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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: J Neuropathol Exp Neurol. 2012 Apr;71(4):348–359. doi: 10.1097/NEN.0b013e31824ea078

Moderate Traumatic Brain Injury Triggers Rapid Necrotic Death of Immature Neurons in the Hippocampus

Hongzhen Zhou 1,6, Liang Chen 2,3,4,6, Xiang Gao 2,3,4, Bingde Luo 5, Jinhui Chen 2,3,4
PMCID: PMC3311037  NIHMSID: NIHMS361845  PMID: 22437344

Abstract

Traumatic brain injury (TBI) causes cell death predominantly in the cerebral cortex but there is additional secondary cell death in the hippocampus. We previously found that the majority of the dying cells in the mouse hippocampus are newborn immature granular neurons in a mouse model of lateral controlled cortical impact (CCI) injury with a moderate level of impact. It is not known how long this selective cell death in the hippocampal dentate gyrus lasts, and how it is induced. Using Fluoro-Jade B and immunohistochemistry, we show that most of the neuron death in the hippocampus occurs within 24 hours post-TBI and that cell death continues at low level for at least another 2 wks in this lateral CCI model. The majority of the dying immature granular neurons did not exhibit morphological characteristics of apoptosis and only a small subpopulation of the dying cells was positive for apoptotic markers. In contrast, most of the dying cells co-expressed the receptor-interacting protein-1, a marker of necrosis, suggesting that immature neurons mainly died of necrosis. These results indicate that moderate TBI mainly triggers rapid necrotic death of immature neurons in the hippocampus in a mouse CCI model.

Keywords: Apoptosis, Cell Death, Hippocampus, Mouse model, Necrosis, Traumatic Brain Injury

INTRODUCTION

Traumatic brain injury (TBI) is a leading cause of death and disability in children and young adults (1, 2). Various experimental TBI models, including controlled cortical impact (CCI) injury (3–7), fluid percussion (8, 9), and stretch injury (10, 11), have shown that TBI induces cell death in the hippocampus. Thus, the hippocampus is one of the most vulnerable brain areas post-TBI in both humans and animals (12). Secondary injury to the hippocampus following TBI correlates with impairment of major cognitive functions and the development of seizures (13).

Hippocampal-associated learning and memory impairment is one of the most significant residual deficits following TBI, and is one of the most frequent patient symptoms (14–16). In part, because of a lack of understanding of the cellular and molecular mechanisms that lead to secondary cell death, there are currently no FDA-approved treatments available to prevent hippocampal cell death post-TBI. Thus, a detailed profile of TBI-induced neuronal injury in the hippocampus, including the time course and mechanism of cell death, and an analysis of the cells that are dying, may suggest potential therapeutic targets and a therapeutic window for treatment.

The distribution and degree of cell death in the hippocampus varies among TBI animal models (17, 18) and with injury severity (6, 19, 20) and age of the animals used (21). For adult mice, we have recently shown that when the CCI model is used and the animals are subjected to a moderate level of injury, the majority of the dying hippocampal cells are largely found in the hippocampal dentate gyrus, where neural stem cells that produce new neurons throughout life reside. The majority of the dying cells in the hippocampal dentate gyrus were found to be newborn immature granular neurons that express the neural cell adhesion molecule (NCAM), but not NeuN, a marker for mature neurons (22). The death of immature neurons can compromise neurogenesis in the hippocampus (22, 23) and contribute to learning and memory impairment following TBI (24).

Excitotoxicity may cause cell death via either apoptosis or necrosis, depending on the intensity of the initiating stimulus and the characteristics of the cell population (25–27). To determine the best time window to facilitate a therapeutic approach that targets immature neuron death in the hippocampus following TBI, we assessed its time course and cell death mechanisms using the apoptotic markers caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunostaining with an antibody against receptor-interacting protein-1 (RIP-1), which has been shown to play critical role in necrotic cell death (28–33).

MATERIALS AND METHODS

Animals

Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were group-housed and subjected to a 12-hour/12-hour light/dark cycle. Access to food and water was ad libitum. All procedures were performed under protocols approved by Indiana University’s Animal Care and Use Committee.

CCI Traumatic Brain Injury

Male mice, 8 to 11 weeks old, were subjected to unilateral moderate lateral CCI injury, as previously described (34–36), with the following exceptions: the depth of deformation was set at 1.0 mm and the piston velocity controlled at 3.0 m/sec. These modifications result in a moderate level of injury using an electromagnetic model (37). Briefly, the mice were anesthetized with Avertin and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) prior to TBI. Using sterile procedures, the skin was retracted and a 4-mm craniotomy centered between the lambda and bregma sutures was performed. A point was identified midway between the lambda and bregma sutures and laterally midway between the central suture and the temporalis muscle. The skullcap was carefully removed without disruption of the underlying dura. Prior to the injury, the impacting tip with a diameter of 5 mm was angled perpendicularly to the exposed cortical surface. Sham (non-injured) animals were subjected only to craniotomy, but not CCI injury. The core body temperature of the animals was maintained at 36–37°C during all surgical procedures and throughout the recovery phase.

Tissue Processing

At 4, 24, 48 and 72 hours and on days 7 and 14 after TBI surgery, the animals (n = 6 per group) were deeply anesthetized with an overdose of Avertin and then perfused transcardially with 0.9% saline, followed by an ice-cold fixative containing 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brains were removed and fixed overnight in PFA, then cryoprotected for 48 hours in 30% sucrose. Serial coronal sections (30 µm thick) were cut using a cryostat (Microm HM 500 M) and stored at −20°C. The sections were then processed for Fluoro-Jade B (FJB) staining and immunohistochemistry.

FJB Staining

Staining was carried out as previously described (38). Briefly, the sections were incubated for 20 minutes in a solution of 0.06% KMnO4 and then rinsed for 5 minutes in distilled water. They were then incubated for 20 minutes in a 0.0004%-solution of FJB (Histo-Chem, Inc., Jefferson, AR), counterstained for 5 minutes with 4',6-diamidino-2-phenylindole ([DAPI]; Sigma, St. Louis, MO), followed by rinsing in distilled water, and overnight air-drying. The dry slides were mounted with DPX (Fluka-Sigma).

Combined FJB/Immuno Staining Analysis

All sections were rinsed 3 times in PBS and then incubated for 1 hour at room temperature in blocking solution (0.1% Triton X-100, 1% bovine serum albumin [BSA], 5% normal goat serum in PBS), followed by overnight incubation at 4°C with primary antibodies. Sections were then washed 3 times with PBS and incubated for 1 hour at room temperature with secondary antibody. After counterstaining with DAPI for 2 minutes, the sections were washed 3 times with PBS. Finally, they were mounted on slides, rinsed with distilled water (5 minutes) and pretreated with 0.06% KMnO4 (5 minutes). After rinsing with distilled water and incubation in a 0.0004% solution of FJB (5 minutes), they were counterstained with DAPI. Primary antibodies and their final concentrations were as follows: anti-NCAM antibody (1:200, mouse, Millipore, Billerica, MA), anti-NeuN antibody (1:100, mouse, Chemicon, Temecula, CA), anti-activated caspase-3 antibody (1:50, rabbit, Abcam, Cambridge, MA), and anti-RIP antibody (1:200, mouse, BD Transduction Laboratories, Franklin Lakes, NJ). Secondary antibodies (Jackson ImmunoResearch, West Grove, PA) conjugated with cy3 were applied in a dilution of 1:800.

TUNEL Assay

TUNEL assays were performed using a commercial kit (In Situ Cell Death Detection Kit, TMR Red; Roche Diagnostics, Mannheim, Germany). Briefly, tissue sections were fixed for 20 minutes at 25°C with 4% PFA in PBS, pH 7.4, followed by washing with PBS (30 minutes), and incubation (30 minutes on ice, 2–8°C) with permeabilization solution (0.1% Triton X–100, 0.1% sodium citrate, freshly prepared). All sections were then incubated with TUNEL reaction mixture (60 minutes at 37°C, in the dark). Positive control sections were pretreated for 10 minutes with DNase I (10 U/mL in 50 mM Tris-HCl, pH 7.5, with 1 mg/ml BSA; Roche Diagnostics) at 25°C before application of the TUNEL reaction mixture. The sections were then counterstained with DAPI, rinsed with PBS and then mounted on slides.

Microscopy

All sections were analyzed by fluorescence microscopy at 10×–63× primary magnification with an inverted microscopy system (Zeiss Axiovert 200 M) interfaced with a computer-controlled Zeiss Axio Cam MRc5 digital camera (both from, Carl Zeiss MicroImaging, Inc., Thornwood, NY). Images were captured with Zeiss imaging software (AxioVision, v4.0), and assembled and labeled in Photoshop 7.0 (Adobe Systems, Adobe Systems Inc., San Jose, CA).

Quantification

We first quantified the number of FJB-positive cells in the entire hippocampus and in the different subregions of the hippocampus in each animal in 3 epicenter sections 180 µm apart. The anatomical boundaries of hippocampus and each hippocampal subregion (CA1 and CA3; granular cell layer, [GCL]; molecular layer, [ML], hilus) were identified as described by Amaral and Witter (39). The total number of FJB-positive cells in the entire hippocampus and the numbers of FJB-positive cells in each subregion were determined using a 40× objective. DAPI counterstaining allowed us to exclude cell fragments and cell debris in the counts. The percentages of FJB-positive cells in each subregion were calculated.

We then determined the density of FJB-positive cells in the GCL across the entire hippocampus in 1 of every 6 sections across the entire hippocampal formation (from bregma −0.94 to −3.80 mm). The total number of FJB-positive neurons in the GCL was determined in a double-blinded quantitative histological analysis using a 40× objective. The GCL area (µm2) was measured using the imaging software and densities of FJB-positive cells in the each GCL were calculated using the cell count divided by section GCL volume. NCAM-positive newborn neurons, NeuN-positive mature neurons, and RIP-1-postive necrotic cells were counted under a fluorescent microscope and percentages of double-positive populations calculated.

Statistical Analysis

All data are presented as mean ± SEM and analyzed using either Student t-test (22, 23) or 1-way ANOVA followed by post hoc comparison of the Dunnett T3 test when the homogeneity of the variances was unequal. The significance level was set at p < 0.05.

RESULTS

Spatial distribution of secondary neuronal death in the hippocampus after moderate CCI injury

It has previously been shown that the hippocampus is particularly vulnerable to TBI and that this may contribute to subsequent cognitive impairment (12, 40). Using FJB staining (41, 42), we identified dying neurons in the hippocampal epicenter 24 hours after moderate CCI injury (n = 7); there were dying neurons in the ipsilateral hippocampus (Fig. 1a) and in the neocortex around the lesion (Fig. 1a, inset). The average number of FJB-positive cells in the hippocampus of each epicentral section was 359 ± 21. No FJB-positive cells were seen in the contralateral hippocampus or the cortex (not shown). Higher magnification showed that FJB stained the cell bodies and processes of the dying neurons (Fig. 1b–d). FJB-positive cells were predominantly located in the dentate gyrus (Fig. 1a–d). Because the different regions of the hippocampus are composed of different types of cells that may exhibit varying susceptibilities to traumatic insults (9, 43), we further assessed the distribution of FJB-positive cells in the subregions at 24 hours after TBI. In the GCL, 291 ±19 FJB-positive cells were counted. This represented 80.9% of the dying neurons in the hippocampus overall, whereas there were 0.15 % in CA1, 11.42% in CA3, 5.04% in the hilus, and 1.65% in the molecular layer (ML) (Fig. 1e). Thus, neuronal death in the hippocampus predominantly occurred in the GCL within 24 hours after moderate TBI.

Figure 1.

Figure 1

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Distribution of dying neurons in hippocampal subregions after traumatic brain injury (TBI). (a–d) Dying neurons 24 hours after TBI are visualized by Fluor-Jade B (FJB) staining (green) in the hippocampus of the ipsilateral hemisphere (a). Cell nuclei are counterstained with DAPI (blue). The area indicated by a square is at higher resolution and outlines FJB-positive cells (b) and their colocalization with the nuclei in the granular cell layer (c,d). (e, f) Pie charts demonstrate the average percentage of FJB-positive cells in hippocampal subregions 24 hours (e) and 3 days (f) after TBI. The column graph illustrates the distribution of FJB-positive cells in the granule cell layer along the rostral-caudal axis at 24 hours (blue bars) and 3 days (red bars) after TBI. GCL, granule cell layer; MCL, molecular layer.

At 3 days after TBI, the total number of FJB-positive cells in the hippocampal epicenter was significantly reduced to 55 ± 3 cells/section. The distribution of FJB-positive cells was: 83.02% in the GCL, 2.37% in CA1, 12.80% in CA3, 0.84% in the hilus, and 0.96% in ML (Fig. 1f). To assess rostral and caudal distribution patterns, we quantified the density of FJB-positive cells in the GCL at the epicenter as well as rostrally and caudally to it. The highest density of dead cells was found in the epicenter; from there, it gradually decreased rostrally and caudally (Fig. 1g).

Temporal profile of neuronal death in the GCL after TBI

Because previous studies suggested that cell death in the hippocampus may last for approximately 2 weeks after TBI (3, 44–46), we assessed neuronal death in the GCL at 4 hours, 24 hours, 2 days, 3 days, 7 days, and 14 days after TBI. At 4 hours there were 32008.5 ±5584.3 cells/mm3 (n = 6) in the GCL. The number of FJB-positive cells peaked at 24 hours (38131.9 ± 4858.2 cells/mm3, n = 7), dropped sharply at 48 hours (9150.3 ± 1763.8 cells/mm3, N = 4, p = 0.008 vs. the 24-hour group), and finally decreased to a very low level after 14 days (3 days, 5840.9 ± 736.1 cells/mm3, n = 6; 7 days, 5945.0 ± 1509.8 cells/mm3, n = 6; 14 days, 921.8 ± 666.6 cells/mm3) (Fig. 2). The average number of dying neurons in each section was less than 2 after 14 days. Thus, neuronal death in the hippocampal dentate gyrus occurs very rapidly following TBI

Figure 2.

Figure 2

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Temporal dynamics of dying neurons in the granular cell layer (GCL) following traumatic brain injury (TBI). (a) Representative images of Fluor-Jade B (FJB)-positive neurons (green) in the granular cell layer at multiple time points (4 hours, 24 hours, 48 hours, 72 hours, 7 days, 14 days) after TBI. Sections were counterstained with DAPI to visualize nuclei in the hippocampal dentate gyrus. (b) Graph demonstrates the changes of the density of the FJB-positive cells (N/mm3) over the time course (4 hours to 14 days). Most of the neurons die within 24 hours after TBI (p < 0.009, vs. 24 hours).

Immature newborn neurons are persistently vulnerable to TBI

The GCL contains immature and mature granular neurons that might respond differently to an insult at different times post-trauma. Indeed, we previously showed that immature neurons are the cells most vulnerable to TBI and start to die within 4 hours post injury (20). To determine whether immature granular neurons are persistently vulnerable at a later time point, we determined the type of GCL cells that were dying at 4 hours and 7 days after TBI (n = 6 for each group) using a combination of FJB and immunostaining with the cell-type specific markers NCAM for immature neurons and NeuN for mature neurons (47). As expected, NeuN-positive mature granular neurons were located across most of the GCL (Fig. 3a), while NCAM-positive immature granular neurons were found in the inner one-third of the GCL (Fig. 3d). Most FJB-positive cells were located in the inner one-third of the GCL (Fig. 3a, b, d, e), where the majority of the newborn immature granular neurons are located (48). Higher resolution and 3-dimensional reconstruction imaging showed that 40.15% of FJB-positive cells could be co-stained with either NeuN or NCAM antibodies. At later stages the FJB-positive cells could not be stained by either antibody. Among co-labeled cells, only 4.96% colocalized with NeuN (Fig. 3b, c), whereas 35.19% colocalized with NCAM (Fig. 3e, f), which represented 88% of the co-labeled FJB-positive cells. Thus, in agreement with our previous report (22), most of the dying cells in the GCL at 24 hours post-TBI are immature granular neurons (22).

Figure 3.

Figure 3

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Colocalization of NeuN- and neural cell adhesion molecule (NCAM)-positive neurons with Fluor-Jade B (FJB)-positive cells. (a–l) Representative images of NeuN/FJB (a–c, g–i) and NCAM/FJB (d–f, j–l) colocalization in hippocampal dentate gyrus 4 hours (a–f) and 7 days post-traumatic brain injury (TBI) (g–l). The combination of FJB staining (green) with immunostaining using NeuN antibody (red) detects FJB-positive dying cells and mature neurons in the dentate gyrus 4 hours and 7 days after TBI (a, g). The combination of FJB staining (green) with immunostaining using NCAM antibody (red) detects FJB-positive dying cells and immature neurons in the dentate gyrus 4 hours and 7 days after TBI (d, j). Enlarged images from the white boxes in (a, d, g, j) are shown in (b, e, h, k) respectively. Enlarged confocal images from the white boxes in (b, e, h, k) are shown with 3-dimensional reconstruction in (c, f, i, l), respectively.

At 7 days after TBI, the number of FJB-positive cells in the GCL was visibly reduced (Fig. 3 g, j). Higher resolution images (Fig. 3h, k) and 3-dimensional reconstruction (Fig. 3i, l) showed that most of the FJB-positive cells did not colocalize with NeuN, but with NCAM-positive cells. Thus, at 7 days after TBI the majority of the dying cells in the GCL are immature granular neurons.

Hippocampal neurons predominantly die of necrosis following moderate TBI

To further characterize the dying immature neurons, we used 2 recognized hallmarks of apoptosis, the cleaved active form of caspase-3 and DNA fragmentation, after sham surgery and at multiple time points post trauma (4 hours, 24 hours, 3 days, and 7 days after TBI; n = 6 in each group). We observed a very small number (258 ± 64/mm3) caspase-3 positive cells in the GCL of sham-treated mice (Fig. 4a, b). The density of caspase-3-positive cells in the GCL did not change significantly at 4 hours after injury (326 ± 57/mm3) (Fig. 4c, d), but was substantially increased 24 hours post injury (2495 ± 312/mm3, p < 0.05 vs. the sham control (Fig. 4e, f). These apoptotic cells represented 6% of the dying FJB-labeled cells. Furthermore, most of the caspase-3-positive cells were located in the outer two-thirds of the GCL, which is mainly composed of mature granular neurons. The density of caspase-3-positve cells returned to a basal level at 3 days (426 ± 82/mm3 (Fig. 4g, h) and 7 days (304 ± 98/mm3) (Fig. 4I, j) post-injury. Thus, numbers of caspase-3-positive cells in the GCL increased only slightly and transiently within the first 24 hours after injury. We also observed some caspase-3-positive cells in the neocortex of the same sections (data not shown).

Figure 4.

Figure 4

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Caspase-3 immunostaining following traumatic brain injury (TBI). (a–j) Caspase-3 immunostaining was performed to examine apoptotic cells in the hippocampal dentate gyrus. Cleaved caspase-3 (red) was detected in the ispilateral side of the sham control (a, b) and the injured granular cell layer at 4 hours (c, d), 24 hours (e, f), 3 days (g, h), and 7 days (i, j) after TBI. The sections were counterstained with DAPI to visualize the nuclei in the hippocampal dentate gyrus.

To confirm the above results we used TUNEL staining, a widely used method to detect DNA fragmentation, as another indicator of apoptotic cell death (Figure, Supplemental Digital Content 1, http://links.lww.com/NEN/A322). We observed a transient increase in the number of TUNEL-positive cells in the hippocampal GCL after 24 hours (sham, 126 ± 73/mm3; 4 hours, 145 ± 75/mm3; 24 hours, 1433 ± 498/mm3; 3 days, 170 ± 108/mm3; 7 days, 189 ± 18/mm3; p<0.05 24 hours vs. sham control). TUNEL-positive cells were also observed in the cortex (data not shown). Together, these results suggest a small and transient increase of apoptosis in the GCL 24 hours after moderate CCI-TBI. Most of the FJP-positive cells in the GCL exhibited processes with slightly condensed nuclei but no visible fragmentation (Fig. 5), indicating that the immature granular neurons in the GCL predominantly do not undergo apoptosis.

Figure 5.

Figure 5

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Morphological analysis of dying neurons in the hippocampal dentate gyrus following traumatic brain injury. (a–c) Dying neurons with processes in the inner layer of the granular cell layer (b) were stained with Fluor-Jade B (FJB). Arrows in (a) and (c) indicate green fluorescence. Nuclei counterstained with DAPI identified after image colocalization are indicated by arrows in (b). (d–f) A single FJB-positive neuron with its process (d, f, green) and nucleus (e, blue) under higher magnification.

Necrotic cell death is characterized by increased cell volume with organelle swelling and plasma membrane rupture and has been considered to be an uncontrolled form of cell death (49). However, there is increasing evidence that the execution of necrotic cell death may be finely regulated by signal transduction pathways and catabolic mechanisms (50, 51). Because RIP-1 phosphorylation is a marker of necrotic cell death (49), we assessed RIP-1 expression in granule cells 24 hours after injury. We did not observe FJB or RIP-1 in the contralateral hippocampal GCL (Fig. 6a, c, e), but detected abundant FJB-positive granule cells and RIP-1 expression in the ipsilateral dentate gyrus (Fig. 6b, d). The merged image in Figure 6 shows colocalization of FJB-positive cells with the RIP-1 signal. In addition, 3-dimensional reconstruction under high amplification indicated that FJB-positive cells expressed high levels of RIP-1 (Fig. 6 g–j). Quantification revealed that 78.6 ± 5.1% of FJB-positive cells co-expressed RIP-1, suggesting that a high percentage of the cells died of necrosis.

Figure 6.

Figure 6

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Receptor interacting protein (RIP-1) expression in injured hippocampal granule neurons after moderate traumatic brain injury. (a–f) Distribution of injured neurons (a. b, green) and RIP-1 (c, d, red) in the contralateral (a, c, e) and ipsilateral (b, d, f) hippocampal dentate gyrus 24 hours post injury after combined Fluoro-Jade B (FJB) immunostaining. (g–j) A representative 3-dimensional image of the injured granule neurons (g) with expression of RIP-1 in the cytoplasm (h) and condensed nuclei (i) confirms colocalization (j) at the single-cell level.

DISCUSSION

Persistent memory dysfunction is associated with hippocampal neuron loss following experimental TBI (24, 52–54). We show that the majority of cell death in the hippocampal GCL peaks at 24 hours and persists up to 14 days after TBI in a lateral CCI mouse model

Previous studies suggest that CCI-TBI causes long-term secondary injuries after the immediate primary impact on the brain (3, 46). Silver staining of degenerating neurons has shown that the area of the brain affected by secondary injury is extensive and long-lasting (3). Although silver staining allows one to outline the areas of neuron degeneration, accurate cell counting and immunostaining of other markers remains difficult (44). The patterns of dying neurons we observed using FJB are quite consistent with those detected using silver techniques. Because at the peak injury time point of 24 hours post-TBI, most dying neurons were in the GCL, the cells in this region appear to be more vulnerable to TBI insult than cells in other subregions in the hippocampus; however, this may reflect the high density of cells in the GCL (55).

The cell death peak within 24 hours post-injury is followed by secondary injury. Interestingly, after the early peak of neuronal degeneration, the density of FJB-positive cells remained at a relatively constant level for several days. This suggests that the therapeutic time window to target neuronal death in the hippocampus would be very narrow, i.e. not later than 24 hours post injury.

The mechanisms of hippocampal neuron vulnerability to TBI are not understood. In rodents and primates, including humans, the hippocampus can support neurogenesis throughout life (56–60). New neurons are continuously generated from neural stem/progenitor cells in the subgranular zone of the dentate gyrus (61, 62). Thus, there are both immature and mature neurons in this area. We previously found that the majority of the dying cells in the hippocampal dentate gyrus in the acute phase post-TBI are 2- to 3-week-old newborn immature granular neurons (22). In the present study we found the ratio of immature to mature degenerating neurons was consistently higher at both acute (4 hours) and subacute (7 days) post-injury time points. This indicates that immature neurons are the major hippocampal neuron population vulnerable to TBI and suggest that immature neurons are potential therapeutic targets.

It is currently not known why immature neurons are so vulnerable to TBI. Recent studies of newborn neurons in the dentate gyrus show that they are electrophysiologically distinct from mature granular neurons in the dentate gyrus (63–68). Newborn immature neurons can be more easily excited than mature neurons due to their unique ion channel expression (69–71). It will be interesting to determine whether newborn neurons display exaggerated excitotoxic responses to neurotransmitters released by the damaged neurons after TBI because of this expression.

Calcium influx-mediated excitotoxicity has been considered a major cause of neuron death when neurons are challenged post-TBI with the excitatory amino acid glutamate (72–74). Excitotoxicity seemingly overlaps with other types of cell death such as apoptosis and necrosis depending on the intensity of the initiating stimulus (75–77). The molecular mechanisms underlying secondary neuronal death in the cortex are complex, overlapping, and temporally variable (78). Triggers of secondary TBI (e.g. glutamate-mediated excitotoxicity) can cause both massive necrosis and apoptosis in the cortex (79). In the present study, FJB staining of degenerating neurons in the hippocampus revealed spindle-shaped cell bodies with clear dendritic processes that did not exhibit the typical nuclear condensation of apoptosis, which suggests that most of them did not undergo apoptotic cell death. Moreover, we assessed cell death by immunostaining with antibodies against caspase-3 or using TUNEL staining and found that at although numbers of caspase-3-positive cells were transiently increased in the hippocampal dentate gyrus over the period of 4 to 24 hours, the density of caspase-3-positive cells was much less than the density of the dying FJB-positive cells in the GCL. Even taking into consideration that caspase-3 and TUNEL staining may only label certain stages of apoptotic cell death cell our data still suggest that only a small portion of the cells in the GCL had apoptotic cell death in this model.

Dying FJB-positive neurons in the GCL co-expressed RIP-1, a marker for a subtype of necrotic cell death. RIP-1 has been shown to play a critical role in necrotic cell death induced by death domain (80–83) and Toll-like receptors (84–86). The upregulated expression of RIP-1 in the hippocampus suggests the presence of necrosis. Moreover, necrostatin-1, an inhibitor that targets RIP-1, has been shown to exert neuroprotective functions and to shield neurons from cell death following CCI-TBI (87). This supports our contention that immature hippocampal neurons mainly die of necrosis and not apoptosis following TBI with a moderate level of impact. When animals are subjected to a higher level of impact to increase the severity of the injury, the numbers of caspase-3- (88, 89) and TUNEL-positive cells (89–91) in the GCL dramatically increase. Thus, numbers of apoptotic cells in the GCL also vary depending on the severity of the injury.

In conclusion, this study provides a quantitative assessment of the changes occurring in the hippocampus after TBI. In particular, we show that moderate TBI triggers rapid necrotic death of immature neurons in the hippocampus. These results suggest that the development of therapeutic approaches should focus on preventing cell death in the hippocampus, in particular the necrotic death of immature neurons. The optimal window for therapeutic intervention appears to be within 24 hours post-injury.

Supplementary Material

1

Acknowledgments

This work was supported through funding from the Indiana Spinal Cord & Brain Injury Research Grants, the Ralph W. and Grace M. Showalter Research Award, Indiana University Biological Research Grant, and NIH grants RR025761 and 1R21NS072631-01A.

Footnotes

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