Ischemia-reperfusion WILLIAM
L. SEXTON,
Department
of Veterinary
Columbia, Louisiana
injury RONALD
in isolated rat hindquarters J. KORTHUIS,
AND
M. HAROLD
Biomedical Sciences and Dalton Research Center, Missouri 65211; and Department of Physiology and Biophysics, State University Medical Center, Shreveport, Louisiana 71130
SEXTW, WILLIAM L., RONALD J. KORTHUIS, AND M. HAROLD LAUGHLIN. Ischemia-reperfusion injury in isolated rut hindquarters. J. Appl. Physiol. 68(1): 387-392, 1990.-The purpose of this study was to determine the suitability of the maximally vasodilated (papaverine) isolated rat hindquarters preparation to study the effects of ischemiaand reperfusion on the microvasculatureof skeletal muscle.The osmotic reflection coefficient for plasmaproteins (a) and total vascular resistance ( RT, mmHg*ml-l ‘min +100 g-l) were determined before ischemicperiods of 30, 60, 120, 180,and 240 min in intact (with skin) and 30, 60, and 120 min in skinned hindquarters and again after 60 min of reperfusion. In both intact and skinned hindquarters, reductionsin c and increasesin RT wereobserved during reperfusionand werecorrelated with the ischemicperiod duration. After 120 min of ischemia in intact and skinned hindquarters, u was reducedfrom preischemiavalues of 0.92 t 0.02 and 0.89+ 0.02 to 0.61 t 0.03and 0.57t 0.03,respectively, whereasRT was increasedfrom preischemialevels of 8.9 k 0.3 and 8.1 & 0.1 to 28.4 k 2.9 and 74.2 t 16.8, respectively. The increasesin RT were associatedwith proportional increasesin skeletal musclevascular resistance.Thus, in isolated rat hindquarters, increasing the duration of ischemia results in progressiveincreasesin the permeability to plasma proteins (decreaseda) and RT, which are associatedprimarily with skeletal muscle. osmoticreflection coefficient; vascular resistance;muscleblood flow; skin blood flow; microspheres;vascular permeability; noreflow phenomenon
ischemic skeletal muscle results in significant damage to the vasculature and myocytes (4, 6-10, 14-16). Clinically, skeletal muscle ischemia is often a consequence of conditions such as compartment syndrome, atherosclerosis, peripheral vascular disease associated with diabetes mellitus, reattachment of severed limbs and/or digits, tourniquet application, and transplantation of muscles in reconstructive or plastic surgery. Although it is obvious that blood flow must be reestablished to limit the extent of ischemic injury, recent evidence indicates that reperfusion may also produce injury (3, 9). Thus delineating the mechanisms underlying the development of vascular injury
LAUGHLIN University
of Missouri,
Vascular injury associated with ischemia and reperfusion in skeletal muscle is characterized by marked increases in the microvascular permeability to plasma proteins (4, 7-9) and subsequent edema formation. In addition, some regions of previously ischemic skeletal muscle fail to reperfuse upon the reinstitution of flow, thus exhibiting the no-reflow phenomenon (14, 15) and increased vascular resistance (7-g). The purpose of these experiments was to develop and characterize a model to investigate the effects of ischemia and reperfusion on the microvasculature of rat skeletal muscle in which consistent microvascular damage could be demonstrated. Other models that have been employed to assess the effects of ischemia and reperfusion on the microvasculature of skeletal muscle include the rat cremaster muscle (E), the isolated perfused canine gracilis muscle (7-10,16), and the isolated perfused canine hindlimb (4). An attractive feature of the rat hindquarters as a model to study ischemia and reperfusion injury in skeletal muscle is that muscle samples representative of each muscle fiber type can be easily identified (1). Therefore it is possible to assess the potential relationships between muscle fiber type composition and the regional distribution of flow within and among skeletal muscles subjected to ischemia and reperfusion. Furthermore, the muscle of the rat hindquarters is -76% fast-twitch glycolytic (FG), and it has been suggested that FG muscle may be more susceptible to the effects of ischemia and reperfusion injury (6).
REPERFUSION OF previously
after reperfusion of previously ischemic skeletal muscle is vitally important. 0161-7567/90
$1.50 Copyright
METHODS Experimental animals. Sixty male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 470 t 9 g, were used in these experiments. The rats were housed two per cage (8 x 7 x 17 in.) under controlled temperature (22 t 2*C) and light (12-h light-dark cycle) conditions. The rats were allowed free access to water and commercial rat chow. Experiments were completed in 30 intact (i.e., with skin) hindquarters preparations with ischemic periods of 30 (n = 7), 60 (n = 4), 120 (n = 7), 180 (n = 3), and 240 (n = 8) min and followed by 60 min of reperfusion. Experiments were also completed in
18 skinned hindquarters
0 1990 the American
Physiological
preparations
with ischemic pe-
Society
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387
388
ISCHEMIA-REPERFUSIUN
riods of 30, 60, and 120 min (n = 6 each) and followed by 60 min of reperfusion. Hindquarters preparation. The rats were anesthetized using pentobarbital sodium (65 mg/kg ip), and a cannula (PE-240) was placed in the trachea to ensure a patent airway during surgery. After surgical isolation of the rat hindquarters as described previously (13), the hindquarters were perfused with a peristaltic pump (Gilson Minipuls Z), and the venous effluent was directed to the perfusate reservoir. In experiments using skinned hindquarters, the skin of anesthetized rats was carefully removed from the hindquarters using thermal cautery before beginning the surgical preparation. The cautery was applied to the dermal surface of the retracted skin to avoid contact with the underlying tissues. Superficial vessels supplying the skin were effectively cauterized during this procedure such that little or no hemorrhage was observed during the experiments. The hindquarters were coated with mineral oil and covered with plastic wrap to prevent desiccation during the experiment. Arterial and venous pressure were monitored from side branches of the respective catheters using pressure transducers (Statham P23 AC). Arterial perfusion pressures were corrected for catheter resistance after each experiment. The hindquarters preparation was placed on a grid and suspended from a strain-gauge transducer (Grass FT03C) to measure weight changes during the experiments. The sensitivity of the weight-recording system was adjusted so that 1 g of weight produced a deflection of 3.0-3.5 cm on the recording paper. Arterial and venous pressures and hindquarters weight were recorded continuously on a Grass model 7D polygraph. The hindquarters were maintained at 37°C with an infrared heat lamp. The hindquarters were perfused with fresh human blood cells (obtained from the American Red Cross Blood Donor Center, Columbia, MO) suspended in horse serum (Pel-Freeze). To prepare the blood cells, blood was centrifuged (10 min at 2,000 rpm), and the plasma supernatant was removed. The cells were resuspended in fresh saline and centrifuged, and the saline wash was drawn off. This procedure was repeated three times, after which the cells were suspended in horse serum (average protein concentration of 6 g/dl) with 1,000 IU heparin added per 150 ml of perfusate. The cells were washed ~24 h before each experiment and stored in a refrigerator at 4°C. At the outset of each experiment, papaverine (average of 15 mg in 50 ml of perfusate) was titrated into the perfusate reservoir to achieve and maintain maximal vasodilation of hindquarters. Maximal vasodilation was presumed when the addition of 1 mg of papaverine caused no further reduction in perfusion pressure at the same flow. The perfusate was bubbled with 95% 02-5% COz, stirred continuously, and maintained at 37°C. Measurements. The capillary filtration coefficients (Kf) were determined as described by Eliassen et al. (5). Arterial and venous pressures were each increased 10 mmHg by increasing hindquarters flow and elevating the venous outflow level, respectively. The temporal pattern of weight gain in the hindquarters consisted of an initial fast component representing vascular pooling followed
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by a slow component representing fluid filtration from the capillaries into the interstitium. The rate of slow weight gain was used to calculate the rate of fluid filtration in ml. min-l JO0 g perfused tissue? Since the increase in mean capillary pressure resulting from similar increases in both arterial and venous pressures is equal to the increases in arterial and venous pressures (assuming no change in the pre-to-postcapillary resistance ratio) (5), Kf was determined by dividing the rate of fluid filtration per 100 g of tissue by the increment in venous pressure. Total vascular resistances were calculated by dividing the isogravimetric perfusion pressure by the total hindquarters flow, inasmuch as venous pressures were maintained equal to zero in these experiments. The osmotic reflection coefficients for total plasma proteins (B) were determined by the integral mass balance modification (17) of the original filtered volumes technique (11). Before the determination of CT,the reservoir volume was reduced to 15-20 ml. Four separate determinations of the initial hematocrit (Hi) and total protein concentration (Ci) of the perfusate were made. Hematocrit was determined by microhematocrit method and the protein concentration of the plasma supernatant was determined using a clinical refractometer (American Optical). Venous pressure was then elevated 20 mmHg, and flow was maintained constant such that there was a net fluid filtration. Filtration was allowed to continue for 15-20 min to ensure that the increases in hematocrit and total protein concentration of the reservoir perfusate were greater than the error incurred in their measurement. The perfusate in the reservoir was stirred continuously throughout the entire procedure to ensure a homogeneous mixing during the filtration period. Four samples were taken from the perfusate reservoir for final hematocrit (Hf) and total protein concentration (C,) determinations immediately before the venous pressure was lowered back to zero. Perfusate removed before the determination of 0 was returned to the reservoir, and -15 min were allowed for restabilization of the preparation. 0 was calculated using the following relationship (17)
Determination of the regional distribution of flow. The regional distribution of flow within hindquarters was determined during reperfusion using radiolabeled (l13Sn) microspheres (16.5 t O.&pm diam, NEN-Trac Microspheres, New England Nuclear Research Products, Boston, MA). The microsphere suspensions consisted of -200,000 microspheres in 0.10 ml of saline with 10% dextran and 0.01% Tween 80. Before infusion, the microsphere suspensions were sonicated and well mixed using an electromagnetic stirrer and stir bar contained in the suspension vial. Total flows were increased transiently to 10 ml/min (6.0-6.5 ml. min-1 100 g-‘) to ensure adequate distribution of the microspheres. Microsphere suspensions were injected over lo-15 s into a mixing chamber interposed in the arterial line immediately proximal to the hindquarters. After each experil
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ISCHEMIA-REPERFUSION
ment, the hindquarters were dissected and the tissue samples were weighed. The radioactivity of the tissue samples was determined using a gamma counter (Tracer Analytic 2250). Tissue flows (ml min-’ -100 g-l) were calculated from the ratio of the counts per minute in the tissue sample to the total counts per minute contained in the hindquarters multiplied by the total flow. Experimental protocol. After the institution of flow to the hindquarters, flow was adjusted and papaverine was added to the perfusate reservoir to achieve the maximally vasodilated isogravimetric state. & coefficient was determined between 20 and 30 min after perfusion was initiated, after which total vascular resistance and 0 were determined. Ischemia was produced in the hindquarters by turning off the perfusion pump and simultaneously clamping both the arterial and venous catheters just proximal to the aorta and vena cava. Perfusate within the arterial and venous lines was backflushed into the reservoir with small volumes of saline to prevent settling and clumping of the blood cells within the tubing during the ischemic period. Immediately before the onset of reperfusion, this saline was removed and discarded and the arterial lines were primed with perfusate from the reservoir. The temperature of the hindquarters and perfusate were maintained at 37°C throughout the ischemic period. After ischemic periods of 30, 60, 120, 180, or 240 min, reperfusion was begun. Reperfusion was initiated with low flows (Cl ml/min) to avoid the possibility of vascular damage associated with high flows at the onset of reperfusion. Flows were increased during the initial lo-15 min of reperfusion to attain isogravimetric flow conditions. Papaverine (2-3 mg) was again titrated into the reservoir to ensure maximal vasodilation. Determinations of & were made -30 min after the initiation of reperfusion, whereas B and total vascular resistance were determined after 60 min. Microspheres were infused for assessment of the regional distribution of flow after the final determination of CT. StatisticaL analysis. The data are presented as means + SE and compared using an unpaired Student’s t test. Tissue flows were compared using a one-way analysis of variance and Duncan’s new multiple range test. The significance of differences between groups was based on a probability level of P 5 0.05.
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T
* 1
l
RESULTS
The effects of varying the duration of the ischemic period on intact postischemic hindquarters flow and total vascular resistance under isogravimetric conditions are depicted in Fig. 1. Data presented for 0 min of ischemia are mean t SE (n = 30) values determined before ischemia for all hindquarters. After ischemia and reperfusion, isogravimetric perfusion pressures (with venous pressure equal to zero) were similar to preischemia values (36 t 1 mmHg), whereas isogravimetric flows were reduced as a function of the ischemic period duration (Fig. 1). Total vascular resistance increased as a function of ischemic period duration from 8.9 $- 0.3 (n = 30) before ischemia to 44.8 t- 4.5 mmHg* ml-’ min 100 g-l after 240 min of ischemia and reperfusion (Fig. 1). Figure 2 depicts data obtained for B and & before and l
l
0
30
Duration
60
120
of Ischemia
180
240
(min)
FIG. 1, Isogravimetric flow (A) and total vascular resistance (B) data (means t SE) determined in maximally vasodilated isolated perfused rat hindquarters before (0 min) and after ischemic periods of 30,60, 120, 180, and 240 min. Postischemic data were determined after 60 min of reperfusion. * Significant (P I 0.05) difference from preischemic values.
after different durations of ischemia followed by reperfusion in intact hindquarters. Preischemia CTwere not different between groups and averaged 0.92 t 0.02 (n = 30). After 30 min of ischemia and 60 min of reperfusion, CTwas not different from preischemia (0.93 t 0.04). However, g were significantly reduced after ischemic periods of 60 (0.82 t 0.02), 120 (0.67 t 0.06), 180 (0.55 +- O.lO), and 240 (0.61 t 0.07) min (Fig. 2). Kf for all groups were similar and were not altered by ischemia and reperfusion of any duration (Fig. 2). Since Kf is the product of the perfused microvascular surface area and the hydraulic conductivity of the exchange vessels, these results may be attributable to the offsetting effects of a decreased microvascular surface area [suggested by the increases in total (Fig. 1) and skeletal muscle (Fig. 3) vascular resistances] and an increased hydraulic conductivity of the exchange vessels after ischemia and reperfusion (6). To test for time-dependent deterioration of this preparation, two rat hindquarters preparations were perfused for 9 and 5 h with no ischemic periods. CJaveraged 0.91 t 0.05 (4 determinations) and 0.90 t 0.06 (2 determinations) over 9 and 5 h, respectively, whereas isogravimetric total vascular resistance did not change. These data indicate that the observed reductions in c and increases
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ISCHEMIA-REPERFUSION
A 1.0
* -
a-
i
0.4
+,
ing perfusion pressure was elevated in proportion to the duration of the ischemic period. The flow data presented in Fig. 3 indicate that the increase in total vascular resistance observed after ischemia and reperfusion was attributable to increases in the vascular resistance of skeletal muscles regardless of the muscle fiber type. -Under these flow conditions, the vascular resistance of the skin was reduced after ischemia and reperfusion (Fig.
3).
$ c? 0.6
O.OZO
.z
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B 1
i
Preischemia
ES3
Reperfusion
l
I
I.
The effects of varying the duration of the ischemic period on flow and total vascular resistance in the isogravimetric state for skinned rat hindquarters are depicted in Fig. 4. Values for 0 min of ischemia are means z!z SE (n = 18) for all hindquarters determined before ischemia. Preischemia isogravimetric flows (3.9 & 0.2 ml* min-l 100 g-‘) and total vascular resistances (8.1 t 0.3 mmHgoml-l *min. 100 g-l) were not different between the intact and skinned hindquarters. After ischemia and reperfusion, isogravimetric flows were less as the ischemic duration increased. After 30 min of ischemia and 60 min of reperfusion, total vascular resistance values in skinned hindquarters (11.5 t 0.9 mmHg . ml-’ min 100 g-‘) were similar to values observed in intact rat hindquarters. However, total vascular resistances were markedly elevated after 60 and 120 min of ischemia and reperfusion (29.0 t 3.8 and 74.2 t 16.8 mmHg* ml-‘. l
60 120 180 30 Duration of Ischemia (min)
240 T
FIG. 2. Osmotic reflection coefficients for plasma proteins (A) and capillary filtration coefficients (B) determined in isolated perfused rat hindquarters before (0 min) and after ischemic periods of 30, 60, 120, 180, and 240 min. Postischemic data were determined after 60 min of reperfusion. Values are means -c-SE. * Significant (P 5 0.05) difference from preischemic values.
0 ESI eZa EB2l
T
Control 30 min lschemia 60 min lschemia 120 min lschemia
1
S
Gr
Gm
l
Gw
001B
Skin
3. Flows to soleus (S) and red (Gr), mixed (Gm), and white (Gw) portions of gastrocnemius muscle and skin of rat hindquarters after 30, 60, and 120 min of ischemia and 60 min of reperfusion compared with nonischemic control flows. Values are means -t- SE. * Significant fP I 0.05) difference from nonischemic control values. FIG.
in total vascular resistance were attributable to the effects of ischemia and reperfusion in this preparation. Radiolabeled microspheres were infused into control (nonischemic) rat hindquarters (rt = 5) and during reperfusion after 30 (n, = 4), 60 (n = 4), and 120 (n = 6) min of ischemia. Total flow was briefly increased over
the isogravimetric
levels as described above. The result-
0
30
60
120
Duration of Ischemia (min) 4. Isogravimetric flow (A) and total vascular resistance (B) determined in skinned rat hindquarters before (0 min) and after ischemic periods of 30, 60, and 120min. Postischemic data were determined after 60 min of reperfusion. Values are means Ifr SE. * Significant (P 5 0.05) difference from preischemic values. FIG.
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ISCHEMIA-REPERFUSION
min 100 g-l, respectively) compared with intact hindquarters. Data obtained from skinned hindquarters for Q and & before and after different durations of ischemia are depicted in Fig. 5. Preischemia IT values for skinned hindquarters (0.89 t 0.02) were not different from intact hindquarters. g was decreased after 30 (0.80 t 0.4) and 60 (0.61 t 0.05) min of ischemia and reperfusion in skinned hindquarters and was not further reduced (0.57 t 0.03) after 120 min of ischemia and reperfusion. Kf (ml. min-’ gmmHg-‘. 100 g-l) measured after 30 (0.0086 -t 0.0005) and 60 (0.0088 t 0.0010) min of ischemia and reperfusion were reduced compared with preischemia but were not different after 120 min (0.0134 t 0.0011) (Fig. 5) . l
DISCUSSION
Reperfusion of previously ischemic skeletal muscle results in vascular injury characterized by increases in microvascular permeability to plasma proteins (measured as a decrease in CT)and increases in vascular resistance (4, 7). The increased vascular resistance has been attributed to failure of some vessels to reperfuse after the reinstitution of flow after an ischemic episode, the no-reflow phenomenon (7-9, 14, 15). Korthuis et al. (7-
B
1
Preischemia
ElS3 Reperfusion
1
U
60 30 Duration of Ischemia (min) FIG. 5. Osmotic reflection coefficients for plasma proteins (A) and capillary filtration coefficients (B) determined in skinned rat hindquarters before (0 min) and after ischemic periods of 30, 60, and 120 min. Postischemic data were determined after 60 min of reperfusion. Values are means & SE. * Significant (P 5 0.05) difference from preischemic values.
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9) noted significant reductions in g and increases in total vascular resistance in isolated canine gracilis muscle preparations after 4 h of ischemia and 0.5-2 h of reperfusion. Using anesthetized rats, Strock and Majno (14) noted substantial areas of no-reflow in rat hindlimbs after 2.5 h of ischemia followed by l-3 h of reperfusion. Recently, Suval et al. (15) reported marked increases in the extravasation of fluorescein-labeled dextran from postcapillary venules in rat cremaster muscles during reperfusion after ischemic periods of 30 and 120 min, suggesting an increase in the vascular permeability to large macromolecules. In addition, they noted that ~30% of the total examined cremaster microcirculation demonstrated no-reflow after ischemia and reperfusion (15). The results of the present study are in agreement with the aforementioned observations and revealed that reperfusion of ischemic isolated perfused rat hindquarters preparations is associated with increases in both the microvascular permeability to plasma proteins (decreased a) and total vascular resistance. Recent data indicate that much of the observed microvascular injury associated with ischemia and reperfusion actually occurs during reperfusion (9). However, our results also indicate that the magnitude of the changes in 0 and total vascular resistance observed in rat hindquarters was correlated with the duration of the ischemic period in both intact and skinned hindquarters preparations. Thus characteristic microvascular injury (decreased CTand increased vascular resistance) was demonstrated after reperfusion of ischemic isolated perfused rat hindquarters preparations. Furthermore the isolated rat hindquarters appears to be more sensitive to microvascular injury associated with the reperfusion after ischemia than the isolated canine gracilis muscle preparation (7-9) or the isolated perfused canine hindlimb (4) in which microvascular injury does not become apparent until the ischemic period duration exceeds 3 h. The magnitude of the decreases in 0 and increases in total vascular resistance measured in isolated rat hindquarters preparations after 60 min of ischemia and reperfusion in the present study was similar to those changes reported for the canine gracilis muscle preparation after 4 h of ischemia and 2 h of reperfusion (7-9). Jennische and Hansson (6) have suggested that FG muscle fibers are more susceptible to ischemic vascular injury than fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) muscle fibers. The canine gracilis muscle is composed exclusively of FOG and SO muscle (2). On the other hand, the rat hindquarters musculature is composed of 76% FG, 19% FOG, and 5% SO muscle fibers by mass (1). Therefore the apparent greater susceptibility of isolated rat hindquarters to postischemia reperfusion injury observed in this study may be related to the fiber-type composition of the muscle tissue. Consistent with this notion, Suval et al. (15) reported significant extravasation of fluorescein-labeled dextran after ischemic periods of 30 and 120 min in the rat cremaster muscle, which is composed of 47% FG muscle fibers (12). Another attractive feature of the isolated perfused rat hindquarters model is that muscles composed primarily of SO, FOG, and FG muscle fibers can be easily identified
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because of the unique stratification of muscle fiber types in rat muscles (1). Therefore it is possible to assess changes in vascular resistance within and among muscles of different fiber types after ischemia and reperfusion. The results of this study revealed that flow to muscles of all fiber types was decreased as a function of the ischemic period duration (Fig. 3). Although resistance to flow in skeletal muscle increased as a function of the ischemic period duration, skin vascular resistance was less affected (Fig. 3). Inasmuch as each hindquarters preparation was maximally vasodilated with papaverine, the increase in flow to the skin probably represents distension of compliant vessels rather than further vasodilation. The differential response to ischemia and reperfusion seen in skeletal muscle and skin in the isolated rat hindquarters model is in accord with the observations of Strock and Majno (14). They noted that during reperfusion the skin of postischemic hindlimbs was more resistant to the development of no-reflow than was skeletal muscle. They also observed significant increases in the water content of the skin, indicative of edema, during reperfusion (14). Finally, the greater sensitivity of the skinned hindquarters preparations to ischemia and reperfusion compared with the intact hindquarters seen in this study (Figs. 1 and 2 vs. Figs. 4 and 5) further demonstrates the differential responses of the microcirculation of skin and skeletal muscle to ischemia and reperfusion. In summary, we have demonstrated marked increases in the microvascular permeability to plasma proteins (decreased a) and total vascular resistance indicative of microvascular injury after ischemia and reperfusion in isolated perfused rat hindquarters. Significant microvascular damage was demonstrable after 60 min of ischemia and 60 min of reperfusion in intact hindquarters preparations, whereas notable microvascular damage was apparent after 30 min of ischemia and 60 min of reperfusion in skinned hindquarters. Furthermore the magnitude of the changes in CTand total vascular resistance was directly dependent on the duration of the ischemic episode. These results indicate that the isolated perfused rat hindquarters preparation can be used as a model to assess the effects of ischemia and reperfusion on the microvasculature of rat skeletal muscle. The authors thank the American Red Cross Blood Donor Center, Columbia, MO, for their kind cooperation during these studies. This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-36088 and HL-36069. W. L. Sexton, R. J. Korthuis, and M. H. Laughlin are recipients of NHLBI National Research Service Award HL-07187. American Heart Association Es-
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tablished Investigator Award 880197, and NHLBI Research Career Development Award HL-01774, respectively. Address for reprint requests: W. L. Sexton, Dept. of Physiology, Kirksville College of Osteopathic Medicine, 800 West Jefferson St., Kirksville, MO 63501. Received 14 November 1988; accepted in final form 28 August 1989. REFERENCES 1. ARMSTRONG, R. B., AND R. 0. PHELPS. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984. 2. ARMSTRONG, R. B., C. W. SAUBERT, H. J. SEEHERMAN, AND C. R. TAYLOR. Distribution of fiber types in locomotory muscles of dogs. Am. J. Anat. 163: 87-98,1982. 3. BRAUNWALD, E., AND R. A. KLONER. Myocardial reperfusion: a double-edged sword? J. CZin. Inuest. 76: 1713-1719,1985. 4. DIANA, J. N., AND M. H, LAUGHLIN. Effect of ischemia on capillary pressure and equivalent pore radius in capillaries of the isolated dog hind limb. Cl’rc. Res. 35: 77-101, 1974. 5. ELIASSEN, E., B. FOLKOW, S. M. HINTON, B. OBERG, AND B, RIPPE. Pressure-volume characteristics of the interstitial space in the skeletal muscle of the cat. Acta Physid. Scud 90: 583-593, 1974. 6. JENNISCHE, E., AND H.-A. HANSSON. Postischemic skeletal muscle injury: patterns of injury in relation to adequacy of reperfusion. Exp. Mol. Pathol. 44: 272-280, 1986. 7. KORTHUIS, R. J., D. N. GRANGER, M. I. TOWNSLEY, AND A. E. TAYLOR. The role of oxygen-derived free radicals in ischemiainduced increases in canine skeletal muscle microvascular permeability. Circ. Res. 57: 599-609, 1985. 8. KORTHUIS, R. J., M. B. GRISHAM, AND D. N. GRANGER. Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H823-H827,1988. 9. KORTHUIS, R. J., J. K. SMITH, AND D. L. CARDEN. Hypoxic reperfusion attenuates postischemic microvascular injury. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H315-H319, 1989. 10. LABBE, R., T. LINDSAY, AND P, M. WALKER. The extent and distribution of skeletal muscle necrosis after graded periods of complete ischemia. J. Vast. Surg. 6: 152-157, 1987. 11. MARON, M., AND C. F. PILATI. Calculation of the reflection coefficient from measurements of endogenous vascular indicators. J. Appl. PhysioZ. 64: 1746-1748, 1988. 12. SARELIUS, I. H., L. C, MAXWELL, S. D. GRAY, AND B. M. DULING. Capillarity and fiber types in the cremaster of rat and hamster. Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H368-H374, 1983. 13. SEXTON, W. L., R. J. KORTHUIS, AND M. H. LAUGHLIN. Highintensity exercise training increases vascular transport capacity of rat hindquarters. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H274-H278,1988. 14. STROCK, P. E., AND G. MAJNO. Vascular responses to tourniquet ischemia. Surg. Gynecol. Obstet. 129: 309-318, 1969. 15. SUVAL, W. D., W. N. DURAN, M. P. BORIC, R. W. HOBSON, P. B. BERENDSEN, AND A. B. RITTER. Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury. Am. J. Surg. 154: 211-218, 1987. 16. WALKER, P. M., T. F. LINDSAY, R. LABBE, D. A. MICKLE, AND A. D. ROMASCHIN. Salvage of skeletal muscle with free radical scavengers. J. Vast. Surg. 5: 68-75, 1987. 17. WOLF, M. B., P. D. WATSON, AND D. R. C. SCOTT. Integral-mass balance method for determination of the solvent drag reflection coefficient. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): Hl94H204,1987.
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L. SEXTON,
Department
of Veterinary
Columbia, Louisiana
injury RONALD
in isolated rat hindquarters J. KORTHUIS,
AND
M. HAROLD
Biomedical Sciences and Dalton Research Center, Missouri 65211; and Department of Physiology and Biophysics, State University Medical Center, Shreveport, Louisiana 71130
SEXTW, WILLIAM L., RONALD J. KORTHUIS, AND M. HAROLD LAUGHLIN. Ischemia-reperfusion injury in isolated rut hindquarters. J. Appl. Physiol. 68(1): 387-392, 1990.-The purpose of this study was to determine the suitability of the maximally vasodilated (papaverine) isolated rat hindquarters preparation to study the effects of ischemiaand reperfusion on the microvasculatureof skeletal muscle.The osmotic reflection coefficient for plasmaproteins (a) and total vascular resistance ( RT, mmHg*ml-l ‘min +100 g-l) were determined before ischemicperiods of 30, 60, 120, 180,and 240 min in intact (with skin) and 30, 60, and 120 min in skinned hindquarters and again after 60 min of reperfusion. In both intact and skinned hindquarters, reductionsin c and increasesin RT wereobserved during reperfusionand werecorrelated with the ischemicperiod duration. After 120 min of ischemia in intact and skinned hindquarters, u was reducedfrom preischemiavalues of 0.92 t 0.02 and 0.89+ 0.02 to 0.61 t 0.03and 0.57t 0.03,respectively, whereasRT was increasedfrom preischemialevels of 8.9 k 0.3 and 8.1 & 0.1 to 28.4 k 2.9 and 74.2 t 16.8, respectively. The increasesin RT were associatedwith proportional increasesin skeletal musclevascular resistance.Thus, in isolated rat hindquarters, increasing the duration of ischemia results in progressiveincreasesin the permeability to plasma proteins (decreaseda) and RT, which are associatedprimarily with skeletal muscle. osmoticreflection coefficient; vascular resistance;muscleblood flow; skin blood flow; microspheres;vascular permeability; noreflow phenomenon
ischemic skeletal muscle results in significant damage to the vasculature and myocytes (4, 6-10, 14-16). Clinically, skeletal muscle ischemia is often a consequence of conditions such as compartment syndrome, atherosclerosis, peripheral vascular disease associated with diabetes mellitus, reattachment of severed limbs and/or digits, tourniquet application, and transplantation of muscles in reconstructive or plastic surgery. Although it is obvious that blood flow must be reestablished to limit the extent of ischemic injury, recent evidence indicates that reperfusion may also produce injury (3, 9). Thus delineating the mechanisms underlying the development of vascular injury
LAUGHLIN University
of Missouri,
Vascular injury associated with ischemia and reperfusion in skeletal muscle is characterized by marked increases in the microvascular permeability to plasma proteins (4, 7-9) and subsequent edema formation. In addition, some regions of previously ischemic skeletal muscle fail to reperfuse upon the reinstitution of flow, thus exhibiting the no-reflow phenomenon (14, 15) and increased vascular resistance (7-g). The purpose of these experiments was to develop and characterize a model to investigate the effects of ischemia and reperfusion on the microvasculature of rat skeletal muscle in which consistent microvascular damage could be demonstrated. Other models that have been employed to assess the effects of ischemia and reperfusion on the microvasculature of skeletal muscle include the rat cremaster muscle (E), the isolated perfused canine gracilis muscle (7-10,16), and the isolated perfused canine hindlimb (4). An attractive feature of the rat hindquarters as a model to study ischemia and reperfusion injury in skeletal muscle is that muscle samples representative of each muscle fiber type can be easily identified (1). Therefore it is possible to assess the potential relationships between muscle fiber type composition and the regional distribution of flow within and among skeletal muscles subjected to ischemia and reperfusion. Furthermore, the muscle of the rat hindquarters is -76% fast-twitch glycolytic (FG), and it has been suggested that FG muscle may be more susceptible to the effects of ischemia and reperfusion injury (6).
REPERFUSION OF previously
after reperfusion of previously ischemic skeletal muscle is vitally important. 0161-7567/90
$1.50 Copyright
METHODS Experimental animals. Sixty male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 470 t 9 g, were used in these experiments. The rats were housed two per cage (8 x 7 x 17 in.) under controlled temperature (22 t 2*C) and light (12-h light-dark cycle) conditions. The rats were allowed free access to water and commercial rat chow. Experiments were completed in 30 intact (i.e., with skin) hindquarters preparations with ischemic periods of 30 (n = 7), 60 (n = 4), 120 (n = 7), 180 (n = 3), and 240 (n = 8) min and followed by 60 min of reperfusion. Experiments were also completed in
18 skinned hindquarters
0 1990 the American
Physiological
preparations
with ischemic pe-
Society
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riods of 30, 60, and 120 min (n = 6 each) and followed by 60 min of reperfusion. Hindquarters preparation. The rats were anesthetized using pentobarbital sodium (65 mg/kg ip), and a cannula (PE-240) was placed in the trachea to ensure a patent airway during surgery. After surgical isolation of the rat hindquarters as described previously (13), the hindquarters were perfused with a peristaltic pump (Gilson Minipuls Z), and the venous effluent was directed to the perfusate reservoir. In experiments using skinned hindquarters, the skin of anesthetized rats was carefully removed from the hindquarters using thermal cautery before beginning the surgical preparation. The cautery was applied to the dermal surface of the retracted skin to avoid contact with the underlying tissues. Superficial vessels supplying the skin were effectively cauterized during this procedure such that little or no hemorrhage was observed during the experiments. The hindquarters were coated with mineral oil and covered with plastic wrap to prevent desiccation during the experiment. Arterial and venous pressure were monitored from side branches of the respective catheters using pressure transducers (Statham P23 AC). Arterial perfusion pressures were corrected for catheter resistance after each experiment. The hindquarters preparation was placed on a grid and suspended from a strain-gauge transducer (Grass FT03C) to measure weight changes during the experiments. The sensitivity of the weight-recording system was adjusted so that 1 g of weight produced a deflection of 3.0-3.5 cm on the recording paper. Arterial and venous pressures and hindquarters weight were recorded continuously on a Grass model 7D polygraph. The hindquarters were maintained at 37°C with an infrared heat lamp. The hindquarters were perfused with fresh human blood cells (obtained from the American Red Cross Blood Donor Center, Columbia, MO) suspended in horse serum (Pel-Freeze). To prepare the blood cells, blood was centrifuged (10 min at 2,000 rpm), and the plasma supernatant was removed. The cells were resuspended in fresh saline and centrifuged, and the saline wash was drawn off. This procedure was repeated three times, after which the cells were suspended in horse serum (average protein concentration of 6 g/dl) with 1,000 IU heparin added per 150 ml of perfusate. The cells were washed ~24 h before each experiment and stored in a refrigerator at 4°C. At the outset of each experiment, papaverine (average of 15 mg in 50 ml of perfusate) was titrated into the perfusate reservoir to achieve and maintain maximal vasodilation of hindquarters. Maximal vasodilation was presumed when the addition of 1 mg of papaverine caused no further reduction in perfusion pressure at the same flow. The perfusate was bubbled with 95% 02-5% COz, stirred continuously, and maintained at 37°C. Measurements. The capillary filtration coefficients (Kf) were determined as described by Eliassen et al. (5). Arterial and venous pressures were each increased 10 mmHg by increasing hindquarters flow and elevating the venous outflow level, respectively. The temporal pattern of weight gain in the hindquarters consisted of an initial fast component representing vascular pooling followed
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by a slow component representing fluid filtration from the capillaries into the interstitium. The rate of slow weight gain was used to calculate the rate of fluid filtration in ml. min-l JO0 g perfused tissue? Since the increase in mean capillary pressure resulting from similar increases in both arterial and venous pressures is equal to the increases in arterial and venous pressures (assuming no change in the pre-to-postcapillary resistance ratio) (5), Kf was determined by dividing the rate of fluid filtration per 100 g of tissue by the increment in venous pressure. Total vascular resistances were calculated by dividing the isogravimetric perfusion pressure by the total hindquarters flow, inasmuch as venous pressures were maintained equal to zero in these experiments. The osmotic reflection coefficients for total plasma proteins (B) were determined by the integral mass balance modification (17) of the original filtered volumes technique (11). Before the determination of CT,the reservoir volume was reduced to 15-20 ml. Four separate determinations of the initial hematocrit (Hi) and total protein concentration (Ci) of the perfusate were made. Hematocrit was determined by microhematocrit method and the protein concentration of the plasma supernatant was determined using a clinical refractometer (American Optical). Venous pressure was then elevated 20 mmHg, and flow was maintained constant such that there was a net fluid filtration. Filtration was allowed to continue for 15-20 min to ensure that the increases in hematocrit and total protein concentration of the reservoir perfusate were greater than the error incurred in their measurement. The perfusate in the reservoir was stirred continuously throughout the entire procedure to ensure a homogeneous mixing during the filtration period. Four samples were taken from the perfusate reservoir for final hematocrit (Hf) and total protein concentration (C,) determinations immediately before the venous pressure was lowered back to zero. Perfusate removed before the determination of 0 was returned to the reservoir, and -15 min were allowed for restabilization of the preparation. 0 was calculated using the following relationship (17)
Determination of the regional distribution of flow. The regional distribution of flow within hindquarters was determined during reperfusion using radiolabeled (l13Sn) microspheres (16.5 t O.&pm diam, NEN-Trac Microspheres, New England Nuclear Research Products, Boston, MA). The microsphere suspensions consisted of -200,000 microspheres in 0.10 ml of saline with 10% dextran and 0.01% Tween 80. Before infusion, the microsphere suspensions were sonicated and well mixed using an electromagnetic stirrer and stir bar contained in the suspension vial. Total flows were increased transiently to 10 ml/min (6.0-6.5 ml. min-1 100 g-‘) to ensure adequate distribution of the microspheres. Microsphere suspensions were injected over lo-15 s into a mixing chamber interposed in the arterial line immediately proximal to the hindquarters. After each experil
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ment, the hindquarters were dissected and the tissue samples were weighed. The radioactivity of the tissue samples was determined using a gamma counter (Tracer Analytic 2250). Tissue flows (ml min-’ -100 g-l) were calculated from the ratio of the counts per minute in the tissue sample to the total counts per minute contained in the hindquarters multiplied by the total flow. Experimental protocol. After the institution of flow to the hindquarters, flow was adjusted and papaverine was added to the perfusate reservoir to achieve the maximally vasodilated isogravimetric state. & coefficient was determined between 20 and 30 min after perfusion was initiated, after which total vascular resistance and 0 were determined. Ischemia was produced in the hindquarters by turning off the perfusion pump and simultaneously clamping both the arterial and venous catheters just proximal to the aorta and vena cava. Perfusate within the arterial and venous lines was backflushed into the reservoir with small volumes of saline to prevent settling and clumping of the blood cells within the tubing during the ischemic period. Immediately before the onset of reperfusion, this saline was removed and discarded and the arterial lines were primed with perfusate from the reservoir. The temperature of the hindquarters and perfusate were maintained at 37°C throughout the ischemic period. After ischemic periods of 30, 60, 120, 180, or 240 min, reperfusion was begun. Reperfusion was initiated with low flows (Cl ml/min) to avoid the possibility of vascular damage associated with high flows at the onset of reperfusion. Flows were increased during the initial lo-15 min of reperfusion to attain isogravimetric flow conditions. Papaverine (2-3 mg) was again titrated into the reservoir to ensure maximal vasodilation. Determinations of & were made -30 min after the initiation of reperfusion, whereas B and total vascular resistance were determined after 60 min. Microspheres were infused for assessment of the regional distribution of flow after the final determination of CT. StatisticaL analysis. The data are presented as means + SE and compared using an unpaired Student’s t test. Tissue flows were compared using a one-way analysis of variance and Duncan’s new multiple range test. The significance of differences between groups was based on a probability level of P 5 0.05.
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T
* 1
l
RESULTS
The effects of varying the duration of the ischemic period on intact postischemic hindquarters flow and total vascular resistance under isogravimetric conditions are depicted in Fig. 1. Data presented for 0 min of ischemia are mean t SE (n = 30) values determined before ischemia for all hindquarters. After ischemia and reperfusion, isogravimetric perfusion pressures (with venous pressure equal to zero) were similar to preischemia values (36 t 1 mmHg), whereas isogravimetric flows were reduced as a function of the ischemic period duration (Fig. 1). Total vascular resistance increased as a function of ischemic period duration from 8.9 $- 0.3 (n = 30) before ischemia to 44.8 t- 4.5 mmHg* ml-’ min 100 g-l after 240 min of ischemia and reperfusion (Fig. 1). Figure 2 depicts data obtained for B and & before and l
l
0
30
Duration
60
120
of Ischemia
180
240
(min)
FIG. 1, Isogravimetric flow (A) and total vascular resistance (B) data (means t SE) determined in maximally vasodilated isolated perfused rat hindquarters before (0 min) and after ischemic periods of 30,60, 120, 180, and 240 min. Postischemic data were determined after 60 min of reperfusion. * Significant (P I 0.05) difference from preischemic values.
after different durations of ischemia followed by reperfusion in intact hindquarters. Preischemia CTwere not different between groups and averaged 0.92 t 0.02 (n = 30). After 30 min of ischemia and 60 min of reperfusion, CTwas not different from preischemia (0.93 t 0.04). However, g were significantly reduced after ischemic periods of 60 (0.82 t 0.02), 120 (0.67 t 0.06), 180 (0.55 +- O.lO), and 240 (0.61 t 0.07) min (Fig. 2). Kf for all groups were similar and were not altered by ischemia and reperfusion of any duration (Fig. 2). Since Kf is the product of the perfused microvascular surface area and the hydraulic conductivity of the exchange vessels, these results may be attributable to the offsetting effects of a decreased microvascular surface area [suggested by the increases in total (Fig. 1) and skeletal muscle (Fig. 3) vascular resistances] and an increased hydraulic conductivity of the exchange vessels after ischemia and reperfusion (6). To test for time-dependent deterioration of this preparation, two rat hindquarters preparations were perfused for 9 and 5 h with no ischemic periods. CJaveraged 0.91 t 0.05 (4 determinations) and 0.90 t 0.06 (2 determinations) over 9 and 5 h, respectively, whereas isogravimetric total vascular resistance did not change. These data indicate that the observed reductions in c and increases
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A 1.0
* -
a-
i
0.4
+,
ing perfusion pressure was elevated in proportion to the duration of the ischemic period. The flow data presented in Fig. 3 indicate that the increase in total vascular resistance observed after ischemia and reperfusion was attributable to increases in the vascular resistance of skeletal muscles regardless of the muscle fiber type. -Under these flow conditions, the vascular resistance of the skin was reduced after ischemia and reperfusion (Fig.
3).
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O.OZO
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B 1
i
Preischemia
ES3
Reperfusion
l
I
I.
The effects of varying the duration of the ischemic period on flow and total vascular resistance in the isogravimetric state for skinned rat hindquarters are depicted in Fig. 4. Values for 0 min of ischemia are means z!z SE (n = 18) for all hindquarters determined before ischemia. Preischemia isogravimetric flows (3.9 & 0.2 ml* min-l 100 g-‘) and total vascular resistances (8.1 t 0.3 mmHgoml-l *min. 100 g-l) were not different between the intact and skinned hindquarters. After ischemia and reperfusion, isogravimetric flows were less as the ischemic duration increased. After 30 min of ischemia and 60 min of reperfusion, total vascular resistance values in skinned hindquarters (11.5 t 0.9 mmHg . ml-’ min 100 g-‘) were similar to values observed in intact rat hindquarters. However, total vascular resistances were markedly elevated after 60 and 120 min of ischemia and reperfusion (29.0 t 3.8 and 74.2 t 16.8 mmHg* ml-‘. l
60 120 180 30 Duration of Ischemia (min)
240 T
FIG. 2. Osmotic reflection coefficients for plasma proteins (A) and capillary filtration coefficients (B) determined in isolated perfused rat hindquarters before (0 min) and after ischemic periods of 30, 60, 120, 180, and 240 min. Postischemic data were determined after 60 min of reperfusion. Values are means -c-SE. * Significant (P 5 0.05) difference from preischemic values.
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T
Control 30 min lschemia 60 min lschemia 120 min lschemia
1
S
Gr
Gm
l
Gw
001B
Skin
3. Flows to soleus (S) and red (Gr), mixed (Gm), and white (Gw) portions of gastrocnemius muscle and skin of rat hindquarters after 30, 60, and 120 min of ischemia and 60 min of reperfusion compared with nonischemic control flows. Values are means -t- SE. * Significant fP I 0.05) difference from nonischemic control values. FIG.
in total vascular resistance were attributable to the effects of ischemia and reperfusion in this preparation. Radiolabeled microspheres were infused into control (nonischemic) rat hindquarters (rt = 5) and during reperfusion after 30 (n, = 4), 60 (n = 4), and 120 (n = 6) min of ischemia. Total flow was briefly increased over
the isogravimetric
levels as described above. The result-
0
30
60
120
Duration of Ischemia (min) 4. Isogravimetric flow (A) and total vascular resistance (B) determined in skinned rat hindquarters before (0 min) and after ischemic periods of 30, 60, and 120min. Postischemic data were determined after 60 min of reperfusion. Values are means Ifr SE. * Significant (P 5 0.05) difference from preischemic values. FIG.
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ISCHEMIA-REPERFUSION
min 100 g-l, respectively) compared with intact hindquarters. Data obtained from skinned hindquarters for Q and & before and after different durations of ischemia are depicted in Fig. 5. Preischemia IT values for skinned hindquarters (0.89 t 0.02) were not different from intact hindquarters. g was decreased after 30 (0.80 t 0.4) and 60 (0.61 t 0.05) min of ischemia and reperfusion in skinned hindquarters and was not further reduced (0.57 t 0.03) after 120 min of ischemia and reperfusion. Kf (ml. min-’ gmmHg-‘. 100 g-l) measured after 30 (0.0086 -t 0.0005) and 60 (0.0088 t 0.0010) min of ischemia and reperfusion were reduced compared with preischemia but were not different after 120 min (0.0134 t 0.0011) (Fig. 5) . l
DISCUSSION
Reperfusion of previously ischemic skeletal muscle results in vascular injury characterized by increases in microvascular permeability to plasma proteins (measured as a decrease in CT)and increases in vascular resistance (4, 7). The increased vascular resistance has been attributed to failure of some vessels to reperfuse after the reinstitution of flow after an ischemic episode, the no-reflow phenomenon (7-9, 14, 15). Korthuis et al. (7-
B
1
Preischemia
ElS3 Reperfusion
1
U
60 30 Duration of Ischemia (min) FIG. 5. Osmotic reflection coefficients for plasma proteins (A) and capillary filtration coefficients (B) determined in skinned rat hindquarters before (0 min) and after ischemic periods of 30, 60, and 120 min. Postischemic data were determined after 60 min of reperfusion. Values are means & SE. * Significant (P 5 0.05) difference from preischemic values.
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9) noted significant reductions in g and increases in total vascular resistance in isolated canine gracilis muscle preparations after 4 h of ischemia and 0.5-2 h of reperfusion. Using anesthetized rats, Strock and Majno (14) noted substantial areas of no-reflow in rat hindlimbs after 2.5 h of ischemia followed by l-3 h of reperfusion. Recently, Suval et al. (15) reported marked increases in the extravasation of fluorescein-labeled dextran from postcapillary venules in rat cremaster muscles during reperfusion after ischemic periods of 30 and 120 min, suggesting an increase in the vascular permeability to large macromolecules. In addition, they noted that ~30% of the total examined cremaster microcirculation demonstrated no-reflow after ischemia and reperfusion (15). The results of the present study are in agreement with the aforementioned observations and revealed that reperfusion of ischemic isolated perfused rat hindquarters preparations is associated with increases in both the microvascular permeability to plasma proteins (decreased a) and total vascular resistance. Recent data indicate that much of the observed microvascular injury associated with ischemia and reperfusion actually occurs during reperfusion (9). However, our results also indicate that the magnitude of the changes in 0 and total vascular resistance observed in rat hindquarters was correlated with the duration of the ischemic period in both intact and skinned hindquarters preparations. Thus characteristic microvascular injury (decreased CTand increased vascular resistance) was demonstrated after reperfusion of ischemic isolated perfused rat hindquarters preparations. Furthermore the isolated rat hindquarters appears to be more sensitive to microvascular injury associated with the reperfusion after ischemia than the isolated canine gracilis muscle preparation (7-9) or the isolated perfused canine hindlimb (4) in which microvascular injury does not become apparent until the ischemic period duration exceeds 3 h. The magnitude of the decreases in 0 and increases in total vascular resistance measured in isolated rat hindquarters preparations after 60 min of ischemia and reperfusion in the present study was similar to those changes reported for the canine gracilis muscle preparation after 4 h of ischemia and 2 h of reperfusion (7-9). Jennische and Hansson (6) have suggested that FG muscle fibers are more susceptible to ischemic vascular injury than fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) muscle fibers. The canine gracilis muscle is composed exclusively of FOG and SO muscle (2). On the other hand, the rat hindquarters musculature is composed of 76% FG, 19% FOG, and 5% SO muscle fibers by mass (1). Therefore the apparent greater susceptibility of isolated rat hindquarters to postischemia reperfusion injury observed in this study may be related to the fiber-type composition of the muscle tissue. Consistent with this notion, Suval et al. (15) reported significant extravasation of fluorescein-labeled dextran after ischemic periods of 30 and 120 min in the rat cremaster muscle, which is composed of 47% FG muscle fibers (12). Another attractive feature of the isolated perfused rat hindquarters model is that muscles composed primarily of SO, FOG, and FG muscle fibers can be easily identified
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because of the unique stratification of muscle fiber types in rat muscles (1). Therefore it is possible to assess changes in vascular resistance within and among muscles of different fiber types after ischemia and reperfusion. The results of this study revealed that flow to muscles of all fiber types was decreased as a function of the ischemic period duration (Fig. 3). Although resistance to flow in skeletal muscle increased as a function of the ischemic period duration, skin vascular resistance was less affected (Fig. 3). Inasmuch as each hindquarters preparation was maximally vasodilated with papaverine, the increase in flow to the skin probably represents distension of compliant vessels rather than further vasodilation. The differential response to ischemia and reperfusion seen in skeletal muscle and skin in the isolated rat hindquarters model is in accord with the observations of Strock and Majno (14). They noted that during reperfusion the skin of postischemic hindlimbs was more resistant to the development of no-reflow than was skeletal muscle. They also observed significant increases in the water content of the skin, indicative of edema, during reperfusion (14). Finally, the greater sensitivity of the skinned hindquarters preparations to ischemia and reperfusion compared with the intact hindquarters seen in this study (Figs. 1 and 2 vs. Figs. 4 and 5) further demonstrates the differential responses of the microcirculation of skin and skeletal muscle to ischemia and reperfusion. In summary, we have demonstrated marked increases in the microvascular permeability to plasma proteins (decreased a) and total vascular resistance indicative of microvascular injury after ischemia and reperfusion in isolated perfused rat hindquarters. Significant microvascular damage was demonstrable after 60 min of ischemia and 60 min of reperfusion in intact hindquarters preparations, whereas notable microvascular damage was apparent after 30 min of ischemia and 60 min of reperfusion in skinned hindquarters. Furthermore the magnitude of the changes in CTand total vascular resistance was directly dependent on the duration of the ischemic episode. These results indicate that the isolated perfused rat hindquarters preparation can be used as a model to assess the effects of ischemia and reperfusion on the microvasculature of rat skeletal muscle. The authors thank the American Red Cross Blood Donor Center, Columbia, MO, for their kind cooperation during these studies. This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-36088 and HL-36069. W. L. Sexton, R. J. Korthuis, and M. H. Laughlin are recipients of NHLBI National Research Service Award HL-07187. American Heart Association Es-
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tablished Investigator Award 880197, and NHLBI Research Career Development Award HL-01774, respectively. Address for reprint requests: W. L. Sexton, Dept. of Physiology, Kirksville College of Osteopathic Medicine, 800 West Jefferson St., Kirksville, MO 63501. Received 14 November 1988; accepted in final form 28 August 1989. REFERENCES 1. ARMSTRONG, R. B., AND R. 0. PHELPS. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984. 2. ARMSTRONG, R. B., C. W. SAUBERT, H. J. SEEHERMAN, AND C. R. TAYLOR. Distribution of fiber types in locomotory muscles of dogs. Am. J. Anat. 163: 87-98,1982. 3. BRAUNWALD, E., AND R. A. KLONER. Myocardial reperfusion: a double-edged sword? J. CZin. Inuest. 76: 1713-1719,1985. 4. DIANA, J. N., AND M. H, LAUGHLIN. Effect of ischemia on capillary pressure and equivalent pore radius in capillaries of the isolated dog hind limb. Cl’rc. Res. 35: 77-101, 1974. 5. ELIASSEN, E., B. FOLKOW, S. M. HINTON, B. OBERG, AND B, RIPPE. Pressure-volume characteristics of the interstitial space in the skeletal muscle of the cat. Acta Physid. Scud 90: 583-593, 1974. 6. JENNISCHE, E., AND H.-A. HANSSON. Postischemic skeletal muscle injury: patterns of injury in relation to adequacy of reperfusion. Exp. Mol. Pathol. 44: 272-280, 1986. 7. KORTHUIS, R. J., D. N. GRANGER, M. I. TOWNSLEY, AND A. E. TAYLOR. The role of oxygen-derived free radicals in ischemiainduced increases in canine skeletal muscle microvascular permeability. Circ. Res. 57: 599-609, 1985. 8. KORTHUIS, R. J., M. B. GRISHAM, AND D. N. GRANGER. Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H823-H827,1988. 9. KORTHUIS, R. J., J. K. SMITH, AND D. L. CARDEN. Hypoxic reperfusion attenuates postischemic microvascular injury. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H315-H319, 1989. 10. LABBE, R., T. LINDSAY, AND P, M. WALKER. The extent and distribution of skeletal muscle necrosis after graded periods of complete ischemia. J. Vast. Surg. 6: 152-157, 1987. 11. MARON, M., AND C. F. PILATI. Calculation of the reflection coefficient from measurements of endogenous vascular indicators. J. Appl. PhysioZ. 64: 1746-1748, 1988. 12. SARELIUS, I. H., L. C, MAXWELL, S. D. GRAY, AND B. M. DULING. Capillarity and fiber types in the cremaster of rat and hamster. Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H368-H374, 1983. 13. SEXTON, W. L., R. J. KORTHUIS, AND M. H. LAUGHLIN. Highintensity exercise training increases vascular transport capacity of rat hindquarters. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H274-H278,1988. 14. STROCK, P. E., AND G. MAJNO. Vascular responses to tourniquet ischemia. Surg. Gynecol. Obstet. 129: 309-318, 1969. 15. SUVAL, W. D., W. N. DURAN, M. P. BORIC, R. W. HOBSON, P. B. BERENDSEN, AND A. B. RITTER. Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury. Am. J. Surg. 154: 211-218, 1987. 16. WALKER, P. M., T. F. LINDSAY, R. LABBE, D. A. MICKLE, AND A. D. ROMASCHIN. Salvage of skeletal muscle with free radical scavengers. J. Vast. Surg. 5: 68-75, 1987. 17. WOLF, M. B., P. D. WATSON, AND D. R. C. SCOTT. Integral-mass balance method for determination of the solvent drag reflection coefficient. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): Hl94H204,1987.
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