Obstruction Requiring Surgery - NCBI

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The pro- gression of mucosal epithelial injury may lead to complete mucosal necro- sis, with disruption of the mucosal barrier; this favors migration of lumi-.
Plasma Myeloperoxidase Level and Polymorphonuclear Leukocyte Activation in Horses Suffering from Large Intestinal Obstruction Requiring Surgery: Preliminary Results S. Grulke, H. Benbarek,

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Caudron, G. Deby-Dupont, M. Mathy-Hartert, F. Farnir, C. Deby, M. Lamy and D. Serteyn

ABSTRACT

Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a significantly higher plasma level of MPO in horses operated for strangulation obstruction of the large intestine (n = 6) than in horses suffering from a non-strangulating displacement of the large intestine (n = 9). For the 2 groups, 3 phases were distinguished: reception (P1), intensive care (P2) and terminal phase (P3). The mean peak values of MPO for these phases were 121.6 ng/mL (P1), 168.6 ng/mL (P2), and 107.0 ng/mL (P3) for the non-strangulating group, and 242.6 ng/mL (P1); 426.0 ng/mL (P2), and 379.5 ng/mL (P3) for the strangulation group. The variations of the mean peak values of plasma MPO were significantly different between the 2 groups and between the different phases. A significant increase of the least square means of MPO was observed between P1 and P2. A significant decrease of the least square means of the number of circulating leukocytes was observed between P1 and P3. Polymorphonuclear neutrophil activation could play a major role in the pathogenesis of acute abdominal disease and endotoxic shock.

des polymorphonucleaires neutrophiles (PMN) et contribue au stress oxydatif observe lors de l'activation des PMN. A l'aide d'un dosage radio-immunologique, les auteurs ont observet un taux plasmatique significativement superieur chez les chevaux operes d'une obstruction etranglee du gros intestin (n = 6) que chez ceux atteints d'un deplacement simple du gros intestin (n = 9; groupe de controle). Pour les deux groupes trois phases ont ete differentiees (reception, soins intensifs et phase terminale). Les moyennes des valeurs pics de MPO etaient respectivement de 121,6 ng/mL (P1); 168,6 ng/mL (P2) et 107,0 ng/mL (P3) pour le groupe de controle et de 242,6 ng/mL (P1); 426,0 ng/mL (P2) et 379,5 ng/mL (P3) pour le groupe presentant une obstruction etranglee. Les variations des valeurs moyennes de MPO e'taient statistiquement differentes entre les deux groupes et entre les differentes phases. Une augmentation de la MPO (least square means) entre les phases P1 et P2 ainsi qu'une diminution du taux moyen de globules blancs circulants (least square means) entre les phases P1 et P3 e'taient observees. L'activation des polymorphonucle'aires neutrophiles pourrait jouer un role majeur dans la pathogenie des pathologies intestinales aigues et le choc endotoxinique chez le cheval. (Traduit par les auteurs)

RESUME

INTRODUCTION

La myeloperoxidase (MPO) est une enzyme localisee essentiellement dans les granules azurophiles

Strangulation obstruction of the small intestine and large colon are common causes of acute abdominal

disease in the horse. Mortality associated with intestinal strangulation in horses is quite variable but usually ranges between 50% and 80% (1-6). Despite surgical correction, intensive medical therapy and supportive care, the prognosis of horses with strangulating intestinal lesions is guarded. During a small intestinal strangulation obstruction, a complete necrosis of the mucosal epithelium extending to the base of the crypts occurs by 3 to 4 h, and by 6 to 7 h the degeneration has progressed outwards beyond the muscular layers (7,8). It also appears that 3 to 4 h of complete ischemia of the large colon caused by arteriovenous occlusion results in irreversible damage of the mucosa (9). The progression of mucosal epithelial injury may lead to complete mucosal necrosis, with disruption of the mucosal barrier; this favors migration of luminal bacteria and endotoxin into the systemic circulation, resulting in endotoxemia (5,10,1 1). Furthermore, the intestinal injury that occurs during strangulation may progress after detorsion due to irreversible cellular alterations that occurred during the ischemic period, or due to exacerbation of cellular injury as a result of the cascade of events initiated on reperfusion (12,13). Reactive oxygen metabolites produced by the system xanthine/xanthine oxidase or by the alteration of the mitochondrial chain seem to play a major role in the cellular lesions. Moreover, as observed in rabbit colons, polymorphonuclear neutrophils (PMN) accumulate in the colonic mucosa of horses during lowflow arterial ischemia and especially on reperfusion (14,15). Activated neutrophils produce reactive oxygen

Anaesthesiologie gdn6rale, Pathologie chirurgicale des grands animaux (Grulke, Benbarek, Caudron, Serteyn), Genetique quantitative (Farnir), Faculte de m6decine vet6rinaire; Centre de biochimie de l'oxygene (Deby-Dupont, Mathy-Hartert, Deby, Lamy); Universite de Liege, B41 4000 Sart Tilman, Belgium. Please address correspondence and reprint requests to Dr. S. Grulke, telephone: (+32 4) 366 41 03; fax: (+32 4) 366 41 08. Received December 9, 1997.

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Can J Vet Res 1999; 63: 142-147

species, lipid mediators such as leukotrienes, platelet-activating factors and prostanoids and release several enzymes such as proteases and myeloperoxidase from their granules (16). A high activity of myeloperoxidase (MPO) was found in the intestinal mucosa after experimental lowflow ischemia and reperfusion (15). MPO is an enzyme located exclusively in the neutrophil azurophilic granules of neutrophils. Eosinophils contain eosinophil peroxidase (EPO), a peroxidase which shares similar functions with MPO. However, human MPO and EPO are immunologically different. Indeed, antisera obtained against MPO do not cross react with EPO (17). MPO produces hypochlorous acid from chloride anions and hydrogen peroxide, the latter being produced by superoxide anion dismutation (18). MPO can be measured by enzymatic assays or immunological techniques. Enzymatic assay measures easily the peroxidasic activity in tissues (19). Peroxidasic activity is not specific for MPO, but is the witness of the activity of different peroxidases (MPO and EPO). Moreover, plasma albumin is an inhibitor of MPO activity. In order to perform enzymatic measurement of MPO in plasma, a gel filtration or an ion-exchange chromatography of the plasma sample is necessary to isolate the MPO from the other plasma proteins, such as albumin, which are considered to be inhibitors of MPO activity. Furthermore, it appears that MPO is inactivated when released by degranulation in the extracellular milieu (20). In human medicine, the immunological technique for MPO assays has been applied to plasma. A high level of plasma MPO is considered to be a proof of neutrophil activation and degranulation, but not correlated to the number of neutrophils in plasma (21). Activated neutrophils trapped in specific organs release their granulocyte content in the extracellular milieu. The granulocyte enzyme can therefore increase in plasma. One of the main objectives of this study was to follow the variation of MPO plasma levels to demonstrate that equine strangulation obstruction was not only characterized by local influx of neutrophils, but also by the extension of inflammation and the disper-

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sion of the granulocytic enzymes to the general circulation. Equine myeloperoxidase was previously purified from equine neutrophils by detergent extraction followed by 2 chromatographic steps (ion-exchange and gel filtration chromatography). This pure equine MPO consists of a mixture of the precursor and mature forms of the enzyme, with a respective molecular mass of 88 and 140 KDa, similar to the human MPO. The spectral characteristics, the enzymatic properties and the pH dependency were also identical to those of human MPO (22). A specific antiserum was obtained by immunizing rabbits with pure equine MPO. A spe-

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cific radioimmunoassay (RIA) was designed using this antiserum to measure MPO in horse plasma obtained from ethylene-diamine-tetraaceticacid (EDTA)-anticoagulated blood (23). In a previous study we determined the physiological mean value of plasmatic MPO level as 69.5 19.4 ng/mL for normal horses (50 warmblood horses, healthy mares and geldings with an age ranging between 4 and 20 y). A physiological range can ±

be considered as mean ± 3 standard deviations (SD), that is to say a maximal value of 128 ng/mL. This choice assures a probability of error in determination of abnormally high values lower than 0.01%. In the same study

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TABLE I. Gravity index recovery time and peak values of myeloperoxidase (MPO) of each horse during each phase on the strangulation and the non-strangulation groups Horse

Gravity index

1 2 2 1 3 1 4 1 5 2 6 2 7 2 8 1 9 2 Mean values Standard deviation

Recovery (h) (P1) Control group 20 56 32 64 24 200 15 56 36 300 18 65 18 172 63 82 23 100 27.6 121.6 84.9

MPO peak value (ng/mL) (P2) (P3) 92 160 230 130 220 184 220 142 140 168.6 47.7

64 56 80 64 190 280 76 100 53 107.0 77.2

732 342 440 312 371 359 426.0 155.8

570 231 413 269 270 524 379.5 144.6

Str. Obs. group

10 3 11 3 12 3 13 3 14 3 15 2 Mean values Standard deviation

MPO was determined for horses immediately after the transport, before, during and after general anesthesia for minor surgeries not affecting the general condition and variation during 24 h showing that there was no significant increase of MPO in these conditions (23). In a clinical study, the variations of the MPO plasmatic level of 6 horses with large intestine strangulation obstruction were compared with the values obtained in nine other horses operated for a non-strangulating displacement of the large colon. These variations were compared to the variations of the number of circulating leukocytes.

MATERIALS AND METHODS Fifteen horses in 2 groups, referred for an acute abdominal disease were used in this study. The first group considered as control consisted of 9 horses suffering from a nonstrangulating obstruction of the large intestine (left dorsal displacement or right displacement of the left colon). This group consisted of 8 mares and 1 gelding (1 thoroughbred and 8 warmblood horses), ranging between 4 and 18 y. The follow up was successful with no major complications. The second group consisted 144

642 136 147 108 264 159 242.6 202.7

of 6 horses suffering from a large intestine strangulation obstruction leading to severe complications and death (complete volvulus of ascending colon, torsion of cecum or left colon). This group consisted of 2 mares, 3 geldings (warmblood horses) and 1 stallion (thoroughbred) with an age ranging between 3 and 25 y. A clinical evaluation was performed on admission of the horse in our clinic. The horses considered in this study did not present signs of severe shock at reception (heart and breathing rates higher than normal, commensurate pain, normal pulse quality, normal systolic pressure, no increase in packed cell volume or lactate). A gravity index (GI) was established and was based on the intensity of 5 parameters classified in 3 categories estimating intestinal obstruction: abdominal distension (no distension, distension located in one flank, severe distension of both flanks), organ distension on rectal examination (only located distension of one organ, more severe distension but exploration possible, no exploration possible), amount of gastric reflux (no reflux, less than 4 L, more than 4 L), intestinal borborygmies (located modification, more extended modification, generalized absence of borborygmies) and pain (response to slight analgetics, response to strong analget-

ics, no response to analgetics). A rank sum test-Wilcoxon was performed on the gravity index of the 2 groups. Before premedication, blood samples were drawn for biochemical analysis (packed cell volume, leukocytes, lactate, blood gases, ions) and for MPO plasma assay (phase 1). Premedication consisted of xylazine administration (0.6 mg/kg). Anesthesia was induced with guaifenesin (10%) and thyamilal (2 g/500 mL of guaifenesin), and isoflurane was used to maintain general anesthesia. After a surgical reduction of the obstruction, the horses were transferred to

recovery boxes and placed in intensive care until recuperation of normal intestinal function. During this period (surgery and recuperation), corre-

sponding to phase 2, blood samples were drawn every 2 h for biochemical evaluation and MPO RIA. For Group 1 (non-strangulating obstruction), a third phase 3 to 5 d after surgery was also considered. Blood samples for MPO RIA were drawn twice a day during this period. For the second group (strangulation obstruction), the third phase was the time when irreversible complications appeared, leading to euthanasia. Total leukocyte count (WBC) was determined with a Coulter counter (Coulter Electronics Ltd.) on each blood sample (EDTA-anticoagulated blood) used for MPO assay. Plasma MPO was measured by a radioimmunoassay using a rabbit antiserum raised against equine MPO, at a dilution of 1/40 000 (antibody). The tracer molecule (= labelled antigen) was pure equine MPO labelled with '25iodine (Amersham, place). Incubation medium was made with 100 ,uL of the diluted antiserum, 100 ,uL of the tracer (± 30 000 cpm), and 100 ,uL of the plasma sample. A standard curve was established with increasing concentrations of equine MPO solution (from 2 to 200 ng/mL) (= unlabelled antigen). Samples were incubated at room temperature for 24 h. The reaction was stopped by precipitation of the antibody-antigen complex with a second antigen directed against rabbit gammaglobulins. After centrifugation, the supernatant was discarded and the precipitate was counted. All the reagents were prepared in phosphate buffer (0.05 M, pH 7.4) and adjusted with 0,9% NaCl, 0.5%

bovine serum albumin (BSA) and 0.05% sodium azide. The results were expressed in ng/mL of plasma. In order to be able to compare the plasma MPO values between the 2 groups, the peak values obtained during each phase were taken into account: phase 1 (P1): reception; phase 2 (P2): surgery and intensive care period; phase 3 (P3): terminal phase was recuperation phase for group 1 and complication phase for group 2. The statistical analysis of the data was studied by a general linear model procedure (analysis of variance) to test the group effect, the phase effect and the interaction between the 2 factors (24). The least square means were used to compare these differences and a significance was considered when P < 0.05.

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The results of the rank sum testWilcoxon between the 2 groups and calculated on the gravity index assessed at the admission gave a probability value inferior to 10%. The classification of the horses in 2 groups was especially based on the lesions observed during surgery and the recovery period. Figure 1 shows all the variations of MPO values observed for each horse. The plasma MPO values measured in the non-strangulating group are illustrated on Figure a and the MPO values for the strangulation group on Figure lb. Maximal physiologic values of MPO were also reported (23). These graphs show a high level of plasma MPO in the strangulation group compared with the physiological value and compared with horses suffering from a non-strangulating obstruction. Table I reports for the gravity index each horse, the time needed for recuperation of a normal intestinal function (recovery), the peak values of MPO at each phase (P1, P2, P3), the mean values of MPO of each group in each phase and the standard deviation (SD). The variations of the mean values of plasma MPO were significantly different between the 2 groups and between the different phases (P < 0.05). The interaction between the different groups and the different

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Figure 2. (A) Variations of the mean peak values of plasma myeloperoxidase obtained at each phase in the strangulation group (St) and the non-strangulation group (nS). * significant difference of the least square means between phase 1 and phase 2 (P < 0.05). (B) Variations of the mean values of total leukocyte count at each phase in both groups. * significant difference of the least square means between phase 1 and phase 3 (P 0.05).