This article has Open Peer Review reports available.
Morphological, histochemical, and interstitial pressure changes in the tibialis anterior muscle before and after aortofemoral bypass in patients with peripheral arterial occlusive disease
© Albani et al; licensee BioMed Central Ltd. 2002
Received: 11 October 2001
Accepted: 25 February 2002
Published: 25 February 2002
Morphological and electrophysiological studies of ischemic muscles in peripheral arterial disease disclosed evidence of denervation and fibre atrophy. The purpose of the present study is to describe morphological changes in ischemic muscles before and after reperfusion surgery in patients with peripheral occlusive arterial disease, and to provide an insight into the effect of reperfusion on the histochemistry of the reperfused muscle.
Muscle biopsies were obtained from the tibialis anterior of 9 patients with chronic peripheral arterial occlusive disease of the lower extremities, before and after aortofemoral bypass, in order to evaluate the extent and type of muscle fibre changes during ischemia and after revascularization. Fibre type content and muscle fibre areas were quantified using standard histological and histochemical methods and morphometric analysis. Each patient underwent concentric needle electromyography, nerve conduction velocity studies, and interstitial pressure measurements.
Preoperatively all patients showed muscle fibre atrophy of both types, type II fibre area being more affected. The mean fibre cross sectional area of type I was 3,745 μm2 and of type II 4,654 μm2 . Fibre-type grouping, great variation in fibre size and angular fibres were indicative of chronic dennervation-reinnervation, in the absence of any clinical evidence of a neuropathic process. Seven days after the reperfusion the areas of both fibre types were even more reduced, being 3,086 μm2 for type I and 4,009 μm2 for type II, the proportion of type I fibres, and the interstitial pressure of tibialis anterior were increased.
The findings suggest that chronic ischemia of the leg muscles causes compensatory histochemical changes in muscle fibres resulting from muscle hypoxia, and chronic dennervation-reinnervation changes, resulting possibly from ischemic neuropathy. Reperfusion seems to bring the oxidative capacity of the previously ischemic muscle closer to normal.
Structural and functional adaptations take place in working and resting muscles when the blood flow is insufficient to satisfy energy requirements [1–3]. Partial ischemia for short periods of time may cause cell damage in humans [4, 5], while metabolic changes in leg muscles have been reported after 16 hours of ischemia . Morphological and electrophysiologic studies of ischemic muscles in peripheral occlusive arterial disease disclosed evidence of denervation and type II fibre atrophy [7–9]. Several studies have demonstrated an increase in the oxidative capacity of ischemic muscles in patients with atherosclerotic peripheral occlusive arterial disease [8, 10–12] while other authors reported reduced oxidative capacity in leg muscles of patients with intermittent claudication [13, 14].
The crucial role of increased blood flow in striated muscles after an ischemic period has been studied extensively in the heart muscle. Recent publications [15, 16] have shown biochemical, morphological and ultra structural changes in the heart muscle after reperfusion in experimentally induced prolonged global ischemia or after the use of free oxygen radical inhibitors.
Although biochemical changes occurring in other organs during reperfusion following ischemia have been described in numerous studies [17–21], there is hardly any information about changes in human muscle fibres during reperfusion after bypass operations. The purpose of the present study is to identify the histochemical profile of ischemic skeletal muscles before and after the revascularization operation in patients with peripheral occlusive arterial disease.
Materials and Methods
Nine 64 ± 12 year-old men with atherosclerotic lesions at the aortoiliac level (Fontaine stage II) were investigated. None of them showed clinical signs of peripheral neuropathy, and any history of diabetes mellitus. All patients were submitted to a reconstructive surgical procedure, which consisted of uni- or bilateral aortofemoral bypass grafting, and were given 100 mg acetylsalicylic acid per day as postoperative treatment. The patients were informed on the nature of the study, with the possible risks of the procedures, and they gave consent prior to inclusion in the study. The Human Subjects Committee of the Aristotle University of Thessaloniki approved the experimental protocol.
All patients underwent standard electromyographic examination of both lower extremities, before the first biopsy to exclude peripheral neuropathy. Motor nerve conduction velocities with corresponding F-waves of the posterior tibial and peroneal nerves and sensory nerve conduction velocities of the sural nerves were obtained bilaterally, using standard technique, and a Neuropack 2® (Nihon Kohden). Concentric needle electromyographic examination was limited to the tibialis anterior, extensor digitorum brevis and abductor hallucis brevis bilaterally. None of the patients showed electrophysiologic evidence of a peripheral neuropathy.
Two tissue samples were obtained from each patient during each biopsy under local anaesthesia (1 % lidocaine), from the tibialis anterior muscle. The samples were dissected out in blocks of 5 × 5 × 5 mm3, were taken from a well-defined portion of the muscle belly, to a depth of about 2 cm beneath the fascia, at the half distance between the ankle and the knee. The first biopsy was taken during the reconstructive surgery procedure and the second biopsy 7 days after the operation. Each sample was then frozen in melting isopentane (-60°C) cooled in liquid nitrogen, and stored in a freezer (-80°C).
Intramuscular pressure measurement
Muscle compartmental pressure was measured pre- and postoperatively, at the 1st & 7th postoperative day, by inserting a special sterile 18 gauge side ported needle 6.4 cm long, in the tibialis anterior muscle, which was connected to the Stryker intra-compartmental pressure monitor system (Stryker, 295–1 Pressure), a hand held monitor system specifically designed for measuring tissue fluid pressures.
Histology and Enzyme histochemistry
Transverse serial sections of 9 μm – thick of each sample were cut in a cryostat microtome at -20°C. The sections were processed with eosin-hematoxylin, hematoxylin-van Gieson for routine histologic examination; with succinic dehydrogenase (SDH) and NADH-trichrome reductase  for oxidative enzyme activity ; with myosin-ATPase for fibre typing ; and with periodic acid-Schiff ((PAS) for glycogen .
Morphometric parameters were determined by projecting the microscope slides (Zeiss) via a camera lucida onto a calibrated grid (Huston Instrument TG 1011 digitizer) linked to a desktop computer. A program was developed in GW-BASIC for digitation and further calculations. Fibre-type cross-sectional area and fibre type proportion were determined by counting 200 contiguous fibres of the central part of each tissue sample.
Continuous data were reported as mean value ± standard deviation and categorical data as percentages. Paired Student's test was used for comparing differences between the mean values before and after reperfusion, and chi-square was used to compare the differences between the fibre-type proportions before and after the reperfusion. Differences were considered significant at P values < 0.05.
First biopsy (before bypass grafting)
Fibre type areas, proportions and age of subjects as reported by other authors
Type I area (μm2)
Type I (%)
Type II area (μm2)
Type II (%)
Age and sex (yrs – M/F)
Jakobsson F et al 
4,050 ± 8,900
84 ± 9
5,700 ± 1970
15 ± 8
Angquist et al 
59, M 47, F
Friden et al 
5,430 ± 883
8,132 ± 1747
Henriksson-Larsen et al 
Sjøstrøm et al 
59, M 47, F
Muscle fibre cross-sectional areas and fibre type proportions of tibialis anterior biopsy samples before and after the reconstructive surgical procedure (values are means ± sd)
3546.8 ± 1413
3264.8 ± 769
P < 0.001**
NON OXIDATIVE FIBRES
4003.5 ± 1388
3771.3 ± 1238
P < 0.05**
Second Biopsy (7 days after bypass grafting)
Morphometric data of tibialis anterior muscle fibres (mean ± SD), in the muscle biopsy samples before and after reperfusion.
TYPE I FIBRES
3,745 ± 2,094
3,086 ± 1,340
P < 0.001**
TYPE II FIBRES
4,654.5 ± 2626
4,009 ± 1,917
P < 0.001**
Interstitial pressure measurements
The interstitial pressure was 6.5 ± 3.3 mm Hg in the tibialis anterior of ischemic limbs before the reconstructive surgery procedure, but was increased to 10.30 ± 5 mm Hg on the 1st postoperative day, and to 14.38 ± 9.8 mm Hg on the 7th postoperative day. Two-tailed paired t-test showed difference only between the preoperative and postoperative values for P < 0.05; no statistically significant difference was found between the 1st and the 7th postoperative day values.
The main morphological characteristics of ischemic muscles in peripheral occlusive arterial disease patients with peripheral occlusive arterial disease observed in the present study are similar to those reported by Sjøstrøm et al . The presence of muscle fibre grouping in all biopsies suggests a process of chronic dennervation-reinnervation, a possibility previously disputed. The question of whether fibre-type grouping and nerve-ending degeneration might be age-related rather than the result of ischemia is still open to debate. The effect of ischemia and reperfusion on motor nerves has been described in humans [9, 27] and in experimental animal models [28–32], nevertheless the underlying mechanism is incompletely understood. During reperfusion of the acutely ischemic tibialis anterior of rabbits, the deep peroneal nerve's conduction was impaired and the action potential was abolished . Tourniquet ischemia for 20 min prevents paw plantar flexion following stimulation of the tibial nerve , and causes changes in sympathetic or parasympathetic activity, when applied for longer periods . The electromyographic findings of the present study, i.e. normal conduction velocities with evidence of chronic dennervation-reinnervation are in agreement with previous reports in patients with peripheral occlusive arterial disease , and would be consistent with the notion of a parallel neuropathic process, likely ischemic in etiology . The fact that no overt neuropathy was apparent in our patients, either on clinical examination, or on nerve conduction testing, is most likely the result of the relatively early peripheral occlusive arterial disease stage of our patients.
Groups of small regenerated muscle fibres were noted in this study on light microscope both before and after 7 days reperfusion with no change in appearance, whereas Karpati et al  on electron microscope after 7 days of experimentally induced ischemia on Sprague Dawley rats observed mitochondrial debris and large membrane-lined space. The same authors reported no change in the appearance of regenerating fibres found in ischemic muscle for up to 3 months. In this study the presence of regenerated and atrophic muscle fibres is also illustrated by the heterogeneity found within both fibre populations. Similar findings were reported by Sjøstrøm  who suggested that these changes were due either to loss of large efferent nerves, and possibly of anterior horn cells, or to the cumulative effect of incidental diseases.
Ischemia affects each muscle fibre type in different ways according to its particular metabolic and functional properties. The type I predominance found in our study, as well as the muscle fibre areas of tibialis anterior muscle are in accordance with the findings of other authors of age-matched human subjects [24, 34, 35]. However, as a result of conflicting reports regarding muscle oxidative capacity in patients with peripheral occlusive arterial disease [1–3, 8, 10, 11, 13, 14, 21, 23, 34] the relationship between local pathology and clinical symptomatology remains elusive. Jennische  found a selective vulnerability of the fast glycolytic fibres to experimental ischemia in rats. By contrast, slow-oxidative fibres were spared. The author attributed the difference to the higher content of free-radical scavenger compounds in the more tolerant fibres, and to their different capability for calcium uptake. Other investigators reported an increase in mitochondrial volume in chronically ischemic muscles [25, 32], while, in an apparent contradiction, decreased muscle oxidative capacity has been reported in patients with intermittent claudication , and decreased dehydrogenase activity following 6 hours of ischemia in dogs .
A possible explanation to this discrepancy was given by Clyne et al , who stated that during the initial stages of ischemia skeletal muscle tries to compensate for the oxygen deficiency, while at later stages as blood flow is further decreased and functional demands are increased, it looses the functional adaptations.
The morphometric, histological and histochemical observations of our study suggest that type I fibres are at least somewhat vulnerable to ischemia, although not to the same degree as type II fibres. The proportion of type I fibres of the ischemic tibialis anterior increased to 66.2% after reperfusion (Table 3), a number closer to that reported for normal subjects [8, 13, 22–24], whereas the proportion of type II fibres proportionally decreased (Table 3), eventhough these changes in proportions of fibre types were not statistically significant. The mean cross-sectional area of both muscle fibre types was decreased after reperfusion, 17.6% for type I and 13.8% for type II, conceivably as a result of a postbypass efflux of intracellular compounds to the interstitial space and a subsequently increased interstitial fluid volume. Such statistically significant increase in interstitial fluid volume would also explain the observed postoperative increase in the interstitial fluid pressure.
The authors thank Dora Kapoucranidou for her technical assistance. We also thank Professor Olga Hudlicka for her comments on the manuscript.
- Elander A, Idstrom JP, Holm S, Schersten T, Bylund-Fellenius AC: Metabolic adaptation to reduced muscle blood flow. II. Mechanisms and beneficial effects. Am J Physiol. 1985, 249: E70-6.PubMedGoogle Scholar
- Elander A, Idstrom JP, Schersten T, Bylund-Fellenius AC: Metabolic adaptation to reduced muscle blood flow. I. Enzyme and metabolite alterations. Am J Physiol. 1985, 249: E63-9.PubMedGoogle Scholar
- Hoppeler H, Hudlicka O, Uhlmann E, Claasen H: Skeletal muscle adaptations to ischemia and severe exercise. Clin J Sport Med. 1992, 2: 43-51.View ArticleGoogle Scholar
- Perry MO, Fantini G: Ischemia: Profile of an enemy. J Vasc Surg. 1987, 6: 231-4. 10.1067/mva.1987.avs0060231.View ArticlePubMedGoogle Scholar
- Perry MO: Historical Perspective. In: Ischemia-Reperfusion Injury of skeletal Muscle. Edited by: Fantini GA. 1994, R G Landes Company, 1-4.Google Scholar
- Eklof B, Neglen P, Thompson D: Temporary incomplete ischemia of the legs induced by aortic clamping in man. Ann Surg. 1981, 93: 89-98.View ArticleGoogle Scholar
- Malan E, Tatoni G: Physio-and Anato-pathology of acute ischemia of the extremities. J Cardiovasc Surg. 1963, 4: 2-10.Google Scholar
- Sjøstrøm M, Angquist KA, Rais O: Intermittent claudication and muscle fibre fine structure: Correlation between clinical and morphological data. Ultrastruct Pathol. 1980, 1: 309-26.View ArticlePubMedGoogle Scholar
- England JD, Regensteiner JG, Ringel SD, Carry MR, Hiatt WR: Muscle denervation in peripheral occlusive arterial disease. Neurology. 1992, 42: 994-9.View ArticlePubMedGoogle Scholar
- Bylund AC, Hammarsten J, Holm J, Schersten T: Enzyme activities in skeletal muscles from patients with peripheral arterial insufficiency. Eur J Clin Invest. 1976, 6: 425-9.View ArticlePubMedGoogle Scholar
- Janssen E, Johansson J, Sylven C, Kaijer L: Calf muscle adaptation in intermittent claudication. Side-differences in muscle metabolic characteristics in patients with unilateral arterial disease. Clin Physiol. 1988, 417: 67-72.Google Scholar
- Lundgren F, Dahllof AG, Schersten T, Bylund-Fellenius AC: Muscle enzyme adaptation in patients with peripheral arterial insufficiency: spontaneous adaptation, effect of different treatments and consequences on walking performance. Clin Sci. 1988, 77: 485-93.View ArticleGoogle Scholar
- Henriksson J, Nygaard E, Andersson J, Eklof B: Enzyme activities, fibre types and capillarization in calf muscles of patients with intermittent claudication. Scand J Clin Lab Invest. 1980, 40: 361-369.View ArticlePubMedGoogle Scholar
- Regensteiner JG, Wolfel EE, Brass EP, Carry MR, Ringel SP, Hargarten ME, Stamm ER, Hiatt WR: Chronic changes in skeletal muscle histology and function in peripheral occlusive arterial disease. Circulation. 1993, 87: 413-21.View ArticlePubMedGoogle Scholar
- Hegstad AC, Ytrehus K, Lindal S, Myklebust R, Jorgensen L: The initial phase of myocardial reperfusion is not associated with aggravation of ischemic-induced ultrastructural alterations in isolated rat hearts exposed to prolonged global ischemia. Ultrastruct Pathol. 1999, 23 (2): 93-105. 10.1080/019131299281743.View ArticlePubMedGoogle Scholar
- Welt K, Fitzl G, Mark B: Lipoxygenase inhibitor FLM an effective protectant of myocardial micro vessels against ischemia-reperfusion injury? An ultrastructural-morphometric study. Exp Toxicol Pathol. 2000, 52 (1): 27-36.View ArticlePubMedGoogle Scholar
- Del Maestro RF: An approach to free radicals in medicine and biology. Acta Physiol Scand Suppl. 1980, 492: 153-68.PubMedGoogle Scholar
- Granger DN: Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol. 1988, 255: H1269-75.PubMedGoogle Scholar
- McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med. 1985, 312: 159-63.View ArticlePubMedGoogle Scholar
- Davis JM, Fantini GA: Remote effects. In: Ischemia-Reperfusion Injury of skeletal Muscle. Edited by: Fantini GA. 1994, R G Landes Company, 1-4.Google Scholar
- Welbourn CR, Goldman G, Paterson IS, Valeri CR, Sherpo D, Heehtman HB: Pathophysiology of ischemia reperfusion injury: central role of the neutrophil. Br J Surg. 1991, 78: 651-5.View ArticlePubMedGoogle Scholar
- Dubowitz V, Brooke MA: Muscle Biopsy: A modern Approach. 1985, London, Philadelphia, Toronto: Saunders, 5-33.Google Scholar
- Nemeth P, Pette D, Vrbova G: Comparison of enzyme activities among single muscle fibres within defined motor units. J Physiol Lond. 1981, 311: 489-95.View ArticlePubMedPubMed CentralGoogle Scholar
- Jakobsson F, Borg K, Edstrom L, Grimby L: Use of motor units in relation to muscle fibre type and size in man. Muscle & Nerve. 1988, 11: 1211-8.View ArticleGoogle Scholar
- Angquist KA, Sjøstrøm M: Intermittent claudication and muscle fibre fine structure: Morphometric data on mitochondrial volumes. Ultrastruct Pathol. 1980, 1: 461-70.View ArticlePubMedGoogle Scholar
- Friden J, Sfakianos PN, Hargens AR, Akeson WH: Residual muscular swelling after repetitive eccentric contractions. J Orthop Res. 1988, 6: 493-8.View ArticlePubMedGoogle Scholar
- England JD, Ferguson MA, Hiatt WR, Regensteiner JG: Progression of neuropathy in peripheral occlusive arterial disease. Muscle & Nerve. 1995, 18: 380-7.View ArticleGoogle Scholar
- Badier M, Guillot C, Laggier-Tessonnier F, Jammes Y: EMG changes in respiratory and skeletal muscles during isometric contraction under normoxic, hypoxemic, or ischemic conditions. Muscle & Nerve. 1994, 17: 500-8.View ArticleGoogle Scholar
- Chervu A, Moore WS, Hosher E, Quinones-Baldrich WJ: Differential recovery of skeletal muscle and peripheral nerve function after ischemia and reperfusion. J Surg Res. 1989, 47: 12-19.View ArticlePubMedGoogle Scholar
- Christ F, Moser CM, Niklas M, Gartside IB, Gamble J, Refior HJ, Peter K, Messmer K: Prevalence of cyclic changes in limb volume (volumotion) of male patients with knee injury and the effects of ischemia/reperfusion due to tourniquet. Int J Microcirc Clin Esep. 1995, 15: 14-20.View ArticleGoogle Scholar
- Lundborg G: Ischemic nerve injury: Experimental studies on intraneural micro vascular pathophysiology and nerve function in a limb subjected to temporary circulatory arrest. Scand J Plast Reconstr Surg Suppl. 1970, 6: 3-113.PubMedGoogle Scholar
- Zollman PJ, Awad O, Schmelzer JD, Low PA: Effect of ischemia and reperfusion in vivo on energy metabolism of rat sciatic-tibial and caudal nerves. Exp Neurol. 1991, 114: 315-20.View ArticlePubMedGoogle Scholar
- Karpati G, Carpenter S, Melmed C, Eisen AA: Experimental Ischemic Myopathy. J Neurol Sci. 1974, 23: 129-61. 10.1016/0022-510X(74)90148-8.View ArticlePubMedGoogle Scholar
- Henliksson-Larsen KB, Lexell J, Sjøstrøm M: Distribution of different fibre types in human skeletal muscles. I. Method for the preparation and analysis of cross sections of whole human tibialis anterior muscles. Histochem J. 1983, 15: 167-78.View ArticleGoogle Scholar
- Albani M, Kiskinis D, Natsis K, Megalopoulos A, Gigis P, Guiba-Tziampiri O: Histochemical and ultrastructural characteristics of leg muscle fibres in patients with repairative abdominal aortic aneurysm (AAA). Anat Rec. 2000, 260: 1-15. 10.1002/1097-0185(20000901)260:1<1::AID-AR10>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
- Jennische E: Ischaemia-induced injury in glycogen-depleted skeletal muscle. Selective vulnerability of FG-fibres. Acta Physiol Scand. 1985, 125: 727-34.View ArticlePubMedGoogle Scholar
- Blebea J, Kerr JC, Shumko JZ, Feinberg RN, Hobson RW: Quantitative histochemical evaluation of skeletal muscle ischemia and reperfusion injury. J Surg Res. 1987, 43: 311-21.View ArticlePubMedGoogle Scholar
- Clyne AC, Mears H, Weller RO, O'Donnell TF: Calf muscle adaptation to peripheral vascular disease. Cardiovasc Res. 1985, 19: 507-512.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/3/8/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.