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.
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References
- Transgenic expression of human complement regulators reduces skeletal muscle ischaemia/reperfusion injury in mice.Clin Sci. 2005; 108: 47-53
- Murine hindlimb reperfusion injury can be initiated by a self-reactive monoclonal IgM.Surgery. 2004; 136: 401-406
- Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement.J Exp Med. 1996; 183: 2343-2348
- Intestinal ischemia-reperfusion injury is mediated by the membrane attack complex.Surgery. 1999; 126: 343-348
- Reperfusion injury to skeletal muscle affects primarily type II muscle fibers.J Surg Res. 2004; 122: 54-60
- A noninvasive murine model of hind limb ischemia-reperfusion injury.J Surg Res. 2004; 116: 55-63
- Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle.J Immunol. 2005; 174: 7285-7291
- Ischaemia-reperfusion is an event triggered by immune complexes and complement.Br J Surg. 2003; 90: 1470-1478
- Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle.Eur J Pharmacol. 2001; 424: 59-67
- Moderation of skeletal muscle reperfusion injury by a sLe(x)-glycosylated complement inhibitory protein.Am J Physiol Cell Physiol. 2001; 281: C224-C230
- Comparison of six methods for the assessment of ischemia-reperfusion injury in skeletal muscle following composite tissue allotransplantation.J Reconstr Microsurg. 2004; 20: 253-259
- Polyadenosine diphosphate-ribose polymerase inhibition modulates skeletal muscle injury following ischemia reperfusion.Arch Surg. 2005; 140 (discussion 351-2): 344-351
- [Controlled reperfusion of the extremities for preventing local and systemic damage after prolonged ischemia. An experimental study with the swine model].Zentralbl Chir. 1996; 121: 774-787
- Reduced ischemia-reperfusion injury in muscle.Acta Orthop Scand. 1997; 68: 369-373
- The role of mast cells in ischaemia-reperfusion injury in murine skeletal muscle.J Pathol. 2000; 191: 443-448
- Induction of heat-shock protein 72 in rat skeletal muscle does not increase tolerance to ischemia-reperfusion injury.Muscle Nerve. 1999; 22: 390-393
- Phosphoenolpyruvate/adenosine triphosphate enhances post-ischemic survival of skeletal muscle.J Reconstr Microsurg. 1995; 11: 415-422
- Targeted disruption of the nitric oxide synthase 2 gene protects against ischaemia/reperfusion injury to skeletal muscle.J Pathol. 2001; 194: 109-115
- The effect of ischemic preconditioning on the recovery of skeletal muscle following tourniquet ischemia.Plast Reconstr Surg. 1997; 100: 1767-1775
- A murine skeletal muscle ischemia-reperfusion injury model: differential pathology in BALB/c and DBA/2N mice.J Appl Physiol. 1998; 85: 1676-1683
Article info
Publication history
Published online: November 30, 2007
Accepted:
June 8,
2007
Identification
Copyright
© 2008 Mosby, Inc. Published by Elsevier Inc. All rights reserved.
