a vascular hypersensitivity model of acute multifocal gastrointestinal infarction

Digestive Diseases and Sciences, VoL 39, No. 3 (March 1994), pp. 534-539

A Vascular Hypersensitivity Model of Acute Multifocal Gastrointestinal Infarction

MARK HUDSON, CHRISTOPHER PIASECKI, ANDREW J. WAKEFIELD, ELIZABETH A. SANKEY, AMAR P. DHILLON, MARTIN OSBORNE,

ROSALIND SIM, and ROY E. POUNDER

We have investigated the hypothesis that submucosal vasculitis may account for the patchy transmural inflammation observed in Crohn's disease. Test ferrets (N = 11) were sensitized to human albumin. Five days after the last sensitization injection, human albumin microspheres (15-150 p.m diameter) were injected intraarterially into the mesenteric circulation o f a defined loop o f mid-gut. Six control ferrets showed no histological abnormality at either 48 hr or two weeks after intraarterial injection. A t 48 hr, five o f six presensitized ferrets demonstrated submucosal vasculitis with fibrinoid necrosis. In two cases there was transmural inflammation and mucosal ulceration. A further five presensitized ferrets received weekly subcutaneous human albumin injections following the mesenteric intraarterial injection o f albumin microspheres: after two weeks one animal demonstrated mild perivascular inflammatory changes and another demonstrated vasculitis. One o f the two animals with transmural inflammation and mucosal ulceration at 48 hr, and the animal with vasculitis at two weeks, had precipitating antibodies to human serum albumin. This model demonstrates that an immune- mediated submucosal vasculitis can sometimes result in discontinuous transmural inflammation o f the intestinal wall.

KEY WORDS: submucosal vasculitis; fibrinoid necrosis; transmural inflammation.

Crohn's disease is characterized by patchy mucosal ulceration and transmural inflammation (1). Many animal models of inflammatory bowel disease produce mucosal inflammation that involves a direct continuous injury to the epithelium (2-7). Based upon the evidence for submucosal vasculitis in Crohn's disease, we have recently developed an animal model to explain the distribution of lesions in this condition. Using nonbiodegradable plastic

Manuscript received November 17, 1992; accepted August 11, 1993.

From the University Departments of Medicine, Histopathol- ogy, and Surgery; and Department of Anatomy, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK.

Address for reprint requests: Professor R.E. Pounder, Univer- sity Department of Medicine, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK.

microspheres, selected specifically to occlude the submucosal vessels, we were able to produce focal mucosal ulceration with morphologically normal adjacent mucosa (8). These experimental observa- tions provide a possible anatomical explanation for "skip lesions."

Having demonstrated that mechanical obstruction of the submucosal vessels can result in focal mucosal ulceration, the aim of this study was to observe the macroscopic and microscopic changes in the intestine of the ferret following an immune-mediated inflammatory injury, localized principally in the submucosal vessels. In particular, we sought to determine whether submucosal vasculitis induced by the local- ization of human albumin microspheres to the submucosal vasculature in presensitized animals can result

534 Digestive Diseases and Sciences, Vol. 39, No. 3 (March 1994) 0163-2116/94/0300-0534507.00/0 © 1994 Plenum Publishing Corporation

FERRET MODEL OF SUBMUCOSAL VASCULITIS

in focal, discontinuous transmural inflammation and mucosal ulceration.

MATERIALS AND METHODS

Sensitization to Albumin Microspheres. Eleven test ferrets were sensitized to the biodegradable human albumin microspheres that are used in clinical practice for perfusion lung scans. The microspheres are made from aggregated human albumin, stannous tin, sodium chloride, and poioxamer 188 (Amersham International plc, Amersham, Berks, UK). The microspheres were injected subcutaneously with Freund's complete adjuvant (Difco, East Molesey, Surrey, UK). Two weeks later a further subcutaneous injection of the microspheres was given with Freund's incomplete adjuvant (Dif- co). Seven days later the sensitization regimen was com- pleted by a final intramuscular injection of the microspheres alone. On each occasion the animals received 600,000 microspheres (0.1 mg albumin).

Embolization of Mesenteric Vessels. Five days after completing the course of sensitization injections, the human albumin microspheres (diameter 15-150 I~m) were injected into the mesenteric circulation. Seventeen ferrets [six controls (group 1) and 11 sensitized (groups 2 and 3)] underwent a laparotomy under general anesthesia (continuous halothane and oxygen 2 liters/min, after premed- ication with atropine 90 ~g and ketamine 30 mg/kg). At operation, suitable loops of mid-gut were identified and measured [median length 16 cm (range 10-25 cm)]. Prior to the injection of microspheres, the selected test loop was isolated with noncrushing clamps to prevent microspheres escaping beyond the test loop. The presence of an adequate collateral circulation to the loop of bowel, by means of the marginal artery, was ascertained visually; the feeding artery was identified and dissected. Two silk ties were applied loosely further downstream and the artery was then dilated with topical Marcaine (Astra Ltd., Kings Langley, England). The vessel was penetrated with a 19-gauge butterfly needle (Abbott Ireland Ltd., Sligo, Republic of Ireland) under stereomicroscopic control. The sterile microspheres were injected into the artery (30,000 spheres/cm bowel length). The vessel was flushed with 2 ml 0.9% saline, and the artery tied to prevent bleeding by backflow from the marginal artery. The noncrushing clamps were released and the intestine was observed until normal color returned. The peritoneum was closed with prolene sutures (Ethicon, Edinburgh, Scotland) and the skin with interrupted silk sutures. The test loops were marked by making a hole in the transpar- ent mesentery near each end of the loop and by tying a silk suture in the mesentery.

Terminal Procedure. Ferrets were killed at 48 hr, two and three weeks. Six sensitized ferrets were killed 48 hr after mesenteric embolization with the albumin spheres (group 2), but five similar animals went on to receive twice weekly subcutaneous injections of human serum albumin 0.1 mg in the form of microspheres (group 3); three of these animals ~¢ere killed at two weeks and two at three weeks. Three control (nonsensitized) ferrets were killed at 48 hr, and also three at two weeks, after mesenteric embolization. The terminal procedure was performed under general anesthe-

sia. Blood for serum was taken from the inferior vena cava; the superior mesenteric artery was identified, dissected, and cannulated with a 3FG size polyethylene catheter (Portex, Hythe, England). The mesenteric vessels were flushed clear with isotonic heparinized saline, and the effluent drained through a large-bore cannula in the portal vein. The mesenteric vasculature was then perfusion-fixed at a pressure of 100 cm of water (mean ferret arterial pressure 79 mm Hg) with 10% formol-saline for 15 rain. The bowel was removed subsequently en bloc for histological analysis.

Histological Processing and Analysis. Representative blocks of formalin-fixed tissues from both normal and experimental loops of bowel were paraffin-processed, and sections from these blocks were stained with hematoxylin and eosin. A total of 236 sections were examined. In addition, selected sections from these blocks were trypsinized and stained immunohistochemically using polyclonal antibodies against fibrinogen (Dako Ltd., High Wycombe, Bucks, England) at a dilution of 1:900 and monoclonal antibody against type IV collagen, a constit- uent of vascular basal lamina (Dako Ltd.), at a dilution of 1:50. Horseradish peroxidase was used as the label, and hydrogen peroxide with diaminobenzidine as the colori- zation step. Sections were counterstained with Mayer's hematoxylin. All histological sections were examined by two experienced histopathologists blinded to the experimental procedure.

Demonstration of Precipitating Antibody to Human Se- rum Albumin. Precipitating antibody to human serum albumin was detected using the Ouchterlony double im- munodiffusion technique (9). In this method, antigen and antibody placed in wells cut in agar gel diffuse towards each other and precipitate to form an opaque line in the region where they meet in optimal proportions. Six wells were cut, equidistant from a central well. The test sera (8 I~1) was placed in the central well and doubling dilutions of the antigen (human serum albumin; Sigma, Poole, Dorset, UK) at a concentration of 1 mg/ml to 0.0625 mg/mi in the surrounding wells. At 48 hr the agar plates were stained with Coomassie brilliant blue, and the re- suits were read.

RESULTS

No animal developed anaphylaxis during the sensitization process or after embolization with the albumin microspheres. The histological findings in the perfusion-fixed embolized small intestine are summarized in Table 1. Sections of intestine away from the test loop (N = 11) and from the test loop of the nonsensit ized (group 1) control animals (N = 6) were normal both macroscopical ly and microscop- ically at 48 hr or two weeks.

No microspheres were seen histologically at either 48 hr or two weeks . The most marked histological changes were seen in the test loop in group 2 animals at 48 hr. Occasional loci of vasculitis and fibrinoid necrosis (Figure 1A and B) with predominantly submucosal inflammation, but in two cases

Digestive Diseases and Sciences, Vol. 39, No. 3 (March 1994) 535

H U D S O N E T A L

TABLE 1. HISTOLOGICAL FEATURES OF FERRET MID-GUT LOOP AFTER EMBOLIZATION WITH HUMAN ALBUMIN MICROSPHERES

Control animals (Group 1)

48 hr 2 weeks

Presensitized animals

Group 2, 48 hr Group 3, 2-3 weeks Histological features (N = 3) (N = 3) (N = 6)* (N = 5)

Vasculitis nil nil 5 1 Hemorrhage nil nil 4 1 Mucosal inflammation nil nil 3 nil Submucosal inflammation nil nil 5 2 Transmural inflammation nil nil 2 nil Mucosal ulceration nil nil 2 nil

"Two of the presensitized animals at 48 hr showed all histological features.

transmural inflammation (Figure 2), were observed. The inflammatory response centered predominantly around vessels and consisted mainly of macro- phages and lymphocytes. Mucosal epithelial ero- sion was demonstrated in two ferrets (Figure 3). Histological findings at two and three weeks were less marked: only two animals showed histological

changes, both at two weeks. In one animal the predominant histological finding was of a slight in- crease in perivascular lymphocytes and eosinophils (Figure 4). Another animal, however, had evidence of submucosal vasculitis, inflammation, and hemorrhage. This animal, and the animal with the most severe changes at 48 hr (transmural inflammation,

• - ~ - ~ ._.~-:~0~, ~ - . , , , ~

Fig 1. (A) Fibrinoid necrosis of a submucosal vessel 48 hr after injection of human albumin spheres in a presensitized animal. H&E, x350. (B) The same area as in A immunostained for fibrinogen demonstrates the area of fibrinoid necrosis, x350 (reduced to 92% for reproduction).

536 Digestive Diseases and Sciences, Vol. 39, No. 3 (March 1994)


Fig 2. Transmural inflammation in a ferret 48 hr postemboliza- tion. The inflammation is predominantly submucosal but extends into the mucosa and muscularis propria. H&E, x 140 (reduced to 92% for reproduction).

submucosal vascular fibrinoid necrosis, and mucosal ulceration), had precipitating antibodies to human serum albumin at 62.5 and 125 v.g/ml, respec- tively. None of the control or other test animals had evidence of a precipitating antibody to human serum albumin.

DISCUSSION

This model did not set out to reproduce Crohn's disease, nor does it seek to provide an animal model for future therapeutic studies. However, the results do support the hypothesis that an immune-mediated submucosal vasculitis can produce focal, transmural inflammation with transient mucosal ulceration. The presence of submucosal arteritis, fibrinoid necrosis, and both an intramural and perivascular cellular infil- trate (polymorphonuclear cells, lymphocytes, macro- phages, and eosinophils) would suggest a mixed hypersensitivity reaction. The failure to demonstrate precipitating antibody in the majority of the test ani-

Fig 3. Incipient mucosal ulceration in a ferret at 48 hr. Discrete area of mucosal and submucosal inflammation with early loss of overlying epithelium (arrow). H&E, x 140 (reduced to 92% for reproduction).

mals suggests that mechanisms other than humoral immunity are involved. Freund's complete adjuvant activates predominantly the mononuclear cell/ macrophage system, and the histological findings would be compatible with a cell-mediated immune response in addition to an Arthus-type hypersensitivity reaction (10-12). The lack of capillary thrombosis favors an Arthus reaction rather than a local Shwartz- man phenomenon (12, 13). The histological features of this model exhibit certain interesting characteristics including focal, discontinuous transmural inflammation and mucosal ulceration. Such findings are consis- tent with vasculitides, including Bechet's disease and Wegener's granulomatosis (14, 15), in addition to other intestinal hypersensitivity reactions and necrotizing enteritis (16).

It has been reported that Crohn's disease is characterized by a granulomatous vasculitis (17). Wake- field's study (17) demonstrated the intimate association of granulomas with the intestinal micro-

Digestive Diseases and Sciences, Vol. 39, No. 3 (march 1994) 537

Fig 4. Chronic perivascular lymphocytic aggregation within the submucosa of a ferret two weeks after embolization and weekly subcutaneous injection of human aggregated albumin. H&E, x350 (reduced to 92% for reproduction).

vasculature, the majority of these lesions affecting vessels within the submucosa. We have recently demonstrated that occlusion of the submucosal vessels of the intestinal wall of the ferret with inert plastic microspheres can result in focal mucosal ulceration at 24-48 hr (8). At 72 hr the mucosa had regenerated, and this regeneration was dependent upon the integrity of the intact submucosal collateral plexus. It must be emphasized that the six control ferrets in group 1 of the present study, embolized without presensitization to albumin, showed no evidence of mucosal damage, thus dem- onstrating the efficiency of this submucosal collateral plexus. Most striking at this time point is the lack of ischemic damage in comparison with the model of mechanical occlusion using latex nonbiodegradable microsphere of similar size (8). The half- life of the albumin spheres is only 2 hr and the ischemic insult only temporary. The present model shows that acute submucosal vasculitis (after deg-

HUDSON ET AL

radation of these albumin microspheres in presensitized animals) can result in acute transmural inflammation and mucosal ulceration in some animals. Furthermore, it suggests that a sustained or re- peated immunological attack directed against the microvasculature of the intestinal wall may result in chronic ischemia, persistent inflammation, and ulceration of the intestine.

It is probable that cellular immunity plays a role in the pathogenesis of Crohn's disease, but attempts to demonstrate a mucosal antigen have been unpro- ductive. The hypothesis of an immunological response directed against an antigen on or in the intestinal endothelial cell in mesenteric vasculiti- dies, including Crohn's disease, is attractive. We now have anatomical evidence to suggest that such an attack could explain the pattern of the disease: a relatively ischemic intestine could also account for the increased permeability (18) and, in addition, the tendency for disease to recur at the anastomosis following surgical resection (19-21).

Although we are still some way from an ideal animal model of Crohn's disease, systems that are directed towards chronic immunological damage to the submucosal microvasculature may help the lab- oratory investigation of this enigmatic disease.

ACKNOWLEDGMENTS

Dr. Mark Hudson is the Leslie Parrott Research Fellow supported by the National Association of Colitis and Crohn's disease. Mr. A.J. Wakefield is supported by the Wellcome Trust. The authors wish to thank Mrs. Deirdre Grennan for her technical assistance and Miss Doris Elliott for processing the manuscript.

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a vascular hypersensitivity model of acute multifocal gastrointestinal infarction

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