Elsevier

Neuroscience

Volume 93, Issue 2, July 1999, Pages 537-549
Neuroscience

The time-course of DNA fragmentation in the choroid plexus and the CA1 region following transient global ischemia in the rat brain. The effect of intra-ischemic hypothermia

https://doi.org/10.1016/S0306-4522(99)00181-5Get rights and content

Abstract

The time-course of DNA fragmentation in the CA1 region of the hippocampus and the choroid plexus was studied following induction of transient forebrain ischemia under lethal normothermic (37°C), or sublethal hypothermic (33°C) conditions. Oligonucleosomal- and high-molecular-weight DNA fragmentation were analysed by conventional agarose gel electrophoresis and pulsed-field gel electrophoresis, respectively. DNA breaks were visualized by the terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridinetriphosphate nick-end labeling method. At 48 h of recovery following normothermic ischemia, in situ labeling of DNA breaks were widespread in medial CA1 and high-molecular-weight DNA cleavage was seen. In contrast, at the same time-point in lateral CA1, many pyknotic but few cells displaying in situ labeling of DNA breaks were observed. Major oligonucleosomal DNA fragmentation was not seen until 72 h of recovery. Following hypothermic ischemia, DNA fragmentation was absent in CA1. DNA fragmentation was seen in the choroid plexus at 24 h of recovery following normothermic ischemia, which was diminished by 48 h of recovery.

In conclusion, oligonucleosomal and high-molecular-weight DNA fragmentation at 10–50 kilobase pairs, occur in CA1 after morphological signs, and acidophilia signifying neurodegeneration appear. DNA fragmentation and cell death in the choroid plexus precede neuronal death in CA1 and may play a causative role.

Section snippets

Surgical procedures

Ninety-six male Wistar rats from Møllegaard Avlslaboratorium (Copenhagen, Denmark) were used for this study. Each experimental group consisted of at least three animals if not otherwise indicated. All animal experiments were approved by the Malmö–Lund Ethical Committee (M 304-97), and carried out in accordance with the National Institute of Health guide for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridinetriphosphate nick-end labeling in the CA1 region

The occurrence of double-stranded DNA breaks in situ were examined by the TUNEL method (n=3). TUNEL is absent in the CA1 pyramidal neurons at 24 h of recovery following 15 min of both normo- and hypothermic ischemia (Fig. 1A and B, respectively). At 48 h of recovery, following normothermic ischemia, a major portion of the cells in the medial part of the CA1 region are TUNEL-positive (Fig. 1C). At 48 h of recovery following hypothermic ischemia, TUNEL is not seen in the CA1 pyramidal neurons (Fig. 1

Discussion

Our study confirms the results of earlier investigations showing that delayed neuronal death of the CA1 pyramidal neurons is accompanied by oligonucleosomal DNA fragmentation.26., 32., 47., 57., 59., 74. We have extended the analysis of DNA fragmentation in this region by investigating HMW DNA fragmentation. We show that in addition to oligonucleosomal DNA fragmentation, the CA1 neurons undergo DNA fragmentation at the level of 10–50 kbp. However, our investigation neither reveals early 10–50 kbp

Conclusions

We conclude that DND following transient forebrain ischemia under normothermic (37°C) conditions results in both low-molecular-weight (oligonucleosomal) and high-molecular-weight (10–50 kbp) DNA fragmentation in the CA1 region, which occur at a time close to but presumably after cell death. DNA fragmentation patterns in CA1 are not seen following a sublethal insult, induced under hypothermic (33°C) conditions. Furthermore, DNA fragmentation as a result of cell degeneration in the choroid plexus,

Acknowledgements

We wish to thank J. P. MacManus for valuable discussions and suggestions. We also thank Ulrika Sparrhult-Björk for excellent technical assistance, and Oskar Hansson for help with the Fluoro-Jade staining. This work was supported by the Swedish Medical Research Council (grant nr. 8644), the European Union BIOMED II (grant BMH4-CT96-0851), the Bergendahl Foundation, and the Juvenile Diabetes Fund.

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