Moderate Traumatic Brain Injury Triggers Rapid Necroticdeath Of Immature Neurons In The Hippocampus

Background and objectiveTraumatic brain injury (TBI) not only causes cell death in the cortex, but it also induces secondary cell death in the hippocampus. The hippocampus is among the most vulnerable brain areas following TBI in both humans and experimental animal models. We previously found that the majority of the dying cells in the hippocampus following TBI at the moderate level of impact are largely constrained in the hippocampal dentate gyrus, where neurogenesis is continuous throughout life. Cell type and birthday analysis found that those dying cells in the hippocampal dentate gyrus are newly born immature granular neurons at the age of 2 to 3 weeks old. Here we showed that most of the newborn neuron death in the hippocampus after TBI occurred within 24 hours, and then remained at a low level for at least 2 weeks. The dying immature granular neurons did not exhibit morphological characteristics of apoptosis, and were negative to apoptotic markers. In contrast, they exclusively co-expressed receptor interacting protein (RIP-1), a marker of necrosis, suggesting that the immature neurons died of necrosis. These results indicate that moderate traumatic brain injury triggers rapid necrotic death of immature neurons in the hippocampus. They also suggest future development of therapeutic approaches aimed at preventing cell death in the hippocampus should target immature neurons by blocking their necrosis process optimally within 24 hours after injury.Traumatic brain injury (TBI) is a leading cause of death and disability in children and young adults. Hippocampal-associated learning and memory impairment is one of the most significant residual deficits following TBI and is among the most frequent complaints heard from patients and their relatives (cite). Partially due to the lack of understanding about the cellular and molecular mechanisms that lead to secondary cell death, no FDA-approved treatment is available to prevent cell death in the hippocampus following TBI. It has been demonstrated using various experimental injury models, including controlled cortical impact (CCI) injury, fluid percussion, and stretch injury that TBI induces cell death in the hippocampus, making the hippocampus one of the most vulnerable brain areas following TBI in both humans and experimental animal models. Secondary injury to the hippocampus following TBI correlates with major cognition functions and seizure development. Thus, a detailed profile of TBI-induced neuronal injury in the hippocampus, including time course of cell death, type of dying cells, and mechanisms of cell death, may suggest a potential therapeutic time-window, as well as targeted cell type, and a specific cell death pathway for therapeutic targeting.The distribution and amount of cell death in the hippocampus varies by injury model, injury severity, and age differences. Our recent data has shown that the majority of the dying cells in the hippocampus are largely constrained in the hippocampal dentate gyrus, where neural stem cells reside and produce new neurons throughout life, using CCI model at the moderate level of impact. Further cell type analysis found that those dying cells in the hippocampal dentate gyrus are newly born immature granular neurons, whose death can compromise neurogenesis in the hippocampus, and contribute to impairment of learning and memory following TBI. In order to determine the best therapeutic time window to facilitate the possibility of a therapeutic approach targeting immature neuron death in the hippocampus in the future, we assessed the time course and mechanism of immature neuron death. We found that, although the excitotoxicity of newborn neuron death in the hippocampus lasted about 2 weeks after TBI, most died within 24 hours after injury. 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 (cite). Further analysis showed that the immature neurons in the hippocampus died exclusively from necrosis following TBI.MethodsC57BL/6 male mice,8～11 weeks old, were subjected to moderate CCI injury as previously described with the following exceptions:the amount of deformation was set at 1.0 mm and the piston velocity controlled at 3.0m/s. Male C57BL/6 mice (The Jackson Laboratories) were group housed with a 12/12-hr light/dark cycle and had access to food and water ad libitum. They were used in experiments at the age of 8-11 weeks. All procedures were performed under protocols approved by the Animal Care and Use Committee of Indiana University.These modifications result in a moderate level of injury using an electromagnetic model. 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 midway between the central suture and the temporalis muscle laterally. The skullcap was carefully removed without disruption of the underlying dura. Prior to the injury, the head of the animal was angled on a medial to lateral plane so that the impacting tip was perpendicular to the exposed cortical surface. This was accomplished by rotating the entire stereotaxic frame on the transverse plane. The mouse CCI model uses an electromagnetic model with which the experimenter can independently control the contact velocity and the level of cortical deformation, thus altering the severity of the injury. In these experiments, the contact velocity was set at 3.0 m/sec and the amount of deformation was set at 1.0 mm, which results in an injury of moderate severity. Sham (non-injured) animals received the craniotomy, but no CCI injury. During all surgical procedures and recovery, the core body temperatures of the animals were maintained at 36-37℃.Results1. Secondary cell death in the hippocampus after moderate TBI predominantly occurred in the granular cell layer. It has been reported that the hippocampus is particularly vulnerable to TBI, which can contribute to hippocampal-dependent cognitive impairment. When we examined the cell death in the hippocampus 24 hours following moderate TBI using Fluoro-Jade B (FJB) staining, a method widely used to detect the dying neurons, the dying cells stained by FJB in green were mainly seen in the neocortex around the lesion area (not shown) and in the hippocampus of ipsilateral side (Figure 1 a). No FJB-positive cells were seen in the contralateral cortex (data not shown). A higher magnification image showed that FJB stained the cell bodies and the processes of dying neurons (Figure lb-d). The FJB-positive cells did not evenly distribute within the hippocampus, instead they predominantly located in the hippocampal dentate gyrus (Figure 1 a-d). Different regions in the hippocampus are composed of different types of cells, which may have varying susceptibilities to traumatic insults. We further assessed the distribution of FJB-positive cells in the different subregions of the hippocampus 24 hours after moderate TBI injury. Twenty-four hours after TBI, there were FJB-positive cells in the granular cell layer (GCL), which represented 80.9% of total number of dying neurons in the hippocampus, whereas, there were 0.15 % in the CA1,11.42% in the CA3,5.04% in the hilus, and 1.65% in the molecular layer (ML) (figure 1, pie graph e). These results showed that the secondary cell death in the hippocampus predominantly occurred in the granular cell layer within 24 hours after moderate TBI. To further determine whether this distribution pattern occurred only during the 24 hours after TBI or continued consistently past the 24 hour mark, we examined the cell death and its distribution at the hippocampus 3 days after injury. The results showed that despite the total number of FJB-positive cells in the hippocampus being significantly reduced from at 24 hours to at 3 days post TBI, the distribution of the FJB-positive cells in the different subregions in the hippocampus 3 days after TBI was similar to the distribution at 24 hours after TBI. The distribution was 80.02% in the GCL,2.37% in the CA1,12.80% at CA3,0.84% in the hilus, and 0.96% in the ML (figure 1, pie graph f). To assess the distribution pattern of dead cells across the hippocampal dentate gyrus, we quantified the density of FJB-positive cells at the epicenter as well as rostral and caudal to the epicenter. The epicenter contained the highest density of dead cells, and the density decreased gradually toward both the rostral and caudal directions (Figure 1 g). These results support the notion that the secondary cell death in the hippocampus predominantly occurs in the granular cell layer after CCI injury at the moderate level of impact.2. Temporal profile of neuronal death in the dentate gyrus granular cell layer (GCL) after TBI. We further examined the temporal profile of neuronal death in the hippocampal dentate gyrus. Based on the evidence from previous studies suggesting that cell death occurring in the hippocampus may continue for about two weeks after TBI, we assessed the neuronal death in the dentate gyrus GCL 4 hours,24 hours,2 days,3 days,7 days and 14 days after TBI. We observed a significant number of FJB-positive cells in the dentate gyrus GCL as early as 4 hours after injury (32008.5±5584.3 cells/mm3, N= 6). 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, compared with the 24 hr group), and then reduced to very low level at 14 days (3d,5840.9±736.1 cells/mm3, N= 6; 7d,5945.0±1509.8 cells/mm3, N= 6; 14d,921.8±666.6 cells/mm3, N= 2) (figure 2). These results indicate that cell death in the hippocampal dentate gyrus occurred very rapidly following TBI. More than 80% of the cell death in the GCL occurs within 24 hours after moderate TBI injury. The temporal profile of neuronal death in the hippocampal dentate gyrus suggests that the therapeutic time window for treatments targeted to prevent neuron death should optimally be carried out within 4 hours and at least not later than 24 hours after injury.3. Immature newborn neurons are persistently vulnerable to TBI insult. There are different cell types in the dentate gyrus granular layer, including immature granular neurons and mature granular neurons. They might respond differently to insult at different times post-trauma. Indeed our previous study has shown that immature neurons are the most vulnerable cell type to TBI injury and are induced to death immediately after injury. It is not yet known whether immature granular neurons are vulnerable only at the acute phase or if they are persistently vulnerable for days after TBI. To address this question, we examined the cell type of dying cells in the GCL 4 hours and 7 days after TBI. We used FJB-staining combined with immunostaining with different cell-type specific markers, including NCAM for the immature neurons and NeuN for the mature neurons, to assess the cell type of dying neurons in the GCL. The results showed that, at 4 hours after TBI, there were a significant number of FJB-positive cells in the GCL (Figure 3 a, b, d, and e). Most of the FJB-positives were located in the inner one-third of GCL (Figure 3 a, b, d, and e), where the majority of the newborn immature granular neurons reside. As expected, NeuN positive mature granular neurons located across most of the thickness of the GCL (Figure 3 a). In contrast, NCAM-positive immature granular neurons located at the inner-one third of GCL (Fgiure 3 d). Higher power imaging (figure 3 b) and 3-dimensional reconstruction imaging (Figure 3 c) showed that most of the FJB-positive cells did not colocalize with the NeuN marker. These results indicate that most of the FJB-positive cells in the GCL are not mature granular neurons. In contrast, higher power imaging (figure 3 e) and 3-dimensional reconstruction imaging (Figure 3f) showed that a large portion of the FJB-positive cells co-localize with NCAM-positive cells, indicating that most of the dying cells in the GCL at 4 hours after moderate TBI are immature granular neurons. This result agrees with our previous report. Seven days after TBI, the number of FJB-positive cells in the GCL was obviously reduced (Figure 3 g and j), which supports the conclusion above that the cell death in the dentate gyrus predominantly occurs within the first 24 hours, and is significantly reduced two days after TBI. The higher power image (figure 3 h) and 3-dimensional reconstruction image (Figure 3 i) show that most of the FJB-positive cells did not colocalize with NeuN, but colocalize with NCAM-positive cells. These data show that at 7 days after TBI, the majority of the dying cells in the GCL are immature granular neurons, and only a small portion of these are mature granular neurons. These results suggest that the immature neurons are highly vulnerable to TBI insult not only in the first few hours after TBI, but also several days after TBI when the rate of cell death has slowed.4. The dead cells in the hippocampus predominantly die of necrosis following moderate TBI. To further determine the type of newborn neuron death in order to facilitate possible therapeutic approaches in the future, we first examined two recognized hallmarks of apoptosis, cleaved active form of caspase-3 and fragmentation of DNA, in the degenerating neurons at multiple time points (4 h,3 d, and 7 d after TBI). We did not see cleaved caspase-3-positive cells in the GCL (Figure 4), although we observed cleaved caspase-3-postive cells in the neocortex of the same section. A subpopulation of dying neurons in the neocortex have been shown to be caspase-3-positive, and the caspase-3-positive cells in the neocortex serve as a positive control to indicate the caspase staining is working. To further confirm that the negative caspase-3 signal-in the injured hippocampal GCL was not a false negative, post-ischemic brain sections taken 7 days after injury when there is large number of apoptotic cells in the CA1 region of hippocampus, were immunostained with cleaved caspase-3 as positive control. As expected, we observed large numbers of cleaved caspase-3-postive cells in the CA1 region (Supplemental figure 1). These data suggest that it is unlikely the immature granular neurons in the GCL died of apoptosis.TUNEL staining is widely used to detect the fragmentation of DNA, which is another critical characteristic phenomena in the process of apoptotic cell death. We did not observe any TUNEL-positive cells in the hippocampal GCL (Supplemental figure 2), however, there were a significant number of TUNEL-positive cells in the neocortex of the same sections (data not shown). In contrast, also as a positive control, the DNase I pre-treated brain sections showed a large amount of TUNEL-positive cells both in the hippocampus and in the neocortex (Supplemental figure 3). We further examined the cellular and nucleus morphologies of FJB-positive cells in the HDG, we found that most of them exhibited processes and their nuclei were slightly condensed but not obviously fragmented (Figure 5). These data indicate that there is no detectable apoptosis in the GCL and likely that the immature granular neurons in the GCL do not die of apoptosis following moderate head trauma. Previous studies suggest that CCI-TBI insults cause long-term secondary injury after the immediately primary impact on the brain. By silver staining of degenerating neurons, the secondary injury can be shown to inundate an extensive area of the brain, extending out to long hairline degenerations of neural fibers. Despite this illumination of the degenerating areas, accurate cell counting and immunostaining of other markers remains difficult by silver staining. In the present study, we stained the dying neurons with Fluoro-jade B, a fluorescent dye that can be easily combined with other markers under a fluorescent microscope to show both the outline and cell type of degenerating neurons. The patterns of degenerating neurons are quite consistent whether they are stained by either silver or FJB. We counted and calculated the percentages of FJB-positive cells in each subregion of the hippocampus to identify the hardest-hit area. At the peak injury time point of 24 hr post TBI, the majority of the dying neurons were located in the granular cell layer of the dentate gyrus. The aggregation of neuronal death in the granular cell layer continued for days after injury. A number of reports have demonstrated the aggregation in several variable TBI models including lateral fluid percussion, closed head injury, and controlled cortical impact. These results suggest that the hippocampal dentate gyrus is among the most vulnerable areas of the brain after traumatic brain injury.Secondly, we counted and calculated the total number of Fluoro-jade B positive cells in the granular cell layer of the dentate gyrus at multiple time points (4 hr,24 hr,48 hr,3 d,7 d and 14 d) after moderate traumatic brain injury. The peak of cell death occurs within 24 hrs from the time of injury indicating that secondary injury is an acute reaction. Interestingly, after the early peak of neuronal degeneration, the FJB positive cell density maintained at a relatively constant level for several days. The temporal profile suggests that the effective therapeutic time window targeting neuronal death in the hippocampus is pretty narrow. The optimal approach should be applicable within 4 hours but not later than 24 hours following injury.In conclusion, this study provided a quantitative assessment to show that moderate traumatic brain injury triggers rapid necrotic death of immature neurons in the hippocampus. These results suggest that future development of therapeutic approaches aiming to prevent cell death in the hippocampus should target immature neurons by blocking their necrosis process, optimally within 24 hours after injury.