Transvascular protein movement in the intact ischemic hindlimb

JOURNAL

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Transvascular

RESEARCH

43, 351-359 (1987)

Protein Movement

in the intact lschemic Hindlimb’*

P. E. BURKE, FRCS(I), C. F. HARVEY, FRCS(ED.), L. M. MURPHY, M.S., C. A. GERVIN, PH.D., AND L. J. GREENFIELD, M.D. Department of Surgery. Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0001 Submitted for publication November 6, 1986 Postischemic limb swelling following reperhusion may be related to microvascular changes associated with &hernia. We used lymph-to-plasma total protein concentration ratios (L/P) and lymph flow (Qr) as an index of transvascular exchange in the intact dog hindlimb during steady state (C) (1 hr), &hernia (I) (6 hr), and reperfusion (R) (3 hr). Central pressures, femoral arterial and venous pressures (PA, Pv), and Qr were recorded every 15 min. Lymph was collected from a femoral lymphatic in the passively flexed leg (50 cycles/min). Three groups of animals were studied GI, sham-operated (Iv = 5); GII, moderate &hernia (N = 7, PA = 30-45% C); and GIII, severe &hernia (N = 7, PA = 5-204 C). In GI, Qr gradually increased over 10 hr without change in L/P. Moderate &hernia produced a decrease in Qr, 3.55 ? 2.02 mg/hr to 0.92 + 0.53 mg/hr (P < O.OOOl), and Qr remained below baseline during R with no change in L/P over the 10 hr. Severe ischemia produced a similar decrease in QL, 1.9 1 + 2.05 mg/hr to 0.15 + 0.1 mg/hr (P < 0.01); however, an increase to 2.56 + 2.14 mg/hr occurred during R. Severe ischemia increased L/PO.42 f 0.08 to 0.64 + 0.23 (P < 0.001) and remained elevated during R at 0.63 + 0.18 (P < 0.001). An increase in the wet-to-dry weight ratio of ischemic to nonischemic muscle after reperfusion was noted only in GIII, 3.82 + 1.17 vs 2.60 f 0.45 (P < 0.04). Severe &hernia produces changes in vascular integrity which augment protein flow. Prevention of these vascular changes may help to minimize the muscle swelling of reperfmion. Q 1987 Academic Press, Inc.

therefore, in the development of both microvascular permeability and postischemic muscle edema is not clearly defined. The concentration of protein in lymph relative to plasma is determined by the rate of protein flow across the capillary and by the degree of permeability to protein of the particular capillary. The most accurate measurement of capillary permeability is the osmotic reflection coefficient which distinguishes between transvascular movement due to increased capillary permeability and that due to increased capillary filtration [5]. Granger and Taylor have shown that lymph-toplasma protein concentration ratios (L/P) that are not changed by increasing lymph flow may be used to express osmotic reflection coefficients [6]. The L/P is then said to be “filtration independent,” implying that the maximal volume of protein transferred by increasing capillary filtration alone has been reached and any subsequent increase in L/P can then be attributed to an alteration in

INTRODUCTION

Recent studies have confirmed that reperfusion after 4 hr of &hernia induced by occluding arterial inflow will produce significant increases in capillary permeability in an isolated canine hindlimb model [l]. However, demonstration of capillary permeability and postischemic muscle swelling is more difficult in the intact hindlimb [2]. Five and one-half hours of tourniquet ischemia followed by 2 hr of reperfusion in the rat hindlimb increased muscle water content by only 10% with little evidence of vascular leakage [3]. The tourniquet technique also produces venous stasis which may persist during reperfusion and thus contribute to limb edema in this model [4]. The role of ischemia alone, ’ Presented at the Annual Meeting of the Association for Academic Surgery, Washington, D.C., November 5-8, 1986. * This work was supported in part by NIH-NHLBI Grant HL 326 19. 351

0022-4804187 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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permeability. In our intact hindlimb preparation it was not possible to produce a “filtration-independent” state without causing significant venous obstruction, which obviously was not desirable. The analysis of changes in L/P, lymph flow, and limb arterial and venous pressure change is, we believe, a good index of microvascular permeability. If an increase in L/P occurs while the indices of filtration pressure are normal or low, then it can be discerned that a change in permeability has occurred. In an intact forelimb preparation, Miller et al. showed a small transient rise in L/P levels upon reperfusion after 2 hr of ischemia [2]. After 4 hr of tourniquet ischemia which produced muscle damage histologically, Courtice showed that reperfusion was associated with an increase in L/P [7]. It is not clear from these and other studies [ 1, 81, however, whether changes in capillary permeability are a direct result of the ischemic period or are related to the restoration of blood flow [l]. Also, a relationship between postischemit muscle swelling and increased transvascular protein movement due to altered capillary permeability has not been shown. The objectives of our study were (1) to develop an intact, nontourniquet model of limb ischemia for measurement of transvascular protein exchange; (2) to determine the effects of acute arterial occlusion and reperfusion on transvascular fluid and protein flux; and (3) to determine its possible role in postischemic limb swelling. METHODS

Mongrel dogs of either sex (20-25 kg) were anesthetized with sodium pentobarbital (30 mg/kg) supplemented as required. The animals were intubated with a cuffed endotracheal tube and allowed to breathe room air. Dogs were ventilated (Edco Scientific, NC) if their arterial oxygen tension fell below 80 Torr. Body temperature was maintained at 39°C with a thermal heat blanket. Fluid loss was replaced by l-2 liters of saline (0.9%) over the 12-14 hr of the study. All animal experiments were approved by the Animal

1987

Care Committee of the Medical College of Virginia and were in accordance with the Principles of Laboratory Animal Care and the Guide for the Use of Laboratory Animals (NIH Publication No. 80-23, revised 1978). Surgical Preparation The right hindlimb was used as the experimental limb in all studies. The infrarenal aorta, the iliac, and the femoral artery were exposed through a long retroperitoneal incision, taking care to preserve all adjacent lymphatics. In order to direct all blood flow in the right hindlimb through the iliofemoral segment, all infrarenal branches of the aorta, excluding both external iliac arteries, and all branches of the right external iliac and femoral artery to the level of the caudal femoral branch were ligated. The distal iliac vessels were cannulated via a large medial side branch (the deep femoral artery and vein) and the venous cannula was directed down the femoral vein to midthigh level. The common carotid artery and the internal jugular vein were cannulated for the purpose of monitoring central pressures and obtaining blood samples. All pressures were recorded using Statham pressure transducers connected to a Gould recorder. All cannulas were kept patent with nonheparinized saline infused under high pressure using a Sorenson valve infusion system as the animals received no heparin. Iliac artery blood flow was determined using an electromagnetic flow probe (Narco Bio-systems Inc., TX). Central and hindlimb arterial and venous blood were sampled for blood gas determinations (Coming, MA, Model 158), hematocrits, and plasma protein concentrations. All hemodynamic parameters were recorded at 15-min intervals, and blood gas determinations were made every hour. Femoral arterial blood was sampled every 15 min for total protein concentration and hematocrits. Muscle needle biopsies of both hindlimbs were taken at 3 and 6 hr of ischemia and at 3 h of reperfusion for pathological evaluation. Larger sections of muscle (1 cm2) were also taken at the end of reperfusion,

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weighed immediately, stored in an oven at 40°C for 1 week, and reweighed. Wet-to-dry muscle weight ratios were determined. Lymphatic

Cannulation

According to Pflug and Calnan [9], the large lymphatic vessels between the femoral artery and vein represent prenodal femoral lymphatics of the deep medial system. These lymphatic vessels principally drain the muscle and deep structures from the thigh, calf, and paw and do not communicate with the superficial system unless either is obstructed. We confirmed this lymphatic drainage in early studies. Injection of Evans blue dye into the muscle compartments of the hindlimb caused staining of the deep medial lymphatics; whereas, no dye appeared in these channels after subcutaneous injection alone. All vessels entering and exiting from the popliteal node were ligated to reduce the theoretical possibility of skin drainage to the deep medial lymphatics, as both superficial and deep lymphatics were ligated in the iliofemoral region. Two to three lymph trunks in a discrete sheath between the femoral vessels were isolated and ligated separately, and one of these was cannulated with a 15-mm length of a modified 22 G Jelco iv catheter (Critikon, FL) connected to Silastic tubing (0.025 in, in internal diameter, 0.047 in. in external diameter). Lymph flow was maintained by passive dorsiflexion of the hindpaw using a pulley system (50 cycles/min). Lymph was allowed to flow for 30 min prior to baseline collections. Lymph flow was recorded at 15-min intervals unless the volume was less than 0.25 ml when 30- and 60-min intervals were used. The lymph flow was measured as weight change over time (mg/hr) in preweighed, graduated vials containing 20 ~1 of sodium citrate (3.8%). Experimental

Protocol

All animals were studied during 1 hr of steady state, 6 hr of ischemia, and 3 hr of reperfusion. The animals were divided into three groups.

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353

Group I (n = 5) controls. Sham-operated animals in which lymph was collected over 10 hr without alterations in femoral pressure. Group II (n = 7) moderate &hernia. Complete occlusion of the infrarenal aorta and both external iliac arteries reduced femoral artery pressure to 50-35% of steady-state levels. Maintenance of this pressure by right external iliac occlusion was sufficient in six of the seven animals. Group III (n = 7) severe &hernia. The femoral artery pressure was reduced to 5-20% of steady-state levels by occlusion of the right external iliac artery. Additional aortic and left external iliac artery occlusions were required in four of the seven animals. Blood and Lymph Analysis Blood samples were centrifuged at 3000 rpm for 10 min. at -4°C and the plasma was stored at -70°C. Lymph was centrifuged before storage only if red cells appeared to be present. Total protein concentration was determined by Bradford’s method [lo]. Lymph protein concentrations were mathematically corrected according to the volume ratio of lymph-to-citrate that was present, and the corrected values were consistent with dilution curves for hindlimb lymph in citrate determined in our laboratory. The osmotic pressures of plasma and lymph were determined on the basis of their total protein concentrations using Navar’s formula [ 1 I], which is a modification of that originally described by Pappenheimer and SotoRivera [ 121. Statistics Data are expressed as means and standard deviations from the mean for each hour for each group. Comparisons were made between steady-state (Hour 1) and subsequent hours in each group and between similar hours in the different groups. The analysis of variance repeated-measures model with Bonferroni’s correction was used to compare all data recorded more than once each hour [ 131. The Student t test was used when appropriate.

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Figures 2, 3, and 4 demonstrate the changes in femoral arterial and venous pressures, lymph flows, and lymph-to-plasma protein ratios in the three groups. Baseline femoral artery pressure was significantly lower in GII as compared to the other groups (P < 0.001). Lymph-to-plasma total protein ratios were significantly lower in the control group (P < 0.00 1) despite an identical preparation in all animals. Steady-state values of limb venous pressure and lymph flow were not significantly different, although lymph 0 1 2 3 4 5 6 7 8 9 10 TIME (hours) flow was high in GII because of one dog that had a baseline lymph flow rate of 7 mg/hr. In FIG. 1. A comparison of pH levels (upper panel) and Group I (Fig. 2), the L/P remained unvenous oxygen tensions (lower panel) in the three groups, control (GI), moderate &hernia (GII), and se- changed, despite a 50% increase in lymph vere &hernia (GIII) over 10 hr. flow and a gradual decrease in femoral artery and venous pressures over the 10 hr. Lymph flow fell during arterial occlusion in GII and RESULTS gradually returned to baseline during reperCentral arterial pressure (CAP) was ele- fusion while the L/P remained unchanged vated during the ischemic period in GIII (Fig. 3). Femoral artery pressure remained compared to GI and GII, respectively, 175 below baseline during reperfusion while limb +- 12 mm Hg to 143 + 19 mm Hg, P < 0.01 venous pressure, which decreased during (GI) to 137 + 17 mm Hg, P -c 0.001 (GII). ischemia, returned to baseline. In contrast to This was a result of cross-clamping the dog’s GII animals, the lymph flow decreased duraorta in four of the seven animals. There was ing arterial occlusion in GIII (Fig. 4) and exa gradual fall in CAP in controls over the 10 ceeded baseline by the second hour of reperhr with a similar fall in the ischemic groups fusion. Lymph-to-plasma protein ratios inupon reperfusion. Central venous pressure creased gradually during severe ischemia, (CVP) and arterial blood gases did not were significantly elevated by the fifth hour change significantly between groups over the (Hour 6), and remained elevated throughout reperfusion despite the return of lymph flow. 10 hr. Femoral venous pH (Fig. 1) dropped significantly in both ischemic groups during Femoral arterial pressure decreased to less arterial occlusion with a greater decrease, than 20 mm Hg during arterial occlusion in 7.38 + 0.07 to 7.16 f 0.22 (P < 0.05) occur- GIII with no change in venous pressure. ring in GIII over the 6 hr of ischemia. FemoHowever, during the first 2 hr of reperfusion, ral venous oxygen tension (Fig. 1) decreased limb venous pressure was significantly elesignificantly over 6 hr of ischemia in GII and vated above baseline. Figure 5 shows that GIII and returned to baseline levels upon re- iliac artery blood flow, like limb venous pressure, did not exceed baseline in GII upon perfusion. Table 1 summarizes the protein changes. reperfusion; however, after severe ischemia a significant reactive hyperemia occurred in Plasma protein concentration decreased gradually in all three groups, despite no association with an elevation in limb venous changes in hematocrit levels. The increase in pressure. Serial needle biopsies showed no changes lymph protein concentrations in GIII contributed to the 40% decrease in the oncotic on electron microscopy during ischemia or pressure gradient by the fifth hour of severe reperfusion in GI or GII. Myocytolysis was ischemia in contrast to a 16% decrease in GI evident only in two animals of GIII after 6 hr and a 9% decrease in GII. of ischemia with no further changes seen 7.5

ikchemia

,

repelfus10n

I

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355

LIMB

TABLE 1 SUMMARY Group Hour I 4 6 8 10

5.0 4.6 4.2 4.2 4.1

+ k f + +

I

0.4 0.4; 0.4** 0.5** 0.5t

1.6 1.4 1.2 I .3 1.2

DATA

Group A*

cl.

G

OF PROTEIN

IT 0.1 k 0.3* f 0.2t f 0.28. + 0.2t

10.4 9.1 8.2 8.1 7.9

f k k k +

CP 1.3# 1.0 1.4** 1.6** 1x**

4.1 3.9 3.7 3.8 3.6

f k k k k

Group

II A*

CL

0.7 0.6 0.6* 0.7* 0.5t

1.8 1.5 1.4 1.4 1.3

+ 0.6 * 0.5 f 0.6 Lk 0.5 + 0.6

6.7 6.6 6.1 6.3 5.7

k * + + 2

G 1.5 1.4 1.4 I.6 1.0

Nofe. C,, plasma total protein concentration (gjdl). CL, lymph total protein concentration (mm W. # P < 0.00 I Group I vs Group II. * P < 0.0 I, **P < 0.00 1, tP < 0.000 1; experimental compared to baseline.

upon reperfusion. Only wet-to-dry muscle weight ratios showed a significant increases in muscle fluid contents in the severe ischemia group, increasing from 2.60 + 0.65 in the control limb to 3.82 f 1.17 in the ischemit limb (P < 0.04) (Fig. 6).

Much information has been learned from isolated limb models about microvascular hemodynamics, interstitial pressure and fluid content, and microvascular permeabil-

b-w/h0

(mm43 F.A.P.

6. 5432

T

+ f + k +

CL 0.6 0.01 1.2: 0.8 0.6;

1.9 2.0 2.3 2.4 2.1

k 0.5 * 0.7 + 0.8 3~ 0.9 f 0.4

7.9 6.0 4.8 5.1 5.3

(g/dl). AT, oncotic pressure gradient

T -p
~~~~

,xig,

0

1

2

3

4

k 1.9 + 5.0 + 4.0’ k 3.0* IL 2.9*

ity during different physiological and pathological states [5, 6, 8, 121. However, studies of the effects of ischemia on microvascular permeability in isolated hindlimb and skeletal muscle models are limited in being able to provide information only during the reperfusion period [ 1, 81. Sparks has also noted other disadvantages in using a vascularly isolated, denervated organ system to determine microvascular exchange [ 141, including alteration in the state of hydration of the organ during its surgical preparation, production of some tissue ischemia and injury, and pro-

DISCUSSION

QL

4.6 4.2 3.9 4.1 4.0

III

5

6

7

6

9

10

TIME (hours)

FIG. 2. Changes in control animals (GI) over 10 hr in femoral artery pressure pressure (FVP), lymph flow (QL), and lymph-to-plasma total protein ratios (L/p).

(FAP),

femoral

venous

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steady LYMPH PLASMA PROTEIN RATIO

F.A.P. (mmh3

F.V.P. (mm%)

o!

0

12

, 3

4

5

6

7

6

9

10

I TIME (hours)

FIG. 3. Changes during moderate &hernia (GII) and reperfusion in femoral artery pressure (FAP), femoral venous pressure (FVP), lymph tlow (Qr), and lymph-to-plasma total protein ratios (L/P).

duction of microvascular pressure values higher than those that occur under normal in vivo conditions. Microvascular exchange of fluids and proteins in the completely intact hindlimb has not been extensively studied because of the difficulty in measuring capillary and interstitial pressure and accurately estimating changes in capillary filtration [5]. Nevertheless, any data derived from a nontourniquet

,,.

steady state t

ischemia

intact ischemic limb model may be more easily applied to acute arterial occlusion in the clinical setting because of the similarity in the pathological events induced. The development of an intact ischemic hindlimb model in the dog without the use of a tourniquet is more difficult because of the animal’s extensive collateral circulation [ 151. Nevertheless, changes in lymph protein flux and microvascular perfusion induced by a

,

GROUP

reperiusion

III

F.A.P. (mmW

0123456769

10

TIME (hours)

4. Changes during severe &hernia (GIII) and reperfusion in femoral artery pressure (FA ,P), femoral venous pressure (FVP), lymph flow (Qr), and lymph-to-plasma total protein ratios (L/P). FIG.

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partial reduction in antegrade flow should not differ from changes produced by arterial occlusion and retrograde collateral flow as observed in the “moderate ischemia” group in our study. The range of L/P in our study is consistent with values found by others cannulating lymphatics that drain the deep structures of the limb. Linden et al. reported L/P values of 0.22 to 0.29 [16]. Jacobsson and Kjellmer reported values of 0.47 to 0.49 and claimed that no difference existed between skin and muscle lymph [ 171. Paw lymph is associated with a L/P lower than that of femoral lymph [ 181 and with a higher lymph flow rate [ 171. Thus, the lower L/P in the control group in our study probably represents a larger paw contribution in this lymph, which is still draining from the deep structures of the hindlimb as described by Pflug and Calnan [9]. It is unlikely, therefore, that the pattern of lymph flow and L/P seen over the 10 hr in the control group would have differed greatly had the protein ratio been higher. Much of the literature on capillary permeability and skeletal muscle ischemia describes changes in permeability in the reperfusion period which are assumed to have resulted, in part, from the ischemic insult [ 1,2, 7, 81, yet there appears to be little information on the changes in L/P and lymph flow during the ischemic interval itself in the intact nontourniquet model of limb ischemia.

ILIAC ARTERIAL FLOW (ml/min)

l *

p
\ ~P<0.0001

vs hr 1

Oi’,‘~‘l”‘,‘,‘I,“,,i 0 1 2 3 4

5 6 TIME (hours)

7

6

9

10

FIG. 5. Comparison of iliac artery blood flow during steady state and reperfusion in three groups: control (GI), moderate ischemia (GII), and severe ischemia (GIII).

IN THE ISCHEMIC

WET TO DRY MUSCLE WEIGHT RATIO

4

‘p<0.04

357

LIMB

t

“S control

3 2 1

ii

0 control

limb

ischemic

limb

FIG. 6. Histogram showing weight ratios of wet and dry muscle in control and ischemic limbs in the three groups: control (GI), moderate ischemia (GII), and severe &hernia (GIII).

In the clinical situation, ischemia of the lower limb followed by reperfusion may lead to compartment syndrome [ 193 or postoperative leg edema after peripheral vascular reconstruction procedures which may be associated with graft occlusion due to high intracompartmental pressures [20, 211. Use of tourniquet models of limb ischemia to study the formation of limb and muscle edema is unsatisfactory. The associated venous stasis may prevent accurate determination of the degree to which ischemia contributes to the problem [3, 4, 141. We produced a relatively small, although significant increase in muscle water content after 6 hr of ischemia and 3 hr of reperfusion and detected little evidence of interstitial edema on microscopy. Other authors have demonstrated edematous changes after 90 min of ischemia and 60 min of reperfusion in a similar type of ischemic hindlimb model [22]. The edema observed in our study may have been reabsorbed by the third hour of reperfusion as maximal edema is suggested to occur within 90 min of reperfusion [3, 221. The rise in lymph flow in the control group may be the result of increased capillary filtration in the passively flexed limb. Net filtration pressure when low will reduce lymph flow because of Starling’s forces [23] and this is illustrated during the ischemic periods in these studies. Similarly, the relatively rapid increase in lymph flow upon reperfusion after a severely ischemic interval (GIII) in contrast to the slow rise after moderate

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&hernia (GII) probably resulted from a tourniquet ischemia caused an increase in higher microvascular pressure. The high transvascular protein movement which is limb venous pressure and reactive hyperemia maintained during reperfusion; and (3) the seem to support this. The decreasing oncotic alterations in capillary integrity resulting pressure gradient during severe ischemia from the ischemia along with the elevated would also contribute to a high filtration limb venous pressure and arterial flow durrate. ing early reperfusion are associated with a The high lymph protein concentration significant increase in interstitial fluid conduring late ischemia could represent local tent during reperfusion despite a functioning tissue breakdown due to prolonged &hernia lymphatic system. The alterations in micro[24] or, as we believe, a movement of protein vascular integrity due to ischemia may be a from the intravascular to the extravascular significant determinant of postischemic limb space as a result of a change in microvascular swelling. This may have clinical application permeability. The persistent elevation of L/P in the protection of the microvasculature in GIII animals after restoring lymph flow during ischemia. indicates that a transvascular flow of protein is occurring, reflecting increased permeabiiREFERENCES ity in the microvasculature during both isch1 Kotthuis, R. J., Granger, D. N., Townsley, M. I., emia and reperfusion. Further studies will be and Taylor, A. E. The role of oxygen-derived free necessary to determine whether the changes radicals in ischemia induced increases in canine skeletal muscle vascular permeability. Circ. Rex 57: induced are reversible and to determine the 599, 1985. relative contributions of increased microvas2. Miller, G. L., Kline, R. L., Scott, J. B., Haddy, F. J., cular pressure and microvascular permeabiland Grega, G. J. Effects of ischemia on forelimb ity to the development of postischemic weight and lymph protein concentration. Proc. Sot. edema. Recent clinical work suggests that Exp. Biol. Med. 149: 581, 1975. 3. Strock, P. E., and Majno, G. Vascular response to local vasoregulatory mechanisms may preexperimental tourniquet ischemia. Surg. Gynecol. vent elevated pressure levels in the capillaries Obstet. 129: 309, 1969. during reactive hyperemia 120, 251. Our 4. Pochin, E. E. Edema following ischemia in the rabstudies suggest that changes in capillary perbit’s ear. C/in. Sci. 4: 341, 1942. meability may be playing a more significant 5. Taylor, A. E. Capillary fluid filtration Starling forces and lymph flow. Circ. Rex 49(3): 557, 1981. role than was previously thought in the pro6. Granger, D. N., and Taylor, A. E. Permeability of duction of interstitial filtrate during reperfuintestinal capillaries to endogeneous macromolesion. cules. Amer. J. Physiol. 238: H457, 1980. Edema was not evident in the control I. Courtice, F. C., Adams, E. P., and Dempsey, J. The effect of ischemia on acid phosphatase, fi-glucuronigroup despite 10 hr of passive exercise, sugdase, and lactic acid dehydrogenase in lymph from gesting that the intact lymphatic system rehind paw of the rabbit. Lymphology 5: 67, 1972. moved excess capillary filtrate produced by 8. Diana, J. N., and Laughlin, H. Effect of ischemia on increasing capillary surface area. In the secapillary pressure and equivalent pore radius in capvere ischemic group, however, the combinaillaries of the isolated dog hindlimb. Circ. Res. 35: 71, 1974. tion of increased microvascular permeability 9. Pflug, J. J., and Calnan, J. S. Lymphatics: Normal and increased filtration pressure may have anatomy in the dog hind limb. J. Anat. 105(3): 457, produced a volume of capillary filtrate which 1969. exceeded the drainage capacity of the lym10. Bradford, M. M. A rapid and sensitive method for phatic system and permitted significant fluid the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. accumulation to occur. Biochem. 72: 268, 1976. From our studies the following are sugNavar, P. D., and Navar, L. G. Relationship beII. gested: (1) an intact hindlimb model of ischtween colloid osmotic pressure and plasma protein emia can be used to determine changes in concentration in the dog. Amer. J. Physiol. 233(2): transvascular fluid and protein movement H295, 1977. using lymph flux protein analysis; (2) non12. Pappenheimer, J. R., and Soto-Rivera, A. Effective

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osmotic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimb of cats and dogs. Amer. J. Physiol. 152(3): 471, 1948. 13. Steel, R. G. D., and Torrie, J. M. Principles and Procedures

14.

15.

16. 17. 18.

of Statistics.

A Biometrical

Microvasc.

20.

Res. 14: 243, 1977.

19. Rollins, D. L., Bernhard, V. M., and Towne, J. B.

21.

22.

23. 24.

25.

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359

Fasciotomy: An appraisal of controversial issues. Surg. 116: 1474, 1981. Eickholf, J. H., and Engell, H. C. Local regulation of blood flow and the occurrence of edema after arterial reconstruction of the lower limbs. Ann. Surg. 195(4): 474, 1982. Qvarfordt, P., Christenson, J. T., Eklof, B., and Ohlin, P. Intramuscular pressure after revascularization of the popliteal artery in severe &hernia. Brit. J. Surg. 70: 539, 1983. Buchbinder, D., Karmody, A. M., Leather, R. P., and Shah, D. M. Hypertonic mannitol. Its use in the prevention of revascularization syndrome after acute arterial ischemia. Arch. Surg. 116(4): 414, 1981. Starling, E. H. On the absorption of fluids from the connective tissue spaces. J. Physiol. (London) 19: 312, 1896. Eklof, B., Neglen, P., and Thomson, D. Temporary incomplete &hernia of the legs induced by aortic clamping in man. Ann. Surg. 193( 1): 89, 198 1. Eichoff, J. H. Normalization of local blood flow regulation in the ischemic forefoot after arterial reconstruction. Surgery 97( 1): 72, 1985. Arch.

Approach,

2nd ed. New York: McGraw-Hill, 1980. Sparks, H. V., Korthuis, R. J., and Scott, J. B. Pharmacology of hemodynamic factors in fluid balance. In N. C. Staub and A. E. Taylor, (Eds.), Edema, pp. 425-439. New York: Raven Press, 1984. Brooks, B. Pathological changes in muscle as a result of disturbances of circulation: An experimental study of Volkmann’s ischemic paralysis. Arch. Surg. 5: 188, 1922. Linden, J., Kupper, W., and Trautschold, I. Enzymatic composition of canine leg lymph. Enzyme 28: 18, 1982. Jacobsson, S., and Kjellmer, I. Flow and protein content of lymph in resting and exercising skeletal muscle. Acta Physiol. &and. 60: 278, 1964. Brace, R. A., Taylor, A. E., and Guyton, A. C. Time course of lymph protein concentration in the dog.

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