THE AMERICAN JOURNAL OF ANATOMY 191:48-56 (1991)

Proximal Skeletal Muscle Alterations in Streptozotocin-Diabetic Rats: A Histochemical and Morphometric Analysis MARIA MEDINA-SANCHEZ, CARMEN RODRIGUEZ-SANCHEZ, JOSE ANTONIO VEGA-ALVAREZ, ARMAND0 MENENDEZ-PELAEZ, AND ANTONIO PEREZ-CASAS Departamento de Morfologia y Biologia Celular, Facultad de Medicina, Uniuersidad de Ouiedo, 33006 Ouiedo, Spain

The response of rat quadriceps ABSTRACT muscle fibers to chronic streptozotocin (STZ) diabetes was studied. Transverse sections of rectus femoris muscle from diabetic and weightmatched control rats were assayed for myofibrilar adenosine triphosphatase (ATPase)and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR).A quantitative analysis was carried out by an automatic interactive analysis system focused on the fiber type size and distribution. STZ-induceddiabetes caused important effects in this muscle, with changes in the distribution of oxidative enzyme reactions, type I fiber hypertrophy, and type I1 fiber atrophy, which was greater in type IIB than in type IIA. It is concluded that hypoinsulinism produces morphological alterations in proximal skeletal muscle fibers that are similar to those of neurogenic myopathy. Thus the pathological changes in these mammalian muscle fibers could explain the clinical syndrome seen in diabetic patients called “diabetic symmetrical proximal motor neuropathy,” perhaps the least understood of the major neuropathic complications of diabetes. INTRODUCTION

Diabetic amyotrophy as well as distal symmetric neuropathy and focal and multifocal neuropathies are the three mayor syndromes related to diabetic neuropathy (Brown and Asbury, 1984). When Garland (1955) first described diabetic amyotrophy, i t was defined as progressive asymmetrical proximal leg weakness and atrophy, with relatively little sensory loss. The term has subsequently been used for two similar-appearing but probably distinct disorders in diabetes. The first is acute proximal asymmetric motor neuropathy, also called “femoral neuropathy” (Calverley and Mulder, 1960). Clinically, severe pain with a n abrupt or subacute onset, unilateral weakness of pelvifemoral muscles, and reduction or absence of the knee jerk are described (Casey and Harrison, 1972; Flatow and Michelsen, 1985). The pathologic basis for this syndrome could be ischemic in nature; several studies have found intraneural infarcts of the vasa nervorum at lumbar plexus, femoral, and obturator nerves (Raff and Asbury, 1968; Raff et al., 1968). The second disorder is the diabetic proximal motor neuropathy, perhaps the least understood of the diabetic neuropathies. Clinical symptoms differ from the asymmetrical syndrome. They progress slowly and in0 1991 WILEY-LISS, INC

clude a n ill-defined aching, proximal bilateral thigh weakness, and severe atrophy of involved muscles, if the disease has been present at least for 3 weeks. Iliopsoas and quadriceps muscles are the most frequently affected, but the thigh adductors, glutei, and hamstring muscles can also be involved. It shows diffuse electrophysiological changes, whereas in the assymmetrical syndrome the alterations are localized (Subramony and Wilbourn, 1982). The pathogenesis of this syndrome is not known. Although it seems to be neurogenic, the site of nerve involvement is not yet known (Locke e t al., 1963; Chokroverty e t al., 1977). Furthermore, several researchers (Redwood, 1962; Armstrong et al., 1975; Chao e t al., 1976; Ianuzzo and Armstrong, 1976; Asbury, 1977, 1987; Grodsky et al., 1982) prefer the hypothesis of metabolic damage to explain the muscle pathology in experimental a s well a s clinical studies. Animal models of diabetes can develop peripheral neuropathy (Sharnia and Thomas, 1987) arid lherefore are useful to study the physiological and pathological changes produced in muscle by experimental diabetes (Challiss et al., 1989).The purpose of this study was to describe the morphological damage in proximal striated muscle in streptozotocin diabetic rats. Three different myofiber types were examined histochemically and morphometrically to contribute to the understanding of the pathology of skeletal muscle in diabetes. MATERIALS AND METHODS

Twenty Wistar adult male rats ranging from 230 to 280 gm in body weight, (13-18 weeks old) were injected with streptozotocin (STZ) solution, adjusted to pH 4.5 with 10 mM citrate buffer (60 mg/kg body weight), into the tail vein to induce experimental diabetes mellitus. All rats were individually housed in cages under a 1ight:dark cycle of 12:12 hr, with the lights automatically turned on at 0700 hr. The temperature was controlled (20” ? 2”C), and the rats were given free access to water and Sandermus rat chow (following the institution guide for the care and use of laboratory animals). Weight-matched male rats receiving the vehicle were used for controls throughout the experiment. We did not use age-matched control rats because age-associated changes were not observed in relative fiber size distribution in normal rats (Medina-Sanchez, 1989). The mammalian rectus femoris muscle was selected

Received February 12, 1990. Accepted November 16, 1990

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PROXIMAL MUSCLE HISTOCHEMISTRY IN DIABETIC RATS

TABLE 1. Blood glucose mean values in control and diabetic rats and weight change in diabetic rats Group Control rats Diabetic rats

Determination Blood glucose (mgidl) Blood glucose (mg/dl) Weight change (grnl2

10 171 621' - 29

Duration of diabetes (dam) 20 30 150 220 762' 705l -45 - 52

60 220 642l - 74

' P < 0.01 vs. control. 'Weight change of diabetic rats throughout the experiment compared with their initial weight.

RESULTS for this study because: 1)one of the major muscle disorders in diabetic patients is the proximal symmetrical Rats injected with streptozotocin remained diabetic motor neuropathy, with clinical features including throughout the experiment as evidenced by hyperglyweakness of iliopsoas and quadriceps muscles; 2) mor- cemia and significant loss in body weight (Table 1). phological studies on this muscle in experimental diaEnzyme Histochemistry betes mellitus a r e scarce; and 3) detailed analyses of its normal structure and histochemical fiber type profile Figures 1-4 illustrate the fiber types (types I, IIA, are available (Vega e t al., 1989). Animals from both and IIB) of the control rectus femoris muscle. The diabetic and control groups (n = 5/group/time point) ATPase in the zone IV of the rectus femoris from the were killed 10, 20, 30, and 60 days after STZ injection. diabetic rats showed the staining same pattern as that Blood samples were collected for glucose assay and in the control muscle sections. The mosaic distribution body weight was determined. of dark and light fibers was similar to that of normal The rectus femoris muscle was removed under pen- muscle. The fiber sizes seemed to be different in the tobarbital anesthesia (30-50 mgikg), and its middle diabetic muscle, with less of a difference in fiber crossone-third was frozen in isopentane cooled in liquid ni- sectional area between types I, IIA, and IIB fibers trogen. Eight micrometer serial sections were cut in a (Figs. 5, 6). cryostat at -20°C. The sections were stained using the The sections reacted for NADH-TR in the diabetic following histochemical t,echniques: 1 ) alkaline d a b l e muscle showed the same enzyme activity distribution myosin adenosine triphosphatase (ATPase) preincu- as that in the normal muscle: there were fibers heavily bated at pH 9.4,2) acid-stable ATP-ase preincubated at stained (type IIA and type I) and slightly stained (type pH 4.6 and pH 4.2 (Brooke and Kaiser, 1970a,b), and 3) IIB) (Figs. 7,s). Nevertheless, there were differences in nicotinamide adenosine dinucleotide tetrazolium re- the distribution of the reaction product. The diabetic ductase (NADH-TR) (Dubowitz and Brooke, 1985). muscle had a more diffuse particle distribution in the Blood sugar was determined using a routine glucose- animals with longer diabetic state, whereas the control oxidase method, and blood sugar levels above 250 mg/ fibers displayed a greater accumulation of particles in dl were considered pathologic. the subsarcolemmal region. Although we did not perform any statistical analysis, Quantitative Analysis type I fibers seemed to be increased in number, with Sections processed for ATPase preincubated a t pH more fibers per area than in the control muscle (Figs. 9, 4.6 were chosen because the three myofiber types could 10). In some sections reacted for ATPase preincubated be differentiated reproducibly. On the basis of this at pH 4.2 from rats 60 days diabetic, three types of method, muscle fibers were classified as type I stained fibers, instead of the two expected, were ob(strongly stained), type IIA (pale), and type IIB (inter- served. This third type corresponded to type IIC (Fig. mediate) (Brooke and Kaiser, 1970a,b). The muscle sec- 11). tion was divided into four zones around the central Morphometric Analysis connective-tissue fascia, and the zone with the highest number of type I myofibers (5.6%)was selected for morThe sizes of the three types of fibers were compared phometric analysis. Quantitative evaluations of fiber with those of fibers from weight-matched control mustypes were made with the help of a Videoplan (Kon- cles (Table 2). Type I fiber size increased in diabetic tron), by which myofiber area (pm2) was counted and muscle in comparison with controls. This increment measured automatically. was significant a t 30 and 60 days after onset of diabetes (P < 0.001). Additionally, STZ-diabetes had a differenStatistical Analysis tial effect on type I1 (glycolytic) fibers. Type IIA fibers A minimum of 50 fibers of type I, more than 350 of showed a significant decrease in size after 10 and 20 type IIA, and more than 500 of type IIB were counted days of diabetes ( P < 0.001). After 30 and 60 days of per animal. Data were analyzed with a n one-way STZ injection, these fibers were smaller than controls ANOVA following a Student-Newman-Keuls test. (P < 0.01). Type IIB fibers were the most affected by Data are presented a s means -t standard errors (SEM). the diabetic condition, with a decrease in mean area ( P Fiber size distribution was compared by means of a x2 < 0.001) for all the experimental groups. In addition, some differences could be observed analysis. At the same time, basic statistical data (count number, number of size classes, and maximal and min- within the histograms of each diabetic experimental group. The percent of type I fibers with a n area >1,000 imum values) were compiled.

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Figs. 1-4. Photomicrographs of serial transverse sections from 10 days control myofibers in rectus femoris. Note the different fiber types, indicated as I, IIA, and IIB. x 100. Fig. 1. Myosin ATPase histochemical reaction (pH Fig. 2. ATPase preincubated a t pH 4.6.

=

9.4).

Fig. 3. ATPase preincubated a t pH 4.2. Fig. 4. NADH-TR histochemical reaction using the Dubowitz technique (Dubowitz and Brooke, 1985).

PROXIMAL MUSCLE HISTOCHEMISTRY IN DIABETIC RATS

Figs. 5, 6. Photomicrographs of 60 day control (Fig. 5) and 60 day diabetic (Fig. 6) rectus femoris muscle stained for the ATPase histochemical reaction preincubated a t pH 4.6. In the diabetic muscle, all fibers appear to be the same size as a result of type I hypertrophy and type I1 atrophy. x 200. Fig. 7. NADH-TR histochemical reaction from 60 day control muscle. Note the greater accumulation of reaction product in the subsar-

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colemmal region and a progressive reduction in particle density toward the axis of the fiber. x 200. Fig. 8. NADH-TR reaction in 60 day diabetic fibers. The particle distribution is more diffuse and the subsarcolemmal aggregation is less apparent. x 200.

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TABLE 2. Muscle fiber area (pm') f SEM of individual fiber types for each diabetic group and their respective control in zone IV from rectus femoris muscle

Duration of diabetes (days)

Fiber tvDe Control I IIA IIB

10

20

30

60

943 t 21 935 k 40 930 t 37 927 t 29 1,296 ? 22 1,280 t 28 1,296 ? 11 1,282 k 27 2,477 2 42 2,436 5 34 2,454 t 41 2,459 t 38

Diabetic I IIA IIB

970 t 59 999 t 34 1,150 k 54' 1,088 t 38' 1,152 t 22' 1,046 ? 21' 1,247 t 25l 1,191 ? 23l 1,902 t 30' 1,338 2 34' 1,646 t 23' 1,650 t 352

' P < 0.05. 2P < 0.001.

did not change, the percent of fibers with larger areas decreased with the diabetic state (P < 0.001) (Fig. 13). Finally, there was a consistent decline in type IIB fibers in the diabetic groups, especially the larger (>2,000 km2) fibers (P < 0.001) (Fig. 14). DISCUSSION

Figs. 9-1 1. Photomicrographs of ATPase histochemical reaction, preincubated a t pH 4.2. X 200. Fig. 9. Sixty day control muscle. Note the scarcity of type I fibers (arrows). Fig. 10. Rectus femoris myofibers after 60 days of diabetes. Note the increase in number and size of the type I fibers. Fig. 11. Myofibers after 60 days of diabetes. Some fibers (asterisks), which were slightly stained, could be type IIC fibers in response to denervation.

km2 increased with the evolution of the disease (P < 0.002) (Fig. 12). Type IIA fibers showed a n irregular distribution: Whereas the smaller groups (

Proximal skeletal muscle alterations in streptozotocin-diabetic rats: a histochemical and morphometric analysis.

The response of rat quadriceps muscle fibers to chronic streptozotocin (STZ) diabetes was studied. Transverse sections of rectus femoris muscle from d...
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