Apoptosis in perinatal hypoxic–ischemic brain injury: How important is it and should it be inhibited?

Brain Research Reviews 50 (2005) 244 – 257 www.elsevier.com/locate/brainresrev

Review

Apoptosis in perinatal hypoxic–ischemic brain injury: How important is it and should it be inhibited? Frances J. Northington a,*, Ernest M. Graham b, Lee J. Martin c a

Department of Pediatrics, Eudowood Neonatal Pulmonary Division, Dept. of Pediatrics, CMSC 6-104, Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287, USA b Department of Gyn-Ob, Div. of Maternal-Fetal Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA c Departments of Pathology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA Accepted 14 July 2005 Available online 10 October 2005

Abstract The discovery of safe and effective therapies for perinatal hypoxia – ischemia (HI) and stroke remains an unmet goal of perinatal medicine. Hypothermia and antioxidants such as allopurinol are currently under investigation as treatments for neonatal HI. Drugs targeting apoptotic mechanisms are currently being studied in adult diseases such as cancer, stroke, and trauma and have been proposed as potential therapies for perinatal HI and stroke. Before developing antiapoptosis therapies for perinatal brain injury, we must determine whether this form of cell death plays an important role in these injuries and if the inhibition of these pathways promotes more benefit than harm. This review summarizes current evidence for apoptotic mechanisms in perinatal brain injury and addresses issues pertinent to the development of antiapoptosis therapies for perinatal HI and stroke. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Perinatal brain injury; Stroke; Hypoxia – ischemia; Programmed cell death; Cell death continuum; Excitotoxicity

Contents 1.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Are there appropriate animal models of perinatal HI and stroke for evaluating antiapoptosis therapies as a prelude to human trials? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Does apoptosis contribute significantly to the death of neurons following perinatal hypoxia – ischemia or stroke?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Which cell death pathways are involved, and do these pathways provide rational opportunities for mechanism-based interventions?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Is the delayed apoptotic neurodegeneration following perinatal HI a form of target deprivation induced cell death? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Will neuronal rescue with antiapoptosis therapies restore function?. . . . . . . . . . . . . . . . . . . . . 1.6. What are the possible downsides of interfering with apoptosis in the developing brain? . . . . . . . . . .

* Corresponding author. Fax: +1 410 614 8388. E-mail address: [email protected] (F.J. Northington). 0165-0173/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2005.07.003

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2. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Inhibition of apoptosis, one of the forms of programmed cell death, has been repeatedly proposed as a novel and promising therapeutic direction for neuronal rescue following neonatal hypoxia – ischemia (HI) or stroke. Before considering clinical use of antiapoptosis therapies, we must first determine whether apoptosis is important in brain injury following neonatal HI and stroke. Despite numerous articles showing biochemical evidence that apoptotic pathways are activated early after neonatal HI, the majority of the initial cell death is necrosis in models of neonatal HI in which cell death structure has been examined [54,76,88,113]. This divergence of pathology and biochemistry appears to be much more exaggerated in the immature brain compared to the adult brain. We will examine this pathologic/biochemical dichotomy in addition to related questions of whether apoptotic mechanisms should be considered rational targets for therapy for neonatal HI. We posed questions that address issues that must be faced in the development of antiapoptosis therapies for perinatal HI brain injury. These questions were adapted from Waldmier, who wrote about whether antiapoptosis therapies are realistic goals for the treatment of adult neurodegenerative diseases [122]. The importance of apoptosis in normal brain development and the fundamental differences in the response of the developing brain versus mature brain to injury demands that data be gleaned from immature models prior to any attempt to utilize antiapoptosis therapies in the immature brain. Thus, although there is abundant literature on some of the following subjects in the context of the adult brain, we will generally confine our discussion to data generated in immature animal models of neonatal HI. The questions to be addressed in this article are: 1. Are there appropriate animal models of perinatal HI and stroke for evaluating antiapoptosis therapies as a prelude to human trials? 2. Does apoptosis contribute significantly to the death of neurons following neonatal hypoxia – ischemia or stroke? 3. Which cell death pathways are involved, and do these pathways provide rational opportunities for mechanismbased interventions? 4. Is the delayed apoptotic neurodegeneration following perinatal HI a form of target deprivation-induced cell death? 5. Will neuronal rescue with antiapoptosis therapies restore function?

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6. What are the possible downsides of interfering with apoptosis in the developing brain? 1.1. Are there appropriate animal models of perinatal HI and stroke for evaluating antiapoptosis therapies as a prelude to human trials? Prior to any discussion of the appropriateness of animal models for evaluating antiapoptosis therapies in perinatal HI, two fundamental issues need clarification: (1) What is the relative brain-maturity of the model compared to that of the human term newborn and (2) how well does the model approximate neonatal HI or stroke in humans? There are relatively few sources for information regarding developmental comparative anatomy. Dobbing and Sands’ comparison of rates of brain growth across species has been the most widely used tool for estimation of relative degree of brain maturity [23]. A more recent multivariate analysis of histological and functional maturity of multiple brain regions and neural systems has provided perhaps the best available tool for comparison of the perinatal brain development of 8 widely used mammalian species to the newborn human [16]. The data demonstrate reasonably that the most widely used model of neonatal HI, the 7 day old rat pup, is quite immature and has brain maturity less than that seen in near-term humans [16]. These data are in accord with earlier estimations that the 7 day old rat is a preterm model [100] and in direct contradiction to studies that have arbitrarily used it as a term model. A large amount of biochemical and pathological data on perinatal HI brain injury has been derived from the adaptation of the Levine model of unilateral carotid ligation plus hypoxia [97] to the postnatal day (p)7 rat pup. The original authors, as well as many others, have provided reviews of this and related models of perinatal HI and stroke [4,47,101,116 –118,120,124]. While this model has supplied abundant biochemical and neuropathologic data, its limitations must be recognized before attempting to extrapolate data to other species and related forms of injury. The p7 rat pup is quite immature and should not be utilized as a model for term-human brain injury. The small size of the animal generally prohibits intensive monitoring of relevant physiologic variables either during or after the insult [40]. Small variations in postischemic temperature have a major impact on the amount of brain damage [125]. The modified Levine model has also been criticized as having a non-clinical distribution of injury, somewhat between the pattern seen with global asphyxia and that of a true stoke [40]. With the emergence of true models of

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neonatal stroke [21,96,123], there is no need to use the model as one of neonatal stroke. Despite the limitations of the modified Levine model, large amounts of data from this relatively inexpensive and easily mastered model of perinatal brain injury continue to accrue. Of particular note, this model is one of only two models of perinatal brain injury in which there is any understanding of the types of cell death leading to neurodegeneration following the insult. Extensive neuropathologic study of the model has been done, in marked contrast to the relative lack of such studies in other models of perinatal HI [111 –114]. These studies along with more recent ones have shown that cell death early after injury is largely a form of necrosis, followed by apoptotic degeneration at more delayed points after the injury [54,88]. It is largely because of these studies that we can endeavor to ask the more detailed and controversial questions about the contribution of apoptosis and the practical implications of antiapoptosis therapies. The only other model of perinatal brain injury in which detailed neuropathologic studies have been done is the neonatal piglet model of global asphyxia [74,76]. We have also gained significant knowledge about the marked strain dependence of perinatal brain injury from modifying the FVannucci_ model to mice [22]. The p7 FVannucci_ model is now being utilized for the assessment of functional and long-term outcomes following perinatal HI [5,7,109,127]. These studies answer a long-standing criticism of perinatal HI research and are demonstrating that the model is relevant to clinical neonatal brain injury because the survivors do show late functional sequelae. Importantly for the present topic, having long-term outcome studies will allow antiapoptosis therapies to be tested for neuropathologic, biochemical and functional outcomes, and for safety of use in the immature brain. Non-rodent models offer the advantage of many functional and pathologic similarities to human HI brain injury. The most recently described model of perinatal brain injury, preterm fetal hypoxia – ischemia in the rabbit, greatly expands the possibility for testing antiapoptosis therapies following in utero HI events [20]. These animals display persistent hypertonia and motor deficits, providing a striking phenotype of cerebral palsy against which to test potential therapies. The piglet model of asphyxic cardiac arrest is particularly relevant to the investigation of HI in the full term newborn and young infant. The basal ganglia and somato-sensory cortical injury created in this model [74] is strikingly similar to that seen on MR studies of full-term human neonates with perinatal asphyxia [98]. It can be argued that, at present, the well established piglet models of neonatal HI used to show the efficacy of hypothermic neuroprotection [2,44,110] are the most relevant models in which to test drug therapies, because of the corresponding success of hypothermia in clinical trials [24,41]. With the plethora of models available, the importance of choosing the appropriate model in which to test antiapoptosis therapies for perinatal brain injury cannot be overstated. In addition to

consideration of similarities in degree of neuronal maturity and neuropathology, and the ability to test for functional outcomes, choice of an appropriate model for the testing of antiapoptosis therapies will also have to be determined by similarity of pharmacokinetics and drug metabolism pathways between the animal model and human neonate. 1.2. Does apoptosis contribute significantly to the death of neurons following perinatal hypoxia– ischemia or stroke? This particularly vexing question has yet to be resolved satisfactorily. Depending on the species, model of injury, the age of the animal, and the severity of the injury, apoptosis may play either no, minor, or major roles in the pathophysiology of cell death following HI or stoke in the neonatal period. Although the role of apoptosis in neonatal HI can be debated, there is no doubt that necrosis plays a major role in the neuronal cell death following neonatal HI and stroke. Striatal damage following HI in 1 week old piglets is one of the best examples of essentially pure necrotic neurodegeneration occurring following neonatal HI [76]. In this model, 80% of neurons in the putamen are dead by 24 h after injury, and they die with a necrotic ultrastructure. During the first 24 h following HI, glutathione is depleted, and peroxynitrite damages membrane proteins, the golgi apparatus and the cytoskeleton; in addition, hydroxyl radical damage to DNA and RNA accumulates [76]. Mitochondrial failure occurs following a transient burst of activity at 6 h following the end of HI [76]. In rodent models of neonatal HI, the cell death phenotypes are more heterogeneous than that found in piglets, but necrotic cell death still remains most prominent [88]. Necrosis is the most abundant form of cell death 1– 2 days following neonatal HI in rat and mice, despite the appearance of non-necrotic cell death phenotypes. Investigation of the ultrastructure of these injured brains fully reveals the complexity of the damage (Fig. 1). Some structural characteristics of dying neurons in the first 24 h after neonatal HI suggest that some apoptotic mechanisms are operative; however, the phenotypic diversity of the cell death suggests a mixture of apoptosis, necrosis and intermediate forms of cell death similar to the Fcontinuum_ cell death elicited by kainic injection in the neonatal forebrain [94]. This complexity in the cell death phenotypes suggests that apoptosis inhibitors alone will be inadequate to ameliorate most of the early brain damage following neonatal HI and, according to the cell death continuum concept [94], could simply push cells toward necrotic cell death as seen in vitro with caspase inhibitors applied following chemical hypoxia [33]. These studies show that morphologic appearance of the dying cell is a valuable tool for providing hints about the biochemical and molecular events responsible for the cell death [94]. It will be extremely important to use clues from cell death structure following different degrees and forms of perinatal brain

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Fig. 1. Gallery of cell death. Electron micrographs of forebrain neurons at mid-to end-stages of degeneration. Developmental programmed cell death (PCD) of neurons in the early postnatal brain is a ‘‘gold standard’’ for neuronal apoptosis. Neonatal HI induces the degeneration of neurons with several phenotypes. The most common forms of cell death are apoptosis, hybrids of apoptosis and necrosis (continuum cells), classical necrosis, and possibly autophagy. The spectrum of cell death morphologies that can be identified in the HI neonatal brain supports the existence of a continuum for cell death.

injury to better understand which injuries are most likely to respond to antiapoptosis therapies and whether antiapoptosis therapies actually ameliorate injury or simply change the phenotype of injury. Suggesting that neonatal HI brain injury causes neurodegeneration which lies along an apoptosis –necrosis continuum is highly controversial. Olney and colleagues classify the rapid death of neurons in the excitotoxically injured developing rodent brain as a form of necrosis which they term excitotoxic neurodegeneration [54]. The major difference in the two classification schemes is whether the ‘‘apoptotic-like’’ features seen in early cell death following neonatal excitotoxicity or HI predict involvement of programmed cell death pathways. The electron micrographs offered in their paper [54] differ from the complete cellular and organelle disruption usually seen in pure necrotic cell death and correspond closely to the Fcontinuum_ phenotype observed by Portera-Cailliau et al. [94] and shown in Fig. 1. We propose that the ultrastructural appearance and the biochemistry of neurodegeneration following neonatal HI and excitotoxic injury suggest involvement of programmed cell death mechanisms. At one level, the differences in these classification schemes are largely semantic; however, the Fcontinuum_ scheme was formulated with the idea that the cell death structure could suggest the molecular mechanisms responsible for the cell death [94]. Therefore, by defining the continuum cell as a degenerating cell that appears as an

incomplete form of apoptosis, but not classically apoptotic, we allow for the co-activation of more than one mechanism for cell death. This type of nomenclature, described in the cell death continuum concept [72,75], helps to predict that emphasizing one form of cell death over another might be counter-productive and anticipates the possibility that antiapoptosis therapies will be only partial and incomplete strategies for treatment of HI injury. We believe that the concept of the cell death continuum is particularly relevant to the degeneration of neurons. The existence of the continuum is shown by the structural diversity of neuronal cell death induced by a variety of injuries including excitotoxicity, HI, and axonal trauma. The fundamental mechanism driving the continuum is thought to be gradations in the responding cells to stress. Some specific mechanisms thought to be driving the continuum are the developmental expression of different subtypes of glutamate receptors, the propinquity of developing neurons to the cell cycle, and the degree of axonal collateralization [71,72]. The concept of the cell death continuum has been challenged and deemed confusing by some investigators [36,54,104]. Arguments against the cell death continuum are based in part on the assumption that morphology and underlying biochemical processes are discrete [36]. While this is likely the case at the extremes of the cell death continuum, absolute discreteness ignores the cellular appearances that occur in response to injury and

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disease. Steadfast arguments have been proposed by opponents of the cell death continuum concept. These arguments include (1) excitotoxic neuronal death in vivo is necrotic, regardless of age, and (2) apoptosis of neurons in the adult nervous system is extremely infrequent [36]. Rigid conceptualization regarding cellular pathology may hinder our goal of the identification of real and relevant molecular mechanisms in complex biological systems, such as the injured perinatal brain, and ultimately limit the realization of therapeutic opportunities. For example, motor neuron degeneration in amyotrophic lateral sclerosis (ALS) was not considered to be a variant of apoptosis until a few years ago when the concept of the cell death continuum was applied [70], and now, antiapoptosis therapies are being considered for the treatment of this disease [10,73]. Importantly, as discussed later, there is substantial agreement that connectivity mediated-target deprivation contributes substantially to the overall brain injury following perinatal HI [88,89,131], and this injury is most likely to be true apoptosis. There remains, however, a fundamental question of whether or not apoptotic mechanisms are directly activated by perinatal HI. There is biochemical evidence for both necrosis and apoptosis following neonatal HI. Studies have been done on the perinatal brain to determine whether apoptotic mechanisms are activated directly by injury. The strongest biochemical evidence for the presence of an intermediate Fcontinuum_ form of cell death comes from Blomgren et al. [6]. They demonstrated that markers for both apoptosis and necrosis are co-expressed in neurons in the injured forebrain at 3 h following neonatal HI in rat. The significance of this finding becomes evident by the demonstration that caspase3 inhibition provides complete blockade of caspase activation but only partial neuroprotection. Caspase-3 inhibition, presumably, does not prevent the necrotic component of cell death induced by HI because it does not prevent the appearance of necrosis specific markers, and, thus, the forebrain is still moderately injured [48]. A disconnect in the degree of caspase-3 activation and severity of injury is also found in the reperfusion model of neonatal stroke developed by Vexler et al. [69]. Their findings show that classic markers for activation of apoptotic pathways fail to coincide with the severity of injury thus suggesting a prominent role for necrosis. The idea that a single stimulus can precipitate dual modes of cell death is also found in in vitro studies. Components of active cell death programs include the release of cytochrome c and the cleavage of caspase-9 and caspase-3 following chemical hypoxia in hippocampal dentate gyrus neurons dying with a necrotic morphology [86]. Cell killing via activation of Fas death receptor pathways also reveals that cells are capable of simultaneously activating both apoptotic and necrotic mechanisms [78,119]. Caspase inhibition allows accumulation of reactive oxygen radicals and subsequent necrotic cell death [78], and both caspase inhibition and antioxidants are required to prevent Fas

death receptor mediated cell death [119]. These in vitro findings are of particular importance in neonatal brain injury because activation of Fas death receptor signaling pathways has been demonstrated in both human neonatal and infant brain injury [27,77,79] and in neonatal animal models of HI injury [25,26,87]. Despite the propensity of the neonatal brain to activate programmed cell death mechanisms and the evidence for activation of components of programmed cell death pathways following neonatal HI, apoptosis is not the major cause of acute cell death following a severe HI insult. It is much more likely that the contribution of apoptosis to neonatal neurodegeneration is most significant in the delayed phases of injury following neonatal HI and stroke and in milder degrees of neonatal HI. For this reason and because of the large amount of data demonstrating activation of apoptotic mediators following neonatal HI, we feel it is important to continue the review of current data on apoptosis in perinatal HI brain injury. 1.3. Which cell death pathways are involved, and do these pathways provide rational opportunities for mechanism-based interventions? There is evidence for involvement of multiple caspases in perinatal HI brain injury. The biochemistry of pathways leading to apoptosis, especially those involving caspase activation, has been extensively reviewed [11,81,101] and will be defined only briefly here. The intrinsic apoptotic pathway is driven by the formation of oligomeric channels composed of Bax or Bak and the permeability transition pore. These channels permit release of cytochrome c that subsequently participates in the formation of the apoptosome (Fig. 2). Alternatively, the extrinsic pathway is driven by activation of plasma membrane death receptors and activation of caspase-8. Both pathways converge on caspase-3. There is abundant evidence that pathways leading to caspase-3 cleavage and activation are engaged following neonatal HI [6,26,52,87]. Interestingly, both in vivo and in vitro studies show that the extent of caspase-3 cleavage and activation following brain injury appears to be maximal in the neonatal period, declining with maturation [52,64]. Further evidence for the involvement of both intrinsic (mitochondrial) and extrinsic (death receptor) apoptosis pathways in perinatal brain injury is accumulating with the use of caspase inhibitors in the setting of neonatal HI. Neuroprotection with non-specific caspase inhibition was first demonstrated with intracerebroventricular injection of a pan caspase inhibitor and intraperitoneal injection of a serine protease inhibitor 3 h after HI [15,30]. Subsequent studies have shown 30 – 50% decreases in tissue loss following neonatal HI with non-selective inhibitors of caspase-8 and caspase-9 [28,29,48]. In these studies, neuropathological examination of the brains was performed up to 15 days after injury and administration of the caspase inhibitors.

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Fig. 2. Mitochondrial regulation of apoptosis. The intrinsic cell death signaling pathway is regulated by mitochondria and involves Bcl-2 family members, cytochrome c release, and apoptosome formation. Bcl-2 family members regulate apoptosis by modulating the release of cytochrome c. Bax and Bak are proapoptotic. They physically interact and form channels that are permeable to cytochrome c. BH3-only members (Bad, Bid, and others not shown) are proapoptotic and can sequester antiapoptotic proteins to allow conformational changes in Bax or Bak. Functional antagonism of Bax and Bak could provide protection against neonatal HI brain damage. Bcl-2 and Bcl-XL are antiapoptotic and can block the function of Bax/Bak. Mimicking the actions of negative regulators could also protect neurons. In the cytosol, cytochrome c, Apaf1, and procaspase-9 interact to form the apoptosome that drives the activation of caspase-3. Caspases are pursued as important targets for neuroprotection in neonatal HI brain injury.

The lack of selectivity of the drugs used for caspase inhibition in most studies prevents an accurate assessment of the role of apoptosis in the resulting brain injury. The calcium-activated, neutral, cytosolic cysteine proteases, known as calpains, are highly activated following neonatal HI [6,91]. Cathepsins, cysteine proteases concentrated in the lysosomal compartment, are also likely to be activated based on electron microscopy evidence of lysosomal and vacuolar changes found following neonatal HI [76]. Importantly, the class of irreversible tetra-peptide caspase inhibitors covalently coupled to chloromethylketone, fluoromethylketone, or aldehydes efficiently inhibits these other classes of cysteine proteases [102]. The efficacy of Z-DEVD-fmk, a widely used caspase inhibitor, to potently inhibit calpain I activity has been demonstrated both in vivo and in vitro [59]. Fortunately, more potent, selective, and reversible nonpeptide caspase-3 inhibitors are now being developed [49]. One of these newer drugs, M826, has been shown to decrease injury following neonatal HI [48], but not nearly as

robustly as the initial reports using non-selective pan caspase inhibition [15]. Conclusions drawn based on the use of non-selective caspase inhibitors must be reevaluated; however, use of combinations of more selective cysteine protease inhibitors may provide valuable information about the overlap of necrotic and apoptotic cell death following neonatal HI. The role of Bcl-2 family proteins in regulating apoptosis is increasingly understood in normal brain development and neonatal brain injury. The baseline expression of most Bcl-2 proteins in the brain varies greatly from E19 to postnatal week 96 in the rat [103]. Importantly, expression of several of the proapoptotic Bcl-2 family proteins is most abundant in the first 2 postnatal weeks [103]. Specifically, immature mitochondria may be Fprimed_ for apoptosis by significant amounts of proapoptosis Bax resident within the mitochondrial fraction even in naı¨ve brain (Fig. 2). As the normal brain matures, Bax levels in the mitochondrial fraction change from high to low [66]. Several studies have shown

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significant alterations in the balance of pro- and antiapoptosis Bcl-2 family protein expression following neonatal hypoxia [19], HI [87], and excitotoxicity [90]. In all cases, expression of proapoptosis Bax is markedly enhanced in relationship to expression of antiapoptosis Bcl-2 or Bcl-XL following the injury. Studies in bax / mice suggest that the major changes in relative Bax protein levels after neonatal HI may have less functional importance than thought originally. Important findings in the bax / mice are that caspase-3 activation still occurs in some dying hippocampal neurons, despite the lack of Bax protein, and caspase-8 activation is not affected by lack of Bax protein [39]. Additionally, although Bax deletion rescues neurons from axotomy-induced apoptosis, the neurons are structurally abnormal, and there is no evidence that they function properly ([106], Martin et al. unpublished observations). These data are consistent with the finding that absence of Bax protein affords partial, only modest neuroprotection in hippocampus and no protection in cerebral cortex in bax / mice exposed to HI at p7 and evaluated at p14 [39]. The role of the Bax homolog Bak needs to be investigated in neonatal HI. Cytokines and death receptor activation likely participate in both brain injury and recovery following perinatal HI and stroke. The clinical association of inflammation and cytokines in perinatal brain injury has been demonstrated repeatedly in studies of both preterm and term neonates with cerebral palsy [18,34,35]. The levels of death receptors such as Fas death receptor and TNFR1 are increased following perinatal HI in humans [9,26,42,87]. The TNF super-family of receptors, which include both Fas death receptor and TNFR1, may signal for either cell survival or cell death. Thus, their role in the injury or recovery process following perinatal brain injury is likely to be both important and complex. Galasso et al. have demonstrated nicely that depending on the region involved, blockade of TNF can either ameliorate or exacerbate excitotoxic injury in the neonatal rat [37]. Specifically, they show that co-injection of a TNF binding protein with NMDA reduces striatal injury but exacerbates hippocampal damage in the p7 rat. Recently, we have shown that there are significant regional differences in the basal expression of FLICE-inhibitory protein (FLIP) between the severely injured hippocampus and the less vulnerable thalamus in neonatal mice [42]. FLIP is a caspase-8 decoy protein that functions as an endogenous caspase inhibitor by blocking caspase-8 activation when engaged by binding to the Fas death receptor-FADD complex [105]. Examination of regional differences in basal expression of other proteins that interact with death receptor cell death cascades will be important. Whether other pathways associated with HI-induced inflammation, such as the complement cascade [17], participate in apoptotic signaling is unknown at present. There is no doubt that the apoptosis signaling pathways examined to date are only a few of many that must be studied for a complete understanding of the contribution of apoptosis to perinatal HI

brain injury. We are left with the conclusion that extensive examination of these multiple pathways are likely to reveal subtle yet important details that need to be understood to provide effective antiapoptosis therapies for neonatal brain injury. Endoplasmic Reticulum (ER) stress can also initiate apoptosis (Fig. 3). Evidence for initiation of apoptosis via signals from lysosomes, Golgi, and the endoplasmic reticulum has been reviewed and a comprehensive integration of cell death signals discussed [31]. The ER is capable of initiating a cell death program in response to ‘‘stress’’ such as hypoxia, hypoglycemia, perturbations in calcium homeostasis, and beta-amyloid protein [84,129]. These stressors initially cause accumulation of misfolded proteins within the lumen of the ER. The initial response to accumulation of misfolded or unfolded proteins in the lumen of the ER is known as the unfolded protein response and results in increased transcription of genes encoding ER molecular chaperones [81]. Excess unfolded proteins are then translocated from the ER to be degraded in the cytosol. ER-directed cell death programs exist for the purpose of responding to an overwhelming accumulation of misfolded or unfolded proteins [81]. Multiple potential apoptotic pathways stimulated by ER stress have been recently identified along with possible crosstalk mechanisms for communication between the ER and mitochondria [13,81]. Several of these ER-directed cell death programs involve caspase-12 [31,82,83] (Fig. 3). Though the caspase-12 gene exists in human, no functional protein is expressed [32]. It is likely that caspase-4, which is similar to caspase-12 and is resident on the ER and activated by ER stress, serves a similar purposes in humans [50]. The Bcl-2 family of proapoptotic proteins, Bax and Bak, previously associated with mitochondria-directed apoptosis, has now been shown to be localized to the ER and participate in activation of caspase-12 and an alternative apoptotic pathway [133]. One possible mechanism is that ER stress induces conformational changes in Bax and Bak, which then interrupts ER membrane integrity and depletes the ER of Ca2+ [133] producing excess cytosolic Ca2+. Increased cytosolic Ca2+ then triggers activation of calpains, which are cytosolic calcium-activated neutral cysteine endopeptidases. Ca2+-activated calpain is capable of activating caspase-12 [83] (Fig. 3). Increased cytosolic Ca2+ also triggers mitochondrial import of Ca2+ leading to mitochondrial-driven apoptosis [133]. These mechanisms are operative in vitro following oxygen –glucose deprivation [83]. Detailed in vivo work in models of neonatal brain injury has not been done, but electron microscopy shows that the ER is structurally compromised early after excitotoxic and HI injuries [76,94]. Most information demonstrating ER involvement in ischemic brain injury is found in adult models. However, one recent study in the neonatal rat model of HI showed that the potent free radical scavenger edaravone conveyed neuroprotection [128], apparently by attenuating ER dysfunction and caspase-12 activation [95].

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Fig. 3. The endoplasmic reticulum (ER) functions in apoptosis. Under conditions of ER stress, such as events resulting in protein misfolding, Bax and Bak regulate the release of Ca2+ from the ER into the cytosol. Increased cytosolic Ca2+ triggers enhanced Ca2+ import into mitochondria and subsequent mitochondrial dysfunction and release of apoptogenic factors that activate caspase-3. Bcl-2 and Bcl-XL can block the release of Ca2+ from the ER. Procaspase12 is localized to the ER and can be cleaved by calpain into the active form in response to prolonged stress. Activated caspase-12 can in turn activate caspase-9 and caspase-3.

This result suggests involvement of the ER in the orchestration of cell death following perinatal HI and indicates that non-mitochondrial mechanisms should be studied when attempting to limit apoptosis following perinatal brain injury. 1.4. Is the delayed apoptotic neurodegeneration following perinatal HI a form of target deprivation induced cell death? The delayed neurodegeneration following perinatal HI is much more likely to be apoptosis and may possibly be due to target deprivation. It is well established in perinatal HI that there is a period of prolonged cell death, especially following mild-moderate initial injury [38]. Geddes et al. dramatically demonstrated that even though cerebral atrophy is not evident at 2 weeks post-mild HI, atrophy evolves significantly over the next 6 weeks so that by 8 weeks postHI there is no neuropathologic difference in the degree of cerebral atrophy between mildly and severely injured animals [38]. Even in the most severely injured animals, we have shown that subacute progression of injury occurs following the initial appearance of injury [88]. Using silver staining to label injured cellular elements, we find a second significant increase in degeneration in the cortex at 48 h

after HI and at 6 days after HI in the striatum and thalamus indicating that cell death continues during the first week postinsult [88]. In a very similar study, ongoing cell death with an apoptotic phenotype was seen in the cortex and basal ganglia throughout the first 7 days following HI, and this occurred in conjunction with continued expression of the cleaved form of caspase-3 [85]. Based on retrograde labeling studies and examination of injury in remote regions of the brain following perinatal HI, it is most likely that ongoing cell death is secondary to loss of essential trophic support for regions with significant interconnections to the area of primary injury. Olney and colleagues argue that apoptosis occurs only during a second wave of cell death following neonatal HI, as a consequence of target deprivation resulting from loss of synaptic inputs and targets but not as a direct consequence of HI [131]. We have made similar conclusions that apoptosis after HI is triggered by target deprivation [75,87,88]. Specifically, we find that retrograde labeling of cortical neurons prior to neonatal HI, identifies selectively vulnerable ventral basal thalamus neurons that die with an apoptotic phenotype after HI [88]. This degeneration may be triggered by loss of target-derived support. We also find that structures in the brain stem with descending connections from the ipsilateral cortex die with evidence of degenerating processes and an

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apoptotic phenotype [88]. This degeneration may be triggered by loss of afferent-derived support. These hypotheses of connectivity mediated delayed cell death following neonatal HI are consistent with evidence that both nerve growth factor (NGF) and BDNF (brain derived neurotrophic factor), known target-derived retrograde transported neurotrophins or afferent derived neurotrophins [12], provide neuroprotection following neonatal HI [14,51], and that BDNF is protective against spatial memory impairments resulting from neonatal HI [3]. There is a growing consensus that apoptotic cell death is responsible for much of the delayed cell death following perinatal HI and that the responsible mechanisms are related to target deprivation and loss of neurotrophic support. Much work remains to be done in this area, including more detailed dissection of the apoptotic pathways activated during delayed phases of injury, further examination of the possible contribution of death receptors and neurotrophin receptors, and detailed neuropathologic examination of remote brain structures with important interconnections to the sites of primary injury. 1.5. Will neuronal rescue with antiapoptosis therapies restore function? It is unlikely that antiapoptosis therapies alone will be adequate to restore function following perinatal brain injury. The primary reason for this supposition is that necrotic – excitotoxic, or non-apoptotic cell death occurs rapidly and abundantly in most forms of neonatal brain injury due to HI and stroke. Towfighi et al. described a rapid increase in necrotic – exicitotoxic cell death following neonatal HI [113], and this finding has been confirmed repeatedly in the neonatal rodent [54,88] and other species [76]. Focusing too much attention on preventing apoptosis and neglecting the search for efficacious antiexcitotoxic and antioxidant therapies might actually delay the identification of effective therapies for neonatal HI. To date, hypothermia is the only therapy available that seems to interrupt the initial rapid and overwhelming necrotic/excitotoxic process following acute severe neonatal HI in both large and small animals [2,8,44 – 46,80, 108,110,115,130]. Research is currently being done to determine whether hypothermia affects apoptotic cell death following HI and whether it can be combined with antiapoptosis therapies for improved efficacy [1,132]. Evidence that early anticonvulsant therapy combined with delayed hypothermia following HI in the p7 rat provides significant improvement in both function and neuropathology is heartening and may answer some of these questions [65]. Data on pathology and function at late time points following antiapoptosis therapies for perinatal brain injury are very limited. Although some of the injury following perinatal HI and stroke is directly attributable to apoptosis, presently there is a paucity of substantial data to determine

whether antiapoptosis therapies restore neurologic function. Caspase inhibition studies performed thus far have shown biochemical and neuropathologic data that suggest efficacy up to 7 [48] and 22 days following the insult [28,29,48]. However, as pointed out earlier, some of the drugs used were non-selective and likely afforded neuroprotection via a variety of pathways. No longer-term neuropathologic or functional outcome studies have been done with any of the caspase inhibitors in any of the models of neonatal brain injury. BDNF is the one potential antiapoptotic therapy for which there is some data regarding outcome. As noted above, BDNF prevented spatial memory impairments in rats following neonatal HI [3]. These encouraging data are limited by additional evidence that BDNF neuroprotection is highly dependent on developmental stage [14,53]. BDNF actually exacerbates excitotoxic lesions in mice at p0, protects at p5, and is without effect at p10 [53]. Other potential concerns about BDNF as a potential antiapoptotic therapeutic are (a) BDNF causes oxidative neuronal necrosis in mixed cortical cell cultures [58], and (b) pretreatment with BDNF markedly increases cell death caused by oxygen – glucose deprivation and N-methyl-d-aspartate in murine cortical cell cultures [60]. The latter result is a cause for concern given the central role of excitotoxicity in the pathogenesis of brain injury following perinatal HI and stroke. At the very least, although promising, BDNF may only be a useful treatment for HI during a limited period of brain development. A promising potential role for antiapoptosis therapies is to provide a bridge to regenerative therapies. Antiapoptosis therapies may be used as a temporizing measure to keep neurons from dying of target deprivation, while initiating tissue engineering strategies. The capacity of the neonatal brain to respond with enhanced endogenous neurogenesis following neonatal HI is controversial and may depend on timing and severity of insult [19,93,99]. It is uncertain if endogenous neurogenesis can be sufficient to provide brain injury repair and functional improvement. It may be that neural tissue transplants or exogenous stem cells are required for structural repair and recovery sufficient to produce an improved functional outcome [56]. Steps are being taken toward this goal. When CD 34+ umbilical cord blood stem cells were systemically administered 48 h after middle cerebral artery occlusion in an adult mouse model, there was enhanced angiogenesis, neurogenesis, and morphological and functional recovery [107]. Unfortunately, because of the many differences between the brains of developing neonatal and adult animals, extrapolation of these data to the neonatal condition is tenuous. However, grafting studies have shown a benefit in models of neonatal HI. Transplantation of fetal neocortex in neonatal rats with HI brain damage promotes functional recovery [55]. Furthermore, grafted GDNF-secreting cells appear to have profound neuroprotective effects in neonatal rats following HI injury [57].

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Supporting or replacing lost neurons should not be the only goal for neuroregenerative therapies following HI brain injury. Another idea is to support or stimulate endogenous axonal sprouting as a mechanism for structural repair. This idea has its basis in important works in the area of axon growth, cortical connectivity, and synapse reinnervation following experimental cortical ablation in the neonatal period [68,92,121]. In this context, apoptotic inhibitors might be advantageous by preventing the death of target-deprived, potential sprouters. However, it will be critical to ensure beneficial sprouting as opposed to chaotic sprouting. 1.6. What are the possible downsides of interfering with apoptosis in the developing brain? Apoptosis is critical for normal brain development and function. The most dramatic evidence for this is that mice with complete absence of caspases-3 and caspase-9 exhibit severe brain malformation, including marked expansion of the ventricular zone, exencephaly, and ectopic brain growth [61,62]. Recently, this severe phenotype has been shown to be strain dependent because caspase-3 deficient mice backcrossed for 7 – 10 generations on pure C57BL/6J strains grown into adulthood with minimal brain pathology [63]. These experiments only speak to the effects of permanent blockade of caspase activity and are not relevant to the question of whether temporary pharmacologic inhibition of caspases in the setting of acute neonatal brain injury would be deleterious. In the studies of acute caspase inhibition, it is not known whether there are subtle changes in normal brain development or function as a result of the single intracerebroventricular doses of caspase inhibitors [28,29,48]. Transient inactivation may interfere with ongoing, normal non-death related functions of caspases. Because perinatal brain injury occurs in the setting of brain development, it is interesting to note that caspase-3 appears to have non-apoptotic functions which would presumably be interrupted by pharmacologic inhibition. These non-apoptotic functions include modulation of synaptic plasticity via involvement in long-term potentiation [43] and cleavage of AMPA receptor subunits [67], and normal differentiation and migration of neurons to the olfactory bulb [126]. It is unknown whether inhibition of caspase activity in the acute setting of injury impairs similar functions or interferes with axonal sprouting, and whether there are long-term changes in brain structure or function as a result of acute, transient inhibition of caspases. Because of the potential importance of the non-apoptotic functions of caspase-3, we must consider whether it may be more appropriate to block the formation of Fstress-induced_ cleaved caspase-3 following injury with selective inhibitors of caspase-8 and caspase-9, which do not interfere with basal levels of caspase-3 activity.

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2. Summary Is preventing apoptosis in the neonatal brain after HI wise? In addition to the question of whether caspase inhibition is beneficial or deleterious in the setting of neonatal brain injury, we also must question whether preventing apoptosis following target deprivation is desirable. Before searching for therapies to provide support and prevent the delayed death of neurons following neonatal brain injury triggered by loss of their primary targets, we must fully question whether it is desirable to keep these neurons alive. Is there a sound rationale for rescuing a neuron when its target is gone? Could this apoptosis after brain injury be a beneficial process that recapitulates normal development, functioning to eliminate cells and prune connections that are at best non-functional and possibly even harmful? Interfering with this mechanism could have long-term consequences because it allows neurons to be present when they should be eliminated. An important conclusion that can be made is that apoptosis contributes variably to the pathology of perinatal brain injury depending on insult, severity, region, model, species, and time following the injury. Nevertheless, we conclude that apoptosis contributes significantly to delayed cell death following perinatal HI, especially as a mechanism of target deprivation mediated neurodegeneration. The molecular mechanisms are complex but definable. Importantly, there are significant differences in the mechanisms leading to apoptosis in the newborn brain compared to the adult that demand attention as antiapoptosis therapies are developed. It is unlikely that antiapoptosis therapies alone will be efficacious in the treatment of most forms of neonatal brain injury. At present, it is not known whether antiapoptosis therapies restore neuronal function, but this issue can be addressed with the ongoing development of models of perinatal brain injury and the ability to test function and behavior in chronic studies. Accordingly, it may be appropriate to inhibit apoptosis as adjuvant therapy to antinecrosis/antiexcitotoxic strategies to prevent massive target deprivation induced cell death while neuroregenerative therapies are initiated. Ideally, the regenerative therapy will provide necessary structural reconstruction and synaptic plasticity to allow for possible reconnection of the targetdeprived neuron to an appropriate target. Finally, as we delve into the application of antiapoptosis therapies to the developing brain, we bear a unique burden to not interfere with normal brain development and normal compensatory mechanisms in a potentially harmful manner.

Acknowledgments Portions of this work were originally presented as a lecture for the Brain Club at the Pediatric Academic Societies Meeting, May 2, 2004 in San Francisco, CA. Special thanks are due to Drs. Donna Ferriero, Estelle

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Gauda, Declan O’Riordan, Jeffrey Perlman, Edward Lawson and John Kattwinkel, Ms. Debbie Flock, Ms. Ann Sheldon, and Mr. Devin Mack. FJN is supported by NS45059. LJM is supported by AG16282, NS34100, and NS20020.

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