Genitalia , Female Experimental inoculation model of Walker 256 carcinoma into vagina and cervix uteri of female rats 1

Purpose: To establish an inoculation model of Walker 256 carcinoma on cervix uteri and vagina of rats. Methods: Fifteen female rats were used, and assigned to three groups each one with fi ve rats: group A rats with 4x106 cells of Walker 256 carcinoma without acid acetic inoculation; group B rats with 2x106 cells of Walker 256 carcinoma with acid acetic inoculation and group C: rats with 4x106 cells of Walker 256 carcinoma with acid acetic inoculation. The day before tumor cells inoculation the rats from groups B and C were anaesthetized with diethylether and 0,3 ml of acetic acid was inoculated into their vaginas. Tumor cell inoculation into the vagina and cervix was done under general anesthesia with diethylether. Then a endocervical brush was used to scrape the vaginal wall and after that 0,3 ml of the liquid containing tumor cells was inoculated on the vagina and cervix. For the tumor analysis, animals were euthanized at day 12 following tumor cell implantation by an excessive inhalation of diethylether. Tumor was resected entirely and weighed and the tumors were then sectioned and counter stained with hematoxylin and eosin for histopathologic evaluation. It was also calculated the percentage of tumor equivalent to the body weight by the formula: P= tumor weight / body weight x 100. Data were analyzed by one-way analysis of variance ANOVA. P values < 0.05 were taken to indicate statistical signifi cance. Results: Implantation and growth on GB and GC was 100% and on GA 20%. There was no statistical difference between GB and GC averages. Conclusion: According to the methods used, the Walker 256 carcinoma inoculation model into vagina and cervix have an implantation and growth rate of 100% when associated with previous acid acetic inoculation and there is no behavioral difference between using 2x106 or 4x106 cells on its inoculation.


Introduction
Cervical cancer is the third most common type of cancer in Brazilian women¹ and it is 30 to 60 times more common than vaginal cancer.Considering the facts that the vagina and the cervix are contiguous structures from the female reproductive tract, that both organs are composed by Malphigi epithelium, and both structures are subject to the same environmental stimulation, the difference between the cancer incidences can be attributed to the fact that glands are present on most external cervical areas. 2,3rimary vaginal neoplasm is rare.Martins (2002) showed that only 2.3% of female reproductive organs' cancers originate from the vagina².In the other hand, it is known that the vagina is a frequent site of endometrial, ovarian, urethral, bladder and rectal cancer's metastases (insert reference).The most common type of metastases found is epidermoid, originated from the cervix or vulva.²Aiming at reducing the deaths caused by cancer, cytotoxic and hormonal drugs, as well as biological agents, have been used on cancer treatment.However, such drugs often have pitfalls, due the fact they have high toxicity against noncancerous cells, and also because various types of cancer cells are resistant against therapy.Therefore, development and test of new anti-cancer substances is necessary. 4One of the best ways to test novel substances is to use it in experimental tumors.The induction model of Walker 256 carcinoma has been largely described in the literature.(insert references) It is a well described neoplasm, easily maintained in laboratory conditions, has fast and uniform growing, rarely regresses, and its effi ciency has been already confi rmed in therapeutical tests. 5There are numerous Walker 256 carcinoma implant site models, such as skin, 6 muscle, 5 subcutaneous tissue, 5 gastric walls, 7 lungs, 8 kidneys, 9 among others.However, to the best of our knowledge, there are no reports of gynecological Walker carcinomas.Since the incidence of gynecological cancers is high (insert reference), we believe that it is necessary to develop a vaginal tumor model in order to test novel drugs and phytotherapics against gynecological cancer in the future.

Methods
The Animal and Human Research Committee of the Pará State University approved all animal experiments.In addition, all animals used in this study received the humane care in compliance with the Brazilian law of animal vivisection, the rules of Brazilian College of Animal Experimentation.Fifteen female rats weighing 200 -250g were obtained from Evandro Chagas Institute (Belém -PA).Animals were kept under standard rodent laboratory housing at conditions with 12 hours day/night cycles and given standard rodent chow diets and tap water ad libitum.The Walker 256 carcinoma cell line was obtained from Federal University of Ceará (Fortaleza -CE).The cell line was maintained with successive transplantations by intramuscular inoculation on the paw of Wistar rats.The tumor was removed from donors and put on a Petri dish containing 4 ml of saline and 1 ml of gentamicin.The tissue was then shredded into pieces into a solution which was then fi ltered and diluted to concentrations of 4x10 6 or 2x10 6 cells.Five animals were assigned to each group and treated as following: Group A: rats inoculated with 4x10 6 cells of Walker 256 carcinoma without previous acid acetic inoculation Group B: rats inoculated with 2x10 6 cells of Walker 256 carcinoma with previous acid acetic inoculation Group C: rats inoculated with 4x10 6 cells of Walker 256 carcinoma with previous acid acetic inoculation The day before tumor cells inoculation on the assigned groups, the rats were anaesthetized with diethylether and 0,3 ml of acetic acid was inoculated into the rats' vaginas.Tumor cell inoculation into the vagina and cervix was done under general anesthesia with diethylether.Then a endocervical brush was used to scrape the vaginal wall (Figure 1) and after that 0,3 ml of the liquid containing tumor cells was inoculated on the vagina and cervix.For the tumor analysis, animals were euthanized at day 12 following tumor cell implantation by an excessive inhalation of diethylether.A laparotomy was performed in order to analyze the presence of macroscopic metastasis sites and to allow the removal of uterus, vagina, cervix and rectum for further analysis and separation of cervix and vagina.Tumor was resected entirely and weighed, representative photographs were taken and the tumors were then sectioned and counter stained with hematoxylin and eosin for histopathologic evaluation.It was also calculated the percentage of tumor weight equivalent to the body weight by the formula: P= tumor weight / body weight x 100.

Results
Walker 256 carcinoma inoculated on vagina and cervix had an implantation and growth rate of 100% on groups B and C and only 20% on group A. There were no distant metastasis sites and near structures were affected by contiguity (Table 1).The tumor affected the entire organ, obstructing the path (Figure 2).In addition there was expansive growth with urethra and rectum compression, causing urine and feces retention (Figure 3).Microscopically, the entire organs' walls were affected by the neoplasm, with loss of their normal characteristics.The cell line was undifferentiated (Figure 4).There were no statistical difference between groups B and C (Table 2).

Discussion
According to Brazil Cancer Incidence Estimative for 2006 cervical cancer was the third most common type of cancer in Brazilian women¹.However, to the best of our knowledge, there are no reports of gynecological Walker carcinomas.On this model, inoculation of acetic acid before Walker 256 carcinoma inoculation was done in order to induce local infl ammation which with vaginal wall scraping causes local lesion, therefore allowing Walker 256 carcinoma implantation and growth on vagina and cervix.Without previous acid acetic inoculation the lesion is inconsistent and uneven, with tumor growth on few rats.That agrees with Oliveira et al 7 fi ndings where the inoculation on gastric mucosa was only made possible with a previous lesion and Dornelas et al 10 fi ndings where tumor implantation happened only on the spot where the bladder was injured, demonstrating that unharmed mucosa does not allow tumor implantation.On both groups B and C the tumor implantation rate was 100%, without difference between concentrations, similar to other inoculation Walker 256 carcinoma models, like kidney, 9 bladder, 10 stomach, 7 mouth 11 and others.In addition there was no difference between tumor behaviors on different concentrations, demonstrated with the absence of statistical relevance between tumor weight percentages.Despite the fact that on our work cells concentration does not infl uence on tumor behavior it is known that different concentrations infl uence on tumor bearing rats' longevity. 5However, on this study the survival rate was not evaluated.It is described that original Walker 256 carcinoma was an adenocarcinoma.However the organs on this work had an undifferentiated kind of tumor cells, without any possibility of distinguishing its type with hystological traditional techniques.That might have happened because of successive inoculations and difference between lineages of different laboratories.That undifferentiation would be also responsible for the tumor agressivity which invaded all vagina and cervix area, extending itself by contiguity to rectum.

Conclusion
According to the methods used, the Walker 256 carcinoma inoculation model into vagina and cervix have an implantation and growth rate of 100% when associated with previous acid acetic inoculation and there is no behavioral difference between using 2x10 6 or 4x10 6 cells on its inoculation.

TABLE 1 -
Implantation and growth rate of Walker 256 carcinoma on vagina and cervix uteri Y-macroscopic growth ; N -no macroscopic growth

TABLE 2 -
Percentage of tumor weight equivalent to the body weight