Abstract In this study we examined the time course ofapoptotic cell death after photochemically induced focalischemia of the rat cerebral cortex. For unequivocal dif-ferentiation between apoptosis and necrosis two criteria ofprogrammed cell death were used: terminal deoxyribonu-cleotidyl transferase-mediated dUTP-digoxigenin nick endlabeling (TUNEL) and morphological evidence of frag-mentation and marginalization of nuclei. After photothrom-bosis, many TUNEL-positive cells were found within theinfarct region from 12 h to 3 days. By day 6 they werepreferentially located in the boundary zone of the infarct,and by day 14 they had disappeared. A high proportion ofTUNEL-positive cells displayed fragmentation or margin-alization of their nuclei, indicating apoptosis. Neurons,but not T cells and macrophages, were apoptotic. Inflam-matory infiltrates were in close contact to apoptotic neu-rons throughout the infarct areas at day 1 and in theboundary zone between days 2 and 6 after photothrombo-sis. In summary, our study shows that neuronal apoptosisafter cerebral ischemia is a prolonged process to whichleukocyte-derived cytokines may contribute. In contrast toautoimmune diseases of the nervous system, terminationof the local inflammatory response after cerebral ischemiadoes not involve apoptosis.

Key words Focal ischemia · Apoptosis · Necrosis · Inflammation · T cells

Introduction

The pathogenic mechanisms that give rise to ischemicbrain damage have not been definitely determined, but

there is good evidence that excitatory amino acids, acido-sis, increases in calcium concentration and production offree radicals are critically involved (reviewed in [31]). Inrecent years it has become clear that, under the influenceof these neurotoxic factors, neurons die via two differentpathways: by necrosis and by apoptosis [4, 15, 17, 19,22]. While necrosis represents passive degeneration ofcells, apoptosis is an active form of programmed suicidalcell death which is energy dependent [11, 18]. Apoptosisis biochemically characterized by activation of cellularendonucleases and cleavage of nuclear DNA between nu-cleosomes into fragments of approximately 180–200 bp.Apoptotic cells can be distinguished from cells undergo-ing necrosis by positive terminal deoxyribonucleotidyltransferase (TNT)-mediated dUTP-digoxigenin nick endlabeling (TUNEL) [5, 9] and typical morphological alter-ations of their nuclei, which undergo condensation, mar-ginalization, segregation and fragmentation.

We have recently shown that focal cerebral ischemia inthe rat induced by photothrombosis or occlusion of themiddle cerebral artery leads to a profound inflammatoryresponse with lymphomonocytic infiltration, predomi-nantly in the boundary zone of the infarcts [10, 29]. Sinceleukocytes and activated microglia can release factors thatare toxic for neurons [6, 7], and since leukocyte-derivedcytokines can induce apoptosis [1, 3, 12, 30, 34], we ex-amined the time course of apoptotic cell death after pho-tothrombosis in relation to this inflammatory response.We show that apoptotic cell death after photothrombosisis a prolonged process continuing for several days after is-chemia and is spatially related to the presence of inflam-matory cells in the boundary zone of the infarcts.

Materials and methods

Induction of photothrombosis

Focal cerebral ischemia was induced in male Wistar rats accordingto the method of Watson et al. [32], as described in detail else-where [10]. Briefly, illumination of the rat skull with a white lightbeam during intravenous injection of the photosensitive dye Rose

Johann S. Braun · Sebastian Jander ·Michael Schroeter · Otto W. Witte · Guido Stoll

Spatiotemporal relationship of apoptotic cell death to lymphomonocytic infiltration in photochemically induced focal ischemia of the rat cerebral cortex

Acta Neuropathol (1996) 92 :255–263 © Springer-Verlag 1996

Received: 5 February 1996 / Revised, accepted: 5 April 1996

REGULAR PAPER

J. S. Braun1 · S. Jander · M. Schroeter · O. W. Witte · G. Stoll (Y)Department of Neurology, Heinrich Heine University, PO Box 101007, D-40001 Düsseldorf, Germany Tel.: +49-211-81-17881; Fax: +49-211-81-18485

Present address:1 Department of Psychiatry, Albert Ludwig University Freiburg, Freiburg, Germany

Fig. 1 Temporal occurrence of terminal deoxyribonucleotidyl trans-ferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) inthe infarct region 4 h (A), 12 h (C) and 24 h (D) after photothrom-bosis. While TUNEL is absent after 4 h (A), some nuclei are alreadystained in superficial cortical layers by in situ nick translation(ISNT) (B). At 12 h (C), a weak TUNEL can be detected over the

whole ischemic focus. Staining is fully developed after 24 h (D). E,F show that after 24 h a large proportion of TUNEL-positive cellsundergo apoptosis: many TUNEL-positive cells (marked by doublearrows in E) show fragmentation or marginalization (single arrows)as revealed by 4′,6-diamidino-2-phenyl indoledihydrochloride(DAPI) staining (F). Bars A–D = 1 mm; E, F = 50 µm

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Fig. 2 TUNEL at later stages; 3 (A), 6 (B) and 14 (C) days afterinfarction. In comparison to 24 h (Fig. 1D) the number of TUNEL-positive cells has decreased at day 3 (A). At 6 days after pho-tothrombosis (B) TUNEL-positive cells are preferentially locatedin the boundary zone of the infarct, while at 14 days TUNEL is vir-

tually absent (C). At day 3, a considerable number of TUNEL-pos-itive cells (arrows in D) still undergo apoptosis, as shown by frag-mentation of their nuclei by DAPI staining (E). Bars A–C = 1 mm;D, E = 50 µm

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Bengal leads to photoperoxidation of the illuminated endotheliumwith ensuing thrombotic infarction. With this method focal corticalinfarcts were induced 3.5–4 mm posterior to the bregma and 3.5–4mm lateral from the midline in the sensory cortex of Wistar rats.Thereafter groups of animals were perfused, under deep anesthe-sia, at 4 h, 12 h, and 1, 2, 3, 6, and 14 days with 4% paraformalde-hyde, and brains were embedded in paraffin.

Detection of apoptotic cell death

In situ labeling of fragmented DNA on paraffin sections was per-formed by two methods, in situ nick translation (ISNT) with DNApolymerase I [33] and the TUNEL assay [5, 9]. Sections (5 µm)were predigested with 20 µg/ml proteinase K (Sigma) for 15 minat room temperature (RT) before incubation with the reaction mix-ture for 1 h (TUNEL) or 2 h (ISNT) in a moist chamber at 37°C.The TUNEL reaction mixture consisted of 10 µl 5x tailing buffer(Boehringer, Mannheim, Germany), 0.5 µl digoxigenin (DIG)DNA labeling mixture (Boehringer), 0.5 µl cobalt chloride (25mmol, Boehringer), 4 U terminal transferase (Boehringer) and 39µl distilled water. The ISNT reaction mixture consisted of 5 µl 10xnick translation buffer [26], 0.5 µl DIG DNA labeling mixture, 1.6U DNA polymerase I (Boehringer) and 44 µl distilled water. Afterrinsing in TRIS-buffered saline (TBS) and blocking with 3% nor-mal goat serum (NGS) in TBS (30 min, RT) sections were incu-bated with alkaline phosphatase (AP)- or fluorescein isothio-cyanate (FITC)-labeled anti-DIG antibody (Boehringer) diluted1:500 or 1:100 in TBS with 3% NGS for 1 h at RT. The incorpo-rated nucleotides were visualized using 5-bromo-4-chloro-3-in-dolyl-phosphatase/4-nitro blue tetrazolium chloride (BCIP/NBT;Boehringer) or New Fuchsin substrate system (Dako, Hamburg,Germany) as substrate for AP. Following TUNEL or ISNT, all sec-tions were stained with 2 mg/ml 4′,6-diamidino-2-phenyl indoledi-hydrochloride (DAPI) to reveal nuclear morphology. Because ofthe limits of specificity of the TUNEL and ISNT reactions, whichalso label nuclei in later stages of necrosis (after approximately12–24 h) due to random DNA fragmentation [13], we defined onlythose cells as apoptotic that fulfilled both the morphological(DAPI) and molecular (TUNEL or ISNT) criteria of apoptosis.

Immunocytochemistry

After blocking with 3% NGS, sections were incubated with thefollowing primary antibodies before or after TUNEL and ISNT:mouse monoclonal antibody (mAb) ED1, a macrophage marker(Serotec, Oxford, UK), mAb 15-6A1, a pan-T cell marker (Hol-land Biotechnology, Leiden, The Netherlands), rabbit polyclonalantibody against neuron-specific enolase (NSE; Dako) and anmAb against NeuN (a gift from Dr. Kuhn, La Jolla [20]), whichrecognizes neurons. As secondary antibody, affinity-purified bi-otinylated rat adsorbed anti-mouse or rabbit IgG was used, fol-lowed by the avidin-biotin-peroxidase complex (Vectastain Elite,Camon, Wiesbaden, Germany). The color reaction was developedwith diaminobenzidine. In one set of experiments sections were

stained by immunocytochemistry for T cells or macrophages, andphotos were taken to document the distribution of the infiltrates.The sections were then processed further for TUNEL before thesame region was photographed again (see Fig. 3). Comparison ofthe micrographs allowed an analysis of the spatial relationship be-tween apoptotic cells and inflammatory infiltrates.

Results

No important differences were seen between TUNEL andISNT staining with respect to the local distribution andtime of occurrence of apoptotic cells. Positive TUNELand ISNT staining was restricted to ischemic areas. In thecontra- and ipsilateral hemisphere remote from the is-chemic focus no or rare nuclei – one to three in each tis-sue section – were labeled with TUNEL or ISNT. Thespecificity of the signal was checked by omitting eitherthe terminal transferase or polymerase I in the TUNEL orISNT assay, which led invariably to loss of staining.

At 4 h after photothrombosis, nuclei were not labeledwith TUNEL (Fig. 1A). However, ISNT (Fig. 1B) stainedsome nuclei located at the surface of the ischemic focus.With DAPI staining, slight nuclear rarification and con-densation, but no fragmentation, was seen. Lymphomono-cytic infiltration was absent.

After 12 h (Fig. 1C), the first TUNEL-positive nucleiwere detected and roughly 20% of them were fragmented.The intensity of TUNEL labeling was weak. Fragmentedand non-fragmented TUNEL-positive nuclei were scat-tered diffusely over the ischemic focus, but increasedgradually from the basis to its surface. Inflammatory cellswere still absent.

At 1 day after photothrombosis (Fig. 1D), most nucleiwithin the ischemic focus were labeled with TUNEL orISNT. Approximately 30% of the TUNEL-positive nucleishowed fragmentation and marginalization, as revealed bydouble labeling with DAPI staining (Fig. 1E, F). Thisshowed that these cells underwent apoptosis. Apoptoticnuclei were more frequent in the periphery than in the is-chemic core. Nuclear DAPI staining began to disappearfrom the center of the lesion. Most TUNEL-positive cellscould be identified morphologically as neurons aftercounterstaining sections with hematoxylin and eosin. Inan attempt to confirm that neurons undergo apoptosis, westained sections for NSE and NeuN, a neuron-specific nu-clear protein. Neurons in intact brain regions were stainedby both markers. In contrast, neurons in the lesion wereinvariably NSE negative, but were labeled with antibodiesagainst NeuN (not shown). However, when sections werepretreated for TUNEL, NeuN immunoreactivity was lost,probably due to the destruction of the nuclear antigen;similarly, no TUNEL positivity was seen when immuno-cytochemistry was performed first. At this early time pointT lymphocytes were diffusely scattered over the whole is-chemic focus, although more prominently on its surface(Fig. 3A). These T cells were often in close contact toapoptotic cells (Figs. 3B, 4A). Double labeling revealedthat T cells were not apoptotic (Fig. 4A). Only few ED1-positive macrophages/microglia were found at that time.

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Fig. 3 Spatial relation of lymphomonocytic infiltration to apopto-sis at day 1 (A, B) and day 3 (C, D) after photothrombosis. Thesurface of the cortex is at the top in each case. Sections werestained for T cells (A) and macrophages (C), and photographstaken to document the distribution of infiltration. TUNEL was thenperformed to identify apoptotic cells and finally the same sectionswere rephotographed (B, D). The comparison between early andfinal micrographs allows a reconstruction of the spatial relation-ship between inflammatory cells and dying neurons. Note thatearly after infarction T cells are scattered over the entire infarct (A)in regions with many TUNEL-positive cells (marked by arrows inB). At day 3 ED1-positive macrophages/microglia are present inthe boundary zone, where many apoptotic cells stain positively forTUNEL (arrows in D). Bars A–D = 200 µm

F

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At 2 days after photothrombosis, the number ofTUNEL-positive nuclei was decreased by about 60%. Asafter 1 day, more apoptotic nuclei were detected in the pe-riphery than in the center of the lesion. In addition, nu-clear DAPI staining was decreased further in the core ofthe infarct.

At 3 days after photothrombosis (Fig. 2A), a furtherdecrease of TUNEL-positive nuclei was obvious. Theproportion of TUNEL-positive nuclei showing fragmenta-tion indicative of apoptosis was higher in the periphery ofthe lesion (Fig. 2D, E) than in the core. Overall, the num-ber of nuclei as revealed by DAPI staining had decreased,especially in the core of the lesion, reflecting necrosis.Macrophages and lymphocytes now formed a ring aroundthe lesion (Fig. 3C), as shown in our previous study [10].In the boundary zone of the lesions, T cells and macro-phages were in close contact to apoptotic cells (Figs. 3C,D; 4B, C). T cells and ED1-positive macrophages/micro-glia were invariably TUNEL negative at any stage afterphotothrombosis (Fig. 4B–D).

At 6 days after photothrombosis (Fig. 2B), TUNEL re-action product was primarily localized peripherally in theregion of lymphomonocytic infiltration (Fig. 4D), and thenumber of ED1-positive macrophages/microglia had in-creased further. DAPI staining disappeared from the centerof the lesion, and in the periphery the proportion of nucleishowing fragmentation was lower than that seen on day 3.

At 14 days after photothrombosis (Fig. 2C), TUNELreaction was virtually absent in the lesion area, whichnow was entirely covered by ED1-positive macrophages/microglia (not shown), accounting for the reappearance ofnuclear DAPI staining in this region. Macrophage infiltra-tion was maximal at this time point. Nuclear fragmenta-tion could no longer be detected.

Discussion

This study confirms and extends previous investigationsshowing that neurodegeneration resulting from cerebralischemia has an apoptotic component [4, 15, 17, 19, 22].The occurrence of apoptotic cell death in photochemicallyinduced focal ischemia of the rat cerebral cortex could beshown by a positive TUNEL reaction in combination withmorphological criteria such as condensation, marginaliza-tion and fragmentation of nuclei. This is a crucial issuesince, in advanced stages of necrosis, dying cells may alsoshow a positive TUNEL reaction due to the degradationof DNA [8, 13, 24].

In an extension of previous studies, we have analyzedthe spatiotemporal relationship of apoptosis to lympho-

monocytic infiltration. Infarcts were covered by TUNEL-positive cells; this was most pronounced between days 1and 3 after photothrombosis. At day 6 TUNEL-positivecells were concentrated in ring-like fashion around the in-farct. Most of them were identified as neurons on mor-phological grounds. TUNEL-positive cells consisted oftwo populations: a high proportion of apoptotic cells dis-playing typical features with fragmentation and marginal-ization of their nuclei, and necrotic cells. A similar com-position of apoptotic and necrotic cells has recently beendescribed after experimental traumatic brain injury in therat [24]. Considering that, in vitro, the process of apopto-sis from the initial structural changes to complete cellularfragmentation takes about 4 h [1], the presence of apop-totic cells at 3 days and to a lesser extent at 6 days afterphotothrombosis suggests that cell death after ischemia isa dynamic ongoing process not simply caused by the ini-tial ischemic insult. In keeping with our findings, Li et al.[15] found a prolonged persistence of apoptotic neurons,for up to 4 weeks, predominantly in the boundary zone ofthe ischemic region after transient middle cerebral arteryocclusion in rats.

We recently described a profound inflammatory re-sponse to focal cerebral ischemia after photothrombosisand middle cerebral artery occlusion in rats [10, 29]. Infil-trates consisted of CD5+ T cells, ED1-positive macro-phages/microglia and a not yet fully identified populationof CD8+ cells which most likely represents a mixture ofcytotoxic/suppressor T cells and natural killer lympho-cytes. In the present study inflammatory infiltrates werein close contact to apoptotic neurons throughout the in-farct at early stages and in the boundary zone at later timepoints after photothrombosis. Apoptosis does not usuallyinduce such an inflammatory response [27] unless it oc-curs on a large scale as in certain phases of embryonic de-velopment (reviewed in [18]). Therefore, it is conceivablethat leukocytes are attracted to ischemic lesions bychemokines, and that leukocyte-derived cytokines thencontribute secondarily to the delayed apoptosis of neuronsseen in the boundary zone of infarcts. In keeping with thishypothesis, CD8+ cytotoxic T cells can kill other cells byinducing programmed cell death [2]. Moreover, lympho-cytes and macrophages are major sources of the lympho-toxin and tumor necrosis factor that have been shown toinduce apoptosis in a variety of target cells [3, 30, 34]. In-terestingly, even immunosuppressive cytokines such astransforming growth factor-beta, which is highly express-ed after stroke [14], can induce apoptosis [1]. However,further studies are necessary to identify unequivocally thepathogenic factors responsible for the delayed apoptosisof neurons after focal ischemia seen in the present andprevious studies.

The functional contribution of programmed cell deathto ischemic brain damage has been indirectly shown byLinnik et al. [16]. The size of the infarction after middlecerebral artery occlusion was dramatically reduced bytreatment with a protein synthesis inhibitor. Since apopto-sis is an energy-dependent process, it was concluded thatthe reduction in infarct volume was due to the reduction

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Fig. 4A–D T cells and macrophages do not undergo apoptosis.Colocalization of TUNEL (red) and T cells (A, B; brown) ormacrophages, (C, D; brown) by immunocytochemistry on thesame section at day 1 (A), 3 (B, C) and 6 (D) after photothrombo-sis. Note that T cells and macrophages have infiltrated areas ofapoptotic cell death similar to Fig. 3, but are always TUNEL neg-ative. In particular T cells are often in intimate contact to TUNEL-positive cells (arrows in A, B). Bars = 50 µm

F

of apoptotic neuronal loss. Similar results using an en-donuclease inhibitor have been obtained in a neuron cul-ture system of hypoxia-induced apoptosis [25].

The identification of cells undergoing apoptosis de-serves further comment. Many TUNEL-positive cellsshowed the typical morphology of neurons after counter-staining with hematoxylin and eosin. Although we wereable to stain degenerating neurons in the infarct area withantibodies against the neuron-specific nuclear protein,NeuN [20], this immunoreactivity was lost when sectionswere pretreated for the TUNEL reaction. Moreover, de-generating neurons were always negative after stainingfor NSE. In contrast, T cells and macrophages could beeasily identified by immunocytochemistry on the samesections after performance of the TUNEL reaction. Theywere always TUNEL negative. In experimental autoim-mune encephalomyelitis, programmed cell death leads toelimination of infiltrating T cells and macrophages, amechanism considered important for the induction of clin-ical recovery [21, 23, 28]. Thus, in focal cerebral ischemiaother mechanisms must be involved in terminating the lo-cal inflammatory response.

Acknowledgements We thank U. Vollmer for photographic as-sistance. This work was supported by the Deutsche Forschungsge-meinschaft (SFB 194 B2 and B6). G. S. holds a Hermann- andLilly-Schilling professorship.

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