Path. Res. Pract. 187, 857-863 (1991)

Capillaries within Human Skeletal Muscle Fibers R. Wolf and H. H. Goebel Division of Neuropathology, University of Mainz, Mainz, FRG

L. Gutmann Department of Neurology, West Virginia University Medical Center, Morgantown, USA

S. Schochet Division of Neuropathology, West Virginia University, Medical Center, Morgantown, USA

SUMMARY

Internalized capillaries, i.e. capillaries within muscle fibers, represent a rare myopathological feature. This was systematically studied in 923 muscle biopsy specimens and found in 24, chiefly in the gastrocnemius muscle, more rarely in the biceps and quadriceps muscles affecting males more often than females and most frequently associated with juvenile spinal muscular atrophy or Becker's muscular dystrophy. Internalized capillaries, often multiple, ran along the long axis of the muscle fiber within an "internalized" extracellular space and were almost exclusively seen in type I myofibers. Internalization seems to start at the site of fiber splitting while penetration through the intact sarcolemma and invasion into transverse tubules were never observed. The presence of internalized capillaries within type I myofibers and increased density of intramuscular capillaries/fiber though not per muscle fiber area suggested hypoxia to playa possible if not crucial role in the formation of internalized capillaries. Our findings do not distinguish between active proliferation of capillaries into myofibers at the site of myofibers and internalization by fusion of vicinal myofibers as the morphogenetic principles suggest that both of these mechanisms may occur.

Introduction The skeletal muscle fiber in the healthy human adult may have a diameter of 60 to 70 ~m and is surrounded by 2 to 8 capillaries which in the endomysium run mostly parallel to the long axis of the respective muscle fiber. In the early seventies 1,18 capillaries were first described as situated within muscle fibers. Since then, the intracellular presence of muscle capillaries has been dealt with infrequently and largely in a cursory fashion 7, 9,10,15, e.g. discussed within the framework of a case report 8, 12. © 1991 by Gustav Fischer Verlag, Stuttgart

Although this morphological phenomenon has now been mentioned in several myopathological textbooks3,4, 11, 13, 16 we are aware of only three systematic studies6, 8,19. Our report evaluates the morphological phenomenon of capillaries within muscle fibers, called internalized capillaries, studied in more than 900 muscle biopsy specimens derived from three medical centers using enzyme histochemistry, immunohistochemistry, electron microscopy, and morphometry for analysis. 0344-0338/9110187-0857$3.50/0

858 . R. Wolf et al.

Material and Methods The biopsied muscles - quadriceps, deltoid, biceps, gastrocnemius, soleus - were collected from three different neuropathological laboratories between 1973 and 1986. Altogether, 24 muscle specimens revealed internalized capillaries, e.g. within muscle fibers, completely surrounded by muscle fiber sarcoplasm and sarcomeres. Age, gender, nosological diagnosis, and biopsied muscles of these 24 patients are listed in Table 1. For light microscopy, the following morphological and enzyme histochemical techniques were employed: modified trichrome, hematoxylin-eosin, elastica van Gieson, menadione-linked alpha-glycerophosphate dehydrogenase, NADH tetrazolium reductase, adenosine triphosphatase (ATPase) after alkaline and acid pre-incubations, according to routine techniques s,17. These were applied to transverse sections of unfixed frozen and/or paraffin-embedded muscle tissue blocks. As invagination of capillaries may be erroneously interpreted as "internalization" longitudinal sections of muscle tissue were not helpful in evaluating the frequency and mechanisms of the internalization. Step sections, i.e. every third section, were prepared from two muscle specimens (number 18 and 19) at 8 [-lm thickness, resulting in an examination of the muscle specimen at every 16 [-lm to follow the course of an internalized capillary within a muscle fiber enabling pursuit of the capillary course over a distance of 400 [-lm. Unfixed frozen sections were stained with the modified trichrome method while a paraffin block (from patient 24) sectioned into 3 or 6 [-lm thick step sections was stained with the elastica van Gieson stain. Immunohistochemically, Ulex europaeus agglutinin I (UEA I) receptors of endothelial cells were visualized according to the technique of Borisch et aV, using the UEA I lectin in a concentration of 1 : 60, and the UEA I antibody in a concentration of 1: 100 at room temperature for 45 min each.

Quantitative and morphometric evaluation entailed: a) Percentage of muscle fibers with internalized capillaries. b) Diameters of cross sectioned myofibers with internalized capillaries, according to Dubowitz and BrookeS, using the modified trichrome and elastica van Gieson stains as well as the ATPase preparations. c) One [-lm thick resin-embedded sections stained with paraphenylene-diamine or methylene-blue to evaluate capillary density in relation to the number of muscle fibers. Similar data were obtained from age- and site-matched controls. Two ratios were determined: ratio 1, the number of capillaries (nc): the number of fibers (nf), i.e. r 1 = ne: nf; and r 2 = nc: A (A, cross sectioned area of muscle fibers). For measurements, a Kontron Videoplan instrument was used. For electron microscopy muscle specimens embedded according to routine procedures, which in one micrometer semi thin sections had revealed internalized capillaries, were prepared for ultrathin sections, contrasted with uranyl acetate and lead citrate and studied in a Philips 301 Electron Microscope.

Results The myofibers in 24 muscle specimens with internalized capillaries (Table 1) revealed varying myopathological processes of neurogenic, myopathic and inflammatory nature (Table 2). Internalized capillaries were densest in specimens from patients afflicted with spinal muscular atrophy ofthe Kugelberg-Welander type (patient4; 2.36% of all fibers contained internalized capillaries), Becker's muscular dystrophy (patients 18 and 24), and non-specific

Table 1 Nr.

age/sex

Diagnosis

Muscle biopsed

1 2 3 4 5 6 7 8

21, m 39, f 65, f 35, m 64, f 16, m 13, f 30, m 53, f 50, m 13,m 66,f 41, m 72, f 37, m 62, m 29, f 32, m 35, m 54, m 53, m 16, m 46, m 34, m

centronuclear myopathy chronic neurogenic atrophy neurogenic atrophy spinal muscular atrophy Kugelberg-Welander neurogenic atrophy Becker's muscular dystrophy (or limb girdle dystrophy) limb girdle muscular dystrophy neuromyotonia neurogenic atrophy spinal muscular atrophy spinal muscular atrophy Kugelberg-Welander granulomatous myositis endstage myopathy oculopharyngeal muscular dystrophy neurogenic atrophy neurogenic atrophy neurogenic atrophy Becker's muscular dystrophy ruptured Achilles tendon myopathy dystrophic myotonia myopathy myopathy Becker's muscular dystrophy

gastrocnemius gastrocnemius gastrocnemius deltoideus gastrocnemius quadriceps femoris quadriceps femoris biceps brachii biceps brachii gastrocnemius quadriceps femoris biceps brachii biceps brachii quadriceps femoris gastrocnemius gastrocnemius gastrocnemi us gastrocnemius soleus quadriceps femoris gastrocnemius quadriceps femoris gastrocnemius gastrocnemius

9

10 11 12 13 14 15 16 17 18 19

20 21

22

23 24

Number of capillaries per section

1 3

1

>10 4

2

1 3 1 1 3 1 1 2 1 1 2 >10 >10 1 2 1

>10 > 10

Capillaries within Muscle Fibers . 859 Table 2 Nr.

1

2 3 4 5 6 7 8

9

10 11

12 13

14 15 16 17 18 19 20 21 22 23 24

fiber size

internal nuclei

HlA H/A

+ + + + + + + + + + + + + + +

A

H/A H/A H/A HlA HlA HlA

A (1)

H/A H/A HlA H/A

H (2) A partial

H/A H/A HlA HlA HlA H/A H/A H/A

H= hypertrophy, A = atrophy, 1 + = frequently present.

+ + + + + + +

= single

fiber splitting

necrosIs

+ + + + + +

(+)

+ + + + +

+ + + + + + + fiber atrophy, 2 =

(+ ) + + (+) (+)

inflammatory infiltra tes

(+) + + (+ ) (+) (+) (+ ) +

(+)

+ (+ )

(+) atrophic fibers,

NADH-related defects

+ + (+) + + + + + +

(+ )

+ + + + + +

not present, ( +)= occasionally present,

Fig. 1. a: An internalized capillary within a type I fiber among numerous other type I fibers, ATPase preparation after pre-incubation at pH 4.2. X 164. - b: Three-dimensiona l pattern of a single muscle fiber with an internalized capillary at three step section levels showing entrance, co urse and exit of the capi ll ary.

860 . R. Wolf et al.

myopathies (patients 19 and 23) (Table 1). The age of the patients at the time of biopsies ranged between 13 and 72 years showing a male preponderance of 2 : 1. Internalized capillaries - 1.1 % in the quadriceps muscle, 1.3% in the deltoid muscle, 2.1 % in the biceps muscle, and 6.4 % in the gastrocnemius muscle - were most often seen in the

gastrocnemius muscle, actually three times more frequen than in the biceps brachii muscle, and six times mor frequent than in the quadriceps femoris muscle. Internal ized capillaries were almost exclusively seen within type myofibers (Fig. 1 a), more than half of the muscle spec mens showing type I fiber predominance, and they wer

Fig. 2. Series of five level of the ame muscle fiber containing different a peets of internalized capillaries, modified trichrome tain. - a: A inwe capillary, X 290. - b: Two separate capillary region , the left one howing twin capillaries, x 290. - c: Two separate capillary region, each howing twin capillaries, x 290. - d: One capillary region with twin capillaries, x 290. -e: Entrance/exit of a capillary int%ut of the muscle fiber, i.e. incipient internalization, x 290.

Capillaries within Muscle Fibers . 861

present in myofibers measuring 50-160!-tm (average 114 !-tm). Capillary density, i.e. number of capillaries/ myofiber in muscle specimens containing internalized capillaries was increased by 50% compared to controls, i.e. the average of 6 capillaries/myofiber versus an average of 4 capillaries/myofiber, respectively. However, capillary density related to myofiber area revealed only 629 capillaries/mm 2 in specimens with internalized capillaries compared to 1219 capillaries/mm2 in control specimens, partly due to marked hypertrophy in the former group. Muscle fibers measuring an average 160 !-tm and above, as demonstrated in our study, may be surrounded by an increased number of capillaries within the endomysium, but as related to myofiber area a deficit in capillary density compared to normal control was evident. Apart from hypertrophic fibers, muscle specimens with internalized capillaries also contained atrophic fibers, fiber splitting, internalized nuclei, endomysial fibrosis, occasional necrotic fibers and substrate-deficient myofibers in NADH-preparations, findings compatible with the above mentioned neuromuscular conditions. Based on step sections, internalized capillaries usually ran along the longitudinal axis of the myofibers, mostly for more than 300 !-tm (Fig. 1 b). Internalized capillaries showed branching and loops demonstrated by repeatedly-seen double barrel appearance (Fig. 2) - as a matter of fact, about 36%

Fig. 4. An electron micrograph of a muscle fiber contains a nucleus (arrow) and an adjacent aggregate of mitochondria (M) both extending between the central extracellular space close to an internalized capillary (not shown, but seen in Fig. 6a) and the peripheral extracellular space with a non-internalized capillary (C), x 10200.

Fig. 3. A muscle fiber shows splitting on the right, and several capillaries with connective tissue in the center, modified trichrome stain, x 400.

of the internalized capillaries showed two or more lumina. Capillary sprouts may have ended blindly or may have branched within muscle fibers surmised from step sections of a single muscle fiber (Figs. 2a-e). Internalized capillaries measured between 4 and 26 ~lm thereby actually comprising precapillary arterioles or venules. Capillaries apparently entered myofibers at the site of fiber splitting (Fig. 3). Affected myofibers did not show, at least in the immediate vicinity of internalized capillaries, any obvious pathological features. Sarcomeres appeared in register, necrosis was absent and NADH reductase-related substrate defects were only seen in one instance. Aggregates of mitochondria were infrequently seen inside a myofiber between the subsarcolemmal site close to the internalized capillary and the periph era l sarcolemma (Fig. 4). At the light microscopic level internalized capillaries were unremarkable, and only occasionally surrounded by an increased amount of connective tissue (Fig. 3) or even inflammatory cells (Fig. 5). Electron microscopically, only thickening and splitting of the capillary basement membranes were apparent while fusion of muscular lamina basalis and capillary basement membrane was never observed. Penetration by capillaries through an intact sarcolemma, e.g. muscular basal lamina

862 . R. Wolf et al.

Fig. 5. An internalized capillary is surrounded by numerous inflammatory cells, hematoxylin-eosin stain, x 400.

and plasma membrane, or through a dilated transverse tubule could not be seen. Ultrathin step sections gave evidence of blind ending of internalized capillaries within the muscle fiber (Figs. 6a-c). Integrity of the muscle fiber remained preserved, internalized capillaries, therefore, were situated in the extracellular' space within the muscle fibers. Discussion Internalized capillaries, based on frequency and scant description in the literature, represent a very rare myopathological feature. Their presence, therefore, may be related to a specific pattern of pathological findings of the skeletal muscle. The higher frequency of internalized capillaries in the male patients may be due to the frequent finding in Becker's muscular dystrophy while the preponderance of internalized capillaries in the gastrocnemius muscle has been documented in earlier reports 6, 8, 15, 19. The observation of internalized capillaries, especially in the gastrocnemius muscle, a peripherally located and especially strained muscle and, on the other hand, in almost exclusively type I fibers, the oxidative type of fibers rich in mitochondria, suggests that chronic hypoxic stimuli may result in internalization of capillaries within muscle fibers. Especially hypertrophic fibers the center of which may

Fig. 6. Series of electron micrographs from ultrathin step sections through the same muscle fiber, contrast at the surface of the muscle fiber and the capillaries enhanced by using Lanthanum during the embedding procedure. - a: Two separate capillaries surrounded by redundant basement membrane material (arrows). Presence of an erythrocyte (E) in one of the capillaries is evidence of connection with systemic circulation, step one, x 3450. - b: A single capillary with fibroblast processes (arrow), step two, 100: 200 !lm distant to step one, previous electron micrograph, x 3700. - c: Internalized extracellular space without capillaries, step three, 100: 200!lm distant to step two, previous electron micrograph, X 4700.

receive a lower supply of oxygen contained internalized capillaries. 71 % of myofibers with internalized capillaries had diameters above 100 !-lm. An earlier report gave a density of internalized capillaries of up to 20% 10. "Hypoxic" stimuli, however, may not be the sole reason for a muscle capillary to sprout into a muscle fiber. Pathological alterations of or within the muscle fibers,

Capillaries within Muscle Fibers . 863

based on the underlying neuromuscular conditions, seem to facilitate internalization of the capillary. Fiber splitting, to us, has crucial significance in this process of internalization because step sections have repeatedly shown the site of fiber splitting as the spot of entry by a capillary into the muscle fiber, an assumption that has been refuted by some 19 , while association of fiber splitting and internalization has not been excluded by others S• We may at least safely state that internalized capillaries do not penetrate the sarcolemma of muscle fibers or enter the interior of a muscle fiber through transverse tubules. Within the muscle fibers, internalized capillaries are still located within an "internalized" extracellular space, marked by a separate muscular basal lamina and a capillary basement membrane as well as collagen fibrils and an occasional fibroblast. Conversely, internalization of capillaries may conceivably also occur during fusion of vicinal muscle fibers. This assumption may be supported by our finding areas rich in mitochondria which extended through the myofiber from close to the internalized capillary to the peripheral sarcolemma, as evidenced by electron microscopy in our and others'14 muscle specimens. Sulaiman and Kinder19 suggest proliferation of capillaries into primarily damaged muscle fibers as the cause of internalization of the capillaries as internalized capillaries were remarkably frequently associated with target or targetoid lesioned myofibers or similar pathological findings. Internalized capillaries, according to these authors 19 , therefore, within normal appearing muscle fibers indicate a preceding repair of muscle fiber damage. Our findings may support such a suggested pathomechanism although defects of substrate in the NADH preparation within fibers containing internalized capillaries were only seen in one instance. The neuromuscular conditions of our patients almost exclusively represented chronic or slowly progressive entities compatible with the above mentioned processes of repair and restoration. There is evidence of capillary proliferation within muscle specimens containing internalized capillaries inside muscle fibers based on our finding of an increased capillary density within such muscle specimens. This capillary proliferation may aim at preventing hypoxia or facilitating increase and supply of oxygen and substrates by muscle fibers being in the process of regeneration or repair. Acknowledgements We are grateful for assistance to the following individuals: Mrs. M. Schlie for light microscopic preparations, Mrs. 1. Warlo for electron microscopic preparations and for photography, and Mrs. M. Messerschmidt for edition and typing of the manuscript. Parts of this paper were submitted as a thesis to obtain the degree of "Doctor of Medicine" at the Johannes Gutenberg-Universitat,

Mainz, and presented at the Annual Meeting of the "Deutsche Gesellschaft fur Neuropathologie und Neuroanatomie", Munich 1990.

References 1 Afifi AK, Rebeiz J, Mire J, Andonian SJ, Der Kaloustian VM (1972) The myopathology of the Prune Belly syndrome. J Neurol Sci 15: 153-165 2 BorischB, Moller P, Harms D (1983) Lektin Ulexeuropaeus I als Marker in der Differentialdiagnose von GefaBtumoren. Pathologe 4: 241-243 3 Brumback RA, Leech RW (1984) Color Atlas of Muscle Histochemistry. PSG Publishing, Littleton (Mass.) p 73 4 Carpenter S, Karpati G (1984) Pathology of Skeletal Muscle. Churchill Livingstone, New York, pp 125-132 5 Dubowitz V, Brooke MH (1973) Muscle Biopsy: A Modern Approach. Saunders, London 6 Gutmann L, Wolf R, Nix W, Goebel HH, Schochet SS, Hopf HC (1989) Internalized myofiber capillaries and exercise induced myalgias. Muscle and Nerve 12: 191-196 7 Hartlage PL, Soudmand R (1981) Internalized capillaries in hypokalemic periodic paralysis (letter). Arch Neurol 38: 602 8 Hastings BA, Groothuis DR, Vick NA (1980) Dominantly inherited pseudohypertrophic muscular dystrophy with internalized capillaries. Arch Neurol 37: 709-714 9 Hastings BA, Groothuis DR, Vick NA (1981) (letter). Arch Neurol 38: 601 10 Huntington HW (1978) Skeletal muscle with numerous intrafiber capillaries (abstract). J Neuropathol Exp Neurol 37: 632 11 Karpati G (1979) Diseases of muscle. In: Vinken PJ, Bruyn GW (Eds.) Handbook of Clinical Neurology. North Holland Publishing, Amsterdam 40: 15-18 12 Kuhn E, Fiehn W, Schroder JM, Assmus H, Wagner A (1979) Early myocardial disease and cramping myalgia in Becker-type muscular dystrophy: A kindred. Neurology 29: 1144-1149 13 Sarnat HB (1983) Muscle pathology and histochemistry. Am Soc Clin Pathol Press, Chicago, pp 23, 66-67 14 Schmalbruch H (1979) Manifestations of regeneration in myopathic muscles. In: Mauro A et al. (Eds.) Muscle Regeneration. Excerpta Medica Foundation, New York, pp 201-214 IS Schmitt HP (1981) Internalized capillaries (letter). Arch Neurol 38: 601 16 Schochet SS (1986) Diagnostic Pathology of Skeletal Muscle and Nerve. Appleton-Century-Crofts, East Norwalk (Conn.) pp 39-48 17 Schroder JM (1982) Pathologie der Muskulatur. In: Doerr W, Seifert G, Uehlinger E (Eds.) Spezielle pathologische Anatomie, Bd. 15, Springer, Berlin 18 Shafiq SA, Sande MA, Carruthers RR, Killip T, Milhorat AT (1972) Skeletal muscle in idiopathic cardiomyopathy. J Neurol Sci 15: 303-320 19 Sulaiman AR, Kinder DS (1989) Vascularized muscle fibers: etiopathogenesis and clinical significance. J Neurol Sci 92: 37-54

Received August 29, 1990 . Accepted September 19, 1990

Key Words: Capillaries - Myofibers - Internalization - Type I myofibers - Electron microscopy Hans H. Goebel, M.D., Division of Neuropathology, University of Mainz, LangenbeckstraBe 1, D-6500 Mainz, FRG

Capillaries within human skeletal muscle fibers.

Internalized capillaries, i.e. capillaries within muscle fibers, represent a rare myopathological feature. This was systematically studied in 923 musc...
3MB Sizes 0 Downloads 0 Views