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The Journal of Neuroscience, June 1, 1999, 19(11):4200-4210
Electron Microscopic Evidence against Apoptosis as the Mechanism
of Neuronal Death in Global Ischemia
Frederick
Colbourne1,
Garnette R.
Sutherland2, and
Roland N.
Auer1, 2
Departments of 1 Pathology and 2 Clinical
Neurosciences, Neuroscience Research Group, Faculty of Medicine,
University of Calgary, Calgary, Alberta, Canada, T2N 4N1
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ABSTRACT |
It has been repeatedly claimed that neuronal death in the
hippocampal CA1 sector after untreated global ischemia occurs via apoptosis. This is based largely on DNA laddering, nick end labeling, and light microscopy. Delineation of apoptosis requires fine structural examination to detect morphological events of cell death. We studied the light and ultrastructural characteristics of CA1 injury after 5 min
of untreated global ischemia in gerbils. To increase the likelihood of
apoptosis, some ischemic gerbils were subjected to delayed postischemic
hypothermia, a treatment that mitigates injury and delays the death of
some neurons. In these gerbils, 2 d of mild hypothermia was
initiated 1, 6, or 12 hr after ischemia, and gerbils were killed 4, 14, or 60 d later. Ischemia without subsequent cooling killed
96% of CA1 neurons by day 4, whereas all hypothermia-treated groups
had significantly reduced injury at all survival times (2-67% loss).
Electron microscopy of ischemic neurons with or without postischemic
hypothermia revealed features of necrotic, not apoptotic, neuronal
death even in cells that died 2 months after ischemia. Dilated
organelles and intranuclear vacuoles preceded necrosis. Unique to the
hypothermia-treated ischemic groups, some salvaged neurons were
persistently abnormal and showed accumulation of unusual,
morphologically complex secondary lysosomes. These indicate selective
mitochondrial injury, because they were closely associated with normal
and degenerate mitochondria, and transitional forms between
mitochondria and lysosomes occurred. The results show that untreated
global ischemic injury has necrotic, not apoptotic, morphology but do
not rule out programmed biochemical events of the apoptotic pathway
occurring before neuronal necrosis.
Key words:
ischemia; hypothermia; gerbil; CA1; necrosis; apoptosis; electron microscopy; mitochondria; lysosome
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INTRODUCTION |
The mechanisms by which postischemic
hippocampal CA1 neurons die remain controversial. Although initial
reports suggested that ischemic cell death was by necrosis, recent
papers propose an apoptotic death. Brief global cerebral ischemia in
rodents (Kirino, 1982 ; Pulsinelli et al., 1982) and humans (Petito et al., 1987; Horn and Schlote, 1992) results in a selective loss of
hippocampal CA1 neurons that typically succumb over a 24-72 hr period,
a time course that could be consistent with apoptosis. However,
ultrastructural reports describe an early proliferation of endoplasmic
reticulum (Kirino, 1982; Kirino and Sano, 1984; Yamamoto et al., 1990),
disaggregation of polyribosomes (Kirino and Sano, 1984; Deshpande et
al., 1992), and selective dendritic swelling (Johansen et al., 1984;
Yamamoto et al., 1990), findings that are not indicative of apoptosis
as a mechanism.
Nevertheless, it has been repeatedly suggested, largely on the basis of
biochemical criteria, that untreated neuronal death in the CA1 zone is
attributable to apoptosis (MacManus et al., 1993, 1995; Okamoto et al.,
1993; Kihara et al., 1994; Iwai et al., 1995; Nitatori et al., 1995;
Honkaniemi et al., 1996; MacManus and Linnik, 1997; Ni et al., 1998).
In this study we examined the ultrastructural events associated with
delayed CA1 neuronal death in global cerebral ischemia in gerbils.
Because there is evidence that milder insults are more likely to result
in apoptosis than necrosis (Kerr, 1971; Portera-Cailliau et al., 1977;
Wyllie et al., 1980; Harmon et al., 1990; Fukuda et al., 1993; Bonfoco et al., 1995; Choi, 1996; Du et al., 1996; Endres et al., 1998), we
used prolonged postischemic hypothermia, which results in some CA1
neurons dying over weeks to months (Colbourne and Corbett, 1994, 1995),
to mitigate the insult and create experimental conditions possibly more
favorable for apoptosis over necrosis.
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MATERIALS AND METHODS |
Animals. This experiment conformed to the guidelines
of the Canadian Council on Animal Care and was approved by a local
animal care committee of the University of Calgary. Female Mongolian gerbils were obtained from High Oak Ranch (Baden, Ontario, Canada) and
used at 16 weeks of age (~56 gm). Gerbils were subjected to either
normothermic ischemia (ISCH, HYPO-1, HYPO-6, and HYPO-12) or sham
operation (SHAM, SHAM+HYPO). Cooling was instituted 1 (HYPO-1,
n = 12), 6 (HYPO-6, n = 13; SHAM+HYPO,
n = 3), or 12 hr (HYPO-12, n = 12)
after the end of ischemia/sham occlusion. The SHAM (n = 7) and ISCH (n = 15) groups were not subjected to postischemic hypothermia. One animal died during ischemia, and two were
excluded because of poor reflow, established by direct visual
inspection of the carotid artery. One HYPO-12 gerbil died approximately
2 months after ischemia of unknown cause. These animals were not
included in the data analysis.
Brain temperature. Similar to previous work (Colbourne et
al., 1996), brain temperature (dorsal striatum) was measured by wireless AM probes (model XM-FH, Mini-Mitter Co., Sunriver, OR) that
were secured inside a guide cannula. A 5.0 mm cannula was implanted at
the dural surface 4 d before ischemia/sham occlusion. Two days
after cannula placement, probes were inserted, and brain temperature
was collected up to 4 d after the time of ischemia/sham surgery.
Temperature was collected (DataQuest IV, DataSciences, St. Paul, MN)
while animals were individually housed in cages that rested on
telemetry receivers. Animals had free access to food and water and were
housed under diurnal lighting conditions.
Ischemia. Surgical techniques were similar to those reported
in previous work (Colbourne and Corbett, 1995). Briefly, gerbils were
anesthetized with 2.0% halothane (70% N2O, 30%
O2) followed by maintenance at 1.5%. They were then
wrapped in a homeothermic body blanket (Harvard Apparatus, South
Natick, MA). After a ventral neck incision, the carotid arteries were
isolated and occluded for 5 min under 0.5-1% halothane (except SHAM
and SHAM+HYPO gerbils). The neck wound was sutured, and a large portion
of the back and abdomen was shaved to facilitate cooling. The duration
of surgery was ~20 min. At the end of surgery, animals were
individually housed in cages for up to 4 d of postischemic
temperature measurement/control.
Temperature control. Core temperature was held near 37.0°C
during ischemia, and brain temperature was maintained close to a
desired 36.4°C. The latter was regulated by feedback control of an
infrared lamp (250 W) placed ~50 cm above the gerbil (Colbourne et
al., 1996). All groups except SHAM, which freely thermoregulated, were
subjected to postischemic temperature control. In an effort to minimize
variability, the untreated ischemia groups (ISCH) were regulated for 24 hr to mimic a mild hyperthermic pattern taken from an average of 37 untreated ischemic gerbils who spontaneously developed this
hyperthermia (Colbourne and Corbett, 1994, 1995). After this,
hypothermia (<35.5°C) was prevented for an additional 24 hr. The
cooled groups were similarly maintained until the initiation of
hypothermia at 1, 6, or 12 hr after ischemia. At that time, groups were
slowly (1.0°C/30 min) cooled to 32.0°C and held at that temperature
for 24 hr. They were then gradually rewarmed to 34.0°C where they
were kept for 24 hr. Finally, they were warmed to 35.0°C and kept
within the 35.0-36.0°C range for 12 hr, after which they were
monitored until 96 hr after ischemia. The slow rates of cooling/warming
were chosen to mimic the clinical situation. Postischemic temperature
control was achieved in the awake, freely moving animal by a
thermoregulator program that controlled a lamp (100 W incandescent or
175 W infrared lamp), fan, and water-misting system for each animal
(Colbourne et al., 1996). Temperature was typically held to within
0.25°C of the desired value except for one SHAM+HYPO gerbil in
which induction of hypothermia was ~1 hr slower than desired for no
apparent reason.
Light microscopy. Gerbils were killed at 4, 14, or 60 d
after ischemia by an overdose of sodium pentobarbital. They were then perfused with ~15 ml of heparinized saline followed by ~60 ml of
Karnovsky's fixative (3% glutaraldehyde, 4% formaldehyde). Brains
were refrigerated (~4°C) in situ overnight before
extraction and blocked into anterior and posterior sections (~2 mm
posterior to bregma). The anterior block, initially fixed with
Karnovsky's solution, was soaked in 10% formalin for several days
before being embedded in paraffin, sectioned at 6 µm, and stained
with hematoxylin and eosin. Glutaraldehyde was replaced with
formaldehyde to improve paraffin embedding and tissue-sectioning
quality. The number of viable CA1 neurons (Colbourne and Corbett, 1995)
in each of medial (adjacent to subiculum), middle (apex of CA1), and
lateral sectors (next to CA2) was then examined at ~1.7 mm posterior
to bregma (Loskoto et al., 1975). CA1 counts were summated over the six sectors (three per hemisphere).
The ISCH and HYPO groups were killed at 4, 14, and 60 d, whereas
the SHAM gerbils were killed at 60 d only and the SHAM+HYPO gerbils were killed at 14 d. There were four or five gerbils per survival time for each of the HYPO groups. The ISCH groups had five
gerbils at each survival time. These numbers were deemed sufficient
because untreated gerbils subjected to 5 min of normothermic ischemia
consistently have near-total CA1 loss (Colbourne and Corbett, 1994,
1995).
Electron microscopy. Immediately after the removal of the
brain from the skull, the posterior hippocampi at ~2.0-2.3
mm posterior to bregma were dissected free, and small slices of CA1
were taken and placed in 2.5% glutaraldehyde solution. This level of
CA1 has a similar degree of neuronal injury as that taken for cell counts (Colbourne and Corbett, 1994, 1995; Nurse and Corbett, 1994).
Care was taken to discard adjacent subiculum and CA2/CA3 regions.
Usually, four to eight semithin (1 µm) sections were stained with
toluidine blue, whereas trimmed ultrathin (600 Å) sections were
stained with uranyl acetate and lead citrate and examined under a
transmission electron microscope (Hitachi M600). Approximately 40,000 neurons or cell remnants were examined in this study, and 638 photographic plates were prepared.
As a positive control for apoptosis, three naive postnatal day 6 (PD6)
gerbils were similarly processed, and semithin and ultrathin sections
of the striatum and midbrain colliculi were examined for signs of
developmental apoptosis.
Statistics.The CA1 cell counts and the temperature data were
analyzed with ANOVA and specific contrasts as performed previously (Colbourne and Corbett, 1994, 1995). When heterogeneity of variance was
detected by a significant Levene's test, we used separate t
tests instead of the usual pooled t tests (BMDP
statistical package). Electron microscopy (EM) findings were not
statistically analyzed because these data were qualitative.
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RESULTS |
Temperature and weight data
Brain temperature (Table 1) during
occlusion or in the first hour thereafter was maintained close to the
baseline temperature of 36.4°C, with no notable differences between
ischemic groups (ISCH, HYPO-1, HYPO-6, HYPO-12). Brain temperature
after ischemia (Fig. 1) was regulated to
the desired profiles outlined in Materials and Methods. There were no
notable differences between subgroups (i.e., different survival
times).
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Table 1.
Mean (±SD) brain temperature (°C) during the day before
ischemia (baseline), during ischemia/sham occlusion, and in the
first hour
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Figure 1.
Brain temperature averaged every hour
starting after ischemia/sham occlusion to 4 d later. Data were
collected every 20 or 30 sec but plotted as averaged 1 hr intervals.
All ischemic groups and the SHAM+HYPO group were regulated for 2 d
after ischemia (see Materials and Methods), whereas SHAM animals were
not manipulated. Servo-control was achieved in awake, freely
moving gerbils by independent use of fan, lamp, and a water-misting
system. See Table 1 for baseline, occlusion, and first hour mean
temperatures.
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All gerbils weighed ~56 gm on the day of ischemia/sham occlusion.
ISCH gerbils lost 2 gm on average by day 4, whereas the hypothermic
animals lost ~5 gm, similar to previous findings (Colbourne and
Corbett, 1995). Animals eventually regained this weight but the HYPO-12
group was slower to normalize than the HYPO-1 and HYPO-6 groups.
Quantitative CA1 cell counts (hematoxylin and
eosin-stained tissue)
CA1 cell counts 1.7 mm posterior to bregma are presented in Figure
2. There was no damage (i.e.,
eosinophilia) in the SHAM and SHAM+HYPO gerbils, and thus they were
combined for subsequent statistical analyses. Damage in the untreated
ISCH group was near total (~96% loss) by 4 d, and because cell
counts were the same at 4, 14, and 60 d these groups were combined
for statistical comparisons. Hypothermia significantly reduced CA1
injury in all groups at all survival times. The near-total protection
in the HYPO-1 groups was similar (t < 1) at the 4, 14, and 60 d survival times. There was a significant decline in cell
counts from 4 to 14 d in the HYPO-6 groups
(t7 = 3.55, p = 0.0094), whereas
the other comparisons were not statistically significant
(p  0.1236). Although the decline in cell
counts with longer survival times in the HYPO-12 groups indicated a
continued loss of CA1 neurons, this was not significant
(p 0.2309).
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Figure 2.
CA1 cell counts expressed as a mean percentage of
normal (SHAM and SHAM+HYPO groups) ±SD. Cell counts in SHAM and
SHAM+HYPO groups were the same and thus were combined. Likewise, cell
counts in the three ISCH sub groups were very similar (differed by only
a few cells) and were combined into one group for statistical
comparisons. Hypothermia significantly blunted injury in all groups
(*p < 0.05, **p < 0.01, ***p < 0.001), but the greatest benefit occurred
with the 1 hr intervention delay where CA1 injury was almost totally
abolished. There was no overlap between CA1 counts in any HYPO group
and the ISCH groups.
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Light microscopy of semithin sections (toluidine
blue staining)
A few dark neurons were noted in toluidine blue-stained semithin
sections from the SHAM group (Fig.
3A). Dark neuronal profiles are known to occur independently of brain regions of ischemia (Garcia
et al., 1995) and even to occasionally appear in aldehyde-fixed tissue
of normal CNS tissue (Cohen and Pappas, 1969).
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Figure 3.
Light microscopic CA1 neuronal findings. Dark
neurons were rare but were found in all groups, as is characteristic of
aldehyde-fixed tissue. The one illustrated (A) is
from a SHAM animal. Hypothermia-treated ischemic animals had robust CA1
neuroprotection (B), but occasional neurons
(arrow) had granular inclusions (also see Figs. 7, 8).
Untreated ischemia produced near-total CA1 loss by 4 d after
ischemia (C), and neuronal cytolysis and
karyorrhectic debris (arrow) predominate in the field.
Necrotic neurons from HYPO groups had similar light microscopic
morphology as from ISCH groups. Large clumps of karyorrhectic debris
might be confused with apoptotic bodies under lower magnification.
However, no apoptotic body was found in any ISCH or HYPO group. For
control, an apoptotic body (compacted chromatin enclosed within a
membrane) is shown (D) from the tectum of a
neonatal gerbil. The same apoptotic body in D is shown
with briefer exposure to illustrate the chromatin mass within the dense
body (inset). Tissue was embedded in Epon, and sections
were stained with toluidine blue. Scale bar (shown in D
applies to A-D): 20 µm.
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Almost all neurons in the CA1 sector of ISCH animals had died by 4 d, as indicated by the quantitative cell counts (Fig. 2). Although many
of these neurons were in an advanced state of degeneration, clumping
chromatin in aggregates of various sizes was still quite evident by
light microscopy (LM) (Fig. 3C). Concomitant with extensive degeneration was a marked gliosis at 4 and 14 d survival.
Hypothermia treatment significantly reduced the number of injured or
killed cells, but some protected neurons showed cytoplasmic granules
(Fig. 3B). Regardless, LM signs of degeneration in HYPO gerbils (e.g., clumped chromatin), which occurred at all survival times, were similar to those seen in ISCH animals. Apoptotic bodies (Fig. 3D) seen in our positive control of developmental cell
death were not found in any adult ischemic animal. However, under low magnification the coarse, punctate chromatin and globular karyorrhectic aggregates of necrotic cells could be misconstrued as apoptotic bodies.
Electron microscopy
Although there were no damaged neurons in the SHAM and SHAM+HYPO
groups (Fig. 4A), an
occasional dark neuron was found (Fig. 4B). These
were shrunken, but there were no signs of degeneration. Occasionally,
small intranuclear vacuoles (Fig. 4A) were noted in
the SHAM group, but these were never as extensive as that found in ISCH
and HYPO groups. ISCH gerbils at day 4 revealed near-total CA1
pyramidal cell disintegration of nuclear and cell membranes and
amorphous cytoplasm (Fig. 4C) with phagocytic microglia and macrophages. At later survival times, such neurons consisted only of
debris, whereas some other cells, which presumably had recently died,
had earlier ultrastructural signs of necrosis. The qualitative ultrastructural characteristics of ischemic CA1 injury were generally identical in ISCH and HYPO groups at all survival times, but
quantitatively, there were fewer injured cells in the hypothermia
groups.
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Figure 4.
CA1 neurons from control animals (SHAM) showing
small lysosomes and some intranuclear vacuoles
(A) and a dark but undamaged neuron
(B). CA1 cell necrosis in untreated ischemia
(ISCH) showed nuclear and plasma membrane breaks, clumped tigroid
chromatin, and amorphous organelles with mitochondrial flocculent
densities after 4 d survival (C). Although
less prevalent after hypothermic-treated ischemia (D,
HYPO-12, 4 d survival), neuronal necrosis has an identical
ultrastructural appearance. Necrotic neurons had variable electron
density. Apoptotic bodies, like those found in neonatal gerbil striatum
(E), were never found in any adult gerbil
examined by light and electron microscopy. Scale bars, 5 µm.
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Lethally injured neurons had clumped tigroid chromatin, discontinuous
nuclear and plasma membranes, and amorphous organelles (Fig.
4C,D). Mitochondrial flocculent densities, which are another sign of cell death, were also observed (Fig. 4C).
Surrounding tissue was highly vacuolated with numerous membranous
whorls within neurons and in the neuropil. Necrotic neurons that had an
electron-dense and often microvacuolated appearance were easily
distinguished from dark, uninjured neurons (e.g., from SHAM) (Fig.
4B) because the latter did not have organelle or
membrane damage. Morphological signs of apoptosis (i.e., apoptotic
bodies containing chromatin) were never found in any ischemic or
hypothermia-treated ischemic gerbil. Conversely, apoptotic bodies were
easily found by LM (Fig. 3D) and EM (Fig.
4E) in the neonatal gerbil brain.
Most of the few remaining CA1 neurons in the ISCH groups were injured,
whereas a much smaller percentage of the remaining CA1 neurons in HYPO
groups were damaged. Prelethal signs of injury in ISCH and HYPO groups,
which were similar at 4, 14, and 60 d, included dilated organelles
[i.e., Golgi apparatus, rough endoplasmic reticulum (RER), and
mitochondria] (Fig. 5A,B),
massive intranuclear vacuoles, and membranous whorls. The occurrence of
intranuclear vacuoles was much more common in ischemic than in SHAM
animals, where it was rare. Large lysosomal aggregates (Fig.
5C) were occasionally found in viable neuronal processes
in the ISCH and HYPO groups. These aggregates contained both primary
and secondary lysosomes. Rarely, surviving neurons showed a
proliferation and lamellar appearance of RER (Fig. 5D).
Extensively dilated axons, dendrites, and astrocytes were also a common
finding.
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Figure 5.
In contrast to the terminal necrotic changes shown
in Fig. 4, some of the remaining neurons showed sublethal alterations
that were either unique or present far in excess of their incidence in
controls. Dilated organelles, including RER, Golgi apparatus, and
mitochondria as well as extensive intranuclear vacuoles
(arrows) (A, ISCH 4 d
survival; B, HYPO-6, 14 d survival) and large
clusters of primary and secondary lysosomes (C, HYPO-12,
2 month survival) were seen in untreated and hypothermia-treated
ischemic groups. Rarely, neurons contained stacks of proliferated RER
(D, HYPO-12, 4 d survival). Scale bars, 5 µm.
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Densely stained inclusions were frequently found in dendrites (Fig.
6A) and axons (Fig.
6B) at 4 d and especially 14 and 60 d
survival times. These inclusions were admixed among dense, pyknotic, or
degenerate mitochondria. These structures were deemed to be mitochondria from their double-membrane structure and visible internal
cristae. These pyknotic mitochondria and other small inclusions were
found in both ISCH and HYPO gerbils as well as in neonatal gerbils
(Fig. 6C).
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Figure 6.
Dendritic (A, HYPO-12, 14 d
survival) and axonal (B, HYPO-1, 2 month survival)
pyknotic inclusions as found in HYPO and ISCH groups. Similar clusters
were found in PD6 gerbils (C, colliculus). Because
normal (A, arrow) and degenerate mitochondria can be
seen with these clusters, these dense inclusions are likely
degenerating mitochondria. Scale bars, 1 µm.
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The granules seen with LM in hypothermia-treated ischemic gerbils
proved to be lysosomal bodies on EM (Fig.
7, 8). The few CA1 sector neurons
containing these complexes were otherwise healthy looking. Adjacent
neurons were often normal (Fig. 7). These inclusions were sometimes
membrane bound and frequently tubulovesicular in appearance (Fig.
8A). Transitional forms
between mitochondria and lysosomes suggested a progression between the
two. These differed from, or were a more advanced form of, the more
typical mitochondrial autolysosome (Fig.
9B,D) yet were distinct from
the large lysosomes found in some cells (Fig. 5C) and also
from the pyknotic mitochondrial clusters (Fig. 6) found in axons and
dendrites.
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Figure 7.
Some CA1 neurons of all HYPO groups at all
survival times showed large numbers of accumulating bodies, probably
derived from mitochondria (see Fig. 9). The neuron containing this
large number of autolysosomes was otherwise healthy looking (HYPO-1,
14 d survival). Adjacent neurons look normal. Scale bar, 5 µm.
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Figure 8.
View of early (A) and late
(B) mitochondrial evolution into lysosomes. On
the left are seen a group of mitochondria with variably
disorganized cristae and a tubulovesicular internal structure. The
appearance of homogenous electron dense streaks suggests lipid
concentration within them. Other groups of cytoplasmic bodies
(B) appear more electron dense throughout most of
their internal structure, apart from the small, round body left
of center, which has a double membrane (arrows)
indicating a mitochondrial origin. Scale bars, 1 µm.
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Figure 9.
Progression of mitochondrial injury to
autolysosomes. All of these forms were found only in
hypothermia-treated ischemic animals at all survival periods. Double
membranes (broad arrows, A, B, F) as well as
remnant cristae (white arrows, A, B, E, F) attest
to the mitochondrial origin of these structures. Collapsing internal
membranes are seen (C, D, G). Increased internal
electron density (F-I) suggests eventual
accumulation of lipid or lipofuscin-like material. Scale bars, 1 µm.
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Glial apoptosis was looked for (Petito et al., 1998), but no evidence
for this was found here.
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DISCUSSION |
It has been repeatedly suggested that global cerebral
ischemia can produce apoptosis of CA1 sector cells (MacManus et al., 1993, 1995; Okamoto et al., 1993; Kihara et al., 1994; Iwai et al.,
1995; Nitatori et al., 1995; Honkaniemi et al., 1996; MacManus and
Linnik, 1997; Ni et al., 1998). This mode of cell death is thought to
be more prevalent after milder insults (Kerr, 1971; Wyllie et al.,
1980; Harmon et al., 1990; Fukuda et al., 1993; Bonfoco et al., 1995;
Choi, 1996; Du et al., 1996; Endres et al., 1998). However, the present
examination of an estimated 40,000 neurons failed to find any
morphological evidence of apoptosis. Attenuation of the massive CA1
ischemic neuronal death using hypothermia led to mitigated neuronal
injury and the discovery of new inclusion bodies in neurons, but still
no evidence of apoptosis, even in neurons that died more slowly.
Apoptosis and necrosis have distinct morphology, and EM is necessary to
distinguish these. Necrosis is seen as a primarily cytoplasmic event.
Thus, the present morphological findings of early organelle
(mitochondria, RER, Golgi apparatus) swelling, disaggregation of
polyribosomes, and subsequent lethal findings of mitochondrial
flocculent densities, and cell and nuclear membrane breaks are clearly
indicative of neuronal necrosis, not apoptosis. Other ultrastructural
studies in ischemia either have found features that do not argue for
either apoptosis or necrosis, such as proliferation of rough
endoplasmic reticulum (Kalimo et al., 1977; Kirino and Sano, 1984;
Yamamoto et al., 1990; Tomimoto et al., 1993) or have found early
features of excitotoxic necrosis, such as dendritic swelling (Johansen
et al., 1984; Yamamoto et al., 1990). Indeed, most ultrastructural
studies of global ischemic CA1 neuronal death have found features of
necrosis, not apoptosis (Kirino and Sano, 1984; Yamamoto et al., 1990;
Deshpande et al., 1992; Tomimoto et al., 1993). Only a single (Nitatori
et al., 1995) electron microscopic study has claimed apoptotic
morphology after transient forebrain ischemia, but the cell remnant
labeled as apoptotic resembles our Figure 4C,D and is
necrotic debris, because it lacks a cell membrane.
We show here that the features of necrosis were constant, regardless of
the time of cell death or the application of postischemic hypothermia.
The novel cytoplasmic inclusions in CA1 neurons were limited to the
hypothermia-treated ischemic gerbils. Although CA1 neurons containing
these bodies were otherwise normal in appearance, the existence of
these massive numbers of secondary lysosomes, and transitional forms
from mitochondria, indicates that they received a borderline or
sublethal insult perhaps affecting mitochondria selectively. The
selective involvement of mitochondria per se does not argue against
apoptosis or necrosis, because mitochondria are involved in both modes
of cell death (Zamzami et al., 1996, 1997). Although these inclusions
were distinct from the more classic autolysosomes, the latter may
eventually form the larger structures (Ghadially, 1988). Although
primary mitochondrial pathology has been proposed to contribute to
ischemic cell death (Abe et al., 1995), mitochondrial autolysosomes
were only found if ischemia was followed by hypothermia. Perhaps this
is because hypothermia allowed ischemic neurons to survive long enough
for turnover and accumulation of degenerate mitochondria. The duration
of hypothermia or the species (rat vs gerbil) might also be a factor,
because autolysosomes were not reported in rats subjected to global
ischemia with and without treatment with 3 hr of immediate postischemic hypothermia (Dietrich et al., 1993).
Mitochondrial injury was also commonly observed in axons and dendrites
of ischemic and hypothermic groups in variably sized clusters of small
inclusions. Similar figures have been illustrated previously, but not
identified, within the hippocampus (Bonnekoh et al., 1990) and cortex
(Nishikawa et al., 1989) of ischemic animals. These small inclusions
intermixed with dense mitochondria appear to be nonspecific signs of
mitochondrial cellular injury, because they resemble the pyknotic
mitochondria in renal necrosis induced with D-serine
(Wachstein and Besen, 1964), in cortical neurons treated with glycine
antagonist (Auer, 1997), in Wallerian degeneration (Symmens, 1986), and
in neonatal gerbils (present study). Thus, these small whorled
membranous bodies found intermingled with dense mitochondria probably
derive from mitochondria undergoing involution (Ghadially, 1988) after
cell death or serious cellular injury.
Biochemical, genetic (MacManus and Linnik, 1997), and pharmacological
(Goto et al., 1990; Chen et al., 1998; Himi et al., 1998) studies have
been interpreted to show that CA1 sector death after global ischemia
occurs by an apoptotic pathway. Caspase activation has been shown to
occur in CA1 cells in ischemia (Chen et al., 1998; Ni et al., 1998),
but whether such activation is causal or merely a concomitant of
neuronal death remains to be shown. Some of these results may merely
reflect DNA breaks occurring with necrosis (Petito et al., 1997).
Apoptotic-like DNA fragmentation can occur in necrotic cells (Collins
et al., 1992; Bicknell and Cohen, 1995; Dong et al., 1997). The
widespread reliance on only terminal deoxynucleotidyl
transferase-mediated biotinylated UTP nick end labeling (TUNEL)
staining to label apoptotic cells is fraught with hazard, because TUNEL
also tags necrotic neurons (Gold et al., 1994; Grasl-Kraupp et al.,
1995; de Torres et al., 1997).
In summary, only our positive control for apoptosis (i.e., PD6 gerbil
brain) showed membrane-delimited apoptotic bodies. Demonstration of
membrane delimitation requires EM. We found no neuron subjected to
ischemia or inadequately protected by delayed hypothermia that showed
features other than prelethal or lethal signs of necrosis. However, a
novel feature in hypothermically salvaged neurons was the cytoplasmic
aggregation of lysosomal-like bodies in persistently surviving CA1
neurons. Given that these large inclusions appear to originate from
mitochondria, a prolonged mitochondrial pathology may contribute to the
very slow cell death that can occur after postischemic hypothermia.
| |
FOOTNOTES |
Received Jan. 21, 1999; revised March 11, 1999; accepted March 15, 1999.
We gratefully acknowledge research support by the Medical Research
Council of Canada (MT-9935) to R.N.A. and by the Heart and Stroke
Foundation of Canada to G.R.S. F.C. was the recipient of a
fellowship from the Heart and Stroke Foundation. We are also grateful
to the Foothills Hospital electron microscopy support staff and to Dr.
D. Raki for histology processing and Drs. D. Corbett and
K. A. Sharkey for their helpful comments on this manuscript.
Correspondence should be addressed to Dr. Roland N. Auer, Department of
Pathology, Faculty of Medicine, University of Calgary, 3330 Hospital
Drive N.W., Calgary, Alberta, Canada, T2N 4N1.
| |
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M. Fujimura, Y. Morita-Fujimura, N. Noshita, T. Sugawara, M. Kawase, and P. H. Chan
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T. L. Briones and B. Therrien
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TOP
ABSTRACT
INTRODUCTION
