Post operative neutrophilic inflammation in equine small intestine after manipulation and ischaemia

REASONS FOR PERFORMING STUDY: Post operative ileus (POI) remains an important cause of post operative morbidity and mortality in the horse. However, clinical progression of naturally occurring cases of POI in both horse and man does not entirely support the 'neurogenic' hypothesis as the sole mechanism of POI; and the hypothesis that inflammation plays a major role at 12-24 h after surgery requires validation. HYPOTHESIS: An inflammatory infiltrate in the muscularis externa and myenteric plexus of equine jejunum is present 18 h following a period of ischaemia. METHODS: Samples of normal jejunum, jejunum from the proximal resection margins of clinical cases and jejunum obtained 18 h after 1 or 2 h ischaemia or manipulation alone were evaluated for neutrophil infiltration. Samples obtained 18 h after surgery were additionally evaluated for leucocyte activation using calprotectin immunohistochemistry. Results were evaluated by ANOVA and P < 0.05 was considered significant. RESULTS: Significant neutrophilic inflammation was identified in the samples from the proximal resection margins of clinical cases compared to uninjured jejunum. In experimental cases, neutrophilic inflammation appeared to be increased further by 18 h and was identified through all intestinal layers, particularly in the serosa, fascial planes around circular and longitudinal muscle fibres, and myenteric plexus. This elevated level of neutrophilic inflammation was mirrored by an increased number of calprotectin-positive cells in these intestinal layers, indicating leucocyte activation. CONCLUSIONS: Significant neutrophilic inflammation occurs in equine jejunal myenteric layers 18 h after surgery. POTENTIAL RELEVANCE: This neutrophilic inflammation coincides with the clinical time point at which POI is identified and may indicate that inflammatory pathways, rather than solely neurogenic pathways, are responsible for POI in the horse.     

Campylobacter jejuni infections in gnotobiotic pigs

At 3 days of age, 12 gnotobiotic pigs were inoculated orally with broth cultures of Campylobacter jejuni. One pig was euthanatized and evaluated each day for 12 days. In the cecum and colon, there was diffuse edema, neutrophilic infiltration, and sloughing of epithelial cells from the mucosa on postinoculation days (PID) 2 through 5. Dysplastic colonic crypt epithelial cells were observed in the submucosa of the colon on PID 5 through 12. Curved, rod-shaped bacteria were detected on the surface of ileal, cecal, and colic absorptive and glandular epithelial cells. Bacteria also were found around small submucosal vessels on PID 3 and 4 and were associated with numerous perivascular neutrophils. The gnotobiotic pig appears to provide a simple, well-controlled in vivo model for the study of the pathogenesis of C jejuni infections in human beings, pigs, and other mammals.     

Effect of a novel solution for organ preservation on equine large colon in an isolated pulsatile perfusion system

REASONS FOR PERFORMING STUDY: Several therapeutic agents have been tested in models of ischaemia and reperfusion injury (IRI) in equine jejunum, with mixed results. This study was based on the use of an organ perfusion solution (OPS) designed to protect human allografts from IRI. HYPOTHESIS: A modified OPS can preserve the integrity of equine large colon during 12 h of isolated pulsatile perfusion, in the absence of oxygen and blood. METHODS: Segments of large colon were removed from anaesthetised horses, the contents removed and the mucosa rinsed with 0.9% saline. Experimental segments were perfused for 12 h with one litre modified OPS (n = 7) delivered by pulsatile flow through an extracorporeal circuit. Control segments (n = 4) were perfused on the same circuit with one litre of autologous blood. Vascular resistance, flow and pressure were measured serially, and aliquots of OPS and blood drawn hourly for routine biochemical analyses. Mucosal biopsies of the experimental and control segments were taken at 0, 6 and 12 h and in vivo mucosal tissue at 0 h for baseline comparison. All biopsies underwent histomorphometric analysis and immunohistochemical assessment of calprotectin activity. RESULTS: All colon segments were machine perfused without technical complications. Vascular and biochemical indices remained constant over 12 h in the OPS group, and were constant over 6 h in the control group, but deteriorated later. Mucosal integrity, expression of cyclooxygenases-1 and -2, and expression of mucosal calprotectin were unchanged in the OPS group compared with the baseline tissues, and mucosal integrity was superior to the control tissues. CONCLUSIONS: A modified OPS designed to target specific pathways of damage from IRI can preserve colonic mucosal integrity for 12 h in the absence of blood and oxygen.     

The Effects of Ischemia and Reperfusion on Mucosal Respiratory Function, Adenosine Triphosphate, Electrolyte, and Water Content in the Ascending Colon of Ponies

Objective- The purpose of this study was to examine the effects of ischemia and reperfusion on the biochemical integrity of equine colonic mucosa to assess the relative roles of ischemic- and reperfusion-induced damage.
Study Design- Two hours of no-flow ischemia experimentally induced by 720° counterclockwise ascending colon volvulus followed by 2 hours reperfusion after derotation.
Animals- Ten ponies.
Methods- Ascending colon biopsies were obtained every hour for measurement of mucosal adenosine triphosphate (ATP), water, sodium, and potassium content. Additional samples were homogenized for assay of mitochondrial respiratory function.
Results- ATP content diminished 92% after ischemia and recovered to only 44% of control levels ( P <.001 versus controls) after 2 hours reperfusion. Reperfusion increased mucosal water and decreased sodium and potassium content for the duration of the experiment. Both NADH- (pyruvate) and FADH-linked (succinate) respiration decreased after ischemia and did not recover during reperfusion indicating electron transport chain dysfunction.
Conclusions- Two hours ischemia induced severe metabolic dysfunction in equine colon mucosa which persisted throughout reperfusion. Unequivocal evidence of injury specific to reperfusion was not observed in this study suggesting that much of the damage observed during reperfusion may be a continuation of injury induced during the ischemic period and not specific to reperfusion per se.
Clinical Relevance- This study suggests that greater efforts to metabolically support ischemically injured mucosa may be an important aspect of obtaining improved survival of horses affected by ascending colon volvulus (ACV).     

Failure to demonstrate reperfusion injury following ischaemia of the equine large colon using dimethyl sulphoxide

A study was undertaken to evaluate the significance and mechanism of reperfusion injury in the equine large colon following 1 h of haemorrhagic strangulation obstruction (HSO) or ischaemic strangulation obstruction (ISO) and to assess the effect of treatment with dimethyl sulphoxide (DMSO). ISO or HSO were created 40 cm from the pelvic flexure and maintained for 60 mins under general anaesthesia. Normal saline or 20 per cent DMSO (1 g/kg bodyweight) was administered intravenously 10 mins prior to the end of the ischaemic period. Four groups of four horses in a 2 x 2 factorial design were used. Treatments of HSO or ISO and DMSO given (yes or no) were utilised. Intestinal wall biopsies and right colic arterial and venous blood samples were taken at 0, 60, 90 and 120 mins following initiation of the obstructions. Histological evaluation of the intestine using haematoxylin and eosin stained sections and immunohistochemical staining for albumin were performed. Mucosal and serum reduced glutathione (GSH) and oxidised glutathione (GSSG) levels and the amount of lymphatic dilatation with albumin and submucosal pooling of albumin were used as indirect measures of oxygen free radical production. Histopathological changes were minimal after 1 h of either type of ischaemia. Progressive changes during the post ischaemic period were minimal for ISO and moderate for HSO. Serum GSH and GSSG levels were not detectable. There was no demonstrable benefit of DMSO treatment as assessed by histology, immunohistochemistry or preservation of GSH levels in the mucosa. In conclusion, a reperfusion injury following 60 mins of ischaemia could not be detected in this study.     

Mucosal distribution of eosinophilic granulocytes within the gastrointestinal tract of horses

OBJECTIVES: To establish reference values for the range of the number of eosinophils found in equine gastrointestinal mucosa and to describe the distribution of this cell within the equine gastrointestinal mucosa. SAMPLE POPULATION: Gastrointestinal mucosal specimens from 14 adult horses euthanatized for reasons other than gastrointestinal disease. PROCEDURES: Gastrointestinal mucosal specimens were collected and grouped according to their anatomic regions. For histologic examination slides were stained with Luna's eosinophil stain to determine eosinophil accumulation and distribution. The mucosa was divided into 5 sections for each anatomic location, and the percentage of eosinophils in each of the 5 sections relative to the total eosinophil count in all sections was determined. Additionally, the number of eosinophils per square millimeter of mucosa was calculated as a measure of the degree of eosinophil accumulation. RESULTS: Lowest numbers of eosinophils were found in the stomach, and numbers increased from there to the cecum, then decreased from the ascending colon (right ventral colon, left ventral colon, pelvic flexure, left dorsal colon, and right dorsal colon) to small colon. In all gastrointestinal sections, most eosinophils were located near the muscularis mucosae and were rarely found near or on the luminal surface of the mucosa. CONCLUSIONS AND CLINICAL RELEVANCE: The distribution of eosinophils in the gastrointestinal tract of horses followed a pattern within the mucosa and between different sections of the gastrointestinal tract. The derived reference values and distribution data could be used to detect changes in eosinophil response in the equine gastrointestinal mucosa caused by diseases states.     

Histopathological and ultrastructural changes in simulated large colonic torsion and reperfusion in ponies.

This investigation examines the histological and ultrastructural lesions of the colonic mucosa during terminal experimental infarction and subsequent reperfusion. Four ponies were anaesthetised and subjected to surgical torsion of the colon. Biopsies were collected at hourly intervals for 3 h, at which point the torsions were corrected. Circulation was re-established for 2 h and the bowel was re-biopsied at hourly intervals. The ponies were killed while under anaesthesia. During the 3 h experimental infarction, the bowel became macroscopically thickened and dark purple. Histologically, the mucosa degenerated from Grade 0 to Grade 3. Ultrastructurally, there was progressive micro-vascular distension with erythrodiapedesis and damage to the interstitial cells. Spaces developed between the bases and sides of the columnar epithelial cells and sloughing followed subsequently. During the 2 h reperfusion interval, the mucosa continued to degenerate rapidly to a Grade 5, and was characterised by extensive interstitial damage, oedema, cellular swelling, necrosis and mitochondrial damage. The results showed that the experimentally infarcted colonic mucosa degenerated sequentially. Following circulatory reestablishment, continued rapid mucosal degeneration characteristic of reperfusion injury occurred. Reperfusion injury is probably responsible, at least in part, for the often poor outcome of infarcted bowel in horses following surgical correction.     

Ischaemia/Reperfusion Injury in Experimentally Induced Abomasal Volvulus in Sheep

The purpose of the study was to evaluate ischaemia/reperfusion injury in simulated abomasal volvulus in sheep. Sixteen ewes were randomly allocated to three groups. The control group (n = 4) served as sham-operated controls. The animals of the ischaemia group and reperfusion group (n = 6, each) underwent a simulated ‘abomasal volvulus’. The abomasum was exteriorized under general inhalation anasthesia and forced into a 180^∘ anticlockwise rotation around its longitudinal axis, followed by another 270^∘ anticlockwise rotation around its transectional axis. All ewes were monitored for 4 h. In the reperfusion group, volvulus was released after 3 h (i.e., 1 h of reperfusion). In the ischaemia group, the volvulus remained for 4 h (no reperfusion). Vital signs were monitored and some haematological and biochemical parameters were measured, without any significant differences. Full-section biopsy specimens were taken at the 3rd and 4th hours from the greater curvature of the abomasum. Histopathological lesions were scored according to the severity of mucosal oedema, submucosal oedema, haemorrhage submucosal and submuscularis layers, and polymorphonuclear infiltration on a scale of 0 to 4 (nil, mild, moderate, severe, and extreme). Another biopsy specimen was taken at the 4th hour for transmission electron microscopic examination. The scored lesions in light-microscopic examination were significantly different at the 3rd and 4th hours between the control and the experimental groups (p<0.05). There was no significant difference between the reperfusion and ischaemia groups (p>0.1). Within-group comparisons (3rd hour with 4th hour) revealed no significant differences. In transmission electron microscopic examination there were no remarkable changes in the control group, but in the ischaemia and reperfusion groups there were remarkable cellular (epithelial and goblet cells), mitochondrial and microvillous changes that strongly implied the occurrence of ischaemia (p<0.05). In transmission electron-microscopic examination of abomasal samples the lesions were more remarkable in reperfusion group than in the ischaemia group. It is concluded that ischaemia/reperfusion injury occurred in this model of simulated abomasal volvulus in sheep and that ischaemia/reperfusion injury should be considered as a potential determining factor in the outcome of cattle with abomasal volvulus.     

The effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study

Phenylbutazone, a non-steroidal anti-inflammatory drug known to produce intestinal erosions, was administered intravenously (13.46 mg/kg bodyweight) to 12 horses which were killed after 24, 48, 72 and 96 h. Eight untreated horses served as controls. Annular erosions in the duodenum and mucosal necrosis in the colon were seen after 48 h which progressed in severity. The erosions were characterised by sloughing of the surface epithelium, subepithelial cleft and bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries and microthrombosis. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Ultrastructurally, there was swelling of the endothelium of capillaries and small vessels, and of pericyte and smooth muscle cytoplasm in arterioles. In capillaries and post capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and oedema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury associated with the formation of duodenal and colonic erosions in horses. The duodenal and colonic mucosa were assayed at 48 and 96 h for prostacyclin and PGE2. There was no statistically significant difference between prostaglandin levels in the mucosa of control and treated horses. It was concluded that there was no correlation between mucosal prostaglandin levels and intestinal erosions after 48 h.     

Morphologic and physiologic changes induced by Clostridium perfringens type A alpha toxin in the intestine of sheep

OBJECTIVE: To evaluate the morphologic and physiologic changes induced by Clostridium perfringens type A (alpha toxin in the ileum and colon of sheep. SAMPLE POPULATION: 16 ligated intestinal loops in 4 Merino lambs and 18 explants of ileum and colon from slaughtered lambs. PROCEDURE: alpha Toxin-induced fluid accumulation was evaluated in ligated ileal and colonic loops of sheep. Tissues were evaluated morphologically by use of gross and histologic examination. Effects of toxin on in vitro intestinal net water transport were tested in modified Ussing chambers. RESULTS: Ovine ileal and colonic loops incubated with C perfringens type A alpha toxin retained more fluid than control loops. Histologically, in the ileum of lambs inoculated with 300 LD50 of alpha toxin/mL, there was a mild to moderate multifocal infiltration of neutrophils in the lamina propria and submucosa. The colonic loops of lambs inoculated with 30 or 300 LD50 of alpha toxin/mL had excessive mucus in the lumen, a moderate amount of neutrophils mixed with mucus in the intestinal lumen, and moderate multifocal infiltration of the lamina propria and submucosa with neutrophils; the blood vessels of these layers were engorged with neutrophils. In vitro measurements of water transport also revealed inhibition of net epithelial water absorption in ileum and colon incubated with alpha toxin on the mucosal side. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that alpha toxin induces alterations in sheep intestine. Clostridium perfringens type A organisms that produce alpha toxin could be responsible for diseases of intestinal origin in some ruminants.     

Attempts to produce granulomatous colitis in Boxer dogs with a mycoplasma.

Four of six Boxer dogs with granulomatous colitis had mycoplasmas in the colon and three in the draining lymph node as well. One isolate identical with Mycoplasma strain HRC-689 was inoculated into the colon of five Boxer dogs at eight weeks of age; four controls were inoculated with medium. Animals were killed at two week intervals and examined. The mycoplasma strain produced changes of the colon characterized by lymphocytic infiltration of mucosa, lymphocytic submucosal perivasculitis, and enlargement of colonic lymphoid nodules but granulomatous colitis did not occur. The experimental dogs developed antibodies, and dogs from a kennel with a high prevalence of colitis cases had high antibody titers against strain HRC-689.     

Local and remote lesions in horses subjected to small colon distension and decompression

The purpose of this study was to observe and characterize colonic and lung lesions in horses subjected to experimental distension and decompression of the small colon. Sixteen healthy adult horses were divided into 2 groups: 9 horses that were subjected to distension of the small colon by means of a latex balloon surgically implanted in the lumen and inflated to a pressure of 40 mm Hg for 4 h, and 7 horses in which the balloon was implanted but not inflated. Colonic biopsy specimens were collected before balloon implantation, at the end of the period of obstruction, and 1.5 and 12 h after decompression and were examined for hemorrhage, edema, and neutrophil infiltration; myeloperoxidase (MPO) activity and hemoglobin concentration were measured as well. At the end of the experiment, lung samples were also collected and examined for neutrophil accumulation and MPO activity. The mucosa was not affected by luminal distension; lesions were restricted to the seromuscular layer. Neutrophil accumulation and edema were observed in the samples from both groups of horses but were greater in those from the distension group, in which there was also hemorrhage, fibrin deposition, and increased MPO activity in the seromuscular layer. Similarly, there was greater accumulation of neutrophils in the lung samples from the distension group than in those from the sham-operated group, as determined by histologic evaluation and MPO assay. These findings provide new evidence of reperfusion injury and a systemic inflammatory response, followed by remote lesions, in horses with intestinal obstruction.     

The characteristics of intestinal injury peripheral to strangulating obstruction lesions in the equine small intestine.

Recent studies suggest that horses requiring surgical correction of strangulating intestinal obstruction may develop post operative complications as a result of ischaemia/reperfusion injury. Therefore, the mucosal and serosal margins of resected small intestine from 9 horses with small intestinal strangulating lesions were examined for evidence of ischaemia/reperfusion injury. Severe mucosal injury and marked elevations in myeloperoxidase activity were detected at ileal resection margins (n = 4), whereas the mucosa from proximal jejunal (n = 9) and distal jejunal (n = 5) resection margins was normal. However, the serosa from jejunal resection margins had evidence of haemorrhage and oedema, and the proximal jejunal serosa had significantly increased numbers of neutrophils. Histological injury in ileal stumps is indicative of the inability fully to resect the ileum in horses with distal small intestinal strangulations. One of 4 horses subjected to ileal resection was subjected to euthanasia and found to have a necrotic ileal stump. Evidence of serosal injury and neutrophil infiltration in the proximal jejunal resection margins may predispose horses to post operative adhesions. Four of 8 horses discharged from the hospital suffered from recurrent colic in the post operative period.     

Serosal injury in the equine jejunum and ascending colon after ischemia-reperfusion or intraluminal distention and decompression

OBJECTIVE: To document morphologic changes that occur in equine intestinal serosa after experimentally induced ischemia and subsequent reperfusion (jejunum, ascending colon) or after intraluminal distention and decompression (jejunum). STUDY DESIGN: Morphologic effects of ischemia-reperfusion or intraluminal distention-decompression determined on the serosal layer of the equine jejunum. The large colon serosa was evaluated after ischemia-reperfusion injury. ANIMALS OR SAMPLE POPULATION: Seven adult horses. METHODS: After induction of general anesthesia and ventral median celiotomy, ischemia was created by arteriovenous (AVO) and lumen occlusion of a 20-cm segment of jejunum and ascending colon for 70 minutes, followed by a 60-minute reperfusion period. Intraluminal distention (25 cm H2O) was created in a second 20-cm jejunal segment and maintained within the abdomen for 120 minutes, followed by a 120-minute decompression period. Seromuscular biopsies were obtained upon entering the abdomen and after the ischemic and reperfusion periods, and after the distention and decompression periods along with corresponding control seromuscular biopsies. Samples were processed and examined by light microscopy, transmission electron, and scanning electron microscopy. RESULTS: Ischemia and reperfusion, and intraluminal distention and decompression, resulted in severe morphologic changes in the seromuscular layer of equine jejunum. A similar period of ischemia-reperfusion caused minimal changes in the ascending colon serosa. CONCLUSION: Adult equine jejunum sustains more serosal damage than the ascending colon after similar periods of ischemia-reperfusion injury. Intraluminal distention and subsequent decompression causes serosal damage in the equine jejunum. CLINICAL RELEVANCE: The small intestine is more susceptible to seromuscular layer damage than the ascending colon.     

Characterisation of the inflammatory reaction in equine idiopathic focal eosinophilic enteritis and diffuse eosinophilic enteritis

REASONS FOR PERFORMING STUDY: Idiopathic focal eosinophilic enteritis (IFEE) and diffuse eosinophilic enteritis (DEE) are primary eosinophilic intestinal conditions without a known cause that are associated with an increasing number of surgical colic cases. Histology may be helpful in defining disease aetiology and pathogenesis. OBJECTIVES: To characterise further the inflammatory infiltrate in equine IFEE and to compare the condition with DEE. METHODS: Twenty-three IFEE cases and 5 DEE cases were examined by light microscopy including immunohistology to identify infiltrating leucocytes. Inflammatory infiltrates in mucosa and submucosa were characterised in IFEE lesions (Group 1), the intestine distant from the lesions in IFEE (Group 2) and DEE (Group 3). RESULTS AND CONCLUSIONS: IFEE lesions represented an accumulation of leucocytes in submucosa and muscularis, with dominance of eosinophils and macrophages and smaller numbers of lymphocytes, plasma cells and neutrophils. T cells represented the dominant lymphocytes. The mucosa overlying the lesion and both mucosa and submucosa in IFEE nonlesion sites and in DEE exhibited a similar composition, with different prevalence of various cell types. Macrophages were significantly more prevalent in the mucosal and submucosal infiltrates in IFEE nonlesion sites than in DEE, and lymphocytes significantly more prevalent in the mucosa in DEE than in IFEE nonlesion sites. The findings confirm IFEE as a primary eosinophilic intestinal disorder and indicate that IFEE represents a focally exacerbated inflammatory reaction in horses with DEE, possibly due to functional changes in the macrophage and T cell components, with subsequent excessive recruitment of both eosinophils and macrophages.     

Prostaglandin E2 and reactive oxygen metabolite damage in the cecum in a pony model of acute colitis

The objective of this project was to determine early tissue biochemical events associated with increased colonic secretion during the acute stage of castor-oil-induced colitis by measuring cecal mucosal and submucosal malondialdehyde (MDA) and prostaglandin E₂ (PGE₂), levels in ponies. Intestinal tissue (inflamed or healthy) samples were obtained from 4 age- and sex-matched Shetland ponies. Biochemical methods were used to determine MDA and PGE₂ levels in intestinal tissue samples from inflamed and healthy equine intestine. Inflamed tissue MDA and PGE₂ levels increased with time after castor oil challenge and correlated with granulocyte infiltration, as determined by myeloperoxidase levels in a companion study. Elevated intestinal tissue MDA levels suggest that lipid peroxidation could be attributed to reactive oxygen metabolites (ROM) released from stimulated, recruited, and resident granulocytes. Tissue levels of MDA and PGE₂ suggest a role for granulocyte-derived mediators of intestinal inflammation in the massive secretory response in cases of acute equine colitis. Tissue MDA and PGE₂ levels may be useful laboratory tools to quantify and characterize intestinal secretory inflammatory responses in acute inflammatory conditions in the equine colon.     

Effects of carprofen on the integrity and barrier function of canine colonic mucosa

OBJECTIVE: To measure effects of carprofen on conductance and permeability to mannitol and histologic appearance in canine colonic mucosa. SAMPLE POPULATION: Colonic mucosa from 13 mature mixed-breed dogs. Procedures-Sections of mucosa from the transverse colon and proximal and distal portions of the descending colon were obtained immediately after dogs were euthanized. Sections were mounted in Ussing chambers. Carprofen (400 microg/mL) was added to the bathing solution for treated sections. Conductance was calculated at 15-minute intervals for 240 minutes. Flux of mannitol was calculated for three 1-hour periods. Histologic examination of sections was performed after experiments concluded. Conductance was graphed against time for each chamber, and area under each curve was calculated. Conductance X time, flux of mannitol, and frequency distribution of histologic findings were analyzed for an effect of region and carprofen. RESULTS: Carprofen significantly increased mean conductance X time, compared with values for control (untreated) sections for all regions of colon. Carprofen significantly increased mean flux of mannitol from period 1 to period 2 and from period 2 to period 3 for all regions of colon. Carprofen caused a significant proportion of sections to have severe sloughing of cells and erosions involving >or= 10% of the epithelium, compared with control sections. CONCLUSIONS AND CLINICAL RELEVANCE: Carprofen increased in vitro conductance and permeability to mannitol in canine colonic mucosa. Carprofen resulted in sloughing of cells and erosion of the colonic mucosa. These findings suggested that carprofen can compromise the integrity and barrier function of the colonic mucosa of dogs.     

Erosive colitis in experimental canine Leishmaniasis

Six 4 month-old beagles were inoculated with Leishmania infantum, three of them intraperitoneally (Group A) and the other three intravenously (Group B). The animals from Group A were killed at 109, 433 and 592 days after inoculation and animals from Group B 109, 171 and 334 days after inoculation. At 350 days (Group A) and 275 days (Group B) after inoculation, dogs started to develop a chronic large bowel diarrhoea. At necropsy a diffuse colonic and rectal wall thickening of gradually increasing severity was observed. Histological examination of the colon showed a diffuse inflammatory infiltration of the mucosa and submucosa by macrophages with amastigotes, lymphocytes, plasma cells and some neutrophils and eosinophils. The surface epithelium developed increasingly extensive degeneration, which caused the development of erosions on the mucosal surface. The crypts of Lieberkühn decreased in number and showed degeneration of the crypt epithelium.     

Effect of Platelet-Activating Factor Antagonist L-691,880 on Low-Flow Ischemia-Reperfusion Injury of the Large Colon in Horses

Objective—To determine the effect of platelet-activating factor (PAF) antagonist L-691,880 on low-flow ischemia and reperfusion (I-R) of the large colon in horses. Animals —12 adult horses. Experimental Design—Horses were anesthetized, and the large colon was exteriorized through a ventral median celiotomy and instrumented. Colonic arterial blood flow was reduced to 20% of baseline (BL) and maintained for 3 hours; flow was then restored, and the colon was reperfused for 3 hours. One of two solutions was administered intravenously 30 minutes before reperfusion: group 1, 10 mL/kg 0.9% NaCl; and group 2, 5 mg/kg PAF antagonist L-691,880 in 0.9% NaCl. Hemodynamic variables were monitored and recorded at 30-minute intervals. Systemic arterial and colonic venous blood were collected for measurement of blood gas tensions, oximetry analyses, packed cell volume, and total plasma protein concentrations. Colonic venous blood was collected for determination of lactate, 6-keto prostaglandin F_1α (6-kPG), prostaglandin E₂ (PGE₂), and thromboxane B₂ (TXB₂) concentrations. Full-thickness biopsy specimens were harvested from the left ventral colon for histological evaluation. Results—There were no significant differences between the two groups for any hemodynamic or metabolic variables. Colonic venous pH decreased, and carbon dioxide tension and lactate concentration increased during ischemia but returned to BL values during reperfusion. Colonic venous 6-kPG concentration was significantly increased above BL value at 2 hours and remained increased through 6 hours in horses of both groups. Colonic venous PGE₂ concentration was significantly greater in group 2 compared with group 1 throughout the study. Colonic venous PGE₂ concentration was increased above BL value from 3 to 6 hours in horses of both groups. Colonic venous TXB₂ concentration was not different between groups but was significantly increased above the BL value for the first hour of reperfusion. Low-flow I-R of the large colon caused significant mucosal necrosis, hemorrhage, edema, and neutrophil infiltration; however, there were no differences in histological variables between vehicle-control and PAF antagonist-treated horses. Conclusion—No protective effects of PAF antagonist L-691,880 were observed on colonic mucosa associated with low-flow I-R. Additionally, deleterious drug-induced effects on hemodynamic and metabolic variables and colonic mucosal injury were not observed.     

Neutrophil migration induced by equine respiratory secretions, bronchoalveolar lavage fluids and culture supernatants of pulmonary lavage cells

Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty housing conditions induced migration of neutrophils in the same order of magnitude. The number of migrated neutrophils and the procoagulant activity (PCA) within respiratory secretion specimens was positively correlated; however, the meaning of this finding is not yet clear. None of the nine cell-free supernatants of bronchoalveolar lavage fluid, which were assayed undiluted, induced significant neutrophil migration, although some samples contained up to 4.0 x 10(5) neutrophils/ml. In vitro culture of lung lavage cells, which mainly comprised macrophages and lymphocytes, without stimulation or with the addition of low doses of phytohemagglutinin (PHA) resulted in the secretion of NMPA which was in great part chemotactic. However, culture supernatants of lung cell preparations which were stimulated by lipopolysaccharide (LPS) or by PHA-prestimulated lymphocytes reduced the migration of neutrophils compared with the supernatants of control cells. NMPA within culture supernatants had a highly significant negative correlation with the PCA of macrophages within the lung cell preparations. Our results imply that a complicated and sophisticated regulation underlies neutrophil accumulation within the airways of horses affected with chronic pulmonary disease. Future experiments are required to assess the biological significance of the factors modulating neutrophil migration which are present in the respiratory secretions and in the culture supernatants of equine lung lavage cells.