Quantitative morphometric study of the skeletal muscles of normal and streptozotocin-diabetic rats

Abstract

Objectives: Quantitative morphometry under light microscope was applied to analyze changes in the number and the diameters of skeletal muscle fibers and their myonuclei in the extensor digitorum longus and rectus femoris muscles of normal and streptozotocin-diabetic rats.

Animals: Twelve adult male albino Fischer rats each weighing 300 g were used in the study.

Interventions: Streptozotocin (STZ)-diabetes was induced by a single intravenous injection of STZ (75 mg/kg body weight) via the tail vein. Six normal and 6 STZ-diabetic rats were sacrificed; samples of the extensor digitorum longus and rectus femoris muscles were taken, fixed in modified Bouin's fluid and processed for paraffin sectioning. The muscle samples were properly oriented during paraffin embedding for cross and longitudinal sectioning. Sections from each block were cut, processed and stained with hematoxylin and eosin. Randomly selected samples from normal and STZ-diabetic rats were analyzed using a 100x objective lens of a light microscope.

Main outcome measures: The diameters of the fibers and the length of the myonuclei were determined from the longitudinal sections while the diameters and number of fibers and myonuclei were measured from the cross sections. A length measuring 10x reticule fitted to the microscope eye piece was used for the direct measurement of the fibers and myonuclei profiles. Morphometric measurement from each rat was determined and the data were pooled for the control and diabetic groups of rats. All data presented are means with standard error and were statistically analyzed using the Student's t-test.

Results: Histological examination of the stained sections from diabetic rats revealed the presence of areas of inflammation and necrosis in the myofibers of both muscles. The estimated mean diameter of the muscle fibers in the STZ-diabetic rats was reduced by 36% and 31% respectively in the longitudinal and cross sections of the extensor digitorum longus. Similarly, the diameter of the fibers of the rectus femoris in the longitudinal and the cross sections were reduced by 44% and 31%, respectively. On the other hand, a corresponding increase in the number of fibers per unit area was recorded in both muscles of the STZ-diabetic rats which amounted to 13% and 16%, respectively as compared to those of normal rats. Analysis of the myonuclei in normal and diabetic rats revealed a slight decrease in their length and diameter which amounted to 4% and 6%, respectively for the extensor digitorum longus and to 4% and 18%, respectively for the rectus femoris. The estimated numerical density of myonuclei per unit area was 10% lower in both muscles of the diabetic rats.

Conclusions: Skeletal muscular atrophy is a well-documented complication in longstanding diabetes and has been attributed to the direct effect of low serum insulin on the motor end plates and on the synthesis of contractile proteins. The present morphometric study illustrates a reduction in the diameter of the myofibers of the extensor digitorum longus and rectus femoris muscles of STZ-diabetic rats. A slight but significant decrease in the length and diameters of the myonuclei between the diabetic and the normal rats was recorded. The results also indicate more evident morphologic changes in the myofibers of the hindlimb muscle.