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Letters to the Editor |
a Department of Surgery, University of California at San Diego, San Diego, CA, USA
b The Children's Hospital of Philadelphia, Department of Anesthesiology and Critical Care Medicine, Philadelphia, PA, USA
c Department of Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia, PA, USA
Received 4 June 2009; accepted 5 June 2009.
* Corresponding author. Address: Division of Cardiovascular Surgery, University of California, San Diego, Rady Children's Hospital, 3030 Children's Way, Suite 202, San Diego, CA 92123, USA. Tel.: +1 858 966 8030; fax: +1 858 966 8032. (Email: ppastuszko{at}rchsd.org).
Key Words: Alpha-stat • Apoptosis • Cardiopulmonary bypass • Circulatory arrest • Oxygen • pH-stat
The authors thank Dr Sakurai for his comments [1] concerning their study.
It has long been recognised that survivors of heart surgery involving deep hypothermic cardiac arrest (DHCA) face a variety of central nervous system deficits and the identification of neuroprotective strategies that would guard the brain from the negative sequelae of DHCA is therefore of great importance. To test for the protection of the brain we have made a rather deliberate decision to measure the critical regulators of programmed cell death by using the Western blot analysis. The accumulating evidence indicates that the increased expression of Bcl-2 provides protection against apoptosis and ischaemic neuronal death, whereas an increase in the pro-apoptotic protein Bax has been shown to promote cell death by caspase activation. Our observation that mild hypothermia increases the Bcl-2/Bax ratio following DHCA is consistent with cerebral protection via decreased apoptotic cell death.
Dr Sakurai is thinking ahead—given that there is protection, what cell types would be affected? In the experiments reported, we did not attempt to determine whether neurons were the primary site of brain injury but focussed on whether hypothermia can decrease the injury [2]. The extensive literature on this subject shows that cardiopulmonary bypass and deep hypothermic circulatory arrest induce neuronal injury. In our publication, we discussed neuronal injury; however, our data did not distinguish among the different cell types.
In an identical experimental protocol, we used the terminal dUTP nick-end labelling to detect apoptotic cells (TUNEL) stain on the perfused piglet brain following 6 h of post-bypass recovery (unpublished data). We observed that there were increased numbers of TUNEL-positive cells following DHCA but, because of the short recovery period, the numbers were too low to allow testing for an effect of hypothermia. While TUNEL cannot distinguish apoptosis from necrosis, it is a valuable marker of neuronal cell damage. Experiments using 12-h recovery periods, where the number of TUNEL-staining cells is substantially higher, are in progress. TUNEL measurements are being combined in our long-term experiments (12-h recovery) with haematoxylin and eosin (H&E) staining to analyse the morphological characteristics of cell death and with immunochemical staining for caspase-3. The preliminary results indicate that there is an increase of caspase-3 immunostaining cells in different regions of the brain, and this effect is diminished by mild hypothermia, particularly in the striatum.
We are aware that it is possible to observe the distribution of Bax, Bcl-2 or other proteins involved in cell injury or protection in tissue slices as well as through other measurements suggested by Dr Sakurai. We have used in situ hybridisation and autoradiograms to show that hypoxia induces expression of the 72-kDa heat-shock protein (hsp72) mRNA in different regions of the newborn piglets’ brain and that this was significantly diminished, particularly in striatum, by the depletion of dopamine prior to hypoxia [3]. In another model of hypoxia – repetitive apnoea – we have shown that striatum of apnoeic piglets has a larger number of fluoro-Jade-positive neurons than that of sham-operated animals [4]. Such studies can identify the type of cells affected and the regional specificity of the injury, but at the expense of quantification.
Acknowledgments
This research was supported in part by grants HL-58669 and NS031465 from the National Institutes of Health, USA.
References
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