Isquemia e reperfusão experimental no cólon menor eqüino

Experimental ischemia and reperfusion in equine small colon

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Experimental ischemia and reperfusion in equine small colon

[Isquemia e reperfusão experimental no cólon menor eqüino]

R.R. Faleiros^1,2, G.E.S. Alves², R.L. Santos², A.P. Marques Junior², D.G. Macoris³

¹Doutorando em Cirurgia Veterinária na FCAV/UNESP

2Escola de Veterinária da UFMG

Caixa Postal 567

30123-970 – Belo Horizonte, MG

3Faculdade de Ciências Agrárias e Veterinárias da UNESP

Recebido para publicação, após modificações, em 23 de março de 2001.

E-mail: [e-mail]

RESUMO

Sob anestesia geral, com constante controle sobre a pressão arterial e a saturação de oxigênio da hemoglobina arterial, realizou-se celiotomia em 12 eqüinos. No cólon menor exposto foram demarcados três segmentos de 25cm, separados entre si por igual distância. Dois desses segmentos foram submetidos à isquemia arteriovenosa completa por 90 (grupo A) ou 180 minutos (grupo B). O terceiro segmento foi o grupo-controle. Amostras para histopatologia foram colhidas ao final dos períodos de isquemia e após 90 e 180 minutos de reperfusão no grupo A e após 90 minutos de reperfusão no grupo B. No controle, colheram-se amostras no início e final do procedimento. Avaliaram-se as lesões produzidas na mucosa e na submucosa pelos métodos semiquantitativos-escores para desprendimento de epitélio, edema, hemorragia e infiltrado de neutrófilos, e pelos quantitativos-porcentagem de perda de mucosa (PM) e razão cripta:interstício (C:I). As lesões isquêmicas foram mais intensas no grupo B do que no A para PM, C:I, desprendimento de epitélio e edema de mucosa. As amostras obtidas após a reperfusão revelaram que houve agravamento na PM, C:I, desprendimento de epitélio e edema de submucosa em ambos os grupos. Concluiu-se que a reperfusão agravou as lesões isquêmicas no cólon menor e que o modelo proposto é viável para produção dessas lesões.

Palavras-chave: Eqüino, isquemia, injúria de reperfusão, cólon menor, abdome agudo

ABSTRACT

The effects of ischemia and reperfusion were studied in the small colon of 12 horses. Under general anesthesia, arterial pressure and arterial hemoglobin oxygen saturation values were maintained constant and within the normal physiological range. After celiotomy, the small colon was exposed and three segments were demarcated. Total arterial venous ischemia was induced in two segments during 90 (group A) and 180 (group B) minutes. The third segment was the control group. Full-thickness biopsy specimens, for histopathology, were obtained at the end of the ischemia periods and at 90 (groups A and B) and 180 minutes (group A) of reperfusion. The mucosa and the submucosa were evaluated by semiquantitative morphological assessment such as epithelial detachment, edema and hemorrhage, and by quantitative morphological assessment such as percentual depth of mucosal loss (ML) and mucosal crypt:interstitium ratio (C:I). After ischemia, lesions such as ML, C:I, epithelial detachment and mucosal edema were more intense in group B when compared to group A. In both groups after reperfusion there were significant increases in ML, C:I, epithelial detachment and submucosal edema. The results showed aggravation of the mucosal lesions in the reperfusion period in equine small colon, and the model was considered adequate for experimental purposes.

Keywords: Equine, ischemia, reperfusion injury, small colon, acute abdomen

INTRODUCTION

Despite recent advances in anesthetic and surgical techniques, colic is still considered the main cause of death in horses. Several factors are related to equine acute abdominal problems, but retrospective studies showed that bowel ischemia indicates a poor prognosis (Ducharme et al., 1983; Lacerda Netto et al., 1994). Intestinal ischemia may cause systemic complications such as hypovolemia, endotoxemia, peritonitis, lameness and renal failure (Moore & Bertone, 1992) and a variety of sequels like fibrosis, abscess, adhesions and mucosal ulceration (Freeman et al., 1988).

Furthermore, it is important to consider that ischemic lesions can be worsened after restoration of normal blood flow. This kind of lesion has been generally denominated reperfusion injury and has already been verified in many organs and tissues of several species (Forsyth & Guilford, 1995). Considering the studies done with laboratory animals, the reperfusion injury is considered to be caused by the return of blood supply to an ischemic tissue resulting in an intense production of free oxygen radicals (Flaherty & Weisfeldt, 1988). The progression of the lesions during reperfusion had already been described in the horse jejunum (Horne et al., 1993; Vatistas et al., 1996; Kooreman et al., 1998) and large colon (Vatistas et al., 1993; Moore et al., 1994a; Kooreman et al., 1998).

In addition to the lack of information about equine small colon reperfusion problems, the other factor that motivated the present study was the greater incidence of small colon diseases in Brazil. The Bolshoi's multicenter study indicated a prevalence of 3.5% for simple obstructions and 0.4% for strangulative obstructions in the small colon (White, 1990). However, in large urban centers in developing countries the incidence of small colon diseases may increase up to 30.9% of the surgical diseases affecting the equine large intestine (Silva et al., 1998). The objective of the present study was to evaluate the effects of reperfusion in equine small colon submitted to experimental ischemia.

MATERIALS AND METHODS

Twelve undefined breed healthy horses (six gelding and six mares) aging 8.9± 3 years were medicated with ivermectin and were maintained for at least seven days eating hay and commercial equine food.

After 12 hours of fasting, acepromazine (Acepran; Univet) 0.05 mg/kg was injected intramuscularly (IM). Thirty minutes latter, anesthesia was induced with 5% thiamylal (Thiamylal; Boehringer) 12.5 to 15 mg/kg intravenously (IV). After tracheal intubation the horses were kept on dorsal recumbency with spontaneous breathing of halothane (1.5 to 3.0%) in 15 ml/kg of oxygen. The mean arterial blood pressure was monitored continuously by using a facial artery catheter (Hubbell, 1991) and sodium chloride 0.9% solution and/or Ringer lactate were administered intravenously in a rate of 5 to 10 ml/kg/h to maintain the mean arterial pressure between 70 and 100mm Hg. When the fluid therapy was no longer enough to maintain the blood pressure, ethylefrine (Cardioton; Boehringer) 0.2 mg/kg IV was used (Fantoni et al., 1994). A pulse oximeter (Dixtal) was used to assess the oxygen saturation, and controlled ventilation was used when necessary to prevent oxygen saturation from going below 95%.

Celiotomy was performed through a median incision. The small colon was exposed and three intestinal segments with 25cm were demarcated. Two of those segments underwent arteriovenous ischemia during 90 (segment of group A) or 180 minutes (segment of group B) by placing clamps made with number 2 Penrose tubing in both branches of the correspondent mesenteric artery and vein. Number 3 Penrose tubing was also used to hold the whole intestinal wall and the mesenteric vessel anastomosis to promote a complete luminal and vascular occlusion. The third segment was not submitted to ischemia (segment of the group control). The sequence of the segments was changed from animal to animal so that in the final data there was a similar number of the segments A, B and control in the oral, intermediate and aboral positions.

A portable Doppler ultra-sound (EU 700; Embracrios) was used according to the procedure described by Freeman et al. (1988) to verify either the absence of bloodstream in the beginning of the ischemia or the flow return after the removal of the Penrose tubings in the beginning of the reperfusion period.

During the sample procedure the exposed small colon was humidified with lactate Ringer solution at 38º C. Full-thickness biopsy specimens (3x6 cm) were taken from the antimesenteric side (parallel to the taenia). In segment A the biopsies were obtained at the end of the ischemia period (AI90) and after 90 (AR90) and 180 (AR180) minutes of reperfusion. In the segment B, samples were taken at the end of the ischemia (BI180) and after 90 minutes of reperfusion (BR90). In control segment a sample was taken after instrumentation corresponding to the initial control (C0) and another at the end of the whole procedure (C270).

Biopsy sites were sutured with a continuous simple pattern and Cushing's seromuscular suture. The bowel was returned into the abdominal cavity and the medium incision was approached with Backhaus tongs and protected with humidified compresses until the next sampling time. At the end of the last sampling and under deep anesthesia the animals were killed with vein infusion of a magnesium sulphate saturated solution.

Biopsy specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned (5mm), and stained with hematoxylin-eosin (Luna, 1968). One section of each sample (seven sections per animal) was examined under light microscope.

Epithelial cell detachment, hemorrhage and edema in the mucosa and submucosa were established as parameters for semiquantitative evaluation of degenerative changes and scored by two independent examiners, in a blinded schedule. A specific scale from 0 to 5 was established to the epithelial cell detachment (Table 1) based in a previous scale for equine large colon mucosal degeneration (Meschter et al., 1986). The scores 0 to 4 for edema and hemorrhage were used (Snyder et al., 1988).

The percentage depth of mucosal loss and the ratio between crypt and interstitium (C:I) were quantitatively evaluated. For percentage depth of mucosal loss a 100 micrometric reticule divisions (POL AT 4x; AO Scientific Instruments) and a 10x objective were superimposed across 10 randomly select microscopic fields in each section. The percentage was obtained by subtracting the height of the intact mucosa (distance of the muscularis mucosa to the position where the epithelium was gone) of the total mucosa height (distance between the muscularis mucosa and the lumen). This number was then divided by the total mucosa height and multiplied by 100 (Moore et al., 1994a). The crypt:interstitium ratio was obtained using an ocular with 25-point grid (KPL 25 points 10x; Zeiss) and a 40x objective superimposed to the middle of the mucosa. Fifteen measurements were performed in each section and the microscopic fields were selected by choice. The calculation of the ratio C:I was made by dividing the number of points that reached the crypts by the number of points that reached the interstitium (Elias et al., 1971).

Variance analysis in randomized blocks followed by Duncan test were performed to compare the mean differences. Semiquantitative morphology was evaluated by Kruskal-Wallis non-parametric test (Sampaio, 1998) based on the average scores of the two examiners. Correlation tests were carried out to verify the accuracy between the scores of each examiner. The significance level was established in P£ 0.05 for all the tests.

RESULTS

The animals were kept under anesthesia for 6 hours. Forty five minutes were taken for the beginning of the surgery, 15 minutes for the instrumentation (exposure of the colon, demarcation of the segments and placement of the Penrose tubings), 4.5 hours for ischemia and reperfusion, and 30 minutes for sutures of the colon and the abdominal wall.

The administration of isotonic fluids was efficient for maintenance of the mean arterial blood pressure above 70mm Hg in most of the horses, whereas in two horses ethylefrine administration was necessary. The oxygen saturation rarely decreased below 95% in all horses, and when it did occur it was immediately corrected by the improvement of the oxygen flow or by controlled ventilation.

In all horses the absence of circulation in the ischemic period and the return of blood flow in the antimesenteric border were observed at the beginning of the reperfusion periods by the Doppler ultrasonografic exam.

With 90 minutes of ischemia, the segments A and B showed purple discoloration. The colonic serosa and mesentery were congested, with multifocal hemorrhagies and clots of fibrin scattered on the serosa surface. After 180 minutes of ischemia, the segment the B showed bluish to purple color, with several hemorrhagic spots and a small amount of a gelatinous material in the serosa and mesentery. In the segment A, after removal of the Penrose tubings (90 min), the serosa turned back to the original color. In contrast, in the segment B after removal of the Penrose tubings (180 min), there was little change in the serosa color, which remained bluish.

After 270 minutes of the beginning of the procedure, the small colon had a thickened brilliant wall characteristic of edema and areas of hemorrhage. There was also fibrin adhered all over the serosa surface particularly in those segments that underwent ischemia. Segments of group A showed normal color and a slight increase in the thickness of the wall. Segments of group B still showed a slightly bluish mucosa and a thickened wall.

A moderate infiltration of eosinophils in the mucosa and submucosa was verified nearly in all the sections. At C0 the microscopic changes were negligible (Fig. 1), however there was a mild neutrophilic accumulation in the mucosa and/or in the submucosa and mild hyperemia and edema of the submucosa and/or in the serosa in 50% of the horses. Only one horse had hyperemia and mild edema in the mucosa with low degree of luminal epithelium detachment and the intensity of these lesions did not change significantly until the end of the procedure.

At the C270 the serosa lesions were characterized by hyperemia, discreet to moderate edema, neutrophils infiltration, and discreet hemorrhage. Neutrophilic infiltration and edema were also observed in the muscular layers. In the submucosa a discreet edema was observed.

In the segments of group A, the most evident microscopic change at the end of the ischemia (AI90) was severe congestion of all layers of the intestinal wall and hemorrhage ranging from discreet to moderate in the mucosa and discreet to light in the serosa. There was also basal vacuolation of the luminal epithelial cells and these cells started to detach (Fig. 2). At the AR90, hyperemia, neutrophilic infiltration and edema in the intestinal wall and discreet hemorrhage in the serosa were observed. Progression of the luminal mucosa epithelium detachment and exposure of the lamina propria were also observed (Fig. 3). Similar lesions were observed at AR180, except for the decrease in the degree of hyperemia.

In the segments of group B were noticed at BI180 intense congestion in the whole wall with severe mucosa and submucosa hemorrhage, associated with discreet to light edema. Hemorrhage and edema in the serosa were also observed. The detachment of the epithelium was noted, with exposure of lamina propria and beginning of cryptic epithelial cell loss (Fig. 4). After 90 minutes of reperfusion (BR90) there were hyperemia, neutrophilic infiltration and edema all over the intestinal wall and hemorrhage from light to intense in the serosa. There was progression in the epithelial detachment towards the deeper regions of the crypts (Fig. 5). In some areas the cryptic epithelium was almost completely lost (Fig. 6).

The quantitative morphology data are summarized in Tables 2 and 3, and the semiquantitative morphology data is summarized in Table 4. The coefficients of correlation between scores of the two examiners were 0.93 for epithelial detachment, 0.84 for mucosal hemorrhage, 0.69 for submucosal hemorrhage, 0.52 for mucosal edema and 0.80 for submucosal edema.

The BI180 lesions were rather more intense than AI90 considering the following parameters: mucosal depth percentual loss, ratio C:I, epithelial detachment and edema in the submucosa.

The histopathologic comparison between samples obtained at the end of the ischemia (AI90 and BI180) and samples obtained after reperfusion (AR90, AR180 and BR90) showed that reperfusion was associated to an increase in the degrees of epithelial detachment, mucosal depth percentual loss, C:I ratio and edema in the submucosa.

DISCUSSION

The described ischemic mucosal lesions were similar to the lesions observed after occlusion of the branches of the mesenteric veins in the small colon (congestive ischemia) for three to six hours (Ruggles et al., 1993). Similar ischemic alterations were also observed in large colon submitted to experimental arteriovenous ischemia (Snyder et al., 1988; Kooremam et al,. 1998) and also in clinical cases of colic due to small and large intestine diseases (Meschter et al., 1986; Sequeira et al., 1994b).

The most important lesions observed in the present study occurred in the reperfusion period and were characterized in the mucosa by progressive luminal and cryptic epithelial cell detachment and degeneration of the lamina propria. The clinical relevance of these findings is related to the fact that histophatological examination of frozen sections of equine colonic segments is considered an objective method for intraoperative evaluation of bowel viability. A segment of large colon is considered to be nonviable if there is more than 97% superficial cell loss and more than 50% crypt cell loss (Moore, 1999). Based on this statement, it has been shown especially in group B, that a small colon segment that could be judged viable at the end of ischemia period (Fig. 4) may be considered nonviable after 90 minutes of reperfusion (Fig. 6).

The progression of the lesions during reperfusion of the small colon occurred as had already been demonstrated in equine large colon after 60 minutes of complete arteriovenous ischemia (AVI) by Vatistas et al. (1993) and Kooreman et al. (1998). However, other authors did not demonstrate it using models of AVI for 60, 180, or 270 minutes (Reeves et al., 1990; Wilkins et al., 1994; Kawcak et al., 1995). Otherwise, Moore et al. (1994a) using a ischemic model with low arterial flow compared large colon segments submitted to three hours of ischemia followed by three hours of reperfusion, with segments submitted to six uninterrupted hours of ischemia. They verified that some parameters such as the index of cellular debris and the estimated percentage of mucosal loss were significantly higher in the reperfused segments.

In the present study the comparison between segments submitted to 90 minutes of ischemia and 90 minutes of reperfusion (AR90) with segments submitted to 180 minutes of ischemia (BI180) showed that the mucosal lesions were more severe in the segments that underwent uninterrupted ischemia. In this case, it can be assumed that reperfusion worsened the ischemic lesions, but it is impossible to affirm whether this worsening was caused by a new pathologic factor that was added during reperfusion (like free oxygen radicals) or it was due to the lesions caused by ischemia itself.

It was verified that in both groups, even after 90 minutes of reperfusion there was still congestion. In other models of ischemia with low flow a 2-fold increase in the bloodstream during reperfusion of the large colon (Moore et al., 1994b; Moore et al., 1995b) and jejunum (Vatistas et al., 1996) was documented. It was described as reactive hyperemia and it may be explained by an increase in the local blood pressure caused by the release of the vascular clamps or vessel dilatation due to the accumulation of potassium and lactate (Moore et al., 1994b) and/or inflammation mediators (Moore et al., 1995a).

In equine jejunum the detection of blood flow by the Doppler ultrasound test and evoked contractions were considered clinical indicators of viable intestine (Freeman et al., 1988). In the present study these signs were verified in all reperfusioned segments of the small colon, even in those that presented scores from 3 to 5 for epithelium detachment. It contrasts with post mortem studies in horses suffering from acute abdomen that, using a similar scale, observed an association between grade lesions above 2 and no survival (Meschter et al., 1986; Sequeira et al., 1994a). These contradictions are difficult to be addressed because specific studies do not exist on Doppler ultrasound evaluation of small colon viability.

There was a high and positive correlation between the two independent examiners, RRF and RLS. It suggests that the score employed was adequate and satisfactory to evaluate differences between groups. The semiquantitatives methods used for evaluation of mucosal lesions showed results similar to the quantitative methods. The proportionality between the quantitative and semiquantitatives methods was also reported by Moore et al., (1994a).

The measurement of the proportion between crypts and interstitium was intended to estimate the lesions associated to the increase of the interstitial volume, which may be related to edema and/or hemorrhage and loss of cryptic epithelium. A similar approach was initially proposed by Snyder et al. (1988) based on the visual estimation of the lamina propria width divided by the crypts width. These authors defined a value £ 1 as a normal interstitium:crypt ratio. Moore et al. (1994a) modified this procedure by using a computer program to calculate the interstitial and cryptic areas in one square millimeter of mucosa. According to their data an interstitium:crypt ratio below 3 was considered normal. In the present study, due to normal mucosa of the small colon presented a larger proportion of crypts in relation to the lamina propria (Pfeiffer & MacPherson, 1990), quantifying that proportion as crypt:interstitium ratio was adopted obtaining the normal values (initial control) ³ 1.60 ± 0.27.

In contrast to our findings, other authors using ischemic models with more than one segment per horse had observed the occurrence of significant mucosal lesions in control segments in jejunum (Freeman et al., 1988) and in small colon (Ruggles et al., 1993). Keeping an intermediate segment with mesenteric irrigation between the experimental segments may avoid this fact. The edema occurrence and discreet hemorrhage in the mucosa and submucosa of the controls are attributed to the manipulation.

In conclusion, there is progression of the ischemic lesions after 90 minutes of the reestablishment of the blood flow in equine small colon and it can be relevant when considering the determination of bowel viability. The present model was appropriate to study the ischemia/reperfusion lesions in equine small colon.

ACKNOWLEDGEMENT

The authors are grateful to Dr. I. B. M. Sampaio for statistical advice and to Dr. M. R. Souza for support with translating.

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Datas de Publicação

• Publicação nesta coleção
  07 Jun 2002
• Data do Fascículo
  Jun 2001

Histórico

• Recebido
  23 Mar 2001