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Apoptosis contributes to delayed neuronal cell death in traumatic brain injury (TBI). To investigate if Bax plays a role in neuronal cell death and functional outcome after TBI, Bax gene disrupted (null) mice and wild-type (WT) controls were subjected to the controlled cortical impact (CCI) model of TBI. Motor function in WT and Bax null mice was evaluated using the round beam balance and the wire grip test on days 0–5. Spatial memory was assessed using a Morris Water Maze adopted for mice on days 14–18 post-injury. For histopathological analysis, animals were sacrificed 24h and 21 days post-injury. In all three behavioral tests, the sham and TBI-injured Bax null mice performed significantly worse than their WT sham and TBI-injured counterparts. However, Bax null mice exhibited a higher percentage of surviving neurons in the CA1 and CA3 regions of hippocampus measured at 21 days post-injury. At 24h after trauma, Bax null mice had fewer TUNEL positive cells in the CA1 and dentate regions of hippocampus as compared to WT mice, suggesting that deletion of the Bax gene ameliorates hippocampal cell death after TBI. Sham-operated Bax null mice had significantly greater brain volume as compared to WT mice. Thus, it is possible that Bax deficiency in the transgenic mice produces developmental behavioral effects, perhaps due to Bax's role in regulating cell death during development.
Neuronal cell death following traumatic brain injury (TBI) has been attributed to direct mechanical injury or to post-traumatic secondary mechanisms that are activated weeks to months following brain injury (Bayir et al., 2003; Faden et al., 1992). While cellular necrosis has been shown to be the prominent cause of cell death, recent evidence suggests apoptosis as a key mechanism that is involved in secondary or delayed neuronal cell death. There is increased expression, cleavage, and activation of caspases after TBI (Clark et al., 1997, 2000; Yakovlev et al., 1997). Moreover, DNA fragmentation and TUNEL staining in a pattern consistent with apoptosis has been demonstrated following brain injury (Clark et al., 2001; Conti et al., 1998; Wennersten et al., 2003). Furthermore, cell death with necrotic morphology may be mediated by activation of apoptotic pathways as Fas or TNFα under certain conditions (Degterev et al., 2005).
The Bcl-2 family consists of an ever increasing number of members, including the anti-apoptotic proteins Bcl-2, Bcl-x, Bcl-w, and mcl-1 and the pro-apoptotic proteins Bax, Bad, Bim, and Bok (Adams, 1998). While the cellular mechanisms underlying neuronal cell death in TBI are not fully understood, many studies have found that expression of several members of the Bcl-2 family is altered after brain injury (Graham, 2000). These Bcl-2 family members may play an important role in determining whether neurons survive or die after TBI.
Bax is the prototypic pro-apoptotic Bcl-2 family member and plays a major role in regulating cell death in variety of cell types including the neuron (Krajewski et al., 1994; Oltvai et al., 1993). Bax expression is upregulated in response to injury and in neurodegenerative diseases (Vila and Przedborski, 2003). Bax mRNA and protein levels are increased in the cortex following TBI injury (Raghupathi et al., 2003; Wennersten et al., 2003). Decreased immunoreactivity for Bcl-2 and increased Bax immunoreactivity have been shown in injured neurons after TBI and in models of ischemia (Gillardon et al., 1996; Raghupathi et al., 2003). Neurons that are injured yet survive, such as CA3 hippocampal neurons after global ischemia, expressed increased levels of Bcl-2 but did not express Bax (Chen et al., 1996, 1997). These observations support the idea that neuronal cell death may be dependent on the cellular ratio of anti-apoptotic and pro-apoptotic Bcl-2 proteins.
Knudson et al disrupted the Bax gene and produced a line of Bax null mice that have been used in a number of studies (Knudson et al., 1995). Gibson et al. (2001) found that neonatal Bax null mice were less susceptible to ischemia induced by unilateral carotid occlusion and hypoxia than wild-type (WT) controls. Young et al. (2003) reported that Bax null mice were protected against ethanol-induced neuronal apoptosis and that ethanol toxicity was mediated through activation of the intrinsic apoptotic cell death pathway. Synthetic small molecules that inhibit the Bax channel have been shown to be neuroprotective in a global ischemia model (Hetz et al., 2005). Bax null mice have also been shown to be protected against cardiac ischemia (Hochhauser et al., 2003). In a recent study, Shi et al. (2007) found that Bax null mice have less hippocampal cell loss and increased hippocampal neurogenesis after cortical contusion injury. Although this work suggests that Bax expression after TBI results in a decreased number of surviving neurons and thus worsens long-term outcome. Long-term behavioral outcome was not studied.
The present study was designed to determine the role of Bax expression in activating the intrinsic pathway of mitochondrial apoptosis in neurons and to directly test the hypothesis that Bax exacerbates neuronal injury and long-term functional outcome in trauma.
Bax null mice (C57BL/J6-BAXtm/Sjk) were obtained from Jackson Laboratories (Jackson Laboratories, Bar Harbor, ME). This transgenic line was generated by Dr. Stanley Korsmeyer and has been backcrossed with C57BL/6 WT mice for more than eight generations (Knudson et al., 1995). WT C57BL/6 mice from the same breeding litter were used as controls. In all experiments, mice were age-matched males.
All experiments were performed in accordance with guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. TBI was performed using the controlled cortical impact (CCI) model in mice as previously described (Sinz et al., 1999; Statler et al., 2006; Tehranian et al., 2006) with minor modifications. Briefly, mice (12 weeks of age) were anesthetized with 4% isoflurane (in 50/50 O2/N2O) for induction and then with 1–2% isoflurane throughout the surgical procedure via a nose cone. Animals were positioned in a stereotaxic frame, and a brain temperature probe was inserted into a burr hole drilled in the left frontal cortex. A 5-mm craniotomy was performed over the left parieto-temporal cortex, the bone flap removed, and brain temperature was maintained at 37±0.5°C for 5min. Body temperature was monitored by a rectal probe (Physitemp Instruments, Clifton, NJ). Mice were then subjected to vertically directed CCI using a pneumatic cylinder with a 3-mm tip impounder at a velocity 6.0±0.2m/sec and a depth of 1.2mm. Immediately after the injury, the bone flap was replaced, sealed with dental cement (Vernon Benshoff, Albany, NY), and the wound sutured closed. Isoflurane was discontinued, and the mice were placed in an oxygen hood for 30min, after which they were returned to their cages. Sham surgeries were identical to the above, but without CCI.
Western blot analysis of tissue from injured hippocampus was performed as described previously (Tehranian et al., 2006). Briefly, animals were sacrificed at 24h post-sham or post-TBI, and the hippocampus was removed (n=4 per group). Total proteins were extracted by homogenization in lysis buffer (30mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, and 1% triton X-100 supplemented with protease and phosphatase inhibitors). Protein content was assayed using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). An aliquot (30–40μg) of protein was separated on 12% SDS gel and electroblotted onto PVDF (Bio-Rad) membranes, and then incubated with anti-Bax primary antibody (1:400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed with Enhanced Chemiluminescence Detection Kit (Amersham, UK). Transfer efficacy was verified using Ponceau staining. Membranes were stripped and reprobed with mouse monoclonal GAPDH antibody (Ambion, Austin TX) to verify for equal loading.
Mice were transcardially perfused via the left ventricle with 20mL of 0.9% NaCl, followed by 20mL of 4% paraformaldehyde at 24h after TBI or sham surgery (n=3 for TBI; n=2 for sham). The brains were removed, sectioned into 2-mm intervals, processed, and embedded in paraffin. Sections were cut 5μm thick on a microtome, mounted on glass slides, and processed for immunohistochemical staining. The following primary antibodies were used: anti-Bax monoclonal antibody (no. sc-7480,1:500; Santa Cruz Biotechnology), anti-caspase-3 monoclonal antibody (no. 9664, 1:100; Cell Signaling Technology, Danvers, MA), and anti-NeuN mouse monoclonal antibody (MAB 377; 1:200, Chemicon, Temecula, CA). Immunofluorescence labeling was carried out with the use of secondary antibodies conjugated to Cy3 or FITC (Zymed Laboratories, Invitrogen, CA). After secondary antibody application, sections were rinsed in PBS, mounted with Vectashield Hard Set Mounting Medium (Vector Labs, CA), counterstained with 4′-6-Diamidino-2-phenylindole (DAPI) and coverslipped. On control sections, primary antibodies were omitted and no staining was observed. Images were captured with a confocal microscope (Olympus Fluoview). Immunoreactivity in CA1, CA3, and dentate was semiquantified using a previously described scale: 0= no cells stained; 1+=1–25% cells stained; 2+=26–50% cells stained; 3+=51–75% cells stained; and 4+=76–100% cells stained (Pulsinelli et al., 1982).
Mice were sacrificed 24h post-TBI, and injured cortex and hippocampal tissue was processed for caspase activity assay similar to Western blot, but without addition of protease and phosphatase inhibitors (n=3–4 per group). Caspase-3 activity was measured using the CaspACE assay system (no. G7220; Promega Madison, WI) according to manufacturer's instructions. Caspase activity in the tissue was expressed as percent increase in absorbance relative to WT sham animals.
DNA fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick-end-labeling (TUNEL) staining performed on 5-μm paraffin-embedded sections located in the center of the contusion from brains of sham and TBI injured mice (n=3 per group) at 24h post-injury using ApopTag*plus Fluorescence In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA) following manufacturer's instructions. Briefly, sections were incubated with the labeling solution containing terminal deoxynucleotidyl transferase (TdT) enzyme in a humidified chamber for 1h at 37°C. To stop the reaction, slides were rinsed in PBS. FITC-conjugated digoxygenin antibodies were added, incubated at room temperature for 30min and counterstained with DAPI. TUNEL-positive neurons were counted in CA1, CA3, and dentate gyrus regions of hippocampus in ipsilateral hemispheres using a fluorescence microscope with the appropriate filter. TUNEL-positive cells with TUNEL staining restricted to the nuclei as confirmed by DAPI double staining were considered apoptotic (TUNEL-positive); diffusely TUNEL-stained cells with no apoptotic morphology were excluded (Portera-Cailliau et al., 1995). Data are expressed as percent DAPI-stained cells.
To assess lesion and brain volume, mice were deeply anesthetized with isoflurane and brains perfused with 20mL of 0.9% NaCl, followed by 20mL of 4% paraformaldehyde via the left ventricle 21 days after TBI or sham surgery (n=15 per group). Brains were removed and coronal brain sections (10μm) were cut on a cryostat at 0.5-mm intervals and mounted on gelatin-coated slides. Sections were stained with cresyl violet and coverslipped. Contralateral and spared ipsilateral hemispheric area was measured using MCID (MCID Imaging Research, St. Catharines, ON, Canada). Volumes were calculated by multiplying slice area×slice interval thickness and adding together all slices. Lesion volume was then quantified as follows:
(Contralateral volume – ipsilateral volume)/contralateral volume
and it is expressed as percent contralateral. Total brain volume was calculated by adding the ipsilateral and contralateral hemispheric volumes together and is expressed as mm3. Animals without intact hippocampi were removed from the study (resulting in n=10–12 per group).
Hippocampal cell survival was assessed in mice 21 days after trauma. The sections (prepared as described above) to be counted were obtained by choosing the slide having those sections with the largest contusion area (n=10–12/group). The number of surviving hippocampal neurons in CA1 and CA3 regions were counted in the ipsi- and contralateral hemispheres using methods previously described (Tehranian et al., 2006). Only cells with visible nuclei were counted; shrunken or pyknotic cells were excluded from cell counts. The number of surviving hippocampal neurons in the ipsilateral hemisphere was normalized to the contralateral side. Additionally, total hippocampal cell counts were performed on sham animals from above, counting all CA1 and CA3 neurons on both the ipsilateral and contralateral sides of the brain at the center of contusion.
Analysis of motor function and Morris Water Maze (MWM) spatial memory acquisition was performed as previously described (Sinz et al., 1999; Whalen et al., 1999). WT and Bax null mice underwent sham or TBI surgery (n=15 per group). Motor function was assessed at days 1–5 after injury with mice trained on day 0 (pre injury) using a round beam balance and wire grip test by observers unaware of the experimental groups. Spatial memory acquisition was assessed using the MWM hidden platform on days 14–18 and visible platform on days 19–20 after TBI. Performance in the MWM was quantified by measuring latency in finding the platform. Animals whose 20-day visible platform scores were greater than two standard deviations from the sham group 2-day average were removed from the study.
Data are expressed as mean±SE, except for caspase-3 activity assay, which is graphed as mean±SD. Data from caspase-3 activity assay and TUNEL staining was analyzed by one-way analysis of variance (ANOVA). Data from lesion volume, total brain volume and hippocampal cell counts were analyzed using two-way ANOVA with homogeneity testing performed prior to post hoc analysis. Motor and MWM data were analyzed by repeated-measures analysis of variance (RMANOVA). Differences were considered significant when p<0.05.
Expression of Bax was studied with Western blotting and immunohistochemistry in sham and TBI-injured WT mice. There was no difference in the expression of Bax in hippocampus in sham or TBI-injured WT animals at 24h post-trauma (Fig. 1A). Additionally, no Bax expression was detected in Bax null mice by Western blotting (data not shown). However, immunohistochemistry detected an increase in Bax expression in select neurons within the ipsilateral CA3 subfield of hippocampus in WT mice at 24h post-trauma (Fig. 1B). A few Bax-positive cells were also present within CA3 on the contralateral side. These data suggest that expression of Bax is increased in some neurons within hippocampus of WT mice as a result of TBI. There were few or no neurons expressing Bax in CA1, another area where there is extensive cell death.
There was no significant difference between caspase-3 activation in cortices of injured WT and injured Bax null mice (data not shown). Caspase activity was increased in the hippocampus of TBI injured WT mice as compared to sham-injured WT mice (Fig. 1C), but there was no significant difference in caspase-3 activity between WT TBI and Bax null TBI mice nor between Bax null sham-operated and Bax null TBI mice. Immunofluorescent staining of activated caspase-3 demonstrated similar results. In WT mice subjected to TBI, activated caspase-3 immunoreactivity in CA3 neurons is graded as 2+(26–50% of neurons stained), while there was 1+(1–25%) immunoreactivity in CA3 of Bax knockout mice after TBI. Immunoreactivity in CA1 and dentate in both Bax null and WT mice after TBI was 0 to 1+. Sham-injured mice from both groups had 0 to 1+ caspase-3 staining (Fig. 1D). A difference was noted in the pattern of activated caspase-3 staining in traumatized TBI CA3 neurons: Caspase-3 immunoreactivity was present in both the cytosol and nucleus in WT CA3 neurons, but caspase-3 immunoreactivity was restricted to the cytosol of neurons in Bax null mice in CA3 neurons. Thus, Bax expression does not appear to be necessary for activation of caspase-3 and induction of apoptosis after TBI at this time point, but Bax deletion altered the subcellular distribution of caspase-3 immunoreactvity.
TUNEL-positive cells displaying apoptotic morphology were present in the CA1, CA3, and dentate regions of hippocampus in brains from WT and Bax null mice at 24h after TBI (Fig. 2A). TUNEL-positive cells were counted and expressed as a ratio of total cells in sections counterstained with DAPI for each region (Fig. 2B). In the CA3 region, there was no significant difference in the TUNEL-positive cells displaying apoptotic morphology between injured WT and Bax null mice. A significantly higher ratio of apoptotic cells was present in the dentate of WT mice as compared to Bax null mice. In the CA1 subfield, there was an increase in the mean number of TUNEL-positive cells in injured WT mice as compared to injured Bax null mice, but this change did not quite reach statistical significance (p=0.054). These findings demonstrate that deletion of Bax protein can prevent or delay DNA damage in hippocampal neurons after TBI.
To examine whether deletion of Bax protein could attenuate injury induced by TBI, brain and lesion volumes were measured in Bax null and WT mice at 21 days after TBI or sham surgery. The mean lesion volume in traumatized Bax null mice was less than injured WT mice (Fig. 3A, left); however, this difference did not quite reach statistical significance (p=0.055). Sham-operated Bax-null mice had significantly larger brains than sham-operated WT mice (Fig. 3B, left), suggesting that Bax deletion may produce significant developmental changes in brain resulting in megaencephalopathy.
To examine if disruption of the Bax gene could protect hippocampal neurons at later time points following TBI injury, mice were sacrificed 21 days post-TBI, and the number of surviving neurons in the hippocampus were counted in cresyl violet–stained sections in WT and Bax null mice after sham or TBI surgery. The mean number of surviving CA1 hippocampal neurons in TBI Bax null mice was more than in WT TBI animals (Fig. 3A, right), but this difference did not reach significant levels (p=0.08). However, while there was no difference in the number of CA3 neurons in the TBI-injured Bax null mice versus TBI-injured WT mice, our analysis showed a significant difference in the number in CA3 hippocampal neurons in Bax null mice after sham surgery as compared to WT mice. The latter data are consistent with the finding that the brain volumes and hippocampal cell counts are increased in Bax null mice (Fig. 3B), and suggest that the Bax null mice may have abnormalities in neuronal development. Representative cresyl violet–stained sections are shown in Figure 3C.
Motor function (round beam balance and wire grip test) was studied in WT and Bax null mice on days 1–5 after TBI. Both WT and Bax null mice exhibited decreased latencies in the round beam balance on day 1 post-injury (Fig. 4A). While latencies improved for both groups on days 2–5, there was a significant difference in the overall performance between the WT-injured mice as compared to injured Bax null mice as determined by RMANOVA. When mice were studied for wire grip test (Fig. 4B), both injured and sham-operated Bax null mice had significantly lower scores than WT sham or traumatized WT mice at all time points tested. Similar results were found when spatial memory acquisition was assessed with the MWM on days 14–18. There were significant differences in the submerged platform latencies for both the sham and traumatized Bax null mice compared to both sham-operated and traumatized WT mice (Fig. 4C) at several time points. Visible platform latencies on days 19–20 followed a similar pattern. Swim speeds were equal between groups, indicating that there was no motor impairment in the spatial memory testing (data not shown). Thus, the developmental behavioral effects attributable to Bax deletion were more severe than the trauma itself.
TBI produces direct mechanical injury to the brain, resulting in immediate necrotic cell death of neurons directly impacted by the trauma at the center of the contusion. While this initial injury may cause acute damage to the brain, neurons continue to die hours to weeks after the initial injury, and a substantial number of these neurons may die via apoptotic mechanisms (Clark et al., 1997, 2000; McIntosh et al., 1998; Yakovlev et al., 1997). Growing evidence suggests a role for Bcl-2 proteins in regulating delayed neuronal cell death after TBI (Clark et al., 1997; Graham et al., 2000; Springer et al., 2001).
Bax is an important initiator of the intrinsic apoptotic pathway. Expression of the mitochondrial protein Bax results in increased permeability of the mitochondrial outer membrane (Eskes et al., 1998; Narita et al., 1998). This increased permeability leads to the release of proteins that normally reside between the inner and outer mitochondrial membranes, including cytochrome C, AIF and other pro-apoptotic proteins into the cytoplasm (Danial and Korsmeyer, 2004). The apoptotic properties of Bax can be antagonized by anti-apoptotic proteins such as Bcl-2 and Bcl-xL (Cheng et al., 1996; Reed et al., 1998; Schlesinger et al., 1997). Thus, it has been hypothesized that the ratio of Bcl-2/Bax determines whether a cell undergoes apoptosis or survives. Both Bcl-2 and Bcl-xL can form heterodimers with Bax and prevent Bax from forming active oligomers (Gross et al., 1998; Krajewski et al., 1994; Oltvai et al., 1993). Other pro-apoptotic Bcl-2 family proteins such as Bak, can also form heterodimers with Bax or homodimers with other Bak molecules and initiate apoptosis (Chittenden et al., 1995; Holinger et al., 1999; Sattler et al., 1997).
In the current study, increased Bax expression was detected with immunohistochemistry within CA3 neurons of hippocampus in mice at 24h after CCI as compared to sham-operated. Increased Bax expression was not detectable in immunoblots from whole hippocampi, consistent with the observation that increased Bax expression was found in only a small fraction of cells within the hippocampus. These results are also consistent with other studies. Raghupathi et al. (2003) found increases in Bax mRNA and protein in cortex of rats subjected to fluid percussion injury; however, there were no changes in Bax mRNA or protein expression in hippocampus. Wennersten et al. (2003) reported that Bax mRNA and protein were increased within the cortex of rats subjected to CCI which peaked at 6 and 10 days post-injury.
Decreased immunoreactivity for Bcl-2 and increased Bax immunoreactivity has been shown in injured neurons after TBI and in models of ischemia (Gillardon et al., 1996; Raghupathi et al., 2003; Vlodavsky et al., 2005). Similar findings have also been reported in studies of CNS and tissue samples from human trauma victims. There is increased expression of Bax and decreased expression of Bcl-2 in patients with poor outcomes (Clark et al., 2000). Therefore, although the increased expression of Bax in this study and others has been limited to a relatively small subset of injured neurons, the global decrease in expression of Bcl-2 and resultant increase in the Bax/Bcl-2 ratio has been proposed to be an important factor in initiating delayed apoptotic death.
Another major finding in the current study was a lack of difference in caspase-3 activation between injured WT and injured Bax null mice. There was an increase in caspase-3 activity within the hippocampus in the WT mice after trauma compared to sham-operated controls. We found no significant difference in total caspase-3 activity in WT and Bax null mice as result of trauma; however, there was a difference in the subcellular distribution of activated caspase-3. The lack of an effect of Bax deletion on total caspase-3 activity after CCI suggests that caspase-3 may be activated by mechanisms other than Bax. One such mechanism is binding of the Fas ligand to its receptor which results in activation of caspase-8, and cleavage and translocation of the pro-apoptotic protein Bid to the mitochondria. Expression of Fas ligand and activation of Fas-mediated apoptosis has been reported in both traumatic and ischemic CNS injury (Martin-Villalba et al., 1999; Raoul et al., 1999; Rosenbaum et al., 2000). Excitatory amino acids are released after TBI resulting in increased intracellular calcium levels via activation of the NMDA receptors (Faden et al., 1989). Calcium may be sequestered in the mitochondria resulting in mitochondrial depolarization and opening of the mitochondrial permeability transition pore (Fiskum, 1983). Shi et al. (2007) found that there was increased neurogenesis in Bax null mice subjected to CCI. They found that there was increased expression of the Kv4.1 potassium channel within the neural stem and progenitor cells in dentate of Bax null mice compared to WT controls. These mechanisms may be more important than caspase-3 in Bax's role in determining the ultimate outcome after trauma. On the other hand, the differences in the cellular distribution of caspase-3 in Bax null mice could also be important. Activated caspase-3 was not detected in the cytosol of juvenile Bax null mice, but was found in the cytosol of WT juvenile mice treated with ethanol (Young et al., 2003). In the current study we found that there was not activated caspase-3 immunoreactivity in the nucleus of traumatized Bax null neurons as was seen in WT mice subjected to CCI. The significance of this finding is uncertain since Bax's site of action is believed to be the mitochondria (Narita, 1998).
The current study found evidence of improved histological outcome after CCI. Injured Bax null mice had fewer TUNEL-positive neurons in the dentate gyrus and a strong trend toward fewer TUNEL-positive neurons in CA1 at 24h post-injury than injured WT control mice. In Bax null mice, histological assessment revealed an increased number of surviving neurons in the CA1 and CA3 region at 21 days post-injury. Shi et al. (2007) found that there was an increase in total hippocampal volume at 30 days after CCI; thus, the results are somewhat comparable. Mean lesion volume in the Bax null mice was less than WT controls although this change did not quite reach statistical significance. This change was similar to the decrease in lesion size found when apoptosis is inhibited by overexpression of the anti-apoptotic Bcl-2 gene in mice (Nakamura et al., 1999; Tehranian et al., 2006). The current data suggest a modest protective effect upon histological outcome after TBI in hippocampus afforded by disruption of the Bax gene.
The unexpected finding of this study is the severe behavioral impairment of both sham-operated and injured Bax-null mice. The effect of Bax disruption in nontraumatized animals produced a larger effect on behavior than CCI itself, making any comparison between the WT and Bax null mice after CCI meaningless. These results, however, do suggest that Bax disruption may have profound developmental effects resulting in cognitive impairment. Programmed cell death of neurons during development is an important mechanism by which excess neurons and synapses are removed as the animal matures. The results from our behavioral tests suggest that Bax may be very important in normal developmental cell death and its disruption may result in cognitive impairment. During early development, Bax is localized primarily within the mitochondria but it resides primarily in the cytosol in the adult neuron (Polster et al., 2003). Thus, Bax may play a greater role in neuronal apoptosis in newborn animals concomitant with its important role in programmed cell death during development.
There are a number of limitations associated with this study. The lifelong disruption of the Bax gene in this mouse model may allow for compensatory changes such as expression of other Bcl-2 family members. For example, Bak is another member of the Bcl-2 family with functions overlapping those of Bax. Bak is structurally similar to Bax and may regulate cytochrome C release and apoptotic cell death in a variety of cell types. Bak has been proposed to compensate for decreased Bax expression in other models of Bax deficiency (Krajewski et al., 1996). In addition, Bax deficiency clearly has profound developmental effects that impair cognitive function. These effects overshadow the behavioral effects of trauma in this model and thus mask any protective effect upon behavioral outcomes that Bax deficiency might have. Anesthesia may also influence performance on behavioral tests, although this was controlled for by the use of sham operated animals (Statler et al., 2000). Other means of altering expression of Bax in trauma models, such as conditional knockout mice, are needed to determine the role of Bax long term functional outcomes after TBI.
We thank Ms. P. Strickler for secretarial support. This work was supported by grants from National Institute of Health (grant number NS30318).
No conflicting financial interests exist.