title: Protoporphyrin fluorescence induced by methyl-ALA in skin healing.
abstract: BACKGROUND
  Estimation of the time period that precedes an injury is critical in forensic medicine. However, there is no reliable method that can be used to evaluate the oldness of a lesion. The aim of this work is to develop a fluorimetric method that can be used to follow the aging process of lesions by applying methyl-ALA (MAL) on wounds and by quantifying protoporphyrin IX (PPIX) fluorescence during the healing process. We also aim to understand the changes in PPIX fluorescence by establishing a correlation with histological evaluations during the healing process.
  
  
  METHODS
  Standardized linear wounds were made on the dorsum of 72 mice, which were divided in control (MAL -) and experimental (MAL +) groups. In vivo fluorescence spectra (FS) were collected from normal and wound skin sites of control and experimental groups, corresponding to four groups of FS spectra: (a) FS of skin wound after MAL (+/+); (b) FS of normal skin after MAL (-/+); (c) FS of skin wound without MAL (+/-) and (d) FS of normal skin without MAL (-/-). Animals were monitored periodically for 3 months and euthanized. Tissue specimens were processed for histological analysis using design-based stereological methods. Serial cross-sections were analyzed to evaluate the organization of the dermis and epidermis, collagen deposition and cellular proliferation.
  
  
  RESULTS
  FS of skin wound with MAL (+/+) showed an expressive intensity increase from the beginning of the experiment to the 34th day, with maximum fluorescence being observed on the ≈ 11 th day after wounding. There was preferential PPIX accumulation in healing sites as compared to adjacent normal skin (+/-) in the early stage of healing. Histological findings allowed correlation of the fluorescence increase mainly with cell proliferation. The drastic decrease in the FS intensity observed in the end of the healing process was correlated with the decrease in the proliferation rate as well as with the presence of new extracellular fibrous materials.
  
  
  CONCLUSIONS
  In the mice wound-healing model tested here, it was possible to distinguish whether the injury was in early or advanced stages by using PPIX fluorescence induced by MAL. We conclude that this method is a promising approach to evaluate the age of skin wounding and we hope this work will stimulate human studies to allow this technique to become standardized in forensic medicine.
authors.affiliation: Departamento de Medicina Legal, Ética Médica, Medicina Social e do Trabalho da Faculdade de Medicina, Universidade de São Paulo, Brazil.
authors.forename: Roberta M F
authors.initials: RM
authors.lastname: Gondim
authors.forename: Vinicius C C
authors.initials: VC
authors.lastname: Vieira
authors.forename: Mariana M
authors.initials: MM
authors.lastname: Veras
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Ferreira
authors.forename: Élia Tamaso Espin Garcia
authors.initials: ÉT
authors.lastname: Caldini
authors.forename: Daniel Romero
authors.initials: DR
authors.lastname: Muñoz
authors.forename: Maurício S
authors.initials: MS
authors.lastname: Baptista
mesh_terms: D000375:Aging / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D007982:Levulinic Acids / Q000032:analysis; D051379:Mice; D008819:Mice, Nude; D017319:Photosensitizing Agents; D011524:Protoporphyrins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry* / Q000293:injuries / Q000473:pathology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014945:Wound Healing / Q000502:physiology*; D014950:Wounds, Penetrating / Q000378:metabolism* / Q000473:pathology
chemical_list: D015415:Biomarkers; D007982:Levulinic Acids; D017319:Photosensitizing Agents; D011524:Protoporphyrins; C119867:methyl levulinate; C028025:protoporphyrin IX
keywords: Dermatology; Experimental surgery; Legal medicine; Lesion age; Spectroscopy; Traumatic lesion