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Visualization of Irreparable Ischemic Damage in Brain by Selective Labeling of Double-Strand Blunt-Ended DNA Breaks

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Abstract

Background

Double-strand DNA breaks with blunt ends represent the most serious type of DNA damage, and cannot be efficiently repaired by cells. They are generated in apoptosis or necrosis and are absent in normal or transiently damaged cells. Consequently, they can be used as a molecular marker of irreparable cellular damage. We evaluated the effects of focal brain ischemia using selective labeling of blunt-ended DNA breaks as a marker of irreversible tissue damage. A new approach permitting such analysis in situ is introduced.

Materials and Methods

Rat brain sections taken 6, 24, 48 and 72 hr after the onset of focal brain ischemia were used. Double-strand DNA breaks were detected directly in the tissue sections via ligation of blunt-ended hairpin-shaped oligonucleotide probes. The probes were attached to the ends of the breaks by T4 DNA ligase. Conventional cresyl violet co-staining and terminal transferase based labeling (TUNEL) were employed to analyze the distribution of labeled cells.

Results

Double-strand blunt-ended DNA breaks rapidly accumulate in brain cells after focal brain ischemia. At 24 hr, they concentrate in the peripheral areas of stroke, which are prone to ischemia-reoxygenation. By 48–72 hr, this type of DNA damage spreads inward, covering the internal areas of the ischemic zone.

Conclusions

Selective labeling of blunt-ended DNA breaks delineates the dynamics of stroke-induced irreversible DNA damage and provides highly specific detection of brain cells with irreparable DNA injury. It can be used for comparing the efficiency of various anti-ischemic drugs, particularly those that target DNA damage, as well as for monitoring stroke-induced damage.

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References

  1. Didenko VV, Wang X, Yang L, Hornsby PJ. (1999) DNA damage and p21WAF1/CIP1/SDI1 in experimental injury of the rat adrenal cortex and trauma-associated damage of the human adrenal cortex. J. Pathol. 189: 119–126.

    Article  CAS  PubMed  Google Scholar 

  2. Lips J, Kaina B. (2001) DNA double-strand breaks trigger apoptosis in p53-deficient fibroblasts. Carcinogenesis 22: 579–585.

    Article  CAS  PubMed  Google Scholar 

  3. Lieber MR. (1998) Pathological and physiological doublestrand breaks: roles in cancer, aging, and the immune system. Am. J. Pathol. 153: 1323′1332.

    Article  Google Scholar 

  4. Didenko VV, Hornsby PJ. (1996) Presence of double-strand breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis. J. Cell Biol. 135: 1369–1376.

    Article  CAS  PubMed  Google Scholar 

  5. Al-Lamki RS, Skepper JN, Loke YW, et al. (1998) Apoptosis in the early human placental bed and its discrimination from necrosis using the in-situ DNA ligation technique. Hum. Reprod. 13: 3511–3519.

    Article  CAS  PubMed  Google Scholar 

  6. Al-Lamki RA, Skepper JN, Loke YW, et al. (1998) Apoptosis in the early human placental bed and its discrimination from necrosis using the in situ DNA ligation technique. Hum. Reprod. 13: 3511–3519.

    Article  CAS  PubMed  Google Scholar 

  7. Morita-Fujimura Y, Fujimura M, Yoshimoto T, Chan PH. (2001) Superoxide during reperfusion contributes to caspase-8 expression and apoptosis after transient focal stroke. Stroke 32: 2356–2361.

    Article  CAS  PubMed  Google Scholar 

  8. Moroni F, Meli E, Peruginelli F, et al. (2001) Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia. Cell Death Differ. 8: 921–932.

    Article  CAS  PubMed  Google Scholar 

  9. Fukuda T, Wang H, Nakanishi H, et al. (1999) Novel non-apoptotic morphological changes in neurons of the mouse hippocampus following transient hypoxic-ischemia. Neurosci. Res. 33: 49–55.

    Article  CAS  PubMed  Google Scholar 

  10. Didenko VV, Tunstead JR, Hornsby PJ. (1998) Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells. Am. J. Pathol. 152: 897–902.

    PubMed  PubMed Central  CAS  Google Scholar 

  11. Didenko VV, Boudreaux DJ, Baskin DS. (1999) Substantial background reduction in ligase-based apoptosis detection using newly designed hairpin oligonucleotide probes. Biotechniques 27: 1130–1132.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Walker PR, Leblanc J, Carson C, et al. (1999) Neither caspase-3 nor DNA fragmentation factor is required for high molecular weight DNA degradation in apoptosis. Ann. N.Y. Acad. Sci. 887: 48–59.

    Article  CAS  PubMed  Google Scholar 

  13. Kontos HA. (2001) Oxygen radicals in cerebral ischemia: the 2001 willis lecture. Stroke 32: 2712–2716.

    Article  CAS  PubMed  Google Scholar 

  14. Li X, Darzynkiewicz Z. (1995) Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28: 571–579.

    Article  CAS  PubMed  Google Scholar 

  15. Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, et al. (1995) In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21: 1465–1468.

    PubMed  CAS  Google Scholar 

  16. Sloop GD, Roa JC, Delgado AG, et al. (1999) Histologic sectioning produces TUNEL reactivity. A potential cause of false-positive staining. Arch. Pathol. Lab. Med. 123: 529–532.

    PubMed  CAS  Google Scholar 

  17. Dikomey E, Lorenzen J. (1993) Saturated and unsaturated repair of DNA strand breaks in CHO cells after X-irradiation with doses ranging from 3 to 90 Gy. Int. J. Radiat. Biol. 64: 659–667.

    Article  CAS  PubMed  Google Scholar 

  18. Velier JJ, Ellison JA, Kikly KK, et al. (1999) Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J. Neurosci. 1: 5932–5941.

    Article  CAS  PubMed  Google Scholar 

  19. Martin LJ, Al-Abdulla NA, Brambrink AM, et al. (1998) Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis. Brain Res. Bull. 46: 281–309.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This research was supported by grant R01 CA78912 from the National Cancer Institute, National Institutes of Health (D.S.B.), by grant 004949-054 from the Texas Higher Education Coordinating Board (D.S.B. and V.V.D.), and by grants from DeBakey Medical Foundation (V.V.D.), Baylor College of Medicine (V.V.D.), The Taub Foundation, The Henry J.N. Taub Fund for Neurosurgical Research, The George A. Robinson, IV Foundation, The Blanche Greene Estate Fund of The Pauline Sterne Wolff Memorial Foundation, and The Seigo Arai and Koppelman Funds of The Neurological Research Foundation (D.S.B.).

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Authors and Affiliations

  1. Department of Neurosurgery, Baylor College of Medicine and VAMC, Houston, TX, USA

    Vladimir V. Didenko, Hop Ngo, Candace L. Minchew, Denise J. Boudreaux, Marsha A. Widmayer & David S. Baskin

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  1. Vladimir V. Didenko
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  2. Hop Ngo
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  3. Candace L. Minchew
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  4. Denise J. Boudreaux
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  5. Marsha A. Widmayer
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  6. David S. Baskin
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Correspondence to David S. Baskin.

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Didenko, V.V., Ngo, H., Minchew, C.L. et al. Visualization of Irreparable Ischemic Damage in Brain by Selective Labeling of Double-Strand Blunt-Ended DNA Breaks. Mol Med 8, 818–823 (2002). https://doi.org/10.1007/BF03402086

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  • DOI: https://doi.org/10.1007/BF03402086

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