THE ANATOMICAL RECORD 199341-347 (1981) Histochemical Patterns of Dehydrogenase Activity in the Development of Free Muscle Grafts in the Rat

Patterns of activity of six dehydrogenase enzymes were studied histochemically in 42 free muscle grafts in the rat. Within hours, the surviving peripheral muscle fibers can be distinguished from the central ischemic muscle fibers. The surviving muscle fibers retain their characteristic pattern of staining throughout the post-transplantation period. The central ischemic muscle fibers stain abnormally and by five or six days they lose their enzymatic activity. The zone of regeneration, between the surviving and the ischemic muscle fibers, initially shows little dehydrogenase activity, but as the regenerating muscle fibers mature, they develop first a homogeneous staining pattern and, later, differences in staining intensity among different types of muscle fibers. In a free muscle graft, the muscle is removed from its bed; all tendinous, vascular, and neural connections are severed, and the muscle is then replaced into its own bed or into another site. Although the tendons are resutured, no attempt is made to reanastomose blood vessels or nerves. Following transplantation, the muscle is characterized by a thin peripheral rim of surviving muscle fibers and a larger central area, in which ischemic muscle fibers are destroyed and replaced by newly regenerating muscle fibers. The destruction of old and the regeneration of new muscle fibers follows a centripetal gradient that is spatially and temporally correlated with the ingrowth of new blood vessels into the graft (Carlson et al., '79). One of the major persisting questions in muscle grafting concerns the nature of the metabolic activities within the graft. Although pure biochemical studies allow the quantitation of total enzyme activity (Wagner et al., '771, they do not allow accurate localization of the metabolically active regions. Histochemical studies, although quantitatively less accurate, permit the precise localization of substrates or enzyme activity to specific areas within the graft. It is for this reason that we undertook a histochemical survey of dehydrogenase enzymes in early free muscle grafts. MATERIALS AND METHODS In 42 male Sprague-Dawley rats (150-200 g) one extensor digitorum longus (EDL) muscle was completely removed from the leg and then freely grafted back into its own bed. The contralateral muscle served as a normal control. At 2 and 4 hours and 1-10,15, 16,20,26,30,35,36, 38, 42, and 44 days after transplantation, grafted and control muscles were removed from the rats. The proximal half of each graft was frozen in a mixture of dry ice and isopentane and cross-sectioned at 7 pm on a cryostat. The distal halves of the grafts were fixed in Bouin's solution, embedded in paraffin, and crosssectioned at 7 p m for subsequent staining with Ehrlich's hematoxylin and eosin for morphological comparison with the histochemical preparations. Histochemical activity of the following dehydrogenase enzymes was examined: lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), hydroxybutyrate dehydrogenase (BDH), aglycerophosphate dehydrogenase (a-GPDH), and glucose-6-phosphate dehydrogenase (GGPDH). The procedure followed for demonstrating SDH activity was that of Nachlas et al. ('57), with malonate controls. Pearse's ('72) standard method (pp. 1342-1343) with the nitro-blue tetrazolium salt was used for aGPDH and BDH. Activity of the soluble enzymes LDH, MDH, and G-6-PDH was also demonstrated by Pearse's standard method Received February 2, 1979 accepted July 28, 1980 Davindra K. Magon's present address is Department of Zwlogy, Kenyatta University College, Nairobi, Kenya. Address reprint requests to Bruce M Carlson, Dept. of Anatomy, Univ. of Michigan, Ann Arbor, MI 48109. 0003-276X/81/19934341$02.5