Angela L F Gibson1, Shalini Shatadal2. 1. Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin. Electronic address: Gibson@surgery.wisc.edu. 2. Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin.
Abstract
BACKGROUND: Cell viability is paramount to wound healing in burn injury. Current methods to determine depth of burn injury in the research setting are based on the subjective visualization of cell viability using hematoxylin and eosin staining. The purpose of this study was to develop a simplified method of lactate dehydrogenase (LDH) staining to identify viable cells in frozen sections of human burn tissue that can be used in the research setting. MATERIALS AND METHODS: After surgical excision, human burn tissue was processed for histologic evaluation. Tissues were fixed and protected with sucrose incubation before cryopreservation. An LDH staining method was developed and evaluated for prolonged stain stability. To evaluate cellular viability in the tissues as demonstrated by enzymatic activity of LDH, digital images of tissue sections were obtained immediately after and 1 mo after staining. RESULTS: The cryopreserved sections of deep partial thickness human burn tissue revealed cellular viability throughout the tissue with the exception of the most superficial region of the tissue. Unlike the hematoxylin and eosin-stained sections, clear demarcation of cellular viability was evident in the LDH-stained sections. CONCLUSIONS: Our simplified protocol identifies, without ambiguity, the viability of the cellular elements in deep partial thickness and full thickness burn injured tissue.
BACKGROUND: Cell viability is paramount to wound healing in burn injury. Current methods to determine depth of burn injury in the research setting are based on the subjective visualization of cell viability using hematoxylin and eosin staining. The purpose of this study was to develop a simplified method of lactate dehydrogenase (LDH) staining to identify viable cells in frozen sections of human burn tissue that can be used in the research setting. MATERIALS AND METHODS: After surgical excision, human burn tissue was processed for histologic evaluation. Tissues were fixed and protected with sucrose incubation before cryopreservation. An LDH staining method was developed and evaluated for prolonged stain stability. To evaluate cellular viability in the tissues as demonstrated by enzymatic activity of LDH, digital images of tissue sections were obtained immediately after and 1 mo after staining. RESULTS: The cryopreserved sections of deep partial thickness human burn tissue revealed cellular viability throughout the tissue with the exception of the most superficial region of the tissue. Unlike the hematoxylin and eosin-stained sections, clear demarcation of cellular viability was evident in the LDH-stained sections. CONCLUSIONS: Our simplified protocol identifies, without ambiguity, the viability of the cellular elements in deep partial thickness and full thickness burn injured tissue.