Temporal pattern of internucleosomal DNA fragmentation in the striatum and hippocampus after transient forebrain ischemia

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NeuroseieneeLetters 186 (1995)157-160

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Temporal pattern of internucleosomal DNA fragmentation in the striatum and hippocampus after transient forebrain ischemia B r u c e T. V o l p e a, T h o m a s C. W e s s e l a, B h a s k a r M u k h e r j e e b, H o w a r d J. F e d e r o f f b,* aDepartment of Neurology and Neuroscience, CorneU University Medical School, Burke Institute for Medical Research, 785 Mamaroneck Avenue, White Plains, NY 10605, USA bDepartments of Medicine and Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Received29 September1994;revisedversionreceived5 Deeember1994;accepted11 January 1995

Abstraet

Transient forebrain ischemia in rodents caused internucleosomal DNA fragmentation that appeared in the striatum 24 h after reperfusion, and in the hippocampus 72 h after reperfusion. Gel electrophoresis and an in situ technique to label 3' termini of endonuclease generated DNA fragments demonstrated similar temporal patterns. These data show that endonuclease activation accompanies the demise of selecfively vulnerable neurons following transient forebrain ischemia. Keywords: Apoptosis; Program cell death; Ischemia; Hippocampus; Striatum

In the rat there is a temporal delay between an episode of transient forebrain ischemia and neuron death [17,18]. One to three days after reperfusion, relatively restricted regions in the striaturn and hippocampus undergo cell death [17,18,20,21]. Recent findings in a number of animais models of ischemia suggest that an additional feature of neuron demise is internucleosomal DNA fragmentation [1,7,10,12,14,15,22]. In these experiments, we demonstrate that the temporal course of the appearance of internueleosomal DNA fragmentation and the in situ labeling of 3' termini of DNA in rats exposed to four vessel occlusion is similar to neuropathological death. Internucleosomal DNA fragmentation was detected only in the striatum and hippocampus. Maie Wistar rats (8 weeks of age) were exposed to 20 min of transient forebrain ischemia by a method previously described and modified [17,23]. For electrophoretic analysis, ischemic animais were sacrificed 1 h (n = 2), 24 h (8), 48 h (6), 72 h (3), and 120 h (3) after reperfusion. Brains were rapidly removed, frozen, and suspended in 0.5 ml of a buffer containing 10 mM Tris--Cl (pli 7.5), 20 mM EDTA, 0.5% v/v Triton X-100 for 30 min at 4°C. Tissue suspensions were spun at 13 000 x g for 15 min and the supernatant removed, adjusted to 0.3 M with respect to sodium acetate, extracted with phenol* Correspondingauthor.

chloroform-isoamyl aicohol (1:0.5:0.04), and ethanol precipitated. Entire samples were fractionated on 1% agarose gels containing 10/rg/ml ethidium bromide, photographed and transferred to nylon membranes. DNA was crosslinked to blots by heating at 65°C for 2 h, prehybridized in 50% formamide, 5 x SSC ( I x =0.15 M sodium chloride, 0.018 M sodium citrate, pli 7.5), 1% sodium dodecyl sulfate, 5 x Denhardt's solution ( I x =0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% BSA, Pentax Fraction V) and 100/rg/ml of denatured salmon sperm DNA at 42°C for 3-10 h. Blots were hybridized in the same solution whieh also contained 106 cpm/ml of 32p_ labeled total rat genomic DNA prepared by random priming (Prime-It, Stratagene, Inc.) according to the manufacturers instructions for 12-16 h at 42°C. Blots were washed sequentially in 0.2x SSC, 0.1% sodium dodecylsulfate at room temperature twice for 5 min each and 65°C twice for 10 min each. Blots were then exposed to Kodak X-Omat film for 0.5 h to several days at -70°C with intensifying screens prior to developing. For in situ analysis, there were 8 sham and 8 ischemic animais that were sacrificed 1 h (2), 24 h (3), and 72 h (3) after reperfusion. Their brains were perfused with 4% formaldehyde, 0.1 M phosphate buffer (pli 7.4), via the ascending aorta after brain circulation was washed out with heparinized saline. The brains were post fixed for 1 h in cold perfusate and placed in 30% sucrose buffer

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B.T. Volpe et aL I Neuroscience Letters 186 (1995) 157-160

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Fig. 1. Internucleosomal DNA laddering deteeted by agarose gel eleetrophoresis. Soluble DNAs were prepared from different braln regions at various times after stroke, separated on 1% agarose gels containing ethidium bromide, photographed, Southern blotted to nylon membranes, hybridized to 32p. labeled totaled rat genomie DNA, washed and autoradiographed. (A) Photograph of ethidium bromide stained gel. Lanes 1 and 14 are DNA size standards, the 1 kb and 123 bp ladders (GIBCO-BRL), respectively. Lanes 2-13 represent DNA samples prepared from brain regions. Each group of four lanes (2-5; 6--9; 10-13) are samples from individual animais and are labeled in the order candate, thalamus, frontal cortex, and hippocampus. Two-day post stroke animais, lanes 2-5 and 6--9; 1-day post stroke animal, lanes 10-13. (B) Photograph of ethidium bromide stalned gel. As in (A), lanes 1 and 14 are DNA size standards. Each group of 4 lanes (2-5; 6--9; 10-13) are samples from individual animais and are loaded in file same order as in (A). All lanes are from 3-day post stroke animais. (C) Autoradiogram of blotted gel shown in (B). Note that with autoradiography, DNA laddering is se,en in some hippocampal samples (lane 13) that were not observed on the ethidium bromide stained gel (B).

overnight in the cold. The next day the brain was blocked for coronal sections which were eut at 40/~m on a freezing sliding microtome and placed in 0.1 M phosphate buffer and mounted on gelatin subbed slides. We modified a method to identify DNA fragmentation in situ [5,6]. After air drying, the sections were covered with 80/tl of terminal transferase (TdT) buffer containing cobalt chloride (COC12, 2.5 mM, Boehringer Mannheim), covered with a strip of ParafilmTM and pre-incubated for 15 min at 37°C. The ParafilmTM strip was removed, 10/ri of buffer containing 10 IU TdT and 0.6/tCi [35S]dCTP was applied to every slide, and the tissue section eovered again with the same strip of film. TdT reaction was terminated after 30 min by submerging the slides in 0.1 M phosphate buffer at 37°C. Four successive washes were performed (37°C), slides were air dried and placed adjacent to X-ray film, and exposed overnight. Subsequently, the slides were dipped in photoemulsion (Kodak NTB-2) and plaeed in light-tight boxes for 24 h before development. Tissue sections were counterstained with cresyl violet and photomicrographs were obtained. Internucleosomal DNA fragmentation was observed by ethidium bromide staining from the caudate nucleus at 1 and 2 days after ischemia (Fig. lA, lanes 2, 6, 10). Southern blotting and hybridization with radioaetively labeled DNA was used to increase the sensitivity of deteetion of DNA ladders. With autoradiography of hybridized blots (Fig. 1C) we were able to detect DNA ladders in hippocampal specimens at 2 (data not shown) and 3 days after isehemia (e.g. compare lane 13 in the ethidium stained gel in Fig. lB and the autoradiograph of the blot in Fig. 1C). The data from three laddering experiments are summarized in Table 1. At 1 day after ischemia, DNA ladder-

ing was observed in caudate speeimens from seven of eight animais and in hippocampal tissue from one of eight animais. Of the six animais at 2 days after isehemia, rive were positive within the caudate and four were positive in the hippocampus. No laddering was deteeted in the eaudate or hippocampus 1 h or 5 days after isehemia. Internucleosomai DNA fragmentation was not observed within the thalamus or frontal cortex at any post-isehemic time, or in sham operated animais. In situ detection of fragmented DNA is depicted in Xray autoradiographs (representative animal shown in Fig. 2, but ai1 animais from eaeh rime period demonstrated similar labeling) developed after overnight exposure. There was no labeling in sham operated animais (Fig. 2) or in animais exposed to isehemia, reperfused and sacrificed immediately (data not shown). Furthermore, predigestion with DNase I (Sigma Chemieal), or omission of Table 1 Summary of DNA laddering data Group

N

Caudate

Hippocampus

Thalamus

Frontal cortex

Sham 1 day 2 days 3 days 5 days

8 8 6 3 3

0/8 7/8 5/6 2/3 0/3

0/8 1/8 4/6 1/3 0/3

0/8 0/8 0/6 0/3 0/3

0/8 0/8 0/6 0/3 0/3

Intcrnucleosomal laddering was detected as DNA laddering on agarosc gels run in the prescnce of ethidium bromido and Southcrn blotted as describcd in the methods section. Samples wcre scored positive if they manifcsted multin~rs of nuclcosomal size DNA fragments on eithcr the stained ge|s or autoradiographed blots. All samples that were positive by ethidium bromide fluoœescence weoe also positive by autoradiography. N represents thc numb¢r of animais.

B.T. Volpe et al. I Neuroscience Letters 186 (1995) 157-160

sham

24 h

159

72 h

Fig. 2. Autoradiography of in situ labeled fragmented DNA. In this X-ray autoradiograph, dark areas represent [35S]dCTP labeling of 3' DNA ends that have exposed the X-ray film. The upper and lower rows represent a coronal 40Fm section through the striatum and hippocampus, respectively. The eolumns represent sham anîmals, animais sacrificed 24 h and 72 h after ischemia and reperfusion, respectively. Note the intense punctate appearanee of darkened areas in the dorsal lateral striatum 24 h after ischemia (top row, middle column). No labeling signal is detected in the hippocampus 24 h after isehemia Çoottom row, middle column). Note also at 72 h after ischemia there is a decreased signal in the dorsal lateral eaudate nucleus and globus pallidus area (top row right eolumn). In an animal 72 h after isehemia and reperfusion there is laminar intense darkening in the CA1 area of the hippoeampus (bottom row, ~rightcolumn).

striatum 24 h

hippocampus 72 h

Fig. 3. Dark and bright field photomicrograph of 40/~m coronal tissue sections through the striatum (top row, 24 h survival), and hippocampus (bottom row, 72 h survival). In the left column, dark field photomicrographs demonstrate silver grains densely distributed over neurons in the caudate nueleus or over the CA1 region of the hippocampus. In the right column, high powered inverted bright field photomicrographs show grains over degenerating neurons in the striatum (top) and CA1 hippocampus (bottom). The high power inset reveals the distorted shape of the nucleus of a degenerating neuron in the striatum seen in the eenter of the image. Bar equals 200Fm and 50/zm (20/~m for inset) for the striatum, and 300/~m and 50/~m for the hippocampus.

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B.T. Volpe et al. I Neuroscience Letters 186 (1995) 157-160

TdT prevented labeling o f vulnerable neurons. Labeling is confined to punctate areas bilaterally in the caudate nucleus of ischemic animais sacrificed 24 h after reperfusion. The hippocampal areas in these animais were not labeled and appeared histologicaily normal. Areas labeled with the in situ technique correlated with active histopathological change including neurons that were misshapen, with pyknotic and fragmented nuclei. On dark field microscopy, silver grains were highly concentrated aimost exclusively over neurons and groups of neurons in the caudate nucleus (Fig. 3, top row). Other neurons that were not labelled with the in situ technique, and that were in the caudate nucleus ofien appeared shrunken. X-Ray autoradiographs (Fig. 2 right column) of animais sacrificed 72 h after reperfusion demonstrated labeling bilaterally in the CA1 regions of the hippocampus and the lateral caudate nuclei. On histological examination the lateral caudate nucleus again showed neuron shrinkage and there were qualitatively fewer neurons compared to animais sacrificed 24 h after reperfusion. In the hippocampus, some neurons were shrunken, some appeared fragmented, and some appeared swollen. On dark field microscopy (Fig. 3, bottom row), silver grains were concentrated over the CA1 hippocampal pyramidal neuron layer. In rats exposed to transient forebrain ischemia, we used gel electrophoresis to demonstrate that discrete laddering o f D N A was confined to both brain regions in which neurons have been shown to die selectively. An in situ technique labeled the nicked 3' ends of D N A in the nucleus o f neurons that were destined to die, also in both the striatum and hippocampus. The a m e course for the appearance of the D N A laddering and in situ labeling correlated with the histopathological expectations. To the already established ischemic induction of calcium overload, excitotoxicity, lactic acidosis, and oxygen free radical formation as mechanisms for cell death [2,9,11,13, 19], our data suggest that ischemia also triggers the activation o f endonuclease activity. Endonuclease activation by ischemia m a y be similar to that occurring in programmed cell death [3,4,8,16]. The authors acknowledge support from The Burke Institute for Medical Research (BTV and TCW) and the USPHS-HD27116 (HJF). [1] Charriaut-Marlangue, C., Pollard, H., Heron, A., Marguill, I., Plotkine, M., Zivkovic, B. and Ben-Ari, Y., Apoptotic laddered DNA in global and focal eerebral isehemia, Soc. Neurosci. Abstr., 20 (1994) 114.5. [2] Choi, D.W. and Rothman, S.M., The foie of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Annu. Rev. Neurosci., 13 (1990) 171-182. [3] Clarke, P., Developmental cell death: morphologie diversity and multiple mechanisms, Anat. Embryoi., 181 (1990) 195-213.

[4] Ellis, R.E., Yuan, J. and Horvitz, H.R., Mechanism and functions of ccll dcath, Annu. Rev. Cell Bioi., 7 (1991) 663-698. [5] Gavrieli, Y., Sherman, Y., Ben-Sasson, S.A., Identification of PCD in situ via specific labeling of nuclcar DNA fragmentation, J. Cell Biol., 119 (1992) 493-501. [6] Saji, M., Cohen, M., Blau, A., Wessel, T.C. and Volpe, B.T., Transient forebrain ischemie induces delayed injury in thc substantia nigra reticulata: degeneration of GABA neurons, eompensatory expression of GAD mRNA, Brain Res., 643 (1994) 234-244. [7] Herron, A., Pollard, H., Dessi, S., Moreau, J., Lasbennes, F., BenAri, Y. and Charriaut-Marlangue, C., Regional variability in DNA fragmentation after global ischemia evidence by combined histological and gel electrophoresis observations in rat brain, J. Neurochem., 61 (1993) 1973-1976. [8] Kerr, J.F.R., Wyllie, A.H. and Currie, A.R., Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics, Br. J. Cancer, 26 (1972) 239. [9] Hossmann, K.A., Ischemic mediated neuronal injury, Resuscitation, 26 (1993) 225-235. [10] Kihara,S., Shiraishi, T., Nakagawa, S., Todo, K. and Tabuchi, K., Visualization of DNA double strand breaks in gerbil hippocampal CA1 following transient ischemia, Neurosci. Lett., 175 (1994) 133-136. [1 lI Kristan, T., Katsura, K., Gido, G. and Siesjo, B.K., The influence of pli on cellular calcium influx during ischemia, Braln Res., 641 (1994) 295-302. [12] Linnik, M.D., Zobrist, R.H. and Hatfield, M.D., Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats, Stroke, 24 (1993) 2002-2009. [13] Lipton, S.A. and Rosenberg, P.A., Excitatory amino acids as a final common pathway for neurologic disorders, N. Engl. J. Med., 330 (1994) 613-622. [14] MacMannus, P.J., Buchan, A.M., Hill, I.E., Rasquinha, I. and Preston, E., Global isehemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci. Lett., 164 (1993) 8992. [15] Okamoto, M., Matsumoto, M., Ohtsuki, T., Taguchi, A., Mikoshiba, K., Yanagihara, T. and Kamada, T., Internucleosomal DNA cleavage involved in isehemia induoed neuronal death, Biochem. Biophys. Res. Commun., 196 (1993) 1356-1362. [16] Oppenheim, R., Cell death during development of the nervous system, Annu. Rev. Neurosci., 14 (1994) 453-501. [17] Pulsinelli, W.A. and Brierley, LB., A new model of bilateral hemispheric isehemic in unanesthetized rat, Stroke, 10 (1979) 267-272. [18] Pulsinelli, W.A., Brierley, LB. and Plum, F., Temporal profile of neuronal damage in a model of transient forebraln ischemia, Ann. Neurol., 11 (1981) 491--498. [19] Pulsinelli, W.A. In S.G. Waxman (Ed.), A Moleeular and Cellular Approach to the Treatment of Neurologic Disease, Raven, New York, 1993, pp. 107-120. [20] Petito, C.K. and Pulsinelli, W.A., Sequential development of mversal and irreversible neuronal damage following ceoebral isehemia, J. Neuropathol. Exp. Neurol., 43 (1984) 141-53. [21I Petito, C.K. and Pulsinelli, W.A., D¢layed neuronal recovery in neuronal death in rat hippocampus following severe cerebral ischemia, J. Cerebral Blood Flow Metab., 4 (1984) 194-205. [22] Tominaga, T., Kure, S., Narisawa, K. and Yoshimoto, T., Endonuclease activation following focal ischemic injury in the rat brain, Brain Res., 608 (1993) 21-26. [23] Volpe, B.T., Davis, H.P., Towle, A. and Dunlap, W.P., Loss of hippocampal CA1 pyramidal neurons correlates with memory impairment in rats with ischemic or neurotoxin lesions, Behav. Neurosci., 106 (1992) 457-464.