In Situ DNA Nick-End Labeling Positive Reaction around
Laser Channels

Lutter et al. [1] observed positive TdT-mediated dUTP-biotin nick-end labeling (TUNEL) reaction around all laser channels in porcine hearts. Since they have not discussed this intriguing result, I would like to submit the following comments regarding this subject.

TUNEL reaction is used for histological detection of DNA breaks in apoptosis. It is known for numerous false positive and negative results; one must always inquire into the reason for its positivity and, if need be, to use other methods to identify apoptosis.

It is obvious that the DNA fragmentation observed by Lutter and al. [1] was not caused by a direct action of the laser beam. The fragmentation appeared in non-irradiated areas and the authors used CO₂ laser that emits low energy photons unable to break covalent bonds in the DNA [2]. When a laser of this type is used, myocardial channels are formed by thermoablation at temperatures above 100 °C. As a result, laser channels are surrounded by a rim of carbonized tissue with a narrow zone of necrosis and a larger zone of ill-defined thermal damage. The altered tissue manifests protein denaturation and birefringence loss that is associated with temperatures from 55 °C to 60 °C [3]. At these temperatures, and those approaching 100 °C, DNA complementary strands separate, but their covalent bonds do not break, indicating that heat was not responsible for the DNA breaks visualized by the TUNEL reaction [1].

Having excluded the above possibilities, one must consider a hypothesis that the positive TUNEL reaction [1] detected DNA fragmentation in cardiomyocyte apoptosis or accidental cell death [4]. Apoptosis has already been described in association with laser channels [5] [6]. Its identification was based on the visual detection of apoptotic bodies and on the acute formation of myocardial defects incompatible with accidental death necrosis [7]. Regrettably, the black and white photographs ([1], see Fig. [5]) do not permit a distinction between apoptotic bodies and red cells [7]. In contrast to interstitial spaces enlarged by edema, myocardial defects incriminating apoptosis are surrounded by and/or contain damaged cardiomyocytes. I have found such defects in Fig. [5 a, b] , and d. Moreover, there is a sign indicating apoptosis leaving no room for doubt in Fig. [5 c] - bellow the channel, TUNEL positivity forms confluent amorphous masses, suggesting that the cardiomyocytes in question have fragmented into apoptotic bodies. This would not occur in necrosis caused by accidental cell death.

Lutter et al. [1] observed TUNEL positivity around all laser channels excepting their border zones. This positivity was not influenced by subsequent no-flow ischemia leading to oncosis and inflicted on the lasered tissue 30 minutes after channel formation. This fact also attests that the TUNEL positivity [1] was associated with apoptosis and not accidental cell death. One may be surprised that DNA breaks mediated by enzymes take place in cells dying from heat. Two reasons may explain this situation: (a) some proteins manifest high denaturation temperatures. For example, caspase-activated DNase inhibitor and chaperone [8] is stable at 90 °C for five minutes and resists strong denaturation agents [9], and (b) elevated temperatures around laser channels quickly dissipate allowing protein renaturation. Since the conditions of myocardial laser channel drilling are easily manipulated and controlled, experimental “laser revascularization” may become an excellent model for studying cardiomyocyte death.

Fig. 5Text und Bild fehlt

References

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