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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.

Background Methotrexate treatment has been associated to intestinal epithelial damage. Studies have suggested an important role of nitric oxide in such injury. The aim of this study was to investigate the role of nitric oxide (NO), specifically iNOS on the pathogenesis of methotrexate (MTX)-induced intestinal mucositis. Methods Intestinal mucositis was carried out by three subcutaneous MTX injections (2.5 mg/kg) in Wistar rats and in inducible nitric oxide synthase knock-out (iNOS-/-) and wild-type (iNOS+/+) mice. Rats were treated intraperitoneally with the NOS inhibitors aminoguanidine (AG; 10 mg/Kg) or L-NAME (20 mg/Kg), one hour before MTX injection and daily until sacrifice, on the fifth day. The jejunum was harvested to investigate the expression of Ki67, iNOS and nitrotyrosine by immunohistochemistry and cell death by TUNEL. The neutrophil activity by myeloperoxidase (MPO) assay was performed in the three small intestine segments. Results AG and L-NAME significantly reduced villus and crypt damages, inflammatory alterations, cell death, MPO activity, and nitrotyrosine immunostaining due to MTX challenge. The treatment with AG, but not L-NAME, prevented the inhibitory effect of MTX on cell proliferation. MTX induced increased expression of iNOS detected by immunohistochemistry. MTX did not cause significant inflammation in the iNOS-/- mice. Conclusion These results suggest an important role of NO, via activation of iNOS, in the pathogenesis of intestinal mucositis.

Many natural terpenoid compounds from plants exhibit antinociceptive property but very few studies have addressed their efficacy in visceral models of nociception. The present study evaluated the antinociceptive potential of oleanolic acid, a pentacyclic triterpene in the mouse model of colonic nociception induced by mustard oil. We further examined the possible participation of opioid, alpha2-adrenergic, and transient receptor potential vanilloid 1 (TRPV1)-receptors in its mechanism. Mice were pretreated orally with oleanolic acid (3, 10, 30 mg/kg) or vehicle, and the pain-related behavioral responses to intracolonic injection of mustard oil was analysed. Oleanolic acid significantly suppressed the mustard oil-induced nociceptive behaviors at test doses of 10 and 30 mg/kg, in a dose-related manner. The antinociceptive effect of oleanolic acid (30 mg/kg) was significantly blocked by pretreatment with the opioid antagonist, naloxone (2 mg/kg, i.p.), while the alpha2-adrenoceptor antagonist, yohimbine (2 mg/kg, s.c.), had no effect. Pretreatment with ruthenium red (3 mg/kg, s.c.), a non-competitive TRPV1 antagonist alone caused significant inhibition of mustard oil-induced nociception but its co-administration with oleanolic acid produced neither antagonism nor potentiation of oleanolic acid antinociception. In the open-field test that detects sedative or motor abnormality, mice received 30 mg/kg oleanolic acid did not show any per se influence, but significantly inhibited the mustard oil-induced decrease in ambulation frequency. These data demonstrate the visceral antinociceptive potential of oleanolic acid that involves an opioid mechanism and possibly a modulatory influence on vanilloid-receptors, which needs further study.

To evaluate immunoexpression of cyclooxygenase-2 (COX-2) in primary gastric carcinomas and respective lymph node metastases.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.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).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.

Chemotherapy-induced hemorrhagic cystitis (HC) remains a common and life-threatening clinical complication, mainly due to the increasing usage of alkylating agents during conditioning regimen for hematopoietic cell transplantation. Currently, mesna and hyperhydration are the two more employed preventive measures. However, these prophylactic approaches have been proven not to be completely effective, since cystoscopic and histopathologic bladder damage are evidenced. Therefore, understanding the pathogenesis of HC must be the cornerstone for the development of novel therapeutic strategies. The purpose of this review is to examine the current knowledge regarding the pathogenesis of HC, describing the importance of transcription factors (nuclear factor kappaB), cytokines (tumor necrosis factor-α, interleukin-1β, -4, -6, and -8), enzymes (inducible nitric oxide synthase and cyclooxygenase-2), among other mediators, for the bladder injury. We also discuss the currently available animal models and future perspectives on the management of HC.

Local tissue reactions provoked by Bothrops venoms are characterized by edema, hemorrhage, pain, and inflammation; however, the mechanisms of tissue damage vary depending upon the species of snake. Here, we investigated the mechanisms involved in the local inflammatory response induced by the Bothrops jararacussu venom (BjcuV). Female Swiss mice were injected with either saline, BjcuV (0.125–8 μg/paw) or loratadine (an H1 receptor antagonist), compound 48/80 (for mast cell depletion), capsaicin (for C-fiber desensitization), infliximab (an anti-TNF-α antibody), indomethacin (a non-specific COX inhibitor), celecoxib (a selective COX-2 inhibitor) or fucoidan (a P- and L-selectins modulator) given before BjcuV injection. Paw edema was measured by plethysmography. In addition, paw tissues were collected for the measurement of myeloperoxidase activity, TNF-α and IL-1 levels, and COX-2 immunoexpression. The direct chemotactic effect of BjcuV and the in vitro calcium dynamic in neutrophils were also investigated. BjcuV caused an edematogenic response with increased local production of TNF-α and IL-1β as well as COX-2 expression. Both edema and neutrophil migration were prevented by pretreatment with indomethacin, celecoxib or fucoidan. Furthermore, BjcuV induced a direct in vitro neutrophil chemotaxis by increasing intracellular calcium. Therefore, BjcuV induces an early onset edema dependent upon prostanoid production and neutrophil migration.

Background Intestinal mucositis is one of the major troublesome side effects of anticancer chemotherapy leading to poor patient compliance. In this study we addressed the role of the novel apolipoprotein E (ApoE) COG 133 mimetic peptide in 5-fluorouracil (5-FU)-challenged Swiss mice and IEC-6 cell monolayers. Experiments were also conducted in C57BL6J ApoE knock-out mice to assess the effects of apoE peptide treatment. Methods Experimental groups were as follows: unchallenged controls, 5-FU-challenged mice (450 mg/kg, i.p) with or without the ApoE peptide (0.3, 1, and 3 μM, given twice daily i.p. for 4 days). Mice were sacrificed 3 days after 5-FU challenge. Proximal small intestinal samples were harvested for molecular biology and histological processing. We conducted ELISA assays and RT-PCR to target IL-1β, TNF-α, IL-10, iNOS, and myeloperoxidase (MPO) to assess intestinal inflammation. Cell death and NF-κB assays were also conducted in apoE knock-out mice. In our in vitro models, IEC-6 cells were exposed to 1 mM of 5-FU in glutamine free media with or without the ApoE peptide (0.02, 0.2, 2, 5, 10, and 20 μM). We investigated IEC-6 cell proliferation and migration, 24 h after the 5-FU challenge. Additionally, apoptotic IEC-6 cells were measured by Tunel and flow cytometry. Equimolar doses of the ApoA-I (D4-F) peptide were also used in some experiments for comparative studies. Results Villus blunting and heavy inflammatory infiltrates were seen in the 5-FU-challenged group, findings that were partially ameliorated by the ApoE peptide. We found increased intestinal MPO and pro-inflammatory IL-1β and TNF-α levels, and TNF-α and iNOS transcripts, and reduction of IL-10 following 5-FU treatment, each of which were partially abrogated by the peptide. Improvements were also found in IEC-6 cell apoptosis and migration following ApoE and D-4F treatment. Conclusion Altogether, these findings suggest that the novel ApoE COG 133 mimetic peptide can reduce 5-FU-induced intestinal changes and potentially benefit mucositis.

We previously described the visceral antinociceptive property of alpha, beta-amyrin in a mouse model of cystitis induced by cyclophosphamide (CPM). This study examined the contribution of vanilloid-1 (TRPV1), peripheral NK1 receptors to CPM-evoked nociceptive behaviors and bladder edema, and its possible modulation by alpha, beta-amyrin.The effect of alpha, beta-amyrin (10, 30, and 100 mg/kg, p. o.) and N-acetylcysteine (NAC) on CPM (400 mg/kg, i. p.)-induced cystitis was studied in mice. Sensory deafferentation was done by a high dose capsaicin. The parameters analysed were: CPM-evoked noxious behaviors, bladder edema, vascular permeability, and NK(1) immunoreactivity. To assess the role of K(+) (ATP) channels in alpha, beta-amyrin effect, animals were pretreated with glibenclamide.alpha, beta-amyrin (30 and 100 mg/kg) and NAC significantly (p0.01) suppressed the visceral pain-related behaviors and NK(1) immunoreactivity, but bladder edema was reduced weakly. Glibenclamide reversed the effects of alpha, beta-amyrin. Sensory deafferentation by capsaicin significantly reduced the nociceptive responses and the NK(1) immunoreactivity to noxious stimulation by CPM.alpha, beta-amyrin attenuates CPM-induced visceral pain and bladder edema by mechanisms that involve, at least in part, a block either of Substance P release or its receptor function, and partly by opening K(+) (ATP) channels.