role of polymorphonuclear leukocytes in cardiovascular depression and cellular injury in hemorrhagic...

ELSEVIER

-~ Original Contribution

Free Radical Biology & Medicine, Vol. 21, No. 5, pp. 609-618, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved

0891-5849/96 $15.00 + .00

PII S0891-5849(96)00156-6

ROLE OF POLYMORPHONUCLEAR LEUKOCYTES IN CARDIOVASCULAR DEPRESSION AND CELLULAR INJURY IN HEMORRHAGIC

SHOCK AND REINFUSION

RAKESH KAPOOR and KAILASH PRASAD

Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

(Received 25 April 1995; Revised 2 October 1995; Re-Revised 12 December 1995; Accepted 22 March 1996)

Abs t rac t - -We investigated the role of polymorphonuclear leukocytes (PMNLs) in cardiac depression and cytotoxicity during hemorrhagic shock and reinfusion. The dogs were assigned to four groups: I (sham), 4 h duration; II, 2 h of shock followed by reinfusion for 2 h; III, shock and reinfusion in neutrophils depleted with immune serum; IV, same as III but pretreated with nonimmune serum. Cardiac function and contractility were depressed during shock while plasma creatine kinase (CK), and CK-MB increased. Reinfusion tended to return hemodynamic parameters towards control values while oxygen free radical producing activity of PMNLs, plasma CK, and CK-MB increased further. Cardiac malondialdehyde (lipid peroxidation product) and superoxide dismutase activity were higher while left ventricular chemiluminescence was lower in group II as compared to group I. Despite the increase in the antioxidant reserve and antioxidant enzymes, there was oxidative damage. PMNL depletion attenuated the deleterious effects of shock and reinfusion on the hemodynamic and biochemical parameters. The changes in group IV were similar to those in group II. These results suggest that PMNLs may partly be involved in the deterioration of cardiac function, and contractility and cellular injury during hemorrhagic shock and reinfusion.

Keywords--Hemorrhagic shock, Oxygen free radicals, Cardiac function, Cardiac contractility, Antioxidant enzymes, Malondialdehyde, Polymorphonuclear leukocyte chemiluminescence, Polymorphonuclear leukocytes depletion

INTRODUCTION

Po lymorphonuc lea r leukocytes (PMNLs) are phagocyt ic cel ls that take part in in f lammatory react ions in the body. They must be ac t iva ted to pe r fo rm their phagocyt ic and inf lammatory function. Once act ivated, they undergo a respira tory burst with concomi tan t release o f oxygen free radicals (OFRs) inc luding supe- rox ide anion (O2-), hydrogen perox ide (H202), hy- d roxyl radicals ( 'OH) , and hypoch lorous acid (HOC1). 1 These OFRs are cyto toxic and cause l ip id peroxida t ion and prote in oxida t ion leading to cell death. 2-4 Indis- c r iminate act ivat ion o f P M N L s and resul tant re lease of OFRs may lead to t issue damage. P M N L s may also cause t issue damage by nonoxida t ive processes that in- c lude re lease o f proteolyt ic enzymes , sequestrat ion in the capi l lar ies , venules and arter ioles leading to b lock-

Address correspondence to: K. Prasad, M.D., Ph.D., Department of Physiology, College of Medicine, University of Saskatchewan, 107 Wiggins Ave. Saskatoon, Sask. S7N 5E5, Canada.

ade o f the capi l lar ies , and, hence, the inhibi t ion o f b lood flow to the affected organs/areas. 5 P M N L s have been impl ica ted in many pa thologica l condi t ions in- c luding i s c h e m i a - r e p e r f u s i o n injury. 5-7 It has been shown that ei ther deple t ion o f neutrophi ls pr ior to ischemia 8 or b lockade o f neu t roph i l - endo the l i a l cell in- teract ions with monoc lona l ant ibodies 6 resul ted in at- tenuat ion of injury and myocard ia l infarct size during reperfus ion fo l lowing ischemia.

W e have shown that during HS there is an increase in myocard ia l ant ioxidant reserve and ant ioxidant enzyme [superoxide d ismutase (SOD)]. 9 In spite o f an increase in ant ioxidant enzyme act ivi ty and ant ioxidant reserve, damage was infl icted on heart and other tis- sues. W e have also shown that ant ioxidants [SOD + cata lase (CAT)] at tenuated the oxida t ive stress and as- socia ted changes in ant ioxidant reserve and ant ioxidant enzymes and preserved cardiac function and contrac- til i ty. 9,t° Potent ial sources for increase in the levels of OFRs during HS include the x a n t h i n e - x a n t h i n e

609

610 R. KAPOOR and K. PRASAD

oxidase system,]~ increased prostaglandin synthesis,J2 catecholamine autooxidation] 3'~4 and activation of PMNLs. l During HS and reinfusion, various activators of PMNLs including arachidonic acid metabolites (leukotriene B4; LTB4), interleukin (IL) 1 and 6, tumor necrosis factor (TNF), and platelet activating factor (PAF)~5 17 are produced in increased amounts. Contribution of PMNLs to oxidative stress during HS and reinfusion is not known. It is possible that PMNLs may be the major source of OFRs during shock. I f so, then the depletion of PMNLs should attenuate the deleterious effects of HS and reinfusion on heart and other cells.

The present studies were therefore undertaken to in- vestigate the contribution of PMNLs in oxidative stress during HS and reinfusion. To achieve this objective, we investigated the effects of HS and reinfusion on cardiac function and contractility, OFR-producing activity of PMNLs (PMNL-CL), plasma creatine kinase (CK), and CK-MB activity and cardiac MDA content, chemiluminescence (an index of antioxidant reserve), and activity of antioxidant enzymes [SOD, CAT, glu- tathione peroxidase (GSH-Px)] in control and PMNL- depleted dogs.

MATERIALS AND METHODS

Mongrel dogs of either sex that weighed between 1 6 - 2 4 kg were anesthetized with pentobarbital sodium (30 mg/kg intravenously) and intubated with a closed- cuff endotracheal tube. The lungs were ventilated with room air by means of a Harvard respirator (Harvard Apparatus, Inc., S. Natick, Mass.) at 20 ml/kg and a respiratory rate of 20 breaths/min. Additional pentobarbital sodium (5 mg/kg) was administered intravenously as required.

Experimental protocol

Dogs were randomly assigned to four groups. Group I (sham 4 h, n = 5), dogs were anesthetized, catheter- ized, and hemodynamic and biochemical parameters were measured for 4 h. Group II (shock for 2 h followed by reinfusion for 2 h, n = 14), shock was produced by bleeding the dogs into standard blood bank bags (containing 63 ml of anticoagulant citrate, phosphate, dex- trose, and adenine solution for 450 ml blood) to lower the mean arterial pressure to 50 _+ 5 mmHg. The shock was maintained at 50 + 5 mmHg for 2 h by further bleeding or reinfusing the shed blood. After 2 h of shock the shed blood was reinfused through the right femoral vein over a period of 20 to 30 min and the dogs were observed for a further period of 2 h. Group III (I- Serum + shock 2 h + reinfusion 2 h, n = 6), 5 ml

immune sera (I-Serum) containing antibodies to dog neutrophils was injected intravenously. After 1 h of injection of I-Serum, the dogs were subjected to shock and reinfusion as in group II. Two milliliters of I-serum was given 20 min before reinfusion. Additional amounts of 0.75 ml I-serum were given every 30 rain after reinfusion. Group IV (NI-Serum + shock + reinfusion, n = 6) was similar to group III except for injection of non-immune serum in the amounts similar to those for I-serum in group III. Hemodynamic measurements and collection of blood samples for PMNL-CL, CK, and CK-MB were made before hemorrhage, at 60 and 120 min of induction of shock, and at 15, 60, and 120 min after completion of reinfusion of shed blood. At the end of the protocol, the heart was removed for measurement of MDA, muscle chemiluminescence, and antioxidant enzyme activity.

Hemodynamic measurements

Hemodynamic measurements were made as previously described. 3 A Swan-Ganz triple-lumen catheter (7F) (Baxter Health Care Corp. Edwards Div., Santa Ana, CA) equipped with a thermistor tip was placed in the pulmonary artery through the right femoral vein to measure pulmonary arterial pressure (PA), pulmonary capillary wedge pressure (PAW), and cardiac output. Cardiac output was measured in triplicate with an Ed- wards Cardiac Output Computer (COM-1, Edwards Co. Inc., Farmington, Conn.). A 7F Cournand catheter (Cordis Corp., Miami, FL) was positioned in the left ventricle through the right femoral artery to record left ventricular pressure. The first derivative of left ventricular pressure (dp/dt) was recorded with a differentiating device coupled to left ventricular pressure at a fre- quency response of 100 Hz. A similar Cournand catheter was placed in the right atrium through the external jugular vein to record mean right atrial pressure (mRAP). Third 7F Cournand catheter was positioned in the aorta through the right common carotid artery to record aortic pressure (AO). All pressures were recorded with Viggo-Spectra-Med P 10EZ pressure trans- ducers (Viggo-Spectramed, Oxnard, CA) and a dyno- graph recorder (Backmann R611). All pressures and ECG (Lead II) were monitored simultaneously. The left ventricular end diastolic pressure (LVEDP), and the ra- tio of (+) dp/dt at CPIP (common peak isovolumic pressure) to PAW, which is not affected by preload and afterload 9J° were used as indices of myocardial contractility. Cardiac index (CI), total systemic vascular resistance (TSVR) and pulmonary vascular resistance (PVR) were calculated as previously described. 9 CI was used as an index of myocardial function.

Role of polymorphonuclear leukocytes in cardiovascular depression 611

Isolation of PMNLs

PMNLs were isolated from dog blood by density gradient using Ficoll-Hypaque. 18 This method yielded a population of cells containing 98% neutrophils and 2% other blood cell types. The viability was more than 97% as determined by a dye exclusion test. These neutrophils were used to raise the antibodies in rabbits.

nized zymosan, which results in their activation. On activation, the PMNLs undergo respiratory burst with concomitant release of OFRs. These OFRs emit chemiluminescence, which is amplified with luminol. The luminol amplified PMNL-CL was measured in whole blood using a luminometer (LKB-1251 model, LKB Wallac, Turku, Finland) by previously reported method 4'9 and expressed as mv-min/106 PMNLs.

Preparation of antibodies against dog neutrophils

Antibodies to dog neutrophils were raised in rabbits as previously reported. 8 1 × 108 neutrophils were suspended in Freund's complete adjuvant (containing 1 mg heat killed and dried Mycobacterium tuberculosis, 0.85 ml paraffin, 0.15 ml mannide monooleate per ml of adjuvant) and injected intradermally to the rabbits. After l0 d, a challenge dose of 1 × l 0 7 neutrophils suspended in incomplete Freund's adjuvant (containing 0.85 ml paraffin, 0.15 ml mannide monooleate per ml of adjuvant) was injected intradermally. Ten days after injection of the challenge dose, the rabbits were bled through the mid ear artery into sterile vacutainer tubes. The blood was allowed to coagulate for 1 h. The serum was separated by centrifugation at 3000 rpm for 15 min. The serum was heated at 60°C and termed as immune serum (I-Serum). The nonimmune serum was ob- tained from rabbits that were not injected with dog neutrophils. The immune- and nonimmune sera were collected fresh on the day of experiment.

Cardiac muscle chemiluminescence

This is a technique in which the tissue is challenged with an oxidant (tert-butyl hydroperoxide). The oxidant releases OFRs, which react with the substrates in the reaction mixture (tissue homogenate) and emit chemiluminescence, which is amplified by luminol. Antiox- idants present in the tissue react with OFRs and prevent chemiluminescence. The net chemiluminescence observed depends on the level of antioxidants present in the tissue. The tissue with higher antioxidants will give lower chemiluminescence and vice versa. Hence, this parameter is a measure of antioxidant reserve in the tissue. Luminol dependent cardiac tissue chemiluminescence in the presence and absence of tert-butyl hydroperoxide was determined using a luminometer (LKB-1251 model, LKB Wallac, Turku, Finland). 19 The results were expressed as mv.s/mg protein.

CK and CK-MB

Lipid peroxidation products [thiobarbituric acid- reactive substances, MDA]

The OFRs react with membrane phospholipids and yield malondialdehyde (MDA). Measurement of MDA levels in a tissue indicates the oxidative damage and, hence, MDA is an indirect measure of levels of OFRs. The cardiac tissue lipid peroxidation was measured as thiobarbituric acid reactive substances by previously reported method 9 and expressed as nmol of MDA/mg protein.

PMN leukocyte count and chemiluminescence

Mixed venous blood was collected in ethylenedi- amine tetraacetic acid (EDTA)-containing tubes for measurement of PMN leukocyte counts and chemiluminescence. PMNL counts were made using a Techn- icon H6000 ~ System (Technicon Instruments Corp., Tarrytown, NY).

PMNL-chemiluminescence (PMNL-CL) is a measure of free radical producing activity of PMNLs. In this techniques, the PMNLs are challenged with opso-

CK and CK-MB were measured using S.V.R. CK reagent test kit (Behring Diagnostics, La Jolla, CA). 2° Creatine kinase-isoenzymes were separated by column chromatography. 2j

Measurement of catalase, GSH-Px, and SOD activity

Catalase, GSH-Px, and SOD activity in supernatant was measured as previously described. ~9 Mn-SOD was measured in the presence of potassium cyanide solution. CuZn-SOD activity was determined by subtract- ing Mn-SOD from total SOD.

Statistical analysis

The data was analyzed by two-way analysis of vari- ance (ANOVA) using repeated measure followed by a test of least significant difference (LSD). 22 For comparison within the group, one-way ANOVA was used. Unpaired Student's t-test was used to analyze the cardiac tissue MDA, chemiluminescence, and antioxidant enzymes. The difference was considered significant if p value was less than .05.

200

R. KAPOOR and K. PRASAD

Table 1. Preshock Values of (-)dp/dt, dp/dt at CPIP/PAW, CI, TSVR and PVR in Four Groups of Dogs

Parameter Group I Group II Group III Group IV

(-)dp/dt (mmHg/s) 5130.00 -+ 810.00 4085.00 ± 291.00 3446.00 ± 349.00 4253.00 _+ 613.00 dp/dt at CPIP/PAW (s -t) 194.41 ± 8.81 295.64 + 31.64 247.67 ± 26.57 194.14 ± 35.75 CI (L/rain/m:) 4.26 ± 0.58 3.81 ± 0.25 3.51 _+ 0.50 3.58 ± 0.32 TSVR (dynes.cm -5) 3510.00 ± 341.00 3457.00 ± 213.00 3994.00 ± 575.00 4347.00 _+ 529.00 PVR (dynes.s .cm ~) 424.00 _+ 51.00 483.00 _+ 28.00 625.00 ± 63.00 649.00 ± 76.00

The results are expressed as mean ± SEM. CI, cardiac index; TSVR, total systemic vascular resistance.

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Fig. 1. Changes in the mean aortic pressure (mAo) and left ventricular end-diastolic pressure (LVEDP) in four groups of dogs. The changes in mAo at " 0 " min indicate the pressure after hemorrhage to shock level. The results are expressed as mean _+ SEM. Group I, sham 4 h; Group II, (S + R), shock 2 h followed by 2 h of reinfusion; group lII, (I-Serum + S + R) similar to group II but PMNLs depleted by pretreatment with rabbit immune serum against dog neutrophils; group IV, (NI-Serum + S + R), similar to Group III but treated with nonimmune rabbit serum (containing no antibodies against dog neutrophils). It should be noted that LVEDP and all other parameters in subsequent figures were measured 60 min after shock and not at 0 rain after shock; hence, lines are drawn from 0 min to 60 min shock period. The preshock values are represented at 0 min. *p < .05, comparison of values at various time intervals with respect to preshock values in the respective groups; +l~ < .05, group I vs. groups II, III and IV; ap < .05, group II vs. groups III and IV; bp < .05, group III vs. group IV.

RESULTS

The initial vo lume of b lood wi thdrawn to lower the mean arterial pressure to 50 + 5 m m H g in shock groups was about 27 ml/kg, and the total volume wi thdrawn at the end of 2 h o f shock was about 45 ml/kg. These values in different groups were not different f rom each other.

Hemodynamics

The treatment with I -serum led to a decrease in P M N L count from 6.57 _+ 1.42 × 109 to 1.54 _+ 0.30 × 109/1, which amounted to a decrease of 72.55 + 5.44%. The I -serum produced a fall in mean aortic pressure (mAo) , which recovered by 40 min. I -serum treatment led to a fall in L V E D P (from 20.83 _+ 3.72 to 12.92 _+ 3.84 mmHg) , CI (from 5.34 _+ 0.78 to 3.51 _+ 0.50 I/rain/m2), but PVR was increased (from 473 _+

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Fig. 2. Changes in cardiac index (CI) in four groups of dogs. The results are represented as mean ± SE of percent changes from control at 0 rain taken as 100%. Other notations are similar to those in Fig. 1. *p < .05, Comparison of values at different times with respect to " 0 " rain in the respective groups; ~p < .05, group I vs. other groups; ap < .05, group II vs. groups III and IV; Up < .05, group III vs. group IV.


44 to 625 _+ 63 dynes, s/cm6). The administration of NI-serum (group IV) had no effect on PMNL counts, which were 4.27 _+ 1.73 × 109 initially and 4.29 +_ 1.24 X 109 after 1 h of NI-serum administration. The NI- Serum administration caused a fall in CI (from 4.44 + 0.35 to 3.58 _+ 0.32 1/min/m=).

The preshock (0 min) values for ( - )dp/dt , dp/dt at CPIP/PAW, CI, TSVR, and PVR are summarized in Table 1.The changes in the hemodynamic parameters of the four groups are summarized in Figs. 1 to 4. Sham dogs showed no changes in hemodynamic parameters except for decreases in the CI, ( - ) dp/dt and increases in TSVR and PVR at 135 min and onwards.

Shock produced a decrease in myocardial function (CI), myocardial contractility (dp/dt at CPIP/PAW, LVEDP), rate of relaxation [ ( - ) dp/dt], and an increase in TSVR and PVR in groups II, III, and IV. The rise in TSVR and PVR during shock was less in the PMNL-

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Fig. 3. Changes in dp/dt at common peak isovolumetric pressure/ PAW (dp/dt at CPIP/PAW) and ( - ) dp/dt in four groups of dogs. The results are expressed as mean ± SEM of percent change from control at 0 min taken as 100%. Other notations are similar to Fig. 1. *p < .05, comparison of values at various times with respect to preshock values at " 0 " rain in the respective groups, tp < .05, group I vs. other groups, ap < .05, group II vs. groups III and IV. bp < .05, group III vs. group IV.

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Fig. 4. Changes in the total systemic (TSVR) and pulmonary vascular resistance (PVR) in the four groups of dogs. The results are expressed as mean ± SEM of percent changes from control at 0 minute taken as 100%. Other notations are similar to those in Fig. 1. *p < .05, Comparison of values at various time intervals with respect to " 0 " min in the respective groups; tp < .05, group I vs. other groups; "p < .05, group II vs. groups III and IV; bp < .05, group III vs. group IV.

depleted group compared to that in groups II and IV. TSVR and PVR throughout the shock were higher in group IV than in group III.

All the hemodynamic parameters in group II tended to return towards the preshock values following reinfusion of shed blood except for dp/dt at CPIP/ PAW, which remained unchanged, mAo, CI, and ( - ) dp/dt declined significantly below preshock values after an initial return to preshock values following reinfusion. LVEDP increased progressively during the postinfusion period reaching above preshock values by 240 rain.

Changes in hemodynamic parameters in the PMNL- depleted group III following reinfusion were qualita- tively similar to those in group II. There was complete recovery of mAo and ( - ) dp/dt. LVEDP returned to preshock values after an initial transient rise. CI returned to preshock values but at 240 min it was lower than preshock values. (+) dp/dt at CPIP/PAW showed a progressive rise above shock levels which recovered


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Fig. 5. C h a n g e s in p l a s m a C K and C K - M B act iv i ty in four g roups o f dogs . The resul ts are expressed as mean _+ S E M o f pe rcen t c h a n g e s f rom cont ro l (p reshock) va lues at " 0 " rain taken as 100%. O t h e r no ta t ions are s imi la r to those in Fig. 1. *p < .05, C o m p a r i s o n o f va lues at d i f ferent t imes wi th respect to p r e s h o c k va lues (0 min) in the respec t ive groups ; +p < .05, g r o u p l vs. o ther g roups ; "p < .05, g r o u p II vs. g roups Ill and IV; bp < .05, g r o u p III vs. g r o u p IV.

to preshock levels by 240 min. The values for CI and ( - ) dp/dt throughout the postreinfusion period, and dp/ dt at CPIP/PAW at 240 min were significantly higher in group III than in group II. TSVR and PVR returned

to preshock values. TSVR was lower at 180 min on- ward in group III than in group II.

Reinfusion in group IV tended to return all the hemodynamic parameters to preshock values; however, LVEDP and PVR throughout the postreinfusion period were higher than preshock values, mAo recovered from 180 min onwards, while ( - ) dp/dt and (+) dp/dt at CPIP/PAW did not recover to preshock values. CI and TSVR returned to preshock values immediately following reinfusion but CI declined soon thereafter, while TSVR increased progressively.

(+) dp/dt at CPIP/PAW at 240 min was lower in group IV than in groups II and III. The values for LVEDP were higher and ( - ) dp/dt were lower in group IV than in group III.

In general, the extent of recovery of hemodynamic parameters following reinfusion was less in group II and IV as compared to group III.

Polymorphonuclear leukocyte (PMNL) counts

PMNL counts remained unaltered in the sham group but increased following reinfusion in groups II and IV. These counts in group Ill remained lower than in groups II and IV throughout shock and reinfusion (Table 2).

Plasma CK and CK-MB

The preshock values of plasma CK in U/L in groups I, II, III, and IV were 53.77 _+ 4.04, 47.28 _+ 2.20, 40.32 _+ 2.72, and 42.67 _+ 4.70, while those of CK-MB in U/L were 12.55 _+ 1.45, 8.01 _+ 1.24, 5.00 _+ 0.34, and 3.95 _+ 1.11, respectively. The percent changes are shown in Fig. 5. Plasma CK activity increased from 120 min onwards, while CK-MB activity remained unchanged in group I. CK and CK-MB increased progressively during shock. Reinfusion caused a massive increase in activity of both enzymes in groups II and

Tab le 2. C h a n g e s in P o l y m o r p h o n u c l e a r L e u k o c y t e ( P M N L ) C o u n t in F o u r G r o u p s o f Dogs

S h o c k Rein fus ion

G r o u p 0 rain O D 60 min 120 min 135 rain 180 min 240 min

S h a m 3.91 _+ 0 .86 2 .76 ± 1.02 2.95 + 1.27 3.95 _+ 1.87 4 .72 + 2 .10 5 .54 + 2.26 S + R 3.30 _+ 0 .54 3 .76 + 0 .40 5 .44 ÷ 0 .96 + 5.75 + 0 .79 *~ 6 .26 + 0 .99* 6.78 _+ 1.02" I -Se rum 6.57 _+ 1.42 1.54 _+ 0 .30* 1.33 _+ 0.31 *! 1.78 _+ 0.41 *~" 1.31 _+ 0 .29 *'~ 1.47 _+ 0 .40 *~' 1.48 _+ 0.36"*" N I - S e r u m 4 .30 _+ 1.70 4.05 _+ 1.44 4 .53 + 1.40 h 6 .45 ÷ 2.41 tb 7.05 + 2.45 +b 7 .99 + 2.93 +b 6 .94 +1 .91 +b

The results are exp res sed as mean _+ SE. O D is the t ime af ter 1 h o f i m m u n e or n o n i m m u n e se rum admin i s t ra t ion , pr ior to shock. * p < .05, c o m p a r i s o n o f va lues at var ious t ime intervals wi th respec t to p r e s h o c k (0 min) va lues in the respec t ive g roups . +p < .05, g r o u p I vs. o the r g roups . a p < .05, g r o u p II vs. g r o u p s IIl and IV. b p < .05, g r o u p Ill vs. g r o u p IV.


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Fig. 6. Changes in polymorphonuclear leukocyte chemiluminescence (PMNL-CL) in four groups of dogs. The results are expressed as mean +_ SEM of percent changes from control at " 0 " min taken as 100%. Other notations are similar to those in Fig. 1. *p < .05, Comparison of values at different time intervals with respect to preshock values (0 min) in the respective groups; tp < .05, group I vs. other groups; "p < .05, group II vs. groups III and IV; bp < .05, group III vs. group IV.

Left ventricular chemiluminescence (L V-CL)

The LV-CL of the four groups is summarized in Fig. 7. The LV-CL of PMNL-depleted shock group was similar to that of sham group. It decreased in groups II and IV compared to group I (sham). These results suggest that antioxidant reserve increased in group II and IV but remained unchanged in PMNL-depleted group compared to the sham group.

Left ventricular SOD activity

Changes in the total SOD, CuZn-SOD, and Mn- SOD activity in the four groups are shown in Table 3. Total SOD activity increased in groups II, III, and IV compared to group I; however, the increase in group III was lower than in group II.

CuZn-SOD activity showed similar rise in groups II, III, and IV as compared to group I.

Mn-SOD activity in groups II and IV was higher than that in group I. PMNL-depleted shock group had similar activity to sham group.

Catalase and GSH-Px activity

Catalase and GSH-Px activities were similar in all groups (Table 3).

IV. Activity was significantly lower in group III than in groups II and IV following reinfusion.

PMNL-chemiluminescence (PMNL-CL)

The initial values for PMNL-CL in mv-min/106 PMNL in groups I, II, III, and IV were 1113 _+ 242, 790 _+ 31,753 + 86, and 748 +_ 154, respectively. The changes are summarized in Fig. 6. PMNL-CL activity remained unaltered in the sham and PMNL-depleted shock group (III) throughout, but decreased during shock in groups II and IV. Reinfusion produced a massive and progressive increase in PMNL-CL activity in groups II and IV. The activity in group III was significantly lower than that in groups II and IV from 135 min onwards.

Lipid peroxidation levels [malondialdehyde (MDA )]

The cardiac lipid peroxidation levels (MDA content) of PMNL-depleted dogs were similar to that of sham dogs but were lower than that in dogs subjected to shock and reinfusion in the untreated group or NI-serum-t rea ted group (Fig. 7).

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1 0 0 0 0

5000

Fig. 7. Left ventricular malondialdehyde (LV-MDA) content and chemiluminescent activity (LV-CL) in four groups of dogs. The results are expressed as mean _+ SEM. Other notations are similar to those in Fig. 1. *p < .05, group I vs. other groups, tp < .05, group II vs. group III and IV. ap < .05, group III vs. groups IV.


Table 3. Effect of Shock and Reinfus ion on Cardiac Ant iox idan t Enzyme Act iv i t ies in Four Groups of Dogs

Group Total SOD CuZn-SOD Mn-SOD C A T GSH-Px

Sham 31.28 _+ 2.75 17.65 _+ 2.02 13.63 _+ 1.21 0.00554 _+ 0.00036 0.0175 _+ 0.0020 S + R 52.08 + 3.04* 27.13 _+ 2.58* 24.95 + 1.64" 0.00465 _+ 0.00060 0.0177 _+ 0.0012 I -Serum 41.54 _+ 4.04 ** 27.07 _+ 3.45* 14.48 _+ 1.38 + 0.00452 _+ 0.00054 0.0160 + 0.0043 NI-Serum 46.53 + 3.01" 28.81 _+ 2.55* 17.72 _+ 1.26" 0.00611 _+ 0.00097 0.0190 _+ 0.0035

The results are expressed as mean __+ SE. * p < .05, group I vs. group II, III, and IV.

p < .05, group II vs. group III and IV.

D I S C U S S I O N

Hemorrhagic shock was induced by withdrawal of blood to lower the mAO to 50 _+ 5 mmHg. This method is comparable to previously published methods. 9 ~0,23

The decreases in cardiac function and contractility and increases in systemic and pulmonary vascular resistance were similar to that previously observed. 9-1°'24 The decreases in myocardial function and contractility during shock could be mediated by low circulating blood volume, leukotrienes, and prostaglandins, j7 Ca 2+ overload, 25 cardiac-depressant factors, 26 or OFRs. 34.9-10 PMNLs may be one of the sources of OFRs during shock. In the present studies, the cardiac function during shock in the PMNL-depleted (I-serum) group was higher than that in the NI-serum group, though it was still depressed. Significantly reduced circulating blood volume causes improper filling of ventricles leading to depression of cardiac function during shock.

The peripheral and pulmonary vascular resistances increased during shock in all the groups, but the increase was less in the PMNL-depleted group. This could be due to increased sympathetic tone or increased levels of OFRs. Oxyradicals (02 and H202) produce contraction of the vascular system, 27"2s which could be mediated by their direct action on the vascular smooth muscle or by inactivation of endothelium derived relaxing factor. 29 PMNLs may also aggregate in capillaries during shock 5 and impede the blood flow besides locally releasing OFRs and proteolytic enzymes, which damage the vascular endothelium. PMNL depletion, by preventing the above-mentioned effects, attenuated the rise in TSVR and PVR.

Reinfusion is essential to restore tissue perfusion in HS. However, reinfusion in irreversibly-shocked pa- tients and animals has been shown to produce deterioration after an initial transitory improvement. 23'25'3° This is a classic example of ischemia/reperfusion injury because during shock, splanchnic organs become ischemic immediately and with maintained shock, the heart and brain also become ischemic. Recovery of cardiac function and contractility after reinfusion of blood was significantly greater in the PMNL-depleted group than in the other two shock groups. These observations

suggest that PMNLs contribute to cardiac depression. The PMNLs may become aggregated during hypovo- lemic conditions and permanently clog the capillaries. 5 Following blood reinfusion, the perfusion to the affected organs is not maintained and the trapped PMNLs may release the OFRs locally and cause damage to vascular endothelium and myocytes. LVEDP was higher than preshock values in groups II and IV following reinfusion. This could be due to changes in diastolic compliance of the ventricle. The decrease in cardiac output in spite of increased LVEDP indicates impend- ing cardiac failureY In the PMNL-depleted shock group, LVEDP returned to preshock values following reinfusion, suggesting that this group did not develop myocardial failure.

The decrease in ( - ) dp/dt could be due to a decrease in myocardial adenosine triphosphate (ATP) level or a decreased Ca 2+ uptake by the sarcoplasmic reticular pump. OFRs inhibit calcium uptake by inhibiting the calcium pump. 31'32 Decrease in ( - ) dp/dt reflects a decrease in left ventricular compliance. 33 It, thus, appears that diastolic compliance is decreased in the untreated and NI-serum-treated groups during shock and reinfusion, while in the I-serum-treated group it decreased only during shock.

The true index of myocardial contractility, (+)dp/dt at CPIP/PAW, a parameter that is independent of the effects of pre-and afterload remained depressed following reinfusion in groups II and IV. The the I-serum group showed complete recovery of myocardial contractility at 240 min. These beneficial effects of PMNL depletion further suggest the role of PMNLs in myocardial depression during shock and reinfusion. Rom- son et al. 8 have also shown that PMNL depletion at- tenuates the myocardial injury following myocardial ischemia.

In the 4-h sham group CI decreased while TSVR and PVR increased. Flynn et al.12 also observed a fall in cardiac output and a rise in systemic vascular resistance in sham-operated dogs. Alteration in these parameters could be due to catheterization, which would stimulate the complement system. Components of complement, especially Csa, stimulate PMN leukocytes to


release free radicals (which depress myocardial function and contractility 3"4) and other vasoactive agents. 34 Another possibility is that these animals required more maintenance doses of pentobarbitone, a myocardial depressant, to maintain the anesthesia.

The increase in plasma CK activity without alteration in CK-MB in the sham group suggests that the myocardium was not damaged, the rise being due to tissue trauma and cardiac catheterization. 25 Shock for 2 h produced an increase in plasma CK and CK-MB activities, and these activities increased markedly following reinfusion after 2 h of shock. Similar results have previously been reported. 9-~°'36 CK and CK-MB are intracellular enzymes that are released into plasma following cell injury and cell death. The PMNL depletion by reducing the levels of OFRs attenuated the shock/reinfusion-induced cell injury leading to a lower rise in CK and CK-MB activity.

An increase in cardiac MDA (indirect measure of levels of OFRs) level in groups II and IV suggests oxidative damage. PMNL depletion in group III pre- vented a rise in cardiac MDA levels by eliminating a source of free radicals.

The OFR producing activity of PMNLs (PMNL-CL) decreased during shock in groups II and IV. This decrease cannot be explained at present but may be due to production of inhibitory modulators during shock. An increase in PMNL-CL following reinfusion in groups II and IV could be due to priming of the PMNLs by LTB4, PAF, TNF, and interleukin. 37-39 The PMNL- CL did not change in the I-serum group during shock and following reinfusion. Similar changes would have been expected in PMNL-CL in the I-serum group. It is possible that PMNL-antibodies rendered the unde- pleted PMNLs hypofunctional. This data further suggests that remaining PMNLs failed to produce free radicals during shock and reinfusion in PMNL-depleted group.

A decrease in left ventricular chemiluminescence in groups II and IV as compared to group I indicates an increase in antioxidant reserve during shock and reinfusion in these groups. This is supported by the findings that total, CuZn, and Mn-SOD activity increased in these groups. The increase in cardiac MDA in these groups, despite increased antioxidant reserve and antioxidant enzymes, suggests that OFRs production increased much more than could be handled by increased antioxidants. The increased SOD activity in these groups would have led to an increased production of hydrogen peroxide by dismutation of superoxide anions. The increased hydrogen peroxide levels would fa- vor production of highly toxic hydroxyl radicals by the Haber-Weiss reaction leading to lipid peroxidation. 4°'41 LV-CL in the I-serum group was not signifi-

cantly different than that in sham group, which suggests no change in total antioxidant reserve. There was an increase in total and CuZn-SOD activity in group III, with no change in Mn-SOD activity. The rise in only CuZn-SOD activity suggests that there was a small oxidative stress on the myocardium in this group that could be due to the production of OFRs from other sources. The increase in OFRs levels would then have been metabolized by increased CuZn-SOD and could not mediate oxidative damage.

CONCLUSION

The results of the present study clearly demonstrated for the first time that the PMNLs contribute to oxidative stress on the myocardium during HS and reinfusion. This oxidative stress lead to an increase in cardiac MDA levels and cardiac SOD activity and plasma CK and CK-MB activities. The oxidative cellular injury contributed to cardiac depression during shock and reinfusion. PMNL-depletion attenuated oxidative stress, thereby reducing oxidative cellular injury and preserving the cardiac function and contractility. In- complete protection in the PMNL-depleted group suggests that factors other than PMNLs also contribute to cellular injury and cardiac depression during shock and rein fusion.

Acknowledgements - - This work was supported by a grant from the Heart and Stroke Foundation of Saskatchewan, Saskatoon, Canada.

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