Studies on the vascular drainage system of the hamster buccal pouch

Because some investigators feel that the hamster buccal pouch is an immune privileged site with no lymphatic drainage, this study was done to demonstrate and further elucidate a drainage system from the pouch to the cervical nodes. Eight hamsters were injected with india ink, submucosally in the right pouch. Dissections at one or 2 days revealed ink in macrophages in the right submandibular nodes at 2 days. Histologically, the ink was noted within macrophages which aggregated around blood vessels. No lymph vessels were noted in the pouch. Six hamsters were injected with fluorochrome-labelled latex microspheres. At 2 and at 4 days the microspheres were noted in both the pouches and the ipsilateral submandibular nodes. The results indicate there is a delayed lymphatic drainage from cheek pouches to the submandibular nodes mediated by macrophages.     

人舌淋巴管分布及引流特征的研究

目的　了解舌淋巴管的分布及引流特征,以期为舌部肿瘤的治疗提供可靠的解剖学依据。方法　应用淋巴管间接注射技术结合淋巴管铺片透明显示法、淋巴管铸型及扫描电镜观察来研究人舌淋巴管的分布特点,同时通过颈部解剖学方法观察舌不同区域初级引流淋巴结的分布。结果　舌背黏膜毛细淋巴管和淋巴管构成双层网络结构,其分布不受界沟和中线的影响,分布于整个舌背黏膜。肌间淋巴管与舌背和舌腹黏膜淋巴管相交通,由此构成完整的舌淋巴管网络结构。各注射区域颈部初级引流淋巴结的分布呈现一定的特点,舌前部主要引流至颏下淋巴结、颌下淋巴结和颈肩胛舌骨肌淋巴结;舌侧部和舌中部主要引流至颌下淋巴结、颈二腹肌淋巴结和甲状淋巴结;舌根部主要引流至颈二腹肌淋巴结。各注射区初级引流淋巴结均可出现于双侧颈部,但同侧发生频率高于对侧。结论　舌淋巴管呈网络状分布,舌部任一区域的淋巴引流有广泛性和双侧性的特点,同时又具有一定的趋向性。     

Hamster cheek pouch carcinoma: effect of incision and cortisone on growth, invasion, and metastasis

After cheek pouch carcinomas were induced in hamsters by the application of dimethylbenzanthracene (DMBA) to the right pouch for 13 weeks, the animals were divided into four groups and observed for seven more weeks. The control group received no further treatment, two experimental groups had incisional biopsies performed on tumors in their pouches, one of these also received injections of cortisone throughout the 20-week experimental period, and a fourth group received cortisone only. The wet weights of the cancerous cheek pouches were determined, and the submandibular and parotid salivary glands with associated cervical lymph nodes, the lungs, and the liver were examined with light microscopy. The cancerous pouches of the animals that received cortisone weighed significantly less than those of animals that received no cortisone but had incisional biopsies of the tumors. There was no significant difference in the degree of histodifferentiation of the tumors among the four groups. The animals in the two groups that received cortisone had significantly more tumors that were invasive than did the animals that did not receive cortisone. Cervical lymph node metastasis occurred in 21% to 38% of the animals but was not significantly different in the four groups. Distant metastases to the lungs or the liver were not found. Incisional biopsy of the tumors stimulated local growth of the cheek pouch tumors, and systemic cortisone administration produced more invasive cheek pouch tumors.     

Histologic effects of smokeless tobacco and alcohol on the pouch mucosa and organs of the Syrian hamster

This study's intent was to measure the histologic effects of smokeless tobacco and alcohol on the buccal pouch mucosa and internal organs of male Syrian hamsters. Eighty hamsters were divided into four groups: tobacco only, alcohol only, tobacco and alcohol, and negative control. 200 mg of smokeless tobacco were placed in each pouch of the tobacco groups five times a week. In the alcohol groups, 2 ml of 15% ethyl alcohol were placed in each pouch five times a week. The negative control group had mechanical stimulation of the right pouch to simulate the placement of the tobacco. After 26 wk the animals were sacrificed with pouches and abdominal organs removed. Alterations were observed in the abdominal organs, but not of statistical significance. However, significant acanthosis of the pouch epithelium was noted in the tobacco and tobacco and alcohol groups. This study reaffirms the lack of carcinogenic potential of smokeless tobacco upon the hamster pouch mucosa and internal organs.     

Regional lymph node metastasis created by partial excision of carcinomas induced in hamster cheek pouch with 9,10-dimethyl-1,2-benzanthracene

The feasibility of using the hamster cheek pouch/dimethyl-benzanthracene (DMBA) system as an experimental model of lymphatic metastasis was investigated. Forty male Syrian golden hamsters treated with DMBA were divided into two equal groups--one with surgical excision of their tumors and a control group without tumor excision. In the excision group, the animals received three applications/week to the left cheek pouch of 0.3% DMBA in acetone for 14 weeks. Following a three-week observation period, the tumors in the pouch were excised at their base, and the animals were killed after four weeks of further observation. In the control group, the animals were treated for 14 weeks in a manner similar to that used for the excision group, left for seven weeks without treatment, and then killed. Cheek pouches with tumors and cervical lymph nodes were processed for histological examination. All of the animals, both with and without metastasis, had borne squamous cell carcinomas (SCC) in their treated cheek pouches. Histologically, seven out of 16 animals in the excision group showed metastatic deposits of SCC confined to the left cervical lymph nodes, while in the control group, metastasis was not found in any of the 19 animals with SCC in their cheek pouches. The results demonstrate that surgical excision of the hamster cheek pouch carcinoma is efficient in producing unequivocal lymph node metastasis.     

A model for the study of lymph node metastasis from oral carcinoma by serial transplantation of metastatic tumor in hamsters

A new model of lymph node metastasis was successfully established by serial transplantation of a metastatic tumor to the submandibular lymph node from a chemically induced squamous cell carcinoma of the tongue into the buccal pouch in hamsters. Tongue carcinomas were induced by application of a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. The tongue tumors and metastatic tumors to the submandibular lymph nodes were transplanted to the buccal pouch of recipient hamsters. In the first generation, lymph node metastases were found only in hamsters that received a metastatic tumor. The metastatic tumors to the lymph nodes were maintained by serial transplantation into the buccal pouch from metastatic sites until the eleventh generation. The incidence of lymph node metastasis exceeded 90% in animals after the ninth generation.     

Establishment of an improved implantation technique for hamster mucous membrane irritation testing

Biological evaluation procedures for the acceptance of new dental materials include testing for mucous membrane irritation and inflammation, with the hamster cheek pouch used as a model system. Previous reports have indicated both a low 14-day implant retention rate and poor differentiation between positive and negative controls, requiring the establishment of a new standard procedure for hamster cheek pouch implantations. Groups of 10 female golden Syrian hamsters were implanted with uniform discs (6 mm in diameter and 1 mm thick) of white base-plate gutta percha (GP), gray welding-rod polyvinyl chloride (PVC), low-density polyethylene (LDPE), or test alloys, under Nembutal anesthesia. Each animal's right cheek pouch was everted and cleaned, and the implant was loosely placed at the bottom of the pouch. The pouch was closed by a double-suture technique. Contralateral (left) cheek pouches served as controls for the physical state of each animal. A collar (cable tie) was placed tightly around the animal's neck at a level between the sutures and the implant. Cheek pouches were assessed after 14 days. Implants were kept in contact with oral mucosa longer using the double-suture-plus-collar technique than in hamsters without collars (14-day retention rates of 48.0% and 6.7%, respectively). Average severity indices (ASI) of 5.4 and 1.7 were obtained for PVC and GP, respectively, as compared with 1.6 for contralateral control pouches. LDPE was not a suitable negative control material (ASI = 2.6). Positive and negative controls were clearly delineated when PVC and GP were the control materials.     

Prevention of experimental cancer and immunostimulation by vitamin E (immunosurveillance)

Eighty young adult male Syrian hamsters were divided into four equal groups. Group 1 animals had the right buccal pouches painted with a 0.1 % solution of 7,12 dimethylbenz(a)anthracene (DMBA) three times per wk for 28 wk. Group 2 animals were similarly painted with DMBA for 28 wk but were also given 140 μg vitamin E in 0.4 ml mineral oil three times weekly on days alternate to DMBA painting. Group 3 animals were used as DMBA-vehicle controls. Group 4 animals were vitamin E controls. Animals were killed after 28 wk, the pouches photographed and tumors counted, measured. The pouches were fixed in formalin, sectioned in paraffin and studied histologically and histochemically for tumor necrosis factors alpha and beta. All animals in Group 1 and 3 had gross tumors of the right buccal pouch. None of the animals in Group 2 had grossly visible tumors. Microscopic studies revealed that, while no gross tumors were seen in the Group 2 animals, there was histologic evidence of dysplasia and early carcinoma-in-situ undergoing degeneration. Immunohistochemical staining revealed a dense infiltrate of mononuclear cells adjacent to tumor sites with a large number of cytotoxic T lymphocytes and macrophages. Vitamin E appears to prevent tumor formation by stimulating a potent immune response to selectively destroy tumor cells as they begin to develop into recognizable microscopic foci of carcinoma.     

Improvement of macromolecular clearance via lymph flow in hamster gingiva by topical warming and massage

The lymphatic system is very important for macromolecular clearance in various tissues, especially in the gingiva. However, the kinetics of macromolecular clearance via the lymph flow in the gingiva are poorly understood. The aim of this study was to investigate whether thermal or mechanical stimulation affects macromolecular clearance via the lymph flow in the gingiva. Carbon black suspension was injected into the mandibular gingiva of anesthetized hamsters and its drainage into cervical lymph nodes was examined. Clearance of 14C-methylated bovine albumin and tritiated water from the gingiva and their drainage into submandibular lymph nodes and blood was quantified. The effect of topical warming or massage on clearance of 14C-methylated albumin from the gingiva during a 15 min period was examined. In addition, the influence of neurochemical antagonists on the stimulatory effect of topical warming on albumin clearance was investigated. Submandibular lymph nodes were clearly delineated by carbon black 10 min after the injection. More radiolabeled albumin appeared in submandibular lymph nodes than in serum, while more tritiated water appeared in serum. Topical warming (45 degrees C, 2 min) and warming plus massage (with a silicon rubber brush, 20 s) decreased the radiolabeled albumin in the gingiva 15 min after the injection. There was less radiolabeled albumin in the gingiva after gingival warming plus massage than after warming. Previous injection of HOE140 or propranolol into the gingiva diminished the stimulatory effect of topical warming on albumin clearance. It was concluded that topical warming plus massage improves macromolecular clearance via the lymph flow in hamster gingiva.     

Evaluation of the carcinogenic potential of toluidine blue O in the hamster cheek pouch.

Toluidine blue O has been shown to have clastogenic and mutagenic effects when tested in vitro, suggesting that it may be a carcinogen. Because this might compromise its use for cancer screening, the carcinogenic potential of this dye was investigated in the hamster cheek pouch, an established in vivo carcinogenesis model. Male hamsters were divided into seven groups at age 5 weeks. The right pouches of four groups were painted three times weekly with 2% toluidine blue or the vehicle for toluidine blue, in conjunction with submaximal applications of 9,10-dimethyl-1,2-benzanthracene (DMBA) (groups II and III, 0.5% twice weekly), and groups IV and V, 0.1% three times weekly). The right pouches of two groups received DMBA only (group I, 0.5% three times weekly, the standard maximal amount, and group VI, 0.1% three times weekly). Group VII received toluidine blue (right pouches) and toluidine blue vehicle (left pouches) three times weekly. The extent of carcinomas and other abnormalities (scored histologically) did not differ among groups receiving the same amount of DMBA with and without toluidine blue or vehicle, and no abnormalities were seen in the pouches from group VII. These results demonstrate no effect of toluidine blue as a carcinogen, cocarcinogen, or promoter.     

Lymph and interstitial fluid dynamics in labial gingival tissues of sheep

Lymphatic drainage and circulation in periodontal tissues have been cited as important components of host defence and pathogenic mechanisms, but quantitative data are sparse because of the technical difficulties associated with small animal lymphatic studies. However, the lymphatic vessels draining the periodontal tissues and surrounding region are sufficiently large in sheep to permit surgical placement of lymphatic catheters. Consequently, lymph and recirculating lymphocytes can be continuously collected and this permits the quantitative assessment of local immune responses in these tissues. We have studied the lymphatic drainage pathways from the labial gingival tissues in sheep by two methods. First, in a series of anatomical studies (n=6), a complex of Evan's blue dye and albumin was injected into the labial gingival tissues. One hour after injection the animals were sacrificed and the submandibular and cervical regions were dissected to expose the stained lymphatics. This anatomical study demonstrated 2 major drainage pathways: 1) cervical lymph ducts and; 2) efferent prescapular lymphatics. Secondly, to compare the relative importance of these two drainage pathways, radiolabeled protein (¹²⁵I-albumin) was injected directly into the gingival tissues and its appearance in the cervical and prescapular lymph was measured (n=7). Despite the technical difficulties encountered in the experiments, data collected showed that over 7.5 h, 64.7% of the injected protein was recovered in the prescapular and cervical lymph vessels (31.8±6.5% and 32.9±8.5%, respectively). In addition, 11.9±2.1% of the injected protein was transported to the blood by routes not involving the cannulated cervical and prescapular lymph vessels. With most of the remaining radiolabeled protein (17.9±4.9%) recovered from the injection site, we were able to account for approximately 95% of the injected protein. This study suggests that the lymph drainage from this region in the sheep model could provide one of the best described closed and contained systems and thus, could be a useful system for future continous monitoring of inflammatory responses during experimental periodontal diseases.     

Hexamethylene bisacetamide as a chemopreventive agent in hamster cheek pouch tumorigenesis

The chemopreventive effect of oral and intraperitoneal (i.p.) administration of hexamethylene bisacetamide (HMBA) on 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tumour formation in hamster cheek pouches was investigated. Male Syrian hamsters were treated by painting both cheek pouches with a 0.5% solution of DMBA twice weekly for 11 weeks. In addition to DMBA application, Group 1 hamsters were given 1% HMBA continuously in the drinking water and Group 2 hamsters received i.p. injection of HMBA at a dose of 500 mg/kg three times per week during the experiment. Group 3 animals received DMBA application alone. Thirteen weeks after the start of the experiment, the numbers of cheek pouch tumours and tumour volume were significantly decreased by oral but not i.p. administration of HMBA. Low levels of HMBA were detected in the plasma of the hamsters which were given 1% HMBA in drinking water. These results indicate that oral administration of HMBA can act as a chemopreventive agent against hamster cheek pouch tumorigenesis.     

Diurnal variation of γ-glutamyl transpeptidase activity during DMBA-induced hamster buccal pouch carcinogenesis

OBJECTIVE: The aim of the present study is to assess the diurnal variation of γ-glutamyl transpeptidase (GGT) during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis.
MATERIALS AND METHODS: The right buccal pouches of 108 Syrian golden hamsters, divided into three experimental groups, were treated three times weekly with 0.5% DMBA in mineral oil over an 11-week treatment regimen. The left buccal pouches were untreated and served as the controlS. Within each group, six animals were killed at 4-h intervals (04.00, 08.00, 12.00, 16.00, 20.00 and 24.00) for 24 h. GGT histochemical stain, according to the method of Ruthenberg and coworkers, was applied. The number of GGT-positive foci in the pouch mucosa was recorded at 3, 7 and 11 experimental weeks.
RESULT: Diurnal variation of GGT histochemical activity during DMBA-induced hamster buccal pouch carcinogenesis was substantiated in the present study.
CONCLUSION: This investigation highlights the importance of the diurnal variation in experimental oral carcinogenesis.     

The effect of levamisole on experimental carcinogenesis of rat submandibular gland.

Sixty young adult male albino rats were studied for the effect of a non-specific enhancer of cell-mediated immunity (levamisole) on the development of chemically induced tumours of submandibular gland. Levamisole was previously shown to delay chemical carcinogenesis of hamster buccal pouch and it was felt that these findings should be confirmed in another species and another experimental tumour system. Group 1 animals received a pellet of 9,10-dimethyl-1,2-benzanthracene (DMBA) implanted into the right submandibular glands and were injected with 0.6 mg levamisole once daily on three consecutive days at intervals of two weeks. Group 2 animals were implanted with DMBA but received no levamisole. Groups 3 and 4 were levamisole-treated and untreated controls. Group 1 animals (DMBA-levamisole) showed delayed carcinogenesis of submandibular gland. Group 2 (DMBA) animals developed tumours more rapidly and they demonstrated more anaplasia and greater invasiveness than those developing in the levamisole-injected animals.     

Vitamin E inhibition of hamster buccal pouch carcinogenesis. A gross, histologic, and ultrastructural study

Sixty-four young adult male and female golden hamsters (Mesocricetus auratus) were divided into four equal experimental groups of sixteen animals. In Group 1 animals the left buccal pouch was painted three times weekly with a 0.25 percent solution of 7, 12-dimethylbenz(a)anthracene (DMBA) in heavy mineral oil. In Group 2 animals the left buccal pouch was similarly painted with DMBA, but the animals also received 7 I.U. of vitamin E (alpha tocopherol) twice weekly on days alternate to the DMBA painting. The vitamin E was administered orally via a fine pipette. Group 3 animals were similarly painted with DMBA and received vitamin E vehicle by pipette. Group 4 animals served as untreated controls. Four animals in each group (two male, two female) were killed at 8, 10, 12, and 14 weeks. Buccal pouches were photographed and excised. Tumors were noted and measured in the left buccal pouches. The buccal pouches as well as major organs were fixed in formalin, sectioned in paraffin, and stained with hematoxylin and eosin. In the Group 2 animals receiving vitamin E, there was a significant delay in tumor formation so that by 12 to 14 weeks there were fewer tumors and their average size was smaller than those in the Group 1 and Group 3 animals painted with DMBA but receiving no vitamin E supplement. Microscopic examination revealed that there was less invasion of underlying tissues and less surface necrosis. The tumors in both control and vitamin E groups were well-differentiated epidermoid carcinomas. No differences in the nature of the cellular patterns of the carcinomas in control and vitamin E groups were revealed by electron microscopic studies.     

Effects of norethynodrel on neoplasms in the buccal pouch of the Syrian hamster

This study was instituted to determine the effect of a progestin, norethynodrel, on the induction of experimental carcinogens of the hamster cheek pouch. Thirty-six Syrian hamsters were divided into two groups. One group received 2 mg./kg./day of norethynodrel in subcutaneous injections, and the other group received nothing. The right cheek pouches of both groups were painted three times a week with the chemical carcinogen 9,10-dimethyl-1,2-benzanthracene in a 0.05 percent acetone solution. After 9 weeks of painting, the first tumors developed in the hormone-treated hamsters, while none appeared in the control group until after 10 weeks. After 15 weeks, 100 percent of both groups had tumors. The average latent period was 11.8 weeks for the treated group and 12.7 weeks for the control group (p = less than 0.05 percent, Student's test). Norethynodrel significantly decreased the induction time of the neoplasms.     

C/Gd纳米复合物用于兔舌间质磁共振淋巴造影的应用价值评价

目的： 探讨新型淋巴造影剂C/Gd纳米复合物经兔舌黏膜下注射后间质磁共振颈淋巴显影的应用价值。方法： 选择健康纯种成年新西兰大白兔12只,在大白兔左右两侧舌缘中后1/3交界黏膜下各注射0.2 mL C/Gd纳米复合物,按摩注射部位30 s。分别于注射造影剂前及注射、按摩后5、10、15、20、40 min进行三维增强磁共振淋巴造影成像。测量增强前、后不同时间段引流区颈淋巴结的信号强度,计算对应时间的信号强化率(E%),采用SPSS17.0软件包对数据进行统计学分析。结果： C/Gd纳米复合物迅速进入引流区,可清晰显影兔颈部淋巴结及淋巴管,无明显血管显影。颈部淋巴结在注射造影剂后约10 min信号强度达到峰值,注射后5～20 min可获得最佳显影效果,40 min后影像基本廓清。结论： C/Gd纳米复合物作为间质磁共振显影剂,能以较小剂量快速有效地显示颈部淋巴结及淋巴管的形态及走行特征。     

Effects of benzo(a)pyrene and nicotine on prostaglandin synthesis in buccal pouch and submandibular glands of the Syrian hamster

Adult male Syrian hamsters were treated by swabbing the apex of the buccal pouch with corn oil (control, C), 1 mM benzo(a)pyrene (BP), nicotine (NC), or BP + NC in corn oil, twice daily, 5 days a week. After a 4-week treatment, the pouches and submandibular glands were dissected and used for the determination of endogenous prostaglandin (PG) production and studies on in vitro PG synthesis. Of the three PGs analysed (PGE₂, PGF₂ and 6-keto-PGF₁), PGE₂ was predominant in the pouch and the glands. BP or NC alone had only a weak effect on PG synthesis in both tissues. However, the combination of BP and NC had a synergistic effect, causing diminished PG synthesis in both tissues. In buccal pouch, BP + NC significantly decreased the concentrations of endogenous PGE₂ and PGF₂ (PGE₂: 0.669 + 0.254 versus 1.698 + 0.460, PGF₂: 0.273 ± 0.090 versus 0.625 ± 0.272 ng/g tissue; BP + NC versus C; mean ± SD, n = 5, p < 0.05). Similarly significant results were also found for in vitro PG synthesis (PGE₂: 0.541 ± 0.249 versus 1.399 ± 0.340, PGF₂: 1.045 ± 0.428 versus 2.133 ± 0.510 ng/g tissue; BP + NC versus C; mean ± SD, n = 5, p < 0.05). In submandibular glands, BP + NC significantly diminished the concentration of endogenous PGE₂ (1.183 ± 0.175 versus 2.379 ± 0.488 ng/g tissue; BP + NC versus C; mean ± SD, n = 5, p < 0.05). The synthesis of 6-keto-PGF₁ in both tissues, and the synthesis of PGF₂ in submandibular gland, were slightly decreased with all treatments.     

Effect of DMBA on oral cancer development in hamsters with latent HSV-1 infections in trigeminal ganglia

This study examined the effect of 7, 12-dimethyl-benz(a)anthracene (DMBA), polycyclic aromatic hydrocarbon carcinogen, on pathologic changes in buccal pouch mucosa of hamsters with latent herpes simplex virus type 1 (HSV-1) infections in their trigeminal ganglia. Of the pouches receiving DMBA treatment, the average number of tumors per pouch was significantly higher (p less than 0.01) in pouches of animals with HSV-1 infections as compared to those that received DMBA only. Of the pouches receiving DMBA, the average cumulative tumor diameter (the sum of the tumor diameters in each group divided by the number of pouches in that group) was notably greater in animals with latent HSV-1 than in animals that had not been infected; however, this difference was not significant. The DMBA-treated hamsters that had latent infections also displayed a higher severity and prevalence of histopathologic changes in their pouch mucosa. This study indicates that latent HSV-1 and DMBA show synergism in the development of tumors and precancerous histopathologic changes in hamster buccal pouch mucosa. An unexpected finding was that among the HSV-1 infected animals, 95% of the ganglia from animals treated with DMBA showed latent HSV-1 virus on explanation culture, whereas only 10% of the ganglia from infected animals that received mineral oil, rather than DMBA, contained latent virus.     

舌侧缘淋巴引流与下颌骨舌侧骨膜淋巴管的关系及其临床意义

本文应用十二例新鲜成人尸体的舌－口底－下颌骨－颈部标本，采用淋巴含间接注射法，对舌侧缘淋巴引流及其与下颌骨舌侧骨膜淋巴管的关系进行了观察。初步发现：舌侧缘淋巴引流与下颌骨舌侧骨膜淋巴管没有关系，下颌骨舌则骨膜淋巴管不是舌侧缘向颌下和颈部淋巴结引流的通道因而，舌癌不会通过下颌骨舌侧骨膜淋巴管途径而转移至局部淋巴结。在临床上，对没有直接侵犯至下颌骨舌侧粘骨膜的舌癌病例，保留下颌骨或保持其连续性是可行的。