Cholecystectomy during ceftriaxone therapy. A translational study with a new rabbit model

<div><p>Abstract Purpose: To evaluate the actual incidence of both microlithiasis and acute cholecystitis during treatment with intravenous ceftriaxone in a new rabbit model. Methods: New Zealand rabbits were treated with intravenous ceftriaxone or saline for 21 days. Ultrasound monitoring of the gallbladder was performed every seven days until the 21st day when histopathology, immunohistochemistry for proliferating cell nuclear antigen (PCNA), pro-caspase-3 and CD68, liver enzyme biochemistry, and chromatography analysis of the bile and sediments were also performed. Results: All animals treated with ceftriaxone developed acute cholecystitis, confirmed by histopathology (P<0.05) and biliary microlithiasis, except one that exhibited sediment precipitation. In the group treated with ceftriaxone there was an increase in pro-caspase-3, gamma-glutamyl transpeptidase concentration, PCNA expression and in the number of cells positive for anti-CD68 (P<0.05). In the ceftriaxone group, the cholesterol and lecithin concentrations increased in the bile and a high concentration of ceftriaxone was found in the microlithiasis. Conclusion: Ceftriaxone administered intravenously at therapeutic doses causes a high predisposition for lithogenic bile formation and the development of acute lithiasic cholecystitis.</p></div

Timeline representation of the experimental protocol.

<p>First randomization: pressure-controlled ventilation (PCV) or pressure support ventilation (PSV). Second randomization: intra-abdominal hypertension (IAH) or normal intra-abdominal pressure (nIAP). Start: immediately after surgery (Sham) or IAH induction at PCV or PSV. V_T, tidal volume; PEEP, positive-end expiratory pressure; FiO₂, fraction of inspired oxygen. Mechanics and arterial blood gases were evaluated at Start and End (after 1 h of mechanical ventilation in PCV or PSV).</p

Arterial blood gases at Start and End.

<p>Arterial blood gases at Start and End.</p

Real-time polymerase chain reaction analysis of biological markers associated with inflammation (interleukin [IL]-6), fibrogenesis (type III procollagen [PCIII]), pulmonary stretch (amphiregulin), type II epithelial cell damage (surfactant protein [SP]-B), and endothelial cell damage (vascular cellular adhesion molecule [VCAM-1]) in animals with normal intra-abdominal pressure (nIAP) or intra-abdominal hypertension (IAH) mechanically ventilated in pressure-controlled ventilation (PCV) or pressure support ventilation (PSV) mode.

<p>Values are given as medians, interquartile ranges, and minimum/maximum of 6 animals in each group. Relative gene expression was calculated as a ratio of average gene expression compared with the reference gene (<i>36B4</i>) and expressed as fold change relative to non-ventilated (NV) animals. *Significantly different from NV (p<0.05). #Significantly different from PCV (p<0.05).</p

Respiratory parameters at Start and End.

<p>Respiratory parameters at Start and End.</p

Cumulative DAD score (scores arithmetically averaged from two independent investigators) representing injury from alveolar collapse, interstitial edema, and septal thickening in animals with normal intra-abdominal pressure (nIAP) or intra-abdominal hypertension (IAH) mechanically ventilated in pressure-controlled ventilation (PCV) or pressure support ventilation (PSV) mode.

<p>NV: non-ventilated animals. Values are given as medians, interquartile ranges, and minimum/maximum of 6 animals in each group. Statistical significance was accepted at p < 0.05. *Significantly different from NV.</p

Migration of intraperitoneally and intravenously administered MSCs in experimental animals.

<p>Tec-99m-labeled MSCs were administered to animals and followed for 24 hours using a gamma-camera. Tec-99m-labeled MSCs were injected intraperitoneally in the right lower abdominal quadrant, and migrated towards the inflamed colon on the contra-lateral left lower quadrant, but not to the non-inflamed colon of controls. Skin fibroblasts used as additional controls did not migrate towards the inflamed colon (A). Tec-99m-labeled MSCs injected through the jugular vein accumulated first into the lungs and then gradually migrated towards liver, spleen, kidneys, and bladder. After 24 hours, labelled cells were barely detectable (B). Images are representative of 3 independent experiments. All animals are lying on their backs. Right (R); left (L).</p

Effect of MSCs on apoptotic rates within the colon.

<p>Apoptotic cells were detected by the TUNEL assay. Photomicrographs of the colon show representative samples of a control, and from TNBS-induced colitic animals treated with BM- and AT-MSCs, or vehicle, and an internal negative control without TdT enzyme (original magnification ×100) (A). Percentages of apoptotic cells in the colonic epithelium and in the lamina propria were analyzed in at least 10 different areas per tissue section. Epithelial values of control and MSCs-treated animals were lower compared to vehicle-treated colitis (p<0.008). Lamina propria values of MSCs- or vehicle-treated animals were higher compared with the control group (p<0.04). Horizontal bars represent medians, boxes represent the 25th and 75th percentiles, and vertical bars represent ranges of 10 animals/group. Differences were analyzed using ANOVA on ranks with Dunnett's test (B).</p

Effect of MSCs on the clinical and colonoscopic parameters of the colitis model.

<p>Survival did not differ significantly among experimental groups. Of the TNBS-colitic animals, 2 treated with vehicle died on days 4 and 7, and 1 treated with BM-MSCs died on day 9. Survival was analyzed by the Kaplan–Meier log-rank test (A). Following TNBS-induction, animals presented a progressive weight loss compared with controls (*p<0.001). After receiving AT- or BM-MSCs, animals gradually regained weight, in contrast to vehicle-treated animals (**p<0.046; ***p<0.004) (B). Colonoscopic imaging was obtained after colitis induction at day 4, and after MSCs administration at day 11. In control experiments, colonoscopy was performed following intra-rectal saline enemas, and intraperitoneal administration of vehicle in TNBS-induced animals (C). Differences before and after treatment were evaluated with the Wilcoxon matched-pair signed rank test. Values are mean±S.E.M. of 10 animals/group.</p

Biological characterization of mesenchymal stromal cells.

<p>Phenotypic analysis of MSCs was carried out by flow cytometry, which revealed that BM- and AT-MSCs expressed the cell markers CD90 (Thy-1) and CD29, but did not express lineage markers such as CD45, CD11b and CD34 (A–F). Solid black lines show AT-MSC, dotted black lines show BM-MSC, and gray lines are isotype control. Functionally, MSCs have the capacity to form different cell lineages. AT- (G–I) and BM- (J–K) MSCs were able to differentiate into adipocytes (H, K) and osteocytes (I, L). These cells were used in subsequent experiments. Oil Red O (G–H, J–K) and Von Kossa (I, L) staining. Length bars represent 50 µm.</p