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This study examined the controlled release of cetylpyridinium chloride (CPC) from a tissue conditioner (TC) containing CPC-montmorillonite (CPC-Mont), the associated antimicrobial activity, and oral mucosa irritation. The CPC release test was performed daily for 28 days in three test solutions: distilled water, 0.2 M NaCl, and 0.2 M HCl. The antimicrobial activities for 7, 14, 21, and 28 days against Candida albicans, Staphylococcus aureus, and Streptococcus mutans were assessed according to the JIS Z 2801/ISO 22196 standard. An oral mucosa irritation test was conducted using cheek pouches in five male hamsters according to the ISO 10993-10:2010 standard. The amount of CPC released each day and the cumulative amount released over 28 days (6.12 mg) were less than the daily safe maximum of sore throat medicines (8 mg). Additionally, TC with CPC-Mont could sustain antimicrobial activity against adherent bacteria for 14 days and has no oral mucosa irritation potential.

Cheek pouches have evolved from the oral cavity in rodents and act as temporary food storage repositories. There are two types of opening, internal and external. Details about the complex cutaneous muscles controlling the pouches have still not been fully elucidated. To understand the shared and derived traits of the muscles surrounding the cheek pouch and their innervation, we carried out an evolutionary morphological study using two desert kangaroo rats (Dipodomys deserti) and three plains pocket gophers (Geomys bursarius) from each of the two families equipped with external cheek pouches, and four Syrian hamsters (Mesocricetus auratus) with internal cheek pouches. The most conspicuous derived trait of the muscles between the external and internal cheek pouches, the sphincter sacculi, surrounds almost all of the edge of the outer entrance of the pouch. It is present in both species with external pouches, but not in hamsters, which have internal pouches. Our neurological findings demonstrate that most pouch muscles are innervated by both the facial and the cervical nerves, regardless of the pouch type. In these dually innervated muscles, the ventromedial part of the muscles tends to be innervated dominantly or uniquely by the cervical nerves, which usually enter from the superficial or lateral aspect. As a trait shared with the cervical nerves innervating the propatagial muscles in aerodynamic mammals such as bats and flying squirrels, and panniculus carnosus in most mammals, our neurological evidence suggests that the cervical nerve has the potential to innervate derived cutaneous muscles in the cervicofacial region.

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p

ZrO2-nanotubes, demonstrating good mechanical characteristics and corrosion resistance, however, cell biocompatibility and Streptococcus mutans adhesive capacity haven't been evaluated. This study aimed to evaluate biocompatibility, adhesion to Streptococcus mutans, safety of ZrO2-nanotubes. In this study, proliferation of ZrO2-nanotubes treated cells was determined. Adhesive capacity to cell and safety of ZrO2-nanotubes were evaluated with transmission electron microscopy (TEM) and HE staining/chromosome aberration assay, respectively. Growth of Streptococcus mutans, live/dead bacteria on ZrO2-nanotubes and adhesive capacity to Streptococcus mutans of ZrO2-nanotubes were also evaluated. ZrO2-nanotubes were successfully synthesized exhibiting appropriate monoclinic phase intensity, without obvious transistor-structure and effects on proliferation. More L929 colonies adhered to ZrO2-nanotubes, however, only a few adhered to ZrO2 control. ZrO2-nanotubes didn't induce inflammatory effect on rats' cheek pouch and chromosome aberration of rat bone marrow, and was proven to be safe for important organs. ZrO2-nanotubes inhibited growth, induced dead of Streptococcus mutans, and obviously repelled adhesion of Streptococcus mutans. In conclusion, this study generated a novel and safe implant, ZrO2-nanotube. ZrO2-nanotube could induce Streptococci mutans dead, demonstrating higher cell biocompatibility and lower Streptococci mutans adhesive capacity and apply in treatment of dental caries.

There remain gaps in knowledge concerning how vascular morphology evolves during carcinogenesis. In this study, we imaged neovascularization by label-free dark-field microscopy of a 7,12-Dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch model of oral squamous cell carcinoma (SCC). Wavelength-dependent imaging revealed distinct vascular features at different imaging depths and vessel sizes. Vascular tortuosity increased significantly in high-risk lesions, whereas diameter decreased significantly in hyperplastic and SCC lesions. Large vessels preserved the same trends seen in the original images, whereas small vessels displayed different trends, with length and diameter increasing during carcinogenesis. On the basis of these data, we developed and validated a classification algorithm incorporating vascular features from different vessel masks. Receiver operator curves generated from the classification results demonstrated high accuracies in discriminating normal and hyperplasia from high-grade lesions (AUC > 0.94). Overall, these results provided automated imaging of vasculature in the earliest stages of carcinogenesis from which one can extract robust endpoints. The optical toolbox described here is simple, low-cost and portable, and can be used in a variety of health care and research settings for cancer prevention and pharmacology research. Cancer Res; 77(24); 7109–19. ©2017 AACR.

Dimethylbenza-a-anthracene (DMBA), is a potent carcinogen that has an immunosuppressive effect on hamsters. Nigella sativa whole oil (NSO) and thymoquinone (TQ), are phytochemicals that have an antioxidant and protective action on the hamster cheek pouch epithelium (HCPE/DMBA) carcinogenesis model. The aim is to find which of the two ingredients TQ & NSO has more immune-enhancing activity comparable with its protective and/or chemotherapeutic activity against the carcinogenic effect of DMBA on HCPE, through evaluation of different peripheral monocytes. Material &methods: NS whole oil (NSO), and thymoquinone (TQ) were given before-, with- or after DMBA painting the hamster’s left cheek pouch. Before each animal’s sacrificing, two ml of blood was drawn in a fine heparinized capillary tube to estimate the total WBCs, total lymphocytes, MID cells, and granulocyte’s count by an automatic count system (Cell-DYN1700).Blood results were statistically analyzed using One-way ANOVA test between groups for each phase followed by LSD (Least Square Difference) test for multiple comparisons between different phases. All pouches were surgically-excised and examined microscopically to compare the histopathologic changes of DMBA-painted pouches, NSO-and TQ-treated groups, with untreated pouches as well as pouches of the untreated group.Histopathologic results: the groups given each of the tested ingredients for 2 wks showed normal appearing thin epithelium, with slight hyper-keratinization as compared with untreated pouches. Severe epithelial dysplasia was seen after 6 wks of DMBA, but when NSO or TQ was given for 2wks followed by DMBA for 6wks, mild dysplasia was seen i.e. both have protective effect. When DMBA for 6wks was followed by NSO or TQ for 6wks mild dysplasia was recorded .i.e. both had a therapeutic effect through regressing severe dysplasia to mild dysplasia in 6 weeks. Mononuclear cells results: DMBA for 6wks resulted in statistically significant reduction in total WBCs &lymphocyte counts than control group, as well as when DMBA for 6wks was followed

The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p

To determine whether the potential effects on liver and kidney caused by dental alloys could be reduced or terminated by the removal of nickel-chromium (Ni-Cr) alloy, cobalt-chromium (Co-Cr) alloy, and commercially pure titanium (CP-Ti), they were placed in the cheek pouches of Syrian hamsters according to ISO 10993-10. Then, the peak/plateau and end times of trace metals in the blood were determined with or without the removal of the dental alloys. Based on these time points, the trace metals and their effects on liver and kidney were examined. We found that trace metals released from these dental alloys and titanium were accumulated transiently in the blood, liver, and kidney but had no effect on the histopathology of the liver or kidney. Although the functions of the liver and kidney were compromised, the function of these tissues seemed to be clinically acceptable compared to those in control Syrian hamsters. In addition, the apoptotic effect on renal cells was terminated by removing the Ni-Cr and Co-Cr alloys, and that on hepatocytes was also eliminated by removing the Ni-Cr alloy. In contrast, the effect of the Co-Cr alloy on hepatocytes was temporary and recovered by itself. Taken together, Ni- and Co-based dental alloys and titanium have no effect on the histopathology or function of liver and kidney. Moreover, Ni-Cr and Co-Cr alloys induce transient trace metal accumulation and apoptotic effects in liver and kidney, which can be reduced or terminated by the removal of the alloys, while CP-Ti shows favorable biocompatibility.

This study aims to investigate the immunomodulatory effect of anthocyanins (ANTs) from Oryza sativa L. extracts on 5-fluorouracil (5-FU)-induced oral mucositis, using a rat model and oral keratinocytes. ANTs were detected by high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry. Animals were randomly given varying doses of ANT-rich extract treatment (500 mg/kg and 1000 mg/kg) in the absence or presence of 5-FU-induced mucositis. Buccal mucosae were photographed and scored for macroscopic analysis and incisional biopsies of cheek pouches were collected for microscopic examination of oral mucositis. 5-FU caused marked hemorrhage, extensive ulcerations and abscesses compared to non-treated animals with slight erythema. Histologically, a loss of collagen bundles and inflammatory cell infiltrates was observed. After 29 days of ANT treatment, lesions resolved, and abundant collagen fibers were evident in the lamina propria. Buccal mucosa of 5-FU-injected rats showed increased Nuclear factor-kappa B (NF-κB) p50 and p65 in oral keratinocytes. The administration of ANT reduced NF-κB-positive cells in 5-FU rats (p &lt, 0.001) compared to the non-treatment group. In oral keratinocytes, ANT treatment significantly restored 5‑FU‑induced growth inhibition and impaired the nuclear accumulation of NF-κB p50 and p65. Our study demonstrated that ANT from Oryza sativa L. exhibited effective anti-inflammatory properties against 5-FU-induced oral mucositis by inhibiting NF-κB activation.

Antimicrobial chemotherapy based on ultraviolet-A (UVA) irradiation of polyphenol solution has been proposed as an adjunctive treatment for dental caries. However, the safety of this treatment has not been thoroughly evaluated. Therefore, the aim of the present study was to assess the influence of this treatment on the oral mucosa in hamsters and wounded skin in rats. An oral mucosal irritation test was performed in hamsters. The cheek pouch was everted and treated with UVA irradiation (wavelength: 365 nm) of pure water, 1 mg/mL of caffeic acid, or 1 mg/mL of grape seed extract using a light-emitting diode at an irradiance of 275 mW/cm2. Each treatment was performed for 2 or 5 min and repeated three times. Macroscopic and histological evaluations were performed 24 h after the last treatment. We also examined the effects of the treatment on the healing process of skin wounds in rats. Full-thickness skin wounds created on the back of the rats were treated for 2 min as described above, but only once. The wound area was then assessed daily for 9 days. The results demonstrated that the treatment induced oral mucosal irritation depending on the irradiation time; however, it did not influence the wound healing process. The oral mucosal irritation potential of three treatment sessions performed for 2 and 5 min was minimal and mild to moderate, respectively, according to histological analysis. These findings suggest that the duration of treatment based on UVA irradiation of polyphenols in the oral cavity should be as short as possible, considering the clinical efficacy of the antimicrobial effects and the irritation potential.

In this work, we compare standard wide-field fluorescence lifetime imaging microscopy (FLIM) and structured illumination FLIM (SI-FLIM) as methods for the early detection of oral squamous cell carcinoma (OSCC). Our technique, SI-FLIM, provides depth dependent fluorescence lifetime information of the oral epithelium, isolating the endogenous fluorophore of interest, NADH, from interfering fluorescence generated mainly by collagen in the lamina propria. Male golden Syrian hamsters (Cricetus auratus) were used as the animal model for OSCC. They were treated with a carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), for a twelve-week period by applying the DMBA suspended in mineral oil to their cheek pouches 3 times per week. The progression of OSCC was monitored over a 12-week period with imaging beginning at the 6th week. The cheek pouch with lesions was imaged in a 3x4 grid (twelve total images), with each section of the grid being correlated with histopathological analysis. The NADH fluorescence channel, as a diagnostic indicator, was compared for both SI-FLIM and widefield FLIM. ROC analysis, in the task of distinguishing between mild dysplasia and normal tissue, showed that SI-FLIM (AUCe0.83, see0.07) may be a better indicator for early cases of mild dysplasia when compared to widefield FLIM (AUCe0.63, see0.07) with statistical significance.

Purpose: To evaluate the effects of red propolis on cheek pouch angiogenesis in a hamster new model sponge implant. Methods: Forty eight animals divided into eight groups. (Groups I-IV), the animals were treated for 15 days before and 10 days after sponge implantation. (Groups V-VIII), the animals were treated for 10 days after sponge implantation (GI and GV: red propolis 100 mg/kg, GII and GVI: celecoxib 20 mg/kg, GIII and GVII: 1% gum arabic 5 mL/kg, GIV and GVIII: distilled water 5 mL/kg). On the 11th day of implantation, the animals were anesthetized for stereoscopic microscopic imaging and morphometric quantification of angiogenesis (SQAN), followed by histopathological evaluation (H&E). Results: In the SQAN analysis, no significant difference was found between the groups. However, on histology, propolis was found reduce the population of mastocytes in the qualitative analyses (p = 0,013) in the quantitative analyses to reduce the number of blood vessels (p = 0,007), and increase the macrophage count (p = 0,001). Conclusion: Red propolis inhibited inflammatory angiogenesis when administered before andcontinuously after sponge implant, and was shown to have immunomodulating effects on inflammatory cells (mastocytes and macrophages) in a new sponge implant hamster model.

This study aimed to elucidate the effect of 5-fluorouracil (5-FU) on the histological aspects of the major salivary glands, salivary flow and saliva composition using an established oral mucositis model in hamsters. Oral mucositis was induced by two intraperitoneal administrations of 5-FU in two consecutive days (60 and 40mg/kg), followed by cheek pouch mucosa scratch, on day 4. The Pilocarpine-stimulated salivary flow was measured 4 and 10days after the first 5-FU injection. Salivary glands were harvested for histopathological analysis, measurement of inflammatory cells, quantification of pro-inflammatory cytokines (TNF-α and IL-1β), investigation of cell death and cell proliferation. Oxidative stress and oxidative defense system were also investigated in the salivary gland tissues using MDA (malondialdehyde), nitrite, non-protein sulfhydryl groups (NP-SH), SOD (superoxide dismutase) and CAT (catalase). In addition, the CAT and lysozyme activities and the IgA and SOD levels were evaluated in the saliva samples. 5-FU significantly reduced the pilocarpine-stimulated salivary flow rate on the 4th experimental day, associated with an increase in the SOD levels in saliva. Recovery of the salivary flow and SOD were observed on day 10, when an increase in the saliva lysozyme levels was detected. In addition, 5-FU promoted vacuolization in parotid (P) and periductal edema in submandibular (SM) gland, combined with an increase in the inflammatory cells influx, mostly observed on the 4th day in SM gland and on 4th and 10th days in P. Oxidative stress was found mostly on day 10 in SM, SL and P glands, associated with release of proinflammatory cytokines, observed in SM and SL glands, but not in P. 5-FU induces an inflammatory response in the major salivary glands, most observed ten days after its first injection, which may contribute to the major salivary glands hypofunction, leading to alterations in the salivary flow rate and composition.

Objective To investigate the ability of mucoadhesive films to locally deliver an immune response modifier (imiquimod) to oral mucosa. Study Design After determining the residence time of films in hamster cheek pouches, drug-loaded films and commercially available imiquimod cream were tested for localization of drug in mucosal tissue. The residence time of drug-free films at different locations was also tested in humans. Results Mucoadhesive films delivered imiquimod to the buccal mucosa with no measureable amounts in blood. In contrast, although the cream formulation resulted in higher tissue levels, it also led to significant systemic distribution of imiquimod. In humans, the films resided on tissue for up to 4 hours, increasing in the order of tongue Conclusion Preclinical findings of localized imiquimod delivery in animals and residence in humans support future investigations of the mucoadhesive system in controlled clinical trials for treating oral precancerous lesions.

The goal of this study was to evaluate the biocompatibility of newly developed Zr61Ti2Cu25Al12 metallic glass (denoted ZT1) and its parallel material, commercially pure titanium (CP-Ti), as dental implants. To this end, we evaluated their cytotoxicity in vitro using human umbilical vein endothelial cells (HUVECs) and in vivo by performing an oral mucosa irritation test in hamsters. Specifically, cytotoxicity was determined in HUVECs by evaluating their cell morphology using scanning electron microscopy (SEM) and their cell viability using CCK-8 assays. Moreover, we examined the mRNA levels of vascular endothelial growth factor and von Willebrand factor by quantitative PCR (qPCR). In the early stages of proliferation and differentiation, no differences were observed between HUVECs inoculated on ZT1 compared to those on CP-Ti. However, in the later stages of proliferation and differentiation, the HUVECs inoculated on ZT1 were significantly better than the cells cultured on CP-Ti. In the oral mucosa irritation test, we sutured sample discs into the cheek pouch of hamsters. After 2, 3, and 4weeks, we harvested the corresponding mucosal tissues, stained them with hematoxylin and eosin, obtained blood samples for biochemical analysis, and finally, observed the topography of the sample discs by SEM. Immunohistochemistry and hematology analyses showed no differences in the biocompatibility of ZT1 and CP-Ti, and neither of these compounds caused irritation of the mucosa. In addition, SEM images showed that no pitting occurred on the sample discs. Together, these data indicate that ZT1 may be a good candidate for dental implants and should be further studied.

BACKGROUND: We previously demonstrated the therapeutic success of sequential boron neutron capture therapy (Seq-BNCT) in the hamster cheek pouch oral cancer model. It consists of BPA-BNCT followed by GB-10-BNCT 24 or 48 hours later. Additionally, we proved that tumor blood vessel normalization with thalidomide prior to BPA-BNCT improves tumor control. The aim of the present study was to evaluate the therapeutic efficacy and explore potential boron microdistribution changes in Seq-BNCT preceded by tumor blood vessel normalization. MATERIAL AND METHODS: Tumor bearing animals were treated with thalidomide for tumor blood vessel normalization, followed by Seq-BNCT (Th+ Seq-BNCT) or Seq-Beam Only (Th+ Seq-BO) in the window of normalization. Boron microdistribution was assessed by neutron autoradiography. RESULTS: Th+ Seq-BNCT induced overall tumor response of 100%, with 87 (4)% complete tumor response. No cases of severe mucositis in dose-limiting precancerous tissue were observed. Differences in boron homogeneity between tumors pre-treated and not pre-treated with thalidomide were observed. CONCLUSION: Th+ Seq-BNCT achieved, for the first time, response in all treated tumors. Increased homogeneity in tumor boron microdistribution is associated to an improvement in tumor control. Fil: Molinari, Ana Julia. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Thorp, Silvia Inés. Comisión Nacional de Energía Atómica; Argentina Fil: Portu, Agustina Mariana. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Saint Martin, María Laura Gisela. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina Fil: Pozzi, Emiliano César Cayetano. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina Fil: Heber, Elisa Mercedes. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina Fil: Bortolussi, Silva. Università degli studi di Pavia; Italia Fil: Itoiz, María Elina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Aromando, Romina Flavia. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina Fil: Monti Hughes, Andrea. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Garabalino, Marcela Alejandra. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina Fil: Altieri, Saverio. Università degli studi di Pavia; Italia Fil: Trivillin, Verónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina Fil: Schwint, Amanda Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina

The present study aimed to evaluate the efficacy of krypton laser photodynamic therapy (PDT) with PsD-007 for the treatment of oral mucosa dysplasia in 9,10-dimethyl-1,2-benzanthracene (DMBA)-treated hamsters. A DMBA-induced hamster cheek pouch model of precancerous lesions was created and the resultant 25 hamsters were divided into five groups. The right side was treated with PDT and the left side was used as the positive control. Following systemic anesthesia, an incision was made in the groin area to expose the femoral vein. PsD-007 was administered intravenously through the femoral vein. Various doses of photosensitizer were used to treat groups A–E. Subsequent to closing the incision, the right side of the buccal mucosa was irradiated with light using the krypton laser at a wavelength of 413 nm, a power density of 150 mW/cm2 and an irradiation time of 20 min. At six weeks post-surgery, the response was analyzed using histological examinations of the buccal pouch mucosa. A total of 24 hamsters completed the six-week observation period, as one hamster from group C died in the second week following the PDT. Of all 24 irradiated sides, 15 formed normal mucosal tissues and nine demonstrated mild dysplasia. Of the total control sides, six developed moderate dysplasia, five developed severe dysplasia and 13 progressed to carcinoma in situ or squamous cell carcinoma (SCC). The results revealed a significant difference between the two sides (P10 mg/kg, there was no statistical difference (P>0.05). PsD-007-mediated krypton laser PDT is effective for the treatment of oral mucosa dysplasia in hamsters.

Objective: The aim of this study was to evaluate the effect of boron on 5-fluorouracil (5-FU)-induced oral mucositis in rats. Materials and Methods: Sixty-four male Wistar albino rats were injected with 5-FU on days 1 and 3. The right cheek pouch mucosa was scratched with the tip of an 18-G needle, dragged twice in a linear movement, on days 3 and 5. The animals were randomly divided into two groups of 32: boron group (BG) and control group (CG). Rats in the CG did not receive any treatment, whereas the others were fed boron (3 mg·kg -1·day -1 ) by gavage. The animals were sacrificed on day 3 (n = 8), 6 (n = 8), 9 (n = 8), and 12 (n = 8), and the cheek pouch was removed for histopathological analysis. Results: On day 3, both groups showed necrosis and active inflammation, but the inflammation was mild in CG and moderate in BG. On day 6, both BG and CG showed necrosis; in the CG, there was moderate inflammation, and in the BG, there was severe inflammation and granulation tissue around the necrotic area. On day 9, re-epithelization began in both groups, and there were no differences between groups. Re-epithelization was complete in both groups on day 12. Conclusion: We found no beneficial effect of boron in healing oral mucositis. Additional research is warranted to elucidate the pathogenic inflammatory mechanisms involved in mucositis and the prophylactic and therapeutic roles of antioxidants.

Oral mucositis induced by chemotherapy or radiotherapy has an impact upon quality-of-life, is dose-limiting for chemotherapy, and causes considerable morbidity. We evaluated the effect of royal jelly (RJ) on 5-fluorouracil (5-FU)-induced oral mucositis in hamsters. Oral mucositis was induced in hamsters through a combination of 5-FU treatment and mild abrasion of the cheek pouch. RJ was contained in chitosan–sodium alginate film (RJ film). Films were attached to the oral mucosa and the healing process examined by measuring the area of mucositis, myeloperoxidase (MPO) activity, microscopic aspects, and RT-PCR for detection of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β). Furthermore, we evaluated the radical-scavenging activity of RJ and generation of keratinocyte growth factor from human periodontal ligament fibroblasts. RJ films (10%, 30%) significantly improved recovery from 5-FU–induced damage, reduced MPO activity and the production of pro-inflammatory cytokines. Additionally, RJ showed radical-scavenging activity. These data suggest that topical application of films that contain RJ had a healing effect on the severe oral mucositis induced by 5-FU and that the effect was caused by the anti-inflammatory or anti-oxidative activities of RJ. Keywords:: antioxidant, cancer, 5-fluorouracil, oral mucositis, royal jelly

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.

The buccal route for drug administration has gained importance in the past years due to its accessibility and rapid onset of action. It has been described as a reliable alternative to the oral route, both for local and for systemic delivery of drugs, overcoming some of the drawbacks existent in oral administration, such as the harsh milieu along the gastrointestinal tract. To successfully develop active drugs, the prediction of their absorption profile across the buccal mucosa is of utmost importance. Due to the high costs, time-consuming nature, and ethical considerations of ex vivo and in vivo studies, in vitro models have been the first choice for the screening of the drugs' permeability. Among the several cell lines originating in the buccal mucosa of humans or animals, hamster cheek pouch cells, oral keratinocytes, and TR146 cells have been the most used for permeability studies. However, due to their primary nature, hamster cheek pouch cells can only be used for a low number of tests, which leads to a high variability in the results. Also, the cancerous origin of TR146 makes it less close to human buccal epithelium, as compared to oral keratinocytes derived from human sources, for permeability studies.

Boron neutron capture therapy (BNCT) combines selective accumulation of (10)B carriers in tumor tissue with subsequent neutron irradiation. We previously demonstrated the therapeutic efficacy of BNCT in the hamster cheek pouch oral cancer model. Optimization of BNCT depends largely on improving boron targeting to tumor cells. Seeking to maximize the potential of BNCT for the treatment for head and neck cancer, the aim of the present study was to perform boron biodistribution studies in the oral cancer model employing two different liposome formulations that were previously tested for a different pathology, i.e., in experimental mammary carcinoma in BALB/c mice: (1) MAC: liposomes incorporating K[nido-7-CH(3)(CH(2))(15)-7,8-C(2)B(9)H(11)] in the bilayer membrane and encapsulating a hypertonic buffer, administered intravenously at 6 mg B per kg body weight, and (2) MAC-TAC: liposomes incorporating K[nido-7-CH(3)(CH(2))(15)-7,8-C(2)B(9)H(11)] in the bilayer membrane and encapsulating a concentrated aqueous solution of the hydrophilic species Na(3) [ae-B(20)H(17)NH(3)], administered intravenously at 18 mg B per kg body weight. Samples of tumor, precancerous and normal pouch tissue, spleen, liver, kidney, and blood were taken at different times post-administration and processed to measure boron content by inductively coupled plasma mass spectrometry. No ostensible clinical toxic effects were observed with the selected formulations. Both MAC and MAC-TAC delivered boron selectively to tumor tissue. Absolute tumor values for MAC-TAC peaked to 66.6 ± 16.1 ppm at 48 h and to 43.9 ± 17.6 ppm at 54 h with very favorable ratios of tumor boron relative to precancerous and normal tissue, making these protocols particularly worthy of radiobiological assessment. Boron concentration values obtained would result in therapeutic BNCT doses in tumor without exceeding radiotolerance in precancerous/normal tissue at the thermal neutron facility at RA-3.

OBJECTIVE: Ischemic preconditioning and some drugs can protect tissues from injury by preserving microcirculation. This study evaluated vascular permeability in a hamster cheek pouch preparation using either short ischemic periods or bradykinin as preconditioning stimuli followed by 30 min of ischemia/reperfusion. METHOD: Sixty-six male hamsters were divided into 11 groups: five combinations of different ischemic frequencies and durations (one, three or five shorts periods of ischemia, separated by one or five minutes) with 10 min intervals between the ischemic periods, followed by 30 min ischemia/reperfusion; three or five 1 min ischemic periods with 10 min intervals between them followed by the topical application of histamine (2 µM); bradykinin (400 nM) followed by 30 min of ischemia/reperfusion; and three control groups (30 min of ischemia/reperfusion or histamine or bradykinin by themselves). Macromolecular permeability was assessed by injection of fluorescein-labeled dextran (FITC-dextran, MW = 150 kDa; 250 mg/Kg body weight), and the number of leaks/cm2 was counted using an intravital microscope and fluorescent light in the cheek pouch. RESULTS: Plasma leakage (number of leaks/cm2) was significantly reduced by preconditioning with three and five 1 min ischemic periods, one and three 5 min ischemic periods and by bradykinin. Histamine-induced macromolecular permeability was also reduced after three periods of 5 min of ischemia. CONCLUSION: Short ischemic periods and bradykinin can function as preconditioning stimuli of the ischemia/reperfusion response in the hamster cheek pouch microcirculation. Short ischemic periods also reduced histamine-induced macromolecular permeability.

We previously demonstrated the efficacy of BNCT mediated by boronophenylalanine (BPA) to treat tumors in a hamster cheek pouch model of oral cancer with no normal tissue radiotoxicity and moderate, albeit reversible, mucositis in precancerous tissue around treated tumors. It is known that boron targeting of the largest possible proportion of tumor cells contributes to the success of BNCT and that tumor blood vessel normalization improves drug delivery to the tumor. Within this context, the aim of the present study was to evaluate the effect of blood vessel normalization on the therapeutic efficacy and potential radiotoxicity of BNCT in the hamster cheek pouch model of oral cancer. Blood vessel normalization was induced by two doses of thalidomide in tumor-bearing hamsters on 2 consecutive days. All studies in thalidomide-treated animals were performed 48 h after the first dose of thalidomide, previously established as the window of normalization. Biodistribution studies were performed with BPA at a dose of 15.5 mg (10)B/kg in thalidomide-treated (Th+) and untreated (Th-) tumor-bearing hamsters. The effect of blood vessel normalization prior to BPA administration on the efficacy of BNCT was assessed in in vivo BNCT studies at the RA-3 Nuclear Reactor in tumor-bearing hamsters. Group I was treated with BPA-BNCT after treatment with thalidomide (Th+ BPA-BNCT). Group II was treated with BPA-BNCT alone (Th- BPA-BNCT). Group III was treated with the beam only after treatment with thalidomide (Th+ BO), and Group IV was treated with the beam only (Th- BO). Groups I and II were given the same dose of BPA (15.5 mg (10)B/kg), and all groups (I-IV) were exposed to the same neutron fluence. Two additional groups were treated with the beam only at a higher dose to exacerbate mucositis in precancerous tissue and to explore the potential direct protective effect of thalidomide on radiation-induced mucositis in a scenario of more severe toxicity, i.e. Group V (Th+ hdBO) and Group VI (Th- hdBO). The animals were followed for 28 days. Biodistribution studies revealed no statistically significant differences in gross boron content between Th+ and Th- animals. Overall tumor control (complete response + partial response) at 28 days post-treatment was significantly higher for Group I (Th+ BPA-BNCT) than for Group II (Th- BPA-BNCT): 84 ± 3% compared to 67 ± 5%. Pretreatment with thalidomide did not induce statistically significant changes in overall tumor control induced by the beam only, i.e. 15 ± 5% in Group III (Th+ BO) and 18 ± 5% in Group IV (Th- BO), or in overall tumor control induced by the high-dose beam only, i.e. 60 ± 7% in Group V (Th+ hdBO) and 47 ± 10% in Group VI (Th- hdBO). BPA-BNCT alone (Group II) induced mucositis in precancerous tissue that reached Grades 3-4 in 80% of the animals, whereas pretreatment with thalidomide (Group I) prevented mucositis Grades 3 and 4 completely. Beam-only Group III (Th+ BO) exhibited only Grade 1 mucositis in precancerous tissue, whereas 17% of the animals in beam-only Group IV (Th- BO) reached Grade 2 mucositis. High-dose beam-only group V (Th+ hdBO) exhibited only Grade 2 mucositis, whereas high-dose beam-only group VI (Th- hdBO) reached Grade 3 mucositis in 83% of the animals. In all cases mucositis in precancerous tissue was reversible. No normal tissue radiotoxicity was observed with any of the protocols. Pretreatment with thalidomide enhanced the therapeutic efficacy of BNCT and reduced precancerous tissue toxicity.

There is a paucity of published information regarding spontaneous neoplasms affecting pet hamsters. This article describes a spontaneous anaplastic sarcoma of probable salivary origin in a pet Syrian hamster ( Mesocricetus auratus ). A 1-year-old hamster was presented with a subcutaneous mass along the right ventrolateral region of the neck, resulting in a partial eversion of the right cheek pouch. The mass was surgically removed and the patient recovered without complications. Approximately 157 days later, the patient was presented for a second mass in the same location and the owners elected to euthanize the hamster. Postmortem histopathological and immunohistochemical analysis of both masses demonstrated highly anaplastic neoplasms of mesenchymal origin. The masses were not directly associated with the cheek pouch and were presumed to have a salivary tissue origin.

OBJECTIVE: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

Objective: to describe and demonstrate an oral mucosa irritation and a skin sensitivity test for evaluation of the biological safety of a colored alumina/glass composite (AGC). Methods: colored alumina/glass composite and control materials were sutured to mucine bilateral cheek pouch mucosa, in a position for gross observation and for histological and cytological examination; skin irritation activity of the composite was tested by intradermal injection of a guinea pig test solution, by inducing, enhancing, and stimulating the allergic response observed after local tissue exposure. Results: colored AGC in mouse cheek pouch mucosa stimulated the same reaction as the control, with no significant inflammatory reaction or infiltration of inflammatory cells. Guinea pigs showed no obvious erythema or edema from the irritation test, such that the assessment of colored AGC skin allergy response in mice was 0%. Conclusion: colored AGC was benign towards mouse oral mucosa and elicited no allergic skin inflammation response, indicating that in these aspects this material had good biological safety.

Objective: Given that locoregional recurrences developing from a tissue with potentially malignant disorders (PMD) in oral mucosa are a frequent cause of therapeutic failure, and that tissue with PMD is dose-limiting, the aim of the present study was to develop a model of tissue with PMD to evaluate the long-term therapeutic/toxic effects of different therapeutic modalities. Materials and methods: We evaluated 5 carcinogenesis protocols based on topical application of the carcinogen dimethyl-1,2-benzanthracene in the hamster cheek pouch, twice a week for 4, 6, 7, and 8 weeks and the classical 3 times a week for 12 weeks. Results: Long-term follow-up (8 months after protocol completion) was only possible with the 4- and 6-week carcinogenesis protocols. Tumour development increased progressively with time and aggressiveness of the carcinogenesis protocols. The time at which tumours developed in ≥90% of the animals was at protocol completion (T0) for the 12-week protocol, 1 month post-T0 for the 8-week protocol, 3 months post-T0 for the 7-week protocol and 4 months post-T0 for the 6-week protocol.

In skeletal muscle, oxygen (O(2)) delivery to appropriately meet metabolic need requires mechanisms for detection of the magnitude of O(2) demand and the regulation of O(2) delivery. Erythrocytes, when exposed to a decrease in O(2) tension, release both O(2) and the vasodilator adenosine triphosphate (ATP). The aims of this study were to establish that erythrocytes release ATP in response to reduced O(2) tension and determine if erythrocytes are necessary for the dilation of isolated skeletal muscle arterioles exposed to reduced extraluminal O(2) tension. Rabbit erythrocytes exposed to reduced O(2) tension in a tonometer (n = 5, pO(2) = 27 +/- 3, p0.01) released ATP in response to reduced O(2) tension. ATP release increased in proportion to the decrease in O(2) tension. The contribution of erythrocytes to the response of skeletal muscle arterioles to reduced extraluminal O(2) tension was determined using isolated hamster cheek pouch retractor muscle arterioles perfused with buffer (n = 11, mean diameter 52 +/- 3 mum) in the absence and presence of rabbit erythrocytes. Without erythrocytes, arterioles did not dilate when exposed to reduced extraluminal O(2) tension (pO(2) = 32 +/- 4 mmHg). In contrast, when rabbit erythrocytes were present in the perfusate (hematocrit 15%), the same decrease in O(2) tension resulted in a 20 +/- 4% dilation (p0.01). These results provide support for the hypothesis that erythrocytes, via their ability to release O(2) along with ATP in response to exposure to reduced O(2) tension, can participate in the matching of O(2) delivery with metabolic need in skeletal muscle.

We tested the hypothesis that differential stimulation of nitric oxide (NO) production can be induced in pre- and postcapillary segments of the microcirculation in the hamster cheek pouch.We applied acetylcholine (ACh) or platelet-activating factor (PAF) topically and measured perivascular NO concentration ([NO]) with NO-sensitive microelectrodes in arterioles and venules of the hamster cheek pouch. We also measured NO in cultured coronary endothelial cells (CVEC) after ACh or PAF.ACh increased periarteriolar [NO] significantly in a dose-dependent manner. ACh at 1 microM increased [NO] from 438.1+/-43.4 nM at baseline to 647.9+/-66.3 nM, while 10 microM of ACh increased [NO] from baseline to 1,035.0+/-59.2 nM (P0.05). Neither 1 nor 10 microM of ACh changed perivenular [NO] in the hamster cheek pouch. PAF, at 100 nM, increased perivenular [NO] from 326.6+/-50.8 to 622.8+/-41.5 nM. Importantly, 100 nM of PAF did not increase periarteriolar [NO]. PAF increased [NO] from 3.6+/-2.1 to 455.5+/-19.9 in CVEC, while ACh had no effect.We conclude that NO production can be stimulated in a differential manner in pre- and postcapillary segments in the hamster cheek pouch. ACh selectively stimulates the production of NO only in arterioles, while PAF stimulates the production of NO only in venules.

The adaptive function of cheek pouches in the primate subfamily Cercopithecinae remains unresolved. By analyzing the circumstances of cheek pouch use, we tested two hypotheses for the evolution of cercopithecine cheek pouches proposed in earlier studies: (1) cheek pouches reduce vulnerability to predation, and (2) cheek pouches increase feeding efficiency by reducing competition. We studied two groups of wild blue monkeys (Cercopithecus mitis stuhlmanni) in the Kakamega Forest, Kenya, conducting focal observations of feeding individuals. Monkeys were less exposed while emptying their cheek pouches than filling them, supporting the predation-avoidance hypothesis. We investigated several measures of competitive threat, but only one supported the competition-reduction hypothesis: when the nearest neighbor's rank increased, subjects were more likely to increase than to decrease cheek pouch use. Overall, our findings supported the predation-avoidance hypothesis more strongly than the competition-reduction hypothesis. We suggest that variation in cheek pouch use may reflect differing behavioral strategies used by cercopithecines to mitigate competition and predation, as well as factors such as resource size and distribution, home range size, and travel patterns.

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m2) and non-lethal (2 g/m2) doses were found to inhibit cell-cycle progression primarily by impairing the G1/S transition. The results were obtained by flow cytometric DNA analysis. The inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). The greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. The time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G1 to S phase (at the onset of the dark period). The main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G1 cells. G1 cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)

Gap junction channels formed by connexin (Cx) protein subunits enable cell-to-cell conduction of vasoactive signals. Given the lack of quantitative measurements of Cx expression in microvascular endothelial cells (EC) and smooth muscle cells (SMC), the objective was to determine whether Cx expression differed between EC and SMC of resistance microvessels for which conduction is well-characterized.Cheek pouch arterioles (CPA) and retractor feed arteries (RFA) were hand-dissected and dissociated to obtain SMC or endothelial tubes. In complementary experiments, small intestine was dissociated to obtain SMC. Following reverse transcription, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed by using specific primers and fluorescent probes for Cx37, Cx40, and Cx43. Smooth muscle alpha-actin (SMAA) and platelet endothelial cell adhesion molecule-1 (PECAM-1) served as respective reference genes.Transcript copy numbers were similar for each Cx isoform in EC from CPA and RFA (approximately 0.5 Cx/PECAM-1). For SMC, Cx43 transcript in CPA and RFA (0.1 Cx/SMAA) was less (p0.05) than that in small intestine (approximately 0.4 Cx/SMAA). Transcripts for Cx37 and Cx40 were also detected in SMC. Punctate immunolabeling for each Cx isoform was pronounced at EC borders and that for Cx43 was pronounced in SMC of small intestine. In contrast, Cx immunolabeling was not detected in SMC of CPA or RFA.Connexin expression occurs primarily within the endothelium of arterioles and feed arteries, supporting a highly effective pathway for conducting vasoactive signals along resistance networks. The apparent paucity of Cx expression within SMC underscores discrete homocellular coupling and focal localization of myoendothelial gap junctions.

The aim of this study was to determine longitudinal and radial gradients in oxygen tension (PO(2)) in microvessels of the hamster cheek pouch.We measured PO(2) using the phosphorescence-quenching method in two orders of arterioles (45.8 +/- 5.5 and 19.9 +/- 1.8 micro m diameter), capillaries, and two orders of venules (50.5 +/- 3.4 and 21.4 +/- 2.0 micro m diameter) in order to determine the longitudinal PO(2) gradient. At the arteriolar and venular sites, we also measured PO(2) at four different sites for an analysis of radial PO(2) gradients: centerline, inside wall (larger arteriole and venule only), outside wall, and interstitium. We used 10 hamsters weighing 115 +/- 27 g anesthetized with pentobarbital intraperitoneally and maintained with alpha-chloralose intravenously. The cheek pouch was everted and a single-layered preparation was studied by intravital microscopy. Albumin-bound Pd-porphyrin was infused into the circulation and excited by flash illumination at 10 Hz, with a rectangular diaphragm limiting the excitation field to 5 x 25 micro m.In the longitudinal direction, intravascular PO(2) decreased significantly (P0.01) from large arterioles (39.5 +/- 2.3 mmHg) to small arterioles (32.2 +/- 0.3 mmHg), then to capillaries (30.2 +/- 1.8 mmHg), and on to small venules (27.3 +/- 2.1 mmHg) and large venules (25.5 +/- 2.2 mmHg). In the radial direction, PO(2) decreased significantly (P0.01) in and around larger arterioles, and to a lesser extent, around the smaller ones (P0.05). There was no significant PO(2) gradient, longitudinal or radial, associated with venules. The PO(2) difference from the centerline to the outside wall in large arterioles was 8.3 +/- 1.4 mmHg, and most of the decline in PO(2) in the radial direction was contributed by the intravascular difference (4.7 +/- 2.1 mmHg) and only about 1.0 +/- 2.7 mmHg by the transmural difference.Our data show that there are large intra-arteriolar radial PO(2) gradients, but no large transmural PO(2) differences, suggesting that the oxygen consumption of the microvessel wall is not exceptionally high.

Velafermin (recombinant human fibroblast growth factor-20, rhFGF-20) has been shown to reduce the severity and duration of mucositis in preclinical acute (single dose) radiation and chemotherapy/radiation models of oral mucositis. Our present study assessed the impact of velafermin on the severity and duration of oral mucositis that occurred as a consequence of fractionated radiation.Male Golden Syrian hamsters were exposed to eight doses of radiation (7.5 Gy/dose) to the cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9 that resulted in severe mucositis. Velafermin (4 mg/kg intraperitoneally) was administered on days 3 and 9; days 2, 3, 8 and 9; days 3, 4, 9 and 10; or days 4, 5, 10 and 11.Although all velafermin-treated groups showed some reduction in the degree of mucositis relative to the vehicle control, the most significant reduction (p0.001) was observed in the groups treated on days 3 and 9 or on days 4, 5, 10 and 11. Further histological analysis of resected buccal mucosa revealed improvements in epithelial tissue degradation, connective tissue degradation and inflammation severity after velafermin treatment. Most notably, velafermin treatment reduced inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF) production possibly through nuclear factor-kappaB (NF-kappaB) mediation. The detection of increased NF-E2-related factor-2 (NRF-2) expression in the early onset stage of mucositis in the buccal mucosa suggested additional protective benefits from reactive oxygen species (ROS) generated as a consequence of fractionated radiation treatment.Thus, velafermin provided therapeutic benefit in a hamster model of oral mucositis induced by fractionated radiation therapy.

Recently, Boron neutron capture therapy (BNCT) was successfully applied to treat experimental squamous cell carcinomas (SCC) of the hamster cheek pouch mucosa, with no damage to normal tissue. It was also shown that treating spontaneous nasal planum SCC in terminal feline patients with low dose BNCT is safe and feasible. In an extension of this work, the present study aimed at evaluation of the response of tumor and dose-limiting normal tissues to potentially therapeutic BNCT doses. Biodistribution studies with (10)B-boronophenylalanine (BPA enriched in (10)B) as a (10)B carrier were performed on three felines that showed advanced nasal planum SCC without any standard therapeutic option. Following the biodistribution studies, BNCT mediated by (10)BPA was done using the thermalized epithermal neutron beam at the RA-6 Nuclear Reactor. Follow-up included clinical evaluation, assessment of macroscopic tumor and normal tissue response and biopsies for histopathological analysis. The treated animals did not show any apparent radiation-induced toxicity. All three animals exhibited partial tumor control and an improvement in clinical condition. Enhanced therapeutic efficacy was associated with a high (10)B content of the tumor and a small tumor size. BNCT is therefore believed to be potentially effective in the treatment of spontaneous SCC. However, improvement in targeting (10)B into all tumor cells and delivering a sufficient dose at a greater depth are still required for the treatment of deep-seated, large tumors. Future studies are needed to evaluate the potential efficacy of the dual mode cellular (e.g. BPA-BNCT) and vascular (e.g. GB-10-BNCT) targeting protocol in a preclinical scenario, employing combinations of (10)B compounds with different properties and complementary uptake mechanisms.

Danshen, a Chinese herb, reduces hypertension in Oriental medicine. We hypothesized that Danshen acts partially through endothelial nitric oxide synthase (eNOS) signaling mechanisms. We tested the hypothesis using tanshinone IIA, an active ingredient of Danshen, and the two-kidney, one-clip renovascular hypertension model in hamsters. Oral tanshinone (50 μg/100 g body wt) reduced mean arterial pressure (MAP) from 161.2 ± 6.9 to 130.0 ± 7.8 mmHg (mean ± SE; P < 0.05) in hypertensive hamsters. MAP in sham-operated hamsters was 114.3 ± 9.2 mmHg. Topical tanshinone at 1 μg/ml and 5 μg/ml increased normalized arteriolar diameter from 1.00 to 1.25 ± 0.08 and 1.57 ± 0.11, respectively, and increased periarteriolar nitric oxide concentration from 87.1 ± 11.3 to 146.9 ± 23.1 nM ( P < 0.05) at 5 μg/ml in hamster cheek pouch. NG-monomethyl-l-arginine inhibited tanshinone-induced vasodilation. Hypertension reduced eNOS protein relative to sham-operated control. Tanshinone prevented the hypertension-induced reduction of eNOS and increased eNOS expression to levels higher than sham-operated control in hamster cheek pouch. Topical tanshinone increased normalized arteriolar diameter from 1.0 to 1.47 ± 0.08 in the cremaster muscle of control mice and to 1.12 ± 0.13 in cremasters of eNOS knockout mice. In ECV-304 cells transfected with eNOS-green fluorescent protein, tanshinone increased eNOS protein expression 1.35 ± 0.05- and 1.85 ± 0.07-fold above control after 5-min and 1-h application, respectively. Tanshinone also increased eNOS phosphorylation 1.19 ± 0.07- and 1.72 ± 0.20-fold relative to control after 5-min and 1-h application. Our data provide a basis to understand the action of a Chinese herb used in alternative medicine. We conclude that eNOS stimulation is one mechanism by which tanshinone induces vasodilation and reduces blood pressure.

SummaryHamster cheek pouch epidermis was examined with a thiol-specific fluorescent reagent [7 -(N-dimethylamino-4-methyl-3-coumarinyl) male imide (DACM)], with the Pauly reagent and by electron microscopy. There were many keratohyalin granules in the cells of the stratum granulosum where they were positively stained with the Pauly reagent, and were fluorescent as a whole, rather than partially, with DACM with or without reduction of disulfide bonds. These data indicate that the whole of a keratohyalin granule was rich in both histidine and cystine. Besides keratohyalin granules, the cellular envelope of the lower half of the stratum corneum showed fluorescence before reduction of disulfide bonds and following reduction of disulfide bonds, the cellular envelope of all stratum corneum cells showed fluorescence.Electron microscopically, keratohyalin granules in hamster cheek pouch epidermis were electron-dense and had a homogeneous structure which was identical to small keratohyalin granules in newborn rat epidermis. Structures corresponding to the single granule of Jessen or to dense homogeneous deposits were not seen. When the specimen was fixed in glutaraldehyde alone, keratohyalin granules consisted of electron- dense and electron-translucent parts. However, the profile was different from that of composite granules seen in both rat tongue epithelium and newborn rat epidermis. Several membrane coating granules were seen in stratum granulosum cells, and marginal band formation also occurred.

External carcinogens, such as tobacco and alcohol, induce molecular changes in large areas of oral mucosa, which increase the risk of malignant transformation. This condition, known as ‘field cancerization’, can be detected in biopsy specimens using histochemical techniques, even before histological alterations are identified. The efficacy of these histochemical techniques as biomarkers of early cancerization must be demonstrated in appropriate models. The hamster cheek pouch oral cancer model, universally employed in biological studies and in studies for the prevention and treatment of oral cancer, is also an excellent model of field cancerization. The carcinogen is applied in solution to the surface of the mucosa and induces alterations that recapitulate the stages of cancerization in human oral mucosa. We have demonstrated that the following can be used for the early detection of cancerized tissue: silver staining of nucleolar organizer regions; the Feulgen reaction to stain DNA followed by ploidy analysis; immunohistochemical analysis of fibroblast growth factor‐2, immunohistochemical labeling of proliferating cells to demonstrate an increase of epithelial cell proliferation in the absence of inflammation; and changes in markers of angiogenesis (i.e. those indicating vascular endothelial growth factor activity, endothelial cell proliferation and vascular density). The hamster cheek pouch model of oral cancer was also proposed and validated by our group for boron neutron capture therapy studies for the treatment of oral cancer. Clinical trials of this novel treatment modality have been performed and are underway for certain tumor types and localizations. Having demonstrated the efficacy of boron neutron capture therapy to control tumors in the hamster cheek pouch oral cancer model, we adapted the model for the long‐term study of field cancerized tissue. We demonstrated the inhibitory effect of boron neutron capture therapy on tumor development in field cancerized tissue with acceptable levels of mucositis, a dose‐limiting side‐effect. Fil: Monti Hughes, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Aromando, Romina Flavia. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina Fil: Pérez, Miguel A.. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina Fil: Schwint, Amanda Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Itoiz, María Elina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina

Oral mucositis (OM) is a common complication of treatments for head and neck cancer, particularly radiotherapy with or without chemotherapy. OM is characterised by oral erythema, ulceration, and pain. The aim of this study was to evaluate the effect of azilsartan (AZT), an angiotensin II receptor antagonist, on 5-fluorouracil (5-FU)-induced oral mucositis (OM) in Syrian hamsters. OM was induced by the intraperitoneal administration of 5-FU on experimental days 1 (60 mg/Kg) and 2 (40 mg/Kg). Animals were pretreated with oral AZT (1, 5, or 10 mg/kg) or vehicle 30 min before 5-FU injection and daily until day 10. Experimental treatment protocols were approved by the Animal Ethics Committee Use/CEUA (Number 28/2012) of the UFRN. Macroscopic analysis and cheek pouch samples were removed for histopathologic analysis. Myeloperoxidase (MPO), Malonyldialdehyde (MDA), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF-α) were analysed by Enzyme Linked Immuno Sorbent Assay (ELISA). Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and transforming growth factor (TGF)-α were measured by immunohistochemistry. Analysis of variance followed by Bonferroni's test was used to calculate the means of intergroup differences (p ≤ 0.05). Treatment with 1 mg/kg AZT reduced levels MPO (p

Oral ulcerative mucositis is a common and painful toxicity associated with chemotherapy for cancer. Current treatment for chemotherapy-induced oral mucositis is largely palliative, and no adequate treatment with conclusive evidence exists. The purpose of this study was to evaluate the potential effectiveness of the topical external medicines used in clinical settings, and the authors investigated the effects of 1% azulene ointment, 0.12% dexamethasone ointment, and polaprezinc-sodium alginate suspension on an animal model for oral mucositis induced by chemotherapy. Oral mucositis was induced in hamsters through a combination treatment of 5-fluorouracil and mild abrasion of the cheek pouch. Each drug was administered topically to the oral mucosa of hamsters, and the process of healing of damaged oral mucositis was examined by measuring the size of the mucositis. Azulene ointment did not reduce the size of the mucositis compared with the vaseline-treated control group. Polaprezinc-sodium alginate suspension significantly improved the recovery from 5-fluorouracil-induced damage. In contrast, local treatment with dexamethasone exacerbated the mucositis markedly. These results suggested the healing effect of polaprezinc-sodium alginate suspension and the risk of steroids to severe oral mucositis induced by chemotherapy.

Drug delivery across the buccal mucosa is convenient and safe transport method. The efficiency of the buccal system of peptide delivery is, however, not yet satisfactory. To improve the buccal transport new absorption promoters should be developed to be sufficiently active and at the same time causing no side effects like irritation or unpleasant taste. We have found that lysalbinic acid, a product of the alkaline hydrolysis of egg albumin and a mild detergent, meets those requirements. The preparation and some physicochemical properties of lysalbinic acid are described. Hamster cheek pouch was used as a model for the penetration process studies lysalbinic acid was shown to increase significantly an oral mucosa permeability for alpha-interferon and insulin. So this substance of the natural origin can be applied as an absorption enhancer for the buccal delivery of peptide drugs.

Having demonstrated BPA-BNCT induced control of experimental squamous cell carcinomas (SCC) of the hamster cheek pouch mucosa with no damage to normal tissue we explored the feasibility and safety of treating spontaneous head and neck tumors, with particular focus on SCC, of terminal feline patients with low dose BPA-BNCT employing the thermal beam of the RA-1 Reactor within a preclinical context. The biodistribution studies showed that, in all three cases evaluated, BPA delivered absolute boron values to tumor in the range that proved therapeutically useful in the experimental model of SCC. BPA-BNCT studies showed no radiotoxic effects, partial tumor control in terms of impaired growth and partial necrosis, an improvement in clinical condition and prolonged survival beyond the terminal condition of the feline patients at the time of recruitment.

Diagnostic ultrasound (US) is reported to increase intracellular oxidative stress in vitro. Increased oxidative stress mediated ischemia-reperfusion injury in the microcirculation. To examine the effects of US in hamster cheek pouch microcirculation during baseline and ischemia and reperfusion (I/R), I/R injury was provoked in the cheek pouch under “sham” (transducer off, group 1) and active US irradiation (group 2) at baseline (15 min) and at the beginning (15 min) of the reperfusion after ischemia (30 min). US transmission was delivered in the harmonic mode (2.5 MHz) with 1.3 mechanical index (MI) and 2.0 peak negative pressure. Microvascular damage was evaluated by measuring arterial diameter, red blood cell velocity, wall shear stress, permeability, perfused capillary length and adherent leukocytes in venules. Lipid peroxides were determined in the systemic blood. US increased permeability (baseline: 0.04 ± 0.02; after US 0.30 ± 0.04, p < 0.01) and slightly decreased capillary perfusion by 7% during baseline (p < 0.01). Arteriolar diameter (35 ± 7 μm vs. 20 ± 5 μm, p < 0.05), RBC velocity (2.8 ± 0.4 mm s−1 vs. 0.75 ± 0.05 mm s−1, p < 0.05) and shear stress ( 0.76 ± 0.09 Pa vs. 0.36 ± 0.05 Pa, p < 0.05) decreased significantly after reperfusion. These parameters increased by 40, 64 and 33%, respectively after US. Leukocyte adhesion decreased by 31 % (p < 0.05) after US and lipid peroxides decreased by 26% and 51% during baseline and 15 min of reperfusion after US, respectively. In conclusion, diagnostic US increased microvascular permeability during baseline and reperfusion. Moreover, US enhanced wall shear stress and reduced oxidative stress during postischemic reperfusion; thus, increasing capillary perfusion. (E-mail: sibert@ifc.cnr.it)

Objective: We previously proposed the hamster cheek pouch model of oral cancer for BNCT studies. We herein present the biodistribution of a non-toxic boron compound, GB-10 ( Na 2 10 B 10 H 10 ), in this model to assess its potential for BNCT or BNCT enhanced Fast Neutron Therapy. Materials and methods: We evaluated the uptake and retention of GB-10 in tumour and precancerous tissue and in potentially dose-limiting, clinically relevant normal tissues. Results: Mean tumour boron concentration delivered by GB-10 (50 mg B/kg) peaked to 77.7±28.0 ppm at 20 min post-administration and remained at therapeutically useful values of 31.9±21.4 ppm at 3 h. The clearance rate for normal tissues was faster than for tumour tissue. The consistently low brain and spinal cord values would preclude normal tissue toxicity. The uptake of GB-10 by precancerous tissue may be of potential use in the treatment of field cancerized areas. GB-10 was deposited homogeneously in different tumour areas, an asset when treating heterogenous tumours. The data suggests that the joint administration of BPA and GB-10 may improve the therapeutic efficacy of BNCT. Conclusions: GB-10 is a potential boron carrier for BNCT of head and neck tumours and for BNCT-FNT.

Background and Objectives The first objective of this study was to evaluate the performance of fluorescence spectroscopy for diagnosing pre-cancers in stratified squamous epithelial tissues in vivo using two different probe geometries with (1) overlapping versus (2) non-overlapping illumination and collection areas on the tissue surface. Probe (1) and probe (2) are preferentially sensitive to the fluorescence originating from the tissue surface and sub-surface tissue depths, respectively. The second objective was to design a novel, angled illumination fiber-optic probe to maximally exploit the depth-dependent fluorescence properties of epithelial tissues. Study Design/Materials and Methods In the first study, spectra were measured from epithelial pre-cancers and normal tissues in the hamster cheek pouch and analyzed with a non-parametric classification algorithm. In the second study, Monte Carlo modeling was used to simulate fluorescence measurements from an epithelial tissue model with the angled illumination probe. Results An unbiased classification algorithm based on spectra measured with probes (1) and (2), classified pre-cancerous and normal tissues with 78 and 94% accuracy, respectively. The angled illumination probe design provides the capability to detect fluorescence from a wide range of tissue depths in an epithelial tissue model. Conclusions The first study demonstrates that fluorescence originating from sub-surface tissue depths (probe (2)) is more diagnostic than fluorescence originating from the tissue surface (probe (1)) in the hamster cheek pouch model. However in general, it is difficult to know a priori the optimal probe geometry for pre-cancer detection in a particular epithelial tissue model. The angled illumination probe provides the capability to measure tissue fluorescence selectively from different depths within epithelial tissues, thus obviating the need to select a single optimal probe design for the fluorescence-based diagnosis of epithelial pre-cancers. Lasers Surg. Med. 34:25–38, 2004. © 2004 Wiley-Liss, Inc.