Modifications of the lactate dehydrogenase assay, a histochemical determinant of osteocyte viability – A qualitative study

Abstract

We have refined a technique for assessment of osteocyte viability in a canine and murine model using a modification of the lactate dehydrogenase assay (LDH). With this method, viable osteocytes react to form non-reversible tetrazolium-formazan granules, while non-viable osteocytes are distinguished by a methyl green stain. LDH assay in canine and murine models have not been reported and our initial efforts were not successful. We examined the effect of (a) concentration of coenzyme and tetrazole (b) bone specimen thickness (c) ability to use frozen sections and (d) incubation time/dilution. We concluded that a 1000-fold increase in the concentration of coenzyme and tetrazole were required. Fresh bone produced optimal results and near-complete viability. Special considerations must be taken with smaller, more fragile specimens (e.g., mouse bone), such as increasing specimen thickness, dilution of incubation medium and/or the reduction of incubation time. Sections from thawed frozen bone resulted in a diffuse reaction. Osteocyte viability can be assessed via LDH assay in both dog and mouse bones; however, this approach requires modifications from the previous published method.