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Stroke. 1997;28:1245-1254

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(Stroke. 1997;28:1245-1254.)
© 1997 American Heart Association, Inc.

Articles

DNA Scission After Focal Brain Ischemia

Temporal Differences in Two Species

Masafumi Tagaya, MD; Kai-Feng Liu, MD; Brian Copeland, MD; Dietmar Seiffert, MD; Robert Engler, MD; Julio H. Garcia, MD; Gregory J. del Zoppo, MD

From the Departments of Molecular and Experimental Medicine (M.T., B.C., G.J. del Z.) and Vascular Biology (D.S.), The Scripps Research Institute, La Jolla, Calif; Division of Neuropathology, Department of Pathology, Henry Ford Hospital, Detroit, Mich (K.-F.L., J.H.G.); and Division of Cardiology, Department of Medicine, Department of Veteran's Affairs Medical Center, San Diego, Calif (R.E.).

Correspondence to Gregory J. del Zoppo, MD, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N Torrey Pines Rd, SBR-17, La Jolla, CA 92037.


*    Abstract
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Background and Purpose Species- and model-dependent differences in cell response to focal brain ischemia may underlie differences in adhesion receptor expression. The aim of this study was to quantitatively evaluate the spatial and temporal distribution of dUTP incorporation into damaged DNA, as an indicator of ischemic injury, in the corpus striatum.

Methods Cerebral ischemia was produced in 16 nonhuman primates and 19 rats by occluding the middle cerebral artery (MCA:O) with reperfusion for various periods. In situ dUTP was incorporated into cells with DNA damage by terminal deoxynucleotidyl transferase (TdT), DNA polymerase I, or the Klenow fragment of DNA polymerase. Dual immunolabeling experiments with immunoprobes against neuronal, vascular, or glial marker proteins were performed.

Results Significant topographical differences in dUTP between the two species were seen. In both models the TdT and polymerase I regions changed characteristically during focal ischemia. The number and density of dUTP-labeled cells increased with time from MCA:O and were dramatically different between the species (2P<.001). By 2 hours of ischemia, the density of dUTP label was 48.8±10.3 cells/mm2 in the primate and 2.4±0.8 cells/mm2 in the rat (2P<.05), but these values became nearly identical by 24 hours of reperfusion. In the primate, 80.0±6.6% of labeled cells displayed microtubule-associated protein-2 antigen (at 2-hour MCA:O), while 1.8±0.5% were associated with microvessels at 24 hours of reperfusion.

Conclusions In situ detection of DNA damage, accomplished by three methods, reveals distinct temporal, topographical, and density differences in ischemic injury to cells in the primate and the rat corpus striatum as a result of MCA:O.


Key Words: cerebral ischemia, focal • DNA damage • neuronal damage • primates • rats


*    Introduction
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 *Introduction
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A growing number of reports describe single- and double-strand DNA scission among the mechanisms of neuronal injury after focal cerebral ischemia in the rodent.1 2 3 4 5 DNA scission may occur during processes of DNA repair, programmed cell death (apoptosis), and necrosis. The issue has been recently highlighted by concerns about the nature of DNA scission during neuronal injury after focal or global ischemia and reperfusion.1 2 3 4 5 6 7 8 Evidence of DNA scission may be viewed as a marker of severe cell injury since it is known to occur after a variety of insults including chemical exposure,9 treatment with ultraviolet rays,10 11 and mutagens.12 13 14 In addition, DNA scission has been observed in a number of noncerebral tissues subject to injury.15 16 17 18 19 Techniques for visual detection and localization of DNA injury/repair in situ include TdT-dependent dUTP-labeling of free 3'-OH ends of double-strand DNA20 ; DNA polymerase I incorporation in nicks, gaps, and staggered 3'-OH ends21 ; and Klenow fragment of DNA polymer- ase I–dependent labeling of staggered 3'-OH ends and gaps.21 Of these, only DNA polymerase I has 5'->3' exonucleolytic activity, which allows nick translation.22

In humans and nonhuman primates, tissue injury caused by interruption of the anterograde flow at the proximal (M1) MCA is confined to the basal ganglia and the temporal cortex.23 24 25 Anatomically comparable lesions have been conveniently produced by MCA:O in the rat by occluding the origin of the MCA by intravascular threading of a monofilament.26 After MCA:O in primates, the basal ganglia, which receives its blood supply from perforating branches of the lenticulostriate artery, becomes injured with variable involvement of cortex,23 whereas a strict sequence of injury from basal ganglia to cortex occurs in the rat.26 Differences in the timing and course of the ischemic lesions between the rodent and the primate during MCA:O/R have been suspected.27 While the cerebral arterial supply of the basal ganglia among primates is quite similar to that in humans,28 29 the rat cerebral circulation displays distinct differences.28 30 More fundamentally, differences in DNA sensitivity to methylene chloride between mouse and nonhuman primate hepatocytes suggest interspecies differences in cell reactivity.31 However, it has been assumed that neuronal responses to ischemia/reperfusion are independent of speciation. Subtle differences in the temporal pattern of microvascular endothelial cell intercellular adhesion molecule-1 appearance between the primate32 and rat33 in response to MCA:O/R have hinted at differential responses of the two preparations to focal ischemia. Reports of the late appearance of dUTP label within cells in rodents after transient MCA:O/R have been taken as an indication of programmed cell death,34 although one report has suggested that dUTP labeling may occur much earlier.5

Those observations prompted this study to evaluate the relative distribution and development of DNA scission in cells of the basal ganglia subject to focal ischemia and reperfusion in rat and nonhuman primate models. Because of its particular vulnerability to injury, this territory was chosen for comparison of the extent and time course of DNA strand breaks as an indicator of neuronal/microvascular injury in both species. The hypothesis tested states that evidence of nonvascular cell injury during focal ischemia/reperfusion develops more rapidly in the primate than the rodent and that microvascular injury and parenchymal injury are related.


*    Materials and Methods
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Animal Procedures
All procedures were approved by the Institutional Animal Research Committees and performed in accordance with standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals), the National Institutes of Health Policy on Human Care and Use of Laboratory Animals, and the US Department of Agriculture Animal Welfare Act at the two institutions. In compliance with these standards, every effort was made to ensure that the subjects were free of pain or discomfort. All animals were neurologically normal and apparently free of infections or inflammation during the experiments.

Tissues from 16 adolescent male baboons (Papio anubis/cynocephalus) and 19 adult male Wistar rats (weight, 270 to 310 g) were used in the present study. Preparation of the awake baboon model of reversible MCA:O has been described in detail.23 24 This preparation involved surgical placement of an inflatable balloon catheter assembly extrinsic to the MCA and proximal to the origin of the lenticulostriate arteries under inhalation anesthesia with halothane (2%) or isoflurane (1.25%). A 7-day postoperative intervention-free interval for observation and recovery allowed recovery of anesthesia-associated polymorphonuclear leukocyte dysfunction.35 Each subject underwent MCA:O or MCA:O/R as described. Three animals were subjected to MCA:O for 2 hours, while the remaining 9 underwent reperfusion for 1 hour (n=3), 4 hours (n=2), or 24 hours (n=4) after 3 hours of MCA:O. Three subjects did not undergo implantation or any other intervention (control). One subject underwent implantation and the 7-day interval, without subsequent intervention, and served as a sham-operated control.

Focal cerebral ischemia (and reperfusion) in the Wistar rat was induced according to procedures described by Zea-Longa et al.36 Briefly, each animal was anesthetized with 3% halothane and spontaneously respired with 1.0 to 2.0% halothane in a 2:1 N2O/O2 mixture, with the use of a face mask. Under the operating microscope, the right CCA was exposed through a midline incision. The ECA and the occipital artery were ligated, and the ICA was isolated from the adjacent vagus nerve. Further dissection identified the origin of the pterygopalatine artery, which was not ligated. A microvascular clip was placed across the CCA. An 18- to 19-mm segment of 4-0 nylon monofilament, its tip rounded by heating, was introduced into the ECA at the origin of the occipital artery. The ECA was tied around the intraluminal nylon monofilament to prevent hemorrhage, and the CCA clip was removed. The nylon monofilament was gently advanced from the ECA into the ICA lumen and the skin incision closed. As described above, 3 subjects underwent MCA:O for 2 hours, while the remaining 10 subjects underwent 1-hour reperfusion (n=3), 4-hour reperfusion (n=3), or 24-hour reperfusion (n=4) after 3 hours of MCA:O. Subjects (n=3) that underwent neither surgery nor ischemia served as controls. A separate cohort (n=3) was exposed to the surgical procedures described above, but the nylon monofilament was removed within 1 minute.

Perfusion Procedures
Each experiment in the nonhuman primate and the rodent after MCA:O or MCA:O/R was terminated under thiopental sodium anesthesia by transcardiac perfusion of an isosmotic perfusate containing heparin (2000 IU/L), sodium nitroprusside (6.7 µmol/L), and BSA (50 g/L). In the nonhuman primate, tissue blocks (1x1x0.5 cm) from symmetrically located sites of basal ganglia, parietal cortex, and temporal cortex were embedded in Tissue-Tek OCT compound (Miles, Inc), frozen in 2-methylbutane/dry ice, and stored at -80°C until they were sectioned. In the rats, the brains were removed in toto quickly after decapitation, sectioned coronally, embedded in Tissue Tek OCT compound, frozen in 2-methylbutane/dry ice, and stored at -80°C until they were shipped. Generally, sections were taken from blocks at the level of the anterior commissure37 in the primate and at the level of 0.2 mm to -0.4 mm bregma in the rat.38 All cassettes containing rodent tissues were shipped by overnight courier on dry ice from Henry Ford Hospital to The Scripps Research Institute, where the intact nature of the specimens was confirmed (see below). Ten-micrometer cryosections were cut for histochemical procedures.

Selected primate tissue specimens were subjected to immersion fixation with 2% paraformaldehyde for 24 hours and embedded in paraffin for ultrastructural studies.

DNA Scission
DNA fragmentation was detected on adjacent cryosections from both species by incorporation of digoxigenin-dUTP with the use of TdT,20 DNA polymerase I, and the Klenow fragment of DNA polymerase.21 For the TdT experiments, tissue sections were incubated with TdT (0.3 U/µL) and digoxigenin-11-dUTP (0.5 nmol/L) in 0.1 mol/L sodium cacodylate, 1 mmol/L CoCl2, 0.1 mmol/L dithiothreitol, and 50 µg/mL BSA for 60 minutes at 37°C. For the DNA polymerase I and the Klenow enzyme, adjacent sections were incubated with a mixture of digoxigenin-conjugated dUTP and unlabeled deoxynucleotides, buffer containing 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgSO4, 0.1 mmol/L dithiothreitol, 50 µg/mL BSA, and the polymerase for 2 hours at 37°C. After sections were washed, incubation for 60 minutes with horseradish peroxidase–conjugated anti-digoxigenin antibody (Boehringer Mannheim) was performed to detect the incorporation of digoxigenin-conjugated dUTP. The chromogen 3-amino-9-ethylcarbazole (Biomeda Corp) was used for color development.39 Controls for the TdT and for polymerase-based procedures included (1) exposure of parallel sections to DNase I (Sigma) (positive control) and (2) deletion of the respective enzyme in each experiment (negative control for nonspecific staining). Exposure to DNase I uniformly led to incorporation of digoxigen-dUTP into all cell nuclei.

Localization of dUTP Incorporation and Antibodies
For immunoperoxidase dual-label experiments, cells incorporating dUTP (dUTP+) were visualized with the metal-enhanced diaminobenzidine, which provided a black reaction product. After the TdT procedure, sections were incubated with one of several single immunoprobes. Well-characterized antibodies were used against MAP2 (clone HM-2; Sigma), type IV collagen (clone COL-94; Sigma), or GFAP (rabbit IgG; Sigma) (Table 1Down). After sections were washed with PBS, biotinylated horse serum against mouse IgG or biotinylated goat serum against rabbit IgG (Vector Laboratories) was applied and incubated over 30 minutes at 37°C. Immunoreactive signals were visualized by the avidin-biotin peroxidase method, and 3-amino-9-ethylcarbazole was used as the second chromogen for the development of the red reaction product.39 Some sections were counterstained with Mayer's hematoxylin (Biomeda Corp).


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  		Table 1. Characterization of Antibodies

For dual immunofluorescence experiments, an FITC-conjugated anti-digoxigenin antibody (Boehringer Mannheim) was used to display dUTP incorporation. Sections were then incubated with primary antibodies against GFAP or myeloperoxidase (rabbit IgG; DAKO), a marker antigen for astrocytes or polymorphonuclear leukocytes, respectively (Table 1Up). Incubation with an anti-rabbit TRITC-conjugated secondary antibody (Vector Laboratories) signaled the presence of the antigen of interest.40

Quantitation
The absolute number and density (per square millimeter) of dUTP+ cells was determined by computer-assisted quantitative video-imaging microscopy. The number (and therefore density) of dUTP+ cells was determined within a 25–microscopic field (or 1.51-mm2) region of interest centered within each section from the ischemic and nonischemic territories. In each section the total number of dUTP+ cells and the number of dUTP+/MAP2+ cells were determined. The number of MAP2+ (dUTP+) cells in the ischemic zone was calculated as a percentage of all dUTP+ cells. Topographical localization of dUTP+ cells was achieved in relation to known landmarks in both sections.

Statistical Analysis
Unpaired Student's t tests were performed to assess differences in the number and density of dUTP+ cells at each time point. Two-way ANOVA was used to compute interspecies differences. For series, time-related groups were subject to a square root transform to stabilize variances across groups, followed by a one-way ANOVA. Significance was set as 2P<.05.


*    Results
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 *Results
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In nonischemic control and sham-operated control subjects from both species and in the nonischemic basal ganglia of subjects undergoing MCA:O(/R), only 1 or 2 cells per section displayed dUTP label by either the TdT, DNA polymerase I, or Klenow fragment–based incorporation methods. In the case of the rodent specimens, these experiments confirmed the absence of any detectable injury induced by overnight transport, as well as the absence of injury in nonischemic cerebral territories in both species. dUTP label was detected throughout all frozen cerebral tissue sections from both species pretreated with DNase I.

Extent of Region of DNA Injury
In the ischemic basal ganglia, scattered or clustered nonvascular cells with nuclear dUTP label were observed at all time points in distinct patterns characteristic to each species. In the primate, small confluent and separate regions of nuclear dUTP incorporation were present in the basal ganglia, variously involving the internal capsule, which appeared as early as 2 hours of MCA:O by TdT and DNA polymerase I (Figs 1Down and 2Down). In contrast, in the rat only solitary dUTP+ cells were noted at that time predominantly in the dorsolateral caudate putamen, with a growing region of labeled cells appearing by 24 hours of reperfusion (Figs 2Down and 3Down). In both species the total number of dUTP+ cells increased with time of MCA:O/R (Table 2Down).



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  		Figure 1. Maps of cellular dUTP incorporation in the ischemic basal ganglia of each primate during MCA:O and MCA:O/R by two methods. Hatched zones refer to the regions of dUTP label by TdT. Solid gray zones refer to label developed by DNA polymerase I (pol I) (see text). CC indicates corpus callosum; IC, internal capsule; P, putamen; CH, caudate head; and LV, lateral ventricle.



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  		Figure 2. Change in density of cellular dUTP incorporation in the basal ganglia with time in the nonhuman primate ({blacksquare}) and the rat ({bullet}) normalized for 1.51-mm2 region of interest (2P<.05).



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  		Figure 3. Maps of cellular dUTP incorporation in the ischemic basal ganglia of the rat during MCA:O and MCA:O/R. Zones of label and their relative frequency are described by hatching (eg, 100%=3 of 3 or 4 of 4 subjects displaying label). pol I indicates polymerase I.


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  		Table 2. Total Number of Cells With dUTP Incorporation in the Nonhuman Primate and Wistar Rat

Topographical Distribution of TdT- and DNA Polymerase I–Dependent dUTP
The relative distributions of cells displaying dUTP label by TdT compared with DNA polymerase I in the primate and the rat basal ganglia are shown in Figs 1Up and 3Up, respectively. The regions defined by DNA polymerase I were larger than and circumscribed those identified by the TdT-based method in both settings. With duration of MCA:O, the region defined by TdT became similar in size to the larger areas of dUTP+ incorporation detected with DNA polymerase I by 24 hours of reperfusion. The patterns developed with the Klenow enzyme were very similar to those of the holoenzyme. A positive-control study in which the basal ganglia was pretreated with DNase I allowed dUTP incorporation throughout the section (Fig 4ADown).



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  		Figure 4. Cells with dUTP (TdT) incorporation in the primate basal ganglia. dUTP+ cells were observed throughout the corpus striatum with the use of DNase I. A, Increasing densities of dUTP+ cells were seen with time of MCA:O/R; B, 2-hour ischemia; C, 3-hour ischemia with 1-hour reperfusion; D, 3-hour ischemia with 24-hour reperfusion. Bar=50 µm.

Relative Density of dUTP+ Cells
In the nonischemic primate, there were 271.4±7.6 normal nonvascular cells/mm2 observed within the defined region of interest (n=3).

Within the basal ganglia of the primate, the density of dUTP+ cells defined by the TdT method increased significantly with time of MCA:O/R (Fig 2Up). The mean number of dUTP+ cells within the 25-field region of interest of the basal ganglia was 0.88±0.38 cells/mm2 in the unoperated control (n=3) and 0.66 cells/mm2 in the sham-operated control (n=1). A monotonic increase in the mean density of dUTP+ cells was noted: from 48.8±10.3 cells/mm2 at 2 hours of MCA:O to 98.2±12.6 cells/mm2 at 3 hours of MCA:O and 24 hours of reperfusion (2P<.05) (Fig 2Up). Fig 4Up shows representative photomicrographs of individual dUTP+ cells. A similar significant increase in dUTP label was observed with the DNA polymerase I–based and the Klenow fragment–based reactions (Table 3Down). The mean densities of dUTP+ cells derived from DNA polymerase I, the Klenow fragment, and the TdT methods were not significantly different by 24 hours of reperfusion.


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  		Table 3. Comparison of Cell Density With dUTP Incorporation Mediated by Three Enzymes

A much different temporal pattern of DNA scission defined by the TdT procedure was observed in the rodent. In the nonoperated control and sham-operated control rats, the mean dUTP+ densities in the basal ganglia were 0.66±0.66 cells/mm2 (n=3) and 1.32±0.67 cells/mm2 (n=3), respectively. However, in the ischemic basal ganglia, densities were 2.43±0.76 cells/mm2 at 2-hour MCA:O and 9.71±6.01 cells/mm2 at 4-hour reperfusion (after 3-hour MCA:O). At 24-hour reperfusion, the dUTP+ density was 136.76±69.02 cells/mm2 (Fig 2Up). Similar patterns of dUTP incorporation were observed by both DNA polymerase methods, indicating that free staggered 3'-OH ends increased with time elapsed after MCA:O. However, by 24-hour reperfusion there was no difference among the methods (Table 3Up). Thus, the temporal profile of dUTP+ incorporation in the ischemic basal ganglia after MCA:O differed characteristically and significantly (2P<.001) between the two species and models.

Identification of dUTP+ Cells
Clear identification of dUTP+ cells was possible under limited conditions. For instance, in primate tissues morphologically intact neurons displayed MAP2 antigen. In the ischemic basal ganglia, 80.0±6.6% of cells labeled by dUTP (TdT) also expressed MAP2 antigen at 2-hour MCA:O (Table 4Down and Fig 5ADown and 5BDown). Identifiable MAP2 immunoreactivity of dUTP+ cells decreased progressively with time of reperfusion, consistent with the sensitivity of MAP2 antigen to ischemia.41 Only 1.8±0.5% dUTP+ (TdT) cells were associated with microvascular structures displaying collagen IV antigen by 24-hour reperfusion (Table 4Down and Fig 5CDown). dUTP was incorporated in unidentified cells associated with the microvascular wall no earlier than 1-hour reperfusion (Table 4Down).


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  		Table 4. Localization of dUTP Incorporation in the Nonhuman Primate



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  		Figure 5. Representative photomicrographs of double-label immunoperoxidase studies with dUTP incorporation (black color) and cell markers (red color). Intact MAP2+ neurons (red) were seen with damaged neurons double-labeled with dUTP (black) and MAP2 (red) at 2-hour ischemia (A) and at 1-hour reperfusion (B). A few dUTP+ cells (black) were associated with microvessels identified by collagen IV in the basal lamina (red) (C). Astrocytes defined by GFAP antigen (red) were observed in the region of dUTP+ cells (black) (D). Bar=50 µm.

Fate of dUTP+ Cells
By 24-hour reperfusion in the nonhuman primate, activated astrocytes defined by expression of GFAP antigen were observed in the region of cellular dUTP label (Figs 5DUp and 6ADown and 6B). Most often they circumscribed the region of dUTP+ cells. Similarly, polymorphonuclear leukocytes defined by myeloperoxidase antigen had infiltrated the region of nuclear dUTP incorporation; however, dUTP label was not identified specifically with granulocytes (Fig 6CDown and 6DDown).



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  		Figure 6. Dual-label immunofluorescence comparison of cells displaying DNA damage (dUTP+, FITC) with markers of astrocytes (GFAP, TRITC) or granulocytes (myeloperoxidase, TRITC). Astrocytes (A) were found within the region of dUTP+ cells (B). Surrounding granulocytes (C) were noted in the region of dUTP+ cells (D). Bar=50 µm.


*    Discussion
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 up arrowAbstract
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 *Discussion
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This is the first description of characteristic and significant differences in temporal topographical and quantitative responses of neurons and microvascular cells of the basal ganglia to focal cerebral ischemia and reperfusion between primates and rodents. The incorporation of dUTP by approaches employing TdT, DNA polymerase I, and the Klenow fragment provided convenient reproducible methods for identifying cells subject to ischemic injury. The distribution and the time course of DNA scission displayed in situ by each method was characteristically different in the nonhuman primate and the Wistar rat at identical times after MCA:O(/R). Evidence of neuronal injury by all three techniques was observed significantly earlier in the primate (within 2 hours of MCA:O) than in the rodent. In the primate, cellular injury could not be directly ascribed to invasion by polymorphonuclear leukocytes or the activation of astrocytes. By 24 hours of reperfusion, the region of dUTP incorporation was circumscribed by reactive astrocytes that were for the most part not labeled with dUTP. Precise identification of neurons by MAP2 antigen was difficult by 24 hours of reperfusion in either primate or rodent preparation.

The significantly different distributions and time course of ischemia-related neuronal injury in the primate and the rat corpus striatum are of great interest. Occlusion of the proximal penetrating arterioles of the MCA initiates neuronal injury and eventual necrosis in the basal ganglia and the temporoparietal cerebral cortex in both species.23 26 However, the severity of the ischemic insult to these structures might reflect different neuronal cell properties in the two species. For instance, among different rodent strains, MCA:O induces lesions with significantly different volumes of injury.42 Those differences have remained unexplained. Also, in the developing primate and rat brain, many of the cells described as apoptotic possess a neuronal phenotype as defined by histochemistry.43 During embryonic development, differences with time in the distribution of dying cells within the basal ganglia were noted between the rat and the primate, although its significance for the adult brain remains unclear.43 Furthermore, neurons in the neostriatum present two distinct types by their nuclear morphology: spiny neurons that have an unindented nucleus and aspiny neurons that exhibit enfoldings of their nuclear envelope.44 45 Interestingly, the proportion of neostriatal neurons with nuclear indentation is significantly greater in the nonhuman primate than in the rodent.46 Furthermore, a heterogeneous distribution of neurons with indented nuclei is reported within the primate striatum, whereas such regional differences are not observed in the rodent.46 These morphological findings may underlie differences in neuronal responses to focal ischemia observed between the two species here.

The 20-fold increase in neurons displaying DNA scission in the ischemic primate basal ganglia compared with analogous rat structures at 2-hour MCA:O may further reflect (1) general differences in cell characteristics; (2) differences in microvascular distribution, including collateral development; (3) effects including the impact of anesthetics; and (4) peculiarities of model preparation in both species.

Differences in functional receptor distributions47 48 49 and protein phosphorylation and dephosphorylation (secondary to brain ischemia) known to exist in the two species50 51 52 53 54 underscore potential relevant differences in cellular reactivity and function. Therefore, nonhuman primate and rat neurons in situ may respond differently to focal ischemia, which may lead to distinct effects on neuronal nuclear DNA integrity. Unfortunately, there is little experimental evidence to clarify this.

During ischemia, biochemical processes are initiated that lead to cell injury. In the brain, oxygen free radicals abundantly produced during reperfusion have been implicated in DNA injury.55 56 57 In addition, increased Ca2+ levels in ischemic cells58 may trigger Ca2+-dependent endonuclease activation, which induces DNA injury.4 In cardiomyocytes, reperfusion itself, but not ischemia, may initiate DNA cleavage.15 In the present study the increasing number and density of dUTP+ cells is dependent on the duration of reperfusion, which might exaggerate cellular DNA injury, at least in the rodent. This observation is consistent with previous reports.34 59 In the primate, however, the appearance of heterogeneously distributed dUTP+ cells at 2 hours of MCA:O implies that ischemia itself can induce DNA injury/repair. One interpretation might be that the severity or depth of ischemia in the primate corpus striatum is enough to induce DNA cleavage in vulnerable cells, while ischemia in comparable rat tissues requires reperfusion to further stimulate the appearance of DNA injury in situ. Alternatively, the pace of ischemic injury in the two species and models may be different.

The vascular supply, ie, the anatomic distribution of feeding arteries, of the basal ganglia is probably relevant. In the rat, the perforating arteries stem from the MCA and ACA, but the supply by the ACA to the basal ganglia is more robust than in the primate.27 30 In the Wistar rat, the distribution of dUTP label by each method appeared to follow the territorial outlay of the perforating vessels. The distribution of nuclear dUTP incorporation among individual primates varied within the putamen and/or caudate nucleus, variously involving or sparing the internal capsule early, but involving it late. Persistent flow to the ischemic zone despite MCA:O28 may provide relative protection of ischemic vascular and nonvascular cells, which may be greater in the rat striatum than the primate.

The proximity of neurons to the vascular supply may be very relevant to their sensitivity to focal ischemia, as evidenced by dUTP incorporation. This is implied by the increase in density of cells that incorporated dUTP during ischemia/reperfusion in both species. The consistent and progressive dUTP incorporation over time within the caudate putamen is consistent with the terminal supply of the penetrating arterioles in the rat.27 It is unlikely that the delay in dUTP label in that territory is due to recurrent arteries from the ACA, since the caudal portion of the caudate putamen is predominantly supplied by the MCA in Wistar rats.30 However, microvascular occlusion within the anterior striatal artery distribution cannot be ruled out. In the primate, a heterogeneous distribution of microvascular occlusions during early ischemia24 60 may contribute to the highly varied regional distribution of dUTP label. Progressive interruption of microvascular blood flow with time by mechanisms previously reported24 32 may contribute to the increasing density of cellular dUTP incorporation observed later in reperfusion. Therefore, cellular injury as evidenced by nuclear DNA scission may represent the summation of responses to ischemia, including heterogeneous microvascular lesions, which are unique to each of the two species.

Other factors that may affect the appearance of identifiable DNA scission include the use of halogenated anesthetics during the induction of ischemia in the rat compared with the awake primate. In addition to putative effects on neurons, such anesthetics significantly impair leukocyte chemotaxis in humans61 62 and other primates.35 They may be responsible for differences in ischemic injury among primate models of MCA:O/R.23 63 To what degree these effects perturb established cell vulnerabilities is not known.

The specificity of the TdT enzyme, DNA polymerase I holoenzyme, and the Klenow fragment allow identification of nuclear DNA scission. Because TdT may identify gaps and extend protruding 3'-OH ends of double-strand DNA, this reaction theoretically can detect clean or staggered double-strand DNA breaks.64 However, its reliability to distinguish in situ internucleosomal DNA fragmentation as apoptosis or necrosis is controversial.6 19 20 65 66 67 The TdT technique, in its most simplistic interpretation, which we apply here, is an indicator of severe cell injury. DNA polymerase I holoenzyme exonuclease activity mediates nick translation and therefore allows visualization of randomly occurring single-strand scission of double-strand DNA, as observed in other processes of cell death associated with MCA:O/R.21 The nick is translated along the paired DNA in a 5'->3' direction.68 The Klenow fragment of DNA polymerase I lacks the exonuclease activity and cannot nick translate, although it can detect gaps and recessed 3'-OH ends.69 Theoretically, it would be expected that both the polymerase holoenzyme and Klenow fragment would detect both single- and double-strand DNA scission to a similar degree, but more frequently and intensely than the TdT enzyme. Indeed, here the DNA polymerase holoenzyme detected cellular DNA injury over a larger region in the primate than the TdT method, although by 24 hours of reperfusion the regions of dUTP label defined by the three enzymes were coterminous and the densities were the same in both species. These data do not allow us to judge the relative sensitivity of the three methods because the optimal conditions for each have not been identified for brain tissue.66

Although the precise mechanisms underlying DNA scission and the contribution of repair were not explored in this study, it is known that neurons can repair double-stranded DNA breaks very slowly in comparison with other cells of the central nervous system, such as astrocytes.70 We suggest that most neurons exhibiting DNA scission without an effective DNA repair system are sensitive to ischemia. It may be the relative lack of a rapid DNA repair mechanism that confers selective vulnerability of striatal neurons to focal ischemia compared with other cells in the brain.

Most dUTP (TdT)-labeled cells seen within 2 hours of MCA:O in the primate were neurons, consistent with previous reports in rats.7 71 One recent study suggested that cells displaying similar dUTP incorporation in the rodent brain by 48 hours after MCA:O were neurons.34 This identification was not confirmed in our experiments. More in keeping with our results were the observations in both species and models that DNA scission did occur early after MCA:O.5

The incorporation techniques applied here do not unequivocally detect apoptosis or necrosis.6 34 65 66 67 Discrete quantitated ultrastructural studies including electron microscopy are necessary to examine for nuclear changes consistent with apoptosis or necrosis. The use of time from MCA:O as a feature of apoptosis, especially in the rat, may be misleading in view of the finding of later cell injury relative to the primate.

Finally, the appearance of DNA scission or repair in nonendothelial vascular cells (>1.7%) by 4 hours of reperfusion in the primate is consistent with the lower vulnerability of vascular cells to ischemia compared with neurons. The identity of those cells was not certain in the primate, although they were associated with the basal lamina. The absence of early widespread endothelial cell injury (by dUTP incorporation) suggests that in the primate neurons are more vulnerable. This is supported by evidence of selective neuronal death after ischemia.72 73

The differing patterns and temporal relationships of DNA scission between the nonhuman primate and rodent during MCA:O/R are consistent with reported differences in peak expression of microvascular intercellular adhesion molecule-1 antigen,32 33 suggesting that territorial injury may proceed at a different pace in the two species and models. Of practical interest, these dUTP incorporation techniques allow convenient early identification of regions of mostly nonvascular cell injury after MCA:O. As markers of cell vulnerability, they will have great utility in the correlation with microvascular events in the primate. The differences in cell vulnerability between the two species in relation to their territorial vascular supply are of special interest. They suggest differences in injury/repair mechanisms and flow dynamics that must be taken into account in interpreting outcomes with both species.


*    Selected Abbreviations and Acronyms
 
ACA = anterior cerebral artery
BSA = bovine serum albumin
CCA = common carotid artery
ECA = external carotid artery
FITC = fluorescein isothiocyanate
GFAP = glial fibrillary acidic protein
ICA = internal carotid artery
MAP2 = microtubule-associated protein-2
MCA = middle cerebral artery
MCA:O = middle cerebral artery occlusion
MCA:O/R = middle cerebral artery occlusion/reperfusion
TdT = terminal deoxynucleotidyl transferase
TRITC = tetramethylrhodamine B isothiocyanate


*    Acknowledgments
 
This study was supported by grant NS-26945 from the National Institutes of Health. We would like to thank Dr John MacManus for his many helpful discussions and critical comments during the preparation of this manuscript. We are grateful to Dr James Koziol for his statistical analysis of the results. We also thank Pearl Akamine and Jacinta Lucero for their technical assistance.


*    Footnotes
 
This is publication No. 10431-MEM from The Scripps Research Institute, La Jolla, Calif.

Received October 17, 1996; revision received February 7, 1997; accepted March 5, 1997.


*    References
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 *References
 

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