- Email: [email protected]
Apoptosis in substantia nigra following developmental hypoxic-ischemic injury
Neuroscience Vol. 69, No. 3, pp. 893-901, 1995
~
Pergamon
0306-4522(95)00282-0
Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
APOPTOSIS IN SUBSTANTIA N I G R A FOLLOWING DEVELOPMENTAL HYPOXIC-ISCHEMIC INJURY T. F. OO, C. H E N C H C L I F F E and R. E. B U R K E * Department of Neurology, Columbia University, 710 West 168th Street, Box 67, New York, NY 10032, U.S.A. Abstract--We have previously observed that either hypoxic-ischemic or excitotoxic striatal injury during development is associated with a reduction in the adult number of dopaminergic neurons in the substantia nigra. This decrease occurs in the presence of preserved striatal dopaminergic markers and in the absence of direct nigral injury. We have also observed that natural cell death, with the morphology of apoptosis, occurs in the substantia nigra, and that there is an induced apoptotic cell death event following early striatal excitotoxic injury. We now report that forebrain hypoxic-ischemic injury is also associated with an induced cell death event in the substantia nigra, with both morphological and histochemical features of apoptosis. Induced apoptotic cell death occurs in immunohistochemically defined dopaminergic neurons. While the mechanisms for this induced cell death are not yet known, in the case of the pars compacta it may be related to the loss of normal striatal target-derived developmental support. Since dopaminergic neurons are postmitotic at the time of the injury, we conclude that this induced cell death is responsible for the diminished adult number of dopaminergic neurons. We also conclude that hypoxi~ischemic injury to the developing brain in general causes not only direct, necrotic injury to vulnerable regions, but also induced apoptotic death at remote sites. The significance of this finding is that apoptosis is a distinct death mechanism, with unique regulatory pathways, which can potentially be modified by approaches different from those which might influence cell death in regions of direct injury. In view of the present finding that apoptosis can occur in the setting of hypoxic-ischemic injury, and our previous work demonstrating its occurrence following excitotoxic injury, it seems likely that it may occur following other forms of injury to the immature brain in which excitotoxic injury plays a role, such as seizures, head trauma and hypoglycemia. Key words: programmed cell death, striatum, dopaminergic.
The striatum is particularly vulnerable to developmental hypoxic-ischemic injury. ~5 Given the major role that the striatum plays in m o t o r control, its injury is likely to underlie the disturbances of m o t o r function, such as dystonia, which are observed in the static encephalopathies of childhood due to hypoxic-ischemic injury. 17 We have therefore studied the alterations in the neurochemical anatomy and function of the nigrostriatal pathways following early hypoxic-ischemic injury. In view of the important role that dopaminergic systems play in m o t o r control, they have been of particular interest. We and others have shown in a unilateral rodent model of developmental hypoxic-ischemic injury that striatal markers of dopaminergic systems are relatively preserved. Previously, Johnston is had shown that at two weeks following hypoxic-ischemic injury at postnatal day 7, measures of tyrosine hydroxylase (TH) and dopamine re-uptake were normal in the striatum. Similarly, we found that injury induced no
change in striatal levels of dopamine or its metabolites, or in levels of [3H]mazindol binding, a specific ligand for dopamine re-uptake sites. 27 Morphological studies revealed a relative increase in the striatal abundance of dopaminergic fibers demonstrated by immunoperoxidase staining for T H ) Dopaminergic terminals also appeared to have fully intact T H activity in vivo. 5 In spite of this relative preservation of striatal dopaminergic measures, the development of the substantia nigra (SN) was not normal. Following early striatal hypoxic-ischemic injury, there was a reduction in the adult number of SN pars compacta (SNpc) dopaminergic neurons, and a decrease in its area. 4 These reductions occurred in the absence of apparent direct hypoxic-ischemic injury to the SN; 4 there was, in fact, complete preservation of the normal cytoarchitectonic features of the SN. We have also observed a similar reduction in the adult number of dopaminergic neurons following a developmental axon-sparing excitotoxic lesion to the striatum, again in the absence of any apparent direct injury to the SN. 4 TO explain these observations, we have hypothesized that the SNpc may depend on its target, the striatum, for trophic support during development,
*To whom correspondence should be addressed. Abbreviations: PB, phosphate buffer; PF, paraformalde-
hyde; PBS, phosphate-buffered saline; SN, substantia nigra; SNpc, SN pars compacta; SNpr, SN pars reticulata; TH, tyrosine hydroxylase. 893
894
T . F . Oo et al.
a n d f o l l o w i n g s t r i a t a l i n j u r y d i m i n i s h e d s u p p o r t res u l t s in i n d u c e d cell d e a t h in t h e S N p c . S u c h a sequence of events has been described for a number of developing neural systems with peripheral targets. 2 In s u p p o r t o f this h y p o t h e s i s , we h a v e f o u n d t h a t n a t u r a l cell d e a t h , w i t h t h e m o r p h o l o g y o f a p o p t o s i s , o c c u r s in t h e S N p c , ~4 a n d i n d u c e d cell d e a t h d o e s o c c u r in t h e S N f o l l o w i n g s t r i a t a l e x c i t o t o x i c i n j u r y . 2t W e h a v e s o u g h t h e r e to d e t e r m i n e w h e t h e r a s i m i l a r cell d e a t h e v e n t o c c u r s in t h e S N f o l l o w i n g h y p o x i c ischemic forebrain injury.
EXPERIMENTAL PROCEDURES
Unilateral hypoxia-ischemia Female rats (Sprague Dawley) were 14-16 days pregnant on arrival from Charles River Laboratories (Wilmington, MA). The day of birth was defined as postnatal day 1. On day 7, the left carotid artery of the neonates was ligated with 6-0 silk suture. After surgery, the neonates were returned to the d a m for 1 2 h and then exposed to humidified 8% oxygen at 37°C for 3 . 5 4 h, during which time they were kept in a plastic bell jar immersed in a water bath at 37°C, as described by Rice et alfl 8 This procedure was approved by the Institutional Animal Care and Use Committee of Columbia University. For surgical control, neonates underwent a s h a m ligation (sham control) in which the carotid was manipulated but not ligated, and then the animals were exposed to hypoxia. This procedure does not induce tissue damage in the brain. Silver staining To demonstrate cell death, and to characterize its morphology, representative sections from the SN and striatum were silver stained using the technique described by Gallyas et al. II At selected time points following hypoxia ischemia, rats were anesthetized with Metofane by inhalation and then perfused through the left ventricle with 0.9% saline at 4°C, followed by perfusion with 4% paraformaldehyde (PF) in 0.1 M phosphate buffer (PB; pH 7.2) for 20 min. Brains were carefully removed from the skull and postfixed in the same fixative for at least one week. Blocks were then cryoprotected by immersing in 20% sucrose/4% PF/0.1 M PB overnight. Blocks were rapidly frozen in 2-methylbutane chilled on dry ice, and sectioned in a cryostat at 3 0 p m . Striatal sections comparable to adult planes 10.2, 9.7, 9.2 and 8.7 m m of the Paxinos and Watson atlas 26 (interaural line coordinates) and SN sections representative of planes 4.2, 3.7 and 3.2 m m were obtained and processed for both Nissl and silver staining. For silver staining sections were maintained in serial order and processed free-floating in custom-made plastic grids with nylon mesh bottoms. Sections were collected into cold fixative, and then washed three times in distilled water. Sections were then immersed in pretreatment solution (equal volumes of 9% N a O H and 1.2% NH4NO3) twice for 5 min. They were then immersed in impregnating solution (60ml 9% N a O H , 4 0 m l 16% N H 4 N O 3, 0.5 ml 50% AgNO3) for 10 minutes. Sections were then washed three times in washing solution (1.0 ml of 1.2% N H 4 N O 3 added to 100ml of a solution containing 5.0 g anhydrous Na2CO3, 300 ml 95% ethanol, brought to 1.0 1 with distilled water), followed by immersion in developing solution (1.0 ml 1.2% N H a N O 3 and 100 ml of a solution consisting of 0.5g citric acid in 15.0ml 37% formalin, 100 ml 95% ethanol, 700 ml water brought to p H 5.8~5.1 with 9% N a O H , and finally brought to 1.01 with water). Sections were kept in developing solution for > 1 min. Sections were then m o u n t e d on subbed slides, air dried and immersed in 0.5% acetic acid three times for 10 min each.
Sections were then dehydrated through alcohols, cleared in xylene and coverslipped under Permount. In situ end-labeling Concurrent with compaction of the nuclear chromatin observed morphologically in apoptosis, cleavage of doublestranded genomic D N A occurs between nucleosomes, resulting in a characteristic "ladder" appearance of integral multiples of 185-200 bp fragments on D N A gels. 36 More recently, it has become possible to demonstrate the resulting formation of numerous free 3'-ends using an in situ approach.t2,35 For Y-end-labeling, neonates were perfused with normal saline, followed by 4% PF in 0.1 M phosphatebuffered saline (PBS; pH 7.4), and then the brains were postfixed overnight in fixative at 4°C. The brain was cryoprotected with 20% sucrose/0.1 M PB overnight, sectioned in a cryostat at 1 4 # m , thaw-mounted onto subbed slides and stored at - 8 0 ° C until analysis. For end-labeling, slides were thawed, briefly immersion-fixed in 4% PF and rinsed in PBS. Sections were treated with 0.1% pepsin in 0.01 N HCI for 60 min. After another rinse, sections were incubated with terminal deoxynucleotidyl transferase in the presence of digoxigenin-dUTP (Apoptag, Oncor) following the supplier's instructions. Sections were rinsed and then incubated with peroxidase-labeled anti-digoxigenin antibodies (ApopTag). Following a rinse, sections were incubated with diaminobenzidine in the presence of H202. Sections were then counterstained with Thionin to define intensely basophilic chromatin clumps, characteristic of apoptosis. It is important to note that identification of the morphological features of apoptosis at a cellular level is critical to the interpretation of peroxidase-labeled free Y-ends, because false positive staining occurs in cells undergoing necrotic death in regions of direct hypoxic-ischemic injury. Control sections with omission of enzyme showed an absence of peroxidase labeling. Tyrosine hydroxylase immunostaining At 48 h following hypoxic-ischemic injury, rat neonates were perfused fixed with 4% PF in 0.1 M PBS, and the brains were then postfixed for two weeks. Cryostat-cut sections (30/~m) were incubated overnight at 4°C with a mouse monoclonal anti-TH antibody (Boehringer Mannheim) at 1:I0 in PBS/10% horse serum, followed by incubations with biotinylated horse anti-mouse immunoglobulin G (Vector) at 1:50 in PBS/10% horse serum, and then with avidin-biotinylated horseradish peroxidase complexes (ABC Kit, Vector) at 1 : 600 at room temperature for l h. Sections were then incubated with diaminobenzidine (Aldrich; 5 0 m g / 1 0 0 m l Tris, pH7.6) in the presence of H202. The number of TH-positive neurons undergoing apoptosis was determined by scanning representative sections at × 600, and counting the number of cells with both TH-positive cytoplasm and dark blue Nissl-stained chromatin clumps, characteristic of apoptosis. ~4 Quantitative morphological analysis Naturally occurring cell death with the morphology of apoptosis is present in the SN at this developmental age, so demonstration of an alteration in levels of cell death requires quantitative analysis. Silver-stained sections through the SN were classified by rostrocaudal location: anterior to the medial terminal nucleus and containing the mammillary body (comparable to P a x i n o s - W a t s o n 4.2); containing the medial terminal nucleus (Paxinos Watson 3.7); posterior to the medial terminal nucleus, and containing both third nerve fibers and interpeduncular nucleus (Paxinos Watson 3.2). At least two sections in each plane were counted, and counts for the SNpc and SN pars reticulata (SNpr) were tallied separately, based on the cytoarchitectonic distinctions between these two structures. The mean n u m b e r o f dying cells for each plane was determined, and these means were s u m m e d to provide a measure of the level of cell death for
Apoptosis in the substantia nigra SNpc and SNpr on each side of the brain. For the counts, the entire SN was scanned on each side of the section at x 600. Cells were counted only if they met morphological criteria for apoptosis: one or more rounded, intensely
HI-INDUCED CELL DEATH: SNpc 13 12 O .J "w
o
IO I-
=.
•
HI
[]
SHAM
8
7 4
D. < W O C~
-1
z
-2
I
I
I
I
I
I
I
I
0
I
2
3
4
5
6
7
A
POST-LESION DAY
HI-INDUCED CELL DEATH : SNpr 1514-
"t
12
0. 0 <
•
HI
[]
SHAM
[] 0 a
z_
-1
-2
0
B
I
I
I
1
2
3
4
POST-LESION
I
I
5
6
7
DAY
Fig. 1. Induced apoptotic cell death in the SNpc and SNpr following unilateral hypoxic-ischemic forebrain injury on postnatal day 7. The number of apoptotic cells in the SNpe and SNpr was determined on the experimental (E) and uninjured control (C) sides for each rat, as described in Experimental Procedures. Data for each rat on each day are expressed as number of dying cells on the experimental side in excess of the number observed on the control side ( E - C), because at this developmental age, there was a progressively changing basal level of naturally occurring cell deathJ 4 At 24 h following hypoxia-ischemia, there was a significant elevation in levels of cell death in the SNpc (P < 0.01) and SNpr (P < 0.05). By 48 h, there was a trend for elevated levels to persist in the SNpc, but the difference was not significant. However, in the SNpr a significant difference persists (P < 0.01). Four neonates were examined at postlesion day 2 for hypoxic-ischemic and sham; five at days 0, l, 4-7; six at day 3 (total N = 39). *P <0.05; **P < 0.01.
895
silver-stained chromatin clumps, with a surrounding, rounded cytoplasm. RESULTS
Cell death was induced in the SN at 24 h following unilateral forebrain hypoxic-ischemic injury, as shown in Fig. 1. At 24 h, the mean prevalence of apoptotic cells in injured SNpc was 13.3 + 1.1, in comparison to 7.5 + 0 . 8 on the non-injured side ( P = 0.007, paired t-test). At 48 h, while there is a trend for the prevalence of dying cells to remain elevated, the mean number of apoptotic profiles (6.7 + 1.7) was not significantly different from that on the contralateral uninjured side (3.0 + 1.4) or in sham-operated controls (left: 3.0 + 0.9; right: 3.5 + 0.3). By the third postlesion day, the number of dying cells on the injured side had returned to basal levels. While the increased prevalence of dying cells at 24 h postlesion was not large in absolute terms, with only a mean increase of six cells per SNpc among representative sections, it is important to note that the duration of apoptotic cell death is very brief, 25 indicating that only a small fraction of cells ultimately destined to die are identifiable as dying at any given time. We have, in fact, previously shown that unilateral hypoxic-ischemic injury as performed in this study can be associated with up to a 20% loss of SNpc dopaminergic neurons. 4 In the SNpr, as in the SNpc, at 24 h there was a significant increase in the prevalence of dying cells on the injured side (19.1 + 3.0) in comparison to the non-injured side (13.0 + 1.8) (P < 0.05, t-test), but in relative terms this increase was less than that observed in the SNpc, where a 77% increase was observed. However, in the SNpr, the induction of cell death lasted longer than it did in the SNpc (Fig. l). At 48 h, in the SNpr, there was a significant, persistent increase in the prevalence of apoptotic cells (16.3 + 3.2) in comparison to the contralateral control (6.9 + 1.0) and both the left ( 2 . 4 + 0.6) and right ( 1 . 5 + 0 . 3 ) sides of sham-operated controls (P = 0.004, one-way A N O V A on ranks). While the SNpc showed no evidence of induced cell death on the uninjured side in comparison to sham-operated controls, the SNpr did show a significant elevation in apoptotic profiles on the uninjured side in relation to sham controls (P < 0.05, post hoc analysis). This effect would suggest that the SNpr contralateral to the side of injury participated to some degree in the induced death event following forebrain ischemia, and that data based on a strict comparison between the injured and non-injured sides, as presented in Fig. 1, while controlling for inter-animal variability, may none the less underestimate the magnitude of induced death on the injured side in absolute terms. By postlesion day 4, the prevalence of cell death in the S N p r had fallen to basal levels. We previously observed a more pronounced decrease in dopaminergic neurons in the rostral portion of the SNpc following developmental hypoxic-
896
T . F . Oo et al.
Fig. 2. A typical silver-stained apoptotic cell in the SNpr at 48 h following forebrain hypoxia-ischemia. On silver stain, apoptotic cells are characterized by the presence of one or more intensely argyrophilic chromatin clumps, surrounded by a less dark silver-impregnated nucleo- or cytoplasm, as shown, The numerous punctate silver deposits in the surrounding tissue are degenerating terminals within the SNpr following direct striatal hypoxic-ischemic injury. Scale bar = 10/~m. ischemic injury. 4 However, an analysis of rostral, central and caudal planes of the SNpc and SNpr revealed no significant differences in the levels of induced cell death. The morphological appearance of the dying cells in both the SNpc and S N p r was typical of that of apoptosis, as demonstrated by silver stain, shown in Fig. 2. These argyrophilic cells typically show a rounded appearance, with multiple, rounded, intensely stained, sharply demarcated chromatin clumps. This appearance is identical to that observed during natural cell death in the SN, ~4 and during cell death induced in the SN following developmental striatal excitotoxic injury. 2~ In the latter setting, we have demonstrated that this morphology indicative of apoptosis at the light microscope level is, in fact,
associated with additional features of apoptosis at the ultrastructural level, including formation of electrondense chromatin clumps, preservation of intracellular organelles (such as mitochondria) and the formation of apoptotic bodies. 2~ Sloviter and colleagues 32'33have likewise previously demonstrated a correspondence between this apoptotic morphology as demonstrated by silver staining, and definitive ultrastructural features of apoptosis. Thus, we conclude that the morphology of induced cell death in both the SNpc and SNpr meets criteria for apoptosis. To further confirm the apoptotic nature of the induced cell death in the SNpc and SNpr, we performed & s i t u 3'-end-labeling to demonstrate the presence of free 3'-ends generated by endonuclease cleavage of genomic D N A , characteristic of apoptosis. 12,35F o r this analysis, we performed Nissl counterstaining after the end-labeling reaction to demonstrate, in addition to the 3'-end-labeling, the intensely basophilic chromatin staining characteristic of apoptosis. 14 Demonstration of this morphological criterion is essential for interpretation of the endlabeling reaction because necrotic cell death can be associated with false-positive staining ~° (and unpublished observations). Figure 3 demonstrates two typical cells in the SN meeting both morphological (chromatin clumping) and histochemical criteria (positive 3'-end-labeling) indicative of apoptosis. A quantitative analysis of the prevalence of these cells in the SN at a postlesion interval of two days following hypoxic-ischemic forebrain injury demonstrated a marked increase on the injured side (28.2 + 3.7 cells) in comparison to the contralateral, uninjured side (3.3 + 1.3, N = 4, P = 0.007). Thus, an induction of apoptotic cell death was confirmed by the analysis of cells meeting both morphological and histochemical criteria for apoptosis. We have shown previously that developmental hypoxic-ischemic injury to the striatum is associated with a diminished adult number of dopaminergic neurons in the SNpc. 4 We therefore wished to determine whether the induced apoptotic cell death in the SNpc occurred within phenotypically-defined dopaminergic neurons, such that it could account for a
Fig. 3. (A) An in situ 3'-end-labeled cell in the SN at 48 h following forebrain hypoxia-ischemia. Intensely basophilic apoptotic chromatin clumps are observed within the nucleus (small arrows). Brown peroxidase reaction product within the nucleus (large arrow) indicates the presence of free 3'-ends. This dying cell had the morphological characteristics of a neuron, with an abundant basophilic cytoplasm and tapered dendritic extensions, out of the plane of focus. (B) A second 3'-end labeled cell in the SN at 48 h. Three characteristic chromatin clumps are observed. These clumps are both Nissl-stained and heavily peroxidaselabeled, giving a dark brown appearance. The surrounding nucleus is also peroxidase-labeled. Scale bar = 10 #m. Fig. 4. (A) TH imunostaining with Nissl counterstain reveals that some cells in the SNpc undergoing apoptosis are dopaminergic. One cell (no. 1, arrow) shows a single basophilic chromatin clump. Normal TH-immunostained neurons do not show such intranuclear staining (no. 2, arrow). Another TH-positive neuron (no. 3, arrow) contains three chromatin clumps (arrowheads), some of which are slightly out of the plane of focus. One cell undergoing apoptosis is TH-negative (no. 4, arrow). (B) An additional example of a dopaminergic neuron undergoing apoptosis following forebrain hypoxic~ischemic injury. Scale bar = I0 #m.
Apoptosis in the substantia nigra
Figs 3 and 4.
897
898
T.F. Oo
decrement in the number of these neurons surviving into maturity. To perform this analysis, we demonstrated dopaminergic neurons within the SNpc by immunoperoxidase staining of TH, followed by Nissl counterstain to identify apoptotic chromatin clumps, as described previously) 1 This double-staining procedure identified TH-positive neurons undergoing apoptosis, as shown in Fig. 4, on the lesioned side. In four pups killed at two days postlesion, a mean of 6.3 + 1.0 such cells were identified per nigra on the injured side, based on screening eight sections representative of four rostrocaudal planes. No TH-positive cells with Nissl-defined chromatin clumps were observed on the unlesioned side. Within each SNpc only approximately 10% of Nissl-defined apoptotic profiles were immunoperoxidase-stained positive for TH. While there are several possible reasons for staining only a small portion of these cells, as discussed below, one major possible reason is limited penetration of antibodies and other reagents into these 30-pm-thick sections. Nevertheless, it is clear that some of the cells undergoing induced apoptotic death in the SNpc are of the dopaminergic phenotype. DISCUSSION We have previously observed that excitotoxic injury to the developing striatum is associated with an induced cell death event in the SN, with morphology of apoptosis. 21 This induced death event could be distinguished from the direct cell death caused by the excitotoxic striatal injury on the basis of its location remote from the site of direct injury, and its delay in onset. In addition, the distinct cell death morphology of apoptosis observed in the SN differed from the necrotic pattern observed at the striatal site of direct injury. We now report that hypoxic-ischemic injury to the forebrain is also associated with an induced death event in the SN, relatively remote from the major sites of direct injury, which include not only striatum, but also cortex and hippocampus. 28 This model of brain injury is not only more global than the excitotoxin model, but also more variable. Therefore, some animals subjected to this paradigm do show extensive injury to the affected hemisphere, with injury extending into the midbrain. However, extensive injury in such animals can be identified both by inspection, which reveals brain cavitation, and histological analysis by Nissl and silver stains, which reveal large confluent areas of neuron loss with the morphology of necrotic cell death in regions of direct injury. Taking care to exclude direct injury to the SN, we have found quantitative evidence of induced cell death on the side of hypoxic-ischemic injury. As in the case of induced death following excitotoxic striatal injury, the induced death in the SN following hypoxic-ischemic injury can be distinguished from direct injury on the basis of the regional and cellular characteristics of the death process. Regionally, cells
et al.
in the SN die as single, isolated cells, as shown in Fig. 1. This pattern is characteristic of apoptosis. 3° In addition, at a cellular level the dying cells in the SN are defined by both morphological and biochemical criteria to be universally apoptotic. In regions of direct injury, such as the striatum, while occasional cells may appear apoptotic by morphological criteria, the vast majority, in the acute phase of injury, appear necrotic by histological stains23'2s and silver staining (unpublished observation). The morphological criteria which we used to identify the pattern of cell death as apoptotic included positive silver staining, with rounding and shrinkage of the cell, in the presence of one or more rounded, intensely argyrophilic chromatin clumps. This morphological pattern of cell death was identical to that observed during natural cell death within the SNpc 14 and during induced cell death following striatal excitotoxic injury.21 In the latter setting, definitive ultrastructural evidence of apoptosis was confirmed. Likewise, Sloviter and colleagues32'33 had previously found that an apoptotic appearance on silver stain is associated with definitive ultrastructural features of apoptosis. The pattern of cell death induced in the SN was further confirmed to be apoptotic by the demonstration of a positive 3'-end-labeling reaction 12'35 in conjunction with a morphological demonstration of apoptotic chromatin clumping by Nissl counterstain. Thus, we conclude that the induced cell death in the SN is universally apoptotic, and in this respect, as well as its regional characteristics, it can be distinguished from the predominant form of necrotic cell death, which occurs in the forebrain as a direct result of hypoxic-ischemic injury. This induced cell death event in the SNpc is likely to account for the reduction in dopaminergic neurons in the adult brain observed following developmental forebrain hypoxic-ischemic injury.4 We have shown here that induced apoptotic cell death is observed in phenotypically-defined SNpc dopaminergic neurons. Since these neurons are postmitotic at this developmental age, 18'z2this cell death event in this population will result in a diminished number of these neurons surviving into adulthood. We found that very few (10%) of the apoptotic profiles in the SNpc were TH-positive. It is possible that the relatively large number of unstained neurons is due to limited penetration of immunoreagents into the section or cell compartment. Although it is also possible that some of these cells are non-dopaminergic, such an explanation seems unlikely to account for the considerable number of these cells in the SNpc. It is also possible that at later stages of apoptotic cell death, expression of phenotypic markers is so minimal that immunostaining is no longer possible. While the present study has identified an association between direct hypoxic-ischemic forebrain injury and an induced apoptotic death event in the SN, the mechanism(s) underlying this relationship is (are)
899
Apoptosis in the substantia nigra unknown. Given the global nature of the hypoxicischemic injury in this model, it will be difficult to define the precise anatomical basis. However, several considerations lead us to favor a pivotal role for striatal injury. The striatum is especially vulnerable to hypoxic-ischemic injury. ~5'28The striatum has major anatomical relationships with the SN; the SNpc provides the major dopaminergic afferent projection to the striatum; the SNpr receives a major GABAergic and substance P pathway from the striatum, l In addition, we know from our previous work that a selective striatal excitotoxic lesion is sufficient to cause a comparable degree of induced cell death in the SN. In relation to the SNpc, because the striatum is a major target, it is possible that hypoxic-ischemic injury results in a loss of retrogradely-derived trophic support, with a resulting induced apoptotic death, as described for many other systems.2 It seems unlikely that the induced death in the SNpc is due to injury of dopaminergic terminals with a resulting retrograde degeneration, because we and others have shown that markers of dopaminergic terminals are relatively spared in this model. 3'5'15'27Nevertheless, it remains possible that loss of a subset of dopaminergic terminals in absolute terms was not detected by the prior biochemical and morphological assessments. It is also possible that SNpc neuron death is due to a transneuronal effect, following death of SNpr neurons. However, the peak of cell death in the SNpc appears to be concurrent with or possibly precede that in the SNpr. The time course of induced cell death in the SN following hypoxic-ischemic injury is similar to that observed following focal excitotoxic striatal injury,21 lending support to the concept that the mechanism of induced death may be similar in the two paradigms. As for the SNpc, the mechanism for induced cell death in the SNpr is unknown. The striatum sends major afferent projections to the SNpr, and there are precedents for induction of cell death due to loss of afferent input in the developmental setting. 19 There is also a precedent for induced cell death in the SNpr following striatal injury in adult injury on the basis of a loss of GABAergic inhibitory input. 29 The precise mechanism will require further clarification. On the basis of the hypothesis that direct injury to the brain may result in induced cell death in other, remote regions as a consequence of diminished developmental support, it seems possible that cell death may occur in other structures with important projections to the striatum, such as the cortex and the intralaminar nuclei of the thalamus. 6 However, identification of such events is made more difficult by the direct involvement of these structures in hypoxicischemic injury. Our focus on the SN in this study was facilitated by the greater availability of information about effects on the dopaminergic system in this model,3 5,27and by our prior findings referable to the SN following striatal excitotoxic injury.21
We have not addressed in this study whether apoptosis may occur in those regions of the brain directly injured by hypoxia-ischemia. A number of investigators have previously shown that apoptosis may occur in such regions. In models of transient global ischemia and focal ischemia in adult rats and gerbils, internucleosomal fragmentation of DNA, causing a characteristic 185-200 bp "ladder" appearance on DNA gels, has been identified in extracts from striatal or hippocampal tissue and interpreted as evidence of apoptosis. 13'2°'24'31'34 In some of these studies, j3'j6,31 in situ end-labeling has confirmed the presence of Y-end-labeling, particularly in the CA1 region of the hippocampus, where delayed neuronal death is known to occur. However, the presence of internucleosomal fragmentation alone does not always correlate with the definitive morphological features of apoptosis. 8 In addition, the presence of 3'-end-labeling alone, in the absence of definitive morphological criteria for apoptosis, must be interpreted with great caution, because this technique will label free Y-ends generated by any process of DNA breakdown. In this regard, it is important to note that in an extensive ultrastructural analysis of the CA1 region of the hippocampus at 6-72 h following ischemia, Deshpande and colleagues9 were unable to identify any apoptotic morphology. Ferrer and coworkers l° examined the brains of immature rats following hypoxic-ischemic injury using the in situ end-labeling technique, and identified morphologies indicative of necrotic cell death, but they did not explicitly identify apoptosis. Some of their figures, however, seem to suggest the presence of apoptosis. Thus, it remains uncertain whether apoptosis, as defined by morphological criteria as used in this study, occurs in regions of direct injury. CONCLUSIONS
We have shown that, following hypoxic-ischemic injury to the developing brain, apoptosis occurs as a separate and distinct death mechanism in a brain region which is remote from sites of direct hypoxic-ischemic injury. One important implication of this finding is that this distinct form of cell death has its own unique regulatory pathways, and thus can potentially be modified by methods quite different from those which might influence cell death in regions of direct injury, where an abundance of data has implicated excitotoxic mechanisms as playing a predominant role. 7 It must be understood, however, that at the present time we do not know the functional consequences of this induced cell death. It is conceivable that it is not, in fact, deleterious, but plays a homeostatic role in regulating structural relationships between brain regions. It is likely that the phenomenon of induced apoptotic cell death plays a wide role in the response of the immature brain to injury. Given that induced death is observed after both excitotoxiczl and hypoxic-ischemic injury, it seems likely that it
900
T . F . Oo et al.
may occur following other forms o f injury to the i m m a t u r e brain in which excitotoxic injury is believed to play a role, such as seizures, head t r a u m a and hypoglycemia. 7
Acknowledgements--The authors are White for diligent preparation of the also grateful to Mr William Kelly for This work was supported by NS26836 Disease Foundation.
grateful to Ms Pat manuscript. We are technical assistance. and the Parkinson's
REFERENCES 1. Albin R. L., Young A. B. and Penney J. B. (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci. 12, 366-375. 2. Barde Y. A. (1989) Trophic factors and neuronal survival. Neuron 2, 1525 1534. 3. Burke R. E., Kent J., Kenyon N. and Karanas A. (1991) Unilateral hypoxi~ischemic injury in neonatal rat results in a persistent increase in the density of striatal tyrosine hydroxylase immunoperoxidase staining. Devl Brain Res. 58, 171 179. 4. Burke R. E., Macaya A., DeVivo D., Kenyon N. and Janec E. M. (1992) Neonatal hypoxic-ischemic or excitotoxic striatal injury results in a decreased adult number of substantia nigra neurons. Neuroscience 50, 559-569. 5. Burke R. E. and Reches A. (1991) Preserved striatal tyrosine hydroxylase activity, assessed in vivo, following neonatal hypoxia-ischemia. Devl Brain Res. 61, 277580. 6. Carpenter M. B. (1984) Interconnections between the corpus striatum and brain stem nuclei. In The Basal Ganglia (eds McKenzie J. S., Kern R. E. and Wilcock L. N.), pp. 1~8. Plenum, Bethesda, MD. 7. Choi D. W. (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623~534. 8. Collins R. J., Harmon B. V., Gobe G. C. and Kerr J. F. R. (1992) Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int. J. Radiat. Biol. 61, 451-453. 9. Deshpande J., Bergstedt K., Linden T., Kalimo H. and Wieloch T. (1992) Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Expl Brain Res. 88, 91-105. 10. 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. 11. Gallyas F., Wolff J. R., Bottcher H. and Zaborsky L. (1980) A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system. Stain Technol. 55, 299-306. 12. Gavrieli Y., Sherman Y. and Ben-Sasson S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493-501. 13. Heron A., Pollard H., Dessi F. et al. (1993) Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain. J. Neurochem. 61, 1973 1976. 14. Janec E. and Burke R. E. (1993) Naturally occurring cell death during postnatal development of the substantia nigra of the rat. Molec. Cell. Neurosci. 4, 30-35. 15. Johnston M. V. (1983) Neurotransmitter alterations in a model of perinatal hypoxic-ischemic brain injury. Ann. Neurol. 13, 511-518. 16. Kihara S.-I., Shiraishi T., Nakagawa S., Toda K. and Tabuchi K. (1994) Visualization of DNA double strand breaks in the gerbil hippocampal CA1 following transient ischemia. Neurosci. Lett. 175, 133-136. 17. Kyllerman M., Bager B., Bensch J., Bille B., Olow 1. and Voss H. (1982) Dyskinetic cerebral palsy. Actapaediat. scand. 71, 543-550. 18. Lauder J. M. and Bloom F. E. (1974) Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. J. comp. Neurol. 155, 469-482. 19. Linden R. (1994) The survival of developing neurons: a review of afferent control. Neuroscience 58, 671~582. 20. Linnik M. D., Zobrist R. H. and Hatfield M. D. (1993) Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 24, 2002-2008. 21. Macaya A., Munell F., Gubits R. M. and Burke R. E. (1994) Apoptosis in substantia nigra following developmental striatal excitotoxic injury. Proc. natn. Acad. Sci. U.S.A. 91, 8117-8121. 22. Marchand R. and Poirer L. J. (1983) Isthmic origin of neurons of the rat substantia nigra. Neuroscience 9, 373-381. 23. McGee-Russell S. M., Brown A. W. and Brierley J. B. (1970) A combined light and electron microscope study of early anoxic-ischaemic cell change in rat brain. Brain Res. 20, 193-200. 24. Okamoto M., Matsumoto M., Ohtsuki T. et al. (1993) Internucleosomal DNA cleavage involved in ischemia induced neuronal death. Biochem. biophys. Res. Commun. 196, 1356-1362. 25. Oppenheim R. W. (1991) Cell death during development of the nervous system. A. Rev. Neurosci. 14, 453-501. 26. Paxinos G. and Watson C. (1982) The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA. 27. Przedborski S., Kostic V., Jackson Lewis V., Cadet J. L. and Burke R. E. (1991) Effect of unilateral perinatal hypoxic-ischemic brain injury in the rat on dopamine D1 and D2 receptors and uptake sites: a quantitative autoradiographic study. J. Neurochem. 57, 1951-1961. 28. Rice J. E., Vannucci R. C. and Brierley J. B. (1981) The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann. Neurol. 9, 131-141. 29. Saji M. and Reis D. J. (1987) Delayed transneuronal death of substantia nigra neurons prevented by gamma-aminobutyric acid agonist. Science 235, 66~8. 30. Searle J., Kerr J. F. R. and Bishop C. J. (1982) Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Path. Ann. 17, 229-259. 31. Sei Y., Von Lubitz D. K. J. E., Basile A. S. et al. (1994) Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia. Neurosci. Lett. 171, 179 182. 32. Sloviter R. S., Dean E. and Neubort S. (1993) Electron microscopic analysis of adrenalectomy induced hippocampal granule cell degeneration in the rat apoptosis in the adult central nervous system. J. comp. Neurol. 330, 337 351. 33. Sloviter R. S., Sollas A. L., Dean E. and Neubort S. (1993) Adrenalectomy induced granule cell degeneration in the rat hippocampal dentate gyrus characterization of an in vivo model of controlled neuronal death. J. comp. Neurol. 330, 324-336. 34. Tominaga T., Kure S., Narisawa K. and Yoshimoto T. (1993) Endonuclease activation following focal ischemic injury in the rat brain. Brain Res. 608, 21-26.
Apoptosis in the substantia nigra
901
35. Wijsman J. H., Jonker R. R., Keijzer R., van de Velde C. J. H., Cornelisse C. J. and van Dierendonck J.-H (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J. Histochem. Cytochem. 41, 7-13. 36. Wyllie A. H., Kerr J. F. and Currie A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306. (Accepted 7 June 1995)
~
Pergamon
0306-4522(95)00282-0
Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
APOPTOSIS IN SUBSTANTIA N I G R A FOLLOWING DEVELOPMENTAL HYPOXIC-ISCHEMIC INJURY T. F. OO, C. H E N C H C L I F F E and R. E. B U R K E * Department of Neurology, Columbia University, 710 West 168th Street, Box 67, New York, NY 10032, U.S.A. Abstract--We have previously observed that either hypoxic-ischemic or excitotoxic striatal injury during development is associated with a reduction in the adult number of dopaminergic neurons in the substantia nigra. This decrease occurs in the presence of preserved striatal dopaminergic markers and in the absence of direct nigral injury. We have also observed that natural cell death, with the morphology of apoptosis, occurs in the substantia nigra, and that there is an induced apoptotic cell death event following early striatal excitotoxic injury. We now report that forebrain hypoxic-ischemic injury is also associated with an induced cell death event in the substantia nigra, with both morphological and histochemical features of apoptosis. Induced apoptotic cell death occurs in immunohistochemically defined dopaminergic neurons. While the mechanisms for this induced cell death are not yet known, in the case of the pars compacta it may be related to the loss of normal striatal target-derived developmental support. Since dopaminergic neurons are postmitotic at the time of the injury, we conclude that this induced cell death is responsible for the diminished adult number of dopaminergic neurons. We also conclude that hypoxi~ischemic injury to the developing brain in general causes not only direct, necrotic injury to vulnerable regions, but also induced apoptotic death at remote sites. The significance of this finding is that apoptosis is a distinct death mechanism, with unique regulatory pathways, which can potentially be modified by approaches different from those which might influence cell death in regions of direct injury. In view of the present finding that apoptosis can occur in the setting of hypoxic-ischemic injury, and our previous work demonstrating its occurrence following excitotoxic injury, it seems likely that it may occur following other forms of injury to the immature brain in which excitotoxic injury plays a role, such as seizures, head trauma and hypoglycemia. Key words: programmed cell death, striatum, dopaminergic.
The striatum is particularly vulnerable to developmental hypoxic-ischemic injury. ~5 Given the major role that the striatum plays in m o t o r control, its injury is likely to underlie the disturbances of m o t o r function, such as dystonia, which are observed in the static encephalopathies of childhood due to hypoxic-ischemic injury. 17 We have therefore studied the alterations in the neurochemical anatomy and function of the nigrostriatal pathways following early hypoxic-ischemic injury. In view of the important role that dopaminergic systems play in m o t o r control, they have been of particular interest. We and others have shown in a unilateral rodent model of developmental hypoxic-ischemic injury that striatal markers of dopaminergic systems are relatively preserved. Previously, Johnston is had shown that at two weeks following hypoxic-ischemic injury at postnatal day 7, measures of tyrosine hydroxylase (TH) and dopamine re-uptake were normal in the striatum. Similarly, we found that injury induced no
change in striatal levels of dopamine or its metabolites, or in levels of [3H]mazindol binding, a specific ligand for dopamine re-uptake sites. 27 Morphological studies revealed a relative increase in the striatal abundance of dopaminergic fibers demonstrated by immunoperoxidase staining for T H ) Dopaminergic terminals also appeared to have fully intact T H activity in vivo. 5 In spite of this relative preservation of striatal dopaminergic measures, the development of the substantia nigra (SN) was not normal. Following early striatal hypoxic-ischemic injury, there was a reduction in the adult number of SN pars compacta (SNpc) dopaminergic neurons, and a decrease in its area. 4 These reductions occurred in the absence of apparent direct hypoxic-ischemic injury to the SN; 4 there was, in fact, complete preservation of the normal cytoarchitectonic features of the SN. We have also observed a similar reduction in the adult number of dopaminergic neurons following a developmental axon-sparing excitotoxic lesion to the striatum, again in the absence of any apparent direct injury to the SN. 4 TO explain these observations, we have hypothesized that the SNpc may depend on its target, the striatum, for trophic support during development,
*To whom correspondence should be addressed. Abbreviations: PB, phosphate buffer; PF, paraformalde-
hyde; PBS, phosphate-buffered saline; SN, substantia nigra; SNpc, SN pars compacta; SNpr, SN pars reticulata; TH, tyrosine hydroxylase. 893
894
T . F . Oo et al.
a n d f o l l o w i n g s t r i a t a l i n j u r y d i m i n i s h e d s u p p o r t res u l t s in i n d u c e d cell d e a t h in t h e S N p c . S u c h a sequence of events has been described for a number of developing neural systems with peripheral targets. 2 In s u p p o r t o f this h y p o t h e s i s , we h a v e f o u n d t h a t n a t u r a l cell d e a t h , w i t h t h e m o r p h o l o g y o f a p o p t o s i s , o c c u r s in t h e S N p c , ~4 a n d i n d u c e d cell d e a t h d o e s o c c u r in t h e S N f o l l o w i n g s t r i a t a l e x c i t o t o x i c i n j u r y . 2t W e h a v e s o u g h t h e r e to d e t e r m i n e w h e t h e r a s i m i l a r cell d e a t h e v e n t o c c u r s in t h e S N f o l l o w i n g h y p o x i c ischemic forebrain injury.
EXPERIMENTAL PROCEDURES
Unilateral hypoxia-ischemia Female rats (Sprague Dawley) were 14-16 days pregnant on arrival from Charles River Laboratories (Wilmington, MA). The day of birth was defined as postnatal day 1. On day 7, the left carotid artery of the neonates was ligated with 6-0 silk suture. After surgery, the neonates were returned to the d a m for 1 2 h and then exposed to humidified 8% oxygen at 37°C for 3 . 5 4 h, during which time they were kept in a plastic bell jar immersed in a water bath at 37°C, as described by Rice et alfl 8 This procedure was approved by the Institutional Animal Care and Use Committee of Columbia University. For surgical control, neonates underwent a s h a m ligation (sham control) in which the carotid was manipulated but not ligated, and then the animals were exposed to hypoxia. This procedure does not induce tissue damage in the brain. Silver staining To demonstrate cell death, and to characterize its morphology, representative sections from the SN and striatum were silver stained using the technique described by Gallyas et al. II At selected time points following hypoxia ischemia, rats were anesthetized with Metofane by inhalation and then perfused through the left ventricle with 0.9% saline at 4°C, followed by perfusion with 4% paraformaldehyde (PF) in 0.1 M phosphate buffer (PB; pH 7.2) for 20 min. Brains were carefully removed from the skull and postfixed in the same fixative for at least one week. Blocks were then cryoprotected by immersing in 20% sucrose/4% PF/0.1 M PB overnight. Blocks were rapidly frozen in 2-methylbutane chilled on dry ice, and sectioned in a cryostat at 3 0 p m . Striatal sections comparable to adult planes 10.2, 9.7, 9.2 and 8.7 m m of the Paxinos and Watson atlas 26 (interaural line coordinates) and SN sections representative of planes 4.2, 3.7 and 3.2 m m were obtained and processed for both Nissl and silver staining. For silver staining sections were maintained in serial order and processed free-floating in custom-made plastic grids with nylon mesh bottoms. Sections were collected into cold fixative, and then washed three times in distilled water. Sections were then immersed in pretreatment solution (equal volumes of 9% N a O H and 1.2% NH4NO3) twice for 5 min. They were then immersed in impregnating solution (60ml 9% N a O H , 4 0 m l 16% N H 4 N O 3, 0.5 ml 50% AgNO3) for 10 minutes. Sections were then washed three times in washing solution (1.0 ml of 1.2% N H 4 N O 3 added to 100ml of a solution containing 5.0 g anhydrous Na2CO3, 300 ml 95% ethanol, brought to 1.0 1 with distilled water), followed by immersion in developing solution (1.0 ml 1.2% N H a N O 3 and 100 ml of a solution consisting of 0.5g citric acid in 15.0ml 37% formalin, 100 ml 95% ethanol, 700 ml water brought to p H 5.8~5.1 with 9% N a O H , and finally brought to 1.01 with water). Sections were kept in developing solution for > 1 min. Sections were then m o u n t e d on subbed slides, air dried and immersed in 0.5% acetic acid three times for 10 min each.
Sections were then dehydrated through alcohols, cleared in xylene and coverslipped under Permount. In situ end-labeling Concurrent with compaction of the nuclear chromatin observed morphologically in apoptosis, cleavage of doublestranded genomic D N A occurs between nucleosomes, resulting in a characteristic "ladder" appearance of integral multiples of 185-200 bp fragments on D N A gels. 36 More recently, it has become possible to demonstrate the resulting formation of numerous free 3'-ends using an in situ approach.t2,35 For Y-end-labeling, neonates were perfused with normal saline, followed by 4% PF in 0.1 M phosphatebuffered saline (PBS; pH 7.4), and then the brains were postfixed overnight in fixative at 4°C. The brain was cryoprotected with 20% sucrose/0.1 M PB overnight, sectioned in a cryostat at 1 4 # m , thaw-mounted onto subbed slides and stored at - 8 0 ° C until analysis. For end-labeling, slides were thawed, briefly immersion-fixed in 4% PF and rinsed in PBS. Sections were treated with 0.1% pepsin in 0.01 N HCI for 60 min. After another rinse, sections were incubated with terminal deoxynucleotidyl transferase in the presence of digoxigenin-dUTP (Apoptag, Oncor) following the supplier's instructions. Sections were rinsed and then incubated with peroxidase-labeled anti-digoxigenin antibodies (ApopTag). Following a rinse, sections were incubated with diaminobenzidine in the presence of H202. Sections were then counterstained with Thionin to define intensely basophilic chromatin clumps, characteristic of apoptosis. It is important to note that identification of the morphological features of apoptosis at a cellular level is critical to the interpretation of peroxidase-labeled free Y-ends, because false positive staining occurs in cells undergoing necrotic death in regions of direct hypoxic-ischemic injury. Control sections with omission of enzyme showed an absence of peroxidase labeling. Tyrosine hydroxylase immunostaining At 48 h following hypoxic-ischemic injury, rat neonates were perfused fixed with 4% PF in 0.1 M PBS, and the brains were then postfixed for two weeks. Cryostat-cut sections (30/~m) were incubated overnight at 4°C with a mouse monoclonal anti-TH antibody (Boehringer Mannheim) at 1:I0 in PBS/10% horse serum, followed by incubations with biotinylated horse anti-mouse immunoglobulin G (Vector) at 1:50 in PBS/10% horse serum, and then with avidin-biotinylated horseradish peroxidase complexes (ABC Kit, Vector) at 1 : 600 at room temperature for l h. Sections were then incubated with diaminobenzidine (Aldrich; 5 0 m g / 1 0 0 m l Tris, pH7.6) in the presence of H202. The number of TH-positive neurons undergoing apoptosis was determined by scanning representative sections at × 600, and counting the number of cells with both TH-positive cytoplasm and dark blue Nissl-stained chromatin clumps, characteristic of apoptosis. ~4 Quantitative morphological analysis Naturally occurring cell death with the morphology of apoptosis is present in the SN at this developmental age, so demonstration of an alteration in levels of cell death requires quantitative analysis. Silver-stained sections through the SN were classified by rostrocaudal location: anterior to the medial terminal nucleus and containing the mammillary body (comparable to P a x i n o s - W a t s o n 4.2); containing the medial terminal nucleus (Paxinos Watson 3.7); posterior to the medial terminal nucleus, and containing both third nerve fibers and interpeduncular nucleus (Paxinos Watson 3.2). At least two sections in each plane were counted, and counts for the SNpc and SN pars reticulata (SNpr) were tallied separately, based on the cytoarchitectonic distinctions between these two structures. The mean n u m b e r o f dying cells for each plane was determined, and these means were s u m m e d to provide a measure of the level of cell death for
Apoptosis in the substantia nigra SNpc and SNpr on each side of the brain. For the counts, the entire SN was scanned on each side of the section at x 600. Cells were counted only if they met morphological criteria for apoptosis: one or more rounded, intensely
HI-INDUCED CELL DEATH: SNpc 13 12 O .J "w
o
IO I-
=.
•
HI
[]
SHAM
8
7 4
D. < W O C~
-1
z
-2
I
I
I
I
I
I
I
I
0
I
2
3
4
5
6
7
A
POST-LESION DAY
HI-INDUCED CELL DEATH : SNpr 1514-
"t
12
0. 0 <
•
HI
[]
SHAM
[] 0 a
z_
-1
-2
0
B
I
I
I
1
2
3
4
POST-LESION
I
I
5
6
7
DAY
Fig. 1. Induced apoptotic cell death in the SNpc and SNpr following unilateral hypoxic-ischemic forebrain injury on postnatal day 7. The number of apoptotic cells in the SNpe and SNpr was determined on the experimental (E) and uninjured control (C) sides for each rat, as described in Experimental Procedures. Data for each rat on each day are expressed as number of dying cells on the experimental side in excess of the number observed on the control side ( E - C), because at this developmental age, there was a progressively changing basal level of naturally occurring cell deathJ 4 At 24 h following hypoxia-ischemia, there was a significant elevation in levels of cell death in the SNpc (P < 0.01) and SNpr (P < 0.05). By 48 h, there was a trend for elevated levels to persist in the SNpc, but the difference was not significant. However, in the SNpr a significant difference persists (P < 0.01). Four neonates were examined at postlesion day 2 for hypoxic-ischemic and sham; five at days 0, l, 4-7; six at day 3 (total N = 39). *P <0.05; **P < 0.01.
895
silver-stained chromatin clumps, with a surrounding, rounded cytoplasm. RESULTS
Cell death was induced in the SN at 24 h following unilateral forebrain hypoxic-ischemic injury, as shown in Fig. 1. At 24 h, the mean prevalence of apoptotic cells in injured SNpc was 13.3 + 1.1, in comparison to 7.5 + 0 . 8 on the non-injured side ( P = 0.007, paired t-test). At 48 h, while there is a trend for the prevalence of dying cells to remain elevated, the mean number of apoptotic profiles (6.7 + 1.7) was not significantly different from that on the contralateral uninjured side (3.0 + 1.4) or in sham-operated controls (left: 3.0 + 0.9; right: 3.5 + 0.3). By the third postlesion day, the number of dying cells on the injured side had returned to basal levels. While the increased prevalence of dying cells at 24 h postlesion was not large in absolute terms, with only a mean increase of six cells per SNpc among representative sections, it is important to note that the duration of apoptotic cell death is very brief, 25 indicating that only a small fraction of cells ultimately destined to die are identifiable as dying at any given time. We have, in fact, previously shown that unilateral hypoxic-ischemic injury as performed in this study can be associated with up to a 20% loss of SNpc dopaminergic neurons. 4 In the SNpr, as in the SNpc, at 24 h there was a significant increase in the prevalence of dying cells on the injured side (19.1 + 3.0) in comparison to the non-injured side (13.0 + 1.8) (P < 0.05, t-test), but in relative terms this increase was less than that observed in the SNpc, where a 77% increase was observed. However, in the SNpr, the induction of cell death lasted longer than it did in the SNpc (Fig. l). At 48 h, in the SNpr, there was a significant, persistent increase in the prevalence of apoptotic cells (16.3 + 3.2) in comparison to the contralateral control (6.9 + 1.0) and both the left ( 2 . 4 + 0.6) and right ( 1 . 5 + 0 . 3 ) sides of sham-operated controls (P = 0.004, one-way A N O V A on ranks). While the SNpc showed no evidence of induced cell death on the uninjured side in comparison to sham-operated controls, the SNpr did show a significant elevation in apoptotic profiles on the uninjured side in relation to sham controls (P < 0.05, post hoc analysis). This effect would suggest that the SNpr contralateral to the side of injury participated to some degree in the induced death event following forebrain ischemia, and that data based on a strict comparison between the injured and non-injured sides, as presented in Fig. 1, while controlling for inter-animal variability, may none the less underestimate the magnitude of induced death on the injured side in absolute terms. By postlesion day 4, the prevalence of cell death in the S N p r had fallen to basal levels. We previously observed a more pronounced decrease in dopaminergic neurons in the rostral portion of the SNpc following developmental hypoxic-
896
T . F . Oo et al.
Fig. 2. A typical silver-stained apoptotic cell in the SNpr at 48 h following forebrain hypoxia-ischemia. On silver stain, apoptotic cells are characterized by the presence of one or more intensely argyrophilic chromatin clumps, surrounded by a less dark silver-impregnated nucleo- or cytoplasm, as shown, The numerous punctate silver deposits in the surrounding tissue are degenerating terminals within the SNpr following direct striatal hypoxic-ischemic injury. Scale bar = 10/~m. ischemic injury. 4 However, an analysis of rostral, central and caudal planes of the SNpc and SNpr revealed no significant differences in the levels of induced cell death. The morphological appearance of the dying cells in both the SNpc and S N p r was typical of that of apoptosis, as demonstrated by silver stain, shown in Fig. 2. These argyrophilic cells typically show a rounded appearance, with multiple, rounded, intensely stained, sharply demarcated chromatin clumps. This appearance is identical to that observed during natural cell death in the SN, ~4 and during cell death induced in the SN following developmental striatal excitotoxic injury. 2~ In the latter setting, we have demonstrated that this morphology indicative of apoptosis at the light microscope level is, in fact,
associated with additional features of apoptosis at the ultrastructural level, including formation of electrondense chromatin clumps, preservation of intracellular organelles (such as mitochondria) and the formation of apoptotic bodies. 2~ Sloviter and colleagues 32'33have likewise previously demonstrated a correspondence between this apoptotic morphology as demonstrated by silver staining, and definitive ultrastructural features of apoptosis. Thus, we conclude that the morphology of induced cell death in both the SNpc and SNpr meets criteria for apoptosis. To further confirm the apoptotic nature of the induced cell death in the SNpc and SNpr, we performed & s i t u 3'-end-labeling to demonstrate the presence of free 3'-ends generated by endonuclease cleavage of genomic D N A , characteristic of apoptosis. 12,35F o r this analysis, we performed Nissl counterstaining after the end-labeling reaction to demonstrate, in addition to the 3'-end-labeling, the intensely basophilic chromatin staining characteristic of apoptosis. 14 Demonstration of this morphological criterion is essential for interpretation of the endlabeling reaction because necrotic cell death can be associated with false-positive staining ~° (and unpublished observations). Figure 3 demonstrates two typical cells in the SN meeting both morphological (chromatin clumping) and histochemical criteria (positive 3'-end-labeling) indicative of apoptosis. A quantitative analysis of the prevalence of these cells in the SN at a postlesion interval of two days following hypoxic-ischemic forebrain injury demonstrated a marked increase on the injured side (28.2 + 3.7 cells) in comparison to the contralateral, uninjured side (3.3 + 1.3, N = 4, P = 0.007). Thus, an induction of apoptotic cell death was confirmed by the analysis of cells meeting both morphological and histochemical criteria for apoptosis. We have shown previously that developmental hypoxic-ischemic injury to the striatum is associated with a diminished adult number of dopaminergic neurons in the SNpc. 4 We therefore wished to determine whether the induced apoptotic cell death in the SNpc occurred within phenotypically-defined dopaminergic neurons, such that it could account for a
Fig. 3. (A) An in situ 3'-end-labeled cell in the SN at 48 h following forebrain hypoxia-ischemia. Intensely basophilic apoptotic chromatin clumps are observed within the nucleus (small arrows). Brown peroxidase reaction product within the nucleus (large arrow) indicates the presence of free 3'-ends. This dying cell had the morphological characteristics of a neuron, with an abundant basophilic cytoplasm and tapered dendritic extensions, out of the plane of focus. (B) A second 3'-end labeled cell in the SN at 48 h. Three characteristic chromatin clumps are observed. These clumps are both Nissl-stained and heavily peroxidaselabeled, giving a dark brown appearance. The surrounding nucleus is also peroxidase-labeled. Scale bar = 10 #m. Fig. 4. (A) TH imunostaining with Nissl counterstain reveals that some cells in the SNpc undergoing apoptosis are dopaminergic. One cell (no. 1, arrow) shows a single basophilic chromatin clump. Normal TH-immunostained neurons do not show such intranuclear staining (no. 2, arrow). Another TH-positive neuron (no. 3, arrow) contains three chromatin clumps (arrowheads), some of which are slightly out of the plane of focus. One cell undergoing apoptosis is TH-negative (no. 4, arrow). (B) An additional example of a dopaminergic neuron undergoing apoptosis following forebrain hypoxic~ischemic injury. Scale bar = I0 #m.
Apoptosis in the substantia nigra
Figs 3 and 4.
897
898
T.F. Oo
decrement in the number of these neurons surviving into maturity. To perform this analysis, we demonstrated dopaminergic neurons within the SNpc by immunoperoxidase staining of TH, followed by Nissl counterstain to identify apoptotic chromatin clumps, as described previously) 1 This double-staining procedure identified TH-positive neurons undergoing apoptosis, as shown in Fig. 4, on the lesioned side. In four pups killed at two days postlesion, a mean of 6.3 + 1.0 such cells were identified per nigra on the injured side, based on screening eight sections representative of four rostrocaudal planes. No TH-positive cells with Nissl-defined chromatin clumps were observed on the unlesioned side. Within each SNpc only approximately 10% of Nissl-defined apoptotic profiles were immunoperoxidase-stained positive for TH. While there are several possible reasons for staining only a small portion of these cells, as discussed below, one major possible reason is limited penetration of antibodies and other reagents into these 30-pm-thick sections. Nevertheless, it is clear that some of the cells undergoing induced apoptotic death in the SNpc are of the dopaminergic phenotype. DISCUSSION We have previously observed that excitotoxic injury to the developing striatum is associated with an induced cell death event in the SN, with morphology of apoptosis. 21 This induced death event could be distinguished from the direct cell death caused by the excitotoxic striatal injury on the basis of its location remote from the site of direct injury, and its delay in onset. In addition, the distinct cell death morphology of apoptosis observed in the SN differed from the necrotic pattern observed at the striatal site of direct injury. We now report that hypoxic-ischemic injury to the forebrain is also associated with an induced death event in the SN, relatively remote from the major sites of direct injury, which include not only striatum, but also cortex and hippocampus. 28 This model of brain injury is not only more global than the excitotoxin model, but also more variable. Therefore, some animals subjected to this paradigm do show extensive injury to the affected hemisphere, with injury extending into the midbrain. However, extensive injury in such animals can be identified both by inspection, which reveals brain cavitation, and histological analysis by Nissl and silver stains, which reveal large confluent areas of neuron loss with the morphology of necrotic cell death in regions of direct injury. Taking care to exclude direct injury to the SN, we have found quantitative evidence of induced cell death on the side of hypoxic-ischemic injury. As in the case of induced death following excitotoxic striatal injury, the induced death in the SN following hypoxic-ischemic injury can be distinguished from direct injury on the basis of the regional and cellular characteristics of the death process. Regionally, cells
et al.
in the SN die as single, isolated cells, as shown in Fig. 1. This pattern is characteristic of apoptosis. 3° In addition, at a cellular level the dying cells in the SN are defined by both morphological and biochemical criteria to be universally apoptotic. In regions of direct injury, such as the striatum, while occasional cells may appear apoptotic by morphological criteria, the vast majority, in the acute phase of injury, appear necrotic by histological stains23'2s and silver staining (unpublished observation). The morphological criteria which we used to identify the pattern of cell death as apoptotic included positive silver staining, with rounding and shrinkage of the cell, in the presence of one or more rounded, intensely argyrophilic chromatin clumps. This morphological pattern of cell death was identical to that observed during natural cell death within the SNpc 14 and during induced cell death following striatal excitotoxic injury.21 In the latter setting, definitive ultrastructural evidence of apoptosis was confirmed. Likewise, Sloviter and colleagues32'33 had previously found that an apoptotic appearance on silver stain is associated with definitive ultrastructural features of apoptosis. The pattern of cell death induced in the SN was further confirmed to be apoptotic by the demonstration of a positive 3'-end-labeling reaction 12'35 in conjunction with a morphological demonstration of apoptotic chromatin clumping by Nissl counterstain. Thus, we conclude that the induced cell death in the SN is universally apoptotic, and in this respect, as well as its regional characteristics, it can be distinguished from the predominant form of necrotic cell death, which occurs in the forebrain as a direct result of hypoxic-ischemic injury. This induced cell death event in the SNpc is likely to account for the reduction in dopaminergic neurons in the adult brain observed following developmental forebrain hypoxic-ischemic injury.4 We have shown here that induced apoptotic cell death is observed in phenotypically-defined SNpc dopaminergic neurons. Since these neurons are postmitotic at this developmental age, 18'z2this cell death event in this population will result in a diminished number of these neurons surviving into adulthood. We found that very few (10%) of the apoptotic profiles in the SNpc were TH-positive. It is possible that the relatively large number of unstained neurons is due to limited penetration of immunoreagents into the section or cell compartment. Although it is also possible that some of these cells are non-dopaminergic, such an explanation seems unlikely to account for the considerable number of these cells in the SNpc. It is also possible that at later stages of apoptotic cell death, expression of phenotypic markers is so minimal that immunostaining is no longer possible. While the present study has identified an association between direct hypoxic-ischemic forebrain injury and an induced apoptotic death event in the SN, the mechanism(s) underlying this relationship is (are)
899
Apoptosis in the substantia nigra unknown. Given the global nature of the hypoxicischemic injury in this model, it will be difficult to define the precise anatomical basis. However, several considerations lead us to favor a pivotal role for striatal injury. The striatum is especially vulnerable to hypoxic-ischemic injury. ~5'28The striatum has major anatomical relationships with the SN; the SNpc provides the major dopaminergic afferent projection to the striatum; the SNpr receives a major GABAergic and substance P pathway from the striatum, l In addition, we know from our previous work that a selective striatal excitotoxic lesion is sufficient to cause a comparable degree of induced cell death in the SN. In relation to the SNpc, because the striatum is a major target, it is possible that hypoxic-ischemic injury results in a loss of retrogradely-derived trophic support, with a resulting induced apoptotic death, as described for many other systems.2 It seems unlikely that the induced death in the SNpc is due to injury of dopaminergic terminals with a resulting retrograde degeneration, because we and others have shown that markers of dopaminergic terminals are relatively spared in this model. 3'5'15'27Nevertheless, it remains possible that loss of a subset of dopaminergic terminals in absolute terms was not detected by the prior biochemical and morphological assessments. It is also possible that SNpc neuron death is due to a transneuronal effect, following death of SNpr neurons. However, the peak of cell death in the SNpc appears to be concurrent with or possibly precede that in the SNpr. The time course of induced cell death in the SN following hypoxic-ischemic injury is similar to that observed following focal excitotoxic striatal injury,21 lending support to the concept that the mechanism of induced death may be similar in the two paradigms. As for the SNpc, the mechanism for induced cell death in the SNpr is unknown. The striatum sends major afferent projections to the SNpr, and there are precedents for induction of cell death due to loss of afferent input in the developmental setting. 19 There is also a precedent for induced cell death in the SNpr following striatal injury in adult injury on the basis of a loss of GABAergic inhibitory input. 29 The precise mechanism will require further clarification. On the basis of the hypothesis that direct injury to the brain may result in induced cell death in other, remote regions as a consequence of diminished developmental support, it seems possible that cell death may occur in other structures with important projections to the striatum, such as the cortex and the intralaminar nuclei of the thalamus. 6 However, identification of such events is made more difficult by the direct involvement of these structures in hypoxicischemic injury. Our focus on the SN in this study was facilitated by the greater availability of information about effects on the dopaminergic system in this model,3 5,27and by our prior findings referable to the SN following striatal excitotoxic injury.21
We have not addressed in this study whether apoptosis may occur in those regions of the brain directly injured by hypoxia-ischemia. A number of investigators have previously shown that apoptosis may occur in such regions. In models of transient global ischemia and focal ischemia in adult rats and gerbils, internucleosomal fragmentation of DNA, causing a characteristic 185-200 bp "ladder" appearance on DNA gels, has been identified in extracts from striatal or hippocampal tissue and interpreted as evidence of apoptosis. 13'2°'24'31'34 In some of these studies, j3'j6,31 in situ end-labeling has confirmed the presence of Y-end-labeling, particularly in the CA1 region of the hippocampus, where delayed neuronal death is known to occur. However, the presence of internucleosomal fragmentation alone does not always correlate with the definitive morphological features of apoptosis. 8 In addition, the presence of 3'-end-labeling alone, in the absence of definitive morphological criteria for apoptosis, must be interpreted with great caution, because this technique will label free Y-ends generated by any process of DNA breakdown. In this regard, it is important to note that in an extensive ultrastructural analysis of the CA1 region of the hippocampus at 6-72 h following ischemia, Deshpande and colleagues9 were unable to identify any apoptotic morphology. Ferrer and coworkers l° examined the brains of immature rats following hypoxic-ischemic injury using the in situ end-labeling technique, and identified morphologies indicative of necrotic cell death, but they did not explicitly identify apoptosis. Some of their figures, however, seem to suggest the presence of apoptosis. Thus, it remains uncertain whether apoptosis, as defined by morphological criteria as used in this study, occurs in regions of direct injury. CONCLUSIONS
We have shown that, following hypoxic-ischemic injury to the developing brain, apoptosis occurs as a separate and distinct death mechanism in a brain region which is remote from sites of direct hypoxic-ischemic injury. One important implication of this finding is that this distinct form of cell death has its own unique regulatory pathways, and thus can potentially be modified by methods quite different from those which might influence cell death in regions of direct injury, where an abundance of data has implicated excitotoxic mechanisms as playing a predominant role. 7 It must be understood, however, that at the present time we do not know the functional consequences of this induced cell death. It is conceivable that it is not, in fact, deleterious, but plays a homeostatic role in regulating structural relationships between brain regions. It is likely that the phenomenon of induced apoptotic cell death plays a wide role in the response of the immature brain to injury. Given that induced death is observed after both excitotoxiczl and hypoxic-ischemic injury, it seems likely that it
900
T . F . Oo et al.
may occur following other forms o f injury to the i m m a t u r e brain in which excitotoxic injury is believed to play a role, such as seizures, head t r a u m a and hypoglycemia. 7
Acknowledgements--The authors are White for diligent preparation of the also grateful to Mr William Kelly for This work was supported by NS26836 Disease Foundation.
grateful to Ms Pat manuscript. We are technical assistance. and the Parkinson's
REFERENCES 1. Albin R. L., Young A. B. and Penney J. B. (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci. 12, 366-375. 2. Barde Y. A. (1989) Trophic factors and neuronal survival. Neuron 2, 1525 1534. 3. Burke R. E., Kent J., Kenyon N. and Karanas A. (1991) Unilateral hypoxi~ischemic injury in neonatal rat results in a persistent increase in the density of striatal tyrosine hydroxylase immunoperoxidase staining. Devl Brain Res. 58, 171 179. 4. Burke R. E., Macaya A., DeVivo D., Kenyon N. and Janec E. M. (1992) Neonatal hypoxic-ischemic or excitotoxic striatal injury results in a decreased adult number of substantia nigra neurons. Neuroscience 50, 559-569. 5. Burke R. E. and Reches A. (1991) Preserved striatal tyrosine hydroxylase activity, assessed in vivo, following neonatal hypoxia-ischemia. Devl Brain Res. 61, 277580. 6. Carpenter M. B. (1984) Interconnections between the corpus striatum and brain stem nuclei. In The Basal Ganglia (eds McKenzie J. S., Kern R. E. and Wilcock L. N.), pp. 1~8. Plenum, Bethesda, MD. 7. Choi D. W. (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623~534. 8. Collins R. J., Harmon B. V., Gobe G. C. and Kerr J. F. R. (1992) Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int. J. Radiat. Biol. 61, 451-453. 9. Deshpande J., Bergstedt K., Linden T., Kalimo H. and Wieloch T. (1992) Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Expl Brain Res. 88, 91-105. 10. 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. 11. Gallyas F., Wolff J. R., Bottcher H. and Zaborsky L. (1980) A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system. Stain Technol. 55, 299-306. 12. Gavrieli Y., Sherman Y. and Ben-Sasson S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493-501. 13. Heron A., Pollard H., Dessi F. et al. (1993) Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain. J. Neurochem. 61, 1973 1976. 14. Janec E. and Burke R. E. (1993) Naturally occurring cell death during postnatal development of the substantia nigra of the rat. Molec. Cell. Neurosci. 4, 30-35. 15. Johnston M. V. (1983) Neurotransmitter alterations in a model of perinatal hypoxic-ischemic brain injury. Ann. Neurol. 13, 511-518. 16. Kihara S.-I., Shiraishi T., Nakagawa S., Toda K. and Tabuchi K. (1994) Visualization of DNA double strand breaks in the gerbil hippocampal CA1 following transient ischemia. Neurosci. Lett. 175, 133-136. 17. Kyllerman M., Bager B., Bensch J., Bille B., Olow 1. and Voss H. (1982) Dyskinetic cerebral palsy. Actapaediat. scand. 71, 543-550. 18. Lauder J. M. and Bloom F. E. (1974) Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. J. comp. Neurol. 155, 469-482. 19. Linden R. (1994) The survival of developing neurons: a review of afferent control. Neuroscience 58, 671~582. 20. Linnik M. D., Zobrist R. H. and Hatfield M. D. (1993) Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 24, 2002-2008. 21. Macaya A., Munell F., Gubits R. M. and Burke R. E. (1994) Apoptosis in substantia nigra following developmental striatal excitotoxic injury. Proc. natn. Acad. Sci. U.S.A. 91, 8117-8121. 22. Marchand R. and Poirer L. J. (1983) Isthmic origin of neurons of the rat substantia nigra. Neuroscience 9, 373-381. 23. McGee-Russell S. M., Brown A. W. and Brierley J. B. (1970) A combined light and electron microscope study of early anoxic-ischaemic cell change in rat brain. Brain Res. 20, 193-200. 24. Okamoto M., Matsumoto M., Ohtsuki T. et al. (1993) Internucleosomal DNA cleavage involved in ischemia induced neuronal death. Biochem. biophys. Res. Commun. 196, 1356-1362. 25. Oppenheim R. W. (1991) Cell death during development of the nervous system. A. Rev. Neurosci. 14, 453-501. 26. Paxinos G. and Watson C. (1982) The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA. 27. Przedborski S., Kostic V., Jackson Lewis V., Cadet J. L. and Burke R. E. (1991) Effect of unilateral perinatal hypoxic-ischemic brain injury in the rat on dopamine D1 and D2 receptors and uptake sites: a quantitative autoradiographic study. J. Neurochem. 57, 1951-1961. 28. Rice J. E., Vannucci R. C. and Brierley J. B. (1981) The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann. Neurol. 9, 131-141. 29. Saji M. and Reis D. J. (1987) Delayed transneuronal death of substantia nigra neurons prevented by gamma-aminobutyric acid agonist. Science 235, 66~8. 30. Searle J., Kerr J. F. R. and Bishop C. J. (1982) Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Path. Ann. 17, 229-259. 31. Sei Y., Von Lubitz D. K. J. E., Basile A. S. et al. (1994) Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia. Neurosci. Lett. 171, 179 182. 32. Sloviter R. S., Dean E. and Neubort S. (1993) Electron microscopic analysis of adrenalectomy induced hippocampal granule cell degeneration in the rat apoptosis in the adult central nervous system. J. comp. Neurol. 330, 337 351. 33. Sloviter R. S., Sollas A. L., Dean E. and Neubort S. (1993) Adrenalectomy induced granule cell degeneration in the rat hippocampal dentate gyrus characterization of an in vivo model of controlled neuronal death. J. comp. Neurol. 330, 324-336. 34. Tominaga T., Kure S., Narisawa K. and Yoshimoto T. (1993) Endonuclease activation following focal ischemic injury in the rat brain. Brain Res. 608, 21-26.
Apoptosis in the substantia nigra
901
35. Wijsman J. H., Jonker R. R., Keijzer R., van de Velde C. J. H., Cornelisse C. J. and van Dierendonck J.-H (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J. Histochem. Cytochem. 41, 7-13. 36. Wyllie A. H., Kerr J. F. and Currie A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306. (Accepted 7 June 1995)