[Effects of mesenchymal stem cell transplantation on growth of liver cancer: experiment with rats].

OBJECTIVE: To evaluate the effects of mesenchymal stem cell (MSC) transplantation on the growth of liver cancer. METHODS: MSCs were isolated from the bone marrows of SD rats. Walker-256 cancer cells were isolated from the cancerous ascites of rat and cultured. Forty-five SD rats were randomly divided into 3 equal groups: mixed transplantation group undergoing laparotomy and transplantation of cancer cells mixed with MSCs into the liver, MSC IV transplantation group undergoing injection of MSCs into the caudal vein, and control group undergoing only MSC transplantation into the liver. MR imaging was performed s at days 3, 6, 9 and 12 after modeling to measure the maximum cross section area of the tumor. At day 12 the rats were killed after MR imaging with their livers taken out to undergo HE staining and pathological examination. Immunohistochemistry was used to detect the expression of vascular endothelial cell growth factors (VEGF), nm23 gene, a tumor metastasis inhibiting gene, and proliferating cell nuclear antigen (PCNA), a nuclear polypeptide necessary in the DNA synthesis. RESULTS: No significant evidence of tumor formation was detected by MRI at days 3 and 6 after modeling in all rats and tumor nodules were observed since day 9. The maximum cross section areas of tumor of the mixed transplantation group and MSC IV transplantation group were significantly larger than that of the control group at days 9 and 12 (F = 4.21, P < 0.05; F = 8.52, P < 0.01). Immunohistochemistry showed that VEGF expression levels of the two study groups were both significantly higher than that of the control group (F = 9.58, P < 0.01), while the nm23 gene expression levels of the 2 study groups were both significantly lower than that of the control group (F = 4.61, P < 0.05). The PCNA expression level of the mixed transplantation group was significantly higher than that of the control group (d'((1, 0.05)) = 0.34, d'((1, 0.01)) = 0.63, P < 0.05), however, there was no significant difference in the PCNA expression level between the MSCs IV transplantation group and the control group (d'((1, 0.05)) = 0.32, d'((1, 0.01)) = 0.48, P > 0.05). There was no significant difference in the tumor apoptotic index between the 2 study groups and the control group (F = 1.25, P > 0.05). CONCLUSION: MSC transplantation increases the expression of VEGF and PCNA, while decreases the expression of nm23 gene in cancer cells, thus favoring the tumor growth.

A comparison of the effects of pneumoperitoneum and laparotomy on natural killer cell mediated cytotoxicity and Walker tumor growth in Wistar rats.

BACKGROUND: Surgical stress promotes impaired immunological function, which contributes to tumor growth. Natural killer activity (NKA) has a protective role in immunity to tumors. So, the aim of this experimental study was to assess tumor growth and (NKA) after pneumoperitoneum and laparotomy. METHODS; Sixty male Wistar rats were divided into three groups (anesthesia, CO2 pneumoperitoneum and laparotomy) plus ten controls. All experimental animals were inoculated subcutaneously with 8 x 105 Walker carcinosarcoma 256 cells. Animals were sacrificed on 1st(POD1) and 8th (POD8) postoperative day. Tumors were excised and weighed. RESULTS: On POD1 all animals had diminished NKA when compared to controls; NKA after pneumoperitoneum was significantly greater than after laparotomy. On POD8 all animals, except after laparotomy, reached NKA at controls levels. Tumor weight was significantly greater after laparotomy when compared to pneumoperitoneum. CONCLUSIONS: Pneumoperitoneum causes a less depressed NKA and less tumor growth when compared to laparotomy.

Disposition of WR-1065 in the liver of tumor-bearing rats following regional vs systemic administration of amifostine.

PURPOSE: Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. It was hypothesized that amifostine may be a good candidate for regional drug delivery to the liver because of its large hepatic extraction and total body clearance. METHODS: Rat livers were implanted with Walker-256 tumors. The tumor-bearing rats received 15 min infusions of amifostine (200 mg/kg) via the portal vein or the femoral vein. WR-1065 concentrations in the blood, liver and tumor were measured at various times. RESULTS: The WR-1065 tumor portal dosing AUC15-60 was 40% of systemic dosing, and tumor concentrations following portal dosing were one-fifth of that following systemic dosing. The portal dosing WR-1065 liver AUC15-60 was 60% higher than the values for systemic dosing. The liver/tumor concentration ratios of WR-1065 following portal dosing were up to 8-fold higher than the ratio following systemic administration. Unfortunately, systemic exposure to WR-1065 was greater following portal vs systemic amifostine. CONCLUSIONS: Amifostine may provide increased liver protection and decreased tumor protection from radio- or chemotherapy when administered by the portal vein. However, portal dosing also increases systemic exposure to WR-1065, which is associated with hypotension.

Carcinossarcoma Walker 256 implantado no estômago de ratos: tratamento cirúrgico / Gastric carcinosarcoma Walker 256 in rats: surgical treatment

O câncer gástrico é um problema de saúde em muitos países, especialmente no Japão, Chile e Oriente Médio. No Brasil, é o primeiro em incidência entre os homens e o terceiro entre as mulheres. A mortalidade ainda é alta, principalmente pelo diagnóstico tardio. Os autores apresentam um modelo para o câncer gástrico experimental em ratos, que simula o padrão de invasão e disseminação da mesma neoplasia de ocorrencia natural. A técnica de implante tumoral consistiu da exposição do estômago dos ratos Wistar, da clampeadura da parede gástrica na grande curvatura e da introdução simultânea na cavidade gástrica através de Cânula oro-gástrica de suspensão de células de tumor de Walker 256. Os animais, do grupo controle, foram submetidos a implante tumoral e acompanhados até o óbito ou até 60 dias. Os grupos de teste, portadores do tumor, foram submetidos a ressecção padronizada da lesão com margem de segurança no quarto ou no oitavo dia de evolução da doença. Os autores avaliaram a sobrevida dos animais tratados cirurgicamente em diferentes momentos da evolução tumoral. Os resultados sugerem que o tratamento cirúrgico do tumor de Walker implantado no estômago do rato é comparável ao que acontece com o tumor gástico humano de ocorrência natural, demonstrando-se que a cirurgia realizada na fase precoce (4§ dia de implante) apresentou aumento estatisticamente significativo da sobrevida em relação ao grupo controle. Nos animais operados no 8§ dia, a cirurgia não proporcionou aumento significativo da sobrevida. Esse modelo permite o teste de novos métodos terapêuticos, passíveis de serem transpostos para o ser humano

The effect of ligation of portal branch on the growth of implanted tumor of the liver in rats.

Small masses of Walker 2 56 carcinosarcoma were implanted into the liver lobe of wistar rats. And at same time, a branch of the portal vein of the involved liver lobe was ligated. After 18 days, the growth of implanted tumor in ligation group was much slower than that in control group, namely, the diameter and wet weight of implanted tumor in ligation group were much smaller and lighter than those in control group. Furthermore, there were no ascites and lung metastases in ligation group. These results suggested that simple ligation of portal branch could apparently inhibit the growth and development of implanted tumor in the liver.

The influence of prosthetic materials on the growth of Walker 256 tumour in the rat.

Polypropylene mesh and expanded polytetrafluoroethylene patches implanted into abdominal wall defects did not affect the rate of growth of Walker 256 experimental tumour in the rat. The tumour invaded through the interstices of the polypropylene mesh to involve the peritoneal surface, whereas the expanded polytetrafluoroethylene patch acted as a physical barrier to the spread of the tumour to the parietal peritoneum. These findings will be of help in the choice of replacement material following wide resection of the abdominal wall for malignant disease.

[Sequential MRI and CT monitoring in cryosurgery--an experimental study in rats].

Cryosurgery is one of treatments of cancer, such as carcinomas of the face, oral cavity, prostate, breast, rectum and liver. But the method of cryosurgery seemed to be not yet completely established. The most important problem in this procedure is to define the extent of the cryolesion. But the trial with MRI and CT has not been reported. The purpose of this study is to investigate whether the image diagnosis such as Magnetic Resonance (MR) and CT is useful for the determination of the region during and after cryosurgery. The animal experiments were performed using 13 Wistar rats with inoculated Walker 256 cancer on both sides of femoral regions subcutaneously in the concentration of 1 million cells. After 7 days, cryosurgery was done by contacting for 15 sec with the absorbent cotton ball dipped in liquid nitrogen to the surface of right femoral tumor 3 to 6 times. The left side of tumor was intact. MRI was performed with a 0.1 Tesla ASAHI MR Mark-J and CT images were obtained using GE 9800. From the freezing to thawing, LF image (Low Flip Angle gradient echo image of Tr = 100 msec, Te = 18 msec, 60 degrees of flip angle) of MR and plain CT were taken every one minute. After thawing is over, SE image of Tr = 1500 msec, Te = 90 msec, IR images of Tr = 1200 msec, Td = 400 msec, and plain and contrast enhanced CT were carried out. Two and 7 days after cryosurgery, LF, SE and IR images of MR and CT with and without contrast medium were obtained with corresponding pathological examinations. The frozen cryolesion was of no signal intensity on the LF MR image and hypodensity on plain CT. Identification of cryolesion became possible during the cryosurgery. The cryolesion immediately after thawing showed higher intensity on the SE and LF images and hypodensity on enhanced CT. Therefore, the extent of cryosurgery can be diagnosed easily by these methods during and immediately after cryosurgery. In the follow-up studies after cryosurgery, the histological changes such as necrosis or tissue reaction were well represented by MRI and enhanced CT, but insufficiently by plain CT. From these experimental results, it may be concluded that MRI and CT are useful for monitoring the process of cryosurgery during and after the procedure.

[A comparative study of the efficacy of cryosurgical and operative treatments in transplanted tumors]. / Sravnitel'noe izuchenie éffektivnosti kriokhirurgicheskogo i operativnogo lecheniia perevivnykh opukholei.

Antitumor efficacy of cryosurgery and standard surgical procedures for Lewis carcinoma, melanoma B16, Guerin's carcinoma and Walker's carcinosarcoma was compared in the treatment of 846 experimental animals. The study established the superiority or, at least, similar efficacy of cryosurgery in terms of cure, survival and radicality of primary tumor treatment. Unlike standard surgery, cryosurgery was followed by distant metastases being inhibited in mice. Although there was no significant difference in the incidence of metastases to regional lymph nodes in rats, it was higher than in untreated animals in both treatment groups. Cryonecrosis of tumor tissue is a factor of distant and regional metastases development following cryosurgery.

Desmin-positive stellate cells associated with angiogenesis in a tumour and non-tumour system.

The angiogenesis induced after implantation of fragments of the Walker 256 carcinoma was compared with the angiogenesis following implantation of different amounts of Indian ink. Morphologically and chronologically the tumour system showed no difference from the Indian ink system, provided sufficient amounts of ink were implanted. Both systems were characterized by significant macrophage infiltration. The vascular development, which was clearly concentrated in a dense rim around the tumour, remained present when the tumour enlarged, suggesting an acquisition of vasculature by the tumour through vessel incorporation and not vessel ingrowth. Initially, scattered desmin-positive cells, in contact or encircled by collagen IV, were found in the developing angiogenic rim. Later many desmin-positive cells were found around vessels and could be identified by electron microscopy as pericytes. They exhibited close local contacts with endothelial cells. After incorporation of the peritumour vascular rim into the tumour the number of pericytes decreased and their shape became flattened and elongated.

Effect of PSK on the serum level of immunosuppressive substance in splenectomized rats.

Tumor-bearing rats with and without splenectomy were used for an immunological study on the effects of PSK, an immunomodulator, with respect to the survival rate and serum level of immunosuppressive (IS) substance. There was no significant difference in survival rates between the tumor-resected and tumor-resected-plus-splenectomy groups. The IS substance level in the former group was higher than that in the latter group. Normalization of IS substance level and prolongation of survival were observed in the group administered PSK. PSK can be effective in combination with surgical treatment.

Preparation of "tissue-isolated" rat tumors for perfusion: a new surgical technique that preserves continuous blood flow.

A surgical technique for preparing "tissue-isolated" rat tumors for perfusion was developed that ensures continuous blood flow to the tissue. The tumor may be perfused in situ or ex vivo. Tumor venous blood flow, tumor glucose and lactic acid metabolism, as well as host animal respiration and core temperature, were unchanged during the procedure. Following addition of 14C-D-Glucose to the tumor afferent blood, all radioactivity appeared in the tumor venous blood. None appeared in the host systemic blood, indicating complete separation of tumor and host vasculature during in situ perfusion.

A comparison of cryodestruction with excision or infarction of an implanted tumor in rat liver.

In this study a malignant tumor was implanted in rat livers and treated by infarction, excision, or cryodestruction. Survival and the pattern of metastases was studied in each group. Walker carcinomas were induced by the inoculation of a tumor cell suspension into the livers of male Sprague-Dawley rats. Ten days after inoculation a solitary tumor had formed. This was treated by (i) mobilization of the tumor-bearing lobe (controls); (ii) ischemic infarction by ligation of the vascular pedicle to the lobe; (iii) excision of the tumor-bearing lobe; or (iv) cryodestruction of the tumor and surrounding liver using a clinical liquid nitrogen probe. A double freeze/thaw cycle to - 180 degrees C at a mean cooling rate of 94 degrees C/min was performed. Autopsy was performed at death or after 110 days, when the experiment was terminated. In general, deaths within 5 weeks of treatment were from recurrent tumor growth in the liver and, after this time, from metastatic disease. A statistically significant increase in survival was noted in the cryotherapy group when compared with the other treatment groups (P less than 0.01 logrank ) and controls (P less than 0.001 logrank ). No real difference in local tumor control was noted between the groups. The improved survival in the cryotherapy group was attributed to a statistically significant reduction in deaths from metastatic disease (P less than 0.05 Chi-square). This finding may be related to an immunological response and warrants further study.

Role of operative trauma: explosive metastases of similar size following amputation of the primary leg tumor.

Trauma produced by amputation of a limb bearing the primary tumor influenced pulmonary metastases of the Carcinosarcoma of Walker 256 of the rat. This enhancement was present in 38% of the animals following the removal of a limb bearing the primary tumor; and in 29% of those in which the controlateral limb without tumor was amputated. A significant prolongation of the clotting time, an elevation of fibrinogen factor II and VII and a rapid consumption of factor VIII present soon after trauma may have been the parameter responsible for the increase of pulmonary metastases seen in this study.

Splenectomy and the thymic involution of increasing age.

Forty young female rats, aged about 3 months, were weighed on entry into the study; half were subjected to initial splenectomy. Animals were weighed at monthly intervals therafter. Ten animals from each group were killed at four months, and ten from each group at one year. Thymuses and spleens were dissected out and weighted. Because of progressive weight gain, all groups showed declining thymic index. Absolute thymus weight did not change signficiantly in splenectomized animals. Intact animals showed significantly reduced thymus weight at 4 months (P less than .001) and at 12 months (P less than .005). We conclude that the spleen may be a causative factor in the thymic involution of increasing age. The process is gradual and indolent compared to the rapid thymic weight depletion that occurs in the presence of growing tumor.

Prevention by splenectomy of thymus weight depletion in the presence of progressively growing tumor in rats: Brief communication.

Groups of inbred rats were inoculated with the Walker 256 carcinosarcoma. The weights of their spleens and thymuses were determined on days 16 and 23 after the tumor inoculation. Spleen weights increased progressively as the tumors grew (P less than 0.001 on day 16 as compared to controls, much less on day 23). Thymus weights decreased at the same intervals (P less than 0.05 on day 16 as compared to controls; P less than 0.001 on day 23). In other groups of inbred rats, splenectomy was performed 5 days prior to tumor inoculation. If splenectomized animals were not inoculated with tumor, thymus weight did not change. Previously splenectomized, tumor-bearing animals also showed no decrease in thymus weight on day 16 or 23. Thymus weight remained greater in splenectomized, tumor-bearing animals than in intact, tumor-bearing animals on day 16 (P less than 0.05) and on day 23 (P less than 0.001). Microscopic examination of thymuses on day 23 revealed some depletion of cortical lymphocytes in splenectomized, tumor-bearing animals and profound depletion of these lymphocytes in intact, tumor-bearing animals. Tumor weights and measurements did not differ between splenectomized, tumor-bearing and intact, tumor-bearing animals. Equal rates of tumor growth provided for a roughly equal tumor burden at termination of the experiment.