A new animal model of intestinal mucositis induced by the combination of irinotecan and 5-fluorouracil in mice.

PURPOSE: Intestinal mucositis (IM) is a common side effect of anticancer agents. Despite polychemotherapy use in clinical practice, the pathogenesis of IM has been investigated in single drug injection animal models. However, the progression of IM could vary according to drug regimens. Thus, we aimed to develop a new experimental mucositis model induced by combining irinotecan and 5-fluorouracil (5-FU) treatments. METHODS: IM was induced in male C57BL/6 mice by the intraperitoneal administration of either 0.9 % saline (5 mL/kg), irinotecan (IRI, 30 or 45 mg/kg), 5-FU (25, 37.5, or 50 mg/kg), or the combination of these doses (IRI + 5-FU) for 4 days. Animal survival, body mass variation, and diarrhea scores were evaluated daily. On the 7th day, the mice were euthanized, and intestinal samples were collected for histopathology and morphometric analysis, as well as for the determination of myeloperoxidase activity and cytokine dosage (TNF-α and IL-6). RESULTS: The optimal dose combination that induced IM and presented no substantial mortality on the 7th day was IRI (45 mg/kg) + 5-FU (37.5 mg/kg), which was used for subsequent studies. IRI and 5-FU in combination induced significant diarrhea, body weight loss, intestinal damage, inflammatory cell infiltration, and increased levels of cytokines when compared with other groups (P < 0.05). Neither IRI nor 5-FU alone induced IM. CONCLUSIONS: We developed a new experimental model of IM induced by combining irinotecan and 5-FU treatments, which will allow us to gain a better knowledge concerning the pathogenesis of this disease through the pharmacological modulation of key inflammatory mediators.

The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis.

Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL-1 and IL-18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL-1ß (405%), IL-18 (365%), COX-2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL-18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.

A handcrafted tissue microarray for a matrix arrangement of tissue samples.

INTRODUCTION: Tissue microarray (TMA) was first designed to enable more efficient immunohistochemical screening of antibodies and tissues. However, due to the high cost of commercial TMA builder instrument, such method is not affordable for many pathology laboratories. Then, methodological adaptations have been proposed in order to reduce TMA-associated cost. METHODS: A manual leather puncher with an inner diameter of 2mm was used to collect a tissue sample from the donor paraffin block. The conventional TMA method was adopted as a control group. RESULTS: Empty paraffin recipient blocks were prepared and a standard 2-mm crochet needle was used to create 24 equidistant holes in the recipient block. Tissue cores obtained from the donor blocks were transferred to the holes in the recipient blocks and routine histopathological techniques were then performed. DISCUSSION: In this study we proposed a new approach to produce TMA recipient blocks as an alternative to the conventional TMA.

Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases.

AIM: To evaluate immunoexpression of cyclooxygenase-2 (COX-2) in primary gastric carcinomas and respective lymph node metastases. METHODS: Immunohistochemistry to analyze COX-2 expression was performed on tissue microarray slices obtained from 36 specimens of gastrectomy and satellite lymph nodes from patients with gastric carcinoma. RESULTS: Immunostaining was seen in most cases, and COX-2 expression was higher in lymph node metastases than in corresponding primary gastric tumors of intestinal, diffuse and mixed carcinomas, with a statistically significant difference in the diffuse histotype (P = 0.0108). CONCLUSION: COX-2 immunoexpression occurs frequently in primary gastric carcinomas, but higher expression of this enzyme is observed in lymph node metastases of the diffuse histotype.

An adapted tissue microarray for the development of a matrix arrangement of tissue samples.

The arrangement of tissue samples in a matrix, known as the tissue microarray (TMA) method, is a well-recognized method worldwide. This technique makes it possible to assess the expression of molecular markers on a large scale with high yields in terms of time, costs, and archived material. Some researchers are trying to adapt the technique to expand the research possibilities. This study proposes an adaptive simplification of low-cost instruments for obtaining samples that will be used in the construction of the TMA. The use of a manual leather puncher, which has a very low cost and a long expected life and eliminates the need to use a press machine, is a simple and effective alternative to building blocks of tissue microarrays.