(Stroke. 1997;28:163-170.)
© 1997 American Heart Association, Inc.
Articles |
Subtle Neuronal Death in Striatum After Short Forebrain Ischemia in Rats Detected by In Situ End-Labeling for DNA Damage
the Neuroscience Research Institute (R.S.-K., A.M.H.) and the Department of Physiology (H.F.), Faculty of Medicine, University of Ottawa (Canada).
Correspondence to Dr Rainald Schmidt-Kastner, Cerebral Vascular Disease Research Center, Department of Neurology (D4-5), University of Miami School of Medicine, PO Box 016960, Miami, FL 33101.
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Background and Purpose Neuronal cell death after global brain ischemia occurs predominantly by necrosis, whereas only a minor fraction of cell death may occur through apoptosis. Brief or moderate insults are thought to facilitate apoptosis, which is associated with DNA fragmentation. After 10 minutes of four-vessel occlusion in rats, conventional neuropathological analysis shows neuronal cell death in hippocampal CA1 but not in the striatum. Thus, we compared hippocampus and striatum for occurrence of cells with DNA fragmentation.
Methods A brief insult of 10 minutes of forebrain ischemia was induced in rats using four-vessel occlusion, and groups of brains were studied at 1, 3, 6, and 12 hours and at 1, 3, and 7 days after ischemia. In situ end-labeling (ISEL) was used to detect neurons undergoing DNA fragmentation. The hippocampal CA1 area was compared with the striatum. Conventional staining and immunohistochemical markers served to exclude ischemic neuronal cell death in the striatum.
Results Hippocampal CA1 neurons were ISEL-positive by 3 days after ischemia. In contrast, positive cells became evident in the striatum between 3 hours to 3 days after ischemia. The ISEL-positive cells were scattered throughout the striatum with a preference for the dorsomedial areas and accounted for about 0.2% of the neurons per striatal area at 1 day. Conventional staining and immunohistochemical markers failed to reveal areas of overt cell damage in the striatum.
Conclusions The scattered cell damage in the striatum after brief forebrain ischemia suggests the occurrence of an apoptotic process. The striatum therefore may be prone to subtle cell death due to metabolic insults.
Key Words: apoptosis • cerebral ischemia, global • DNA damage • hippocampus • rats
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Introduction |
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Brain ischemia can damage neurons by a variety of pathobiochemical mechanisms.1 2 3 4 5 Recently, the concept of programmed cell death (apoptosis) has raised increasing interest in the context of ischemic cell death of neurons.6 7 8 9 10 11 12 13 14 15 16 17 18 19 CA1 neurons in the hippocampus die with a considerable delay of 2 to 4 days after transient global brain ischemia, which points to the activation of self-destructive mechanisms in this form of ischemic cell death. It was previously proposed that CA1 neurons might fail to undergo regular programmed cell death at the appropriate time of development and that retention of such mechanisms could place these cells at special risk during ischemia.5 However, it is now believed that in addition to CA1 neurons, the ischemic death of other neurons also contains a component of programmed cell death.6 8 9 12 14 The importance of the apoptosis concept lies in the fact that neurons appear to contain genetic programs for the active execution of cell death.20 More important, since the programmed steps leading to apoptosis may be subject to modulation by extracellular conditions, neuronal apoptosis may be influenced by therapeutic approaches.21 22 Because trophic factors can be used to counteract neuronal loss during programmed cell death in the developing nervous system, it is also believed that use of trophic molecules might play a role in prevention of cell death in adult neurons.23 24
Evidence that ischemic cell death includes a significant component of apoptosis derives mainly from studies using in situ end-labeling (ISEL, also TUNEL) as a marker of DNA fragmentation.25 26 To date, a large number of studies have described the appearance of ISEL-positive cells in ischemic brain.6 7 8 9 10 13 14 15 16 17 18 19 Most of these studies examined rodent brains after a severe ischemic impact, where ischemic damage is entirely predictable. However, the end-labeling techniques do not clearly distinguish between apoptosis and necrosis, since the latter also involves DNA fragmentation.8 Thus, additional support for neuronal apoptosis derives from the distinctive "laddering" in agarose gels with DNA extracted from ischemic brain tissue.6 7 8 9 10 12 13 14 16 18 19 Apoptosis has been studied in a variety of cell systems, and a common feature is that mild insults cause apoptosis, whereas stronger insults produce necrosis.21 22 27 28 Because the known morphological features of programmed cell death are not generally observed during ischemic cell death of neurons, the traditional view that severe ischemia leads primarily to necrosis remains largely unchallenged.4 Nevertheless, it now appears likely that a fraction of cells may also die from apoptosis and could therefore be amenable to a molecular-based treatment approach.
Apoptosis typically occurs in scattered individual cells rather than in all cells in a given area, suggesting that in any given population of cells, some are more susceptible to injury than others.21 22 27 28 Therefore, it is important to establish which brain cells exhibit enhanced susceptibility to apoptosis after mild insults. One way of approaching this problem is to induce brief brain ischemia and to compare vulnerable regions with resistant areas using conventional criteria for ischemic necrosis. Transient global forebrain ischemia of short duration leads to delayed neuronal death in the CA1 area of the hippocampus, whereas the striatum and neocortex remain intact.29 The aim of the present study was therefore to search for ISEL-positive cells in the striatum and cortex after a 10-minute insult of forebrain ischemia induced by four-vessel occlusion in rats. ISEL-positive cells in hippocampal CA1 served as a reference. To avoid a priori judgment of the results, we use the terms "ISEL-positive cells" or "DNA damage," leaving open the issue of whether such cell death is entirely due to apoptosis or also necrosis.
The absence of overt necrotic neuronal death in the striatum after this mild insult was studied using conventional staining of paraffin sections and immunohistochemistry with cell-specific markers for neurons and glial cells on cryostat sections.
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Ischemia Model
Dense forebrain ischemia was induced in male Wistar rats (210 to 220 g body weight) using the four-vessel occlusion model.30 The experiments were approved by the local animal care committee. On day 1, the vertebral arteries were electrocauterized with rats under inhalation anesthesia with 1.5% to 2% halothane in oxygen, and threads were placed around the common carotid arteries. On day 2, animals were reanesthetized with 1.5% to 2% halothane, and the common carotid arteries were occluded with aneurysm clips for 10 minutes. Rectal temperature was measured, and body temperature was maintained above 37.0°C using a heating pad. Tympanic temperature was monitored during occlusion using a miniature probe inserted into the auditory canal. Animals devoid of righting reflex were used in the study. No seizures or unexpected deaths occurred after ischemia. Sham controls had vertebral artery occlusion and a sham exposure of the carotid arteries.
Fixation and Brain Processing
One series of animals was killed while under deep halothane anesthesia at 1, 3, 6, and 12 hours and 1, 3, and 7 days after ischemia (n=4, each group), and the brains were frozen in methylbutane over liquid nitrogen. Control brains were collected in the same way. Sections (12 µm) were prepared in a cryostat, mounted onto silan-coated slides and stored at -80°C. Another series of animals was deeply anesthetized, and the brains were fixed by perfusion with 4% paraformaldehyde in PBS (pH 7.4) after 7 to 8 days of survival (n=10). One set of brains (4 with 7-day survival, 2 controls) was embedded in paraffin, and sections were cut at 6 µm for hematoxylin and eosin (H&E) staining. The other brains (3 at 7-day and 3 at 8-day survival, 2 controls) were cryoprotected in sucrose, frozen, and sectioned at 50 µm in a cryostat for immunohistochemical studies.
In Situ End-Labeling
The protocol for histochemical detection of DNA breaks was based on previously published procedures.16 25 26 Unless otherwise specified, all reagents were from Sigma Chemical Co or BDH. Frozen cryostat sections were thawed and fixed in 1% glutaraldehyde for 15 minutes at room temperature and were then washed twice for 5 minutes each with PBS. The sections were subsequently permeabilized with methanol/acetone (1:1) for 10 minutes at room temperature and washed twice with PBS. They were then incubated with 20 µg/mL proteinase K in 25 mmol/L Tris-HCl (1 mL per section), pH 6.6, for 15 minutes at room temperature. They were washed twice for 15 minutes each with water, then stained with the nuclear dye Hoechst 33258 (0.05 µg/mL) for 30 minutes at room temperature while being protected from light, and washed three times for 1 minute each in PBS. Sections were then incubated in 75 µL of a buffer solution containing 200 mmol/L potassium cacodylate, 2 mmol/L CoCl2, 0.25 mg/mL BSA, 25 mmol/L Tris-HCl pH 6.6, 10 µmol/L biotin-16-dUTP (Boehringer Mannheim Canada), and 25 U terminal transferase (Boehringer) for 1 hour at 37°C in a humidified chamber. The reaction was terminated by washing the sections three times for 1 minute each with PBS at room temperature. The sections were then incubated with 1 mL of solution containing 2.5 µg/mL fluorescein isothiocyanate-avidin (avidin FITC), 4x SSC buffer, 0.1% Triton X-100, and 5% powdered milk for 30 minutes at room temperature while being protected from light. Positive control samples were prepared by incubating sections with 10 U/mL DNAse I for 20 minutes at 37°C before treatment with terminal transferase. Sections were washed three times with PBS and then coverslipped in "anti-fade solution" (1 mg/mL p-phenylenediamine, 90% glycerol in PBS). Fluorescence was examined and photographed with a Zeiss Axiophot microscope. To ensure the validity of the comparative analysis, histochemical staining was performed simultaneously on a series of sections (hippocampus controls, 1 hour to 3 days; striatum controls, 1 hour to 7 days). The striatum series was repeated for confirmation.
Neuropathology
Paraffin sections were prepared from brains of animals surviving ischemia by 7 to 8 days. Sections at three levels through the central portion of the striatum were stained with H&E for analysis of ischemic neuronal damage.31
Immunohistochemistry
A set of antibodies was used to examine the striatum for cell damage after 7 to 8 days of survival. Antibodies to the neuron-specific protein NeuN were used as a general neuronal marker,32 and antibodies to parvalbumin, calbindin, and calretinin served to label subclasses of striatal neurons.33 34 Antibodies to synapsins provided a global labeling of the neuropil and permitted the exclusion of minute infarction.35 Finally, antibodies to glial fibrillary acidic protein (GFAP) were used to document reactive astrocytosis.36 37 Sections were reacted free-floating in immunohistochemical agents and washed in PBS two times for 5 to 10 minutes each between all reactions. They were first exposed to 0.3% H2O2 for 15 minutes to bleach endogenous peroxidase and then to normal serum (1%). They were then reacted overnight at room temperature with a set of primary antibodies: mouse monoclonal anti-NeuN (1:100, clone A60; a kind gift of Dr R. Mullen, Salt Lake City, Utah32 ), anti-parvalbumin (1:2000, clone PA-235; Sigma), anti-calbindin (1:500, clone CL-300; Sigma), and rabbit anti-calretinin (1:500; Swant, Switzerland), anti-synapsin I (1:500; affinity purified antiserum, A-6442; Molecular Probes Inc), and anti-GFAP (1:1000; DAKO). After incubation with appropriate bridge antibodies and peroxidase-antiperoxidase complex, sections were reacted in 3'3-diaminobenzidine and H2O2.
Evaluation
The signals obtained by ISEL were mostly nuclear and were easily identified under the fluorescence microscope when using the FITC optics. The localization of these cells was charted onto plots of a standard rat brain atlas.38 The hippocampus was analyzed at a section level (-2.8 to -3.3 mm with reference to bregma) where typical delayed neuronal death of CA1 neurons is observed. The striatum was studied at a section level through the center of the structure (+0.7 mm to bregma) and at caudal levels (-2.8 mm). Cortical areas were investigated at both levels.
For the quantification of cells in the striatum, the position of each ISEL-positive cell was plotted for all sections using a standard frontal section level (+0.7 mm to bregma). For each time point, the mean±SD of ISEL-positive neurons was calculated per area of striatum (unilaterally). The data were analyzed using ANOVA (P<.01) followed by t tests, with the critical probability values adjusted for multiple comparisons (P<.05). An overlay was then used on the plots to divide the striatum into four subdivisions: dorsomedial, dorsolateral, ventrolateral, and ventromedial areas. ISEL-positive cells were then counted in these subdivisions, and the mean±SD values were calculated. Subdivisions were plotted along with total striatum to estimate the regional distribution of positive cells. To estimate the percentage of ISEL-positive cells per striatal area, the total number of neurons per striatal area was determined. Sections from animals (n=4) with 7-day survival (and no damage) were counterstained with cresyl violet, and neurons were counted at x400 magnification in four fields of 0.12 mm2 each. The area of the striatum (unilaterally) was then determined from a stereotaxic atlas, which is identical to the area used for counting ISEL-positive cells. The average number of neurons per striatal area was then estimated and used as reference for the counts of ISEL-positive cells.
Paraffin sections through the striatum were examined for damage or infarction using pink H&E staining of damaged neurons as the parameter.31 Immunohistochemical sections of striatum were screened for loss of neurons or glial reactions.
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In Situ End-Labeling
Control brains were largely devoid of cellular signals using the ISEL technique. Occasional fluorescent cells were found in the vicinity of the lateral ventricle, within the corpus callosum, and in layer VI of the neocortex.
After ischemia, the most marked finding was the occurrence of strongly fluorescent cells in the medial CA1 after 3 days (Fig 1
). No changes were seen in the hippocampal CA1 sector at earlier time points, but occasionally positive cells were seen within the hilus of the dentate gyrus. This observation is in line with previous descriptions of the hippocampus in global brain ischemia models.6 8 9 16 17 18 19
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However, a novel finding was made in sections through the main body of the striatum (+0.7 mm to bregma), which showed ISEL-positive cells with a distinct time course (Figs 2 and 3). Control animals contained very few or no ISEL-positive cells (0.58±1.38 cells per area; Fig 2a through 2c). No increase was noted at 1 hour (0.88±0.83 cells per striatum). However, ISEL-positive cells were found scattered throughout the striatum at 3 hours (9.10±8.13 cells per striatum; Fig 2d through 2f), 6 hours (12.57±7.63; Fig 2g through 2i), and 12 hours (3.00±3.80; Fig 3a through 3c). ISEL staining in the dorsomedial areas close to the lateral ventricle was particularly elevated, but all other parts of striatum were also affected. By 1 day, the striatum still showed ISEL-positive cells (15±8.73 cells per striatum; Fig 3d through 3f). By 3 days, the frequency of labeled cells declined (5.40±8.26 cells per striatum), and clusters of positive cells were found in the dorsomedial striatum close to the corpus callosum (Fig 3g through 3i). By 7 days, few cells were labeled (1.17±2.73 cells per striatum). The numbers of positive cells per total striatum and per different regions are shown in Fig 4. Statistical analysis showed significant increases in the numbers of ISEL-positive cells per total striatal area at all time points between 3 hours and 3 days (ANOVA, P<.01; t tests, P<.05). This plot shows that the majority of labeled cells occurred in the dorsomedial region at all time points and that the lateral areas also showed a pronounced increase. However, there was no obvious preferential increase in ISEL staining in the dorsolateral or ventrolateral areas, which are the main focus of necrotic damage after 20 minutes of ischemia.29 In cellular stains, there were 96±18 neurons/0.12 mm2 or about 8800 neurons/11 mm2 of total striatal area (unilaterally). At the peak of changes after ischemia, 15 ISEL-positive cells per total striatal area were found, which would account for 0.2% of all neurons in the same area.
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Sections taken through the caudal pole of the striatum showed clusters of ISEL-positive cells in the dorsal tip of the structure, immediately adjacent to the angle formed by the fiber tracts of the external and internal capsule. These cells were observed between 12 hours and 3 days. The only other area with an obvious presence of ISEL-labeled cells was the choroid plexus, which showed some positive cells during the first day. Occasional cells were seen within the corpus callosum and in the lower layer VI of the dorsal neocortex, but this was not a consistent finding after ischemia. In a few animals, scattered cells in the lateral thalamus and dorsal portions of septum were labeled.
Neuropathological Examination
Examination of hippocampal sections showed typical ischemic cell death in medial CA1 and hilus (not shown). After the detection of the ISEL-positive cells in the striatum, the question arose as to whether the 10-minute insult caused detectable morphological damage to this area in our model. Paraffin sections through the striatum stained with H&E revealed overall intactness of the striatum in 7 of 8 hemispheres. At higher magnification, the neuronal architecture was fully intact. Many neurons were regularly stained, as were astrocytes and cells within the white matter bundles. Darkly stained small cells were seen, which could have been microglial cells or endothelial cell nuclei out of the section plane. These cells occurred with the same regularity in controls and postischemic animals. One animal had a small focus of microglial reaction and neuronal loss in the dorsolateral striatum.
A combination of marker proteins was then used to identify either neuronal damage or glial reactions in the striatum. Dense labeling was found with the neuron-specific antibody NeuN, and there was no indication of neuronal loss. There was also no qualitative evidence for loss of parvalbumin-positive or calretinin-positive subclasses of interneurons. Calbindin labeling of projection cells and local circuit neurons was not different between controls and ischemic animals. Synapsin I antibodies revealed an even staining all over the striatal neuropil, indicating the absence of infarction. No signs of astrocyte reactivity, such as locally increased staining of processes, were found with GFAP antibodies. Taken together, the binding pattern of these immunohistochemical markers with the histopathological analysis was indicative of a qualitatively normal striatum in 95% of hemispheres. Thus, animals subjected to a 10-minute insult did not show areas of ischemic damage in the lateral striatum, which were seen after 20 or 30 minutes of ischemia in this model.29 39
Changes in Temperature
In the series of experiments examined with the ISEL method, body temperature at 1 to 2 minutes of ischemia was 37.2±0.4°C, and it remained at 37.3±0.4°C at 9 to 10 minutes of ischemia. Temperature in the auditory canal (tympanic temperature) was 35.8±0.5°C at 1 to 2 minutes and then dropped to 35.1±0.8°C at 9 to 10 minutes. Theoretically, animals with higher temperatures at the onset of ischemia and with no or moderate drop of tympanic temperature should have more damage and more ISEL-positive cells. Animals were divided into those with >10 ISEL-positive cells per total area to indicate severe damage and those with mild damage having fewer positive cells. There were about the same numbers of animals in both groups that had tympanic temperatures above (mild, n=9; severe, n=4) and below (mild, n=10; severe, n=5) the average temperature at the onset of ischemia. Animals with severe damage did not differ in the drop of brain temperature during occlusion from animals with mild damage (t test, P>.2).
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Ischemic cell damage of neurons is associated with severe alterations in several cell components. For example, nuclear alterations can occur during and after ischemia.31 It is therefore not surprising that biochemical or histochemical techniques also reveal changes in DNA integrity after ischemia. Calcium-induced damage in brain ischemia and related conditions includes activation of endonucleases that can subsequently produce DNA damage.2 40 One technique to detect DNA fragmentation is ISEL using biotin-tagged oligonucleotides and terminal transferase.25 26 This technique was initially developed to detect the DNA strand breaks that occur during apoptosis.25 26 ISEL-positive cells have been found in studies of neuronal death in degeneration models, where apoptosis is a possible mechanism of cell death.41 The technique has also been used with ischemic brain tissue, where neurons become ISEL-positive at about the time that obvious cell death is observed with conventional stains.6 7 8 9 11 14 15 16 17 18 19 In fact, CA1 neurons in the present study showed ISEL staining at 3 days, when conventional staining indicated neuronal damage (not shown), confirming previous reports for global ischemia models.6 8 9 16 17 18 19 However, studies with other neuropathological materials showed that ISEL signals can also be observed in necrotic cells.8 Therefore, it is presently difficult to use ISEL staining alone to establish definitively whether CA1 neurons die due to apoptosis or necrosis. Similar problems arise with the interpretation of ISEL staining in tissue undergoing overt infarction,7 13 unless additional cytological criteria are used.11 14 15
We attempted to circumvent this ambiguity by searching for ISEL-positive cells in regions of the brain that are normally not injured by mild ischemia. The rationale for this approach is that the presence of ISEL-positive cells in regions that do not exhibit overt cell damage by normal staining would be indicative of early DNA fragmentation associated with apoptosis. Several studies using in vitro systems showed that apoptosis occurs under conditions of a mild injury, whereas necrosis is produced with stronger insults.27 Accordingly, neurons in areas with mild impact might be prone to apoptosis.
The present study therefore set out to analyze the striatum for ISEL-positive cells, as this structure should not be injured to any significant degree by 10 minutes of ischemia.29 We documented in this study that an insult of 10 minutes of four-vessel occlusion (without heating of the ischemic brain) did not produce overt neuronal damage in the striatum. Overall, the histological studies on paraffin sections with H&E staining and immunohistochemical evaluation using neuron-specific antibodies did not reveal neuronal damage or loss in the striatum at 1 week after 10 minutes of four-vessel occlusion. In 20 hemispheres analyzed, only one showed a focus of damage that was localized to the dorsolateral region. Some classes of interneurons might be vulnerable to ischemia and disappear without changes in conventional staining, but those containing parvalbumin, calbindin, or calretinin appeared to be resistant. Large cholinergic neurons have already been documented as being very resistant to ischemia.29 39 Furthermore, our immunohistochemical studies with antibodies to GFAP revealed the absence of a glial response. Therefore, we conclude that the 10-minute period of ischemia subjects striatum to only a mild impact, which does not result in visible neuronal damage or glial activation. This finding is fully in line with previous data showing that the striatum is not vulnerable to 10 minutes of four-vessel occlusion.29
Despite the apparent absence of overt neuronal injury, we observed scattered ISEL-positive cells in rat striatum at various times after 10 minutes of ischemia. The affected cells were situated outside the myelin bundles and were most likely neurons. The majority of labeled cells were situated in the dorsomedial striatum, but positive cells also occurred in all other subdivisions. Although one case revealed neuropathological damage after 10 minutes of ischemia, this damage was localized to the typical dorsolateral regions, and there was not a single case with such a pattern in the main series used for the ISEL study. Thus, the pattern found here after 10 minutes of ischemia was clearly different from the global ischemic damage seen after 20 or 30 minutes of ischemia that occurs in the dorsolateral striatum.29 39 The number of ISEL-positive cells accounted for about 0.2% of all cells per total striatal area at the peak response time of 1 day. The time course of this response suggests that the wave of cell damage in striatum occurred between 3 hours and 3 days of reperfusion after 10 minutes of ischemia. Few ISEL-positive cells were seen at 7 days. The transient nature of the ISEL response, namely the scattered damage to individual cells and the rapid disappearance of affected cells, is strongly suggestive of apoptosis.27 28 Moreover, the absence of a glial reaction indicates the absence of an inflammatory response. Because apoptotic cells are cleared rapidly from tissue, it is possible that the numbers seen at 1 or 3 days underestimate the total number of neurons that have died up to this time point. In summary, the pattern of minute scattered damage in the striatum after 10 minutes of global ischemia fulfills several criteria for apoptosis and presents a persuasive example of a possible role of programmed cell death. However, it is possible that with shorter periods of ischemia, a similar phenomenon of scattered positive cells would occur in the hippocampus.
We deliberately chose 10 minutes of four-vessel occlusion and allowed for the spontaneous drop of head temperature to avoid overt damage to the striatum, whereas most previous studies used models and ischemia periods that would be expected to damage the striatum. For example, with two-vessel occlusion and hypotension for 12 minutes in rats, cells with DNA fragmentation were seen at 12 hours in the dorsomedial striatum and later extended to the lateral and ventral regions, showing the classic pattern of ischemic damage.8 However, two-vessel occlusion and hypotension with maintained brain temperature produces a strong ischemic insult, and severe striatal damage is seen after this ischemic period.42 In the four-vessel occlusion model used without heating of the head, striatal ischemic damage occurs only after 20 minutes of occlusion.29 Thus, after 20 minutes of four-vessel occlusion, DNA breaks can be seen in the striatum.9 18 In studies using shorter ischemia periods in the gerbil, the analysis was restricted to the hippocampus.16 17 19 It is of interest that 10 minutes of transient focal ischemia, induced by insertion of an intravascular filament, produced DNA fragmentation in the striatum, whereas longer insults produced both necrosis and apoptosis.15
Alternative interpretations must be also considered. The dorsomedial striatum lies closest to the subventricular (or subependymal) zone, which is a source of ongoing neurogenesis and gliogenesis in the adult rodent brain.43 Recent evidence suggests that newly formed precursors may exit from this zone and become part of the neuronal circuitry or form glia.44 45 Stress increases the formation of new cells in this area.43 Theoretically, ischemia could have increased formation and migration of these immature cells from the subventricular zone, which then died by apoptosis within the striatum. However, positive cells were seen in other subdivisions of striatum, which argues against this concept.
It therefore appears that the striatum might be vulnerable to neuronal loss by apoptotic mechanisms. Recently, a report appeared showing the increased occurrence of apoptotic neurons in the aging rat striatum.46 Significantly, a recent study on reserpine toxicity also revealed an increased number of ISEL-positive cells in the striatum, which were preferentially localized to the dorsomedial areas.47 Furthermore, an apoptotic component was proposed for the neuronal degeneration in the striatum in Huntington's disease and in related neurotoxin models.48 It is intriguing that a recent study showed that the defective gene product huntingtin may interfere with a key enzyme in glucose metabolism49 and hence with energy metabolism. This indicates further similarities between ischemic cell damage in the striatum and the spontaneous neurodegeneration in striatum in Huntington's disease. There is recent evidence that a delayed form of neuronal death may occur in the striatum after a 10-minute insult of forebrain ischemia that was induced using two-vessel occlusion and hypotension.50 A delayed neuronal death was also reported for the striatum in transient focal ischemia.51 It is possible that the combined releases of glutamate and dopamine in ischemia52 play a role in causing apoptosis in striatum.
At present, most evidence for an apoptotic process after brain ischemia is indirect, making it difficult to distinguish this form of cell death from necrosis. However, it now seems likely that a component of the observed cell death, such as that demonstrated in the striatum after brief ischemia in this study or in the surrounding of ischemic infarction,7 11 13 14 15 bears many similarities to apoptosis. In programmed cell death, cells kill themselves through the activation of certain "death genes" and by production of "killer proteins."20 21 22 28 41 The downregulation of protective inhibitors of the killer proteins is another potential mechanism. In postmitotic neurons, cell death after ischemia could result from faulty activation of signal pathways, leading to loss of control over apoptosis inhibition. However, it is also possible that partial damage to neurons (eg, cytoskeleton damage53 ) leads to an activation of the apoptotic mechanism. There is, in fact, evidence for alterations in gene expression of proapoptotic and antiapoptotic genes after ischemia.54 55 At the same time, gene expression of trophic factors is increased after brain ischemia, and endogenous trophic factors may play a role in the control of neuronal survival.23
Finally, it should be pointed out that the relatively small number of cells damaged (0.2% of total population) detected by the sensitive ISEL technique in the striatum after brief ischemia may have no significant functional consequences. However, it seems likely that repeated periods of minor ischemia, such as may occur in the clinical setting of transient ischemic attacks, could lead to significant cumulative neuronal loss. Further experiments are needed to determine whether recurrent episodes of energy failure can induce subtle neurodegeneration in the striatum via repeated activation of apoptotic mechanisms.
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Acknowledgments |
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This work was supported by grants from the Heart and Stroke Foundation of Ontario (NA2867 to Drs Hakim and Schmidt-Kastner, NA2936 to Dr Fliss) and London Life Canada. Dr Schmidt-Kastner was supported by Heisenberg Stipendium of DFG Bonn (Schm 776/4-1). Dr R.J. Mullen kindly provided the A60 antibodies to NeuN. We thank Deborah A. Gattinger, Georgette Roy, and Annie Bedard for excellent technical support.
Received April 24, 1996; revision received July 24, 1996; accepted September 17, 1996.
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Editorial Comment
Cerebral Vascular Disease Research CenterUniversity of Miami School of MedicineMiami, Fla
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Observations accruing over the past several years suggest that ischemic neurons may succumb, in part, to genetically determined cell-death programs activated by an ischemic insult. These studies have stimulated a vigorous research initiative throughout the world to characterize more precisely the manner in which apoptosis and processes of DNA damage and repair might contribute to ischemic injury.1R In this meticulously crafted study, Schmidt-Kastner and colleagues focus primarily on the question of whether a 10-minute global ischemic insult carried out at slightly hypothermic cranial temperature (tympanic temperature of 35.1°C to 35.8°C)—and hence too mild to produce neuronal injury in the striatum by conventional histological or immunocytochemical criteria—might nonetheless induce apoptosis in that structure. Their novel and intriguing finding is that approximately 0.2% of striatal neurons (in particular, localized to the dorsomedial striatum) exhibit evidence of DNA fragmentation (positive in situ end labeling) over the time span of 3 hours to 3 days following ischemia, and yet the striatum shows no detectable ischemic neuronal changes when examined by conventional histopathology at 7 to 8 days. Although this burden of putatively apoptotic neurons is admittedly quite small, quantitative assessment of apoptosis is particularly vexacious in that apoptotic cells are apparently cleared from the tissue rather quickly.
The possibility exists that, with very prolonged survival, additional evidence for apoptotic damage might emerge. For example, our group has shown that 10 minutes of normothermic ischemia induces striatal neuropathology that only becomes evident at 2 months of survival, and that postischemic hypothermia plus delayed administration of the N-methyl-D-aspartate antagonist MK-801 aborts these changes.1R
While the bulk of evidence to date favors the view that the majority of neurons succumbing to an acute global or focal ischemic insult do so primarily by necrosis rather than apoptosis, the "last word" is by no means in. Intriguing topics for future study include the possible contribution of apoptotic processes to neuronal loss in traditionally unexpected settings, such as very chronic oligemic states (eg, bilateral carotid artery occlusions), repeated mild hypoxic/ischemic insults (eg, multiple transient ischemic attacks), or following intraoperative interruptions of the cerebral circulation (eg, after conventional hypothermic cardiopulmonary bypass). For investigators of cerebrovascular pathophysiology, these are indeed interesting times.
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1R. Chopp M, Chan PH, Hsu CY, Cheung ME, Jacobs TP. DNA damage and repair in central nervous system injury: National Institute of Neurological Disorders and Stroke workshop summary. Stroke.. 1996;27:363-369.
2R. Dietrich WD, Lin B, Globus MY-T, Green EJ, Ginsberg MD, Busto R. Effect of delayed MK-801 (dizocilpine) treatment with or without immediate postischemic hypothermia on chronic neuronal survival after global forebrain ischemia in rats. J Cereb Blood Flow Metab.. 1995;15:960-968.
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