Use of cryomicrotomy to study gastric diffusion of amoxicillin in guinea pigs

Abstract

Cryomicrotomy has been used as a new technique for removing gastric mucosae from adult guinea pigs for the study of amoxicillin secretion across gastric mucosae. This method allowed a very regular thickness of the removed surface layer of mucosa to be obtained with good reproducibility. Gastric superficial mucosa concentrations and gastric juice concentrations of amoxicillin were determined 1, 2, and 4 h after intramuscular administration (50 mg/kg) in 21 guinea pigs by a microbiological method. No antibiotic was detected in gastric samples at 4 h, except for a low-level mucosal concentration in one animal, thus indicating the short time that amoxicillin is present in gastric samples.Gastric human biopsies may be used to study the diffusion of drugs, such as antibiotics in the stomach (1,8). These biopsies may include deep vascularized layers of the gastric wall and therefore be inadequate for the determination of antibiotic concentrations in the superficial gastric mucosa. On the contrary, the guinea pig is a more convenient model for pharmacological studies since it permits the superficial luminal portion of the mucosa of the whole stomach, free of blood contamination, to be obtained (14). For this purpose, Westblom et al. (14) used glass slide scraping. We propose to modify this technique by using cryomicrotomy, which we have applied to the study of amoxicillin gastric diffusion after parenteral administration.Each adult male guinea pig, after an overnight fast, was given a single intramuscular (i.m.) dose of amoxicillin (50 mg/ kg) or saline (controls) in the thigh. Blood samples were collected on citrate at 1, 2, and 4 h after i.m. administration by intracardiac puncture, and plasma was obtained by centrifugation. Animals were then immediately sacrificed with chloroform and autopsied. After clamping, a total gastrectomy was performed and gastric juice was aspirated. Then the whole stomach was opened by cutting through larger and smaller curvatures and was thoroughly rinsed in citrate buffer.The superficial gastric mucosa was removed by standardized cryomicrotomy (Cryo-microtome Leitz; Frigomat Jung, Nussloch, Germany) of the whole stomach and controlled each time by histological examination. First, tangential sections of mucosae from five guinea pigs at depths from 50 to 800 m were obtained. A depth of 400 m was retained for this study as it permitted the removal of the superficial mucosa. Under these conditions, the muscularis mucosa was always at least 100 m from the section plane (Fig. 1). The weight of each superficial mucosal specimen obtained was 400 Ϯ 50 mg, and samples were always devoid of vascular structures.Mucosae were ground in citrate buffer. Homogenate was centrifuged at 3,000 rpm (GR 412 centrifuge; Jouan) for 10 min, and the supernatant was recovered. All specimens were stored at Ϫ80ЊC until assayed.The concentrations of amoxicillin in plasma, gastric mucosae, and gastric juice were measured by a microbiological method. Gastric juice pH was determined with a pHmeter 28 (Radiometer, Copenhag...

Gastric Penetration of Amoxicillin in a Human Helicobacter pylori -Infected Xenograft Model

“…Freezing allows better stretching of the gastric mucosa, which minimizes gastroplication. This may explain why cryomicrotomy may represent a more reproducible way to obtain gastric superficial mucosa (17). However, in guinea pigs, pharmacokinetic studies were performed with the superficial mucosa of the whole stomach, which permitted retrieval of enough material to detect even low levels of antibiotics, but one animal was killed at each time point.…”

“…This could also be due to contamination of mucosal samples with blood, the risk of which would be increased by the important neoangiogenesis observed in all the infected xenografts. However, this seems unlikely or at least negligible since no macroscopic blood contamination was detected at the time of sampling and since cryomicrotomy prevents any significant contamination from interstitial tissue and plasma (17). Amoxicillin is unstable at normal gastric pH (pH 1 to 2) (9).…”

“…Concomitantly, large gastric antral biopsy specimens (4 by 4 by 1 mm) were surgically obtained from areas devoid of any hemorrhagic lesions and were rinsed in 0.1 M phosphate buffer (pH 7.5). Then, the superficial gastric mucosa was immediately removed at a depth of 300 m by standardized cryomicrotomy at Ϫ20°C (Cryomicrotome HM 500M; Microm, Francheville, France) as described previously (17), with sections of 3 m put into preweighed Eppendorf caps. The caps containing the mucosal material was reweighed without delay and were stored at Ϫ80°C until drug assays.…”

In vitro assessment of gastric mucosal transfer of anti-Helicobacter therapeutic agents

“…This concentration cannot be equated to the amount of antibiotic that is available to the organism on the epithelial surface, as the distribution of antibiotics within tissues is not homogeneous, for example, due to accumulation within cells (1), trapping within the acid lumen of the stomach (18), or high levels of plasma protein binding. Cryomicrotomy, used to remove the superficial cell layer, has been proposed as an improvement (10), excluding contamination from interstitial tissue and plasma, but is still not ideal.…”