ANIMALS

Specific pathogen free male Sprague-Dawley rats weighing 200–250 g were purchased from Centre d'Elevage R Janvier (Le Genest, France). The rats were housed under controlled temperature, humidity, and illumination conditions, and maintained on standard rodent chow (Letica, Barcelona, Spain) and tap water. The local research committee (Comissio de Recerca, Hospital General Universitari Vall d'Hebron) approved all studies.

EXPERIMENTAL MODEL

Rats with a colonic loop excluded from faecal transit were used. The excluded colonic loop provides an in vivo ecological environment for colonisation with preselected bacterial strains, after elimination of the native flora with antibiotics. The colonic loop was surgically prepared as previously described.12 In anaesthetised rats (90 mg/kg ketamine, Parke-Davis, Ann Arbour, Michigan, USA, and 4 mg/kg xylazine, Bayer, Leverkusen, Germany), a colonic segment (ascending to the mid-descending colon) was brought out to the anterior abdominal wall through two colostomies. The caecum was removed, and continuity of the tract was achieved by anastomosis of the terminal ileum to the distal colon. After suture of the abdominal wall, colostomies were washed with broad spectrum non-absorbable antibiotics (5 mg/ml imipenem and 5 mg/ml vancomycin in sterile saline) and closed by ligature to prevent the entry of air into the colonic segment. Thereafter, rats were allowed to recover, and standard chow pellets and water were provided ad libitum. By diversion of faecal transit, bacterial populations within the loop can be controlled and maintained without significant variations for several days.12

EXPERIMENTAL DESIGN

Six groups of rats were surgically prepared as described above. In the control group, the colonic segments were flushed through the colostomies with the solution of broad spectrum antibiotics (imipenem and vancomycin, as above) on days 1 and 4 after surgery, under anaesthesia. The remaining five groups were used for recolonisation with a single bacterial strain. All strains were of rat intestinal origin and included: Escherichia coli, Klebsiella pneumoniae, Streptococcus viridans, Bacteroides fragilis, andLactobacillus brevis. These bacterial strains were identified by standard bacteriological procedures, including biochemical testing with API strips and Vitek system identification cards (bioMérieux, Marcy-l'Etoile, France), and had been used in previous experimental studies using the same model.10 ,12 ,13 Colonic segments were recolonised 24 hours after surgery. Under anaesthesia, colostomies were flushed with the antibiotic mix and kept tightly closed for two hours. Thereafter, colostomies were washed with sterile saline, inoculated with 1.5–2.0 ml of a specific bacterial suspension, and immediately closed by ligature. Bacterial suspensions were prepared from liquid cultures of each individual strain and used after the phase of logarithmic growth, containing approximately 10¹⁰ colony forming units/ml. Four days after recolonisation, segment washings with sterile saline were cultured to check actual bacterial colonisation. Only rats with positive culture for the specific strain and without other contaminant bacteria were used for the permeability studies. All rats given antibiotics on days 1 and 4 after surgery showed negative cultures of segment washings and were included in the control group.

COLONIC PERMEABILITY

Lumen to blood clearance of ¹⁴C-d-mannitol (molecular weight 184 Da; Amersham International, Buckinghamshire, UK) and ³H-dextran (molecular weight 70 000 Da; Amersham) was measured in groups of control and recolonised rats (controls n=10;Streptococcus viridans n=6;Klebsiella pneumoniaen=6; Escherichia coli n=7;Bacteroides fragilis n=7;Lactobacillus brevis n=9). Four days after bacterial inoculation, rats were anaesthetised (90 mg/kg ketamine and 4 mg/kg xylazine), a tracheotomy performed, and the right carotid artery and left jugular vein were cannulated with polyethylene tubing (PE-50; Clay Adams, Becton Dickinson, Parsippany, New Jersey, USA). Bilateral nephrectomy was achieved by ligation of the renal pedicles through right and left lumbotomy. A 500 μl bolus of 10 mmol/ml coldd-mannitol in saline with 0.4 μCi D-[1-¹⁴C]mannitol and 0.3 μCi [³H]dextran was instilled into the lumen of the excluded colonic segment and both colostomies were kept closed for five hours. Total radioactivity administered to each animal was calculated by counting two 50 μl aliquots of the administered solution using a scintillation counter with a dual channel programme for simultaneous ¹⁴C and³H counting (LS 6500; Beckman Instruments, Palo Alto, California, USA). Blood samples (500 μl) were taken through the carotid line at 0, 1, 2, 3, 4, and 5 hours after the intracolonic bolus, and used for measurement of plasma radioactivity levels by means of a scintillation counter. Immediately after sampling, an equal volume of 2% albumin in sterile saline was administered via the jugular catheter to prevent changes in haemodynamics. Finally, the rat was killed by anaesthetic overdose, and two urine samples were obtained from the bladder and counted to exclude leakage of the radioactive probes. Accumulation of the probes in blood was assumed to reflect entry rates (lumen to blood clearance) of the molecules as rats had been nephrectomised and the compounds are not metabolised. Thus data on lumen to blood clearance of d-mannitol were expressed as a percentage of the administered dose that accumulated in the extracellular fluid volume (25% of body weight), normalised by the length (cm) of the excluded colonic segment of each rat. Lumen to blood clearance of dextran was expressed as a percentage of the administered dose that accumulated in the intravascular fluid volume (6% of body weight), normalised by the length of the excluded colonic segment.

Ancillary experiments with controls (n=8) and E coli recolonised rats (n=5) used ⁵¹Cr EDTA (molecular weight 341 Da; Dupont, NEN Research Products, Boston, Massachusetts, USA) as a permeability probe as this compound is biologically inert and cannot be degraded by bacteria. The experiments were performed following the same procedure as with mannitol and dextran. A 500 μl bolus of 0.5 mmol/ml cold EDTA in saline with 5 μCi ⁵¹Cr-EDTA was instilled into the lumen of the excluded colonic segment and both colostomies were kept closed for five hours. Radioactivity in two aliquots of the administered dose and in hourly blood samples was measured by means of a gamma counter. Lumen to blood clearance of ⁵¹Cr-EDTA was expressed as a percentage of the administered dose that accumulated in the extracellular fluid volume (25% of body weight), normalised by the length of the excluded colonic segment.

MICROBIOLOGICAL METHODS

Bacterial suspensions for inoculations were prepared from cultures of each individual species. All strains were isolated from inmate Sprague-Dawley rats and maintained at −80°C. Bacteria were defrosted and allowed to grow in liquid medium (brain-heart infusion with haemin and vitamin K₁) at 37°C until the logarithmic growth phase. The final inoculation suspension contained approximately 10¹⁰ colony forming units/ml.

Saline washings of the excluded colonic segment were incubated under aerobic and anaerobic conditions to check actual bacterial colonisation. Aerobic media consisted of blood agar, blood agar with nalidixic acid, McConkey agar, Cled agar, and Sabouraud agar. Anaerobic media consisted of lacquered blood agar enriched with haemin and vitamin K₁, blood agar with phenyl-ethanol, and agar-aztreonam (20 μg/ml). Plates inoculated for obligate anaerobes were incubated in an anaerobic chamber for 48–72 hours at 37°C, and plates for aerobes in air at 37°C.

STATISTICAL METHODS

Results are expressed as mean (SEM). Statistical differences between groups were determined using overall analysis of variance, and the Student-Newman-Keuls method as post-test.