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Acta Neuropathol (2006) 112:471–481 DOI 10.1007/s00401-006-0108-2 O RI G I NAL PAPE R The fate of Nissl-stained dark neurons following traumatic brain injury in rats: diVerence between neocortex and hippocampus regarding survival rate Hidetoshi Ooigawa · Hiroshi Nawashiro · Shinji Fukui · Naoki Otani · Atsushi Osumi · Terushige Toyooka · Katsuji Shima Received: 13 April 2006 / Revised: 20 June 2006 / Accepted: 26 June 2006 / Published online: 21 July 2006 © Springer-Verlag 2006 Abstract We studied the fate of Nissl-stained dark neurons (N-DNs) following traumatic brain injury (TBI). N-DNs were investigated in the cerebral neocortex and the hippocampus using a rat lateral Xuid percussion injury model. Nissl stain, acid fuchsin stain and immunohistochemistry with phosphorylated extracellular signal-regulated protein kinase (pERK) antibody were used in order to assess posttraumatic neurons. In the neocortex, the number of dead neurons at 24 h postinjury was signiWcantly less than that of the observed N-DNs in the earlier phase. Only a few N-DNs increased their pERK immunoreactivity. On the other hand, in the hippocampus the number of dead neurons was approximately the same number as that of the N-DNs, and most N-DNs showed an increased pERK immunoreactivity. These data suggest that not all N-DNs inevitably die especially in the neocortex after TBI. The fate of N-DNs is thus considered to diVer depending on brain subWelds. Keywords Dark neuron · Nissl stain · Traumatic brain injury · Extracellular signal-regulated protein kinase Introduction Dark neurons are traditionally known to represent a typical morphological change of injured neurons following many kinds of insults, and have been observed H. Ooigawa · H. Nawashiro (&) · S. Fukui · N. Otani · A. Osumi · T. Toyooka · K. Shima Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan e-mail: [email protected] using various staining methods, such as hematoxylin and eosin stain, Nissl stain and silver stain [17, 19, 25, 29, 33, 41]. Since dark neurons show massive shrinkage and abnormal basophilia, they can be clearly distinguished from normal neurons. Some studies have shown the following electron microscopic characteristics of dark neurons: (1) a marked condensation of both karyoplasm and cytoplasm, (2) an intact nuclear membrane, cytoplasmic membrane and cytoplasmic organelles, (3) mitochondrial swelling, (4) a dilatation of the Golgi cisternae, (5) a decrease in the rough endoplasmic reticulum, and polyribosomes, and (6) an aggregation of the nuclear chromatin [2, 3, 11, 39], which resembles the appearance of necrotic neurons. Similarly, in the Weld of traumatic brain injury (TBI), dark neurons have been observed as one kind of feature of damaged neurons. The regions where dark neurons appear at a high rate after TBI, such as neocortex, CA3 subWeld and dentate hilus, coincide with the regions where subsequent neuronal death is induced. These facts appear to indicate that dark neurons inevitably die [10, 25, 33]. On the other hand, some reports have described the possibility that dark neurons recover from such a damaged status [11, 15, 16]. In the rat lateral Xuid percussion injury (FPI) model, numerous dark neurons are observed in the neocortex and the hippocampus in the early phase after TBI [10, 17, 29]. However, it remains unclear whether all dark neurons result in subsequent neuronal cell death. In this study we addressed a numerical relationship between the emergence of Nissl-stained dark neurons (N-DNs) and subsequent neuronal cell loss in the neocortex and the hippocampus after TBI. We counted total neuronal number and N-DNs in the neocortex and the hippocampus from immediately to 24 h after 123 472 TBI. Neuronal death was calculated from the subtraction of total neuronal number in sham brains minus total neuronal number in injured animals at 24 h after TBI. Since at least some of the observed neuronal cell death is due to apoptosis [4, 5, 8, 22], we ensured the neuronal death using terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling (TUNEL) stain. Nissl/acid fuchsin stain was also performed as another staining method to conWrm the presence of damaged neurons [6]. Furthermore, we immunohistochemically studied phosphorylated extracellular signal-regulated protein kinase (pERK) on dark neurons, which plays an important role in signal transduction after TBI [31], and shows a diVerential immunoreactivity between the cortex and hippocampus regarding duration and magnitude [35, 36, 43]. Materials and methods Animal experimental procedure Adult male Sprague-Dawley rats weighing from 300 to 400 g were used. The rats were housed in individual cages under controlled environmental conditions (12/ 12 h light/dark cycle, 20–22°C; room temperature) with food and water available for at least 1 week before undergoing experimental surgery. The Animal Care and Use Committee of the National Defense Medical College approved all animal procedures. All surgical procedures were performed under aseptic surgical conditions. The rats were anesthetized with isoXurane in 30% oxygen and 70% nitrous oxide gas mixture through a facemask. The rats were Wxed in a stereotaxic Xame. A 4.8 mm craniectomy was made over the right parietal cortex (3.8 mm posterior and 2.5 mm lateral to the bregma). A plastic Luer-Loc was placed over the craniectomy site with dental acrylic cement. The next day, the rats were anesthetized and a PE catheter was implanted in the right femoral artery. The rats were intubated with a 14-gauge angiocatheter and maintained on a mechanical ventilator after the infusion of pancronium bromide (tidal volume: 2.5–3.0 ml/ kg; respiratory rate: 70 per min). The rectal temperature was measured with a rectal probe and then it was maintained at a constant level of around 37.0°C using a heating pad. Arterial blood samples were analyzed intermittently. The rats were subjected to FPI at a moderate severity (1.8–2.2 atmosphere, 12–14 ms in duration) using a DragonXy Xuid percussion device (model HPD-1700, DragonXy R&D, Silver Spring, MD) as previously described [27]. The pressure pulse was measured using a pressure transducer (Model 211 123 Acta Neuropathol (2006) 112:471–481 B4, Kistler Instrument Corp., Amherst, NY) and it was recorded on a digital storage computer (MacLab, AD Instruments, NSW, Australia). Following the induction of the injury, the connection cap was removed and the scalp was sutured. The rats were placed on the heating pad to maintain their temperature around 37.0°C until they could move independently. The sham-operated rats (n = 6) were subjected to all the same procedures except for an actual insult, and then were sacriWced 15 min after pseudoinjury. After completing the surgical procedures, the rats were returned to their home cage with food and water available ad libitum. Tissue preparation Under the intrapenetneal injection of pentobarbital sodium, the rats were transcardially perfused with normal saline followed by 4% buVered paraformaldehyde after predetermined survival times (0, 15, 30, 60 min; 3, 6 and 24 h; n = 6–8 per each time point). The brains were removed just after perfusion and immersed in the 4% buVered paraformaldehyde overnight, and then were embedded in paraYn. Three series of 5-m-thick coronal sections were cut every 100 m from 3.3 to 4.3 mm posterior to the bregma. Each series of sections was used for Nissl stain, Nissl/acid fuchsin stain and immunostain against pERK. The existence of morphological changes, such as a gliding contusion, was conWrmed in all sections of the traumatized animals. Combined Nissl/acid fuchsin staining For combined Nissl/acid fuchsin staining, the sections were stained with 0.1% cresyl violet. The sections were then placed in 0.1% acid fuchsin fortiWed with a few drops of acetic acid for 20 s, and then they were subsequently washed with distilled water, dehydrated with ethanol and lemosol, and Wnally coverslipped. Immunohistochemistry Immunohistochemistry was performed using a HISTOFINE SAB-PO (R) Kit (NICHIREI Co, Tokyo, Japan). After deparaVinization and hydration, the sections were boiled in 10 mmol/l sodium citrate buVer (pH 6.0) for 10 min at 95°C, and were cooled for 20 min. Endogenous peroxidase was blocked with 3% hydrogen peroxidase. The sections were incubated with PBS containing 10% normal goat serum at room temperature to eliminate any nonspeciWc binding, and then they were incubated overnight at 4°C with polyclonal antibody against pERK1/2 (Cell Signaling Technology, Danvers, MA1, 1:100). The sections were incubated Acta Neuropathol (2006) 112:471–481 473 with biotinylated secondary antibody and horseradish peroxidase-linked streptavidin for 1 h at room temperature. Peroxidase was then demonstrated with DAB. TUNEL staining APOPTOSIS in situ Detection Kit (WAKO, Osaka, Japan) was used for TUNEL staining, according to the manufacturer’s instruction. After deparaVinization and hydration, sections were incubated with protein digestion enzyme solution for 5 min at 37°C, The sections were treated with TdT solution and with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity, and then treated with peroxidase-conjugated antibody for 10 min at 37°C. Nick end labeling was visualized by DAB solution. For the positive control, sections were treated with DNase I before treatment with TdT. For negative control sections were incubated with TdT buVer that did not contain the enzyme. Counting of neurons Three periodic Nissl-stained sections at the coronal level of 3.8 mm posterior to the bregma were used for cell counts and the average was calculated. In the neocortex, neurons were counted in two diVerent regions circumscribed with a rectangle measuring 0.47 £ 0.35 mm2 (Fig. 1). The centers of both counting boxes were 5.0 mm far from each other. The one counting box was located just under the craniectomy, in which no cerebral contusion was seen. The center of the box was situated in the cortical layer V and 2.5 mm lateral to the midline. The other box was placed in the region close to the gliding contusion. This area is located on the boundary between the area 1 of temporal cortex and the area 1 of parietal cortex [37]. In the hippocampus, neurons were counted in the pyramidal cell layer of the CA3 and the dentate hilus (Fig. 1). The CA3 subWeld was determined as a territory from the CA3/2 border to a point where the pyramidal cells enter the hilus as deWned by the lateral aspect of the granule cell layer’s dorsal and ventral leaves. The dentate hilar neurons were counted as those cells existing within the region deWned by the superior and inferior granular cell layers, thus excluding the CA3 pyramidal cell neurons and transitional neurons within approximately 20 m of the pyramidal neurons [25]. The specimens were scanned at a magniWcation of £200 within the identical areas in the neocortex and the hippocampus, using a microscope (Olympus Optical Co, LTD, Japan) equipped with a digital camera system (Pixera 600CL-CU, Pixera Corporation, Japan). The intact neurons were deWned as Fig. 1 Schematic illustration of regions that were selected for neuronal cell counting (colored with blue). The area colored with red indicates the lesions of a gliding contusion nonbasophilic neurons with both the pale nuclei and the discrete nucleoi. Any neurons that had nucleolar fragments larger than one-half of the average nucleolar diameter were included in the count as well as the neurons that had intact neuronal bodies [23]. The N-DNs were deWned as neurons with abnormal morphologies of massive shrunken, hyperbasophilic features, and corkscrew-like dendrites. No correlation factor was applied because the somata were smaller than the distance between the two sections stained by the same method [23]. In the Nissl/acid fuchsin sections, intensely acidophilic cells were counted. The pERK-positive cells were also counted in the aforementioned areas. All cells were counted as pERK-positive cells regardless of the density of labeling by DAB [30]. We used sections taken by 1 h after TBI for cell counting of pERK-positive cells, because ERK is activated predominantly in non-neuronal cells as early as 2 h postinjury [43]. Fragments of somata that could be clearly identiWed as cells were also counted [30]. All neuronal counting was performed blinded to the treatment conditions. DeWnition of the number of dead neurons The number of dead neurons was deWned as (neuronal cell number in sham-operated rats) ¡ (neuronal cell number in injured rats sacriWced 24 h after TBI) Statistical analysis All data are expressed as mean § SD. The physiological parameters were analyzed using one-way analysis 123 474 Acta Neuropathol (2006) 112:471–481 of variance. Cell counting was analyzed using Kruskal– Wallis test. If any signiWcance was noted, individual comparisons were made using Mann–Whitney’s U test. A P value of less than 0.05 compared to sham values was considered statistically signiWcant. Results Physiological data There were no signiWcant diVerences in the physiological data between each group (Table 1). Table 1 Summary of physiological parameters Preinjury After 5 min After 15 min 7.43 § 0.03 38.2 § 2.4 128.4 § 15.6 108.7 § 8.9 7.41 § 0.02 38.6 § 3.1 125.8 § 15.4 110.0 § 9.4 TBI group sacriWced immediately after TBI pH 7.44 § 0.02 7.43 § 0.03 pCO2 36.1 § 2.8 37.9 § 4.1 pO2 127.0 § 15.1 122.1 § 10.8 MABP 114.2 § 10.7 121.1 § 13.2 7.42 § 0.04 38.9 § 3.4 119.8 § 20.6 115.1 § 12.2 TBI group sacriWced 15 min after TBI pH 7.42 § 0.02 7.42 § 0.04 pCO2 37.8 § 2.2 38.4 § 4.0 pO2 129.4 § 5.4 129.0 § 15.9 MABP 111.4 § 8.2 113.9 § 7.6 7.43 § 0.03 36.8 § 4.8 123.0 § 24.4 111.5 § 8.9 TBI group sacriWced 30 min after TBI pH 7.44 § 0.04 7.44 § 0.04 pCO2 38.2 § 2.7 38.9 § 5.2 pO2 128.3 § 13.6 129.4 § 15.0 MABP 112.1 § 10.2 114.7 § 13.8 7.43 § 0.04 39.6 § 4.8 125.5 § 19.3 113.6 § 10.3 TBI group sacriWced 1 h after TBI pH 7.43 § 0.03 7.42 § 0.04 pCO2 35.6 § 1.1 38.7 § 4.5 pO2 127.2 § 13.5 124.8 § 21.1 MABP 107.9 § 9.2 108.5 § 10.2 7.43 § 0.03 37.9 § 3.8 121.0 § 13.6 106.9 § 10.8 TBI group sacriWced 3 h after TBI pH 7.44 § 0.02 7.43 § 0.03 pCO2 37.1 § 1.4 39.1 § 3.4 pO2 126.5 § 16.6 123.1 § 9.7 MABP 109.9 § 9.4 112.3 § 12.6 7.45 § 0.04 41.0 § 5.0 113.0 § 18.9 108.6 § 13.0 TBI group sacriWced 6 h after TBI pH 7.44 § 0.03 7.42 § 0.02 pCO2 36.4 § 3.6 39.0 § 2.7 pO2 126.8 § 7.8 122.7 § 17.6 MABP 112.2 § 13.9 114.0 § 11.0 7.43 § 0.03 39.1 § 2.8 119.3 § 16.5 114.2 § 10.7 TBI group sacriWced 24 h after TBI pH 7.43 § 0.03 7.42 § 0.05 pCO2 36.5 § 2.6 37.0 § 2.5 pO2 126.3 § 11.5 123.4 § 17.2 MABP 112.1 § 10.4 110.3 § 6.6 7.45 § 0.04 37.1 § 2.8 120.4 § 13.7 111.2 § 8.3 Sham-operated group pH 7.43 § 0.03 pCO2 36.6 § 3.0 PO2 132.1 § 15.6 MABP 112.1 § 10.2 123 Dark neurons in the neocortical region distant from the contusion Nissl-stained dark neurons were not detected in the sham-operated rats (Fig. 2b, c). N-DNs began to appear immediately after TBI, and kept at maximum number for up to 30 min postinjury (Fig. 2b, d). Thereafter, the N-DNs began to decrease at 1 h postinjury and completely disappeared by 24 h after TBI (Fig. 2b, g). Despite the existence of N-DNs in the acute phase, there was no signiWcant decrease in neuronal number at 24 h postinjury in comparison to the sham values (Fig. 2a). Acid fuchsin-positive neurons were also observed in the acute phase after TBI, and coincided with the N-DNs (Fig. 2e). No acid fuchsin-stained neurons were detected 24 h postinjury along with the disappearance of the N-DNs (Fig. 2b, h). pERK-positive neurons also emerged in this area. pERK positive neurons were clearly less than N-DNs in number during the period till 1 h after TBI (Fig. 2b). In fact, most dark neurons did not have pERK immunoreactivity (Fig. 2f). No TUNEL-staining cells were detected in this region 24 h after TBI (Fig. 2i). Dark neurons in the neocortical region close to the contusion Nissl-stained dark neurons were observed from immediately to 6 h after TBI (Fig. 3b, e). The number of N-DNs observed during this period was signiWcantly higher than that in the region distant from the contusion (P < 0.01 at 15 and 30 min after TBI; Figs. 2b, 3b). The number of total neurons at the point of 24 h after TBI was decreased compared to that of sham-operated rats (P = 0.02; Fig. 3a, h). However, the number of dead neurons was signiWcantly less than that of the N-DNs appearing in the acute phase (P = 0.03, Fig. 3c). The acid fuchsin-positive neurons were consistent with N-DNs in the early phase after TBI (Fig. 3f). Although there were no N-DNs in the neocortex close to the contusion at 24 h after TBI (Fig. 3b, h), acid fuchsin-staining cells could be observed (Fig. 3i). Some pERK-positive neurons were observed in the acute phase (Fig. 3b), but most of them were not dark neurons (Fig. 3g). In spite of the diVerence in the number of N-DNs between the two cortical areas, almost the same number of pERK-positive neurons was observed (Figs. 2b, 3b). Many TUNEL-positive cells were observed 24 h after TBI in contrast to the other neocortical regions (Fig. 2k). Acta Neuropathol (2006) 112:471–481 Fig. 2 Neuronal counts and typical patterns of brain tissue in the neocortex distant from the contusion. Bar graphs show the total neuronal number (a) and the number of Nissl-stained dark neurons, acid fuchsin-positive neurons and pERK-positive neurons (b), respectively. Photomicrographs of the following were taken: sham-operated rats (c Nissl stain), injured rats in the acute phase after TBI (d Nissl stain, e Nissl/acid fuchsin stain, f pERK immunostaining) and injured rats at 24 h postinjury (g Nissl stain, h Nissl/acid fuchsin stain, i TUNEL stain) . j indicates a positive control of TUNEL stain. Insets show magniWed 475 views. There were no N-DNs in sham-operated rats (b and c). Despite emergence of many N-DNs in the early phase after TBI (b and d), neuronal cell loss was not observed 24 h after TBI (a and g). Acid fuchsin-positive neurons were also observed in the acute phase (e), and then disappeared by 24 h after TBI. Some pERK-positive neurons were seen in this area. No TUNELstaining cell was observed 24 h after TBI (i). S and I indicate sham-operated and immediately sacriWced groups, respectively. # P < 0.05, *P < 0.01 compared with sham-operated rats. Scale bar = 200 m 123 476 Fig. 3 Neuronal counts and typical patterns of brain tissue in the neocortex close to the contusion. Bar graphs show the total neuronal number (a), the number of Nissl-stained dark neurons, acid fuchsin-positive neurons and pERK-positive neurons (b), and comparison in number between N-DNs appearing 30 min after TBI and dead neurons (c), respectively. Photomicrographs of the following were taken: sham-operated rats (d Nissl stain, j TUNEL stain), injured rats in the acute phase after TBI (e Nissl stain, f Nissl/acid fuchsin stain, g pERK immunostaining) and injured rats at 24 h postinjury (h Nissl stain, i Nissl/acid fuchsin stain, k TUNEL stain). Insets show magniWed views. No N-DNs were observed in the sham-operated rats (b and d). Many N-DNs ap- 123 Acta Neuropathol (2006) 112:471–481 peared in the early phase after TBI (b and e), SigniWcant neuronal cell loss was observed 24 h after TBI (a and h). However, the number of dead neurons was signiWcantly less than that of N-DNs appeared in the acute phase (c). Acid fuchsin-positive neurons were observed 24 h after TBI (i) as well as in the acute phase (f). Most dark neurons do not increase their immunoreactivity for pERK (g). TUNEL-staining cell was observed 24 h after TBI (k). In the bar charts, S and I indicate sham-operated group and immediately sacriWced group, respectively. #P < 0.05, *P < 0.01 compared with sham-operated rats (a and b). #P < 0.05 comparison between two groups (c). Scale bar = 200 m Acta Neuropathol (2006) 112:471–481 Dark neurons in the hippocampus Nissl-stained dark neurons were also detected in the hippocampus in the early phase after TBI both in the dentate hilus (Fig. 4b, e) and the CA3 subWeld (Fig. 5b, d). The number of surviving neurons at 24 h postinjury signiWcantly decreased in comparison to the shamoperated rats in those areas (dentate hilus; P < 0.01, CA3; P = 0.01, Figs. 4a, 5a). The maximum number of N-DNs in the acute phase, which were observed immediately after TBI in the dentate hilus, and 15 min after TBI in the CA3 subWeld (Figs. 4b, 5b), were approximately the same as the number of dead neurons (Figs. 4c, 5c). The acid fuchsin-positive cells coincided with the N-DNs in the early phase (Fig. 4f), and could be detected even after the N-DNs had disappeared (Figs. 4h, i, 5f). pERK-positive cells could also be observed in both of hippocampal regions. The number of the pERK-positive cells reached a maximum 15 min after TBI in the dentate hilus (Fig. 4b), and 30 min postinjury in the CA3 subWeld (Fig. 5b). In the pERK immunostaining, pERK-positive cells are consistent with dark neurons in the acute phase after TBI (Figs. 4g and 5e). Although some TUNEL-positive neurons were observed in the hippocampus 24 h after TBI, there was no proliferation in comparison to sham groups (data not shown). Discussion Methodological considerations The Nissl stain is a very popular technique and clearly supports a neuronal appearance. However, counting of Nissl-stained neurons is aVected by the characteristics of the dye used, time of exposure of the section to dye solution, and the thickness of the section [9, 23]. Many dyes are available for Nissl staining; cresyl violet, thionin, toluidine blue, and methylen blue. We used cresyl violet, because the dye has advantages of both the clarity of background and the intensity of staining of the Nissl substance, nuclei and nucleoi [23]. Among 11 periodic sections, we counted neurons using only three adjacent sections near the level of 3.8 mm posterior to the bregma, at which most large gliding contusions were observed in our model (data not shown). When attempting to count the absolute neuronal number in brain tissue, the use of serial thick sections may be more accurate. However, using serial thick sections may also result in serious problems, since the same neurons may be counted more than once or the dye used does not always inWltrate suYciently in the section 477 [9, 18]. We therefore used periodic 5-m sections to avoid such problems. When intending to observe damaged neurons, some staining methods such as hematoxylin and eosin stain, Nissl stain, acid fuchsin stain, silver stain and FluoroJade stain can be suitable [10, 11, 14–17, 19, 41, 45–47]. The nomenclature of ‘dark neuron’ has been mainly used for a damaged neuron stained with Nissl stain or silver stain because of its morphological feature. Although both of the two staining methods can detect damaged neurons in the early phase after TBI, the characteristics of those stainings are somewhat diVerent from each other [11, 15, 16]. The silver method has been known to be more speciWc to detect damaged neurons than Nissl method [15, 16, 44]. On the other hand, the method fails to demonstrate damaged neuron within the territories of contusion, in spite of the existence of massive shrunken and hyperbasophilic damaged neurons in Nissl-stain preparations [16]. We therefore needed to identify the diVerence in characteristics between each staining methods, even if both of them can show damaged neurons. In the present study, we used the Nissl method to observe dark neurons, which is easier and more routinely performed than the silver method in the Weld of neuropathology. We also employed acid fuchsin staining as another method to detect damaged neurons. There was a clear diVerence in the staining properties between Nissl stain and acid fuchsin stain at 1 h or later of TBI. Two possible hypotheses have been described previously [17]. One is that some neurons become newly damaged and stained with acid fuchsin. The other is that many injured neurons lose their aYnity for Nissl staining by 12–24 h, even though they continue to stain for acid fuchsin. As a result, acid fuchsin staining was able to indicate injured neurons at 24 h after injury, in contrast to Nissl staining. In the neocortex distant from the contusion, no acid fuchsin-positive neurons were detected at 24 h after injury. These results suggest that the neurons, which recovered from N-DNs, lost their aYnity to acid fuchsin. Nissl-stained dark neurons can recover in the neocortex The present study indicates that not all N-DNs necessarily die after TBI. Especially in the neocortex, many damaged neurons, even N-DNs close to the contusion, were able to survive for 24 h after TBI. However, there was a discernible diVerence between the two neocortical lesions regarding the survival ratio of the N-DNs. Almost all N-DNs were able to recover in the neocortex distant from the contusion. On the other hand, in 123 478 Acta Neuropathol (2006) 112:471–481 Fig. 4 The neuronal counts and typical patterns of brain tissue in the dentate hilus. Bar graphs show the total neuronal number (a), the number of Nissl-stained dark neurons, acid fuchsin-positive neurons and pERK-positive neurons (b), and comparison in number between N-DNs appeared immediately after TBI and dead neurons (c), respectively. Photomicrographs of the following were taken: sham-operated rats (d Nissl stain), injured rats in the acute phase after TBI (e Nissl stain, f Nissl/acid fuchsin stain, g pERK immunostaining) and injured rats at 24 h postinjury (h Nissl stain, i Nissl/acid fuchsin stain). Insets show magniWed views. There was no N-DNs in sham-operated rats (b and d). Many N-DNs appeared in the early phase after TBI (b and e), SigniWcant neuronal cell loss was observed 3 h and later after TBI (a and h). The number of dead neurons was approximately the same as that of N-DNs appeared in the acute phase (c). Acid fuchsin-positive neurons were observed 24 h after TBI (i) as well as in the acute phase (f). Most dark neurons increased their immunoreactivity for pERK (g). In the bar charts, S and I indicate sham-operated group and immediately sacriWced group, respectively. # P < 0.05, *P < 0.01 compared with sham-operated rats (a and b). Scale bar = 200 m the neocortex close to the contusion, both the incidence and the death rate of the N-DNs were higher. This result seems to be consistent with the fact that it is diYcult to save dark neurons, when they are induced by severe injury to the brain [19]. In the neocortex, a contusion or disruption of the vascular permeability induced by FPI might aVect the diVerence in the recov- erability of N-DNs. In the hippocampus, the maximum number of N-DNs observed over each time point approximately matched the number of dead neurons. There was an obvious diVerence in the surviving rate of N-DNs between the neocortex and the hippocampus. Hippocampal N-DNs died at a higher rate than those observed in the neocortex. Hippocampal neurons are 123 Acta Neuropathol (2006) 112:471–481 479 Fig. 5 The neuronal counts and typical patterns of brain tissue in the CA3 subWelds. Bar graphs show the total neuronal number (a), the number of Nissl-stained dark neurons, acid fuchsin-positive neurons and pERK-positive neurons (b), and comparison in number between N-DNs appeared 15 min after TBI and dead neurons (c), respectively. Photomicrographs of the following were taken: injured rats in the acute phase after TBI (d Nissl stain, e pERK immunostaining) and injured rats at 24 h postinjury (f Nissl/acid fuchsin stain). Many N-DNs appeared in the early phase after TBI (b and d); signiWcant neuronal cell loss was observed 24 h after TBI (a). The number of dead neurons was approximately the same as that of N-DNs appeared in the acute phase (c). Acid fuchsin-positive neurons were observed 24 h after TBI (f). Most dark neurons increased their immunoreactivity for pERK (e). In the bar charts, S and I indicate sham-operated group and immediately sacriWced group, respectively. #P < 0.05, *P < 0.01 compared with sham-operated rats (a and b). Scale bar = 50 m (e) and 100 m (f ) known to be vulnerable against TBI. Owing to the fact that hippocampal neurons do not tend to recover well after TBI, such injured hippocampal neurons might aVect hippocampal vulnerability. Morphological alterations in neuronal cell after TBI have been reported to be attributed to the loss of cytoskeletal proteins such as neuroWlament 68 (NF68), neuroWlament 200 (NF200) and microtubule-associated protein 2 (MAP-2) [20, 42, 44] with activation of calpain [34]. TBI induces neuroWlament compaction within 5 min after injury, and the compaction persists for at least 6 h [reviewed in 28, 40]. Thereafter, neuroWlament compaction is thought to demonstrate a state of neuroWlament proteolysis and Wnally show neuronal cell death [28]. However, resent studies have shown a loss of cytoskeletal proteins such as MAP-2 in vivo [49, 50], and neuroWlament proteins in vitro [21] occur in the absence of neuronal cell death. The present study also indirectly indicates that neuroWlament compaction does not inevitably lead to neuronal cell death. ERK activation in morphologically diVerent type of neurons One unique Wnding in the present study is the morphological diVerence in pERK-positive neurons between the neocortex and the hippocampus. In the neocortex, ERK was mainly activated in the neurons with a normal feature, but in the hippocampus ERK immunoreactivity was increased in dark neurons. This result indicates that ERK activation in the neocortical neurons, most of which do not die, may play a survival promoting role, whereas the activation of ERK in the hippocampus, where most dark neurons die, may play a death promoting role. ERK activation in the hippocampus might thus be a sign indicating that the 123 480 circumstances are not favorable for the spontaneous recovery of the dark neurons. ERK is known to be activated in response to growth stimuli for proliferation and survival. In addition, a recent study revealed that the ERK pathway might have functions involved in neuronal degeneration and apoptosis [reviewed in 6, 7, 18, 46], in association with cerebral ischemia [1, 32], Parkinson’s disease [24, 51] and Alzheimer disease [13, 38]. Marshall [26] reported that the short-to-moderate term activation of ERK signaling promoted proliferation and diVerentiation, respectively, whereas Subramaniam [48] described that long ERK activation leads to neuronal death. In the Weld of TBI study, some studies have reported its bilateral characteristics of ERK activation. Mori et al. [31] reported a deleterious eVect of this pathway using an inhibitor of ERK pathway, in which the cortical lesion volume of rats treated with the inhibitor was signiWcantly reduced. In contrast, Dash et al. [12] reported that the inhibition of this cascade worsened retrograde amnesia, exacerbated motor deWcit and did not have any overt eVects on cell survival. The role of ERK activation after TBI is still not fully understood. Further investigation will be needed to elucidate the function according to various brain subWelds. To summarize, the present study investigated the fate of N-DNs following TBI. N-DNs are observed in both the neocortex and hippocampus in the early phase after TBI. However, the fate of N-DNs tended to diVer depending on the regions of the brain. In the neocortex, most N-DNs appeared to survive for 24 h after TBI, while N-DNs tended to die in the hippocampus. Acta Neuropathol (2006) 112:471–481 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 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