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.
journal_name
Surgeryjournal_title
Surgeryauthors
McCormack MC,Kwon E,Eberlin KR,Randolph M,Friend DS,Thomas AC,Watkins MT,Austen WG Jrdoi
10.1016/j.surg.2007.06.005subject
Has Abstractpub_date
2008-01-01 00:00:00pages
126-33issue
1eissn
0039-6060issn
1532-7361pii
S0039-6060(07)00354-6journal_volume
143pub_type
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