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Translational animal models for oral mucositis (OM) are necessary to simulate and assess the bioclinical effects and response in humans. These models should simulate high levels of radiation exposure that leads to oxidative stress and inflammatory‐initiated tissue changes. Hamster models have been extensively studied to observe pathological effects of radiation exposure and help in the development of effective treatments. To successfully evaluate the potential for treatment regimens with consistency and relevance, a radiation‐induced OM hamster model was developed using a clinical linear accelerator utilized by cancer patients daily. The dose exposure to the isolated, everted cheek pouch of a hamster, as well as the progression of injury, pro‐inflammatory marker, histological, and elasticity analyses of the buccal pouch were conducted to verify replicability and reproducibility of the injury model. The findings from this model demonstrated its ability to consistently induce injury and resolution over 28 days using an acute dose of 60 Gy. This model was developed to enhance clinical relevance when evaluating potential efficacious treatments and can now be utilized in efficacy studies to better evaluate developed therapeutics in a preclinical model that is easy to translate to clinical studies..

Background/Aim: A three-dimensional (3D) printed tracheostomy tube has potential application for patients who require a specialized tube. The aim of this study was to evaluate the characteristics of various 3D printing materials and determine their use in producing 3D-printed tracheostomy tube. Materials and Methods: Mechanical, chemical, and microbiological in vivo changes in the scaffolds were analyzed using a hamster cheek pouch (HCP) model. Results: The poly methyl methacylate (PMMA)-resin showed superior pre- and post-insertion mechanical properties and a relatively consistent lower biofilm formation compared with other scaffolds. PMMA-resin was successfully 3D-printed with dimensional accuracy without a support system. The use of a 3D-printed PMMA tracheostomy tube in a rabbit trachea showed no definite signs of infection, allergy or foreign body reaction. Conclusion: PMMA-resin can be proposed as an alternative for a 3D-printed tracheostomy tube material. In addition, we suggest HCPs as an in vivo model for evaluating indwelling medical devices.

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.

Boron neutron capture therapy (BNCT) is a promising cancer binary therapy modality that utilizes the nuclear capture reaction of thermal neutrons by boron-10 resulting in a localized release of high- and low-linear energy transfer (LET) radiation. Electrochemotherapy (ECT) is based on electroporation (EP) that induces opening of pores in cell membranes, allowing the entry of compounds. Because EP is applied locally to a tumor, the compound is incorporated preferentially by tumor cells. Based on the knowledge that the therapeutic success of BNCT depends centrally on the boron content in tumor and normal tissues and that EP has proven to be an excellent facilitator of tumor biodistribution of an anti-tumor agent, the aim of this study was to evaluate if EP can optimize the delivery of boronated compounds. We performed biodistribution studies and qualitative microdistribution analyses of boron employing the boron compound sodium decahydrodecaborate (GB-10) + EP in the hamster cheek pouch oral cancer model. Syrian hamsters with chemically induced exophytic squamous cell carcinomas were used. A typical EP treatment was applied to each tumor, varying the moment of application with respect to the administration of GB-10 (early or late). The results of this study showed a significant increase in the absolute and relative tumor boron concentration and optimization of the qualitative microdistribution of boron by the use of early EP + GB-10 versus GB-10 without EP. This strategy could be a tool to improve the therapeutic efficacy of BNCT/GB-10 in vivo.

Background and Aims: Fructose intake is directly related to vascular dysfunction and it is a risk factor for the development of metabolic and cardiovascular diseases, such as obesity, diabetes, and hypertension. Selenium, a component of antioxidant enzymes, improves hyperglycemia and vascular function in diabetic animals. The aim of this study was to evaluate the effects of dietary selenium supplementation on microcirculatory and metabolic parameters of fructose-fed hamsters. Methods and Results: Male hamsters (Mesocricetus auratus) had their drinking water substituted or not by 10% fructose solution for 60 days, during which their microcirculatory function was evaluated in the cheek pouch preparation. Blood glucose and serum insulin levels were also tested. Microcirculatory responses to acetylcholine (an endothelium-dependent vasodilator) and to sodium nitroprusside (SNP, an endothelium-independent vasodilator), and macromolecular permeability increase induced by a 30-min ischemia/reperfusion (I/R) procedure, showed that endothelium-dependent and independent vasodilatation was significantly increased in animals that had high selenium supplementation, in both the control and fructose-fed groups. Selenium supplementation protected against plasma leakage induced by I/R in all control and fructose-fed groups. Conclusion: Our results indicate that dietary selenium supplementation reduces microvascular dysfunction by increasing endothelial-dependent and independent dilatation and reducing macromolecular permeability increase in fructose-fed animals.

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.

Myogenic tone is an important feature of arterioles and resistance arteries, but the mechanisms responsible for this hallmark characteristic remain unclear. We used pharmacological inhibitors to compare the roles played by phospholipase C (PLC; 10 μM U73122), inositol 1,4,5-trisphosphate receptors (IP3Rs; 100 μM 2-aminoethoxydiphenylborane), protein kinase C (10 μM bisindolylmaleimide I), angiotensin II type 1 receptors (1 μM losartan), Rho kinase (10 nM−30 μM Y27632 or 300 nM H1152), stretch-activated ion channels (10 nM−1 μM Gd3+or 5 μM spider venom toxin GsMTx-4) and L-type voltage-gated Ca2+channels (0.3–100 μM diltiazem) in myogenic tone of cannulated, pressurized (80 cmH2O), second-order hamster cremaster or cheek pouch arterioles. Effective inhibition of either PLC or IP3Rs dilated cremaster arterioles, inhibited Ca2+waves, and reduced global Ca2+levels. In contrast, cheek pouch arterioles did not display Ca2+waves and inhibition of PLC or IP3Rs had no effect on myogenic tone or intracellular Ca2+levels. Inhibition of Rho kinase dilated both cheek pouch and cremaster arterioles with equal efficacy and potency but also reduced intracellular Ca2+signals in both arterioles. Similarly, inhibition of mechanosensitive ion channels with Gd2+or GsMTx-4 produced comparable dilation in both arterioles. Inhibition of L-type Ca2+channels with diltiazem was more effective in dilating cremaster (86 ± 5% dilation, n = 4) than cheek pouch arterioles (54 ± 4% dilation, n = 6, P < 0.05). Thus, there are substantial differences in the mechanisms underlying myogenic tone in hamster cremaster and cheek pouch arterioles. Regional heterogeneity in myogenic mechanisms could provide new targets for drug development to improve regional blood flow in a tissue-specific manner.NEW & NOTEWORTHY Regional heterogeneity in the mechanisms of pressure-induced myogenic tone implies that resistance vessels may be able to alter myogenic signaling pathways to adapt to their environment. A better understanding of the spectrum of myogenic mechanisms could provide new targets to treat diseases that affect resistance artery and arteriolar function.

Aim: Determine the potential for cheek pouch buccal mucosa irritation in hamsters following administration of apomorphine hydrochloride film (APL-130277). Methods: Three studies were conducted with Syrian golden hamsters. (First study, four hamsters received APL-130277 three times a day [TID] for 7 days. Second study, four hamsters received APL-130277 once a day [QD] for days 1–3, twice a day [BID] for days 4–7 and TID for days 8–21. Third study, 32 hamsters received either a placebo strip or APL-130277-dosed TID for 28 days). For all the studies, the macroscopic appearance of the buccal cavities was evaluated throughout the study. In the third study, all animals were necropsied on day 29, and macroscopic and histopathological examinations were performed. Results: In the first and second studies, the buccal mucosa of the cheek pouch did not show any signs of irritation. In the third study, administration of APL-130277-dosed TID for 28 consecutive days did not result in observable local irritation of the buccal mucosa. Conclusion: In all the studies, APL-130277 produced no irritation of the cheek pouch buccal mucosa.

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

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.

Boron neutron capture therapy (BNCT) is based on selective accumulation of B-10 carriers in tumor followed by neutron irradiation. We demonstrated, in 2001, the therapeutic effect of BNCT mediated by BPA (boronophenylalanine) in the hamster cheek pouch model of oral cancer, at the RA-6 nuclear reactor. Between 2007 and 2011, the RA-6 was upgraded, leading to an improvement in the performance of the BNCT beam (B2 configuration). Our aim was to evaluate BPA-BNCT radiotoxicity and tumor control in the hamster cheek pouch model of oral cancer at the new “B2” configuration. We also evaluated, for the first time in the oral cancer model, the radioprotective effect of histamine against mucositis in precancerous tissue as the dose-limiting tissue. Cancerized pouches were exposed to: BPA-BNCT; BPA-BNCT + histamine; BO: Beam only; BO + histamine; CONTROL: cancerized, no-treatment. BNCT induced severe mucositis, with an incidence that was slightly higher than in “B1” experiments (86 vs 67%, respectively). BO induced low/moderate mucositis. Histamine slightly reduced the incidence of severe mucositis induced by BPA-BNCT (75 vs 86%) and prevented mucositis altogether in BO animals. Tumor overall response was significantly higher in BNCT (94–96%) than in control (16%) and BO groups (9–38%), and did not differ significantly from the “B1” results (91%). Histamine did not compromise BNCT therapeutic efficacy. BNCT radiotoxicity and therapeutic effect at the B1 and B2 configurations of RA-6 were consistent. Histamine slightly reduced mucositis in precancerous tissue even in this overly aggressive oral cancer model, without compromising tumor control. Fil: Monti Hughes, Andrea. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Longhino, Juan. Comisión Nacional de Energía Atómica. Centro Atómico Bariloche; Argentina Fil: Boggio, Esteban. Comisión Nacional de Energía Atómica. Centro Atómico Bariloche; Argentina Fil: Medina, Vanina Araceli. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Martinel Lamas, Diego José. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Garabalino, Marcela Alejandra. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina Fil: Heber, Elisa Mercedes. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina Fil: Pozzi, Emiliano César Cayetano. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina Fil: Itoiz, María Elina. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Aromando, Romina Flavia. Universidad de Buenos Aires. Facultad de Odontología; Argentina Fil: Nigg, David W.. Idaho National Laboratory; Estados Unidos Fil: Trivillin, Verónica Andrea. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina Fil: Schwint, Amanda Elena. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina

OBJECTIVES: Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation. METHODS: Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of post-capillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (α1-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). RESULTS: The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17-22) leaks/cm2 vs. 124 (123-128) leaks/cm2, respectively, p

Summary Background Over-expression of histamine receptors has been reported in several types of malignancies. Earlier we have successfully demonstrated use of chlorin p6-histamine conjugate (Cp6-his) for improving cellular uptake and photo toxicity of Cp6 in oral cancer cell lines. In the present study, after having confirmed that histamine receptors are over-expressed in tumors of hamster cheek pouch, we investigated the efficacy of Cp6-his for photodynamic treatment (PDT) of tumors in this animal model. Methods Cp6-his (3 mg/kg body weight) was injected intraperitoneally and its accumulation in tumor, surrounding tissue, normal mucosa and abdominal skin was monitored non-invasively by fluorescence spectroscopy. For PDT, tumors at 4 h after Cp6-his administration were exposed to red light (660 ± 25 nm, 100 J/cm2). Tumor damage and regression were assessed by histology and tumor volume measurements, respectively. Expression of histamine H2 receptors in tumor and normal mucosa was assessed by immuno-staining. Results The accumulation of Cp6-his was higher in tumors as compared to normal mucosa at 4 h after its administration. For Cp6 similar preferential accumulation was observed except that in normal mucosa the accumulation of Cp6 was more as compared to Cp6-his. The clearance of Cp6-his from skin was rapid showing ∼80% decrease within 48 h from its peak level at 4 h after drug injection. PDT led to extensive cellular damage and tumors of size up to ∼1000 mm3 regressed completely one week after PDT. Conclusion Higher tumor selectivity of Cp6-his and complete regression of bigger tumors after PDT suggest that conjugating Cp6 to histamine is a promising approach to improve PDT efficacy.

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

The aim of the present study was to assess the in vivo effects of aldosterone topically applied on the hamster cheek pouch microcirculation under baseline conditions or during ischemia-reperfusion. Male Syrian hamsters were anesthetized, tracheotomized and intubated. They were studied under baseline conditions or submitted to ischemia-reperfusion. Cheek pouch microvessels were visualized by fluorescence microscopy. Microvascular parameters were determined by computerized methods. Under baseline conditions, aldosterone (0.2, 0.5, 2.4 μM/L/2 min) induced dose-dependent constriction of all arterioles within 2.0 ± 0.5 min of administration. Diameter reduction was in the same range in smaller arterioles: A3 ones constricted by 24 ± 3% of baseline (at the highest dose). Aldosterone applied prior to ischemia and at reperfusion caused arteriolar constriction, marked microvascular permeability (0.66 ± 0.03 Normalized Grey Level), reduction in perfused capillary (-70 ± 4% of baseline) and leukocyte adhesion. All changes were statistically significant compared with ischemic animals. Potassium canrenoate (mineralcorticoid receptor inhibitor) prior to aldosterone did not abolish the aldosterone-induced effects, while valsartan (angiotensin II AT1 receptor inhibitor) prior to aldosterone ameliorated microvascular ischemia-reperfusion injury. In conclusion, aldosterone determined dose-dependent arteriolar constriction likely by angiotensin II type-1 receptor activation (non-genomic mechanism) worsening the effects of ischemia-reperfusion on capillary perfusion, while protecting from free radical formation.

Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis, and can be targeted for cancer prevention. In order to develop potent topical agents for oral cancer chemoprevention, five known 5-Lox inhibitors from dietary and synthetic sources, Zileuton, ABT-761, Licofelone, Curcumin and Garcinol, were evaluated in silico for their potential efficacy. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be a promising agent based on the calculation of a theoretical activity index. Computer modeling showed that garcinol well fit the active site of 5-Lox, and potentially inhibited enzyme activity through interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. These results demonstrated that topical application of a 5-Lox inhibitor, garcinol, had chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) is the sixth most common human neoplasm worldwide. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is highly expressed in human OSCC and is a target for cancer therapy and prevention. This study investigated the therapeutic and preventive effects of an inhibitor of EGFR (PD153035) on OSCC. METHODS: The effects of PD153035 were examined in human cancer cell lines in vitro, in an athymic nude mouse xenograft model in vivo, and in the 7,12-dimethylbenz[ a]anthracene (DMBA)-induced hamster cheek pouch tumour model in vivo. RESULTS: PD153035 significantly inhibited cell growth, delayed cell cycle progression and induced apoptosis in human OSCC cells in vitro. In vivo, PD153035 inhibited xenograft tumour growth in nude mice in a dose-dependent manner and prevented the development of OSCC at the postinitiation stage in the DMBA-induced hamster cheek pouch tumour model. PD153035 inhibited the DMBA-induced increases in cell proliferation and in levels of phosphorylated EGFR and phosphorylated signal transducer and activator of transcription 3 (STAT3) protein in the hamster cheek pouch. CONCLUSIONS: Inhibitors of EGFR, such as PD153035, have potential value in the treatment and prevention of OSCC.

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

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.

Summary Given the clinical relevance of locoregional recurrences in head and neck cancer, we developed a novel experimental model of premalignant tissue in the hamster cheek pouch for long-term studies and demonstrated the partial inhibitory effect of a single application of Boron Neutron Capture Therapy (BNCT) on tumor development from premalignant tissue. The aim of the present study was to evaluate the effect of a double application of BNCT with a 6 week interval in terms of inhibitory effect on tumor development, toxicity and DNA synthesis. We performed a double application, 6 weeks apart, of (1) BNCT mediated by boronophenylalanine (BPA-BNCT); (2) BNCT mediated by the combined application of decahydrodecaborate (GB-10) and BPA [(GB-10 + BPA)-BNCT] or (3) beam-only, at RA-3 nuclear reactor and followed the animals for 8 months. The control group was cancerized and sham-irradiated. BPA-BNCT, (GB-10 + BPA)-BNCT and beam-only induced a reduction in tumor development from premalignant tissue that persisted until 8, 3, and 2 months respectively. An early maximum inhibition of 100% was observed for all 3 protocols. No normal tissue radiotoxicity was detected. Reversible mucositis was observed in premalignant tissue, peaking at 1 week and resolving by the third week after each irradiation. Mucositis after the second application was not exacerbated by the first application. DNA synthesis was significantly reduced in premalignant tissue 8 months post-BNCT. A double application of BPA-BNCT and (GB-10 + BPA)-BNCT, 6 weeks apart, could be used therapeutically at no additional cost in terms of radiotoxicity in normal and dose-limiting tissues.

In the present study the therapeutic effect and potential toxicity of the novel "Sequential" boron neutron capture therapy (Seq-BNCT) for the treatment of oral cancer was evaluated in the hamster cheek pouch model at the RA-3 Nuclear Reactor. Two groups of animals were treated with "Sequential" BNCT, i.e., BNCT mediated by boronophenylalanine (BPA) followed by BNCT mediated by sodium decahydrodecaborate (GB-10) either 24 h (Seq-24h-BNCT) or 48 h (Seq-48h-BNCT) later. In an additional group of animals, BPA and GB-10 were administered concomitantly [(BPA + GB-10)-BNCT]. The single-application BNCT was to the same total physical tumor dose as the "Sequential" BNCT treatments. At 28 days post-treatment, Seq-24h-BNCT and Seq-48h-BNCT induced, respectively, overall tumor responses of 95 ± 2% and 91 ± 3%, with no statistically significant differences between protocols. Overall response for the single treatment with (BPA + GB-10)-BNCT was 75 ± 5%, significantly lower than for Seq-BNCT. Both Seq-BNCT protocols and (BPA + GB-10)-BNCT induced reversible mucositis in the dose-limiting precancerous tissue around treated tumors, reaching Grade 3/4 mucositis in 47 ± 12% and 60 ± 22% of the animals, respectively. No normal tissue toxicity was associated with tumor response for any of the protocols. "Sequential" BNCT enhanced tumor response without an increase in mucositis in dose-limiting precancerous tissue.

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.

Background In view of the debate on the safety of using ultrasound associated with microbubbles, we investigated the microvascular effects in situations of potential clinical interest. Methods Ultrasound, microbubbles, and their association were evaluated on the cheek pouch microcirculation in 96 male hamsters (n = 6 in each group): control; ischemia–reperfusion, 30 minutes total ischemia followed by reperfusion; hyperinsulinemia and hyperglycemia (substitution of the drinking water by 10% fructose solution for 3 months); and endotoxic shock (induced by Escherichia coli lipopolysaccharide). The heart rate, mean arterial pressure, number of rolling and adhered leukocytes, and arteriolar and venular diameters were analyzed. Results Heart rate and mean arterial pressure were lower in the endotoxic shock group than in the control group. Ultrasound alone or associated with microbubbles decreased the number of rolling and adhered leukocytes in the ischemia–reperfusion and hyperinsulinemia and hyperglycemia groups and increased venular diameter in the ischemia–reperfusion group. Conclusion The use of ultrasound decreases inflammation. Although it has not been established that pseudo-anaphylaxis can be assessed by intravital microscopy, microbubble administration does not increase venular leukocyte adhesion.

Our goal was to characterize changes in flow and diameter with vascular endothelial cell growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Observations were made in arteriolar networks of the cheek pouch tissue in anesthetized hamsters (pentobarbital 70 mg/kg, i.p., n = 45). Local and remote dilation responses to micropipette-applied VEGF or FGF2 yielded similar EC50 values. The role of gap junctions in the remote response was tested by applying sucrose, halothane or 18αGA to the feed arteriole midway between the remote stimulation and upstream observation sites; all remote dilation to FGF2 was prevented, while only the early dilation to VEGF was blocked. The remote dilation to VEGF displayed a second rheologic mechanism. The second mechanism involved an abrupt increase in upstream velocity and shear rate, followed by nitro-arginine sensitive dilation. To test whether the abrupt increase in shear could be caused by other agents known to cause edema, remote responses to histamine and thrombin were tested. Each caused an abrupt increase in velocity followed by nitro-arginine-sensitive dilation. This study shows that VEGF or agents that increase permeability can initiate an upstream velocity increase with dilation that recruits flow to the network; this is in addition to simultaneous gap junction-mediated dilation.

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.

This study was performed to examine the carcinogenic effects of benzo[a]pyrene (B[a]P) and manufactured gas plant (MGP) residues on the hamster cheek pouch (HCP). Syrian hamsters were treated topically with a suspension of 2%, 10%, or 20% B[a]P or 50% or 100% MGP-7 (a mixture of residues from 7 MGP sites) in mineral oil for eight (short-term study) and sixteen, twenty, twenty-eight, and thirty-two weeks (long-term study). The short-term study showed that B[a]P induced p53 protein accumulation, indicative of genotoxic damage, as well as increased cell proliferation, hyperplasia, and inflammation, which is usually associated with promotional activity. In contrast, the MGP-7 presented only marginal p53 accumulation and induction of BrdU incorporation. In the long-term experiments, animals treated with 2% and 10% of B[a]P continued to show p53 protein accumulation as well as hyperplasia and increased cell proliferation and inflammation. By thirty weeks, all the animals treated with B[a]P had a 100% incidence of squamous cell carcinoma (SCC). Animals treated with 50% and 100% MGP-7 showed only weak hyperplasia and a low proliferation rate and accumulation of p53 protein through thirty-two weeks. Benzo[a]pyrene was highly carcinogenic when used at adequate doses. Manufactured gas plant residue, however, was not carcinogenic in this model.

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.

Microspheres formulated from blending poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-grafted-chitosan (PEG-g-CHN), using a modified in-emulsion-solvent-evaporation method, were investigated for the delivery of protein. A model protein, bovine serum albumin (BSA), was incorporated into the PLGA/PEG-g-CHN microspheres and both initial burst and release kinetics could be modulated by varying the PEG-g-CHN content. Basic fibroblast growth factor (bFGF) was formulated into the microspheres containing 5% PEG-g-CHN and the bFGF contents in the releasates were determined by a receptor-based ELISA with their in vitro bioactivities validated by fibroblast cell culture. The in vivo effect of the bFGF microspheres formulation was evaluated in a hamster cheek pouch model using a 7 day exposure (e.g., before significant vascular remodeling was expected). Using intravital microscopy, the tissue showed no evidence of inflammation with any formulation; deliberate activation of a preconditioning response linked to inflammation was attenuated by BSA microspheres alone. Vasoactive responses (receptor-dependant and independent constriction and dilation) linked to nitric oxide were attenuated, and constriction to endothelin was enhanced in bFGF and not BSA containing microspheres. PLGA/PEG-g-CHN blended microspheres were also demonstrated to be non-inflammatory and non-thrombogenic in vivo by observing the vascular changes in the cheek pouch. In conclusion, the addition of PEG-g-CHN to PLGA microspheres can serve as a sustained delivery vehicle for bFGF and the released protein provides vasoactive changes consistent with chronic bFGF exposure.

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.

Oral cancer is a common neoplasm worldwide with tobacco and alcohol being the major etiological factors contributing to its pathogenesis. Epidermal growth factor receptor (EGFR) and ErbB2 are known to be involved in the development of oral cancer with the former up-regulated in up to 90% human cases. The goal of this study was to evaluate the chemopreventive effects of a dual inhibitor of EGFR and ErbB2 tyrosine kinases, GW2974, in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch model. A short-term experiment (3-week topical DMBA followed by 1-week topical GW2974) was conducted to examine the effects of GW2974 on aberrant arachidonic acid (AA) metabolism and cell proliferation in the hamster oral epithelium. Topical application of 0.1 ml GW2974 (160 microM, three times a week) significantly reduced the levels of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 5-, 12-, 15-hydroxyeicosatetraenoic acid (HETE), and cell proliferation (BrdU-labeling index). In a long-term post-initiation experiment (6-week topical DMBA followed by 18-week topical GW2974), GW2974 (4 mM and 8 mM) significantly inhibited the incidence, number and size of visible tumors. Under microscope, the numbers of oral lesions (hyperplasia, dysplasia, carcinoma) and the incidence of squamous cell carcinoma (SCC) were also significantly suppressed by GW2974. In summary, our study indicated that dual inhibition of EGFR and ErbB2 tyrosine kinases by GW2974 was effective in preventing oral carcinogenesis in DMBA-induced hamster cheek pouch model. GW2974 exerted its chemopreventive effects in part by suppressing aberrant AA metabolism.

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.

The purpose of this study was to determine whether protein tyrosine kinase, a ubiquitous family of intracellular signaling enzymes that regulates endothelial cell function, modulates bradykinin- and substance P-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. Using intravital microscopy, I found that suffusion of bradykinin or substance P (each, 0.5 and 1.0 μM) onto the cheek pouch elicited significant, concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-dextran (FITC-dextran; molecular mass, 70 kDa; P < 0.05). These responses were significantly attenuated by suffusion of genistein (1.0 μM) or tyrphostin 25 (10 μM), two structurally unrelated, nonspecific protein tyrosine kinase inhibitors ( P < 0.05). Conceivably, the kinase(s) involved in this process could be agonist specific because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses while the opposite was observed with substance P. Both inhibitors had no significant effects on adenosine (0.5 M)-induced responses ( P > 0.5). Collectively, these data suggest that the protein tyrosine kinase metabolic pathway modulates, in part, the edemagenic effects of bradykinin and substance P in the intact hamster cheek pouch microcirculation in a specific fashion.

Previous experiments in our laboratory, using the hamster cheek pouch microcirculation, have shown that precapillary vessels exhibit spontaneous rhythmic luminal variations, termed vasomotion, a myogenic activity sustained by a balance between membrane currents among which polarizing K + currents play an important role. In these microvessels, endothelium-derived relaxing factors (EDRFs) seem to regulate arteriolar diameter [via nitric oxide (NO) and cyclic GMP] and vasomotion [probably via endothelium-derived hyperpolarizing factor (EDHF)]. Fish or fish oil diet can decrease the risk of cardiovascular diseases, probably by modifying the conductance of selective ion channels, such as K + and/or Ca ++ , and/or increasing the production of vasodilators, such as NO. To investigate its effect on microvascular reactivity, using the same preparation and an intravital microscope coupled to a closed circuit TV system, male hamsters were treated for 14 days, twice a day, with 0.4 mL/100 g body weight with fish or olive oil. An attempt was also undertaken to record in arterioles, in vivo , the membrane potential of smooth muscle cells during their vasomotor activity combining conventional microelectrode and intravital microscopy techniques. The effects of topical application of two vasodilators, acetylcholine [endothelium-dependent one, NO release and membrane hyperpolarization via Ca ++ -activated K + channels (K Ca )] and sodium nitroprusside (endothelium-independent, NO donor and no change on membrane potential) and two vasoconstrictors which elicited membrane depolarization via Ca ++ channels, phenylephrine (α 1 -adrenergic receptor agonist) and serotonin (5-hydroxi-tryptamine) on mean internal diameter of arterioles and venules, arteriolar blood flows, spontaneous arteriolar vasomotion frequency and amplitude and functional capillary density (FCD, number of capillaries with flowing red blood cells per unit area of tissue) were determined. Anesthesia was induced by sodium pentobarbital (i.p.) and maintained with α-chloralose through the femoral vein. In the presence of vasomotion, the membrane potentials are slowly oscillating by about 20 mV around values of approximately − 50 mV in perfect synchrony with vasomotor movements and depolarizing phases coincide with vasoconstrictions while polarizing ones with vasodilatations. Comparing all parameters, in control conditions, only the spontaneous vasomotion frequency was significantly higher (2.37 times higher) on the group treated with fish oil and persisted as such throughout all experiments. With topical application of the drugs mentioned above, the group treated with fish oil showed, for each drug concentration, a balance towards vasodilatation with consequent increase on arteriolar blood flow and on FCD, compared with the olive oil treated one. No significant changes on mean arterial pressure, spontaneous arteriolar vasomotion amplitude or venular diameter could be detected in the two groups. Our results support the concept that, in the hamster cheek pouch microcirculation, fish oil supplementation activates K + channels which act as the EDHF and might also increase the production of vasodilators, probably NO.

Purpose Overexpression of epidermal growth factor receptor (EGFR) is observed in oral squamous cell carcinoma (OSCC) and is associated with increased proliferation, metastasis and therapeutic resistance. We aim to develop a novel drug delivery system comprised of a photosensitizer Chlorin e6 (Ce6) that is encapsulated in a viral envelope and tagged with anti-EGFR antibody to target OSCC. Methods Ce6 was encapsulated in both virosomes (Ce6-Vir) and virosomes tagged with anti-EGFR antibody (Ce6-Vir-EGFR′). In vitro studies were conducted to assess the cellular uptake and bioavailability of the photosensitizer in OSCC cells. Ce6 alone or in constructs was then administered in a hamster cheek pouch model and fluorescence imaging and spectroscopy was performed. Results In vitro results showed that the uptake of Ce6-Vir-EGFR′ was lower than that for Ce6-Vir and Ce6 possibly due to its large size. Nevertheless, in vivo results showed significant tumor specificity of Ce6-Vir-EGFR′ compared to Ce6. The tumor to normal mucosa ratio showed that Ce6-Vir-EGFR′ can successfully target OSCC lesions and therefore shows potential for use in fluorescence diagnosis of OSCC. Conclusions Both the virosome-Ce6 constructs were internalized by OSCC cells and successfully used for fluorescence imaging. Tagging with anti-EGFR antibody further improved the targeting ability toward OSCC.

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

There is compelling evidence for the cancer chemopreventive effects of vitamin E and related compounds. Of all the vitamin E derivatives that have been investigated to date, vitamin E acid succinate is the most effective anti-cancer agent. This report describes the preparation and testing of liposomal formulation of mono alpha-tocopheryl ester of succinic acid (alpha-TOS) for cytotoxicity against hamster cheek pouch carcinoma cell line (HCPC-1). Small unilamellar vesicles (SUV) of phosphatidylcholine incorporating 70 microM alpha-TOS were superior to alpha-TOS alone or SUV without incorporated alpha-TOS, as inducers of apoptosis in HCPC-1 cells. Liposomal alpha-TOS perturbed the lipid structure in cells, promoted apoptosis, and decreased cell viability. The mechanism of action of alpha-TOS appears to involve membrane damage and induction of ceramide mediated apoptosis.

Intracellular calcium concentration ([Ca2+]i) governs the contractile status of arteriolar smooth muscle cells (SMC). Although studied in vitro, little is known of SMC [Ca2+]idynamics during the local control of blood flow. We tested the hypothesis that the rise and fall of SMC [Ca2+]iunderlies arteriolar constriction and dilation in vivo. Aparenchymal segments of second-order arterioles (diameter 35 ± 2 μm) were prepared in the superfused cheek pouch of anesthetized hamsters ( n = 18) and perifused with the ratiometric dye fura PE-3 (AM) to load SMC (1 μM, 20 min). Resting SMC [Ca2+]iwas 406 ± 37 nM. Elevating superfusate O2from 0 to 21% produced constriction (11 ± 2 μm) that was unaffected by dye loading; [Ca2+]iincreased by 108 ± 53 nM ( n = 6, P < 0.05). Cycling of [Ca2+]iduring vasomotion (amplitude, 150 ± 53 nM; n = 4) preceded corresponding diameter changes (7 ± 1 μm) by ∼2 s. Microiontophoresis (1 μm pipette tip; 1 μA, 1 s) of phenylephrine (PE) transiently increased [Ca2+]iby 479 ± 64 nM ( n = 8, P < 0.05) with constriction (26 ± 3 μm). Flushing blood from the lumen with saline increased fluorescence at 510 nm by ∼45% during excitation at both 340 and 380 nm with no difference in resting [Ca2+]i, diameter or respective responses to PE ( n = 7). Acetylcholine microiontophoresis (1 μA, 1 s) transiently reduced resting SMC [Ca2+]iby 131 ± 21 nM ( n = 6, P < 0.05) with vasodilation (17 ± 1 μm). Superfusion of sodium nitroprusside (10 μM) transiently reduced SMC [Ca2+]iby 124 ± 18 nM ( n = 6, P < 0.05), whereas dilation (23 ± 5 μm) was sustained. Resolution of arteriolar SMC [Ca2+]iin vivo discriminates key signaling events that govern the local control of tissue blood flow.

Background and Objectives Optical clearing agents (OCAs) have previously been shown to increase depth penetration within turbid tissue ex vivo. This paper quantifies tissue optical properties of the hamster cheek pouch model in order to provide a means to assess the effect of OCAs quantitatively in vivo. Study Design/Materials and Methods Diffuse reflectance spectra were obtained from both cheeks of 12 hamsters before and after immersion in dimethyl sulfoxide (DMSO), glycerol or a phosphate buffer saline (PBS) control for 20 minutes. A Monte Carlo model was then utilized to derive the wavelength dependent reduced scattering and absorption coefficients. Results DMSO caused a statistically significant decrease in the absorption and reduced scattering coefficients derived by the model. Glycerol caused a statistically significant increase in the wavelength dependent absorption coefficient, but no statistically significant changes in the reduced scattering coefficient. Conclusions DMSO and glycerol act upon tissues differently as reflected by the tissue optical properties, implying that not all OCAs are equally effective in optically clearing tissues. Lasers Surg. Med. 38:920–927, 2006. © 2006 Wiley-Liss, Inc.

Objectives: Systemic retinoic acid (RA) treatment for chemoprevention of squamous cell carcinoma of the head and neck (HNSCC) is limited by RA's toxic side effects at therapeutic doses. The pulsed-dye laser (PDL), through a mechanism of selective vascular targeting, may allow reduction of the RA dose to one that is better tolerated when these treatments are used in combination. This study tests our hypothesis that combination therapy of PDL irradiation and low-dose systemic RA is as effective as high-dose RA therapy alone in the chemoprevention of HNSCC. Study Design: Randomized, prospective study in a hamster model. Methods: Dysplastic lesions were induced in the cheek pouches of 48 hamsters by painting with topical 9,10-dimethl-1,2-benzanthrancene (DMBA). The hamsters were randomly divided into four treatment groups: 1) control (no treatment); 2) PDL irradiation only; 3) 5.0 mg RA (all-trans retinoid, 5.0 mg/kg per day, intraperitoneally [IP]); and (4) PDL + 0.5 mg RA (0.5 mg/kg per day, IP). The PDL irradiation was conducted at day 0 and 15, whereas the RA treatment was continued for 27 days. Tumor burden was measured over time. Results: The lesions in all of three treatment groups grow more slowly than the untreated controls. The combination treatment of PDL and RA had the greatest inhibitory effect on tumors. Conclusion: This study suggests that combination treatment of PDL and low-dose RA is more effective than high-dose RA alone in the chemoprevention of HNSCC in a hamster cheek-pouch model, so that it should allow greatly improved tolerance of this regimen.

We previously reported biodistribution and pharmacokinetic data for GB-10 (Na210B10H10) and the combined administration of GB-10 and boronophenylalanine (BPA) as boron delivery agents for boron neutron capture therapy (BNCT) in the hamster cheek pouch oral cancer model. The aim of the present study was to assess, for the first time, the response of hamster cheek pouch tumors, precancerous tissue and normal tissue to BNCT mediated by GB-10 and BNCT mediated by GB-10 and BPA administered jointly using the thermalized epithermal beam of the RA-6 Reactor at the Bariloche Atomic Center. GB-10 exerted 75.5% tumor control (partial+complete remission) with no damage to precancerous tissue around tumor or to normal tissue. Thus, GB-10 proved to be a therapeutically efficient boron agent in this model despite the fact that it is not taken up selectively by oral tumor tissue. GB-10 exerted a selective effect on tumor blood vessels leading to significant tumor control with a sparing effect on normal tissue. BNCT mediated by the combined administration of GB-10 and BPA resulted in a reduction in the dose to normal tissue and would thus allow for significant escalation of dose to tumor without exceeding normal tissue tolerance.

A new experimental model was developed in hamsters for amoebic abscess caused by Entamoeba histolytica. E. histolytica trophozoites were cultured in a liquid axenic medium, and then injected intradermally into the cheek pouch of the Syrian golden hamster, Mesocricetus auratus. Inoculation consistently resulted in abscess formation at the site in 20 of 22 (91%) study animals. The amoebic nature of the abscesses was confirmed by light microscopy and histopathologic examination. Abscess formation was maximal at day 12 post-inoculation. Potential applications of this simple and reliable model include further elucidation of the pathogenesis of invasive amoebiasis, studies of the host response to amoebae, and in vivo evaluation of chemotherapeutic agents that show in vitro efficacy against E. histolytica.

Betel quid (BQ) chewing is strongly associated with the occurrence of oral leukoplakia, oral submucous fibrosis, and oral cancer. There are about 200-600 million BQ chewers in the world. Previous animal studies support the potential carcinogenicity of BQ in different test systems. However, little animal experiment has let hamsters or rats to chew BQ directly, similar to that in humans. In the present study, we established a hamster model of chewing BQ or areca nut (AN). A total of 81 2-week-old hamsters were randomly divided into three groups: 25 for control group, 28 for BQ-chewing group, and 28 for AN-chewing group. These animals were fed with powdered diet with/without BQ or AN for 18 months. Although the consumption of BQ or AN showed some variations, hamsters fed with powdered diet could chew and grind AN or BQ into small pieces of coarse fibers during the entire experimental period. The survival rate of AN-chewing hamsters decreased significantly after 6 months of exposure. The mean survival time was 15.6 +/- 0.9 months for control animals, 13.6 +/- 0.98 months for AN-chewing animals, and 15.7 +/- 0.55 months for BQ-chewing animals. The body weight of BQ- or AN-chewing animals also decreased after 4-13 months. Hamsters fed with AN for 18 months showed hyperkeratosis in 80% and acanthosis in 50% of cheek pouches. Animals fed with BQ for 18 months also showed hyperkeratosis in 93% and acanthosis in 14% of cheek pouches. These results indicate that AN and BQ components may induce alterations in proliferation and differentiation of oral epithelial cells. Animal model of chewing BQ or AN can be useful for future tumor initiation, promotion and chemoprevention experiments simulating the condition of BQ chewing in humans.