Is reperfusion injury an important cause of mucosal damage after porcine intestinal ischemia?

Is reperfusion injury an important cause of mucosal damage after porcine intestinal ischemia? Anthony Robert

T. Bliislager, A. Argenzio,

DVM, Malcolm PhD, Kaleigfz ard

C. Roberts,

BVSc,

PhD,

J. Marc

Rhoads,

MD,

mzd

Cl~.ap~l Hill, N.C.

Background. Intestinal

ischemic injuly is exacerbated 0)) rept@sion in rodent and feline models because of xanth.ine oxidase--initiated T-ea.&e oqgen metabolite formation and nezttroph.il infiltration. Studies were conducted to determine the relevance of repe$usion injuq in the juven.ile pig, whose low levels of xa.nthin.e oxidase are similar to those of the hu ma:n being. Methods. Ischernia was induced b) mea,ns of complete mesenteric a.rteria.1 occlusion, volvulus, 07 h.emorrhagic shock. Inju.q was assessed by means of histologic examination and measurement of lipid pn-oxidation. In a.ddition, nqeloperoxidase, as a. marker of neuhDphi1 inJiltration, a,nd xan thine oxidase-xanth ine deh#rogenase were measwred. Results.SigniJrcant ischemic injq was evident after 0.5 to 3 hours of complete mesentel-ic occlusion 01 2 hou,rs of shock or volvulus. In none of these models was the ischemic injuq worsened by reperfusion. To maximize superoxide produ.ction, pigs were ventilated on. 100% 02, but on& limited ,reperftLsion in&q (1.2-fold increase in histolog’c grade) was noted. Xanthi,ne oxidase-xanthine dehydrogenase levels were negligible (0.4 ? 0.4 m Lvgm). Conclusions.&pe@ion injuq may not plajl an impol-tant role in intestinal injuq under conditions of complete rnesenteric ischemia and low-flow sta.tes in the pig. This may result f&m lo-w xanthine oxidase-sa~nthine dehydrogen,ase levels, which a:re similar to those found in the humari being. (Surge? 1997;121:526-34.) From the Depa&nents of Anatoq PI2ysiological Sciences, and Radziology, and Food dnimal and Equirle Medicine, College of \%terinnry Illedicine, North Carolina State L’niversity, Ra.leigh, and Depaytmetz t of Prrliatlics, l%;il2iversityof .Vorfh Carolina, Chapel Hill, N C.

THE ROLE OF IXHE~ILA and reperfusion in intestinal microvascular damage has been well characterized,‘” but the mechanisms that lead to intestinal mucosal damage under clinical conditions (complete mesenteric vascular occlusion, shock, and volvulus) are not well established. Severe intestinal mucosal injury accompanies ischemia7-” and may be exacerbated during reperfusion because of generation of reactive oxygen metabolites by the intestinal enzyme xanthine oxidase and neutrophilic reduced nicotinamide adenine dinucleotide phosphate oxidase. ‘l-l4 Tissues are primed for reperfusion injuly during ischemia because xanthine dehydrogenase is converted to xanthine oxidase, and its sub

Supported

by United

States

Department

of.Xgriculture

grant

9-I-37201-

0448. Accepted

for publication

Replint requests: Medicine, North leigh, NC 27606. Copyright

% 1997

0039.6060J'9i.'f5.00

526

YJov. 8, 1996.

AnthonyCarolina

SURGERY

T. Blikslager, State Universiy,

by MosbyYear +O

11/56/79112

Book,

[)\%I. College of \~eterinan 1700 Hillsborough St., RaInc.

strate, hypoxanthine,accumulatesfromcontinuedadenosine triphosphate use. As oxygenated blood rehems to ischemic tissues, xanthme oxidase triggers repelfusion injury by producing superoxide as a by-product of hypoxanthine metabolism. The superoxide radical initiates mucosal injury, as well as inducing chemoattraction of neutrophils. ‘l-l5 Thus the present concept of reperfusion injury hinges on the fact that xanthine oxidase is central to reperfusion injuly, even though neutrophils produce far more reactive oxygen metabolites.‘4, I5 Because of the potent effect of reactive oxygen metabolite-inducedinjuryduringreperfusioninrat”. I7 and feline’3,1’ models, research has focused on preventing ischemia-reperkion injury by inhibiting or scavenging reactive oqgen metabolites.‘-3* 7, ” However, it has been suggested that not all species may be subject to the same degree of repel-fusion injury because of variation in intestinal xanthine oxidase-xanthine dehydrogenase levels. I8 For example, human beings have low levels of xanthine oxidase-xanthine dehydrogenase; therefore the role of reperfusion injury in the human being is unclear.‘”

Blikslager

W

lschemia

et al.

W

lschemia

El

1 Hr Reperfusion

527

40 -2

1

Control Hours

Hours

Fig. 1. Effect

of various periods of complete mesenteric ischemia (0.5 to 3 hours) and reperfusion (0 to 3 hours) on histologic injuq grade. Grades were assigned according to Chiu et al.‘O There was a time-dependent relationship between hours of ischemia and histologic grade. There was no significant repelfusion injuq. *p < 0.05 Yersus control: **1, < 0.01 versus control. h’umbers at bottom of col~nms car-respond to number of animals.

0

Control

1

0.5

Hours

of

2

3

lschemia

Fig. 2. Effect

of various periods of complete ischemia and reperfusion on lel-els of malonaldehyde as an indicator of lipid peroxidation. Significant elevations in malonaldehyde levels were inconsistentlv demonstrated, butt there was evidence of reperfusion injury after 3 hours of ischemia. *:p < 0.05 versus control; -& < 0.05 WI-SLIS Shour ischemic levels.

pig may

reperfusion

sion

injury.

be an ideal

injury

portedly have LVe hypothesized

der a variety

of lschemia

2 Hr Reperiusion

Fig. 4. Effect

xanthine

3

of complete ischemia and repelfusion on myeloperoxidase levels as an indicator of neutrophil infiltration. There was no significant elevation of myeloperoxidase with ischemia or reperfusion.

0.5

Hours

intestinal

2

Fig. 3. Effect

cntr1

The

1

0.5

of lschemia

in

model

buman

for beings

intestinal

ischemia-

because

low levels of intestinal xanthine that the pig would develop

reperfusion oxidase

injury

because

of

levels. This hypothesis

of conditions

designed

low

they

re-

oxidase.” minimal tnucosal

was tested un-

to ampli@

reperfu-

of complete

1

2

3

of lschemia ischemia

and repetfusion

on his-

tologic grade in pigs ventilated with 100% 02. There was a time-dependent relationship between hours of ischemia and histologic injury. In addition, there was significant reperfusion injuc after repelfusion of Z-hour ischemic tissues. “:p < 0.05 versus control; **!I < 0.01 versus control; tfi < 0.05 versus 2 hours of ischemia.

MATERIAL

AND

METHODS

Subjects and surgical procedures. This study was apbv the Institutional Animal Care and Use Committee of North Carolina State University. Six- to eightweek-old Yorkshire crossbreed pigs of both genders were used throughout the study. Pigs were anesthetized with pentobarbital and ventilated with room air via a tracheotom)~ by using a time-cycled ventilator. The jugular vein and carotid arterywere cannulated, and blood gas analysis was performed hourly to confirm normal pH and partial pressure of carbon dioxide values. Lactated Ringer’s solution was administered intravenously during anesthesia at a maintenance rate of 15 ml/kg/ proved

5. Histologic photograph of porcine ileum subjected to 2 hours of complete ischemia (A) and after 1 hour of repeAlsion (B; 1 cm Bnr, 80 pm). Pigs were xrentilated on 100% 02. Immediately after 2 hours of ischemia, there is severe epithelial sloughing from villi (A, urrows) . There was slight worsening of hiscologic injury between ischemic tissue and reperfused tissue, particularly at the level of the crypts (B, crrrows) There was no evidence of extensive neutrophil infiltration in ischemic or reperfused sections, although a mononuclear cell infiltrate is noted in crypts of reperfused tissues. Fig.

hr. Blood pressure was continuously monitored via a transducer connected to the carotid artery. Pigs were placed on a heating pad. After a ventral midline incision was made, the ileum was identified, and 6 cm loops of ileum were constructed with no. 1 silk ligatures. No attempt was made to flush the intestinal lumen. Ischemia was induced by clamping mesenteric vessels approximately 1 cm from the intestinal margin with Johns Hopkins bulldog clamps for 0.5, 1, 2, or 3 hours, followed by 0, 1, 2, or 3 hours of reperfusion. X pulse oximeter was applied to ileal loops immediately before and during reperfusion to confirm that reperfusion had taken place. Pigs were killed with an overdose of pentobarbital at the termination of the experiment. Similar experiments were subsequently performed on pigs ventilated with 100% 02. Mesenteric ischemia was created as before, and tissues were reperfused for up to 6 hours. In further experiments ischemia was induced by creating intestinal volvulus. Ileal loops were twisted 360 degrees around their mesenteric axis and maintained in that position for 2 hours, followed by repel-fusion for 0 or 1 hour. Additional pigs were subjected to hemorrhagic shock by bleeding from rhe carotid catheter into blood collection bags until the mean arterial pressure reached 45 mm Hg. This procedure took approximately 20 minutes. The pressure was maintained at this le\Fel for 2 hours by means of periodic bleeding and reducing the fluid administration rate to 7.5 ml/kg,/hr. After 2 hours pigs were resuscitated

within a 3O-minute period by means of rapid intravenous infusion of 30 ml/kg lactated Ringer’s solution in addition to all collected blood. Pigs were maintained for 1 hour after resuscitation on a fluid administration rate of 15 ml/kg/hr. Samples were collected after 2 hours of shock and 1 hour after resuscitation (corresponding to 2 hours of ischemia and 1 hour of reperfusion). Biochemical assays and histology. After resection of treatment loops, mucosal tissues were scraped with a glass slide from the seromuscular layer and snap frozen in liquid nitrogen. These samples were later thawed and assayed for myeloperoxidase as a marker of neutrophil infiltration and lipid peroxidation as an assessment of reactirTe oxygen metabolite-mediated cellular damage. Myeloperoxidase was assayed according to the method of Krawisz et al.” Acti+ was reported as milliunits myeloperoxidase activity per milligram protein. Lipid peroxidation was determined by measuring thiobarbitulic acid reactive material (nanomoles) with malonaldehyde as the standard, according to the method of Ohkawa et al.*l Activit~was reported as nanomoles malonaldehyde per milligram protein. Xanthine oxidase-xanthine dehydrogenase concentrations were assayed on the basis of methods adapted from Parks et a1.2” Tissues were harvested from neonatal 8day-old pigs and weaned T-week-old pigs. Mucosal tissue was scraped from the jejunum and ileum separately, snap frozen in liquid nitrogen, stored at -70” C, and assayed within 3 weeks. Because Parks et al. showed

Blik.slager

fl

1 Hr Reperfusion

q

2 Hr Reperfusion

Control

0.5 Hours

1 of

2

et al.

52~

3

lschemia

Fig. 6. Effect of complete ischemia and reperfusion on malonaldehyde levels in pigs ventilated with 100% 02. Significant elevations in malonaldehvde after 2 and 3 hours of ischemia were not significantI!- elevated after repelfusion. *‘I < 0.05 \.ersus control. some loss of activity with storage, the assay was repeated on fresh tissue from four 7week-old pigs. Xanthine oxidase-xanthine dehvdrogenase was simultaneously assayed in mucosal tissue from mature rats to validate the assay, because rats reportedly have high levels of these enzymes.“’ One unit of activity was defined as 1 pmol urate formed,/min. Full-thickness tissues were harvested from experimental ileal loops and preserved in 10% neutral buffered formalin. A4fter embedding tissues in paraffin, three 5 pm tissue sections from each experimental intestinal loop were stained with hematohylin-eosin. Histologic intestinal injuty was independently assessed by two blinded investigators according to a grading scale developed by Chiu et a1.l’ (grade 0, normal: grade I, ep ithelial separation at the tip of \illus; grade 2, epithelial sloughing at the tip of villus; grade 3, epithelial sloughing from approximately 50% to 75% of the surface of villus; grade 4, epithelidl sloughing ft-om the entire villus; grade 5, complete epithelial sloughing of villus and damage to the crypts). Neutrophils were counted within four random 10” pm’ areas of the mucosa of select intestinal loops by using a calibrated grid within the evepiece of a light microscope. Permeability studies. In four pigs blood-to-lumen permeability was assessed by using a 51Cr-ethylenediaminetetraacetic acid (EDTA) clearance technique during 2 hours of hemorrhagic shock and compared with clearance during 1 hour of reperfusion. Control values for 51Cr-EDTL% clearance were established in normal pigs during a 3-hour period. The ileum was flushed with 0.9%) saline solution, and ileal loops were constructed as before. Normal saline solution (10 ml) was infused into each loop. The ureters were ligated, and 100 pCi “Cl-= labeled EDTAwas injected intravenously into pigs at the onset of hemorrhagic shock. After injection of 51Cr-

Fig. 7. Histologic appearance of tissues subjected to :! hours of ischemia and 6 hours of reperfusion (1 cm Dnl; 160 pm). Note flattening and extension of epithelium across the denuded portion of the villus as result of restitution (nrrows). In addition, extensive neutrophil infiltration is present within the lamina propria (compared with tissues in Fig. 5).

labeled EDTA, arterial blood samples were obtained at 0, 1, 3, 5, 10, 15, 20, and 30 minutes and every 15 minutes thereafter to determine the mean plasma 51CrEDTA activity. After hemorrhagic shock and after 1 hour of reperfusion, ileal loops wet-e flushed with 10 ml 0.9% saline solution, and the total volume retrieved was recorded. Clearance of EDTA from plastna to loop was calculated by dividing the total loop 51Cr obtained by the mean plasma concentration during the collection period, as previously described.‘” Permeability was expressed as plasma 51Cr-EDTA (milliliters) cleared into each loop per hour. In addition, “‘Cr-EDTA clearance was compared with histologic assessment of tnucosal injury. Statistics. ,%ll datawere expressed as mean 2 standard en-or. Data were analyzed by using paired Student’s 1 tests for continuous data and rank sum test for graded data. A p value of less than 0.05 was considered significant and k value of less than 0.01 highl! significant. RESULTS There was a significant time-dependent histologic injury duting 0.5 to 3 hours of ischemia in isolated intestinal loops ($ < 0.01)) which was not significantly exacerbated with repelfusion (Fig. 1). Ileal loops in some pigs were repel-fused for up to 3 hours to see whether repelf&ion was delayed, but further inju?, was not evident (Fig. 1). To assess the contribution of reactive oxygen metabolites to injury, the level of malonaldehpde (an end-product of lipid peroxidated cell membranes) was measured. Significant elevations in malonaldehyde

530

Blikslager

et al.

Table I. Lipid peroxidation (malonaldehyde, nmol/ mg) , myeloperoxidase (mU/mg) , and histologic injury grade in pigs subjected to 2 hours of hemorrhagic shock and resuscitation (n = 4) Treatment

Control* Shock Resuscitation

Lipid peroxidation

7.1 ? 2.3 6.6 + 1.8 6.6 2 1.8

Values are mean -c SELL *Control samples were obtained

Histologic g-rade

Afyeloperoxidase

from

13.8 2 2.5 11.4 5 2.2 12.2 I 1.0 pigs before

onset

0.25 c 0.1 0.1 2 0.1 0.25 _f 0.1

of shock.

levels were noted after 3 hours of ischemia, and this was marginally but significantly exacerbated after 1 hour of reperfusion (p < 0.05; Fig. 2). However, there were no significant elevations in myeloperoxidase levels during any of the ischemic or reperfusion periods, indicating a lack of notable neutrophil infiltration (Fig. 3). In an attempt to heighten superoxide radical formation, which is partial pressure of oxygen dependent, pigs were subsequently ventilated with 100% oxygen.*’ Intestinal partial pressure of oxygen is directly related to inspired oxygen concentration.25 Time-dependent histologic injury was demonstrated (pi 0.01) during 0.5 to 3 hours of ischemia, and a small but significantly increased histologic grade of injury after reperfusion of 2-hour ischemic ileal loops ($ < 0.05) was noted (Figs. 4 and 5). Although lipid peroxidation levels were minimally but significantly elevated after 2 hours of ischemia ($ < 0.05), there was no significant exacerbation during reperfusion (Fig. 6). To assess whether reperfusion injury was a delayed phenomenon in the pig, experiments in which ischemic tissues were subjected to prolonged reperfusion (2, 3, or 6 hours) were performed. However, there was no further exacerbation of injury with 2 or 3 hours of reperfusion, and surprisingly, there was a remarkable degree of repair in those tissues reperfused for 6 hours. These tissue sections were not graded because of evidence of healing rather than further injury. Repair appeared to be a result of epithelial restitution (Fig. 7). Myeloperoxidase levels did not show significant elevations with &hernia or reperfusion until 6 hours after the ischemic period, when myeloperoxidase reached levels approximately sixfold those of control (p < 0.01; Fig. 8). Because of the importance of neutrophils to the pathogenesis of reperfusion injury, histologic sections were evaluated for the presence of neutrophils. Neutro phils were counted in histologic sections from intestinal loops subjected to 2 hours of ischemia and various periods of reperfnsion (Fig. 9). Neutrophil counts showed similar trends to myeloperoxidase data, except that there were significant increases in neutrophils in all

‘Table II. Lipid peroxidation (malonaldehyde, nmol/mg) , myeloperoxidase (mU/mg) , and histologic injury grade in pigs subjected to intestinal volvulus (n = 4) Treatment

Control Ischemia Reperfusion

Lipid per-oxidation

3.6 2 0.4 6.3 k 1.3 8.3 + 5.0

Histologic Myelopuoxidase

12.4 2 1.8 13.9 + 2.3 14.3 k 2.0

gVaae

0.2 !I 0.1 1.2 ” 0.3* 0.4 L 0.1

Values are mean L SEhC. *,b c 0.05 versus control and reperfusion.

sections compared with control and a significant increase in the number of neutrophils in sections reperfused for 2 and 6 hours compared with 2-hour ischemic tissues. In further experiments shock and intestinal volvulus were created in an effort to establish reperfusion injury because low-flow states in other species produce sub maximal ischemic injury that is markedly exacerbated during reperfusion. ‘l-l5 During shock mean arterial pressure was maintained at 45 mm Hg and was rapidly elevated to 102 + 3 mm Hg (n = 8) by means of resuscitation. Significant histologic injury was demonstrated after 2 hours of intestinal volvulus but not after 2 hours of shock (Tables I and II). Histologic injury was not exacerbated with reperfusion of ischemic tissues, and there were no significant elevations in lipid peroxidation or myeloperoxidase levels. Clearance of 51Cr-EDTA was measured to assess intestinal permeability during shock and resuscitation to further evaluate the absence of reperfusion injury seen on histologic examination. The ischemic level of permeability was lower than its respective control, suggesting that reduced blood flow to intestinal tissues during hemorrhagic shock affected uptake and clearance of 51Cr-EDTA. Although permeability was signiiicantly greater during the resuscitation period compared with the shock period, there was no significant difference between experimental groups and their respective controls (Fig. 10). Intestinal xanthine dehydrogenase-xanthine oxidase was assayed in neonatal and weaned pigs to further evaluate reported low levels of this enzyme system in neonatal pigs. lg As a control for the assay, levels of xanthine dehydrogenase-xanthine oxidase were also assessed in the mature rat, a species with known high levels.‘* Xanthine dehydrogenase-xantbine oxidase levels were minimal in neonatal and ‘I-week-old pigs as compared with mature rats (Table III). Because previous studies suggested that freezing unhomogenized tissues (as was done in this study) may decrease xanthine oxidase-xanthine dehydrogenase levels,*’ we performed

Blikslager et al.

Hours

531

of lschemia

Fig. 8. Effect of complete ischemia and reperfusion on myeloperoxidase levels in pigs ventilated with 100% 02. There were no significant elevations in myeloperoxidase with ischemia but marked increases in activity after 6 hours of reperfusion. *‘*p < 0.01 I rersus control; tp < 0.05 versus 2 hours of ischemia.

18

1

n

lschemia

H

1 Hr Reperfusion

0

2 Hr Reperfusion

q

3 Hr Reperfusion

t **

T

Control

2 Hours

of

lschemia

Fig. 9. Neutrophil counts in tissues subjected to 2 hours of complete ischemia and various periods of reperfusion. Neutrophil counts showed similar trends to results of the myeloperoxidase assay, but there were significant increases in neutrophil counts in all ischemic and repelfused tissues compared with control. In addition, there was significant evidence of neutrophil infiltration in tissues reperfused for 2 and 6 hours compared with 2-hour ischemic tissues. *p < 0.05 versus control; **p < 0.01 versus control; tp < 0.05 versus 2 hours of ischemia. assays on fresh tissues in Y-week-old pigs. This confirmed that low or nonexistent levels of xanthine oxidase-xanthine dehydrogenase were present in the pig.

DISCUSSION This study demonstrated development of severe timedependent histologic injury during 0.5 to 3 hours of complete ischemia and submaximal ischemic injury after induction of volvulus or hemorrhagic shock. Minimal histologic repelfusion injuly was demonstrated after reperfusion of P-hour ischemic loops in pigs ventilated wiyith 100% 02, but this was not associated with significant exacerbation of myeloperoxidase or lipid peroxidation levels. Thus it appears that the classic pathway of repelfusion injury, initiated by reactive oxygen metabolite formation and amplified by neutrophil

infiltration,“, I5 is absent in juvenile pigs. According to neutrophil counts, there was significant neutrophil infiltration after prolonged reperfusion of 2-hour ischemic tissues, but this did not coincide with exacerbation of histologic injury. Furthermore, myeloperoxidase levels and neutrophil counts were far more impressive after 6 hours of reperhlsion at a time when a remarkable degree of repair of the villi had taken place. Reperfusion injury is initiated by the superoxide radical during metabolism of hypoxanthine by xanthine oxidase in the presence of 02.14, I5 Previous experiments in the cat have demonstrated significant attenuation of reperfusion injury by administering allopurinol, a xanthine oxidase inhibitor,‘, 3, 7, I4 confirming an important role for xanthine oxidase in the develop ment of reperfusion injury. Furthermore, xanthine ox-

532 Blikslager et al.

Surge9

l%zJ 1997

1.0 z E g

0.8

2i g

0.6

2 a, 0

0.4

2 +I

0.2

*

q

t r m

Shock/

Resuscitation

Control/

Shock

0.0 2Hr

3Hr

Control/

Resuscitation

Treatment Fig. 10. Intestinal permeability is significantly increased during resuscitation compared with shock. However, this appears to be a result of blood flow-limited tissue availability of 51Cr-EDTA during shock, because clearance of isotope during shock was well below that of the respective control. *k < 0.05 versus shock.

Table III. Intestinal

xanthine

oxidase/xanthine

dehydrogenase

(XO/XDH)

activity (mU/gm

wet weight)

Total XO/XDH Jq’unZL m

Species

Rat (adult) Rat (adult) Pig (8 days old, frozen tissue) Pig (7 weeks old, frozen tissue) Pig (7 weeks old, fresh tissue) Human (neonate) Human (adult) Values

are mean

I SEM. Data from

other

studies

553.6 374 3.0 3.4

2 42.1 2 17 2 1.9 2 1.6 0.0 0 56.0

are reported

Ileum 150.5 13s 1.3 0.4 0.9

k 26.7 -t 5 !I 1.3 + 0.4 2 0.9 0 29.0

Referm

ce

Present data (n = 6) 22 (n = 69) Present data (1~= 6) Present data (~1= 6) Present data (fz= 4) 19 (TZnot recorded) 19 (,n not recorded)

for comparison.

idase is critical for initiating chemoattraction of neutrophils,15 the principal source of reactive oxygen metab olitess5 Significant neutrophil infiltration does not occur if xanthine oxidase is inhibited.“, l5 In one study it was estimated that xanthine oxidase directly or indirectly (via chemoattraction of neutrophils) produced 65% of the reactive oxygen metabolites associated with reperfusion injun;. The source of the remaining reactive oxygen metabolites is unknown.14 In the present study neutrophil infiltration was minimal during 1 to 3 hours of reperfusion but was markedly elevated after 6 hours of reperfusion. This likely relates to subepithelial exposure to luminal bacteria” and coincides with the beginning of the inflammatory phase of healing. We do not believe that this delayed neutrophil infiltration relates to mechanisms responsible for repelfusion injury in other species, namely xanthine oxidase-triggered chemoattraction of neutrophils. The level of xanthine oxidase necessary to exacerbate intestinal injury is unknown.” In the rat, which develops marked reperfusion injury in the small intestine (where very high levels of xanthine dehydrogenasexanthine oxidase are present), a lack of reperfusion in-

jury in the large intestine was attributed to the absence of xanthine oxidase in the colon.” This mechanism was also used to explain the more severe reperfusion injury that occurs in the feline distal stomach compared with the proximal stomach, because pyloric xanthine oxidase level was twice that of the fundic region.?” We hypothesized that the lack of xanthine oxidase would interrupt the pathway of reactive oxygen metab olite-mediated chemoattracnon of neutrophils. It is likely that the lack of reperfusion injury in this study related largely to the lack of neutrophil infiltration or activation during 1 to 3 hours of reperfusion. Comparison of neutrophil county and myeloperoxidase activity suggested that neutrophil counts are more sensitive, because significantly increased numbers of neutrophils were detected in all ischemic tissues despite an insignificant rise in myeloperoxidase levels. Altemativelv, it is possible that myeloperoxidase is a better indicator of neutrophil activity than absolute numbers, because my eloperoxidase is directly responsible for producing the reactive oxygen metabolite hypochlorous acid.” Despite a lack of marked neutrophil infiltration during 1 to 3 hours of reperfusion (compared with ische-

Blikslager

mia), studies on depletion of mucosal neutrophils in the cat have suggested that the resident population of interstitial neutrophils is capable of triggering reperfusion.‘” Interestingly, the basal tissue levels of myeloperoxidase in our study (2.2 ? 0.04 units/gm, measured in units per gram tissue foi- the sake of comparison) were lower than those measured in the aforementioned feline study (12 units/gm) lS This suggests that the pig may have a relatively small population of interstitial neutrophils that is incapable of generating sufficient reactive oxygen metabolites to exacerbate ischemic injury. Further neutrophil recruitment from the circulation Mould then be necessary to develop reperfusion injuy, but this does not occur in the absence ofxanthine oxidase activity.“. l5 An alternative reason for the lack of marked reperfusion injury during our initial experiments is the complete nature of the ischemia. Low-flow ischemia appears to be necessary to prime tissues for reperfusion injury.‘. ’ In rats the marked injury that was noted after reperfusion of intestine subjected to 3 hours of low-flow ischemia was reduced (although still present) after complete ischemia.s,’ The severit+? of ischemic injury is also important, because only submaximal ischemic injuq (developed after low-flow ischemia or short periods of complete ischemia) allows the development of additional injury on reperfusion. However, subm;lsimal injury in pigs after shorter periods of ischemia had no apparent effect on the degree of reperfusion injury. Furthermore, submaximal injury after shock or volvulus did not result in reperfusion injury. In fact, reperfusion injury was more likely to be present after severe ischemic injury (2 hours of ischemia) Ischemic injury in the pig appears very similar to that in the horse in which limited histologic evidence of intestinal reperfusion injury OCCLU-s after 3 hours of complete ischemia and 1 hour of reperfusion.” Allopurinol, a xanthine oxidase inhibitor, failed to attenuate injuly when administered before reperfusion.” Interestingly, the adult horse has low levels of xanthine oxidase in the ileum.*s and the neonatal foal has no detectable xanthine oxidase activity (unpublished observations). Furthermore, the horse has low levels of intestinal myeloperoxidase (0.1 rt 0.04 units,/gm, unpublished observations), suggesting a small residential pool of neutrophils similar to that of the pig. Sl’e believe the pig provides a valuable model of ischemic injuy, because the lack of intestinal xanthine osidase, as in the human neonate, has allowed us to assess the importance of reperfusion injury in the absence of a reactive oxygen metabolite-generating enzyne systern. This model supports previous studies in which xanthine oxidase has been assigned a vital role in the development of reperfusion injury.“, 6. 14, l5 Under conditions where reperfusion injury does not signifi-

et al.

533

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Role

in the cat stomach. MZ. mucosa oxygen. M. -&anof the

mucosa

after