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Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice
Brain Research 856 Ž2000. 93–100 www.elsevier.comrlocaterbres
Research report
Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice a Christelle Guegan , Brigitte Sola ´ a
b, )
Laboratoire de Neurosciences, UniÕersite´ de Caen, CNRS-UMR 6551, 14074 Caen Cedex, France b UniÕersite´ de Caen, UPRES-EA 2128, CHU Cote ˆ de Nacre, 14032 Caen Cedex, France Accepted 9 November 1999
Abstract In experimental models of cerebral ischemia, cells within the damaged territory die by necrosis and by apoptosis that contributes to the expansion of the insult. Apoptotic machinery mobilizes intracellular processes such as induction of Bcl-2 family members, activation of the proteolytic cascade including the caspases, and cleavage of caspase substrates, such as polyŽADP–ribose. polymerase or PARP. Mitochondria play a pivotal role in controlling apoptosis by releasing cytochrome c and modulating redox state, both under the regulation of manganese superoxide dismutase ŽMn SOD. via superoxide anion detoxification. The implication and the kinetics of such events in apoptosis induced after focal permanent ischemia in mice remains to be studied. In a paradigm of ischemic insult induced by occlusion of the middle cerebral artery ŽMCAO. in mice, we showed by immunohistochemistry a constitutive expression of caspase-3 that is enhanced after MCAO in neurons localized within the infarcted zone. As a function of time intervals after MCAO, the cytochrome c amount increased in the cytosolic fraction of ischemic cortical extracts. The kinetics of the release was in concordance with the expression of caspase-3 and the subsequent cleavage of PARP appearing before the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. When the apoptotic markers progressively appeared, no changes of Mn SOD activity or Mn SOD expression were detected after MCAO. We can therefore speculate that the recruitment of Mn SOD did not participate per se in the release of cytochrome c elicited after permanent focal ischemia. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Caspase; Cerebral ischemia; Cytochrome c; Manganese superoxide dismutase; PolyŽADP–ribose. polymerase
1. Introduction Biochemical and morphological characteristics of apoptotic cell death, such as cytoplasmic shrinkage, chromatin condensation and DNA fragmentation, have been detected in cerebral focal and global ischemia models in rodents Žfor review, see Ref. w2x.. After focal ischemia in mice, the predominant localization of apoptotic cells at the inner boundary of the ischemic lesion suggests that the apoptotic process largely contributes to the expansion of the ischemic damage w7,8,18,21x. The apoptotic pathway is composed of at least three functional distinct phases: an initiation phase during which cells receive death stimuli; an effector phase dependent of Bcl-2 family members and of apoptogenic proteins released from mitochondria; and a
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degradation phase, dependent of caspases w16x. The Bcl-2 family proteins could function as channels regulating the mitochondrial permeability transition, andror the release of apoptogenic proteins, such as the cytochrome c Žalso named Apaf-2. or Apoptosis Inducing Factor ŽAIF. w16,29x. The redistribution of cytochrome c from mitochondria to cytosol participates in in vitro neuronal apoptosis w25x, by contributing to the proteolytic activation of caspase-9 and -3 w19,31x. The cytochrome c is released in the cytosol after transient focal w5x and global w26x ischemia in rats. The activation and the cleavage of caspase-3 mediate delayed neuronal death after transient ischemia w1,24x. One of the best characterized substrate of caspase-3 is the polyŽADP–ribose. polymerase ŽPARP., a nuclear enzyme involved in DNA repair and maintenance of genome integrity w27x. The cleavage of PARP by caspase-3 has been demonstrated in a model of transient global ischemia w1x and PARP knock-out mice exhibit a reduction of ischemic damage after a transient focal ischemia w4x.
0006-8993r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 4 7 - 1
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In a model of transient focal ischemia, the volume of infarction was significantly decreased in transgenic mice overexpressing the manganese superoxide dismutase ŽMn SOD., an endogenous mitochondrial antioxidant w13x and mutant mice with Mn SOD deficiency presented an exacerbation of cerebral lesion w22x. Recently, it was shown that knock-out mice deficient in Mn SOD presented an enhanced release of cytochrome c as well as an enhanced DNA fragmentation after the occlusion of the middle cerebral artery ŽMCAO. in mice w6x, suggesting a direct participation of Mn SOD in the apoptotic pathways elicited after ischemic shock. We have analyzed different steps of ischemia-induced apoptosis, namely the subcellular distribution of cytochrome c, the expression of caspase-3, the cleavage of PARP and the DNA fragmentation in function of time intervals after MCAO in a model of focal permanent ischemia in mice. We report here that the kinetics of cytosolic increase of cytochrome c is in good concordance with the induction of caspase-3 expression in the infarcted area, and the delayed cleavage of PARP. Our result confirm the pivotal role of mitochondria as central executioner of the apoptotic machinery. But, we found no modifications of Mn SOD protein content or enzymatic activity indicating that Mn SOD does not seem recruited in this model.
2. Materials and methods 2.1. Surgical procedure and localization of infarcted brain territory All the studies with mice have been conducted in respect to the French and the EU legislations. Male ŽC57Blr6= DBAr2.F1 mice ŽCharles River. weighing 20–25 g were anesthetized intraperitoneally with chloral hydrate Ž500 mgrkg.. Coagulation of the left MCA was realized as previously described w8x. Under low power magnification, a skin incision was made vertically between the eye and the ear. The parotid gland and surrounding soft tissues were pushed downward and the underlying temporalis was incised. The tissue was retracted until the middle cerebral artery ŽMCA. was visible through the surface of the skull. A craniotomy was performed with a small round burr, the dura was opened, and the MCA was electrocoagulated with a bipolar diathermy. The temporalis and the parotid gland were replaced and the incision was sutured. During the surgical procedure and until recovery from anesthesia, the body temperature of mice was maintained at 37–388C. Different time intervals after MCAO, mice were sacrificed and cortices removed for further biochemical or histochemical studies. For the characterization of the infarcted brain territory, frozen ischemic brains were cut into coronal sections Ž15 mm. and stained with Cresyl violet before microscopic examination.
2.2. Determination of Mn SOD actiÕity Contra-lateral and ipsi-lateral cortices were dissected from non operated mice Ž n s 6. or mice Ž n s 6 for each group. sacrificed 3, 6, and 24 h after MCAO, were homogenized in 50 mM NaH 2 PO4 , pH 7.4 and centrifuged at 15,000 = g for 1 h at 48C. Protein content and enzymatic activity determinations were carried out on the supernatants as previously described in detail elsewhere w10x. Mn SOD activity was measured from the inhibition of the auto-oxidation of pyrogallol for 15 min with the addition of 2 mM KCN to inhibit CuZn SOD activity. One unit of Mn SOD activity was defined as 50% inhibition of pyrogallol auto-oxidation ŽUrmg of protein.. Mn SOD activity was determined in triplicate on an automatic Cobias-Bio. 2.3. Isolation of total proteins, and cytosolic and mitochondrial protein fractions Contra-lateral and ipsi-lateral cortices were obtained from animals Ž n s 3. non operated, or sacrificed 3, 6, or 24 h after MCAO Ž n s 3 for each time.. Homogenates were performed in 1 ml of ice-cold buffer Ž50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 ml protease inhibitor cocktail, Sigma., and centrifuged at 48C for 30 min at 12,000 = g. Total proteins present in the supernatant were then collected. Contra-lateral and ipsi-lateral cortices from mice non operated or sacrificed at 30 min, 1, 3, 6, and 24 h post-occlusion Ž n s 3 for each condition. were homogenized in cold buffer containing 10 mM Tris pH 7.4, 320 mM sucrose, 1 mM EDTA. Homogenates were centrifuged at 500 = g for 5 min at 48C. Supernatants were collected and centrifuged again at 4300 = g for 10 min at 48C. Supernatants were then centrifuged at 13,000 = g for 30 min at 48C and resulting supernatants corresponded to the cytosolic fraction. For the mitochondrial fraction, after the first centrifugation, pellets were homogenized in 400 ml of cold buffer Ž50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.5 ml protease inhibitor cocktail inhibitor, Sigma.. Samples were then centrifuged at 600 g for 5 min at 48C and the resulting supernatant corresponded to the mitochondrial fraction. 2.4. Western blot analysis Protein concentrations were determined by the protein assay kit II ŽBio-Rad.. Protein samples Ž30 mg. were electrophorezed through a 12% SDS-polyacrylamide gel. Gels were blotted onto PVDF membranes ŽMillipore. at 400 mA for 2 h at 48C and membranes were blocked for 1 h at room temperature in a buffer containing 0.05% Tween-20 in Tris-buffered saline ŽT-TBS. plus 5% nonfat milk. Washing steps were also done with T-TBS. Mem-
C. Guegan, B. Sola r Brain Research 856 (2000) 93–100 ´
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Fig. 1. Cresyl violet staining of a coronal section of ischemic brain. Stained coronal section corresponding to A4750 mm from the interaural line Žaccording to Lehmann’s atlas w17x. obtained from a mice sacrificed 24 h after MCAO. The infarcted area was identified by the pallor of the damaged tissue relative to the surrounding healthy tissue.
branes were incubated at room temperature for 2 h with primary antibodies diluted in T-TBSr5% milk. Primary antibodies were the following: a mouse monoclonal antibody directed against cytochrome c Žclone 7H8.2C12, Pharmingen, 1r250 final dilution., a rabbit polyclonal antibody directed against Mn SOD Žw10x; 1r1000 final dilution. and a rabbit polyclonal antibody anti-PARP Žclone 7D3-6, Pharmingen, 1r1000 final dilution.. Incubations with the secondary antibodies Žanti-mouse or anti-rabbit IgG biotinylated antibodies, Sigma, 1r80,000. were realized at room temperature for 1 h. After three washes, membranes were incubated for 30 min with avidin– biotin–peroxidase solution ŽABC kit, Vector Labs.. and then for 5 min with DAB plus nickel ammonium. The immunoreaction was visualized by a black precipitate on
Fig. 2. Determination of Mn SOD activity. Mn SOD activity is expressed in U per mg of protein in function of the time intervals after MCAO, in contra- and ipsi-lateral cortices. Values represent means"S.E.M. Ž ns6.. Statistical analysis have been performed by a one-way ANOVA followed by a t-test.
the membranes. Equal loading of proteins was checked by incubating membranes with naphtol blue black. 2.5. Immunohistochemical analysis Immunohistochemistry was performed on brain sections obtained from non operated mice, and mice sacrificed 1, 3, and 24 h after MCAO. After fixation by intracardiac perfusion of 4% PFA-buffered solution, brains were embedded in paraffin blocks and cut in 6 mm coronal sections. Immunohistochemistry was performed following the avidin–biotin–peroxidase method ŽVector laboratories. as previously described w10x. The rabbit polyclonal antibody directed against caspase-3 ŽPharmingen, 65906E. that recognized the 32 kDa unprocessed pro-caspase-3 and the 17 kDa subunit of the active caspase-3, was used at 1r400 final dilution. The specificity of the reaction was tested by
Fig. 3. Expression of Mn SOD protein. Detection of Mn SOD protein was performed by Western-blotting with contra- and ipsi-lateral total extracts obtained from cortices of non operated mice or mice sacrificed 3, 6, 24 h post-occlusion. The size Žin kDa. of the two forms corresponding to the monomers and to the dimers was determined using standard molecular weight markers and is indicated on the left-hand side of the figure Ž24 and 48 kDa, respectively..
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Fig. 4. Western blot analysis of cytochrome c. Protein extracts from cytosolic ŽA. and mitochondrial ŽB. fractions were obtained from the ipsi- and contra-lateral cortices of the same animal. Three animals have been studied, one representative experiment is presented.
incubating adjacent sections with the biotinylated second antibody. 2.6. Genomic DNA electrophoresis Non operated or operated mice were decapitated 3, 6, and 24 h after MCAO and contra-lateral and ipsi-lateral cortices were removed and frozen at y808C. Samples obtained from two animals for each group were homogenized in lysis buffer containing 100 mM NaCl, 25 mM EDTA, 10 mM Tris pH 7.5, 0.5% SDS, 100 mgrml proteinase K and incubated overnight at 508C. After a first extraction with phenol, aqueous phases were treated with RNaseA Ž50 mgrml. for 2 h at 378C, and then with proteinase K Ž100 mgrml. for 3 h at 508C. After two successive extractions Žphenol and phenolrchloroformrisoamylic alcohol Ž25r24r1.., pellets were homogenized in 10 mM Tris pH 8.0, 1 mM EDTA. Then, 20 mg of DNA were run on 1.2% agarose gel containing ethidium bromide and analyzed after UV transillumination.
3. Results
temporo-parietal cortex ŽFig. 1.. The damaged area, visible as early as 30 min, expanded as a function of time interval to reach a maximum at 24 h w10x. Apoptotic cells were only present in the ischemic lesion and were detected as early as 1 h after MCAO in the boundary of ischemia territory w9x. Apoptotic cells were recognized either on the basis of morphological criteria after hematoxylin- or propidium iodide-staining or after identification of DNA fragmentation by the TUNEL procedure as reported in details previously w8,9,11x. 3.2. Determination of Mn SOD actiÕity and protein expression No differences of Mn SOD activity were detected between the contra- and ipsi-lateral cortices whatever the time intervals after MCAO ŽFig. 2.. By Western-blot analysis, two bands of 24 and 48 kDa corresponding, respectively, to the monomeric and dimeric forms of the Mn SOD were observed ŽFig. 3.. No changes of Mn SOD protein content were evidenced, regardless of the cortex analyzed and the time intervals after MCAO. 3.3. Analysis of release of cytochrome c An unique 15 kDa band specific for cytochrome c was detected by Western blotting ŽFig. 4A,B.. When a strong immunostaining was present in mitochondrial fractions of contra- and ipsi-lateral cortices from mice non-operated, or sacrificed different time intervals after MCAO ŽFig. 4B., only a weak band was observed in cytosolic fractions of contra-lateral cortices and in ipsi-lateral cortices of non operated mice ŽFig. 4A.. But, within the cytosolic fraction, the intensity of the 15 kDa band increased as a function of time interval as early as 30 min in the ipsi-lateral side ŽFig. 4A.. These data indicated that a release of cytochrome c occurred strictly in the damaged ipsi-lateral part of the ischemic brain.
3.1. Description of the ischemic lesion
3.4. Analysis of caspase-3 expression
Following MCAO, the ischemic territory was strictly ipsi-lateral Žleft side. and localized exclusively in the
No immunoreactivity was detected in coronal sections incubated with the secondary antibody. In non operated
Fig. 5. Immunodetection of caspase-3 on coronal brain sections. Anti-caspase-3 antibody was assayed on the parietal cortex of non operated mice ŽA. and on the contra- ŽB. and ipsi-lateral ŽC, within the ischemic zone. cortices of mice sacrificed 1 h after MCAO. Pictures were presented with a =1000 magnification.
C. Guegan, B. Sola r Brain Research 856 (2000) 93–100 ´
mice, a weak caspase-3 immunoreactivity was detected in cortical structures ŽFig. 5A.. In the contra-lateral cortex of mice sacrificed 1 ŽFig. 5B., 3, and 24 h, no changes in caspase-3 expression were detected. On the contrary, in the ipsi-lateral cortex, caspase-3 immunoreactivity was increased in neurons within the infarcted territory from 1 h post-occlusion ŽFig. 5C., and persisted up to 3 and 24 h later. The strong expression of caspase-3 was exclusively restricted to the ischemic zone ŽFig. 6.. Immunoreactive cells were numerous at the boundary of the infarcted area ŽFig. 6A,B., and only a few in the ischemic core ŽFig. 6C.. This preferential localization of caspase-3 positive neurons suggests a link between caspase-3 immunostaining and neuronal apoptosis induced by the MCAO, since this immunoreactive pattern is superposable to the TUNEL staining previously reported w8x. 3.5. Analysis of PARP cleaÕage Using a rabbit polyclonal anti-PARP antibody, we detected a band of 116 kDa corresponding to the intact PARP, and to the 85- and 24-kDa fragments generated by PARP cleavage in ischemic brain ŽFig. 7.. The native PARP was present in all samples and the two PARP fragments at negligible levels in contra- and ipsi-lateral cortices of non operated mice, and in contra-lateral cortices of mice sacrificed 3, 6, 24 h post-MCAO. In contrast, an
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Fig. 7. Western blot analysis of PARP cleavage. The expression of the full-length PARP Ž116 kDa. and the two PARP fragments Ž85 and 24 kDa. was analyzed by Western blotting in contra- and ipsi-lateral cortices of non operated mice or ischemic mice as a function of time intervals after MCAO.
increase of the amount of PARP fragments was observed in ipsi-lateral cortices of operated mice as a function of time after occlusion, with a maximum at 24 h ŽFig. 7.. 3.6. Genomic DNA electrophoresis When high molecular weight DNA was isolated from non operated mice or mice sacrificed 3, 6, 24 h post-occlusion, no DNA laddering was detected in contra-lateral samples, and in ipsi-lateral samples of mice sacrificed 3, and 6 h after MCAO. A laddered oligomeric pattern resulting from activation of endonuclease during apoptosis
Fig. 6. Localization of caspase-3 immunoreactive cells within the ischemic zone. Sections corresponding to the A2750 mm coordinate w17x were obtained from animals sacrificed 3 h post-occlusion. Three cortical regions were chosen to illustrate caspase-3 immunoreactivity: ŽA. border of the ischemic area which is delimited by arrows; ŽB. infarcted zone; ŽC. region surrounding the ischemic core Žc. as indicated. Pictures were =250 magnified.
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Fig. 8. Electrophoresis of high molecular weight DNA. DNA was purified from contra- and ipsi-lateral cortices of mice non operated Žcontrol., or mice sacrificed 3, 6, 24 h after MCAO. Molecular weight markers were indicated as m1 Ž lDNA cut with HindIII, Life Technologies. and m2 Ž wX174 DNA cut with HaeII, Life Technologies..
was present only in ipsi-lateral cortices of mice sacrificed 24 h post-occlusion ŽFig. 8.. The ultimate degradation step of apoptosis was detected 24 h after MCAO in ischemic brain.
4. Discussion In this study, we report in a model of focal permanent ischemia in mice that the cytosolic increase of cytochrome c and the induction of caspase-3 immunoreactivity occurs exclusively in the damaged brain, and precedes the cleavage of PARP, a caspase-3 substrate. The kinetics of these events is in concordance with the later apparition of the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. On the contrary, no differences of either Mn SOD protein content or Mn SOD activity are observed, suggesting that Mn SOD is probably not recruited after permanent focal ischemia. During cerebral ischemia, mitochondria are the major site of production of free radicals, specially superoxide anions, that play a role in the expansion of tissular damage w15,22x. In transient focal ischemia, Chan and colleagues have shown that after 90 min of MCAO and 4 h reperfusion, the release of cytochrome c occurs from intact mitochondria w6x, and precedes the loss of mitochondria potential w5,22x. Moreover, after permanent focal ischemia, they also reported an early cytosolic release of cytochrome c w6x. In concordance with these results, we detected an enhancement of the cytochrome c amount as early as 30 min after MCAO. Probably due to a weak contamination of cytosolic preparation with mitochondria extracts, we detected a faint band corresponding to cytochrome c in the cytosolic fraction of the contra-lateral cortices; the intensity of this band did not show any variation whatever the time intervals after MCAO ŽFig. 4.. Thus, we can use this band as an internal control for monitoring, as a function of time, the cytochrome c release in the cytosol in the
ipsi-lateral cortices of ischemic brains. The degeneration of mitochondria, via osmotic lysis or swelling, can occur during cerebral ischemia w23x. We cannot exclude that this degradation participates to the cytosolic increase of cytochrome c in our model but it is largely admitted that mitochondria are generally well-preserved during the apoptotic process w14x and that degeneration of mitochondria appear late after the ischemic shock w2x. Altogether, these data suggest that the cytosolic release of cytochrome c is not the result of a neosynthesis or the degradation of mitochondria. As demonstrated in vitro, mitochondria are involved in apoptosis by releasing apoptogenic proteins, such as cytochrome c w16,20x, which contributes to the activation of caspase-3, an apoptotic executioner w3,12,25x. In vivo, the involvement of cytochrome c in apoptosis has not been proved, but the activation of caspase-3 cleavage mediates neuronal death after global and focal cerebral ischemia w1,24x. In contrast with the study of Namura et al. w23x but in agreement with the report of Chen et al. w1x, we have observed that cortical neurons constitutively contained very weak caspase-3 immunoreactivity. However, the caspase-3 expression is strongly induced as early as 1 h after MCAO, strictly in the damaged area, in particular in the ischemic penumbra. As we used an anti-caspase-3 antibody which recognizes the inactive and active forms, we can speculate that the strong caspase-3 immunoreactivity in the ischemic zone is the reflect of the activation of caspase-3, as previously reported in ischemic models w1,24x. Then, the cytochrome c release and the concomitant expression of caspase-3 in the infarcted cortex indicate their participation in the neuronal apoptosis after focal permanent ischemia. The PARP, an enzyme of DNA repair, is a substrate of caspase-3 in the hippocampus after transient global ischemia w1x. Accordingly, we detected increasing amounts of 85 and 24 kDa PARP fragments exclusively in the ipsi-lateral cortices of mice sacrificed 3, 6, 24 h after MCAO. The cleavage of PARP appears after the induction of caspase-3 expression, and precedes the last step of apoptotic death, namely the DNA degradation, and the formation of apoptotic bodies. Recently, it was proposed that the 24 kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by block-
Fig. 9. Schematic kinetics of the recruitment of some apoptotic effectors and targets after permanent focal ischemia in mice.
C. Guegan, B. Sola r Brain Research 856 (2000) 93–100 ´
ing the access of DNA repair enzymes to DNA strand breaks w28x. The deficiency or inhibition of PARP induces a reduction of ischemic damage after transient MCAO. The activation of PARP may contribute to ischemic cell death by NAD depletion and energy failure w4,30x. After MCAO, knock-out mice deficient in Mn SOD, an endogenous mitochondrial antioxidant, presented an enhanced cerebral lesion, as well as an increase of cytosolic cytochrome c and of DNA fragmentation w6,22x. It has been suggested that Mn SOD prevents the cytosolic release of cytochrome c by blocking the formation of mitochondrial transmembrane pore elicited after ischemia w22x. In the present study, no changes of Mn SOD protein content or Mn SOD activity were observed in mice subjected to MCAO. We have reported previously, the spatio-temporal localization of Mn SOD protein by immunohistochemistry in association with the characterization of cellular types after MCAO in mice w10x. The Mn SOD protein was expressed constitutively in astrocytes and in cortical neurons including neurons of the ischemic territory. An apparent reduction of Mn SOD immunoreactive neurons was seen, which could be the reflect of cellular loss rather an adaptative response. This result was thus in concordance with an unchanged Mn SOD activity and protein content. We can hypothesize either that the constitutive Mn SOD is sufficient to regulate cytochrome c release, or that Mn SOD is not recruited in this phenomenon. In summary, early after the ischemic shock, the release of cytochrome c to the cytosol, the expression of caspase-3, and finally the cleavage of PARP are sequentially observed, all preceding DNA fragmentation Žas schematized in Fig. 9..
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Acknowledgements C.G. is supported by the Ministere ` de l’Education Nationale, de la Recherche, et de la Technologie. We thank Dr. Irene ` Ceballos-Picot ŽCNRS URA 1335, Hopital ˆ Necker, Paris, France. for her help with Mn SOD enzymatic activity determination.
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w28x M.E. Smulson, D. Pang, M. Jung, A. Dimtchev, S. Chasovskikh, A. Spoonde, C. Simbulan-Rosenthal, D. Rosenthal, A. Yakovlev, A. Dritschilo, Irreversible binding of polyŽADP.ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis, Cancer Res. 58 Ž1998. 3495– 3498. w29x S.A. Susin, N. Zamzami, M. Castedo, T. Hirsh, P. Marchetti, A. Macho, E. Daugas, M. Geuskens, G. Kroemer, Bcl-2 inhibits the mitochondrial release of an apoptogenic protease, J. Exp. Med. 184 Ž1996. 1–11. w30x K. Takahashi, J.H. Greenberg, P. Jackson, K. Maclin, J. Zhang, Neuroprotective effects of inhibiting polyŽADP–ribose. synthetase on focal cerebral ischemia in rats, J. Cereb. Blood Flow Metab. 17 Ž1997. 1137–1142. w31x H. Zou, W.J. Henzel, X. Liu, A. Lutschg, X. Wang, Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependant activation of caspase-3, Cell 90 Ž1997. 405–413.
Research report
Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice a Christelle Guegan , Brigitte Sola ´ a
b, )
Laboratoire de Neurosciences, UniÕersite´ de Caen, CNRS-UMR 6551, 14074 Caen Cedex, France b UniÕersite´ de Caen, UPRES-EA 2128, CHU Cote ˆ de Nacre, 14032 Caen Cedex, France Accepted 9 November 1999
Abstract In experimental models of cerebral ischemia, cells within the damaged territory die by necrosis and by apoptosis that contributes to the expansion of the insult. Apoptotic machinery mobilizes intracellular processes such as induction of Bcl-2 family members, activation of the proteolytic cascade including the caspases, and cleavage of caspase substrates, such as polyŽADP–ribose. polymerase or PARP. Mitochondria play a pivotal role in controlling apoptosis by releasing cytochrome c and modulating redox state, both under the regulation of manganese superoxide dismutase ŽMn SOD. via superoxide anion detoxification. The implication and the kinetics of such events in apoptosis induced after focal permanent ischemia in mice remains to be studied. In a paradigm of ischemic insult induced by occlusion of the middle cerebral artery ŽMCAO. in mice, we showed by immunohistochemistry a constitutive expression of caspase-3 that is enhanced after MCAO in neurons localized within the infarcted zone. As a function of time intervals after MCAO, the cytochrome c amount increased in the cytosolic fraction of ischemic cortical extracts. The kinetics of the release was in concordance with the expression of caspase-3 and the subsequent cleavage of PARP appearing before the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. When the apoptotic markers progressively appeared, no changes of Mn SOD activity or Mn SOD expression were detected after MCAO. We can therefore speculate that the recruitment of Mn SOD did not participate per se in the release of cytochrome c elicited after permanent focal ischemia. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Caspase; Cerebral ischemia; Cytochrome c; Manganese superoxide dismutase; PolyŽADP–ribose. polymerase
1. Introduction Biochemical and morphological characteristics of apoptotic cell death, such as cytoplasmic shrinkage, chromatin condensation and DNA fragmentation, have been detected in cerebral focal and global ischemia models in rodents Žfor review, see Ref. w2x.. After focal ischemia in mice, the predominant localization of apoptotic cells at the inner boundary of the ischemic lesion suggests that the apoptotic process largely contributes to the expansion of the ischemic damage w7,8,18,21x. The apoptotic pathway is composed of at least three functional distinct phases: an initiation phase during which cells receive death stimuli; an effector phase dependent of Bcl-2 family members and of apoptogenic proteins released from mitochondria; and a
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degradation phase, dependent of caspases w16x. The Bcl-2 family proteins could function as channels regulating the mitochondrial permeability transition, andror the release of apoptogenic proteins, such as the cytochrome c Žalso named Apaf-2. or Apoptosis Inducing Factor ŽAIF. w16,29x. The redistribution of cytochrome c from mitochondria to cytosol participates in in vitro neuronal apoptosis w25x, by contributing to the proteolytic activation of caspase-9 and -3 w19,31x. The cytochrome c is released in the cytosol after transient focal w5x and global w26x ischemia in rats. The activation and the cleavage of caspase-3 mediate delayed neuronal death after transient ischemia w1,24x. One of the best characterized substrate of caspase-3 is the polyŽADP–ribose. polymerase ŽPARP., a nuclear enzyme involved in DNA repair and maintenance of genome integrity w27x. The cleavage of PARP by caspase-3 has been demonstrated in a model of transient global ischemia w1x and PARP knock-out mice exhibit a reduction of ischemic damage after a transient focal ischemia w4x.
0006-8993r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 4 7 - 1
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In a model of transient focal ischemia, the volume of infarction was significantly decreased in transgenic mice overexpressing the manganese superoxide dismutase ŽMn SOD., an endogenous mitochondrial antioxidant w13x and mutant mice with Mn SOD deficiency presented an exacerbation of cerebral lesion w22x. Recently, it was shown that knock-out mice deficient in Mn SOD presented an enhanced release of cytochrome c as well as an enhanced DNA fragmentation after the occlusion of the middle cerebral artery ŽMCAO. in mice w6x, suggesting a direct participation of Mn SOD in the apoptotic pathways elicited after ischemic shock. We have analyzed different steps of ischemia-induced apoptosis, namely the subcellular distribution of cytochrome c, the expression of caspase-3, the cleavage of PARP and the DNA fragmentation in function of time intervals after MCAO in a model of focal permanent ischemia in mice. We report here that the kinetics of cytosolic increase of cytochrome c is in good concordance with the induction of caspase-3 expression in the infarcted area, and the delayed cleavage of PARP. Our result confirm the pivotal role of mitochondria as central executioner of the apoptotic machinery. But, we found no modifications of Mn SOD protein content or enzymatic activity indicating that Mn SOD does not seem recruited in this model.
2. Materials and methods 2.1. Surgical procedure and localization of infarcted brain territory All the studies with mice have been conducted in respect to the French and the EU legislations. Male ŽC57Blr6= DBAr2.F1 mice ŽCharles River. weighing 20–25 g were anesthetized intraperitoneally with chloral hydrate Ž500 mgrkg.. Coagulation of the left MCA was realized as previously described w8x. Under low power magnification, a skin incision was made vertically between the eye and the ear. The parotid gland and surrounding soft tissues were pushed downward and the underlying temporalis was incised. The tissue was retracted until the middle cerebral artery ŽMCA. was visible through the surface of the skull. A craniotomy was performed with a small round burr, the dura was opened, and the MCA was electrocoagulated with a bipolar diathermy. The temporalis and the parotid gland were replaced and the incision was sutured. During the surgical procedure and until recovery from anesthesia, the body temperature of mice was maintained at 37–388C. Different time intervals after MCAO, mice were sacrificed and cortices removed for further biochemical or histochemical studies. For the characterization of the infarcted brain territory, frozen ischemic brains were cut into coronal sections Ž15 mm. and stained with Cresyl violet before microscopic examination.
2.2. Determination of Mn SOD actiÕity Contra-lateral and ipsi-lateral cortices were dissected from non operated mice Ž n s 6. or mice Ž n s 6 for each group. sacrificed 3, 6, and 24 h after MCAO, were homogenized in 50 mM NaH 2 PO4 , pH 7.4 and centrifuged at 15,000 = g for 1 h at 48C. Protein content and enzymatic activity determinations were carried out on the supernatants as previously described in detail elsewhere w10x. Mn SOD activity was measured from the inhibition of the auto-oxidation of pyrogallol for 15 min with the addition of 2 mM KCN to inhibit CuZn SOD activity. One unit of Mn SOD activity was defined as 50% inhibition of pyrogallol auto-oxidation ŽUrmg of protein.. Mn SOD activity was determined in triplicate on an automatic Cobias-Bio. 2.3. Isolation of total proteins, and cytosolic and mitochondrial protein fractions Contra-lateral and ipsi-lateral cortices were obtained from animals Ž n s 3. non operated, or sacrificed 3, 6, or 24 h after MCAO Ž n s 3 for each time.. Homogenates were performed in 1 ml of ice-cold buffer Ž50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 ml protease inhibitor cocktail, Sigma., and centrifuged at 48C for 30 min at 12,000 = g. Total proteins present in the supernatant were then collected. Contra-lateral and ipsi-lateral cortices from mice non operated or sacrificed at 30 min, 1, 3, 6, and 24 h post-occlusion Ž n s 3 for each condition. were homogenized in cold buffer containing 10 mM Tris pH 7.4, 320 mM sucrose, 1 mM EDTA. Homogenates were centrifuged at 500 = g for 5 min at 48C. Supernatants were collected and centrifuged again at 4300 = g for 10 min at 48C. Supernatants were then centrifuged at 13,000 = g for 30 min at 48C and resulting supernatants corresponded to the cytosolic fraction. For the mitochondrial fraction, after the first centrifugation, pellets were homogenized in 400 ml of cold buffer Ž50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.5 ml protease inhibitor cocktail inhibitor, Sigma.. Samples were then centrifuged at 600 g for 5 min at 48C and the resulting supernatant corresponded to the mitochondrial fraction. 2.4. Western blot analysis Protein concentrations were determined by the protein assay kit II ŽBio-Rad.. Protein samples Ž30 mg. were electrophorezed through a 12% SDS-polyacrylamide gel. Gels were blotted onto PVDF membranes ŽMillipore. at 400 mA for 2 h at 48C and membranes were blocked for 1 h at room temperature in a buffer containing 0.05% Tween-20 in Tris-buffered saline ŽT-TBS. plus 5% nonfat milk. Washing steps were also done with T-TBS. Mem-
C. Guegan, B. Sola r Brain Research 856 (2000) 93–100 ´
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Fig. 1. Cresyl violet staining of a coronal section of ischemic brain. Stained coronal section corresponding to A4750 mm from the interaural line Žaccording to Lehmann’s atlas w17x. obtained from a mice sacrificed 24 h after MCAO. The infarcted area was identified by the pallor of the damaged tissue relative to the surrounding healthy tissue.
branes were incubated at room temperature for 2 h with primary antibodies diluted in T-TBSr5% milk. Primary antibodies were the following: a mouse monoclonal antibody directed against cytochrome c Žclone 7H8.2C12, Pharmingen, 1r250 final dilution., a rabbit polyclonal antibody directed against Mn SOD Žw10x; 1r1000 final dilution. and a rabbit polyclonal antibody anti-PARP Žclone 7D3-6, Pharmingen, 1r1000 final dilution.. Incubations with the secondary antibodies Žanti-mouse or anti-rabbit IgG biotinylated antibodies, Sigma, 1r80,000. were realized at room temperature for 1 h. After three washes, membranes were incubated for 30 min with avidin– biotin–peroxidase solution ŽABC kit, Vector Labs.. and then for 5 min with DAB plus nickel ammonium. The immunoreaction was visualized by a black precipitate on
Fig. 2. Determination of Mn SOD activity. Mn SOD activity is expressed in U per mg of protein in function of the time intervals after MCAO, in contra- and ipsi-lateral cortices. Values represent means"S.E.M. Ž ns6.. Statistical analysis have been performed by a one-way ANOVA followed by a t-test.
the membranes. Equal loading of proteins was checked by incubating membranes with naphtol blue black. 2.5. Immunohistochemical analysis Immunohistochemistry was performed on brain sections obtained from non operated mice, and mice sacrificed 1, 3, and 24 h after MCAO. After fixation by intracardiac perfusion of 4% PFA-buffered solution, brains were embedded in paraffin blocks and cut in 6 mm coronal sections. Immunohistochemistry was performed following the avidin–biotin–peroxidase method ŽVector laboratories. as previously described w10x. The rabbit polyclonal antibody directed against caspase-3 ŽPharmingen, 65906E. that recognized the 32 kDa unprocessed pro-caspase-3 and the 17 kDa subunit of the active caspase-3, was used at 1r400 final dilution. The specificity of the reaction was tested by
Fig. 3. Expression of Mn SOD protein. Detection of Mn SOD protein was performed by Western-blotting with contra- and ipsi-lateral total extracts obtained from cortices of non operated mice or mice sacrificed 3, 6, 24 h post-occlusion. The size Žin kDa. of the two forms corresponding to the monomers and to the dimers was determined using standard molecular weight markers and is indicated on the left-hand side of the figure Ž24 and 48 kDa, respectively..
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Fig. 4. Western blot analysis of cytochrome c. Protein extracts from cytosolic ŽA. and mitochondrial ŽB. fractions were obtained from the ipsi- and contra-lateral cortices of the same animal. Three animals have been studied, one representative experiment is presented.
incubating adjacent sections with the biotinylated second antibody. 2.6. Genomic DNA electrophoresis Non operated or operated mice were decapitated 3, 6, and 24 h after MCAO and contra-lateral and ipsi-lateral cortices were removed and frozen at y808C. Samples obtained from two animals for each group were homogenized in lysis buffer containing 100 mM NaCl, 25 mM EDTA, 10 mM Tris pH 7.5, 0.5% SDS, 100 mgrml proteinase K and incubated overnight at 508C. After a first extraction with phenol, aqueous phases were treated with RNaseA Ž50 mgrml. for 2 h at 378C, and then with proteinase K Ž100 mgrml. for 3 h at 508C. After two successive extractions Žphenol and phenolrchloroformrisoamylic alcohol Ž25r24r1.., pellets were homogenized in 10 mM Tris pH 8.0, 1 mM EDTA. Then, 20 mg of DNA were run on 1.2% agarose gel containing ethidium bromide and analyzed after UV transillumination.
3. Results
temporo-parietal cortex ŽFig. 1.. The damaged area, visible as early as 30 min, expanded as a function of time interval to reach a maximum at 24 h w10x. Apoptotic cells were only present in the ischemic lesion and were detected as early as 1 h after MCAO in the boundary of ischemia territory w9x. Apoptotic cells were recognized either on the basis of morphological criteria after hematoxylin- or propidium iodide-staining or after identification of DNA fragmentation by the TUNEL procedure as reported in details previously w8,9,11x. 3.2. Determination of Mn SOD actiÕity and protein expression No differences of Mn SOD activity were detected between the contra- and ipsi-lateral cortices whatever the time intervals after MCAO ŽFig. 2.. By Western-blot analysis, two bands of 24 and 48 kDa corresponding, respectively, to the monomeric and dimeric forms of the Mn SOD were observed ŽFig. 3.. No changes of Mn SOD protein content were evidenced, regardless of the cortex analyzed and the time intervals after MCAO. 3.3. Analysis of release of cytochrome c An unique 15 kDa band specific for cytochrome c was detected by Western blotting ŽFig. 4A,B.. When a strong immunostaining was present in mitochondrial fractions of contra- and ipsi-lateral cortices from mice non-operated, or sacrificed different time intervals after MCAO ŽFig. 4B., only a weak band was observed in cytosolic fractions of contra-lateral cortices and in ipsi-lateral cortices of non operated mice ŽFig. 4A.. But, within the cytosolic fraction, the intensity of the 15 kDa band increased as a function of time interval as early as 30 min in the ipsi-lateral side ŽFig. 4A.. These data indicated that a release of cytochrome c occurred strictly in the damaged ipsi-lateral part of the ischemic brain.
3.1. Description of the ischemic lesion
3.4. Analysis of caspase-3 expression
Following MCAO, the ischemic territory was strictly ipsi-lateral Žleft side. and localized exclusively in the
No immunoreactivity was detected in coronal sections incubated with the secondary antibody. In non operated
Fig. 5. Immunodetection of caspase-3 on coronal brain sections. Anti-caspase-3 antibody was assayed on the parietal cortex of non operated mice ŽA. and on the contra- ŽB. and ipsi-lateral ŽC, within the ischemic zone. cortices of mice sacrificed 1 h after MCAO. Pictures were presented with a =1000 magnification.
C. Guegan, B. Sola r Brain Research 856 (2000) 93–100 ´
mice, a weak caspase-3 immunoreactivity was detected in cortical structures ŽFig. 5A.. In the contra-lateral cortex of mice sacrificed 1 ŽFig. 5B., 3, and 24 h, no changes in caspase-3 expression were detected. On the contrary, in the ipsi-lateral cortex, caspase-3 immunoreactivity was increased in neurons within the infarcted territory from 1 h post-occlusion ŽFig. 5C., and persisted up to 3 and 24 h later. The strong expression of caspase-3 was exclusively restricted to the ischemic zone ŽFig. 6.. Immunoreactive cells were numerous at the boundary of the infarcted area ŽFig. 6A,B., and only a few in the ischemic core ŽFig. 6C.. This preferential localization of caspase-3 positive neurons suggests a link between caspase-3 immunostaining and neuronal apoptosis induced by the MCAO, since this immunoreactive pattern is superposable to the TUNEL staining previously reported w8x. 3.5. Analysis of PARP cleaÕage Using a rabbit polyclonal anti-PARP antibody, we detected a band of 116 kDa corresponding to the intact PARP, and to the 85- and 24-kDa fragments generated by PARP cleavage in ischemic brain ŽFig. 7.. The native PARP was present in all samples and the two PARP fragments at negligible levels in contra- and ipsi-lateral cortices of non operated mice, and in contra-lateral cortices of mice sacrificed 3, 6, 24 h post-MCAO. In contrast, an
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Fig. 7. Western blot analysis of PARP cleavage. The expression of the full-length PARP Ž116 kDa. and the two PARP fragments Ž85 and 24 kDa. was analyzed by Western blotting in contra- and ipsi-lateral cortices of non operated mice or ischemic mice as a function of time intervals after MCAO.
increase of the amount of PARP fragments was observed in ipsi-lateral cortices of operated mice as a function of time after occlusion, with a maximum at 24 h ŽFig. 7.. 3.6. Genomic DNA electrophoresis When high molecular weight DNA was isolated from non operated mice or mice sacrificed 3, 6, 24 h post-occlusion, no DNA laddering was detected in contra-lateral samples, and in ipsi-lateral samples of mice sacrificed 3, and 6 h after MCAO. A laddered oligomeric pattern resulting from activation of endonuclease during apoptosis
Fig. 6. Localization of caspase-3 immunoreactive cells within the ischemic zone. Sections corresponding to the A2750 mm coordinate w17x were obtained from animals sacrificed 3 h post-occlusion. Three cortical regions were chosen to illustrate caspase-3 immunoreactivity: ŽA. border of the ischemic area which is delimited by arrows; ŽB. infarcted zone; ŽC. region surrounding the ischemic core Žc. as indicated. Pictures were =250 magnified.
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Fig. 8. Electrophoresis of high molecular weight DNA. DNA was purified from contra- and ipsi-lateral cortices of mice non operated Žcontrol., or mice sacrificed 3, 6, 24 h after MCAO. Molecular weight markers were indicated as m1 Ž lDNA cut with HindIII, Life Technologies. and m2 Ž wX174 DNA cut with HaeII, Life Technologies..
was present only in ipsi-lateral cortices of mice sacrificed 24 h post-occlusion ŽFig. 8.. The ultimate degradation step of apoptosis was detected 24 h after MCAO in ischemic brain.
4. Discussion In this study, we report in a model of focal permanent ischemia in mice that the cytosolic increase of cytochrome c and the induction of caspase-3 immunoreactivity occurs exclusively in the damaged brain, and precedes the cleavage of PARP, a caspase-3 substrate. The kinetics of these events is in concordance with the later apparition of the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. On the contrary, no differences of either Mn SOD protein content or Mn SOD activity are observed, suggesting that Mn SOD is probably not recruited after permanent focal ischemia. During cerebral ischemia, mitochondria are the major site of production of free radicals, specially superoxide anions, that play a role in the expansion of tissular damage w15,22x. In transient focal ischemia, Chan and colleagues have shown that after 90 min of MCAO and 4 h reperfusion, the release of cytochrome c occurs from intact mitochondria w6x, and precedes the loss of mitochondria potential w5,22x. Moreover, after permanent focal ischemia, they also reported an early cytosolic release of cytochrome c w6x. In concordance with these results, we detected an enhancement of the cytochrome c amount as early as 30 min after MCAO. Probably due to a weak contamination of cytosolic preparation with mitochondria extracts, we detected a faint band corresponding to cytochrome c in the cytosolic fraction of the contra-lateral cortices; the intensity of this band did not show any variation whatever the time intervals after MCAO ŽFig. 4.. Thus, we can use this band as an internal control for monitoring, as a function of time, the cytochrome c release in the cytosol in the
ipsi-lateral cortices of ischemic brains. The degeneration of mitochondria, via osmotic lysis or swelling, can occur during cerebral ischemia w23x. We cannot exclude that this degradation participates to the cytosolic increase of cytochrome c in our model but it is largely admitted that mitochondria are generally well-preserved during the apoptotic process w14x and that degeneration of mitochondria appear late after the ischemic shock w2x. Altogether, these data suggest that the cytosolic release of cytochrome c is not the result of a neosynthesis or the degradation of mitochondria. As demonstrated in vitro, mitochondria are involved in apoptosis by releasing apoptogenic proteins, such as cytochrome c w16,20x, which contributes to the activation of caspase-3, an apoptotic executioner w3,12,25x. In vivo, the involvement of cytochrome c in apoptosis has not been proved, but the activation of caspase-3 cleavage mediates neuronal death after global and focal cerebral ischemia w1,24x. In contrast with the study of Namura et al. w23x but in agreement with the report of Chen et al. w1x, we have observed that cortical neurons constitutively contained very weak caspase-3 immunoreactivity. However, the caspase-3 expression is strongly induced as early as 1 h after MCAO, strictly in the damaged area, in particular in the ischemic penumbra. As we used an anti-caspase-3 antibody which recognizes the inactive and active forms, we can speculate that the strong caspase-3 immunoreactivity in the ischemic zone is the reflect of the activation of caspase-3, as previously reported in ischemic models w1,24x. Then, the cytochrome c release and the concomitant expression of caspase-3 in the infarcted cortex indicate their participation in the neuronal apoptosis after focal permanent ischemia. The PARP, an enzyme of DNA repair, is a substrate of caspase-3 in the hippocampus after transient global ischemia w1x. Accordingly, we detected increasing amounts of 85 and 24 kDa PARP fragments exclusively in the ipsi-lateral cortices of mice sacrificed 3, 6, 24 h after MCAO. The cleavage of PARP appears after the induction of caspase-3 expression, and precedes the last step of apoptotic death, namely the DNA degradation, and the formation of apoptotic bodies. Recently, it was proposed that the 24 kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by block-
Fig. 9. Schematic kinetics of the recruitment of some apoptotic effectors and targets after permanent focal ischemia in mice.
C. Guegan, B. Sola r Brain Research 856 (2000) 93–100 ´
ing the access of DNA repair enzymes to DNA strand breaks w28x. The deficiency or inhibition of PARP induces a reduction of ischemic damage after transient MCAO. The activation of PARP may contribute to ischemic cell death by NAD depletion and energy failure w4,30x. After MCAO, knock-out mice deficient in Mn SOD, an endogenous mitochondrial antioxidant, presented an enhanced cerebral lesion, as well as an increase of cytosolic cytochrome c and of DNA fragmentation w6,22x. It has been suggested that Mn SOD prevents the cytosolic release of cytochrome c by blocking the formation of mitochondrial transmembrane pore elicited after ischemia w22x. In the present study, no changes of Mn SOD protein content or Mn SOD activity were observed in mice subjected to MCAO. We have reported previously, the spatio-temporal localization of Mn SOD protein by immunohistochemistry in association with the characterization of cellular types after MCAO in mice w10x. The Mn SOD protein was expressed constitutively in astrocytes and in cortical neurons including neurons of the ischemic territory. An apparent reduction of Mn SOD immunoreactive neurons was seen, which could be the reflect of cellular loss rather an adaptative response. This result was thus in concordance with an unchanged Mn SOD activity and protein content. We can hypothesize either that the constitutive Mn SOD is sufficient to regulate cytochrome c release, or that Mn SOD is not recruited in this phenomenon. In summary, early after the ischemic shock, the release of cytochrome c to the cytosol, the expression of caspase-3, and finally the cleavage of PARP are sequentially observed, all preceding DNA fragmentation Žas schematized in Fig. 9..
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Acknowledgements C.G. is supported by the Ministere ` de l’Education Nationale, de la Recherche, et de la Technologie. We thank Dr. Irene ` Ceballos-Picot ŽCNRS URA 1335, Hopital ˆ Necker, Paris, France. for her help with Mn SOD enzymatic activity determination.
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