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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..

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.

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

Objectives Several studies have attempted to prevent or improve oral mucositis (OM) but have not produced a qualified treatment yet. This study evaluates the effects of Carum carvi L. (caraway) hydroalcoholic extract (CHE) as one of the traditional medicinal plants in 5-fluorouracil (5-FU)-induced OM in golden hamsters. Materials and methods OM was induced in 54 male golden hamsters by 5-FU and cheek pouch scratching. Starting from day 12, 500 and 1000 mg kg−1 per day topical CHE were administered. Pouch histopathology score, malondialdehyde and reduced glutathione contents, and activity of myeloperoxidase plus microbial cultures of cheek pouch, antimicrobial properties of CHE, and essential oil constituents were evaluated. Results Lower histopathology score (0, 1, and 2) and malondialdehyde level, higher reduced glutathione level and activities of myeloperoxidase were detected in 1000 and 500 mg kg−1 per day topical CHE and control groups, respectively (P

Fil: Monti Hughes, Andrea. Comisión Nacional de Energía Atómica. Departamento de Radiobiología; Argentina Fil: Pozzi, Emiliano C. C. Comisión Nacional de Energía Atómica. Departamento de Investigación y Producción de Reactores; Argentina Fil: Thorp, S. I. Comisión Nacional de Energía Atómica. Departamento de Instrumentación y Control; Argentina Fil: Curotto, P. Comisión Nacional de Energía Atómica. Departamento de Investigación y Producción de Reactores; Argentina Fil: Medina, Vanina A. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Laboratorio de Radioisotopos; Argentina Fil: Medina, Vanina A. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Laboratorio de Biología Celular y Molecular; Argentina Fil: Fil: Medina, Vanina A. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martinel Lamas, Diego José. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Laboratorio de Radioisotopos; Argentina Fil: Martinel Lamas, Diego José. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Laboratorio de Biología Celular y Molecular; Argentina Fil: Rivera, Elena S. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Laboratorio de Radioisotopos; Argentina Fil: Garabalino, M. A. Comisión Nacional de Energía Atómica. Departamento de Radiobiología; Argentina Fil: Farías, R. O. Comisión Nacional de Energía Atómica. Departamento de Tecnología y Aplicación de Aceleradores; Argentina Fil: González, S. J. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: González, S. J. Comisión Nacional de Energía Atómica. Departamento de Tecnología y Aplicación de Aceleradores; Argentina Fil: Heber, Elisa M. Comisión Nacional de Energía Atómica. Departamento de Radiobiología; Argentina Fil: Itoiz, María E. Comisión Nacional de Energía Atómica. Departamento de Radiobiología; Argentina Fil: Itoiz, María E. Universidad de Buenos Aires. Facultad de Odontología. Departamento de Patología Oral; Argentina Fil: Aromando, Romina F. Universidad de Buenos Aires. Facultad de Odontología. Departamento de Patología Oral; Argentina Fil: Nigg, David W. Idaho National Laboratory; Estados Unidos Fil: Trivillin, Verónica A. Comisión Nacional de Energía Atómica. Departamento de Radiobiología; Argentina Fil: Trivillin, Verónica A. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schwint, Amanda E. Comisión Nacional de Energía Atómica. Departamento de Radiobiología; Argentina Fil: Schwint, Amanda E. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Abstract: OBJECTIVES: Searching for more effective and selective therapies for head and neck cancer, we demonstrated the therapeutic effect of boron neutron capture therapy (BNCT) to treat oral cancer and inhibit longterm tumor development from field-cancerized tissue in the hamster cheek pouch model. However, BNCTinduced mucositis in field-cancerized tissue was dose limiting. In a clinical scenario, oral mucositis affects patients’ treatment and quality of life. Our aim was to evaluate different radioprotectors, seeking to reduce the incidence of BNCT-induced severe mucositis in field-cancerized tissue. MATERIALS AND METHODS: Cancerized pouches treated with BNCT mediated by boronophenylalanine at 5 Gy were treated as follows: control: saline solution; Hishigh: histamine 5 mg kg 1 ; Hislow: histamine 1 mg kg 1 ; and JNJ7777120: 10 mg kg 1 . RESULTS: Hislow reduced the incidence of severe mucositis in field-cancerized tissue to 17% vs CONTROL: 55%; Hishigh: 67%; JNJ7777120: 57%. Hislow was non-toxic and did not compromise the long-term therapeutic effect of BNCT or alter gross boron concentration. Conclusion: Histamine reduces BNCT-induced mucositis in experimental oral precancer without jeopardizing therapeutic efficacy. The fact that both histamine and boronophenylalanine are approved for use in humans bridges the gap between experimental work and potential clinical application to reduce BNCT-induced radiotoxicity in patients with head and neck cancer.

Field-cancerized tissue can give rise to second primary tumours, causing therapeutic failure. Boron neutron capture therapy (BNCT) is based on biological targeting and would serve to treat undetectable foci of malignant transformation. The aim of this study was to optimize BNCT for the integral treatment for oral cancer, with particular emphasis on the inhibitory effect on tumour development originating in precancerous conditions, and radiotoxicity of different BNCT protocols in a hamster cheek pouch oral precancer model.Groups of cancerized hamsters were locally exposed to single or double (2 or 4 weeks apart) applications of BNCT at different dose levels, mediated by the boron compounds boronophenylalanine (BPA) or BPA and decahydrodecaborate (GB-10) administered jointly. Cancerized, sham-irradiated hamsters served as controls. Clinical status, tumour development from field-cancerized tissue and mucositis were followed for 8 months.A double application (4 weeks apart) of BNCT mediated by GB-10+ BPA at a total dose of 10 Gy in two 5-Gy doses rendered the best therapeutic advantage (63-100% inhibition of tumour development from field-cancerized tissue), minimizing dose-limiting mucositis.BNCT can be optimized for the integral treatment for head and neck cancer, considering the implications for field-cancerized tissue.

Please cite this paper as: Schneider M, Broillet A, Tardy I, Pochon S, Bussat P, Bettinger T, Helbert A, Costa M, Tranquart F. Use of intravital microscopy to study the microvascular behavior of microbubble-based ultrasound contrast agents. Microcirculation19: 245–259, 2012. Abstract Purpose: The study describes the use of intravital microscopy (IVM) to assess the behavior of ultrasound contrast agents (UCAs), including targeted UCAs, in the microcirculation of rodents. Materials and Methods: IVM was performed on various exteriorized organs: hamster cheek pouch, rat mesentery, liver, spinotrapezius muscle, and mouse cremaster muscle. A dorsal skin-fold chamber with MatBIII tumor cells was also implanted in rats. Nontargeted UCAs (SonoVue® and BR14) and targeted UCAs (BR55 and P-selectin targeted microbubbles) were tested. IVM was used to measure microbubble size, determine their persistence, and observe their behavior in the blood circulation. Results: Intravenous and intra-arterial injections of high doses of UCAs did not modify the local microvascular hemodynamics. No microbubble coalescence and no increased size were observed. Adhesion of some microbubbles to leukocytes was observed in various microcirculation models. Microbubbles are captured by Kupffer cells in the liver. Targeted microbubbles were shown to adhere specifically to endothelial receptors without compromising local blood flow. Conclusion: These results support the safety of both targeted and nontargeted UCAs as no microvascular flow alteration or plugging of microvessels were observed. They confirm that binding observed with targeted microbubbles are due to the binding of these microbubbles to specific endothelial receptors.

Objectives/Hypothesis: Early glottic cancer has been involuted by treatment with the 532 nm pulsed potassium-titanyl-phosphate (KTP) laser in initial clinical studies. Selective photoangiolysis of the sublesional circulation that allows for relative sparing of surrounding tissue is the presumed mechanism. No prior controlled animal-model study has analyzed the ability of selective coagulation of lesional microvasculature coagulation with the KTP laser to involute malignant lesions. This study tests the efficacy of photoangiolysis with the KTP laser in treating squamous cell carcinoma in an established animal model. Study Design: In vivo. Methods: Malignant lesions were induced unilaterally in the cheek pouches of 21 hamsters by applying 9,10-dimethyl-1,2-benzanthrancene. The contralateral cheek pouch served as a control. Weekly lesion photodocumentation and pulsed KTP laser (30 W, 15 msec pulse width, 2 pulses/sec) treatments were done. The endpoint of each treatment was a uniform white-blanching of the lesion. Hamsters were sacrificed 1 week after the last treatment and cheek pouches were analyzed histologically. Results: Carcinoma was confirmed in 19 hamsters, and lesions that were initially 2 mm (P = .0004). Every lesion (10/10) that initially measured 5 mm resolved after completion of the treatment period. Conclusions: Pulsed KTP laser photoangiolysis can effectively involute small malignant lesions, but may be less effective at involuting larger (>2 mm) lesions.

Background and Objectives: Several studies have suggested that low-level laser therapy (LLLT) can ameliorate oral mucositis; however, the mechanisms involved are not well understood. The aim of this study was to investigate the mechanisms of action of LLLT on chemotherapy-induced oral mucositis, as related to effects on collagen expression and inflammation. Materials and Methods: A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil, followed by surface irritation. Animals were randomly allocated into three groups, and treated with an InGaAIP diode laser at a wavelength of 660 nm and output power of 35 or 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. Collagen was qualitatively and quantitatively evaluated after picrosirius staining. The density of the neutrophil infiltrate was also scored. Results: Peak clinical severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW and control groups. The reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a decrease in the number of neutrophils and an increase in the proportion of mature collagen as compared to the other two groups. The total quantity of collagen was significantly higher in the control (no laser) group at the day 11 timepoint, as compared to the 35 mW laser group, consistent with a more prolonged inflammatory response in the control group. Conclusion: This study supports two mechanisms of action for LLLT in reducing mucositis severity. The increase in collagen organization in response to the 35 mW laser indicates that LLLT promotes wound healing. In addition, LLLT also appears to have an antiinflammatory effect, as evidenced by the reduction in neutrophil infiltrate. Lasers Surg. Med. 42:546–552, 2010. 2010 Wiley-Liss, Inc.

Objective: The therapeutic success of different boron neutron capture therapy (BNCT) protocols employing the hamster cheek pouch oral cancer model has been previously reported by our laboratory. The aim of this study was to explore potential mechanisms of BNCT-induced damage to tumor in terms of potential inhibition in DNA synthesis and induction of apoptosis in the tumors that underwent partial remission following application of the different BNCT protocols in this model. Materials and methods: We evaluated DNA synthesis employing incorporation of 5-bromo-2′-deoxyuridine as an end-point. Apoptosis was evaluated by immunohistochemistry employing the deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling technique and Bax and Bcl-2 labeling. These studies were performed in tumors that underwent partial remission 1–30 days post-BNCT mediated by boronophenylalanine (BPA), GB-10 (Na210B10H10) or (BPA + GB-10). Results: BNCT exerted a marked inhibitory effect on DNA synthesis in tumors for all the protocols under study. The inhibitory effect of BPA-BNCT occurred as soon as 1 day post-treatment (P

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.

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.

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.

Background: Oral chronic traumatic ulcer (CTU) is caused by constant irritation by dental edges or restorations and could behave as a pre-malignant lesion in a field initiated by carcinogens such as tobacco or alcohol. Methods: We developed an experimental model in the hamster cheek pouch, combining the chemical carcinogen 7,12-dimethyl-benzanthracene (DMBA) with CTU. Results: The successive or simultaneous action of both agents induced a significantly larger number of endophytic carcinomas than larger doses of DMBA alone. CTU alone failed to induce tumor development. Ploidy analysis revealed significantly higher malignancy indices in endophytic than in exophytic carcinomas. 5-Bromo-2-deoxyuridine labeling evidenced greater proliferation around the ulcers in chemically cancerized epithelium than around ulcers in healthy epithelium. Conclusions: The results show that CTU acts as a tumor promoter in this model. This finding is clinically relevant in that CTU may increase the risk of malignant transformation in patients with subclinical tumor initiation.

Background and Objective: Oral cancer results in 10,000 U.S. deaths annually. Improved highly sensitive diagnostics allowing early detection of oral cancer would benefit patient survival and quality of life. Objective was to investigate in vivo non-invasive optical coherence tomography (OCT) techniques for imaging and diagnosing neoplasia-related epithelial, sub-epithelial changes throughout carcinogenesis. Study Design/Materials and Methods: In the standard hamster cheek pouch model for oral carcinogenesis (n ¼ 36), in vivo OCT was used to image epithelial and sub-epithelial change. OCT- and histopathology-based diagnoses on a scale of 0 (healthy) to 6 (squamous cell carcinoma, SCC) were performed at all stages throughout carcinogenesis by two blinded investigators. Results: Epithelial, sub-epithelial structures were clearly discernible using OCT. OCT diagnosis agreed with the histopathological gold standard in 80% of readings. Conclusion: In vivo OCT demonstrates excellent potential as a diagnostic tool in the oral cavity. Lasers Surg. Med. 35:269–275, 2004. 2004 Wiley-Liss, Inc.

1. The present study was designed to evaluate the effect of micronization on the protective effect of the purified flavonoid fraction (MPFF) on increases in macromolecular permeability induced by ischaemia-reperfusion in the hamster cheek pouch microcirculation. 2. Male hamsters (Mesocricetus auratus) were treated orally, twice a day, with vehicle (lactose), MPFF and non-micronized purified flavonoid fraction (PFF) at 5, 20, 80 and 320 mg/kg per day for 10 consecutive days. On the 11th day, cheek pouches of anaesthetized animals were prepared for intravital microscopy. 3. Local ischaemia was obtained by clamping the neck of the everted pouch and the increase in microvascular permeability was quantified as leakage (leaks) of intravenously injected fluorescein isothiocyanate-labelled dextran (FITC-dextran 150; MW = 150 000). 4. Reperfusion, after 30 min ischaemia, resulted in an immediate but reversible increase in post-capillary leakage. The MPFF induced a significant dose-related reduction in the increased permeability, with 83.4% inhibition compared with control at 320 mg/kg per day (19.2 +/- 1.9 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001). Non-micronized PFF was significantly less effective: only 47.9% inhibition compared with control was observed at 320 mg/kg per day (60.3 +/- 1.0 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001) and there was no dose-effect relationship. 5. In conclusion, micronization significantly enhances the protective effects of the purified flavonoid fraction on reperfusion injury in the hamster cheek pouch. This improvement is likely to be related to the better absorption of the micronized formulation, which could explain the superior clinical efficacy shown in previous studies.

Conduction of vasodilation triggered by acetylcholine (ACh) in arteriolar networks reflects hyperpolarization and its spread from cell to cell along the vessel wall. The amplitude and distance of the vasomotor response appear greater than can be explained by simple passive decay of the electrical signal. The authors tested the hypothesis that the conduction of hyperpolarization involves active membrane processes as the signal travels along the arteriolar wall.Intracellular recordings of membrane potential were made from either the smooth muscle or endothelial cell layer of arterioles of the hamster cheek pouch in vivo. Acetylcholine was delivered onto an arteriole using microiontophoresis at defined distances from the recording site, and transient hyperpolarizations were recorded. The area enclosed by the transients (voltage x time integral below baseline) was measured and compared to the area expected if the hyperpolarization was spreading passively.In 11 of 15 recordings from smooth muscle and 5 of 7 from endothelium, areas of the transients were larger than expected for purely passive spread of the electrical signal.Conduction of hyperpolarization is enhanced by active membrane processes as the signal travels along the arteriolar wall. Signal augmentation will promote blood flow to tissue regions from which hyperpolarization of arterioles originates.

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.

DepartmentofOralMedicineandDiagnosticScience,CollegeofDentistry,UniversityofIllinoisatChicago,Chicago,IL,USAOBJECTIVES: To compare the morphological changesand quantitative distribution of mitochondria in thehamster cheek pouch (HCP) epithelium treated withsmokeless tobacco (ST).MATERIALS AND METHODS: Archives of experimentalmaterial from previously published studies (Ashrafi et al.,1992) were utilized. Animals in experimental groupreceived moist ST (snuff) in their right pouch, 5 daysweekly for 24 months, while no snuff was given to controlgroup. After 24 months, the epithelial tissues wereprocessed for electron microscopy study. Volume densi-ties of mitochondria were assessed by morphometry.MAIN OUTCOME MEASURES: Mitochondrial volumedensities in the two groups, experimental vs control.RESULTS: In both control and experimental groupsmitochondria were concentrated between the nucleusand basal cell plasma membrane. A decrease in the meanmitochondrial volume density (Vvmit) was observedfrom the basal layer to the more superficial layers in bothgroups. The experimental HCP displayed more mito-chondria than control, and the granular epithelial celllayer in experimental group showed significantly a highermean Vvmit than the control group (P ¼ 0.03). It wasconcluded that greater numbers of mitochondria wereretained in ST-treated granular cells of the hyperplasticepithelia than in the normal epithelium.Oral Diseases (2003) 9, 138–143Keywords: hamster cheek pouch epithelium; smokeless tobacco;mitochondria; mitochondrial volume density; granular cells

Background and Objectives: The development of 5aminolevulinic acid (ALA)-induced tissue fluorescence is optimal 2–4 hours after ALA application. Goal of this work was to develop a means of accelerating oral topical ALAinduced tissue fluorescence. Study Design/Materials and Methods: In 300 hamsters, DMBA (9,10 dimethyl-1,2-benzanthracene) cheek pouch carcinogenesis produced dysplasia in 3–5 weeks. Topical application of 20% ALA in Eucerin was followed by localized ultrasound treatment (1, 3.3 MHz) in 150 animals. In 75 animals, ALA was applied in an Oral Pluronic Lecithin Organogel (OPLO—an absorption enhancer) vehicle. Seventy-five animals received only topical ALA in Eucerin. Hamsters were sacrificed and cryosections underwent fluorescence measurements, histological evaluation, 20–180 minutes after ALA application. One-way ANOVA detected independent effects of pathology on laserinduced fluorescence (LIF). Two-way ANOVA tested for independent effect of pathology and of OPLO, ultrasound, and interaction effects. Results: Ultrasound significantly (P < 0.05) accelerated tissue fluorescence development. Conclusions: Low-frequency ultrasound can accelerate ALA-induced fluorescence development. Lasers Surg. Med. 32:185–188, 2003. 2003 Wiley-Liss, Inc.

The in vitro hemolytic and in vivo mucosal irritation potential of ethylene oxide (EO) was investigated with standard procedures used to determine the biocompatibility of medical devices. Test solutions containing EO at concentrations of 25, 50, 100, 250, 500, 1,250, 2,500, 5,000, or 10,000 microg/mL were prepared in saline to simulate a worst-case aqueous extraction of standard medical devices containing 125, 250, 500, 1,250, 2,500, 6,250, 12,500, 25,000, or 50,000 microg/g of EO, respectively. Concentrations of EO up to 500 microg/mL were not hemolytic (5% hemolysis after a 4-h exposure), whereasor =1250 microg/mL of EO resulted in significant hemolysis. Hamster cheek pouches exposed to cotton pellets saturated with EO at concentrations of up to 2500 microg/mL for 4 h with a recovery period of 14 days were without effects attributable to EO. However, ator =5000 microg/mL of EO, significant histomorphological alterations of the buccal mucosa were observed and attributed to EO exposure. It was concluded that solutions of EO of up to 500 microg/mL representing an aqueous extract of a general medical device containing at least 2500 microg/g of EO residue do not result in significant hemolysis and irritation.

The potency of three sulphonylureas, glibenclamide, glimepiride and gliclazide in antagonizing the vasorelaxant action of openers of adenosine triphosphate (ATP)-regulated K+ channel (KATP) was studied in vivo and in vitro in micro- and macrovessels, respectively. In the hamster cheek pouch, the vasodilatation and the increase in vascular diameter and blood flow induced by diazoxide were markedly reduced by the addition of either glibenclamide or glimepiride (0.8 microm) while they were not affected by gliclazide up to 12 microm. Similarly, in rat and guinea-pig isolated aortic rings, glibenclamide, glimepiride and gliclazide reduced the vasodilator activity of cromakalim. However, the inhibitory effect of gliclazide was considerably less when compared with either glimepiride or glibenclamide. These results suggest that, in contrast to glibenclamide and glimepiride, therapeutically relevant concentrations of gliclazide do not block the vascular effects produced by KATP channel openers in various in vitro and in vivo animal models.

The aim of this study was to characterize the hydrodynamic interactions between rolling and free-stream leukocytes in an in vivo model of selectin-mediated rolling, and to identify those physical mechanisms that influence the dynamics of transient adhesion with the walls of postcapillary venules.Postcapillary venules of diameter 22-37 microm in the cheek pouch of anesthetized hamsters were visualized using intravital microscopy, with selectin-mediated rolling occurring in response to surgical preparation.Rolling velocity was found to be a strong function of the center-to-center separation with the nearest cell, and also was found to correlate strongly with the number of nearby cells. These effects are shown to be beyond that attributable to variations along the length of the vessel. Adherent leukocytes were observed to provide a nucleation site, precipitating further adhesion events of free-stream cells.The dynamics of the transient adhesion of leukocytes to the vessel wall in postcapillary venules is strongly dependent on the local concentration of adherent leukocytes, due to the complex hydrodynamics induced by their presence. The results are shown to agree well with theoretical considerations of the flow field induced by multiple nearby cells, suggesting a need for the future exploration of multicellular effects in the microcirculation.

Background and Objective: New techniques for noninvasive early detection and diagnosis of oral dysplasia and carcinoma are required. Our objective was to determine in the hamster cheek pouch model whether differentiation between the healthy tissue and the different stages of oral premalignancy and malignancy is possible using laserinduced fluorescence after tissue exposure to 5-Aminolevulinic acid (ALA). Study Design/Materials and Methods: DMBA carcinogenesis was applied to one cheek pouch in 18 hamsters for 0–20 weeks. Prior to sacrifice, 20% ALA was applied to the cheek tissues. Excised cheek tissues were cryosectioned and imaged using fluorescence microscopy with excitation at 405 nm, detection at 635 nm. After fluorescence measurement, H&E staining and histopathological evaluation were performed. Results: Fluorescence intensity was significantly lower in healthy tissue than in pathological tissues. Significantly higher intensities and more ‘‘fluorescence hot spots’’ occurred in severe dysplasia and carcinoma than in healthy tissue, hyperkeratosis, mild and moderate dysplasia. Conclusions: Light-induced fluorescence after ALA exposure can differentiate between the different stages of premalignancy and malignancy. Its ability to differentiate between healthy tissue and early pathology is particularly interesting Lasers Surg. Med. 32:17–24, 2003. 2003 Wiley-Liss, Inc.

To test the hypothesis that arteriolar occlusion causes different cellular changes in endothelial and smooth muscle cells.Cheek pouch arterioles (resting diameter 41 +/- 2 microm) of anesthetized hamsters were occluded briefly (60 seconds) either upstream or downstream from an observation site. Changes in membrane potential and intracellular calcium concentration ([Ca(2+)](i)) of the endothelial or smooth muscle cells were determined by using fluorescence microscopy (ratiometric analysis).The pressure in the occluded segment decreased by 17.4 +/- 2.6 cm H(2)O during upstream occlusion and increased by 16.8 +/- 6 cm H(2)O during downstream occlusion (n = 5). Upstream occlusion caused vasoconstriction of the occluded segment by 2.4 +/- 0.4 microm, whereas downstream occlusion produced brief vasodilatation by 1.1 +/- 0.2 microm. The endothelial cells hyperpolarized during upstream or downstream occlusion (ratio change: 2.26 +/- 0.24% and 2.39 +/- 0.42%, respectively; p0.01, n = 5). There were no changes in endothelial [Ca(2+)](i). The smooth muscle cells depolarized (ratio change: -2.08 +/- 0.14%, n = 5) with an increase in [Ca(2+)](i) (ratio change: 2.92 +/- 0.16%, n = 6) during downstream occlusion. However, there was no detectable change in membrane potential or [Ca(2+)](i) of smooth muscle cells during upstream occlusion. All the changes rapidly recovered when occlusion was released. Responses of an in-situ isolated segment on a side branch revealed conducted dilatory signals caused by the occlusions.Our results show that the endothelial and smooth muscle cells respond independently to arteriolar occlusion. The endothelial and smooth muscle cells do not effectively communicate in [Ca(2+)](i) or membrane potential during acute arteriolar occlusion. Hyperpolarizing signals in endothelium cause conducted dilation.

The apoptosis-inducing capacity of S-allylcysteine (SAC), a water-soluble garlic constituent, during 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch (HBP) carcinogenesis was investigated in male Syrian hamsters using DNA fragmentation and the apoptosis-associated proteins, tissue transglutaminase (tTG) and Bcl-2. Hamsters were divided into four groups of six animals each. Animals in group 1 were painted with a 0.5% solution of DMBA in liquid paraffin on the right buccal pouches three times a week for 14 weeks. Group 2 animals painted with DMBA as in group 1, in addition received 200 mg kg−1 body weight SAC orally on days alternate to DMBA application. Group 3 animals received SAC as in group 2. Group 4 animals received neither DMBA nor SAC and served as the control. The experiment was terminated at the end of 14 weeks. Administration of SAC (200 mg kg−1 body weight) to animals painted with DMBA inhibited DMBA-induced HBP carcinogenesis as revealed by the absence of neoplasms, induction of tTG and inhibition of Bcl-2 expression. The results of the present study suggest that SAC may exert its chemopreventive effect by inducing apoptosis. Copyright © 2002 John Wiley & Sons, Ltd.

Leukoplakia in the oral cavity has been used as a putative surrogate marker of head and neck cancer development. A class of chemoprevention compounds, called protease inhibitors, has been shown in vitro and in animal models to effectively suppress premalignant lesions. Bowman-Birk inhibitor (BBI) is a protease inhibitor derived from soybeans that has demonstrated chemoprevention activity in many in vitro and animal systems, including the hamster cheek pouch model. Pilot, Phase I and Phase IIa studies of Bowman-Birk Inhibitor in patients with oral leukoplakia have demonstrated no detectable side effects. In the Phase IIa trial, changes in the protease activity in oral mucosal cells after BBI Concentratec (BBIC) treatment correlated with the changes in neu protein levels. Additionally, evidence for a dose-related treatment effect of BBIC on oral leukoplakia was demonstrated. These results indicate that BBIC should be investigated for chemopreventive activity in a randomized clinical trial.

Squamous cell carcinoma of the oral cavity continues to be a major clinical problem, with about 100,000 new deaths each year worldwide. There is therefore a need to search for new tools to aid oral cancer treatment. We tested the inhibitory activity on chemical carcinogenesis of the three principal protein fractions of about 50, 14, and 8.5 kDa of the mixture UK101 derived from goat liver. These are composed principally of a glycoprotein rich in mannose residues, a protein with analogy to the heat shock protein family, and ubiquitin, respectively. The animal model employed was dimethylbenzanthracene-induced hamster cheek pouch carcinoma. Number of tumours per animal, tumour mass per animal, and proliferating cell nuclear antigen (PCNA) in non-tumour mucosa were quantified: the 14-kDa fraction was the most active; this was also confirmed by testing its corresponding recombinant material. The 50-kDa fraction was inactive, while the ubiquitin showed only low inhibitory activity. It is possible that the technique described and the results obtained could lead to an interesting clinical approach to the treatment of oral cancer.

The purpose of this study in the hamster cheek pouch was to determine whether or not vascular endothelial growth factor (VEGF) induced changes in plasma extravasation and if so, the mechanism(s) involved. The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and the number of vascular leakage sites, as shown by fluorescein isothiocyanate (FITC-dextran, 150 kD) extravasation, was counted. Drugs and VEGF were applied topically. VEGF from 0.05 to 0.5 μg ml−1 (1.2 to 12 nM) produced a dose-dependent increase in the number of microvascular leakage sites from virtually none in basal conditions to up to 250 in some pouches. The effects of VEGF (0.1 μg ml−1 or 2.4 nM) were blocked in a concentration-dependent manner by the non-specific heparin growth factor antagonist TBC-1635 (0.1, 1 and 3μM). The placenta growth factor (PlGF-1: 0.1 and 0.5 μg ml−1 or 3.4 and 17 nM) did not increase plasma extravasation, per se, but abolished the effects of VEGF (2.4 nM). The increases in microvascular leakage produced by VEGF (2.4 nM) were partially but significantly (P

Background and Objective: The hamster cheek pouch carcinogenesis model, using chronic treatments of dimethylbenz[a]anthracene (DMBA) was used as a model system to investigate changes in epithelial tissue autofluorescence throughout the dysplasia-carcinoma sequence. Study Design/Materials and Methods: Fluorescence emission spectra were measured weekly from 42 DMBAtreated animals and 20 control animals at 337, 380, and 460 nm excitation. A subset of data in which histopathology was available was used to develop diagnostic algorithms to separate neoplastic and non-neoplastic tissue. The change in fluorescence intensity over time was examined in all samples at excitation-emission wavelength pairs identified as diagnostically useful. Results: Algorithms based on autofluorescence can separate neoplastic and non-neoplastic tissue with 95% sensitivity and 93% specificity. Greatest contributions to diagnostic algorithms are obtained at 380 nm excitation, and 430, 470, and 600 nm emission. Changes in fluorescence intensity are apparent as early as 3 weeks after initial treatment with DMBA, whereas morphologic changes associated with dysplasia occur on average at 7.5‐12.5 weeks after initial treatment. Conclusions: Fluorescence spectroscopy provides a potential tool to identify biochemical changes associated with dysplasia and hyperplasia, which precede morphologic changes observed in histologically stained

Background Radiation-induced xerostomia is a frequent sequela in patients treated for cancer of the head and neck. One strategy to treat xerostomia would be to relocate portions of salivary tissue to adjacent submucosal sites that lie outside the radiation portals such as the anterior oral vestibule. It is not known whether salivary tissue transplanted as an autogenous free graft can survive, function adequately, and not produce mucoceles. Methods Salivary gland tissue from the parotid and submandibular glands of the Syrian hamster were transplanted into the submucosal layer of the cheek pouch. After 3 months of observation, looking at graft size, graft extrusion, ulceration, infection, and mucocele formation, the graft sites were harvested. The specimens then underwent pathologic analysis by hematoxylin and eosin staining, as well as immunohistochemical methods to determine positivity for cytokeratin, smooth muscle actin (SMA), and amylase. Results Histologic analysis of tissue harvested from Syrian hamsters grafted into the cheek pouch demonstrated intact, viable, organized salivary gland tissue. Eighty percent of the animals in the submandibular group and 63% of the animals in the parotid group had at least 1 graft with viable salivary tissue without undue complications. Conclusions Salivary gland tissue can be transplanted successfully as free autogenous grafts in the Syrian hamster model. Further studies are needed to determine whether the grafts will subsequently become functional and whether growth can be biologically stimulated. This approach may be a useful strategy to protect salivary gland tissue in patients undergoing radiotherapy for head and neck cancer.

Objective: Adenosine and inosine accumulate in tissue during periods of ischemia and both molecules have been shown to degranulate mast cells in the hamster cheek pouch via activation of an A3 receptor. An A2-mediated inhibitory action of adenosine on mast cell degranulation has also been reported (16), and the objective of this research was to investigate the role of adenosine A2 receptors in modulating inosine-induced mast cell degranulation and subsequent vasoconstriction of microvessels. Methods: Cheek pouches of the Golden hamster were prepared for in vivo microscopy. Adenosine, inosine, and other agents were applied either globally in the superfusion solution or to selected regions of the tissue by pipette. Results: Micropipette application of 10−4 M inosine to periarteriolar mast cells caused a vasoconstriction and an associated mast cell degranulation in 71% of the arterioles tested. The average diameter reduction was 29 ± 5%. To establish a modulatory role for the A2 receptor, low doses of adenos...

beta-Endorphin, enkephalins and morphine dilated in vivo the arteriole of the microcirculatory system in hamster cheek pouch. beta-Endorphin had the highest potency and morphine the lowest. The effect was dose dependent and blocked competitively by the opiate antagonist, naloxone. The result demonstrated an effect of the opiates on the arteriole of the cutaneous circulation. This effect probably plays an important role in thermoregulation.

BACKGROUND Various methods of detecting cancer with fluorescence have been developed. One type of fluorescence is based on the tumor-localizing properties of certain dyes. However, the phototoxicity of most known tumor-localizing dyes hinders the safe use of such diagnostic methods. The authors have developed a fluorescence imaging system to detect the distribution of a nontoxic dye, fluorescein, and they have evaluated the feasibility of the system by using it to detect oral dysplastic lesions in hamsters. METHODS Dysplasia was induced in the cheek pouches of hamsters by application of the carcinogen 9,10-dimethyl-1,2-benzantracene. Fluorescein was administered to the hamsters either intravenously or orally before the fluorescence examination. The endoscopic fluorescence system produced dye-distribution images of both treated and control pouches. Two fluorescence images in different spectral regions were processed for each dye image. Biopsy material from both pouches was examined histopathologically. RESULTS The accumulation of fluorescein was detected in 22 of 23 specimens containing dysplastic lesions. CONCLUSIONS These results demonstrate the utility of this fluorescein accumulation method in the detection of dysplasia. The accumulation of fluorescein in dysplastic lesions may point to acidification of interstitial medium in such lesions. Cancer 1999;85:1421–9. © 1999 American Cancer Society.

This study used intravital microscopy to investigate the receptors stimulated by amylin which shares around 50% sequence homology with the vasodilator calcitonin gene-related peptide (CGRP) in the hamster cheek pouch microvasculature in vivo. Receptor agonists dilated arterioles (diameters 20–40 μm). The −log of the concentrations (±s.e.mean; n=8) causing 50% increase in arteriole diameter were: human βCGRP (10.8±0.3), human αCGRP (10.8±0.4), rat αCGRP (10.4±0.3). Rat amylin and the CGRP2 receptor selective agonist [Cys(ACM2,7]-human αCGRP were 100 fold less potent (estimates were 8.5±0.4 and 8.2±0.3 respectively). The GCRP1 receptor antagonist, CGRP8–37 (300 nmol kg−1; i.v.) reversibly inhibited the increase in diameter evoked by human αCGRP (0.3 nM) from 178±22% to 59±12% (n=8; P 0.05). The amylin receptor antagonist, amylin8–37 (300 nmol kg−1; i.v.) did not significantly inhibit the increase in diameter evoked by human αCGRP (0.3 nM) which was 112±26% in the absence, and 90±29% in the presence of antagonist (n=4; P 0.05). The agonist profile for vasodilatation and the inhibition of this dilatation by CGRP8–37, although not the amylin8–37 indicates that amylin causes vasodilatation through interaction with CGRP1 receptors in the hamster cheek pouch. British Journal of Pharmacology (1999) 126, 280–284; doi:10.1038/sj.bjp.0702272

The goal of this study was to determine the role of oxygen radicals in impaired histamine-induced increases in venular macromolecular efflux from the hamster cheek pouch. We used intravital fluorescent microscopy and fluorescein isothio-cynate dextran (FITC-dextran; MW = 70 K) to examine macromolecular extravasation from post-capillary venules in nondiabetic and diabetic (2–4 weeks after injection of streptozoticin) hamsters in response to histamine. Increases in extravasation of macromolecules were quantitated by counting venular leaky sites and by calculating clearance (ml/s × 10−6) of FITC-dextran-70 K. In non-diabetic hamsters, superfusion with histamine (1.0 and 5.0 µM) increased venular leaky sites from 0 ± 0 to 17 ± 6 and 35 ± 6 per 0.11 cm2, respectively. In addition, clearance of FITC-dextran-70 K increased during superfusion with histamine. In contrast, superfusion with histamine did not increase the formation of venular leaky sites (0 ± 0) or clearance of FITC-dextran-70 K in diabetic hamsters....

1 The effects of a purified micronized flavonoid fraction (S5682) on mean internal diameter and blood flow of arterioles and venules, as well as the functional capillary density (FCD) were evaluated in the hamster cheek pouch microvasculature before and after 90 min of total ischaemia. 2 Male hamsters were treated for ten days, twice a day, with oral doses of S5682 (5, 20, 80 and 160 mg kg−1 day−1) or placebo (10% lactose solution). The cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. Local ischaemia was obtained by a cuff mounted around the neck of the everted pouch where it leaves the mouth of the hamster. 3 Measurements were performed before ischaemia, at the onset of reperfusion and 10, 20, 30, 45 and 60 min thereafter. Diameters were measured by means of an image shearing device. Red blood cell (RBC) velocity was analysed by use of the dual-slit photometric technique. Blood flow was calculated from diameters and RBC velocities. FCD, defined as the number of capillaries with flowing blood per field of observation, was also assessed. 4 During reperfusion, placebo-treated animals showed a significant vasodilatation, a decrease in blood flow and FCD and S5682-treated animals showed a clear trend, dose-dependent, towards maintaining these parameters closer to the value found before ischaemia. 5 In conclusion, our results indicate that S5682 improves the microvascular reactivity and FCD after ischaemia/reperfusion. These data suggest that S5682 could function as an antioxidant, which may explain its beneficial therapeutic effect in chronic venous insufficiency where oxidative stress is involved in the pathological mechanism.

1 The present study was designed to investigate a possible role of thromboxane A2 (TXA2) on arteriolar vasomotion (spontaneous rhythmic variations of the vessel diameter). Therefore the microcirculatory effects of the thromboxane-receptor (TP-receptor) agonist, U 46619, as well as the effects of the TP-receptor antagonists S 17733 and Bay U3405 were evaluated in the hamster cheek pouch microcirculation. For comparison some effects of angiotensin II were also investigated. 2 For microcirculatory measurements, the cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. The TV monitor display was used to obtain arteriolar internal diameter measurements by means of an image shearing device. 3 Superfusion (0.1 nM to 1 μM) or bolus application (1 pmol to 10 nmol) of U 46619 concentration- or dose-dependently decreased the arteriolar diameter and induced vasomotion in arterioles with a mean initial diameter of 24±2 μm. Both the vasoconstriction and the vasomotion induced by U 46619 were inhibited by the TP-receptor antagonists S 17733 (100 mg kg−1, i.v.) and Bay U3405 (10 mg kg−1, i.v.). 4 Bolus applications of angiotensin II (0.1 pmol to 1 nmol) induced transient vasoconstriction followed by vasodilatation in the cheek pouch arterioles. The dilatation but not the constriction, was sensitive to treatment with the NO-synthase inhibitor Nω-nitro-L-arginine (L-NOARG; 100 μM). Angiotensin II did not induce vasomotion in control conditions or in the presence of L-NOARG. 5 Bolus application of phenylephrine (10 pmol) induced vasoconstriction but no vasomotion in previously quiescent hamster cheek pouch arterioles. 6 These results indicate that activation of TP-receptors causes vasomotion in the hamster cheek pouch arterioles. These spontaneous rhythmic variations in arteriolar diameter are not observed with equipotent doses of angiotensin II and phenylephrine. Thus, the vasoconstriction by itself cannot explain the occurrence of vasomotion observed with the TP-receptor agonist. British Journal of Pharmacology (1997) 122, 859–866; doi:10.1038/sj.bjp.0701464

Summary Background Previous studies have shown that antihistamities provide little or no protection against the recruitment of leucocytes in allergic inflammation. Objective We wanted to examine if threshold doses of histamine can potentiate chemoattractant-induced leukocyte adhesion and if complete inhibition of histamine-induced microvascular effects is necessary to reduce allergic leucocyte recruitment. Methods The role of histamine in allergic leucocyte recruitment was examined by use of intravital microscopy of the hamster cheek pouch microcirculation. Results We found that topical administration of histamine caused a concentration-dependent increase in microvascular permeability in the cheek pouch; i.e. 0.3 μM histamine caused no detectable plasma leakage, while 1 μM and 10 μM histamine resulted in 29 ± 9.3 and 356 ± 47 leakage sites/cm2 cheek pouch area, respectively. The percentage of postcapillary venules with more than five adherent leucocytes (an index of early leucocyte recruitment) was 1.1 ± 0.51% in the control situation, and did not increase significantly after stimulation with histamine alone (0.3–10μM) or with 1 nM ieukotriene B4 (LTB4). On the other hand, coapplication of 10μM histamine and 1 nM LTB4 increased leucocyte adhesion 24-fold. In fact, the 10 times lower dose of histamine (1 μM) together with 1 nM LTB4 increased leucocyte adhesion to a similar extent (20 fold). The increase in vascular permeabihty evoked by exogenous 10μM histamine (with or without LTB4), or by histamine released from activated mast cells (antigen challenge), was completely reversed by local pretreatment with the H1-receptor antagonist mepyramine. This mepyramine treatment also abohshed the enhanced leucocyte adhesion in response to coapplication of histamine and LTB4. Moreover, mepyramine, which had no effect on leucocyte recruitment evoked by 3 nM LTB4per se, reduced antigen-induced recruitment of leucocytes to the extravascular tissue by 79.5 ± 14.8%. Conclusion We conclude that threshold concentrations of histamine can strikingly potentiate chemoattractant-induced leucocyte responses, and that in order to reduce allergic leucocyte recruitment it may be necessary to use antihistamines in doses high enough to abolish the microvascular actions of histamine.

Following the signal observation that contact with positively charged dextran resin (PCDR) inhibited the growth of cultured mammary (Hs578T and MDA-MB-231), pancreatic (H2T), and myeloma (RR-658) tumor cell lines, studies were developed in the hamster cheek pouch model using hamster H2T pancreatic tumor cells to determine if the antiproliferative effect of PCDR could inhibit tumorigenesis. In these studies, the control population represented groups injected with H2T cells alone or in combination with either neutral or negatively charged resin. When cells (5 x 10(2) to 1 x 10(5)) and PCDR were administered simultaneously, the tumor incidence (percent engraftment) and growth of tumors that already had been established were significantly reduced. When PCDR was injected into already established 1-35-mm2 H2T tumors (engraftment for 21 days = 96%), the resin suppressed the growth of the smallest tumors (< 10 mm2). In none of these trials was the somatic growth of the host hamsters affected. PCDR contact with H2T cells in vitro for 4 days or used to treat growing solid tumors for 72 days significantly reduced cellular ornithine decarboxylase activity. While the mechanism of PCDR action has not been established, the observations have implications for in vivo tumor therapeutic models.

Background and Objective The objective of this study was to determine the relative efficacy in preventing tumor recurrence by photodynamic therapy (PDT), and by ablative CO2 laser surgery followed by PDT, compared to ablative surgery alone (negative control) or ablative surgery followed by a course of hyperthermia (positive control). Study Design/Materials and Methods The cheek pouches of 36 hamsters were treated with 0.5% 9,10 dimethyl-1,2-benzanthracene in acetone three times a week. After 12 weeks all animals showed tumors of their cheek pouches and were divided into four groups. In groups number I, II, and III, all visible tumors were removed by aid of a CO2 laser. Animals of group I did not receive any further treatment. After tumor resection, the cheek pouches in group II were treated with hyperthermia by aid of a Nd:YAG laser and a temperature of 43°C for 30 minutes. In group III after resection of the tumors, the cheek pouches were treated with PDT (75mW/cm2 175J/cm2—3mg/kg Photofrin i.p./24h). In group IV, the tumors were not excised, instead they were only treated with PDT (as above). All animals were observed for 50 days for any signs of tumor recurrence. Results In group I (CO2 all tumors (100%) recurred within 50 days. In group II (CO2) + hyperthermia) 61%, in group III (CO2 + PDT) 27.7%, and in group IV (PDT) 50% of all tumors recurred. The first signs of recurrence could be seen in group I, followed by groups II and IV. Group III was the last one presenting tumor recurrence. Conclusions The combination of CO2 surgery and PDT produced significantly better results than CO2 surgery or PDT alone, and better than the combination of CO2 surgery and hyperthermia. Lasers Surg. Med. 20:188–194, 1997. © 1997 Wiley-Liss, Inc.

In the normal hamster cheek pouch epithelium, cell proliferation takes place with a pronounced circadian rhythm. We tested our previous hypothesis that all cells having S phase DNA content are actively synthesizing DNA and thus participating in the daily cohort of proliferating cells. We found no evidence of resting S phase cells in the normal epithelium. Using labelling with tritiated thymidine followed by fluorescence activated cell sorting according to DNA content and by autoradiography of the sorted nuclei, it was demonstrated that during the 24 h period almost all cells with mid S phase DNA content were active in DNA synthesis.

Hamster cheek pouch mucosa is an accepted model of oral carcinogenesis. We herein examined the value of morphometric evaluation of silver-stained nucleolar organizer regions (AgNOR) in the detection of epithelial foci in malignant transformation following dimethyl-1, 2-benzanthracene-induced carcinogenesis of hamster cheek pouch. AgNOR-related parameters were analyzed at different stages of the process of carcinogenesis (control epithelium, epithelium with no unusual microscopic features, “dysplastic” epithelium, exophytic and endophytic carcinomas). Morphometric evaluation of AgNOR revealed incipient cellular alterations which were not evident in routine preparations and contributed to the characterization of different stages of carcinogenesis in this model.

The aim of the present study was to describe changes which could be regarded as a result of neoplastic rather than inflammatory processes. Fifty-five weeks after 6 weeks of DMBA application to the cheek pouches of 5 male hamsters there were 4 types of lesion : larger ulcerated sessile ; smaller non-ulcerated sessile ; non-ulcerated pedunculated ; conical projections. These and the rest of the pouches were examined by scanning electron and light microscopy. The interlesional mucosa, non-ulcerated sessile lesions and conical projections were covered by flat polygonal cells with either a honeycomb surface pattern of interconnecting microridges, or microridges arranged into more parallel lines. Cell imprints and boundaries were formed by linear ridges, grooves or both. Pedunculated lesions had flat smooth-surfaced cells and cells with a honeycomb surface pattern. None of these lesions were carcinomas by light microscopy but the ulcerated sessile lesions were. The appearance of the cells on the ulcerated lesions varied : flat, with a variable number of short microvilli that were often knob-like, isolated short microridges, or both ; plump, giving a cobblestone appearance with surfaces that were smooth, covered by microvilli, short microridges or both. Such appearances have been described as characteristic for dysplastic and malignant stratified squamous epithelium in a number of sites. Further study of both experimental and naturally occurring mucosal disease is needed to validate this.