (Newcomb et al., 1999). Regardless of the mechanism of cell death, the slow time course of neuronal degeneration described here and by others (Dunn-Meynell and Levin, 1997) may represent an opportunity for treatment after brain injury, although initial results with a protein synthesis inhibitor resulted in only temporary prevention of thalamic neurone death following cortical infarction (Watanabe et al., 1997). Microvessels We have shown in the initial stages of injury that microvessel density was decreased and mean diameter was

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increased in the contusion margin and this resolved to control values by 7 days. This is consistent with a fluid percussion injury study that demonstrated a significant reduction in vessel density at 7 days post injury, but no decrease in the fractional area occupied by the vessels in the contusion margin, indicating that there were large vessels present (Lin et al., 2001), similar to results in other work (Cobbs et al., 1997; Daneyernez, 1999). It is unclear why vessel size was not similarly increased at 7 days in the present study after contusion injury. One possible explanation is that it is a methodological issue, i.e., vessel parameters return to control values because the necrotic rim evolves over time to become part of the contusion cavity, so that later vessel measurements are biased toward less severely injured tissue. However, this appears unlikely since the same trend was noted in less affected, midline cortical tissue (Fig. 9). We are uncertain why mean vessel diameter was increased at 4 days after injury while it was unaffected at 1 day. Brain oxygen consumption (Levasseur et al., 2000) and glucose metabolism (Richards et al., 2001; Yoshino et al., 1991) are increased acutely after experimental trauma so that maximal vessel dilation might have be expected at 1 day as a response to the metabolic demand. However, this was not the case and one possibility is that a simultaneous vasospastic response might occur acutely due to a high concentration of extracellular potassium or other contractile agent that ultimately resolves with time. Endothelial nitric oxide synthase expression, a molecule important for vascular tone (Ma et al., 1996) peaks at around 3 days after weight-drop injury (Lu et al., 2001) and this implies a possible role in vessel dilation at 4 days in the current study. However, other studies have demonstrated only an early peak in endothelial nitric oxide synthase (Gahm et al., 2000) and nitric oxide (Cherian et al., 2000) after contusion injury, consistent with only a very acute increase in metabolic demand followed by a prolonged metabolic suppression after injury in the fluid percussion model (Yoshino et al., 1991), so that a prolonged physiological response may not account for our findings. An alternative hypothesis to explain the expanded vessels is that it is a proliferation response, since vascular growth factors are known to be upregulated following brain trauma (Nag et al., 1997), and dilated cortical vessels lacking uniform spacing result from vascular endothelial growth factor infusion (Rosenstein et al., 1998). In summary, we have shown that the cellular response to CCI injury is highly complex involving a number of pathologies each with a specific regional and temporal response. Against the well-known background of neuronal/axonal injury, we have demonstrated a robust response by NG2- and nestin-positive cells, and the available evidence suggests that they may contribute to the endogenous repair process that follows injury to the CNS. A more detailed investigation of these potential repair processes is required to determine whether they can be manipulated to enhance outcome after injury.

Acknowledgments We thank Dr. James Fawcett for supplying some of the antibodies and Mr. Eduardo Torres for discussions on some of the immunohistology protocols. We are grateful to The Medical Research Council of the United Kingdom for funding and to the Overseas Research Council and Merck Sharp & Dohme who funded SC and NGH, respectively.

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