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Novel non-apoptotic morphological changes in neurons of the mouse hippocampus following transient hypoxic-ischemia
Neuroscience Research 33 (1999) 49 – 55
Novel non-apoptotic morphological changes in neurons of the mouse hippocampus following transient hypoxic-ischemia Takaichi Fukuda a,*, Huaidong Wang b, Hiroshi Nakanishi b, Kenji Yamamoto b, Toshio Kosaka a a
Department of Anatomy and Neurobiology, Faculty of Medicine, Kyushu Uni6ersity, Maidashi, Higashi-ku, Fukuoka 812 -8582, Japan b Department of Pharmacology, Faculty of Dentistry, Kyushu Uni6ersity, Maidashi, Higashi-ku, Fukuoka 812 -8582, Japan Received 5 October 1998; accepted 29 October 1998
Abstract Apoptosis has been recently implicated in the dying process of neurons under several pathological conditions including ischemia. However, although apoptosis was originally defined on the basis of its unique ultrastructural features (Kerr et al., 1972. Br. J. Cancer 26, 239–257; Wyllie et al., 1980. Int. Rev. Cytol. 68, 251 – 306), unambiguous ultrastructural evidence of apoptosis has been rarely demonstrated in the adult brain. In this study, we examined ultrastructural changes in mouse hippocampal neurons after transient hypoxic-ischemia. A small population of dentate granule cells showed typical apoptotic ultrastructures that could be used as internal morphological standards of apoptosis, whereas most other hippocampal neurons consistently showed a distinct form of cellular disintegration. Nuclei of the latter cells shrank and became TUNEL-positive but were distinguishable from apoptotic nuclei by both the presence of characteristic reticular-formed chromatin condensation and the absence of nuclear fragmentation. Perikarya of degenerating neurons also shrank as in apoptosis, but apoptotic bodies were not observed. Although organelles other than mitochondria disappeared almost completely from the perikarya, neither plasma nor mitochondrial membranes were disrupted, indicating that these changes were also different from typical necrosis. The presence of a novel form of cell death suggests the necessity of morphological re-examination of neuronal death, particularly in mature neurons in vivo. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Apoptosis; Necrosis; Electron microscopy; Hippocampus; Mouse.
1. Introduction Apoptosis is a distinctive form of cell death and has been proposed as a mechanism for controlled cell deletion, playing the opposite role to mitosis in the regulation of cell numbers (Kerr et al., 1972; Wyllie et al., 1980). It serves to eliminate excessive or disused cells during development, postnatal cell turnover, and the period after tissue damage. Recent studies have suggested that neurons in the adult brain may also undergo apoptosis under several pathological conditions such as neurodegenerative diseases and ischemia (Li et al., * Corresponding author. Fax: [email protected].
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1995; Nitatori et al., 1995; Portera-Cailliau et al., 1995). However, the vast majority of neurons have no mitotic activity postnatally, and nuclear and cytoplasmic conditions in mature neurons that relate to the regulation of the cell cycle and/or death are assumed to be somewhat different from those in other types of cells, making it necessary to take careful consideration when one applies the concept of apoptosis to death in mature neurons. In fact, it has already been pointed out that several morphological as well as biochemical aspects of ischemic neuronal death are inconsistent with apoptosis (Deshpande et al., 1992; Van Lookeren Campagne and Gill, 1996; Petito et al., 1997). Apoptosis was originally defined by its unique ultrastructural features. According to the classical reviews
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T. Fukuda et al. / Neuroscience Research 33 (1999) 49–55
(Kerr et al., 1972; Wyllie et al., 1980), the ultrastructural sequence in apoptosis can be summarized as follows: (1) aggregation of the chromatin into large discrete masses that abut on the nuclear membrane, (2) fragmentation of both the nucleus and the cytoplasm, leading to formation of apoptotic bodies, and (3) rapid removal of the apoptotic bodies by phagocytosing cells. In the light of these classical criteria, unambiguous ultrastructural profiles of apoptosis in neurons have been demonstrated only in the developing brain and in the very restricted area of the adult brain (Sloviter et al., 1993, 1996) where mitotic activity is maintained postnatally throughout life (Altman and Das, 1967; Kaplan and Hinds, 1977; Bayer and Yackel, 1982). On the other hand, presumptive apoptosis in neurons has been investigated in most studies by combining some of the following methods: light microscopic (LM) observations, gel electrophoresis revealing internucleosomal cleavage of DNA, and terminal dUTP nick-end-labeling (TUNEL). It is now recognized, however, that the latter two biochemical procedures can no longer define cell death as apoptosis (Bicknell and Cohen, 1995; Grasl-Kraupp et al., 1995; Van Lookeren Campagne et al., 1995). Moreover, light microscopic observation, due to its relatively low resolution, does not necessarily discriminate apoptotic cell changes from other degenerating processes. Therefore use of electron microscopy (EM) is still helpful and essential to characterize the form of cell death. For a correct understanding of ischemic neuronal death, both morphological and molecular biological analyses are indispensable and should be correlated with each other. Although mice have been most extensively used in molecular biological research, there have been few morphological studies on brain ischemia in the mouse. Moreover, no appropriate models of cerebral ischemia has been established in the mouse. In the present study we intended to provide basic morphological data on ischemic neuronal death in the mouse hippocampus by a detailed analysis in EM. We adopted a model of hypoxic-ischemia developed by Levine (1960) with some modifications because of the simplicity in its experimental procedures. Using this model we found a novel form of neuronal death that could not be ascribed to either apoptosis or necrosis by the classical morphological criteria.
2. Materials and methods This study was approved by the Animal Research Committee of the Kyushu University. Thirty-seven mice of both sexes (C57BL/6J, 6 – 10 weeks of age) were subjected to a unilateral cut of the right common carotid artery between double ligatures under deep anesthesia with ether. Following a 2 h recovery after
wound closure, they were exposed to a humidified gas mixture (8% O2 –92% N2 at 37°C) in an enclosed chamber for 60 min. The animals were returned to their cages and allowed to survive for 3 h (n=7), 24 h (n= 15), or 4 days (n= 15) prior to fixation. Three normal mice were used as controls. Following the prescribed survival time, the animals were deeply anesthetized with sodium pentobarbital (i.p.; 100 mg/kg body weight) and quickly perfused through the ascending aorta with 10 mM phosphatebuffered saline (PBS), followed by 50 ml of a fixative containing either 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for LM (n= 35), or 2.5% glutaraldehyde and 2% paraformaldehyde in PB for EM (n=3 at 3 h; n=2 at 4 days), both pH 7.2 at room temperature. Serial 50 mm-thick coronal sections were cut with a Microslicer (DTK-3000, Dosaka EM, Japan) and stained with cresyl violet for LM analysis. Sections for EM were postfixed with 1% OsO4 in PB for 1.5 h on ice, stained en bloc with 1.5% uranyl acetate for 1 h, then dehydrated and flat-embedded in Araldite. Serial ultrathin sections were cut and stained with uranyl acetate and lead citrate and examined in a transmission electron microscope (Hitachi H-7100). Some Microslicer sections were stained with propidium iodide (PI) and observed with a confocal laser scanning microscope (BioRad MRC-1000) using a 60× oil immersion objective lens (N.A.=1.40, Nikon) as described previously (Fukuda et al., 1998). Sections from four mice (24 h, n= 2; 4 days, n=2) were processed for TUNEL. They were treated with 2% H2O2 in PBS for 30 min, rinsed several times, and stained by using the ApopTag kit (Oncor, Gaithersburg, USA).
3. Results The present model of forebrain hypoxic-ischemia was assumed to allow various degrees of collateral flow to the ischemic area, depending on the patency of the arterial ring of Willis. As a result the extent of the damage was rather heterogeneous among the animals: 35% (13/37) of the mice did not show any appreciable changes and were excluded from the analysis. Hippocampal neurons in the remaining 65% of animals consistently underwent a distinct set of morphological changes, though the numbers of damaged neurons varied considerably from animal to animal. Degenerating neurons were located exclusively in the side ipsilateral to the resected carotid artery, and their morphological features were basically the same in both pyramidal cells in the hippocampus proper (CA1 and CA3 regions) and granule cells in the dentate gyrus. Among these three regions there was no apparent tendency as to which region was the most vulnerable to ischemic damage. Exceptional profiles were noted in a
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Fig. 1. (A – D) Photomicrographs of pyramidal cells in the CA1 region of the mouse hippocampus stained with cresyl violet (A – C) and TUNEL (D). As compared to normal animals (A), neurons reduced their sizes greatly at 3 h (B) and 4 days (C) after hypoxic-ischemia. Note several clumps (arrows) inside the nucleus of postischemic neurons. These degenerating neurons were TUNEL-positive at 24 h after hypoxic-ischemia (D). (E, F) Confocal laser scanning microscopic images of PI-stained nuclei in the pyramidal cell layer of the CA1 region at 3 h (E) and in the granule cell layer of the dentate gyrus at 24 h (F) after hypoxic-ischemia. The nuclei of postischemic pyramidal cells maintained their round or oval shape and contained several irregular masses as well as minute speckle-like structures (E). On the other hand, the neurons at the crest region of the granule cell layer (F) showed typical apoptotic profiles such as crescentic caps (open arrows) and fragmented nuclei (arrowheads). Scale bars: 10 mm.
very small population of cells in the dentate gyrus, i.e. at the border between the granule cell layer and the hilus (Fig. 3A) and occasionally around the crest region of the granule cell layer (Fig. 1F); in both places typical apoptotic structures were found (see below). Thus the present model allowed us to make a direct comparison of the morphological changes between the novel type of cell degeneration and typical apoptosis within the same materials. Nuclei of degenerating neurons reduced their sizes greatly even at 3 h after ischemia (Fig. 1). The diameters of these nuclei decreased to approximately half to one-third of the normal ones and remained at that size from 3 h up to 4 days after ischemia. These shrunken nuclei were intensely stained by TUNEL (Fig. 1D). Although these features appeared common to apoptosis, one of the discriminating points was that neither the nuclei nor cell bodies were fragmented (Fig. 1B, C, E), contrasting with the formation of apoptotic bodies in apoptosis (Figs. 1F and 3C). In fact electron microscopic observations (Fig. 2) revealed that the nucleus continued to be in its ordinary round or oval shape with the nuclear envelope still existing as the boundary
between the nuclear and cytoplasmic matrices, though nuclear pores appeared dilated and lost diaphragms, possibly allowing the passage of denatured nuclear substances through the pores. EM and CLSM observations revealed that the injured nuclei were further characterized by two types of uncommon structures, presumably resulting from chromatin condensation. The first one was composed of reticular-shaped structures scattered throughout the nucleus (Fig. 2). They appeared as numerous speckles when observed in single EM sections (Fig. 2A and B). When they were three-dimensionally reconstructed in serial EM sections, they were found to be linked to each other and formed a delicate network within the nucleus (data not shown, but see white arrows in Fig. 2B). This reticular structure was clearly distinguishable from the nuclear pattern in apoptosis (Fig. 3), where chromatin was uniformly aggregated into large discrete masses abutting on the nuclear membrane, typically seen as crescentic caps (Figs. 1F and 3B). It was difficult in Nissl-stained sections to recognize the reticular type of chromatin condensation presumably due to its thinner profile and rather weak staining.
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Fig. 2. Electron micrographs of CA1 pyramidal cells at 4 days (A – C) after hypoxic-ischemia and their earlier changes at 3 h (D, E). (A) Five degenerating neurons (d1–d5) are characterized by their small nuclei, novel type of chromatin condensation, and marked cytoplasmic disintegration. By contrast, three neighboring neurons (n1–n3) appear to preserve fairly normal ultrastructures. (B) Cell d2 in (A) is enlarged. Two irregular-shaped masses (asterisks) and numerous speckle-like structures are seen inside the nucleus. Note the interconnection (white arrows) among the speckle-like structures. Three presynaptic axon terminals (s) are in direct contact with this cell, one of which is further enlarged in (C). (C) The axon terminal (s) formed synaptic junction (crossed arrow) with the soma, clearly indicating that this cell was a neuron. Both plasma (arrows) and nuclear membranes (arrowheads) were not disrupted, though the nuclear pores were dilated. Mitochondria (m) also retained their inner and outer membranes together with some cristae-like structures but their internal structures already suffered severe damage. Organelles other than mitochondria disappeared almost completely at 4 days (B), but at 3 h (D) markedly swollen rough endoplasmic reticulum (rER) with numerous ribosomes abutting onto them (E) could be observed. Scale bars: 5 mm (A), 1 mm (B and D), 0.5 mm (C and E).
Another discriminating structure in the nucleus was composed of irregular-shaped masses (Fig. 2B, asterisks), that were also recognizable in Nissl-stained sections as several clumps inside the nucleus (Fig. 1B and C). Though they might appear in LM as if they were fragmented nuclei seen in apoptosis, EM observations clearly revealed that they remained inside the round or oval nucleus (Fig. 2A and B), being entirely different from apoptotic fragmented nuclei (Figs. 1F and 3D). Moreover, these intranuclear masses were also different
from the crescentic caps because they were rather irregular in shape and usually located apart from the nuclear membranes (Fig. 2A and B). These structures were assumed to be derived from denatured nucleoli, although we can not determine their origin at present. Novel morphological changes were also seen in the cytoplasm. Perikarya were initially swollen at 3 h after ischemia (Fig. 2D), then shrunk at 4 days (Fig. 2A–C). In the swollen cytoplasm, rough endoplasmic reticulum (rER) was markedly dilated but still encrusted with
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Fig. 3. Electron micrographs of cells displaying typical morphological features of apoptosis in and around the dentate granule cell layer at 3 h after hypoxic-ischemia. (A) A cell showing crescentic caps (arrows) was located at the border between the granule cell layer (g) and the hilus. (B) Crescentic caps were composed of uniformly aggregated chromatin that abutted on the nuclear membrane (arrowheads). (C) An apoptotic body containing closely packed mitochondria was observed in a position similar to (A). The most part of this apoptotic body was surrounded by a neighboring cell (nb). (D) An example of a cell with a fragmented nucleus. Scale bars: 5 mm (A), 1 mm (B – D).
numerous ribosomes (Fig. 2D and E). The outer membrane of the nuclear envelope underwent similar change to rER. By contrast, the mitochondria were only slightly swollen though the array of cristae appeared somewhat disordered (Fig. 2D). The Golgi apparatus and lysosomes were difficult to identify. At 4 days after ischemia the cytoplasm was more severely degraded and most of the organelles other than mitochondria disappeared from the perikarya; only amorphous material was left there. Even at this stage the mitochondria retained their inner and outer membranes as well as cristae-like structures (Fig. 2C), though integrity of the presumptive cristae was severely damaged. This was different from the usual course of necrotic changes. Moreover, in spite of the disintegrated nucleus and cytoplasm at 4 days, there was no disruption of the plasma membrane, on which several presynaptic terminals occasionally formed synaptic junctions (Fig. 2B,C). The absence of cell lysis was in sharp contrast with the generally depicted profiles in necrosis. On the other hand, the cellular surfaces were not protruded, different from the fragmented cell bodies seen in apoptosis. Profiles of apoptotic bodies were not observed in and around the hippocampal pyramidal cell layers (Fig. 2A) whereas they were occasionally seen in the above-mentioned subregions in the dentate gyrus (Fig. 3C) where typical apoptosis did occur. The vast majority of degenerating somata appeared to escape phagocytosis at least 4 days following ischemia. Although some microglia entered the pyramidal and granule cell layers, most of them were just in partial contact with the degenerating somata (Fig. 2A and B); profiles of microglia engulfing the degenerating perikaryon were seldom encountered. Though it requires further studies to elucidate the ultimate fate of the degenerating neurons shown here, the process of
removing these cells were much slower than might be expected in the known time course of apoptosis.
4. Discussion The present study demonstrated a novel form of neuronal degeneration characterized by distinctive morphological changes. In spite of the apparent resemblance to apoptosis in LM and the positive staining by TUNEL, detailed analysis by EM revealed the structural features clearly distinguishable from those in classical apoptosis. Moreover, neurons displaying these features coexisted with the typical apoptotic cells located in the same sections, indicating that the two forms were distinct morphological entities. On the other hand, the absence of cell lysis and the preservation of mitochondrial membranes was inconsistent with necrotic changes. Since the present form of cell injury was common to the vast majority of degenerating pyramidal and granule neurons, it could be concluded on morphological grounds that the general pattern of hippocampal neuronal death in the current model was not of typically apoptotic or necrotic in form. We also encountered similar profiles in the somatosensory cortex and the thalamus when selective neuronal death occurred in these areas. Furthermore, recent EM study investigating neuronal degeneration after systemic administration of neurotoxic agent 3acetylpyridine also revealed unequivocally non-apoptotic ultrastructures in the inferior olive (Wu¨llner et al., 1997) that closely resembled the ultrastructures shown here. Therefore the present form of neuronal degeneration appears to not be restricted to our model but might be more ubiquitous. Nuclear changes in the degenerating hippocampal neurons were unique. Interestingly, similar profiles were
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also reported in isolated hepatic nuclei treated with Ca2 + , a known activator of endonuclease (Oberhammer et al., 1993). The authors concluded that these hepatic nuclei were non-apoptotic in spite of positive in situ nick labeling and DNA ladder, which is consistent with our present observations. Ca2 + overload has been implicated in mechanisms of ischemic neuronal death in the hippocampal CA1 region (Deshpande et al., 1987; Choi, 1988). Elevated level of Ca2 + might play some roles in the formation of novel ultrastructural changes in the nuclei of postischemic neurons. The morphological changes shown in the present study have some features that differ from those in the wellknown delayed neuronal death in the hippocampus. This was assumed to be derived primarily from the differences in the experimental models used. Ischemia in the hippocampus has been investigated most frequently with either of the following two models: bilateral occlusion of the carotid arteries in gerbils (Kirino, 1982), or four-vessel occlusion (bilateral carotid and vertebral arteries) in rats (Petito and Pulsinelli, 1984; Deshpande et al., 1992), each lasting for brief periods (5 – 20 min). These methods have revealed a particular form of neuronal death that occurs preferentially in the CA1 region after several days of an asymptomatic period, hence the name delayed neuronal death. In the present study we used mice derived from a strain c57BL/6, since modern gene-targeting as well as transgenic techniques have been conducted most frequently on mice belonging to this strain. Because of the difficulty in conducting four-vessel occlusion in mice, we adopted a more simple model of hypoxic-ischemia. Although the extent of the damaged area varied among animals in the present study, the hippocampal neurons underwent consistent morphological changes qualitatively. In contrast with delayed neuronal death, degenerating neurons in the present model were not restricted to the hippocampal CA1 region but distributed more extensively in the CA3 region and the dentate granule cell layer. Another difference between the two forms of neuronal death was concerned with the time course of cell degeneration. The present form was characterized by the early onset and preservation for at least several days of the ultrastructural changes, contrasted with the delayed onset and the rapid disappearance of injured cells in the delayed neuronal death. The ultrastructural profiles in the present study were distinct from those in typical apoptosis, whereas there have been controversial data regarding the apoptotic nature of delayed neuronal death, partly owing to the lack of convincing ultrastructural evidence of apoptosis in delayed neuronal death. Since experimental conditions for the two methods were quite different we cannot conclude at present whether the morphological differences in the two forms of neuronal death reflected distinct mechanisms of cell death. Alternatively, essentially the same mechanisms might underlie
the two forms but the differences in some incidental conditions such as the duration of ischemia could lead to apparent morphological differences. An hypoxic-ischemia model with a shorter ischemic period or a delayed death model with a longer ischemic period would be helpful in elucidating the relationship of the two death patterns. The rapid recognition and removal of dying cells is an important feature of apoptosis, whereas necrotic cells are phagocytosed after the plasma membrane integrity is disrupted. In this sense, one of the distinguishing features in the present form of cell death was that the dying cells appeared to be relatively silent at least for several days, undergoing a slow degradation process inside the plasma membrane and keeping themselves from active scavenging reactions. In the light of the slow process of cell removal, we tentatively name the present form of cell death ‘lathiptosis’, according to the Greek words lathi (escaping notice) and ptosis (falling; a metaphor for death), to facilitate the comparison of several forms of potentially non-apoptotic cell death with the present form. Due to rapid progress in molecular biological research into apoptosis the concept of apoptosis is now fluctuating and may expand or be modified in the future, mainly based on new biochemical standards. Although we preliminarily confirmed that lathiptosis also occurred ubiquitously in the hippocampus of transgenic mice overexpressing bcl-2, the potent inhibitor of apoptosis, it still remains unknown whether lathiptosis can be included in the renewed category of apoptosis, or is totally different to apoptosis. It is necessary at present that morphological features of neuronal death in vivo should be re-examined in detail, in concert with biochemical investigation of the critical molecules such as caspases and related substances.
Acknowledgements We are most grateful to postgraduate student Tetsuya Takahashi and Research Associate Tetsuya Nagashima, Department of Philosophy, Faculty of Letters, Kyushu University, for their erudite suggestion of the Greek words.
References Altman, J., Das, G.D., 1967. Postnatal neurogenesis in the guinea pig. Nature 214, 319 – 335. Bayer, S.A., Yackel, J.W., 1982. Neurons in the rat dentate gyrus granular layer substantially increase during juvenile and adult life. Science 216, 890 – 892. Bicknell, G.R., Cohen, G.M., 1995. Cleavage of DNA to large kilobase pair fragments occurs in fome forms of necrosis as well as apoptosis. Biochem. Biophys. Res. Commun. 207, 40–47.
T. Fukuda et al. / Neuroscience Research 33 (1999) 49–55 Choi, D.W., 1988. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci. 11, 465 – 467. Deshpande, J.K., Siesjo¨, B.K., Wieloch, T., 1987. Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia. J. Cereb. Blood Flow Metab. 7, 89–98. Deshpande, J.K., Bergstedt, K., Linde´n, T., Kalimo, H., Wieloch, T., 1992. Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Exp. Brain Res. 88, 91–105. Fukuda, T., Aika, Y., Heizmann, C.W., Kosaka, T., 1998. GABAergic axon terminals at perisomatic and dendritic inhibitory sites show different immunoreactivities against two GAD isoforms, GAD67 and GAD65, in the mouse hippocampus: a digitized quantitative analysis. J. Comp. Neurol. 395, 177–194. Grasl-Kraupp, B., Ruttkay-Nedecky, B., Koudelka, H., Bukowska, K., Bursch, W., Schulte-Hermann, R., 1995. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21, 1465 –1468. Kaplan, M.S., Hinds, J.W., 1977. Neurogenesis in adult rat: electron microscopic analysis of light radioautographs. Science 197, 1092 – 1094. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257. Kirino, T., 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57–69. Levine, S., 1960. Anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 36, 1 – 17. Li, Y., Sharov, V.G., Jiang, N., Zaloga, C., Sabban, H.N., Chopp, M., 1995. Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat. Am. J. Pathol. 146, 1045 – 1051. Nitatori, T., Sato, N., Waguri, S., Karaswa, Y., Araki, H., Shibatani, K., Kominami, E., Uchiyama, Y., 1995. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J. Neurosci. 15, 1001– 1011.
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Oberhammer, F., Fritsch, G., Schmied, M., Pavelka, M., Printz, D., Lassman, H., Schulte-Hermann, R., 1993. Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J. Cell Sci. 104, 317–326. Petito, C.K., Pulsinelli, W.A., 1984. Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes. J. Cereb. Blood Flow Metab. 4, 194 – 205. Petito, C.K., Torres-Munoz, J., Roberts, B., Olarte, J.-P., Nowak, T.S. Jr., Pulsinelli, W.A., 1997. DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat. J. Cereb. Blood Flow Metab. 17, 967–976. Portera-Cailliau, C., Hedreen, J.C., Price, D.L., Koliatsos, V.E., 1995. Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci. 15, 3775 – 3787. Sloviter, R.S., Dean, E., 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. Sloviter, R.S., Dean, E., Sollas, A.L., Goodman, J.H., 1996. Apoptosis and necrosis induced in different hippocampal neuron populations by repetitive perforant path stimulation in the rat. J. Comp. Neurol. 366, 516 – 533. Van Lookeren Campagne, M., Lucassen, P.J., Vermeulen, J.P., Bala´zs, R., 1995. NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal rat brain. Eur. J. Neurosci. 7, 1627 – 1640. Van Lookeren Campagne, M., Gill, R., 1996. Ultrastructural morphological changes are not characteristic of apoptotic cell death following focal cerebral ischaemia in the rat. Neurosci. Lett. 213, 111 – 114. Wu¨llner, U., Weller, M., Groscurth, P., Lo¨schmann, P.-A., Schulz, J.B., Mu¨ller, I., Klockgether, T., 1997. Evidence for an active type of cell death with ultrastructural features distinct from apoptosis: the effects of 3-acetylpyridine neurotoxicity. Neuroscience 81, 721 – 734. Wyllie, A.H., Kerr, J.F.R., Currie, A.R., 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251 – 306.
Novel non-apoptotic morphological changes in neurons of the mouse hippocampus following transient hypoxic-ischemia Takaichi Fukuda a,*, Huaidong Wang b, Hiroshi Nakanishi b, Kenji Yamamoto b, Toshio Kosaka a a
Department of Anatomy and Neurobiology, Faculty of Medicine, Kyushu Uni6ersity, Maidashi, Higashi-ku, Fukuoka 812 -8582, Japan b Department of Pharmacology, Faculty of Dentistry, Kyushu Uni6ersity, Maidashi, Higashi-ku, Fukuoka 812 -8582, Japan Received 5 October 1998; accepted 29 October 1998
Abstract Apoptosis has been recently implicated in the dying process of neurons under several pathological conditions including ischemia. However, although apoptosis was originally defined on the basis of its unique ultrastructural features (Kerr et al., 1972. Br. J. Cancer 26, 239–257; Wyllie et al., 1980. Int. Rev. Cytol. 68, 251 – 306), unambiguous ultrastructural evidence of apoptosis has been rarely demonstrated in the adult brain. In this study, we examined ultrastructural changes in mouse hippocampal neurons after transient hypoxic-ischemia. A small population of dentate granule cells showed typical apoptotic ultrastructures that could be used as internal morphological standards of apoptosis, whereas most other hippocampal neurons consistently showed a distinct form of cellular disintegration. Nuclei of the latter cells shrank and became TUNEL-positive but were distinguishable from apoptotic nuclei by both the presence of characteristic reticular-formed chromatin condensation and the absence of nuclear fragmentation. Perikarya of degenerating neurons also shrank as in apoptosis, but apoptotic bodies were not observed. Although organelles other than mitochondria disappeared almost completely from the perikarya, neither plasma nor mitochondrial membranes were disrupted, indicating that these changes were also different from typical necrosis. The presence of a novel form of cell death suggests the necessity of morphological re-examination of neuronal death, particularly in mature neurons in vivo. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Apoptosis; Necrosis; Electron microscopy; Hippocampus; Mouse.
1. Introduction Apoptosis is a distinctive form of cell death and has been proposed as a mechanism for controlled cell deletion, playing the opposite role to mitosis in the regulation of cell numbers (Kerr et al., 1972; Wyllie et al., 1980). It serves to eliminate excessive or disused cells during development, postnatal cell turnover, and the period after tissue damage. Recent studies have suggested that neurons in the adult brain may also undergo apoptosis under several pathological conditions such as neurodegenerative diseases and ischemia (Li et al., * Corresponding author. Fax: [email protected].
+ 81-92-642-6059;
e-mail:
1995; Nitatori et al., 1995; Portera-Cailliau et al., 1995). However, the vast majority of neurons have no mitotic activity postnatally, and nuclear and cytoplasmic conditions in mature neurons that relate to the regulation of the cell cycle and/or death are assumed to be somewhat different from those in other types of cells, making it necessary to take careful consideration when one applies the concept of apoptosis to death in mature neurons. In fact, it has already been pointed out that several morphological as well as biochemical aspects of ischemic neuronal death are inconsistent with apoptosis (Deshpande et al., 1992; Van Lookeren Campagne and Gill, 1996; Petito et al., 1997). Apoptosis was originally defined by its unique ultrastructural features. According to the classical reviews
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(Kerr et al., 1972; Wyllie et al., 1980), the ultrastructural sequence in apoptosis can be summarized as follows: (1) aggregation of the chromatin into large discrete masses that abut on the nuclear membrane, (2) fragmentation of both the nucleus and the cytoplasm, leading to formation of apoptotic bodies, and (3) rapid removal of the apoptotic bodies by phagocytosing cells. In the light of these classical criteria, unambiguous ultrastructural profiles of apoptosis in neurons have been demonstrated only in the developing brain and in the very restricted area of the adult brain (Sloviter et al., 1993, 1996) where mitotic activity is maintained postnatally throughout life (Altman and Das, 1967; Kaplan and Hinds, 1977; Bayer and Yackel, 1982). On the other hand, presumptive apoptosis in neurons has been investigated in most studies by combining some of the following methods: light microscopic (LM) observations, gel electrophoresis revealing internucleosomal cleavage of DNA, and terminal dUTP nick-end-labeling (TUNEL). It is now recognized, however, that the latter two biochemical procedures can no longer define cell death as apoptosis (Bicknell and Cohen, 1995; Grasl-Kraupp et al., 1995; Van Lookeren Campagne et al., 1995). Moreover, light microscopic observation, due to its relatively low resolution, does not necessarily discriminate apoptotic cell changes from other degenerating processes. Therefore use of electron microscopy (EM) is still helpful and essential to characterize the form of cell death. For a correct understanding of ischemic neuronal death, both morphological and molecular biological analyses are indispensable and should be correlated with each other. Although mice have been most extensively used in molecular biological research, there have been few morphological studies on brain ischemia in the mouse. Moreover, no appropriate models of cerebral ischemia has been established in the mouse. In the present study we intended to provide basic morphological data on ischemic neuronal death in the mouse hippocampus by a detailed analysis in EM. We adopted a model of hypoxic-ischemia developed by Levine (1960) with some modifications because of the simplicity in its experimental procedures. Using this model we found a novel form of neuronal death that could not be ascribed to either apoptosis or necrosis by the classical morphological criteria.
2. Materials and methods This study was approved by the Animal Research Committee of the Kyushu University. Thirty-seven mice of both sexes (C57BL/6J, 6 – 10 weeks of age) were subjected to a unilateral cut of the right common carotid artery between double ligatures under deep anesthesia with ether. Following a 2 h recovery after
wound closure, they were exposed to a humidified gas mixture (8% O2 –92% N2 at 37°C) in an enclosed chamber for 60 min. The animals were returned to their cages and allowed to survive for 3 h (n=7), 24 h (n= 15), or 4 days (n= 15) prior to fixation. Three normal mice were used as controls. Following the prescribed survival time, the animals were deeply anesthetized with sodium pentobarbital (i.p.; 100 mg/kg body weight) and quickly perfused through the ascending aorta with 10 mM phosphatebuffered saline (PBS), followed by 50 ml of a fixative containing either 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for LM (n= 35), or 2.5% glutaraldehyde and 2% paraformaldehyde in PB for EM (n=3 at 3 h; n=2 at 4 days), both pH 7.2 at room temperature. Serial 50 mm-thick coronal sections were cut with a Microslicer (DTK-3000, Dosaka EM, Japan) and stained with cresyl violet for LM analysis. Sections for EM were postfixed with 1% OsO4 in PB for 1.5 h on ice, stained en bloc with 1.5% uranyl acetate for 1 h, then dehydrated and flat-embedded in Araldite. Serial ultrathin sections were cut and stained with uranyl acetate and lead citrate and examined in a transmission electron microscope (Hitachi H-7100). Some Microslicer sections were stained with propidium iodide (PI) and observed with a confocal laser scanning microscope (BioRad MRC-1000) using a 60× oil immersion objective lens (N.A.=1.40, Nikon) as described previously (Fukuda et al., 1998). Sections from four mice (24 h, n= 2; 4 days, n=2) were processed for TUNEL. They were treated with 2% H2O2 in PBS for 30 min, rinsed several times, and stained by using the ApopTag kit (Oncor, Gaithersburg, USA).
3. Results The present model of forebrain hypoxic-ischemia was assumed to allow various degrees of collateral flow to the ischemic area, depending on the patency of the arterial ring of Willis. As a result the extent of the damage was rather heterogeneous among the animals: 35% (13/37) of the mice did not show any appreciable changes and were excluded from the analysis. Hippocampal neurons in the remaining 65% of animals consistently underwent a distinct set of morphological changes, though the numbers of damaged neurons varied considerably from animal to animal. Degenerating neurons were located exclusively in the side ipsilateral to the resected carotid artery, and their morphological features were basically the same in both pyramidal cells in the hippocampus proper (CA1 and CA3 regions) and granule cells in the dentate gyrus. Among these three regions there was no apparent tendency as to which region was the most vulnerable to ischemic damage. Exceptional profiles were noted in a
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Fig. 1. (A – D) Photomicrographs of pyramidal cells in the CA1 region of the mouse hippocampus stained with cresyl violet (A – C) and TUNEL (D). As compared to normal animals (A), neurons reduced their sizes greatly at 3 h (B) and 4 days (C) after hypoxic-ischemia. Note several clumps (arrows) inside the nucleus of postischemic neurons. These degenerating neurons were TUNEL-positive at 24 h after hypoxic-ischemia (D). (E, F) Confocal laser scanning microscopic images of PI-stained nuclei in the pyramidal cell layer of the CA1 region at 3 h (E) and in the granule cell layer of the dentate gyrus at 24 h (F) after hypoxic-ischemia. The nuclei of postischemic pyramidal cells maintained their round or oval shape and contained several irregular masses as well as minute speckle-like structures (E). On the other hand, the neurons at the crest region of the granule cell layer (F) showed typical apoptotic profiles such as crescentic caps (open arrows) and fragmented nuclei (arrowheads). Scale bars: 10 mm.
very small population of cells in the dentate gyrus, i.e. at the border between the granule cell layer and the hilus (Fig. 3A) and occasionally around the crest region of the granule cell layer (Fig. 1F); in both places typical apoptotic structures were found (see below). Thus the present model allowed us to make a direct comparison of the morphological changes between the novel type of cell degeneration and typical apoptosis within the same materials. Nuclei of degenerating neurons reduced their sizes greatly even at 3 h after ischemia (Fig. 1). The diameters of these nuclei decreased to approximately half to one-third of the normal ones and remained at that size from 3 h up to 4 days after ischemia. These shrunken nuclei were intensely stained by TUNEL (Fig. 1D). Although these features appeared common to apoptosis, one of the discriminating points was that neither the nuclei nor cell bodies were fragmented (Fig. 1B, C, E), contrasting with the formation of apoptotic bodies in apoptosis (Figs. 1F and 3C). In fact electron microscopic observations (Fig. 2) revealed that the nucleus continued to be in its ordinary round or oval shape with the nuclear envelope still existing as the boundary
between the nuclear and cytoplasmic matrices, though nuclear pores appeared dilated and lost diaphragms, possibly allowing the passage of denatured nuclear substances through the pores. EM and CLSM observations revealed that the injured nuclei were further characterized by two types of uncommon structures, presumably resulting from chromatin condensation. The first one was composed of reticular-shaped structures scattered throughout the nucleus (Fig. 2). They appeared as numerous speckles when observed in single EM sections (Fig. 2A and B). When they were three-dimensionally reconstructed in serial EM sections, they were found to be linked to each other and formed a delicate network within the nucleus (data not shown, but see white arrows in Fig. 2B). This reticular structure was clearly distinguishable from the nuclear pattern in apoptosis (Fig. 3), where chromatin was uniformly aggregated into large discrete masses abutting on the nuclear membrane, typically seen as crescentic caps (Figs. 1F and 3B). It was difficult in Nissl-stained sections to recognize the reticular type of chromatin condensation presumably due to its thinner profile and rather weak staining.
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Fig. 2. Electron micrographs of CA1 pyramidal cells at 4 days (A – C) after hypoxic-ischemia and their earlier changes at 3 h (D, E). (A) Five degenerating neurons (d1–d5) are characterized by their small nuclei, novel type of chromatin condensation, and marked cytoplasmic disintegration. By contrast, three neighboring neurons (n1–n3) appear to preserve fairly normal ultrastructures. (B) Cell d2 in (A) is enlarged. Two irregular-shaped masses (asterisks) and numerous speckle-like structures are seen inside the nucleus. Note the interconnection (white arrows) among the speckle-like structures. Three presynaptic axon terminals (s) are in direct contact with this cell, one of which is further enlarged in (C). (C) The axon terminal (s) formed synaptic junction (crossed arrow) with the soma, clearly indicating that this cell was a neuron. Both plasma (arrows) and nuclear membranes (arrowheads) were not disrupted, though the nuclear pores were dilated. Mitochondria (m) also retained their inner and outer membranes together with some cristae-like structures but their internal structures already suffered severe damage. Organelles other than mitochondria disappeared almost completely at 4 days (B), but at 3 h (D) markedly swollen rough endoplasmic reticulum (rER) with numerous ribosomes abutting onto them (E) could be observed. Scale bars: 5 mm (A), 1 mm (B and D), 0.5 mm (C and E).
Another discriminating structure in the nucleus was composed of irregular-shaped masses (Fig. 2B, asterisks), that were also recognizable in Nissl-stained sections as several clumps inside the nucleus (Fig. 1B and C). Though they might appear in LM as if they were fragmented nuclei seen in apoptosis, EM observations clearly revealed that they remained inside the round or oval nucleus (Fig. 2A and B), being entirely different from apoptotic fragmented nuclei (Figs. 1F and 3D). Moreover, these intranuclear masses were also different
from the crescentic caps because they were rather irregular in shape and usually located apart from the nuclear membranes (Fig. 2A and B). These structures were assumed to be derived from denatured nucleoli, although we can not determine their origin at present. Novel morphological changes were also seen in the cytoplasm. Perikarya were initially swollen at 3 h after ischemia (Fig. 2D), then shrunk at 4 days (Fig. 2A–C). In the swollen cytoplasm, rough endoplasmic reticulum (rER) was markedly dilated but still encrusted with
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Fig. 3. Electron micrographs of cells displaying typical morphological features of apoptosis in and around the dentate granule cell layer at 3 h after hypoxic-ischemia. (A) A cell showing crescentic caps (arrows) was located at the border between the granule cell layer (g) and the hilus. (B) Crescentic caps were composed of uniformly aggregated chromatin that abutted on the nuclear membrane (arrowheads). (C) An apoptotic body containing closely packed mitochondria was observed in a position similar to (A). The most part of this apoptotic body was surrounded by a neighboring cell (nb). (D) An example of a cell with a fragmented nucleus. Scale bars: 5 mm (A), 1 mm (B – D).
numerous ribosomes (Fig. 2D and E). The outer membrane of the nuclear envelope underwent similar change to rER. By contrast, the mitochondria were only slightly swollen though the array of cristae appeared somewhat disordered (Fig. 2D). The Golgi apparatus and lysosomes were difficult to identify. At 4 days after ischemia the cytoplasm was more severely degraded and most of the organelles other than mitochondria disappeared from the perikarya; only amorphous material was left there. Even at this stage the mitochondria retained their inner and outer membranes as well as cristae-like structures (Fig. 2C), though integrity of the presumptive cristae was severely damaged. This was different from the usual course of necrotic changes. Moreover, in spite of the disintegrated nucleus and cytoplasm at 4 days, there was no disruption of the plasma membrane, on which several presynaptic terminals occasionally formed synaptic junctions (Fig. 2B,C). The absence of cell lysis was in sharp contrast with the generally depicted profiles in necrosis. On the other hand, the cellular surfaces were not protruded, different from the fragmented cell bodies seen in apoptosis. Profiles of apoptotic bodies were not observed in and around the hippocampal pyramidal cell layers (Fig. 2A) whereas they were occasionally seen in the above-mentioned subregions in the dentate gyrus (Fig. 3C) where typical apoptosis did occur. The vast majority of degenerating somata appeared to escape phagocytosis at least 4 days following ischemia. Although some microglia entered the pyramidal and granule cell layers, most of them were just in partial contact with the degenerating somata (Fig. 2A and B); profiles of microglia engulfing the degenerating perikaryon were seldom encountered. Though it requires further studies to elucidate the ultimate fate of the degenerating neurons shown here, the process of
removing these cells were much slower than might be expected in the known time course of apoptosis.
4. Discussion The present study demonstrated a novel form of neuronal degeneration characterized by distinctive morphological changes. In spite of the apparent resemblance to apoptosis in LM and the positive staining by TUNEL, detailed analysis by EM revealed the structural features clearly distinguishable from those in classical apoptosis. Moreover, neurons displaying these features coexisted with the typical apoptotic cells located in the same sections, indicating that the two forms were distinct morphological entities. On the other hand, the absence of cell lysis and the preservation of mitochondrial membranes was inconsistent with necrotic changes. Since the present form of cell injury was common to the vast majority of degenerating pyramidal and granule neurons, it could be concluded on morphological grounds that the general pattern of hippocampal neuronal death in the current model was not of typically apoptotic or necrotic in form. We also encountered similar profiles in the somatosensory cortex and the thalamus when selective neuronal death occurred in these areas. Furthermore, recent EM study investigating neuronal degeneration after systemic administration of neurotoxic agent 3acetylpyridine also revealed unequivocally non-apoptotic ultrastructures in the inferior olive (Wu¨llner et al., 1997) that closely resembled the ultrastructures shown here. Therefore the present form of neuronal degeneration appears to not be restricted to our model but might be more ubiquitous. Nuclear changes in the degenerating hippocampal neurons were unique. Interestingly, similar profiles were
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also reported in isolated hepatic nuclei treated with Ca2 + , a known activator of endonuclease (Oberhammer et al., 1993). The authors concluded that these hepatic nuclei were non-apoptotic in spite of positive in situ nick labeling and DNA ladder, which is consistent with our present observations. Ca2 + overload has been implicated in mechanisms of ischemic neuronal death in the hippocampal CA1 region (Deshpande et al., 1987; Choi, 1988). Elevated level of Ca2 + might play some roles in the formation of novel ultrastructural changes in the nuclei of postischemic neurons. The morphological changes shown in the present study have some features that differ from those in the wellknown delayed neuronal death in the hippocampus. This was assumed to be derived primarily from the differences in the experimental models used. Ischemia in the hippocampus has been investigated most frequently with either of the following two models: bilateral occlusion of the carotid arteries in gerbils (Kirino, 1982), or four-vessel occlusion (bilateral carotid and vertebral arteries) in rats (Petito and Pulsinelli, 1984; Deshpande et al., 1992), each lasting for brief periods (5 – 20 min). These methods have revealed a particular form of neuronal death that occurs preferentially in the CA1 region after several days of an asymptomatic period, hence the name delayed neuronal death. In the present study we used mice derived from a strain c57BL/6, since modern gene-targeting as well as transgenic techniques have been conducted most frequently on mice belonging to this strain. Because of the difficulty in conducting four-vessel occlusion in mice, we adopted a more simple model of hypoxic-ischemia. Although the extent of the damaged area varied among animals in the present study, the hippocampal neurons underwent consistent morphological changes qualitatively. In contrast with delayed neuronal death, degenerating neurons in the present model were not restricted to the hippocampal CA1 region but distributed more extensively in the CA3 region and the dentate granule cell layer. Another difference between the two forms of neuronal death was concerned with the time course of cell degeneration. The present form was characterized by the early onset and preservation for at least several days of the ultrastructural changes, contrasted with the delayed onset and the rapid disappearance of injured cells in the delayed neuronal death. The ultrastructural profiles in the present study were distinct from those in typical apoptosis, whereas there have been controversial data regarding the apoptotic nature of delayed neuronal death, partly owing to the lack of convincing ultrastructural evidence of apoptosis in delayed neuronal death. Since experimental conditions for the two methods were quite different we cannot conclude at present whether the morphological differences in the two forms of neuronal death reflected distinct mechanisms of cell death. Alternatively, essentially the same mechanisms might underlie
the two forms but the differences in some incidental conditions such as the duration of ischemia could lead to apparent morphological differences. An hypoxic-ischemia model with a shorter ischemic period or a delayed death model with a longer ischemic period would be helpful in elucidating the relationship of the two death patterns. The rapid recognition and removal of dying cells is an important feature of apoptosis, whereas necrotic cells are phagocytosed after the plasma membrane integrity is disrupted. In this sense, one of the distinguishing features in the present form of cell death was that the dying cells appeared to be relatively silent at least for several days, undergoing a slow degradation process inside the plasma membrane and keeping themselves from active scavenging reactions. In the light of the slow process of cell removal, we tentatively name the present form of cell death ‘lathiptosis’, according to the Greek words lathi (escaping notice) and ptosis (falling; a metaphor for death), to facilitate the comparison of several forms of potentially non-apoptotic cell death with the present form. Due to rapid progress in molecular biological research into apoptosis the concept of apoptosis is now fluctuating and may expand or be modified in the future, mainly based on new biochemical standards. Although we preliminarily confirmed that lathiptosis also occurred ubiquitously in the hippocampus of transgenic mice overexpressing bcl-2, the potent inhibitor of apoptosis, it still remains unknown whether lathiptosis can be included in the renewed category of apoptosis, or is totally different to apoptosis. It is necessary at present that morphological features of neuronal death in vivo should be re-examined in detail, in concert with biochemical investigation of the critical molecules such as caspases and related substances.
Acknowledgements We are most grateful to postgraduate student Tetsuya Takahashi and Research Associate Tetsuya Nagashima, Department of Philosophy, Faculty of Letters, Kyushu University, for their erudite suggestion of the Greek words.
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