- Original Articles
- Open Access
BAX Contributes to Apoptotic-Like Death Following Neonatal Hypoxia-Ischemia: Evidence for Distinct Apoptosis Pathways
© Picower Institute Press 2001
- Accepted: 10 August 2001
- Published: 1 September 2001
Hypoxic-ischemic (H-I) injury to the neonatal brain has been shown to result in rapid cell death with features of acute excitotoxicity/necrosis as well as prominent delayed cell death with features of apoptosis such as marked caspase-3 activation. BAX, a pro-apoptotic molecule, has been shown to be required for apoptotic neuronal cell death during normal development but the contribution of endogenous BAX in cell death pathways following H-I injury to the developing or adult brain has not been studied.
Materials and Methods
Bax +/+, +/−, and −/− mice at post-natal day 7 were subjected to unilateral carotid ligation followed by exposure to 45 minutes of 8% oxygen. At different timepoints following H-I, brain tissue was studied by conventional histology, immunohistochemistry, immunofluorescence, Western blotting, and enzymatic assay to determine the extent and type of cell injury as well as the amount of caspase activation.
We found that bax −/− mice had significantly less (38%) hippocampal tissue loss than mice expressing bax. Some of the remaining cell death in bax −/− mice, however, still had features of apoptosis including evidence of nuclear shrinkage and caspase-3 activation. Though bax −/− mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I, the density of cells with activated caspase-8 in the CA3 region of the hippocampus did not differ between bax +/− and bax −/− mice.
These findings demonstrate that endogenous BAX plays a role in regulating cell death in the central nervous system (CNS) following neonatal H-I, a model of cerebral palsy. In addition, while BAX appears to modulate the caspase-3 activation following neonatal H-I, caspase-8 which is linked to death receptor activation, may contribute to apoptotic-like neuronal death in a BAX-independent manner.
Apoptosis or programmed cell death is an important component of nervous system development (1,2). Cell death, which has features of apoptosis, is also prominent in the developing brain following hypoxiaischemia (H-I) (3–10). Many checkpoints of regulation exist in this pathway (reviewed in (11,12)), including the Bcl-2 family checkpoint. Interactions between different BCL-2 family members have the potential to regulate whether a cell lives or dies (reviewed in (13)). Anti-apoptotic family members, such as BCL-2 and BCL-XL, can heterodimerize through homologous domains with pro-apoptotic members, such as BAX (13–15). If a balance does not exist and protective members of the BCL-2 family are in excess, cells can be protected. However, when BAX or BAX homologues are in excess, homodimers can dominate and cells are more susceptible to apoptosis (14).
Studies have shown that endogenous BAX is important in programmed neuronal cell death. In bax −/− mice, sympathetic, peripheral nervous system (PNS), motor, and cerebellar granule neurons are all dramatically protected from apoptosis both in vitro and in vivo (15–19). Although BAX plays an important role under these circumstances and in these cells, few studies have examined a role for endogenous BAX in the cell death which occurs in the normal or injured CNS. Recently, it was shown that BAX regulates apoptosis following ionizing radiation to the CNS (20) and that pathological cell death seen in cerebellar granule neurons but not in Purkinje cells in Lurcher mice is BAX dependent (19). Whether endogenous BAX plays a role following other types of injury to the developing or adult CNS such as H-I has not been defined.
H-I encephalopathy in the prenatal and perinatal period is a major cause of morbidity and mortality and can result in cerebral palsy (21,22). Models of H-I in neonatal animals have been shown to mimic many of the pathological and cognitive abnormalities that are seen in children who have sustained a H-I insult (21–23). The Levine model of H-I includes unilateral carotid artery ligation followed by exposure to hypoxia (24,25). In this model, injury occurs in the hemisphere ipsi- but not contralateral to the ligation, and there are prominent features of both apoptosis as well as necrosis (8–10,26–28). Apoptotic/caspase-dependent injury appears to contribute to as much as 50% of the tissue loss following neonatal H-I (9,29). In recent studies, we have utilized this model in different transgenic and knockout mice to determine the role of specific genes in H-I induced brain damage (30). We find that nNOS and clusterin exacerbates the injury (28,31) while overexpression of BCL-XL is protective (29). We implemented this model in bax −/− mice to study the role of BAX in apoptotic-like death following neonatal H-I.
Animals and Surgical Procedures
Bax +/− male mice and bax −/− female mice on a C57BL/6 background were produced as described (32). Domains BH1 and BH2 were deleted rendering the protein non-functional by a BAX targeting vector, which substituted PGK-Neo for exons 2 through 5. The disrupted BAX allele was transferred through the germ line. All mice were housed under a 12:12 hr light:dark cycle, with food and water available during the duration of the study. The neonatal H-I brain injury followed the Levine procedure (21,24,25, 30,33,34). At postnatal day 7 (P7), pups were anesthetized with 2.5% halothane (balance room air), and the left common carotid artery was surgically exposed and permanently ligated. The incision was sutured and the pups were returned to the mother for a 2 hour recovery and feeding period. Pups were placed in individual containers (37°C water bath to maintain normothermia) through which humidified 8% oxygen (balance nitrogen) flowed for 45 minutes. Following H-I, the pups were returned to their cages and remained with their mother. Tail DNA was prepared and utilized for PCR. The normal allele generated a 304 basepair product amplified using an exon 5 forward primer (0.64 µM: 5′-TGATCAGAACCATCATG-3′) and an intron 5 reverse primer (0.64 µM: 5′-GTTGACCAGAGTGGCGTAGG-3′). The mutant allele generated a 507 basepair product which was amplified with a neo/pgk primer (0.16 µM: 5′-CCGCTTCCATTGCTCAGCGG-3′) and the same intron 5 reverse primer, which together generate a 507 basepair product. The cycling parameters for the reaction were 1 min at 94°C, 55°C, and 72°C each for a total of 30–35 cycles (16).
For histological analyses, either 24 hours or 1 week following H-I, animals were anesthetized with 150 mg/kg of pentobarbital intraperitoneally and then perfused through the left ventricle with phosphate buffer saline (PBS) (pH 7.4). Brains were removed and fixed overnight at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were then cyroprotected overnight at 4°C in a solution of 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Brains were frozen in dry ice and 50 µm coronal sections were cut on a freezing sliding microtome and then stored in 0.1M Tris-buffered saline (TBS, pH 7.5, 4°C). For cresyl violet staining, every sixth section was mounted on a slide beginning with the corpus callosum and ended at the posterior end of the hippocampus. Tissue was stained with cresyl violet at pH 3.6 for 5 min. The slides where then rinsed and dehydrated in ethanol and coverslipped. Damage resulting from H-I was determined by calculating the amount of surviving tissue in coronal sections. For each brain, four sections containing the hippocampus were assessed corresponding to figures 44, 49, 51, and 55 in a mouse brain atlas (35) and seven sections of the cortex were assessed corresponding to figures 24, 31, 37, 44, 49, 51, and 55. The area of each brain region was then calculated and the percent area loss in the lesioned versus the unlesioned hemisphere was determined. The scorer was blinded to genotype of the animals. For analysis comparing % tissue loss in bax +/− vs. bax −/− mice, data are presented as mean +/− SEM and were analyzed with a t-test with a significance cut off at P < 0.05. For DEVD cleavage assay 24 hours after H-I, animals were perfused with PBS (pH 7.4) and brains were removed. Tissue from the cortex and hippocampus were dissected and frozen in dry ice.
To further assess the volume of the hippocampus and number of CA1 and CA3 neurons in bax +/− and bax −/− mice, we utilized unbiased stereological methods. For assessment of hippocampal volume as well as CA1 and CA3 neuron number, ten animals (bax +/− N = 5; bax −/− N = 5) at P14, 7 days following H-I at P7, were assessed. The brains were sectioned in the coronal plane and every sixth section from the beginning to the end of the hippocampus (rostral to caudal) was mounted on a slide. The sections were stained with the fluorescent dye 4′, 6 diamidino-2-phenylindole (DAPI) stain (1:1000). The hippocampal region of every brain section was identified and traced, and the section thickness was determined with a 100X lens (N.A. 1.40). From that information, the volume of the hippocampus was determined with the Cavalieri Estimator technique using Stereo Investigator software (MicroBright-Field, Inc., Colchester, VT). To determine the number of CA1 and CA3 neurons in the hippocampus, the Optical Fractionator technique was utilized. The CA1 and CA3 areas of the hippocampus were traced on the same brain sections utilizing a 4x lens with the image projected onto a computer screen. At a higher power (100x lens, 1.4 N.A.), neuronal nuclei in the CA1 and CA3 region were marked if they fell within a 30 µm x 30 µm (x,y) counting frame within a 15 µm z-depth. A 10 µm guard zone was utilized. Sampling distances were set so that 100–400 neuronal nuclei and 100–300 sites per CA1 and CA3 region were sampled. The number of neurons in each hippocampal region was then calculated from the information obtained utilizing the Stereo Investigator software.
DEVD Cleavage Assay
DEVD-AMC cleavage assay was performed as described (9,36,37). Frozen tissue samples were homogenized in lysis buffer (10 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 2 mm EDTA, 2 mM EGTA, 1 mM PMSF, and protease inhibitor cocktail) and centrifuged at 12,000 g for 10 min at 4°C. Ten µl of the lysate was incubated in a 96-well plate with 90 µL of assay buffer (10 mM Hepes, pH 7.4, 42 mM KCl, 5 mM MgCl2, 1 mM DTT, 10% sucrose) containing 30 µM Ac-DEVD-AMC (Calbiochem, San Diego, CA). The emitted fluorescence was measured every 5 minutes for 30 minutes at room temperature at an excitation wavelength of 360 nm and an emission wavelength of 460 nm using a microplate fluorescence reader (Bio-tek Instruments, Winooski, VT). DEVD activity was obtained from the slope of fluorescence against time. Ac-AMC (Calbiochem, San Diego, CA) was used to obtain a standard curve and the enzyme activity was calculated as the pmol AMC/mg/protein/min. Data are presented as mean +/− SEM. Data were analyzed with ANOVA followed by Newman-Keuls multiple comparisons test with significance cut off at P < 0.05.
Immunohistochemistry, Immunofluorescence, and Western Blotting
Fifty-micrometer free floating sections through the forebrain were processed for peroxidase immunohistochemistry using the rabbit polyclonal antibody CM1 to activated caspase-3 (1:20,000; gift of Idun, Inc., La Jolla, CA) (36,38), the rabbit polyclonal antibody SK440 to activated caspase-8 (1:3000, gift of K. Kikly, Smith Kline Beacham Pharmaceuticals, King of Prussia, PA (39)), or the anti-cytochrome c antibody (1:500; PharMingen, Inc., San Diego, CA) with the Vectastain ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA) as previously described (36). For quantitation of the density of activated casapse-8-immunoreactive (IR) cells in the CA3 region of Bax +/− and Bax −/− mice, 4 hippocampal sections corresponding to figures 44, 49, 51, and 55 in a mouse brain atlas (35) were assessed. Four fields measuring 0.023 mm2 in the CA3 region were assessed in each section. Caspase-8-IR cells were assessed by an observer blinded to genotype and were counted as positive if there was staining of a clearly distinguishable cell body in which the staining intensity was greater than the level of any of the cells observed in the contralateral CA3 region as determined by thresholding utilizing image analysis with an Optiphot digital camera (Nikon, Inc., Melville, NY). For double-labeling immunofluorescence experiments for activated caspase-3, a neuronal nuclear marker, and cytochrome c, brain sections were incubated with the following antibodies: CM1 (1:5,000) along with mouse anti-neuronal nuclei antibody (1:100) (NeuN; Chemicon, Inc., Temecula, CA) or anti-cytochrome c (1:500). Fluorescein isothiocyanate- and indocarbocyanine-labeled secondary antibodies were utilized for fluorescent detection as described (36). For fluorescent double labeling for activated caspase-3 (CM1) and activated caspase-8 (SK440), sections were first blocked with 1% BSA and 0.2% dry milk in Tris-buffered saline (TBS) for one hour and then incubated overnight with SK440 (1:5000 dilution). After washing, sections were incubated with fluorescein-conjugated anti-rabbit antibody using TSA-direct tyramide signal amplification kit (NEN Life Science Products, Boston, MA). Sections were then blocked with 3% goat serum in TBS for one hour, incubated overnight with CM1 (1:5,000 dilution), washed, and then incubated with goat anti-rabbit IgG coupled to Alexa 568 (Molecular Probes, Eugene, OR) for one hour. Controls included omission of each primary antibody. Slides were coverslipped with Vectashield mounting media (Vector Laboratories, Burlingame, CA) and examined with a Nikon FXL fluorescence microscope (Nikon, Inc., Melville, NY) as well as a Bio-Rad Confocal microscope for analysis (Bio-Rad, Inc., Hercules, CA). Western blotting with hippocampal lysates was performed as described (28,36,37). Antibody to FAS was obtained from Upstate Biotechnology (Lake Placid, NY) and antibody to pro-caspase-8 was obtained from Pharmingen, Inc. (San Diego, CA). Quantitation of band density on X-ray film following Western blotting was performed as described (40).
Endogenous BAX Plays a Role in Tissue Loss Following H-I
Hippocampal Volume Loss Correlates with CA1 Neuronal Loss in Bax +/− and Bax −/− Mice Following Neonatal H-I
In this neonatal H-I model, all regions of the hippocampus are damaged and there is not selective vulnerability of only one cellular layer. Coincident with the global neuronal loss in the hippocampus, we previously analyzed CA1 neuronal loss in this model and found that hippocampal volume loss correlates well with CA1 neuronal loss following H-I injury in the P7 rat (34). While it was not practical to count CA1 neurons in the 53 mice analyzed following neonatal H-I, utilizing stereological methods, we determined whether hippocampal volume and percent tissue loss correlates with CA1 cell number and percent CA1 cell loss respectively in a group of bax +/− and bax −/− following H-I. Bax +/− (N = 5) and bax −/− (N = 5) mice were randomly selected and hippocampal volume CA1 cell number were assessed with stereological methods at P14, seven days following unilateral carotid ligation and exposure to 8% oxygen for 45 minutes. We found that in both bax +/− and bax −/− mice, there was a strong correlation (r2 = 0.75, p = 0.017) between hippocampal volume and CA1 cell number (Fig. 3A). Similarly there was a strong correlation (r2 = 0.52, p = 0.019) between percent hippocampal volume loss and percent CA1 neuron loss (Fig. 3B). There was less CA1 cell loss and hippocampal volume loss in the bax −/− versus the bax +/− analyzed in this fashion. In assessing only 5 animals per cohort for stereological analysis, there was not a significant difference in CA1 cell number between these groups (Fig. 3C). Taken together, however, with the data in Figure 1 and Figure 2, these results suggest that in addition to having less hippocampal injury, bax −/− mice have less CA1 neuronal loss following neonatal H-I.
Caspase-3 Activation is Attenuated But Not Absent in Bax −/− Mice
Caspase-8 Activation is not Blocked by the Absence of BAX
In this murine model of neonatal H-I, the absence of BAX resulted in neuroprotection in the hippocampus. The degree of neuroprotection observed in this study was similar to that seen in our previous studies of neonatal H-I in which we used other anti-apoptotic strategies. For example, there was 39% less hippocampal and 61% less cortical damage following H-I in neonatal transgenic mice overexpressing Bcl-XL as compared to age-matched littermate controls (29). In addition, following neonatal H-I in P7 rats, the administration of the pan-caspase inhibitor BAF resulted in approximately 50% less injury to the hippocampus and cortex (9). Other studies also suggest that caspases including caspase-9 and -3 are not only important in programmed cell death (59,60) but also in ischemia-mediated death following brain injury (61,62). Together, these studies indicate that apoptotic-like pathways are not only involved in a significant percent of brain injury following neonatal H-I but also suggest that a variety of anti-apoptotic strategies and molecules could be targeted for potential drug development.
While our study demonstrates that endogenous BAX is involved in apoptotic-like death following neonatal H-I, some death with caspase-3 activation still occurred in the absence of BAX. The amount of protection was similar in this study to that seen following ionizing radiation to the P5 mouse cerebellum where bax −/− mice had an approximately 50% reduction of apoptosis compared with wild-type mice (20). Further, apoptotic cell death in cerebellar granule cells of Lurcher mice was blocked in bax −/− mice (19). The presence of activated capase-3 that we observed in the present study in the absence of BAX suggests that one possibility is that other BAX-like molecules may also be contributing to the cytochrome c dependent death occurring following neonatal H-I. There are several proapoptotic Bcl-2 family members including Bax, Bcl-XS, Nbk/Bik, Bim, Bad, Bid, Harikari, and Bak. Some of these genes have been shown to be expressed in the brain (55–57,63,64), though their expression and role in the developing brain remains to be characterized. Studies by Han et al. (65) and Zhai et al. (66) show that Bid is expressed in the brain and may be a cytochrome c efflux-inducing factor. It is interesting to note that caspase-8 is activated in some neurons in an adult stroke model (39) and can cleave BID which can lead to mitochondrial damage, cytochrome c release, and cell death (67–71). This prompted us to look for caspase-8 activation in the neonatal model.
Caspase-8 was originally identified via proteinprotein interactions and was found to contain two death effector domain-like modules allowing it to interact with FADD (47,48,72–74). FADD is an adaptor protein which directly couples cell surface signaling via death receptors belonging to the TNF receptor gene superfamily to activation of caspase-8, (for reviews see (75,76)). Upon activation, caspase-8 can lead to activation of many other downstream caspases such as caspase-9 and -3 (47–49). It has been proposed that the amount of caspase-8 activation may determine whether a mitochondrial-dependent pathway is required for amplification of the caspase cascade (69). While BAX-regulated mitochondrial events influence a significant amount of cell death following neonatal H-I, our data in mice as well as other data from rats (43,58) suggest that caspase-8 may regulate BAX-independent apoptotic-like death and caspase-3 activation associated with cytochrome c release following H-I (Fig. 9). This could occur via direct caspase-9 and -3 cleavage by caspase-8. Alternatively, caspase-8 has been shown to amplify cytochrome c release via cleavage of BID and its translocation to the mitochondria (67–70). There, caspase-8-activated BID can trigger homooligomerization of both BAX and BAK resulting in cytochrome c release and apoptosis (77,78). While the absence of BAX may lessen the effect of caspase-8 following neonatal H-I, it is possible that the presence of BAK or another BH3-domain only homologue still allows for caspase-8 mediated mitochondrial dysfunction. In light of these results, it is interesting to note that Fas, a death receptor linked to caspase-8 activation, is present in the developing brain and is upregulated following H-I (50). In addition, some apoptosis-inducing agents, such as staurosporine, can result in caspase-8 activation in cultures of cortical neurons (79). It will be interesting in future experiments to further define the role of death receptor ligands/receptors and caspase-8 in neonatal H-I as well as the role of BID in caspase-8 actions in vivo.
This study shows that the absence of BAX is protective against apoptotic-like death following neonatal H-I. Our previous studies have shown that molecules such as brain-derived neurotrophic factor (BDNF) can completely block the apoptotic as well as most of the non-apoptotic components of cell death following neonatal H-I (34,36,37). In addition, BDNF can protect against H-I-induced learning abnormalities (23). Important issues that remains regarding anti-apoptotic strategies in general is determining exactly which pathways are involved, the maximum protection that can be achieved, a detailed timecourse of when in relation to injury therapeutic agents can be administered, and whether different agents can be used in combination to achieve even greater neuroprotection. These issues will be important to understand in the setting of both neonatal H-I as well as following other injuries to the developing brain.
This work was supported by NIH grant NS35902. The authors thank Eugene Johnson, Mohanish Deshmukh, and Jeff Gidday.
- Oppenheim RW. (1991) Cell death during the development of the nervous system. Annu. Rev. Neurosci. 14: 453–501.View ArticlePubMedGoogle Scholar
- Nijhawan D, Honarpour N, Wang XD. (2000) Apoptosis in neural development and disease. Ann. Rev. Neurosci. 23: 73–87.View ArticlePubMedGoogle Scholar
- Ferrer I, Tortosa A, Macaya A, et al. (1994) Evidence of nuclear DNA fragmentation following hypoxia-ischemia in the infant rat brain, and transient forebrain ischemia in the adult gerbil. Brain Path. 4: 115–122.View ArticleGoogle Scholar
- Mehmet H, Yue X, Squier MV, et al. (1994) Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult. Neurosci. Lett. 181: 121–125.View ArticlePubMedGoogle Scholar
- Hill IE, MacManus JP, Rasquinha I, Tuor UI. (1995) DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat. Brain Res. 676: 398–403.View ArticlePubMedGoogle Scholar
- Sidhu S, Tuor UI, Del Bigio MR. (1997) Nuclear condensation and fragmentation following cerebral hypoxia-ischemia occurs more frequently in immature than older rats. Neurosci. Lett. 223: 129–132.View ArticlePubMedGoogle Scholar
- Silverstein FS, Barks JD, Hagan P, Liu XH, Ivacko J, Szaflarski J. (1997a) Cytokines and perinatal brain injury. Neurochem. Int. 30: 375–383.View ArticlePubMedGoogle Scholar
- Pulera MR, Adams LM, Liu HT, et al. (1998) Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia. Stroke 29: 2622–2629.View ArticlePubMedGoogle Scholar
- Cheng Y, Deshmukh M, D’Costa A, et al. (1998) Caspase inhibitor affords neuroprotection with delayed adminstration in a rat model of neonatal hypoxic-ischemic brain injury. J. Clin. Invest. 101: 1992–1999.View ArticlePubMedPubMed CentralGoogle Scholar
- Taylor DL, Edwards AD, Mehmet H. (1999) Oxidative metabolism, apoptosis and perinatal brain injury. Brain Path. 9: 93–117.View ArticleGoogle Scholar
- Holtzman DM, Deshmukh M. (1997) Caspases: A treatment target for neurodegenerative diseases? Nature Med. 3: 954–955.View ArticlePubMedGoogle Scholar
- Chan SL, Mattson MP. (1999) Caspase and calpain substrates: roles in synaptic plasticity and cell death. J. Neurosci. Res. 58: 167–190.View ArticlePubMedGoogle Scholar
- Gross A, McDonnell JM, Korsmeyer SJ. (1999) Blc-2 family members and the mitochondria in apoptosis. Genes and Development 13: 1899–1911.View ArticlePubMedGoogle Scholar
- Oltvai ZN, Milliman CL, Korsmeyer SJ. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog bax, that accelerates programed cell death. Cell 74: 609–619.View ArticlePubMedGoogle Scholar
- Sedlak TW, Oltvai ZN, Yang E, et al. (1995) Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc. Natl. Acad. Sci. USA 92: 7834–7838.View ArticlePubMedGoogle Scholar
- Deckwerth TL, Elliott JL, Knudson CM, Johnson EMJ, Snider WD, Korsmeyer SJ. (1996) BAX is required for neuronal death after trophic deprivation and during development. Neuron 17: 1–20.View ArticleGoogle Scholar
- Miller TM, Moulder KL, Knudson CM, et al. (1997) Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death. J. Cell Biol. 139: 205–217.View ArticlePubMedPubMed CentralGoogle Scholar
- White FA, Keller-Peck CR, Knudson CM, Korsmeyer SJ, Snider WD. (1998) Widespread elimination of naturally occurring neuronal death in Bax-deficient mice. J. Neurosci. 18: 1428–1439.View ArticlePubMedGoogle Scholar
- Doughty ML, De Jager PL, Korsmeyer SJ, Heintz N. (2000) Neurodegeneration in Lurcher mice occurs via multiple cell death pathways. J. Neurosci. 20: 3687–3694.View ArticlePubMedGoogle Scholar
- Chong MJ, Murray MR, Gosink EC, et al. (2000) Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system. Proc. Natl. Acad. Sci. USA 97: 889–894.View ArticlePubMedGoogle Scholar
- Vanucci RC. (1990) Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediatr. Res. 27: 317–326.View ArticleGoogle Scholar
- Volpe JJ (1995) Neurology of the newborn (W. B. Saunders, Philadelphia).Google Scholar
- Almli CR, Levy TJ, Han BH, Shah AR, Gidday JM, Holtzman DM. (2000) BDNF protects against spatial memory deficits following neonatal hypoxia-ischemia. Exp. Neurol. 166: 99–114.View ArticlePubMedGoogle Scholar
- Levine S. (1960) Anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 36: 1–17.PubMedPubMed CentralGoogle Scholar
- Rice JE, Vannucci RC, Brierley JB. (1981) The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann. Neurol. 9: 131–141.View ArticlePubMedGoogle Scholar
- Ikonomidou C, Mosinger JL, Salles KS, Labruyere J, Olney JW. (1989) Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity. J. Neurosci. 9: 2809–2818.View ArticlePubMedGoogle Scholar
- Nakajima W, Ishida A, Lange MS, et al. (2000) Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat. J Neurosci 20: 7994–8004.View ArticlePubMedGoogle Scholar
- Han BH, DeMattos RB, Dugan LL, et al. (2001) Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia. Nat Med 7: 338–343.View ArticlePubMedGoogle Scholar
- Parsadanian AS, Cheng Y, Keller-Peck CR, Holtzman DM, Snider WD. (1998) Bcl-XL is an anti-apoptotic regulator for postnatal CNS neurons. J. Neurosci. 18: 1009–1019.View ArticlePubMedGoogle Scholar
- Lendon CL, Han BH, Salimi K, et al. (2000) No effect of apolipoprotein E on neuronal cell death due to excitotoxic and apoptotic agents in vitro and neonatal hypoxic ischaemia in vivo. Eur. J. Neurosci. 12: 2235–2242.View ArticlePubMedGoogle Scholar
- Ferriero DM, Holtzman DM, Black SM, Sheldon RA. (1996) Mice without neuronal nitric oxide synthase have less injury after perinatal hypoxia-ischemia. Neurobiol. Dis. 3: 64–71.View ArticlePubMedGoogle Scholar
- Knudson CM, Tung KSK, Tourtellotte WG, Brown GAJ, Korsmeyer SJ. (1995) Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270: 96–98.View ArticlePubMedGoogle Scholar
- Johnston MV. (1983) Neurotransmitter alterations in a model of perinatal hypoxic-ischemic brain injury. Ann. Neurol. 13: 511–518.View ArticlePubMedGoogle Scholar
- Cheng Y, Gidday JM, Yan Q, Shah AR, Holtzman DM. (1997) Marked age-dependent neuroprotection by BDNF against neonatal hypoxic-ischemic brain injury. Ann. Neurol. 41: 521–529.View ArticlePubMedGoogle Scholar
- Franklin KBJ, Paxinos G (1997) The Mouse Brain in Stereotaxic Coordinates (Academic Press, Inc., San Diego).Google Scholar
- Han BH, D’Costa A, Back SA, et al. (2000) BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia. Neurobiol. Dis. 7: 38–53.View ArticlePubMedGoogle Scholar
- Han BH, Holtzman DM. (2000) BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J. Neurosci. 20: 5775–5781.View ArticlePubMedGoogle Scholar
- Srinivasan A, Roth KA, Sayers RO, et al. (1998) In Situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death & Diff. 5: 1004–1016.View ArticleGoogle Scholar
- Velier JJ, Ellison JA, Kikly KK, Spera PA, Barone FC, Feuerstein GZ. (1999) Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J. Neurosci. 19: 5932–5941.View ArticlePubMedGoogle Scholar
- Holtzman DM, Bayney RM, Li Y, et al. (1992) Dysregulation of gene expression in mouse trisomy 16, an animal model of Down syndrome. EMBO J. 11: 619–627.PubMedPubMed CentralView ArticleGoogle Scholar
- Selznick LA, Holtzman DM, Han BH, et al. (1999) In Situ Immunodetection of neuronal caspase-3 activation in Alzheimer disease. J. Neuropath. Exp. Neurol. 58: 1020–1026.View ArticlePubMedGoogle Scholar
- Northington FJ, Ferriero DM, Graham EM, Traystman RJ, Martin LJ. (2001) Early neurodegeneration after hypoxiaischemia in neonatal rat is necrosis while delayed neuronal death is apoptotis. Neurobiol. Dis. 8: 207–219.View ArticlePubMedGoogle Scholar
- Northington FJ, Ferriero DM, Flock DL, Martin LJ. (2001) Delayed neurodegeneration in neonatal rat thalamus after hypoxia-ischemia is apoptosis. J. Neurosci. 21: 1931–1938.View ArticlePubMedGoogle Scholar
- Han BH, D’Costa A, Back SA, et al. (2000) BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia. Neurobiol. Dis. 7: 38–53.View ArticlePubMedGoogle Scholar
- Deshmukh M, Johnson EM. (1998) Evidence of a novel event during neuronal death—development of competence-to-die in response to cytoplasmic cytochrome C. Neuron 21: 695–705.View ArticlePubMedGoogle Scholar
- Putcha GV, Deshmukh M, Johnson J. E. M. (1999) Bax translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, bcl-2, and caspases. J. Neurosci. 19: 7476–7485.View ArticlePubMedGoogle Scholar
- Srinivasula SM, Ahmad M, Fernadnes-Alnemri T, Litwack G, Alnemri ES. (1996) Molecular ordering of the Fas-apoptotic pathway: The fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93: 14486–14491.View ArticlePubMedGoogle Scholar
- Muzio M, Chinnaiyan AM, Kischkel FC, et al. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas-APO-1) death-inducing signaling complex. Cell 85: 817–827.View ArticlePubMedGoogle Scholar
- Stennicke HR, Jurgensmeier JM, Shin H, et al. (1998) Procaspase-3 is a major physiological target of caspase-8. J. Biol. Chem. 273: 27084–27090.View ArticlePubMedGoogle Scholar
- Felderhoff-Mueser U, Taylor DL, Greenwood K, et al. (2000) Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain. Brain Pathol. 10: 17–29.View ArticlePubMedGoogle Scholar
- Martinou J-C, Dubois-Dauphin M, Staple JK, et al. (1994) Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 13: 1017–1030.View ArticlePubMedGoogle Scholar
- Chen J, Graham SH, Nakayama M, et al. (1997) Apoptosis repressor genes Bcl-2 and Bcl-x-long are expressed in the rat brain following global ischemia. J. Cerebral Blood Flow Metab. 17: 2–10.View ArticleGoogle Scholar
- Minami M, Jin KL, Li W, Nagayama T, Henshall DC, Simon RP. (2000) Bcl-w expression is increased in brain regions affected by focal cerebral ischemia in the rat. Neurosci. Lett. 279: 193–195.View ArticlePubMedGoogle Scholar
- Yan C, Chen J, Chen D, et al. (2000) Overexpression of the cell death suppressor Bcl-w in ischemic brain: implications for a neuroprotective role via the mitochondrial pathway. J. Cerebral Blood Flow Metab. 20: 620–630.View ArticleGoogle Scholar
- Krajewski S, Mai JK, Krajewska M, Sikorska M, Mossakowski MJ, Reed JC. (1995) Upregulation of Bax protein levels in neurons following cerebral ischemia. J. Neurosci. 15: 6364–6376.View ArticlePubMedGoogle Scholar
- Hara A, Iwai T, Niwa M, et al. (1996) Immunohistochemical detection of Bax and Bcl-2 proteins in gerbil hippocampus following transient forebrain ischemia. Brain Res. 711: 249–253.View ArticlePubMedGoogle Scholar
- MacGibbon GA, Lawlor PA, Sirimanne ES, et al. (1997) Bax expression in mammalian neurons undergoing apoptosis, and in Alzheimer’s disease hippocampus. Brain Res. 750: 223–234.View ArticlePubMedGoogle Scholar
- Cao G, Minami M, Pei W, et al. (2001) Intracellular Bax translocation after transient cerebral ischemia: Implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death. J. Cereb. Blood Flow & Metab. 21: 321–333.View ArticleGoogle Scholar
- Kuida K, Zheng TS, Na SQ, et al. (1996) Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384: 368–372.View ArticlePubMedGoogle Scholar
- Hakem R, Hakem A, Duncan GS, et al. (1998) Differential requirement fo caspase 9 in apoptotic pathways in vivo. Cell 94: 339–352.View ArticlePubMedGoogle Scholar
- Hara H, Firedlander RM, Gagliardini V, et al. (1997) Inhibition of interleukin 1β converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. USA 94: 2007–2012.View ArticlePubMedGoogle Scholar
- Chen J, Nagayama T, Jin K, et al. (1998) Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci. 18: 4914–4928.View ArticlePubMedGoogle Scholar
- Rickman DW, Nacke RE, Rickman CB. (1999) Characterization of the cell death promoter, Bad, in the developing rat retina and forebrain. Brain Res. 115: 41–47.View ArticleGoogle Scholar
- Shimohama S, Fujimoto S, Sumida Y, Tanino H. (1998) Differential Expression of rat brain Bcl-2 family proteins in development and aging. Biochem. Biophys. Res. Comm. 252: 92–96.View ArticlePubMedGoogle Scholar
- Han Z, Bhalla K, Pantazis P, Hendreickson EA, Wyche JH. (1999) Cif (cytochrome c effluxing-inducing factor) activity is regulates by Bcl-2 and caspases and correlates with the activation of Bid. Mol. Cell. Biol. 19: 1381–1389.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhai D, Huang X, Han X, Yang F. (2000) Characterization of Bid-induced cytochrome c release from mitochondria and liposomes. FEBS Lett 472: 293–296.View ArticlePubMedGoogle Scholar
- Luo X, Budihardjo I, Zou H, Slaughter C, Wang XD. (1998) BID, a BCL2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481–490.View ArticlePubMedGoogle Scholar
- Li HL, Zhu H, Xu CJ, Yuan JY. (1998) Cleavage of BID by caspase-8 mediates the mitochondrial damage in the FAS pathway of apoptosis. Cell 94: 491–501.View ArticlePubMedGoogle Scholar
- Kuwana T, Smith JJ, Muzio M, Dixit, V., Newmeyer DD, Kornbluth S. (1998) Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. J. Biol. Chem. 273: 16589–16594.View ArticlePubMedGoogle Scholar
- Gross A, Yin XM, Wang K, et al. (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-X-L prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem. 274: 1156–1163.View ArticlePubMedGoogle Scholar
- Yin XM, Wang K, Gross A, et al. (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400: 886–891.View ArticlePubMedGoogle Scholar
- Boldin MP, Goncharov TM, Goltsev YV, Wallach D. (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803–815.View ArticlePubMedGoogle Scholar
- Medema JP, Scaffidi C, Kischkel FC, et al. (1997) FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16: 2794–2804.View ArticlePubMedPubMed CentralGoogle Scholar
- Bertin J, Armstrong RC, Ottilie S, et al. (1997) Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis. Proc. Natl. Acad. Sci. USA 94: 1172–1176.View ArticlePubMedGoogle Scholar
- Ashkenazi A, Dixit VM. (1998) Death receptors: signaling and modulation. Science 281: 1305–1308.View ArticlePubMedGoogle Scholar
- Budihardjo I, Oliver H, Lutter M, Luo X, Wang XD. (1999) Biochemical pathways of caspase activation during apoptosis. Ann. Rev. Cell Devel. Biol. 15: 269–290.View ArticleGoogle Scholar
- Ruffolo SC, Breckenridge DG, Nguyen M, et al. (2000) BID-dependent and BID-independent pathways for BAX insertion into mitochondria. Cell Death & Different. 7: 1101–1108.View ArticleGoogle Scholar
- Wei MC, Zong WX, Cheng EH, et al. (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292: 727–730.View ArticlePubMedPubMed CentralGoogle Scholar
- Budd SL, Tenneti L, Lishnak T, Lipton SA. (2000) Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons. Proc. Natl. Acad. Sci. USA 97: 6161–6166.View ArticlePubMedGoogle Scholar