title: Development of reproducible histologic injury severity scores: skeletal muscle reperfusion injury.
abstract: BACKGROUND
  Ischemia-reperfusion injury of skeletal muscle is a common clinical and experimental problem. To date, there has been no uniform and reproducible method to assess the extent of histologic injury. In this study, we developed a novel statistical methodology for evaluating injury in individual myocytes and 3 distinct methods for the interpretation of this data.
  
  
  METHODS
  C57/BL6 mice underwent 2 h of hindlimb ischemia followed by reperfusion for 3 (n = 11), 24 (n = 12), or 48 (n = 10) h. The gastrocnemius muscles were harvested, stained, and evaluated under microscopy. Standardized criteria were applied to score individual myocytes as healthy or injured, and injury score was expressed as injured fibers/total fibers %. Three methods of analyzing myocyte data were developed and evaluated with statistical Block-Random Sampling to determine the number of counted fibers required to represent accurately the total injury. The Full-Frame Counting, Fourfold Divided Counting, and Stratified Individual Counting methods differ in the random order in which fibers or microscopic fields are scored.
  
  
  RESULTS
  The 3 methods were found to be statistically sound at all experimental time points. Using the Full-Frame, Fourfold, and Stratified methods, the maximum number of required fibers at all time points was 600, 300, and 100, respectively, to obtain an estimation of injury with a 95% confidence interval.
  
  
  CONCLUSIONS
  These criteria and statistical methods for histologic evaluation of ischemia-reperfusion injury in skeletal muscle are accurate and reproducible. The Fourfold method is the most practical and technically efficient method of assessing injury. Such a quantitative, direct assessment of injury is important and will be useful for future studies.
authors.affiliation: Division of Plastic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02144, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: McCormack
authors.forename: Edwin
authors.initials: E
authors.lastname: Kwon
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Eberlin
authors.forename: Mark
authors.initials: M
authors.lastname: Randolph
authors.forename: Dan S
authors.initials: DS
authors.lastname: Friend
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Thomas
authors.forename: Michael T
authors.initials: MT
authors.lastname: Watkins
authors.forename: William G
authors.initials: WG
authors.lastname: Austen
mesh_terms: D000818:Animals; D006614:Hindlimb / Q000098:blood supply; D008297:Male; D008722:Methods; D051379:Mice; D008810:Mice, Inbred C57BL; D032342:Muscle Cells / Q000473:pathology; D018482:Muscle, Skeletal / Q000098:blood supply*; D015427:Reperfusion Injury / Q000473:pathology*; D015203:Reproducibility of Results; D012720:Severity of Illness Index; D013997:Time Factors