Experimental methods in the pathogenesis of limb ischemia

JOURNAL OF SURGICAL RESEARCH 44, 284-307 (1988)

CURRENT Experimental

REVIEW

Methods in the Pathogenesis

PHILIP S. BARIE, *Department

RESEARCH

of Limb lschemia

M.D.,**’ AND RICHARD J. MULLINS, M.D.t,2

of Surgery, Cornell University Medical College, New York, New York, and t University of Louisville, Louisville, Kentucky

Submitted for publication August 3, 1987 Ischemia of extremities is responsible for considerable morbidity and mortality and the pathophysiology of this condition warrants further study. The purpose of this review is to discusstechniques used in the evaluation of limb &hernia and reperfusion. It is of critical importance to study limb blood flow distribution to the microcirculation where nutritive exchange occurs. Skeletal muscle &hernia progressesto infarction when critical deficits of cellular metabolites develop, which mandates that studies be focused at the cellular level. It is clear that the adverse effects of &hernia can be exacerbated by a reperfusion injury to the endothelium of the microvasculature. Investigators wishing to study limb &hernia have a wide spectrum of methodology and established models available to use in improving the understanding of the complex events of ischemic injury. D 1988 Academic PBS, IIIC.

in 1904. However, experience has shown that inflation of the tourniquet for more than Extremity ischemia, an important clinical 2 hr can result in irreparable injury to the problem, can be divided into chronic and extremity. Because extremity ischemia is an acute ischemia. Many patients present to enormous clinical problem, a breadth of exvascular surgeons with chronic &hernia of perimental methodology has been used to the lower extremities due to gradual obstrucinvestigate how management of these pation by atherosclerosis of large inflow vessels. tients can be improved. The purpose of this These extremities are hypoxic but remain vireview is to critically discuss these experiable because as large artery atherosclerosis mental techniques, while highlighting some progressively obstructs flow, collateral vesof the pathologic and physiologic concepts sels develop and restore blood flow. Patients that relate to extremity ischemia and reperwho present after sudden occlusion of a fusion. major vesseloften have cadaveric extremities and anoxic tissues; immediate restoration of flow is mandatory if amputation is to be PHYSIOLOGY OF BLOOD FLOW TO avoided. Successful reimplantation of exTHE EXTREMITIES tremities can be jeopardized by a reperfusion injury. Surgeons have reduced blood loss and The majority of blood flowing into an eximproved exposure with the pneumatic tremity distributes to skeletal muscle, skin, tourniquet, introduced by Harvey Cushing and bone, with minor fractions going to tendon, periostium, cartilage, and nerve. Using ’ To whom reprint requests should be addressed at microspheres to study resting unanesthetized Department of Surgery, F-1926, New York Hospitaldogs, Rutherford and Valenta reported that Cornell Medical Center, 525 East 68th Street, New 7 1%of blood flowed to muscle, 15%to bone, York, NY 10021. 2 Supported by a research grant from the Veterans and 7% to skin [94]. In these resting dogs, the blood flows were 5 to 6 ml/ 100 g/min of skin Administration Merit Review Board. INTRODUCTION

0022-4804/88 $1SO Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and subcutaneous tissue and 7 to 8 ml/ 100 g/min of skeletal muscle [94]. Redisch et al. estimated 25 to 50% of blood flowing into human forearms and legs distributed to skin and 50 to 75% flowed to muscle [87]. Blood vesselsin skin and muscle adjust to changes in perfusion pressure so that a constant supply of oxygen is delivered to tissues. Guyton defined autoregulation as continuous local adjustment of blood tlow in proportion to the need of the tissue for nutrients [36]. The distribution of blood flow is regulated both by the sympathetic nervous system and local release of vasoactive metabolites. Total extremity blood flow is principally modulated by alterations in the diameter of precapillary arterioles. Local autoregulation occurs when changes in perivascular smooth muscle tone adjust luminal diameter to maintain nutritive flow during constant inflow pressure. The response of arteriolar smooth muscle is heterogeneous, and mediators which produce constriction in the vesselsof one tissue can result in dilation of other vessels. Mechanisms by which skin blood flow are regulated are interdependent. Skeletal muscle blood flow is lessautoregulated compared to skin because the cutaneous circulation contains a rich network of arteriovenous anastomoses (AVA), which are absent in skeletal muscle [67, 1041.Skin blood flow is regulated by adrenergic mechanisms; (Ystimulation produces active cutaneous vasoconstriction [48]. Also active are local adjustments in vascular resistance which respond to changes in skin temperature. A combination of local factors and active sympathetic vasodilation can mediate a IO-fold increase in skin blood flow. Some of these local influences are related to sweat gland activity, although the putative common mediator, speculated to be bradykinin, has not been isolated [ 1041. Baroreceptor influences on cutaneous blood flow are also clearly active [ 51. Increased transmural pressure across the carotid sinuses results in vasodilation, while either shock or exercise will result in cutaneous vasoconstriction. However, barorecep-

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tor-mediated adrenergic vasoconstriction cannot override maximal local or reflex-mediated vasodilation. Baroreceptors compete with thermoregulatory reflexes for control of the cutaneous circulation, and there is strong evidence that cutaneous AVAs are highly specific as thermoregulatory effector organs [48]. These AVAs appear to be under principal control of a-adrenergic efferents [ 16, 17, 27, 1041. cu-Adrenergic ablation in the form of sympathectomy produces dramatic increasesin AVA flow [ 16, 171. Baseline flow through AVAs comprises 4 to 9% of the total hindlimb blood flow, but may shunt as much as 25% of the total flow with CYblockade [27]. In contrast to the virtual absence of padrenergic receptors in control mechanisms for the cutaneous circulation, skeletal muscle capillary beds are under simultaneous control of (Yand /3 receptors [ 1041. Infusion of the P-agonist isoproterenol increases capillary flow two- to fivefold through active vasodilation [27, 1041.Muscle with few if any AVAs in an extensive capillary bed can either vasodilate or vasoconstrict in response to adrenergic activity. However, it is well established that most of the vasodilation in skeletal muscle is mediated by local factors. Although adrenergic vasodilators appear to play an important role in early exercise-induced increases in blood flow, after exercise, local factors may make reflex vasodilation insignificant by comparison. The principle local factor mediating skeletal muscle blood flow appears to be tissue oxygen tension 18, 361. Oxygen transport to the tissues is the most nearly flow-limited of the physiologically important substances transported by blood [36]. Total blood flow to an in situ, isolated, perfused canine hindlimb increasesthreefold when the oxygen tension of the blood perfusate is reduced from 100 to 30 Tot-r [36]. These findings have been confirmed in isolated canine femoral artery, showing that vascular resistance decreasesby nearly 60% as p02 decreasesto 30 Torr [8]. A pertinent question is: to what degree are autoregulatory mechanisms preserved dur-

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ing limb &hernia? It is clear that there is a relationship between skeletal muscle blood flow and oxygen consumption when muscle blood flow exceeds 3 ml/min/ 100 g, leading to the suggestion that autoregulation in skeletal muscle may be a consequence of metabolism rather than a primary factor [25]. The precise point at which autoregulatory mechanisms fail to respond to tissue hypoxia has been difficult to define. Ischemia does not appear to alter the fact that a-adrenergic ablation (sympathectomy) does not increase skeletal muscle capillary blood flow [ 161. Furthermore, autoregulatory mechanisms do not preserve hindlimb oxygen consump tion after sympathectomy when limb blood flow is less than 50% of normal volume, in part because of induced increased shunting of blood through nonnutritive arteriovenous anastomoses [ 171. Reactive hyperemia is a vascular phenomenon, initiated when blood flow is restored to an ischemic limb, that is characterized by a marked but transient increase in blood flow above baseline. Reactive hyperemia will occur after only a few seconds of occluded arterial inflow, and both skin and skeletal muscles are involved [ 1031. In the human forearm when inflow occlusion was less than 100 min, the duration of the period of hyperemia measured by plethysmography was proportional to the duration of the ischemia. However, on reperfusion the volume of hyperemic blood flow (flow greater than baseline flow) was consistently in excessof what was the calculated “debt” incurred during arterial occlusion [8 1, 1221. Kristensen and Henriksen measured by xenon- 133 washout the duration of hyperemia and the cumulative excessblood flow in the skin of normal volunteers. Both of these parameters were proportional to the duration of ischemia when the ischemia was lessthan 24 min [ 571. Reactive hyperemia will occur in extremities without sympathetic or somatic innervation, indicating that local factors mediate reactive hyperemia [ 1031. Evidence indicates that ischemia may produce vasodilator metabolites (adenosine). Guyton et al. have shown

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that the hyperemia after reperfusion will persist if the blood reperfusing the extremity is hypoxic [36]. Also hypothesized to influence reactive hyperemia is myogenic relaxation by the smooth muscle of the vascular wall [ 1031. With a fall in intraluminal pressure during ischemia, the decrease in mural tension in arterioles leads to a decline in myogenie tone. Upon reperfusion there is a delay in the return to baseline of the contraction of the smooth muscle in the media of arteriolar resistance vessels.Evidence in support of the myogenic hypothesis is the experiment of Wood et al., who first distended the limb by venous hypertension before arterial occlusion [ 1221. Using plethysmography in normal humans these investigators found that reactive hyperemia was 2 1 to 5 1% less in limbs with preocclusion congestion produced by a venous tourniquet than the hyperemia measured in normal limbs [ 1221. Reactive hyperemia has been used to evaluate arterial insufficiency. In patients with proximal arterial obstruction there is a delay in the return of arterial pressure in ankle vessels to baseline after a 5-min occlusion of arterial inflow because blood is shunted to skeletal muscle vesselsdilated due to reactive hyperemia [ 1151. This technique uses the rate at which arterial pressure returns to baseline during reactive hyperemia as an indirect manifestation of reactive hyperemia. A noninvasive direct measurement of reactive hyperemia can be made with laserDoppler velocimetry. With this technique Wilkin reported that a distinctive finding in reactive hyperemia after a 6-min period of occlusion in healthy human skin was a rhythmic oscillatory activity that appeared to be unrelated to sympathetic activity [ 1191. Reactive hyperemia is a physiologic response to reversible periods of ischemia that appears to “repay” oxygen debt. In contrast to reactive hyperemia is no-reflow, a pathophysiologic phenomenon characterized by an inability to reperfuse tissues. The duration of ischemia before no-reflow varies depending on the tissue and species.When ischemia exceeded 2 hr for rat skeletal muscle, and

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the canine femoral artery, blood flow from a venous cannula dropped to 20% of baseline within seconds, and then increased to 40% of baseline by 1 hr [23]. In another experiment when femoral artery and external iliac vein flows were simultaneously measured, the vein flows were 80% of the arterial flows. This ratio existed over a wide range of arterial Ilows [ 141. Retrograde blood flow from the femoral artery distal to the ligature is not considered an adequate indication of collateral flow becausethe normal resistance in the microvasculature of tissues is bypassed. Coffman concluded that venous flows, while not precise, are representative of the direction of change in collateral flow [ 141. Rosenthal and Guyton in their studies of collateral blood flow avoided the problem of measuring total limb flow by studying a single artery distal to an occlusion [92]. Pressure and electromagnetic flow probe measurements were simultaneously recorded in canine anterior tibial arteries after acute femoral artery occlusion. Compensatory dilation of the artery occurred within 2 min of femoral occlusion, and they calculated a 70 to 80% fall in collateral resistance. Denervation of the hindpaw, including a complete sympaCOLLATERAL BLOOD FLOW thectomy, had no influence on acute vasodiOcclusion of the major arteries to extremi- lation of collateral arteries, and the authors ties in many casesdoes not result in tissue concluded that tissue ischemia in some way necrosis becauseof compensatory blood flow effected collateral vasodilatation [92]. through collateral arterial vessels.The develThe development of collateral arteries opment of collateral flow around an ob- after femoral artery ligation of I l-weeks dustructed vessel has been characterized as ration in the dog has been documented by having two phases. The initial increase in preparing corrosion casts of the arterial tree flow is due to reflex vasodilation of collat- of hindlimbs. Anesthetized dogs’ femoral arerals which occurs within minutes of the ob- teries distal to ligatures were injected with a structing event. The second phase is an ad- liquid plastic which hardened within the vesjustment over days that involves prolifera- sels. The collateral vessels had both an intion of new vessels and an anatomical creasein number and an enlargment in size, increase in the size of collateral arteries. which returned the total vascular cross-secMeasurement of total extremity collateral tional area to normal [ 151. Williams and flow is problematic because the blood is Saelenshave examined the response to pharflowing through multiple small vessels in macologic manipulation of collateral arteries parallel. Venous outflow can be collected by in dogs [ 1201.Collateral arteries showed difree flow into a container or by shunting or minished contractility to an electrical stimupumping the blood through a flowmeter. lus, but were more responsive to exogenous Eckstein et al. reported that after ligation of norepinephrine than anatomically compara-

exceeded 6 to 8 hr for rat skin, the no-reflow phenomenon was noted [ 106,107,12 11.The phenomenon of no-reflow has been studied by serial biopsy of tissues after reperfusion. Focal injury to the microvasculature has been noted, with endothelial cell swelling apparent on electron micrographs interpreted as the earliest evidence of injury. After 8 hr of ischemia, injured skin showed plugging of small vesselsby white blood cells [ 1211.Capillary plugging by granulocytes appears to be a fundamental pathophysiologic mechanism that causesthe no-reflow phenomenon [99]. One technique for study of the no-reflow phenomenon may be to measure the number of 3-pm radiolabeled microspheres trapped in tissues after reperfusion compared to the number of 15-pm microspheres. Becausethe mean diameter of capillaries is about 8 pm, the 3-pm microspheres will only be trapped if there is occlusion of flow in capillaries. Sasaki and Pang reported that the extent of fluorescein tissue staining after intravenous injection of fluorescein and the penetration into skin flaps of 3-pm microspheres were similar [98].

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aerobic capacity. Fast-twitch-glycolytic fibers are anaerobic cells with a high glycogen content, but few of the enzymes needed for oxygen metabolism. Muscle groups vary in the proportions of these fiber types that they contain. The type of fibers in the muscle group studied during ischemic experiments must be defined if informative comparisons are to be made between reported studies of metabolic changes due to ischemia [46, 561. Six techniques have been used to study the metabolic response to ischemia and reperfusion: (1) biochemical studies of tissue samples, (2) histologic studies of tissue samples, (3) measurements of myocyte transmembrane potential, (4) surface pH monitoring, (5) nuclear magnetic resonance measurements of metabolites, and (6) functional assessmentsof muscle contraction. The metabolic events noted with ischemia and the value of tests showing these changesare summarized in Table 1. The concentration of molecular intermeMETABOLISM diates of metabolism can be measured in small tissue samples biopsied serially during Skeletal muscle performs mechanical work through transformation of the high en- ischemia and after reperfusion. Tissue samergy phosphate bonds of adenosine triphos- ples are collected by the freeze-clamp techphate (ATP) and phosphocreatinine (CP). nique and immediately transferred into liqNot all skeletal muscle cells have the same uid nitrogen to arrest metabolic activity. machinery for energy metabolism, and with After being homogenized in iced perchloric a combination of histochemical, biochemi- acid, the tissue supernatant can be assayed cal, and physiological studies three skeletal for ATP, CP, lactic acid, glycogen, and other muscle fiber types have been identified. His- metabolites using simple spectrophotometric tochemical staining techniq,ues identify cells techniques [37, 39, 45, 891. If the blood flow with oxidative (aerobic) and glycolytic (an- to tissues can be measured, and the arterioveaerobic) capacities. Biochemical tests can be nous concentration differences determined, performed on the supernatants of tissue ho- then metabolite uptake and release can be mogenates to identify substrates and en- calculated and used to quantitate the metazymes. Two of the three types of muscle bolic response [39]. In addition to measuring fibers are categorized as fast because of a metabolites in tissue biopsies, products of the short time-to-peak tension when stimulated ischemia and reperfusion injury, such as to contract, while one fiber type is catego- conjugated dienes produced by oxygen-mediated free radical damage to lipids, can be rized as slow [84]. Fast-twitch-oxidative-glycolytic fibers have a high aerobic capacity measured as an indication of reperfusion inand a high glycolytic capacity. Around this jury 1391. Hematoxylin and eosin-stained skeletal type of muscle fiber there is a dense capillary muscle preparations examined by light minetwork which can deliver large amounts of croscopy do not have clear histologic evioxygen. Slow-twitch-oxidative fibers have low glycogen stores and moderate to high dence of changesafter 2 hr of ischemia. After

ble vessels from the normal contralateral hindpaw. These findings suggest that vascular smooth muscles in dilated collateral arteries are less efficiently innervated by adrenergic fibers than normal vessels,with resultant supersensitivity of postsynaptic cY-adrenergic receptors [ 1201. Studies of collateral circulation to the kidney by Abrams led to the hypothesis that a humoral vasculogenic agent is released from ischemic tissues after arterial occlusion [ 11. With autoradiography, increased tritium-labeled thymidine uptake was evident in enlarging collateral vessels. This observation was interpreted as evidence of vascular neogenesis during the response to ischemia. These studies have been hampered by the availability of only a bioassay to identify the vascular mitogen. Development of a radioimmunoassay should enhance future studies of postocclusive angiogenesis [ 11.

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BARIE AND MULLINS: LIMB ISCHEMIA TABLE 1 Duration of ischemia

Technique

Event

Sequential cellular events in skeletal muscle during ischemia 30 min 30 min

Membrane potential Histology

30 min

Surface pH

90 min

Histology

2 hr

Biochemistry

2 hr

Histology

2 hr

NMR NMR Functional

3 hr >3 hr

5 hr

Histology Histology

7 hr

Histology

2.5 hr

Histology

2.5 <2 hr

Biochemistry

>3 hr >4 hr >5 hr

NMR Histology Biochemistry

4 hr

Histology

Fall in PD in aerobic and anaerobic muscle (46, 45) Focal areas of no reflow; occasional mitochondrial swelling ( 107, 114) Aerobic muscle pH normal; anaerobic muscle pH down (56) Earliest evidence of myolibrillar degeneration (97) Aerobic muscle: ATP and CP down 40%, lactate up; anaerobic muscle: no change in ATP or CP, lactate up (46) Uniform tetrazolium cell staining of skeletal muscle cells indicates viable cells (39); minimally abnormal ultrastructure by electron microscopy (39) Tissue pH 6.6; CP decreased;ATP normal (75) Tissue pH 6.2; CP and ATP undetectable (75) No contraction with muscle or nerve stimulation (100) Myocytes abnormal by light microscopy ( 100) Increased alizarin red S staining of cells indicates injury (44) Many abnormalities on electron microscopy; 75 to 90% of cells have no up-take of vital dyes (39) Focal areas of no-reflow ischemia persist on reperfusion (38, 106) Minimal structural damage (44) Complete regeneration of intramuscular phosphagens (39) With reperfusion ATP repletion slow (75) Maximum edema on reperfusion (107) ATP, CP, and glycogen not repleted despite reperfusion (39)

4 to 7 hr abnormalities become evident [39, jury [39]. When muscle ultrastructure was 441. By 8 to 12 hr of &hernia there is ne- examined with electron microscopy areas of crosis of muscle [95, 1001. Special histo- minimal cellular injury in vascular endothechemical stains can identify damaged cells lial cells and parenchymal muscle cells were despite a grossly normal appearance. Ali- seen after 2 hr of ischemia. The ultrastruczarin red S will demonstrate intracellular cal- ture of skeletal muscle was grossly abnormal cium precipitation which indicates cell in- after 7 hr of ischemia [39]. Measurement of transmembrane potential jury after 5 hr of ischemia [44]. Staining with NADH-nitroblue tetrazolium dye indicates provides a sensitive index of skeletal muscle viable cells, and after 7 hr of ischemia 85% of cell injury [83, 891. As a fine-tipped glass micells do not stain, indicating irreversible in- croelectrode is advanced through muscle,

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myocytes are impaled and the membrane potentials of multiple cells are measured. With this technique there can be repeated measurements over a time interval at different sites with minimal injury to the muscle [45]. Changes in membrane potential occur within 1 hr of tourniquet ischemia. The fall in membrane potential from the normal value of -90 mV has been reported to correspond with the accumulation of tissue lactate, whereas changesin membrane potential have been recorded in ischemic tissue with normal ATP levels [45]. Rapid reversals to normal of deficits in ATP and CP have been noted on reperfusion, while the return to a normal membrane potential is delayed [30, 891. Thus membrane potential is a more sensitive index of tissue injury and repair than measurement of tissue metabolites [83]. A range of decreasedtransmembrane potentials in skeletal muscle which had been reperfused after 3 hr of ischemia confirmed the heterogenous nature of reperfusion, with focal areas of injury amid normal muscle [ 381.Histologic studies of reversibly ischemic muscle also show a pattern of intermittent focal injury, with normal appearing areasimmediately adjacent to abnormal areas [ 1061. The production of lactate by hypoxic skeletal muscle cells through anaerobic glycolysis is proportional to the degree of ischemia [45, 831. Lactate accumulation, which is measured in tissue biopsies, and fall in membrane potential are early indicators of injury. However, lactate measurements are not entirely reliable indicators of viability because reperfusion of severely ischemic muscle has resulted in washout of elevated lactic acid without return of ATP and CP levels to normal in irreversibly injured tissue [37]. An alternative to tissue biopsy for lactate measurement is surface pH measurement, a nondestructive technique that can quantitatively measure tissue acidosis during ischemia. Greater acidosis during ischemia was identified by surface pH measurements in muscle that contained principally anaerobic glycolytic fibers [56].

Nuclear magnetic resonance spectroscopy is an excellent noninvasive technique for evaluation of molecules containing the appropriate isotopes of phosphorous, hydrogen, and carbon, all of which are present in large concentrations in tissues [40, 1181.Relative proportions of tissue phosphate such as CP, ATP, and inorganic phosphate can be determined in viva [43]. Newman has shown that tourniquet ischemia results first in depletion of CP and then ATP. Total depletion of ATP after 2 10 min of tourniquet ischemia in rat calf muscle corresponded to irreversible ischemia. The time course of intramyocellular pH during ischemia and different patterns of pH correction after reperfusion have also been identified [75]. Nuclear magnetic resonance spectroscopy has shown that the benefit of hypothermia in amputated extremities may relate more to protection against severe acidosis than to preservation of ATP levels [78]. The advantages of NMR are that it is quantitative, does not disrupt or injure tissues, and can be repeatedly used in an intact extremity. However, in addition to expense, a disadvantage of this technique is that it provides total limb (skin and muscle) data. This lack of precision may explain why there are differences in reported results of observations during ischemia. For example the fall of NMR-measured ATP occurred only after the phosphocreatine signal was no longer recorded in ischemic rat limbs, where the skeletal muscle is primarily composed of glycolytic fibers, while in cat limbs there was evidence of ATP depletion occurring while phosphocreatine was still detectable [75,78]. Measurements of single twitch contraction force can be made after reperfusion of ischemit extremities as a quantitative index of physiologic recovery. With a force displacement transducer, the maximum contractile force in the rat Achilles’ tendon can be measured. The muscle is stimulated to contract with a square wave stimulator, which delivers a series of escalating voltages. After 3 hr of ischemia, muscle failed to contract by direct electrical and mechanical stimulation [ 1001. However, the failure to respond to

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stimulation in studies that measure limb been attributed to an increase in microvascumotion may reflect an injury to nerve rather lar (arteriole, capillary, and venule) memthan muscle. Nerve injury can be related to brane permeability to both water and pro&hernia or can occur because tourniquet teins [55, 851. We will review the advantages compression produces a direct mechanical and disadvantages of three techniques which injury [77, 1141. Force displacement trans- have been used to measure protein permeducers have also been employed to deter- ability after ischemia. mine responses of muscle afferents to ischMechanical perfusion of an isolated exemia during static contraction of hindlimb tremity or muscle while its weight is continmusculature [ 5 I]. Increased afferent electri- uously recorded is an accurate technique by cal activity has been identified during isch- which the microvascular membrane permeemia produced by transient aortic occlusion ability for both water and plasma proteins in cats, and may be responsible for the reflex can be quantitated. The principle of the techincreases in blood pressure, heart rate, and nique is that while the specimen is perfused myocardial contractility seen during skeletal an isogravimetric state (specimen weight remuscle ischemia and long postulated to arise mains stable) can be sustained if venous from working muscle [5 11. pressure and blood flow are adjusted so that the Starling forces across the microvasculature are balanced [80]. In the isogravimetric INCREASES IN PERMEABILITY specimen there is no net transfer of fluid Skeletal muscle compartment syndromes from the vascular compartment into the inare a major clinical problem which can jeop- terstitium because the net hydrostatic presardize the limb after successfulreperfusion of sure favoring fluid transport out of the vascuseverely ischemic extremities. With the re- lar compartment equals the net oncotic turn of blood flow to ischemic muscle, fluid forces working in the opposite direction. If can accumulate in sufficient volume to raise the pumped flow rate into the artery or the tissue pressure [72]. When tissue compart- venous outflow pressure are changed, a dement pressuresincrease to within 30 mm Hg termination of capillary water permeability of the aortic diastolic pressure, blood flow can be made based upon the rate of change in (measured by xenon- 133 washout determi- specimen weight. The permeability of the nations) stops because the microvasculature microvascular membrane to proteins can be is compressed [ 131.In the anterolateral com- measured by changing the oncotic pressure partment of the canine leg Mubarak et al. of the perfusate and recording what effect compared the wick technique with the nee- that has on the weight of the specimen. The dle manometer technique and a solid-state isogravimetric technique is a precise method microprobe [ 721. These investigators and for measurement of microvascular permeothers have found that the wick technique is ability, and also enables the investigator to superior in terms of accuracy and reproduc- measure hemodynamic parameters which influence capillary hydrostatic pressure [20, ibility [72, 9 I]. A minimum of 3 hr of ischemia has 801. However, it has disadvantages in that if usually been required before tissue swelling the specimen is denervated, the preparation develops on reperfusion [20, 70, 1071. Two will deteriorate (i.e., become edematous) pathophysiologic mechanisms have been after several hours of study and an isograviproposed to explain compartment syn- metric specimen is not suited to repeated observations over an experimental protocol dromes: cellular swelling and interstitial edema. Measurement and analysis of events composed of sequential interventions. Howassociated with cellular swelling have been ever, this technique has been used to make discussed in the metabolism section. The several observations regarding ischemia and postischemic increase in interstitial fluid has reperfusion. With an isolated canine hind-

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paw Diana and Laughlin demonstrated that edema which developed after 1 to 3 hr of ischemia was primarily due to an increase in capillary hydrostatic pressure produced by arterial vasodilatation [20]. In only 5 of the 16 experiments in which the hindpaw was ischemic for 3 hr were these investigators able to show changes consistent with an increase in plasma protein permeability [20]. Korthius et al. used the isolated canine gracilis muscle, and showed that the protein permeability of skeletal muscle microvasculature increased (i.e., reduction in osmotic reflection coefficient and isogravimetric capillary pressure) after 4 hr of inflow occlusion and reperfusion [55]. Furthermore they showed that pretreatment of ischemic muscle with drugs that blocked oxygen radicals prevented the increase in permeability produced by ischemia and reperfusion [55]. Other investigators have also reported that oxygen radicals contribute to the reperfusion injury [ 1161. Analysis of lymph data has been widely used to determine the protein permeability of the microvasculature. Optimal data is obtained when lymph is collected from prenoda1 lymphatics which exclusively drain the tissue of interest. In dogs lymphatics which drain predominately skin and subcutaneous tissue can be readily identified next to larger subcutaneous veins and cannulated [74, 881. Skeletal muscle lymphatics located deep in muscle compartments are inaccessible, small, fragile, and difficult to cannulate [6, 70, 751. In order for the interpretation of lymph data to be reliable, it is critical that the lymph be collected during a steady state. This means that the flow rate and protein composition of lymph are identical to the net transvascular water flow and plasma protein flux at the microvascular membrane in the segment of tissue which the lymphatic drains. To obtain reliable postischemic lymph data which are steady state may require several hours of lymph collection after reperfusion. Because of this need for collecting steady-state lymph, a disadvantage of

lymph analysis is that it is not sensitive to transient changes in permeability. Methods for lymph analysis have been established [88, 1121. One analysis technique requires that venous pressure be increased in a stepwise fashion in order to effect sequential increases in transvascular fluid flow, and thus lymph flow. When further increases in lymph flow are not accompanied by a fall in lymph protein concentration then a filtration-independent state has been achieved. The osmotic reflection coefficient, which is a quantitative index of protein permeability, can be simply calculated as one minus the lymph over plasma total protein concentration ratio determined with filtration-independent lymph [ 1121.The osmotic reflection coefficient has a value between one and zero. The closer the osmotic reflection coefficient is to zero the more permeable the membrane. This technique has the disadvantage of being cumbersome becauseof the long period after reperfusion with venous pressure increases which is required before a filtration-independent state can be reached. Increasesin lymph flow are to be expected after ischemia when the tissues are reperfused because arteriolar vasodilation during reperfusion increases the number of capillaries perfused and the hydrostatic pressure within the capillaries [20]; hence, higher lymph flows without a fall in lymph over plasma total protein concentration ratios do not prove that microvascular membrane permeability has increased [63, 69, 70, 751. Demonstrating a reduction in membrane selectivity is a second technique which can be used to determine whether microvascular membrane protein permeability has increased [88]. Normally for the plasma proteins larger than albumin, the lymph over plasma protein concentration ratios are smaller the larger the molecule. When protein permeability increases because there are more large pathways in the microvascular membrane of sufficient size to not discriminate solute molecules based on size, there should be a fractional increase in the lymph protein concentration of larger molecules (IgG and IgM)

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of blood flow measurement are also available, including laser-Doppler velocimetry of erythrocytes [ 18, 49, 791, transcutaneous measurement of pOZ [7 11,skin fluorescence [98], and thermography. Plethysmographic techniques have been well described and are now part of the standard methodologic armamentarium [ 12, 2 1, 65, 76, 113, 1171.These techniques assume that vessel engorgement with blood distal to a point of venous outflow occlusion will cause the obstructed segment of the extremity to increase in volume directly proportional to the rate of arterial inflow [ 1171. Both strain gauge (resistive) and venous occlusion (capacitance) plethysmographic techniques can be repeatedly used and have a coefficient of variation of less than 15% [ 1131. Venous occlusion plethysmography produced flow values which correlated with measured flow during physiologic roller pump perfusion of human sarcoma-bearing limbs [76], although the correlation was better for perfusion of the lower extremity. Excellent correlation also exists between straingauge and capacitance techniques for measurement of forearm blood flow in both normal [ 121and seriously ill patient populations [21]. Accurate application of capacitance plethysmography requires that the pressure from the inflated cuff does not affect arterial pressure or inflow, that complete venous occluQUANTITATION OF EXTREMITY sion is achieved, and that during the period PERFUSION when extremity volume increases, the inInvestigation into the pathogenesis of limb crease in venous pressure does not reduce ischemia cannot be properly interpreted arterial inflow [76]. Strain gauge techniques without an objective assessment of how may be simpler to apply and more versatile much perfusion has been interrupted, or by comparison with capacitance plethysmogwithout quantitative data regarding reperfu- raphy [21]. It is important to recognize that sion. Experimental techniques successfully plethysmographic techniques measure total used to measure extremity blood flow in- blood flow to the extremity and cannot difclude plethysmography [ 1171, electromag- ferentiate the relative distribution of perfunetic probes [65], tracer washout techniques sion to skin, subcutaneous tissue, and mus[35, 6 11,and injection of radioactive micro- cle [ 1241. spheres [7]. Substantial methodologic validaBlood flow can also be reliably measured tion is available to allow confident applica- with electromagnetic flow probes, which can tion of these standard techniques. Altema- be placed directly around vesselsas small as tive techniques that reflect the effectiveness 0.5 mm in diameter [59]. Plow is measured

greater than that of smaller molecules [73, 881. An advantage of this method is that the first steady-state lymph data obtained after reperfusion can be analyzed. However, the selectivity data usually provide more of a qualitative index of permeability, compared to the quantitative value obtained when the osmotic reflection coefficient is calculated. High permeability edema is characterized by an increase in transendothelial clearance of plasma proteins that results in an increase in interstitial albumin content. Albumin labeled with radioactive tracers or Evans blue dye can be intravenously injected after reperfusion of an ischemic extremity, and then the tissue can be excised and analyzed for albumin content [44]. The amount of tissue albumin after reperfusion in skin and skeletal muscle excised from the experimental extremity can be compared with tissue data from the contralateral control extremity. Accurate measurement of interstitial tracer requires correcting the total tissue albumin content for intravascular tracer content [6]. One limitation of using clearance techniques to judge permeability changes is that it is difficult to distinguish between an increase in permeability and an increase in transvascular protein movement due to higher intravascular hydrostatic pressures caused by reactive hyperemia [20].

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using the principle of electromagnetic induction, where the flow of blood through a magnetic field induces a measurable voltage which is directly proportional to flow [ 191. Flowmeters must be repeatedly calibrated [59] and are subject to error if the circumferentially placed probes are too tight, improperly positioned, or subject to motion during data collection. Flow rates may also be underestimated in small vessels because these vessels have a small internal-to-external diameter ratio, and vascular walls have a different conductivity for blood [24]. However, there are distinct advantages to this methodology, including continuous recording, multiple recordings from the use of multiple probes, and utility in acute and chronic experiments. Direct comparison of blood tlow data using electromagnetic flow probes and strain-gauge plethysmography shows that the two techniques can be correlated but that the flow probes may substantially underestimate flow [65]. Plethysmographic flow was 1.7 times greater during steady-state forearm exercise in humans, perhaps due to collateral blood flow around the flowmeter at the level of skin or bone. Although this seemsa large discrepancy, substantial data suggeststhat as much as 50% of resting extremity blood flow may not go to skeletal muscle [65, 87, 1241. The washout of inert radioactive tracer substances gently injected into tissues is extensively utilized as an estimate of perfusion [102]. The most widely utilized tracer method for assessmentof limb perfusion has been ‘33Xe [6 11,although similar data can be obtained using other freely diffusible (lipophilic) indicators such as “Kr or 13’1-antipyrine. These techniques are highly reproducible and have a distinct advantage that absolute flow values from a specific tissue can be obtained [6 11.In addition to injection into tissue, these highly soluble substances may be administered by inhalation. If injected into skeletal muscle directly, precise measurement of muscle blood flow may be made without interference from other tissues.Lassen et al. [ 6 1] utilized this technique to examine resting and maximal skeletal

muscle blood flow responses to ischemic work performed during tourniquet inflation to 250 Torr, and they found the coefficient of variation to be 13% for blood flow above 30 ml/min/ 100 g of muscle and 20% when flow was below the 30 ml level. As experience with the technique accumulates, it should be possible to achieve reproducibility within the 3 to 5% range [47]. Data collected by xenon133 clearance have also been directly compared with strain-gauge plethysmography [ 1131.The two techniques gave closely comparable measurements of calf blood flow in humans postexercise or after tourniquet ischemia, with a coefficient of variation of 13% between the two techniques [ 1131.While the reliability of xenon- 133 washout in measuring skin blood flow appears good, the technique has not been as consistent when skeletal muscle blood flow was measured [28,57]. In a study of the isolated perfused dog gastrocnemius, the xenon- 133 clearance method of blood flow measurement was found to more than 40% underestimate blood flow when compared to values measured as direct venous outflow or 15-pm microsphere trapping [lo]. Washout techniques which do not require radioisotopic methodology have also been described. Inhalation of molecular hydrogen in oxygen to saturate tissues with hydrogen can be safely accomplished [35]. Oxidation of molecular hydrogen at a platinum electrode generatescurrent proportional to tissue pH [4]. For highly diffusible and lipid-soluble gases such as hydrogen, it may be assumed that the tissue is in instantaneous diffusion equilibrium with venous blood through the entire saturation/desaturation period [52]. Although this assumption appears valid in isolated, perfused myocardium from various speciesas evidenced by a highly linear relationship with measured venous outflows, this assumption may require modification for application to skeletal muscle [4]. These investigators showed that hydrogen washout from isolated, perfused canine gracilis muscle, while linear, was substantially delayed in comparison to disappearance

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from venous blood. This discrepancy may have resulted from inhomogenous perfusion of the isolated muscle preparation. The technique has recently been used to assessviability of reimplanted extremities following traumatic amputation and seems to provide a reliable qualitative assessment of perfusion [35]. Quantitative use of this technique for skeletal muscle blood flow may require further validation, both in vivo and in isolated systems before the methodology can be reliably employed. Two types of instruments which depend upon the Doppler effect are available for blood flow determinations. The techniques differ widely in application despite sharing the underlying principle. One relies on reflected ultrasound to measure blood flow in large vessels[ 11,471, while the other relies on helium-neon laser light reflected from moving erythrocytes in capillaries to measure skin blood flow [ 1849, 791.Both techniques are increasingly employed, and both have methodologic shortcomings. While continuous-wave ultrasonic Doppler devices provide information regarding blood velocity, the pulsed ultrasonic probe uses two piezoelectric transducers so that flow can be calculated from the difference between upstream and downstream signal velocities. This feature makes pulsed ultrasonic velocimetry of particular value in studies of limb ischemia. Femoral artery blood flow has been measured in dogs comparing pulsed Doppler velocimetry with electromagnetic flow probes over a wide range of flows (5 to 300 cm3/min) [ 111. There was a positive, linear, highly significant correlation between the two techniques with overall mean blood flow differing by less than 2%. However, it must be noted that there was a large scattering in relative error about the mean for blood flow measured ultrasonically for in vivo studies in comparison to the error noted during in vitro calibration. This may have occurred because of oscillation in arterial diameter during the cardiac cycle, the interposition of skin and subcutaneous tissue between the vessel and the transducers, or

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imprecision in the electromagnetic flow data used for comparison [ 111. Laser-Doppler velocimetry uses coherent helium-neon light which is directed via a fiberoptic conduit onto an area of skin or exposed tissue. The light reflected from circulating erythrocytes in capillaries undergoes a frequency shift proportional to average redcell velocity [49]. An evaluation of skin blood flow was made by comparing the responses by xenon-133 washout and laserDoppler flowmeter during reactive hyperemia. These investigators and others have concluded that the laser-Doppler flowmeter seemedto measure flow in capillaries as well as in arteriovenous anastomosesbeneath the epidermis, while the xenon-133 method measured only capillary flow [28, 321. The potential advantages of this technique are numerous since the signal is continuous and has an excellent frequency response. However, the technique is limited to tissue depths of 1.5 mm, and skeletal muscle perfusion cannot be measured. The method does not measure actual blood flow per unit of tissue, rather it measures flow in relative terms. Measurements of blood cell velocity are most helpful when the investigator uses an intervention to change flow. For example, the laser-Doppler technique has been utilized to great advantage in assessmentof the reactive hyperemia response to postischemic reperfusion [ 18, 32, 79, 1191. It has recently been proposed that this technique may allow inferential assessmentof skeletal muscle perfusion by analysis of latency in the cutaneous reactive hyperemic response [ 181. LaserDoppler velocimetry has been compared with capacitance plethysmography in humans, which helps investigators put this technique in perspective [47]. In resting normal volunteers who had incremental skin blood flow changes induced by raising body temperature up to 39°C total forearm blood flow correlated well with laser-Doppler flow within each study, but there was wide variability among studies. There were also marked regional variations (up to 5.7-fold) in laser-Doppler blood flow within a single ex-

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tremity, probably related to capillary density in the skin being examined or less likely to regional variation in hematocrit. These data highlight the current difficulty in relying on laser-Doppler velocimetry for quantitation of blood flows. The variability among subjects makes it difficult to convert laser-derived data into conventional blood flow units. Another major problem is insecurity regarding the value of laser-Doppler flow when skin flow is zero during venous occlusion, Estimates of fractional increases (necessary because the technique is only semiquantitative) require accurate knowledge of instrument output at zero flow. Johnson et al. [49] have reported that laser-Doppler blood flow does not fall to zero during occlusive ischemia. Intrastudy comparisons or even region-to-region comparisons of the same individual must be interpreted with caution because of the widely disparate results. A new, noninvasive technique that provides data similar to laser-Doppler’s is dynamic capillaroscopy, in which papillary capillaries in the nailfold are directly observed. In addition to being able to study the dynamics of the skin microcirculation, the relative hematocrit in single skin capillaries can be measured [32]. Measurement of regional blood flow by the injection of radioactive microspheres is a highly reliable, well-described technique [7]. Flow rates will vary less than 20% from flow measured by other techniques [lo]. Coefficients of variation less than 5% can be accomplished with careful attention to theoretical considerations and proper technique. There are distinct advantages to the technique in addition to reliability and reproducibility. Flow may be measured to very small structures (for example canine papillary muscle) and repeated measures can be made by serially injecting spheres labeled with different radionuclides whose energy emissions can be separated by gamma spectroscopy. Disadvantages include the expense of the gamma counter and disposal of radioactive carcasses.The radioactivity renders the technique unsuitable for survival experiments

and requires special precautions when handling the tissues. The successof the method requires that the spheres be thoroughly mixed at the site of injection, that they be injected rapidly in a high flow location upstream (ideally into the left atrium) and distribute downstream in proportion to regional blood flow, that minimal transfer of spheres occurs to the venous circulation (failure of capillary entrapment, as with arteriovenous shunting), and that the injection should not perturb nutritive flow through extensive embolic occlusion of the microcirculation. The reference sample method is the most reliable microsphere technique for blood flow measurement [7]. In this technique a known quantity of arterial blood is withdrawn and assayed for radioactivity during cardiac injection of a well-mixed sample of microspheres. With blood radioactivity, and the measured radioactivity in the tissues, blood flow is measured without having to directly disturb the flow of blood to that organ. The reference sample technique requires that sufficient microspheres reach the target tissue so that radioactivity increases significantly above background. The dose of microspheres delivered to tissues depends upon the fraction of cardiac output it receives. Insufficient embolized microspheres in the counted tissue sample are the largest potential sources of error with this technique. The spheres must be injected into the left atrium or inadequate mixing will introduce error. A reference sample of adequate size must be obtained to ensure accurate counting. Spheres ranging in size from 8-50 pm are available, and sphere size seems to make little difference for measuring blood flow to most organs. Isotope decay does not pose a problem when the investigator accounts for the half-lives of the various isotopes. Weak radioactivity can be compensated for by using longer counting times for each sample or by injecting larger numbers of spheres. In detailed study of the microcirculation smaller spheres are advantageous [85, 981. Spheres 8- 15 pm in diameter have rheologic

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properties similar to erythrocytes. They occlude a smaller percentage of vascular surface area, so that more can be given without compromising perfusion, thereby increasing reliability of flow measurements to smaller regions. Because small microspheres are less variable in size than larger spheres, there is more even mixing and distribution. Use of small spheres increases the likelihood of transcapillary transit into the venous circulation, since maximally dilated capillaries may reach 10 pm in diameter, and such shunting must be quantitated in each system to be studied. Less than 1% of &pm microspheres injected into the coronary circulation pass into coronary venous drainage, but the values for muscle perfusion are more variable [ 10, 851. The partial pressure of oxygen in tissues measured transcutaneously (TcP02) is being increasingly utilized clinically and in the laboratory for assessmentof both normal and low flow states [71, 93, 1231. Measurement of TcP02 assumes that skin blood flow under a heated (42-44°C) oxygen sensor is sufficiently high with respect to cutaneous oxygen consumption and that local tissue oxygen tension is essentially equal to arterial POZ . Although this assumption appears reasonable when oxygenation is adequate and flow rates are normal [7 1,931, the validity of the assumption in low flow state requires examination. Wyss et al. [ 1231 examined the relationship of changing local arteriovenous pressure gradients on TcP02 measurements by progressive lower extremity elevations in normal subjects (leg elevation decreasesarteriovenous pressure difference because venous pressure cannot decrease below interstitial pressure) [66]. There was substantial nonlinearity in the TcPOz response to decreasing arteriovenous pressure differences, with TcP02 falling rapidly to zero below a pressure difference of about 25 Torr, indicating clearly that TcP02 does not reflect P,OZ under conditions of decreased blood flow. The effects of alterations in blood flow and changing P,Oz on TcP02 have been directly examined by Moosa et al. [71] in

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paired canine hindlimbs. TcP02 measured as femoral artery blood flow was decreasedat various inspired oxygen concentrations. Femoral artery blood flow was quantitated electromagnetically after collateral flow was abolished by ligation and tourniquet application. TcP02 again displayed substantial nonlinearity with reduced arterial inflow while the dog ventilated room air. TcP02 dropped precipitously when blood flow was reduced by 80%. In experiments where FiOz was increased during progressive arterial occlusion, TcP02 was dependent on Pa02 for flow reductions up to 50%. When flow was reduced 75% or more, TcP02 became a flow-dependent variable which was refractory to increasing Fi02 [7 11.Although a sensitive indicator of hypoperfusion, the lack of a linear response when blood flow is substantially reduced makes TcP02 of limited value for quantitation of extremity perfusion in low-flow states. The technique may have greater applicability as a sensitive indicator of ischemic damage, however, since severely ischemic muscle may not be able to increase oxygen consumption during reperfusion. This question has been indirectly addressed in a porcine hemorrhagic shock model (systolic blood pressure 50 Torr, mortality 45%) [93]. TcP02 fell dramatically to 10%of baseline levels during hemorrhage and began to recover during the reinfusion of shed blood in both survivors and nonsurvivors. In nonsurvivors, however, peak recovery of TcP02 was only 40% of baseline (at the end of reinfusion) and subsequently deteriorated again while progressing toward normal in surviving animals. Transcutaneous POZ measurements may thus be valuable to assessadequacy of resuscitation for sensitive detection of ischemic injury in a qualitative manner. EXPERIMENTAL PREPARATIONS LIMB ISCHEMIA

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Review of the extensive literature regarding the experimental production and study of the ischemic limb reveals many different approaches. Some of the variations are in-

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consequential to the interpretation of the results. However, critical differences in the available types of experimental preparations can impact on the validity of the comparative observations. It is therefore essential that the investigator precisely define the scientific question before selecting the type of ischemic extremity preparation to be employed. Essentially, the methods for the production of limb ischemia can be divided into four broad categories. Tourniquet ischemia, where both arterial inflow and venous outflow are completely interrupted by inflation of a proximal tourniquet to 200 to 500 Tot-r, has been well described and widely employed in both human and animal studies. Modifications of vascular or neurovascular isolation of anatomical muscles in situ have been increasingly employed. Arterial inflow occlusion in situ utilizing techniques of aortic occlusion with preservation of venous outflow from the ischemic extremity has been described. Studies of muscle microcirculatory dynamics can be carried out with highly specialized ex viva,isolated, perfused muscle systems. These four basic types of preparations are dissimilar and not equally applicable (Table 2). An example of each type of model will be described in detail to describe methodologic strengths and limitations. The literature is replete with descriptions of tourniquet ischemia. This experimental approach remains popular and is the subject of ongoing investigation [33, 97, 1011.Tourniquet ischemia has been studied in cats [45], dogs [30, 75, 971, rats [62, 101, 1061,rabbits [37, 961, various speciesof primates 134, 53, 70, 81,82, 1141,and humans [33,79, 1141.A tourniquet is placed on the proximal extremity and rapidly inflated from a high-pressure gas reservoir to assure complete arterial occlusion and to avoid problems with an increase in extremity volume produced by a transient period of venous engorgement during slow inflation [33]. Application of these techniques should achieve rapid and virtually complete interruption of arterial and venous flows. Tourniquet models have been criticized in the past because the tourniquet

does not interrupt blood flow into and out of the extremity via medullary channels of bone. The flow through bone however is minimal. Klenerman and Crawley [53] have shown in the limbs of monkeys subjected to a 300-TOIT tourniquet that arterial inflow is 1% of control levels as determined with 50-pm spheresand that venous return is only 0.2% of control values as determined by 22Na washout. Ischemic injury distal to an inflated tourniquet has been well described. Early changes of mitochondrial swelling have occasionally been observed within 30 min of occlusion [S 1, 82, 107, 1141,and progressive myofibrillar degeneration related to the duration of ischemia begins after 14 hr [97]. However, histological findings of these types are of questionable significance since there is consensus that tourniquet ischemia of 2-hr duration does not produce irreversible metabolic or histologic abnormalities [33, 971. Brief periods of reperfusion ( 10 min), before irreversible changes may occur (longer than 3 hr of ischemia), rapidly restore metabolic activity and can greatly prolong tissue tolerance to ischemia [97]. Significant irreversible anatomic and functional damage occurs after 4 hr of uninterrupted tourniquet ischemia, including failure of contractile elements, irreversible ischemic neuropathy, and disruption of local temperature autoregulation [ 1141.Six hours of continuous ischemia is necessary before widespread muscle necrosis occurs [97]. Morphological manifestations of the reperfusion injury generally peak within 24 hr [97]. Cellular autolysis after ischemia does not occur immediately, and a significant portion of delayed histopathology may represent progressive injury despite, or becauseof, reperfusion. Delayed physiologic muscle dysfunction has also been demonstrated in primates [Sl]. Although isometric tension development of lower extremity muscles returned to normal within a few minutes of reperfusion, a marked reduction in muscle tension existed after 24 hr of reperfusion, which occasionally persisted for 6 days. Substantial concern regarding tourniquetinduced extremity damage apart from isch-

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BARIE AND MULLINS: LIMB ISCHEMIA TABLE 2 COMPARISONOFANIMALMODELSOFLIMBISCHEMIA Type of preparation

Advantages

Disadvantages

Suitability

Tourniquet

Simple methodology very well characterized Intact fascial compartments

Limb completely excluded from systemic circulation Tissue damage from tourniquet itself, apart from &hernia Blood flow during &hernia difficult to quantitate Entire limb is rendered ischemic

Reactive hyperemia Studies of irreversible &hernia Tourniquet effect studies

In situ isolated muscle

Model focuseson muscle metabolism

Systemic factors cannot be studied if venous effluent is collected Fascial compartments opened during preparation Influence of muscle denervation is unpredictable

Reversible and irreversible ischemia

Surgically prepared contralateral limb can be used as control Autoperfused Blood flow can be precisely measured No crush artifact Partial muscle &hernia, aortic clamping, graded femoral artery occlusion

Collateral circulation intact Metabolic products of ischemia reach central circulation

Aortic clamping and declamping has independent hemodynamic consequenceswhich may influence results Contralateral limb unavailable as control (with cross-clamping)

Studies of partial &hernia or “lowflow” states

Ex vivo isolated muscle

Model focuseson integrity of skeletal muscle microvascular bed Model is extremely sensitive to subtle changes, allowing research to be designed with precision

Technically demanding methodology Shares other disadvantages of isolated preparations Requires heparin and papaverine if permeability studies are performed Pump-perfused

Ideal for studies of microvascular perfusion, and characteristics and quantitation of edema formation

emia-related mechanisms has been raised. There is clear evidence that myocytes directly beneath the tourniquet show greater damage than do distal cells. This compression injury is aggravated both by increasing occlusion pressures and by longer duration

of ischemia [8 1, 821,and must be considered by investigators using this technique. Direct compressive damage to peripheral nerves has been demonstrated [77, 1141. Delayed conduction across the nerve segment compressed has been shown, while nerve con-

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duction of distal ischemic nerves remained normal [34]. The greatest histopathologic changesin nerves were noted in the segments of nerve which were crushed beneath the edge of the tourniquet [77, 1141. The fact that tourniquets completely isolate the ischemic limb from the systemic circulation must be considered as well. While tourniquets appear well-suited for ongoing study of metabolic responses to complete ischemia, or for study of “reperfusion injury,” investigators who make exclusive use of tourniquet models may ignore evidence that significant ischemic damage may also occur in hypoperfused muscle [26, 891. Metabolic products of partial ischemia which gain accessto the systemic circulation may have adverse consequenceson the otherwise intact subject, including abnormalities of coagulation 1261 and prostanoid metabolism [63]. It must also be remembered that tourniquets render the entire limb ischemic, and not just skeletal muscle. Investigators interestedin the systemic responsesto reperfusion of ischemic skeletal muscle should consider that ischemic skin and subcutaneous tissue will also be reperfused with deflation of the tourniquet. Although these tissues are remarkably resistant to ischemia in comparison to skeletal muscle, blood flow to skin during the reactive hyperemic response may actually divert flow from skeletal muscle. A number of experimental preparations of lower extremity skeletal muscles have been described in which the muscle is perfused in situ but otherwise is isolated from surrounding structures. Most of these techniques use canine muscles, which are of sufficient size that the surgical preparation is easy and repeated sampling by sequential tissue biopsies can be performed. The canine gracilis muscle [22, 39, 58, 901 is easily accessible and contains both oxidative (65%) and glycolytic (35%) fibers [3, 581, and through extensive use it has become well characterized. Other canine muscle preparations have been described such as the isolated gastrocnemius/ plantaris preparation [ 1051.The best of these models allows for autologous perfusion via

careful control of blood flow through the ipsilateral femoral artery, so that the contralatera1leg may be used as a control. Rocko et al. [90] have described a gracilis muscle preparation in greyhounds in which the muscle was maintained only on a neurovascular pedicle consisting of gracilis artery, vein, and nerve. The investigators collected all venous effluent, allowing none to return to the systemic circulation, and offered as an advantage that their preparation was innervated and not heparinized. Although intravascular coagulation appears to be an important secondary phenomenon in the pathogenesis of limb ischemia and could certainly disrupt microvascular blood flow in ischemic muscle, the benefits which accrue by avoiding heparin in their models are difficult to quantitate. Other investigators have been unable to document that heparinization influences muscle metabolism [75 1. If metabolic studies are performed with this model it should be recognized that greyhounds have a higher muscle content of oxidative fibers [3]. These muscles had a 17% increase in weight after 3 hr of ischemia, which may have been due to coagulation-mediated changes in skeletal muscle microvascular permeability. Compartment syndromes due to edema formation may have had lesspathogenic influences in this model since during its preparation fascial planes are opened (a problem common with all isolated in situ preparations). The importance of intact muscle innervation is also not clear. Adrenergic control of autoregulatory mechanisms is clearly an important regulator of blood flow in normal extremities, but such may not be the case during ischemia [25]. A study in a canine “isolated thigh” preparation which was neurologically intact but donor perfused and heparinized following hip and knee d&articulation suggests that subsequent nerve sectioning had no effect on skeletal muscle blood flow [50]. An alternative technique for preparing the isolated in situ gracilis muscle has been described [39, 581, in which the muscle is further isolated by division and sutured approximation of tendons at the muscle origin and

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insertion to eliminate collateral flow, while maintaining resting muscle length. This preparation is unsuitable for contractility studies as a result of the tenotomies and the fact that the muscle is denervated. This preparation is also not heparinized and is hemodynamically and metabolically stable for control periods of 6 to 9 hr. Kuzon and colleagues emphasized that metabolic parameters can vary significantly among animals and concluded that an experimental muscle in one extremity should be compared with a control muscle obtained from the contralatera1extremity [58]. Using this model, a 2-hr ischemic injury was demonstrated to be reversible, and a 7-hr period of ischemia produced irreversible injury [39, 581. Recent data suggestthat significant skeletal muscle ischemia can be produced when arterial blood flow is reduced, but not completely stopped [2, 25, 26, 891. Furthermore, evidence supports the conclusion that such “low-flow” injuries may be more likely to exhibit prolonged abnormalities during recovery than muscle rendered completely ischemic by total arterial inflow occlusion for an equivalent period [2, 25, 891. Although partial ischemia studies can be performed using in situ isolated muscle preparations, these studies are typically performed using graded occlusion of the femoral artery in animals [89] or during aortic cross-clamping for vascular reconstruction in humans [2,25, 261.Studies of this type are of interest in view of this recent evidence that there may be important differences in the pathogenesis of skeletal muscle ischemia produced by lowflow or no-flow states. These models are attractive because collateral perfusion and venous drainage of the extremity are preserved. Studies quantitating the amount of extremity blood flow resulting from collateral perfusion are needed but generally have not been rigorously performed. Indirectly, aortic cross-clamping in humans may be expected to decrease femoral venous blood flow by 50 to 70% [25]. A great deal of variability can be expected becauseof differences in the level of occlusion. The extent of isch-

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emia after clamping the aorta is not uniform, with wide variability in the anatomy of pelvic collaterals among species [ 1011,and between patients there will be differences based upon the extent and duration of occlusive diseasepresent in collaterals. A series of patient studies performed during temporary infrarenal aortic occlusion for peripheral vascular reconstruction have been performed to determine the influence of partial ischemia on skeletal muscle metabolism [2, 25, 261. Delayed recovery of human skeletal muscle phosphocreatine stores following 90 min of aortic cross-clamping compared to tourniquet ischemia of the same duration has been observed [2, 261. Roberts et al. showed that resting transmembrane potentials in canine gracilis muscles remain abnormal during reperfusion after 3 hr of extremity perfusion at 50 Torr, in comparison to rapid normalization of transmembrane potentials following complete femoral artery occlusion in the contralateral limb [89]. Assessmentof the systemic effectsof skeletal muscle ischemia may be difficult when using a model of aortic clamping, since placement and removal of the clamp may have profound hemodynamic consequences independent of any effectson skeletal muscle [9,86]. These alterations may be due to acute changes in left ventricular afterload or to large blood volume changes because of sudden exclusion by clamping or sudden reperfusion (declamping) of large beds of vasodilated, ischemic, poorly autoregulated skin and skeletal muscle. The sudden delivery of large amounts of lactic acid to the central circulation as it washesout of ischemic skeletal muscle may also have a negative inotropic effect. Whatever the cause, these changes may be profound and thereby subtle changes of physiologic interest can be obscured. Ex vivo isolated muscle preparations are being described which, although demanding to prepare and maintain in stable states,have particular advantages for certain kinds of studies. Korthius et al. have described techniques for the preparation of isolated canine gracilis muscle [ 551and for preparation of an

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isolated rat hindquarter model [54]. These models are well suited for estimation of capillary perfusion pressures [54] and for studies of alteration in skeletal muscle microvascular permeability [55]. Permeability studies generally require that the perfusate be heparinized to eliminate intravascular coagulation as a mediator of permeability changes and also to eliminate the possibility that alterations in microvascular surface area caused by coagulation-related occlusion could be misinterpreted as permeability changes. The isolated perfused model is sensitive to alterations in microvascular surface area so that papaverine is added to the perfusate to completely abolish autoregulatory mechanisms and maximally vasodilate the skeletal muscle bed [22, 551. This model has been recently used to provide compelling evidence that cellular injury in ischemic skeletal muscle is caused by oxygen-derived free radicals [55]. The source of the free radicals appears to be the action of xanthine oxidase (localized in mitochondria and type II fibers of skeletal muscle [42]) on hypoxanthine produced as a product of ATP catabolism [60]. The importance of free radical-mediated injury has been corroborated in the in vivo isolated muscle preparation [39] by the high-performance liquid chromotographic identification of free fatty acid-conjugated diene products of lipid peroxidation in muscle biopsy specimens. The choice of animal model to be employed may also be influenced by considerations of speciesvariability. Canine preparations are widely employed because the large muscle size allows for repeated biopsy sampling for metabolites and because the metabolic responses of ischemic human and canine muscles are very similar [29]. In contrast, although the metabolic effects of ischemia on rat skeletal muscle have been well studied [75], conclusions may be difficult to draw since the muscle of small mammals is more sensitive to ischemia than is human muscle. For identical periods of ischemia, rat skeletal muscle exhibits a more rapid and severemetabolic deterioration and

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a slower recovery from tourniquet ischemia than has been observed for human or canine muscle [75, 971. Although newer techniques for assessmentof muscle metabolism such as 3’P NMR will make smaller muscles entirely suitable for metabolic studies [75], other methodologic difficulties with rat preparations remain. Seifert et al. [ 1011 point out that femoral artery ligation does not produce significant hindlimb ischemia. Abundant collateral vesselsexist because the profunda femoris artery is absent in the rat. Iliac artery ligation also does not produce ischemia at rest although a 70% flow reduction does occur in the exercising rat. Aortic ligation produces ischemic lesions only in male rats. Seifert et al. [ 1011have described a complex two-stage arterial ligation model suitable for ischemic studies in the rat which has been quantified with xenon- 133 washout studies, but the complex and time consuming preparation has obvious disadvantages. Honig and colleagues [41] have suggestedthere may be important differences in autoregulation of skeletal muscle blood flow between dog and rat as well. Although this review concentrates on methodology for acute ischemia studies, it must be noted that animal models for chronic ischemia have been developed. A canine model of chronic ischemia has been described which requires as many as 14 separate ligations of branches of the iliac and common femoral arteries and ligation of the profunda artery at its origin [ 1081. After a 6-week recovery period, the animals have no pain at rest but claudicate after running less than 50 m. The distal-to-central systolic blood pressure index was 0.60 + 0.02, while resting blood flow approximated normal at 4.2 t- 0.6 ml/min/lOO g. As would be expected, the reactive hyperemic response is impaired. An alternative approach to evaluating the microcirculation during ischemia and after reperfusion has been to directly observe in vivo the arterioles, capillaries, and venules using microscopic preparations. With these techniques isolated tissues such as the rat cremaster or the rat gracilis muscle or the

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hamster cheek pouch are dissected out with a neurovascular pedicle intact [3 1, 64, 1111. The thin tissues are transilluminated and the changes in diameter of arterioles, capillaries and venules are observed. Also amenable to measurement are vascular pressures,red cell velocities, and perivascular oxygen tensions. Becausethe tissues are superfused in a sealed chamber, the surrounding environment can be manipulated. Anesthetic agents can significantly influence the response of microvasculature to hypoxia and hypotension. A decerebrate rat model has enabled investigators to avoid the confounding effectsof anesthetics [3 I]. Evidence of an increase in permeability can be readily observed when fluorescein labeled dextrans are injected intravenously, and sites and extent of extravasation from the microvessels can be observed [ 1IO]. An advantage of this method is that the exact site of permeability change in the microcirculation can be identified, and the time frame of transient changes can be established. The relatively qualitative technique of microscopy has been recently extended to a quantitative technique [68]. The clearance of fluorescein-labeled dextran from the vascular compartment into the extravascular space has been measured by determining after fluorescein-labeled dextran has been intravenously injected the rate and extent of its appearance into the suffusate of a cremaster preparation [ 1093. REFERENCES 1. Abrams, H. L. The collateral circulation: Response to ischemia. AJR 140: 105 1, 1983. 2. Andersson, J., Eklof, B., Neglen, P., and Thomson, D. Metabolic changesin blood and skeletal muscle in reconstructive aortic surgery. Ann. Surg. 189: 283, 1979. 3. Armstrong, R. B., Saubert, C. W., Seeherman, H. J., and Taylor, C. R. Distribution of fiber types in locomotory muscles of dogs.Amer. J. Anat. 163: 87, 1982. 4. Aukland, K., Bower, B. F., and Berliner, R. W. Measurement of local blood flow with hydrogen gas. Circ. Rex 14: 164, 1964. 5. Beiser, G. D., Zelis, R., Epstein, S. E., Mason, D. T., and Braunwald, E. The role of skin and muscle resistance vessels in reflexes mediated by

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