Nitro Blue Tetrazolium (NBT) Staining as an Assessment of Thermal Penetration

Medical devices utilizing hyperthermic tissue injury are increasingly being used in surgical procedures. It is clinically essential to accurately assess the extent of thermal tissue damage these devices have on tissues. Histological stains such as hematoxylin and eosin (H&E) and trichrome staining provide a generic tissue staining, of nucleic acids and proteins. As a result of its generality, the application of these stains in the assessment of tissue injury and viability is similarly limited. In contrast, Nitro Blue Tetrazolium (NBT) staining is requires functional cellular proteins and energy production; as such, this stain has the potential to more accurately establish viability and the extent of tissue injury. Here, we describe a validated methodology for NBT staining to assess the extent of thermal tissue damage/injury using the ex vivo porcine model. Following hyperthermic treatment, the tissues are maintained at room temperature for at least two but not longer than four hours and are then frozen at −80°C. The treatment sites are subsequently dissected and embedded in Optimal Cutting Temperature (OCT) Compound on a liquid nitrogen freezing stage. NBT staining is performed on tissue sections cut between 10–12 microns thick. Histologically, the thermally damaged/injured tissue is quantified by assessing the extent of NBT‐negative (yellow‐tan in color) staining within the treatment zone. In contrast, the thermally unaffected tissue stains NBT‐positive (purple‐blue) due to its preservation of diaphorase protein function and maintenance of energy production. Our results validate NBT staining, as an effective assessment of thermal tissue injury, in several different tissues including muscle, liver, stomach and colon.