Improved staining method for determining the extent of thermal damage to cells.

*Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts 02114Background and Objective: Enzyme histochemicalstains of frozen sections have been used by investigatorsto assess thermal damage. The assessment of thermaldamage to cells in lipid-rich tissues such as subcutaneoustissue and sebaceous glands can be difﬁcult due to thequalityoffrozensectionsofsuchtissues.Thepurposeofthisstudy is to develop an improved method for this type ofevaluation.Study Design/Materials and Methods: Thick frozensections of thermally damaged pig and human skin werestained for lactate dehydrogenase. The sections were ﬁxedin formalin and processed for parafﬁn-embedded sections.Results: The sections showed well-deﬁned localization oftheenzymaticdepositsaswellaspreservationofthe tissuearchitecture.Conclusion: The parafﬁn-embedded lactate dehydrogen-ase stained sections provide improved evaluation ofthermally damaged tissues, particularly the lipid richtissues. Lasers Surg. Med. 39:128–131, 2007.pig and human skin, which were thermally damaged at2006 Wiley-Liss, Inc.Key words: lactate dehydrogenase; pathology; thermaldamageINTRODUCTIONTheassessmentoftheextentofcellulardamageintissueis a frequent endpoint in the initial evaluation of lasertherapies. Unfortunately and despite numerous investiga-tions over several hundred years, there is no deﬁnitivemethod to identify cells that are going to survive from cellsthat are mortally damaged. Table 1 summarizes methodsthatarefrequentlyusedtoevaluatethermalinjuryinducedbylasers.Ourexperiencehasbeenthatthebestmethodisasurvival study where the animals are treated and thenallowed to recover for 48 hours. Most of the cells that arekilled by the thermal damage manifest the morphologicalalterations of necrosis by 48 hours. There can still bedelayedcelldeath;however,inthermalinjury,thisisaverysmall number of cells. In order to limit the number ofanimals used in experiments and to increase the speed atwhich experiments can be completed, there is a need formethods that do not require the survival of the animalsafterthetreatment.Forlaser-inducedthermaldamage,weandothers [1–8] have used enzyme histochemical stains oftissue as an immediate method to assay for cell viability intissue. This method shows decreased to absent stainingwhen there has been thermal denaturation of manyglobular proteins. It has been our observation that thismethod correlates well with the extent of thermal damageas assayed by cell survival for most laser irradiationconditions. This method is not accurate when the laserirradiation is very short (nanosecond and femtosecond) orwhen the tissue is heated slowly (seconds or more) and thetemperature is raised only a few degrees.The typical method for doing enzyme histochemistry ontissue utilizes frozen sections. This method of processingworks well for many tissues; however, we have beeninterested in laser-induced thermal damage to sub-cutaneous tissue and sebaceous glands. Frozen sections ofthese tissues are frequently suboptimal and make this anunreliable approach for evaluation. We have developed amethod to permit better evaluation of thermal damage tothese tissues and which may have wider applications.METHODSInthisstudy,we examined3and4mmpunch biopsies ofvarious depths using Coherent Diamond 200 W CO