Anaesthetic effects in the ferret of alfaxalone alone and in combination with medetomidine or tramadol: a pilot study.

Alfaxalone is a neurosteroid with anaesthetic effects and it has been used successfully in several animal species. However, there are no data, to our knowledge, about its efficacy and safety in ferrets (Mustela putorius furo). We evaluated a variety of anaesthetic regimens in ferrets, namely, alfaxalone at 20, 10 and 5 mg/kg (n = 1, 10 and 9, respectively; intravenously); medetomidine at 20 µg/kg (n = 3; intramuscularly); medetomidine (20 µg/kg, intramuscularly) plus alfaxalone (2.5 mg/kg, intravenously; n = 7); and tramadol (5 mg/kg, intramuscularly) plus alfaxalone (5 mg/kg, intravenously; n = 2). Two animals treated with alfaxalone at 10 mg/kg and 20 mg/kg, respectively, died. At 5 mg/kg alfaxalone produced anaesthesia with a similar onset but a shorter duration of anaesthesia and analgesia than alfaxalone at 10 mg/kg. The medetomidine-alfaxalone combination produced anaesthesia and analgesia of a longer duration than alfaxalone administered alone at 5 mg/kg (P < 0.0001 and P < 0.001, respectively). Under this anaesthetic regimen, there was a progressive decrease in pulse rate during the first 30 min before the pulse rate stabilized. Respiratory parameters were maintained at acceptable levels. When tramadol was administered, all the animals exhibited a strong excitation reaction and in no case was the toe-pinch reflex clearly abolished. Thus, alfaxalone plus medetomidine provided safe and effective anaesthesia in ferrets. Alfaxalone, alone or in combination with tramadol, did not produce satisfactory results for use as an anaesthetic for this species.

Pneumonectomy in isoflurane-anesthetized rats without tracheal intubation: an experimental model.

INTRODUCTION: A few models for pneumonectomy in rats have been described, and in most of these, anesthesia includes orotracheal intubation, which increases morbidity and mortality and also adds technical complexity. Models without tracheal intubation but with injectable anesthesia are difficult to reproduce, however, and lead to a lengthy postoperative-recovery period with high morbidity and mortality rates. OBJECTIVE: The objective of this study was to describe a simple, safe, and effective experimental model for pneumonectomy in rats without tracheal intubation. MATERIALS AND METHODS: A left-sided pneumonectomy was performed on 26 Sprague-Dawley rats anesthetized by isoflurane applied via a mask without tracheal intubation. To avoid dangerous traction movements, the lung pedicle was ligated en bloc using clips. RESULTS AND DISCUSSION: No rat demonstrated cardiorespiratory depression. Of the 26 rats, 1 was dehydrated and had lost more than 10% of its body weight, resulting in death on the third day after surgery. Total mortality was therefore 3.8%. Mean (standard deviation [SD]) anesthesia duration was 9.8 (1.0) minutes, surgery time was 3.0 (0.6) minutes, and open pneumothorax time was 1.2 (0.3) minutes. Mean (SD) weight loss during the early postoperative period was 4.5% (3.5%). These results were more satisfactory than results obtained using ketamine mixtures as anesthetic agents (ketamine plus xylacine, and ketamine plus diazepam). CONCLUSION: Our model for left-sided pneumonectomy in isoflurane-anesthetized rats does not require endotracheal intubation and is effective, safe, quick, and easily reproducible.

Loss of a reporter gene for green fluorescent protein during tumor progression suggests the recruitment of host cells in rats with experimentally induced colon cancer.

The interactions between a host's normal cells and tumor cells appear to be of significant importance during the development of tumors. In the present study, we examined this issue using a cancer model in vivo in which tumor cells were tagged with a reporter gene for green fluorescent protein (GFP). We used a model of colon cancer in immunocompetent rats, which were given a subcutaneous injection of tumor cells that had been transfected with a gene for GFP. We found that the number of fluorescent cells decreased with the progression of the primary tumors and that lymph node and lung metastases were never macroscopically fluorescent. No GFP-encoding sequences were detected by PCR in many of the long-term primary tumors, in most lymph node metastases (86%) and in all lung metastases, whereas the detection of mutated k-ras, which identified such cells as tumor cells, was always positive. To explain these findings, we present a brief review of the literature and postulate that tumor growth did not occur exclusively as a result of the division of the injected cells, but also involved recruitment of host cells.

Circulating nucleic acids in plasma and serum (CNAPS) and its relation to stem cells and cancer metastasis: state of the issue.

The presence of circulating cell-free nucleic acids has been demonstrated both in disease and health. In the last decade, a burst of papers about Circulating Nucleic Acids in Plasma and Serum (CNAPS) have been found in the literature, showing the scientific interest raised by this phenomenon and their putative clinical interest, especially in the field of cancer. Today, the detection of extracellular tumor-derived DNA and/or RNA is considered by many authors as a new molecular marker for situations such as cancer diagnosis, monitoring the outcome of a disease and, even, as a treatment response indicator. Furthermore, in some studies it has been suggested a possible role of tumor CNAPS in the development of metastasis. Specifically, the hypothesis known as the "genometastasis hypothesis" proposes that stem cells might be naturally transfected with dominant oncogenes as a result of dissemination of such genes in the plasma. On the other hand, current studies concerned with the biology of metastatic cells are increasingly being focused on the striking similarities found between these cells and stem cells. In this review we intend to expound and integrate two theories about metastatization: the "genometastasis hypothesis" and the idea of stem cells as cancer stem cells.

Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene.

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT-->GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10(7) normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10(10) normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.

Functionality of circulating DNA: the hypothesis of genometastasis.

The detection of free, circulating tumor DNA in the plasma of cancer patients opens up new possibilities for the diagnosis and prognostication of cancer. Whatever might be the mechanism for the presence of such DNA, it is now clear that oncogenes can circulate in the plasma fraction of blood and we can now ask whether this phenomenon has potentially important implications in cancer patients. The results of our experiments, together with previous observations of other authors, have led us to propose the "Hypothesis of Genometastasis", which suggests that metastases might develop as a result of transfection of susceptible cells in distant target organs with dominant oncogenes that circulate in the plasma and are derived from the primary tumor.

Effects of perioperative treatment with TNP-470 on the resistance of colonic anastomoses in rats.

AIMS: The present study was designed to evaluate the effects of perioperative treatment with TNP-470 on the resistance of colonic anastomoses. TNP-470 is a drug that was developed as an inhibitor of angiogenesis. METHODS: A colonic anastomosis was constructed in Sprague-Dawley rats. From 4 days before surgery to 4 days afterwards, each animal received daily intraperitoneal treatment that differed according to the group to which it belonged: the control group, which received gum arabic, or the TNP group, which received 30 mg/kg TNP-470 in gum arabic. The size of the cecum and the diameters of the pre-anastomotic and post-anastomotic colon were measured at operation and 4 days after surgery, when all animals were sacrificed. At this time the presence of adhesions was also investigated. Each segment containing an anastomosis was removed and the bursting pressure (BP) and bursting wall tension (BWT) were determined. RESULTS: Loss of cecum caliber and decreases in pre-anastomotic diameter were significantly greater in the TNP group than in the control group (p = 0.017 and p = 0.004, respectively). Dilatation and obstruction of the colon were more frequent in the control group, but the difference between the groups was significant only with respect to dilatation (p = 0.005). Moreover, loss of body weight was greater in the TNP group than in the control group (p < 0.001). BP and BWT were significantly lower in animals that had received TNP-470 (p = 0.04 and p = 0.005, respectively). With respect to post-anastomotic diameter, general adhesions and adhesions to the anastomotic line, there were no statistically significant differences between the groups. CONCLUSIONS: Perioperative treatment with TNP-470, an inhibitor of angiogenesis, affects the healing and reduces the resistance of colonic anastomoses.

Experimental evidence does not support use of the "no-touch" isolation technique in colorectal cancer.

PURPOSE: The benefits of the "no-touch" isolation technique usually performed to prevent the circulation of tumor cells are not evident. The aim of this study was to determine whether conventional surgical procedures for treatment of colon cancer could provoke the circulation of tumor cells detected by a genetic technology. METHODS: Sixteen patients undergoing resection for colorectal cancer and two patients with irresectable tumors were studied. No patient showed liver or lung metastasis. With specific primers for carcinoembryonic antigen, we used reverse transcriptase-polymerase chain reaction to analyze tumor biopsy specimens and blood samples obtained from the antecubital vein before and after surgery and from the main drainage vein of the tumor when the tumor had been extracted. Peritoneal fluid was also collected in irrecsectable cases. RESULTS: Amplification of cDNA with carcinoembryonic antigen-specific primers was achieved with all tumor biopsies and samples of peritoneal fluid. In two patients carcinoembryonic antigen reverse transcriptase-polymerase chain reaction products were detected in antecubital vein blood before surgery and in one of them also after surgery. Only in one patient (Dukes C) were carcinoembryonic antigen reverse transcriptase-polymerase chain reaction products detected from the main drainage vein of the tumor. In serial dilution experiments we determined that the limit of detection of this method was ten tumor cells in 2 ml of blood. CONCLUSION: Our data suggest that the use of no-touch isolation techniques in colorectal cancer is not justified, based on lack of evidence indicating the detachment of cells from the tumor at surgery.

Tumor DNA circulating in the plasma might play a role in metastasis. The hypothesis of the genometastasis.

BACKGROUND: Clinical and experimental observations suggest that more than one pathway might be involved in the development of metastases. In the present study, we examined the presence of tumor DNA in plasma using an experimental model in which tumor cells were modified with a genome-associated tag. We also investigated whether plasma of tumor-bearing rats had any effect on cultured cells and healthy animals. METHODS: Transfected cancer cells (DHD/K12-PROb stably transfected with pCDNA3.1CAT.) were injected subcutaneously into the chest of BD-IX rats. Animals were divided into ten groups according to the time between injection of tumor cells and euthanasia. Prior to euthanasia (2-14 week), blood samples were collected by cardiac puncture. To detect circulating tumor cells and CAT-encoding DNA in plasma, we performed PCR with nested primers. Fifty samples of plasma were chosen at random to supplement the medium of fifty cultures of DHD cells for 10-12 days. PCR for the detection of CAT DNA in cells was performed approximately one to two months later. Four healthy rats received an intraperitoneal injection of plasma from a tumor-bearing rat five times at week for 4 to 6 weeks. Animals were sacrificed and samples of liver, kidney, spleen, omentum, blood and lung were processed by PCR for the detection of CAT DNA. RESULTS: Detection of CAT DNA in plasma was slightly more frequent than in the buffy-coat fraction. All surviving cultures that had been supplemented with plasma were positive at some point for CAT DNA. In all four healthy animals injected with plasma of tumor-bearing rats, the marker gene for CAT was found in extracts of lungs. CONCLUSION: Our present observation lead us to propose the following hypothesis. Metastases might develop as a result of transfection of susceptible cells in distant target organs with dominant oncogenes that are present in the circulating plasma and are derived from the primary tumor.

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L.

Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted significant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kappaB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10-1000 ng/ml specifically promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound.

Detection of genomically-tagged cancer cells in different tissues at different stages of tumor development: lack of correlation with the formation of metastasis.

Genetic detection of tumor cells in blood, lymphatic nodes or bone marrow using reverse transcription and polymerase chain reaction (PCR) is quite attractive because it allows the early diagnosis of cancer dissemination. Unfortunately, this type of detection strategy cannot be applied to solid parenchymas, because they usually share with tumor cells the mRNA markers. To avoid this impediment, we have developed an experimental model of cancer using cells with a genome-associated tag. DHD/K12-PROb cancer cells were stably transfected with pcDNA3.1CAT. Approximately 10(6) transfected cells (DHD-CAT cells) were injected subcutaneously into the chest of BD-IX rats. Animals were divided into 11 groups according to the time between injection of tumor cells and euthanasia. An additional 'untagged group' was injected with untransfected cells (DHD-Wild). Blood and tissues samples were collected after euthanasia. Macroscopic and microscopic analysis was done. To detect circulating tumor cells or their presence in peripheral organs, we performed PCR with nested primers to amplify chloramphenicol acetyl transferase-encoding (CAT-encoding) DNA sequences. The minimum number of cells that yielded detectable cells routinely was 2 in 10(6). No modification of cancer aggressiveness was observed in DHD-CAT cells. DHD-CAT cells were detected by PCR in lung from the 1st week after inoculation, in liver, spleen and kidney from the 3rd week and in the blood from the 5th week. All animals analyzed 12 weeks after injection showed lung metastases. Metastases in liver, spleen or kidney, either microscopic or macroscopic, were never detected. We have developed an experimental model of cancer based on genomic tagging of tumor cells that allows the detection of small numbers of cells in all organs and the blood. The presence of cancer cells in parenchymas detected with molecular technology does not correlate with the development of clinically relevant metastases.

Effects of long-term treatment of colon adenocarcinoma with crocin, a carotenoid from saffron (Crocus sativus L.): an experimental study in the rat.

We used an experimental model in the rat to examine the effects of long-term treatment with crocin, a glycosylated carotenoid from the stigmas of the saffron crocus, on colon cancer. BD-IX rats were divided into four groups: Groups G1 and G2, designated "cancer groups," were used to study the effects of crocin on the progression of colon cancer, and Groups G3 and G4, designated "toxicity groups," were used to study the effects of the treatment on metabolic processes and the parenchyma. DHD/K12-PROb cells were injected subcutaneously into the chest of Group G1 and G2 animals. From 1 to 13 weeks after inoculation, animals in Groups G2 and G4 received a weekly injection of crocin (400 mg/kg body wt s.c.). Animals in Groups G1 and G3 received no treatment. In addition, lines of animal and human colon adenocarcinoma cells (DHD/K12-PROb and HT-29) were used to perform assays in vitro to examine the cytotoxicity of crocin. Life span was extended and tumor growth was slower in crocin-treated female rats, but no significant antitumor effect was found in male rats. Acute tubular necrosis was found in all kidney samples from crocin-treated animals, but slight signs of nephrotoxicity were found by biochemical analysis of the serum. In assays in vitro, crocin had a potent cytotoxic effect on human and animal adenocarcinoma cells (HT-29 and DHD/K12-PROb cells, 50% lethal dose = 0.4 and 1.0 mM, respectively). Treated cells exhibited a remarkable loss of cytoplasm and wide cytoplasmic vacuole-like areas. In conclusion, long-term treatment with crocin enhances survival selectively in female rats with colon cancer without major toxic effects. The effects of crocin might be related to its strong cytotoxic effect on cultured tumor cells.

Establishment of an experimental model of colon cancer which replicates the regional extension pattern of human colorectal adenocarcinoma.

OBJECTIVE: The aim of this study was to develop an experimental model of colon adenocarcinoma based on the orthotopic implantation of tumor cells in syngenic rats, which reproduces the regional extension pattern of human colorectal adenocarcinoma. EXPERIMENTAL DESIGN: Cell cultures: cell line DHD/K12-PROb. ANIMALS: BD-IX rats. Tumour implantation: intra-caecal injection containing 1 x 10(6) cells in 0.25 ml of PBS. DESIGN: randomized observation study of three groups until five rats were shown to have cancer implantations in each group: GROUP I: sacrificed one week after the injection (n = 6), GROUP II: sacrificed two weeks after the injection (n = 9), GROUP III: sacrificed four weeks after the injection (n = 10). MEASUREMENTS: macroscopic and histological examination, with particular emphasis on caecal lesions. Tumours were classified according to the TNM system (UICC, 1987). RESULTS: GROUP I: tumors were found in 83% of cases (5/6), 4 of which were classified as T1N0M0 and 1 as T2N0M0. GROUP II: tumour in 55.5% of cases (5/9). One was classified as T1N0M0, 3 as T2N0M0 and 1 as T3N0MPER. GROUP III: tumors were found in 50% of cases (5/10). Two were classified as T4N0M0, 2 as T4N1MPER, and 1 as T3N1MPER. The degree of wall infiltration of the tumor showed statistical differences between groups I and III and groups II and III (p < 0.05). CONCLUSIONS: Our model offers a step by step reproduction of events described for human colorectal adenocarcinoma. It was therefore easy to predict how long it would take to achieve a degree of local extension, which is essential in the design of cancer experiments. Moreover, this model has the advantage that it uses immunocompetent rats, which facilitates the methodology.

Does neostigmine have a deleterious effect on the resistance of colonic anastomoses?

The aim of the present study was to evaluate the effects of neostigmine as a final anaesthetic manoeuvre on colonic anastomoses. A colonic anastomosis was constructed in 40 Sprague-Dawley rats. The animals were divided into two groups: (1) rats receiving intravenous saline solution (placebo); and (2) rats receiving an intravenous injection of neostigmine. The size of the caecum, and the diameters of the pre-anastomotic and post-anastomotic colon were measured during the operation and 4 days after surgery, when all the animals were sacrificed. At this time, the presence of adhesions was also investigated. Each segment containing an anastomosis was removed, and the bursting pressure and bursting wall tension were determined. Loss of caecum diameter was significantly greater in group 2 than in group 1 (P = 0.03). Dilatation and obstruction of the colon were significantly more frequent in group 1 (dilatation, P = 0.01; obstruction, P = 0.047). Also, consumption of water by group 2 was greater than that by group 1 (P = 0.049). No statistically significant differences were found between the diameters of the colon (pre- and post-anastomosis), or with respect to general adhesions and adhesions to the anastomotic line. No significant differences were found between anastomotic resistance (determined in terms of bursting pressure and bursting wall tension) in the two groups. The inclusion of neostigmine in an anaesthetic protocol under experimental setting did not reduce the resistance of colonic anastomoses and did not compromise normal healing. Moreover, obstruction caused by peristaltic weakness might be prevented by the expulsion of stool that is induced by the strong contraction of the colonic smooth muscle.

Orthotopic implantation of colon carcinoma cells provides an experimental model in the rat that replicates the regional spreading pattern of human colorectal cancer.

Our goal was to develop an experimental model of colon adenocarcinoma based on the orthotopic implantation of tumour cells in syngenic rats which would replicate the pattern of regional spreading of human colorectal adenocarcinoma. We used cell line DHD/K12-PROb and 62 BD-IX rats with intra-caecal injection of 1 x 10(6) cells. Macroscopic and histological examinations were made at different times after injection (from 1 to 16 weeks) with particular emphasis on caecal lesions and tumours were classified according to the TNM system (UICC, 1987). Our results suggest that this model provides a step-by-step reproduction of the development of human colorectal adenocarcinoma. It also allows us to predict how long it will take to achieve a certain degree of local spreading, which is essential for the design of cancer-related experiments. Moreover, our model has the advantage that it uses immunocompetent rats, which facilitates its application.

The effects of the pharmacological manipulation of postoperative intestinal motility on colonic anastomoses. An experimental study in a rat model.

INTRODUCTION: The aim of the study was to determine the effects of pharmacological manipulation of postoperative intestinal motility on the resistance of colonic anastomoses. MATERIALS AND METHODS: Seventy-one Sprague-Dawley rats were divided into three groups: Group 1 (n = 20; colonic anastomosis+1 cc of saline solution subcutaneously, daily); Group 2 (n = 29; colonic anastomosis+1.2 mg/100 g body weight metoclopramide in 1 cc subcutaneously, daily); and Group 3 (n = 22; colonic anastomosis+2 mg/100 g body weight hyoscine N-butyl-bromide in 1 cc subcutaneously, daily). Surviving rats (20 in each group) were sacrificed 4 days after surgery and adhesions were evaluated. Each segment containing an anastomosis was removed and the bursting pressure was determined. RESULTS: The cause of death during the early postoperative period was dehiscence in 8 cases (7 in Group 2 and 1 in Group 3). General adhesion scores in Group 2 were higher than in Group 3 (P = 0.003). The score for adhesions to the anastomosis in Group 1 was higher than in Group 2, but no statistically significant difference was found. Bursting-pressure was significantly lower in Group 2 than in other groups (P = 0.001). In all cases leakage of dye was observed at the anastomosis. CONCLUSION: The use of metoclopramide (a gastrointestinal prokinetic agent) during the early postoperative period was associated with an increase in dehiscence in colonic anastomosis and, when animals survived, there was a significant decrease in anastomotic resistance. Hyoscine (an inhibitor of gastrointestinal motility) did not improve the healing of anastomoses.

Role of immediate postoperative feeding on colonic anastomoses resistance. An experimental study in a rat model.

PURPOSE: This study was performed to investigate whether immediate postoperative feeding induced changes on colonic anastomoses resistance. On the other hand, results was compared with a hyoscine-induced deep paralytic ileus status. METHODS: Sixty-three Sprague-Dawley rats were divided into three groups: Group 1 (n = 20; colonic anastomosis + water "ad libitum" + 1 cc of saline solution subcutaneously, daily); Group 2 (n = 21; colonic anastomosis + water and standard rat chow "ad libitum" + 1 cc of saline solution subcutaneously, daily); and Group 3 (n = 22; colonic anastomosis + water "ad libitum" + 2 mg/100 g body weight hyoscine N-butylbromide in 1 cc subcutaneously, daily). Body weight, food intake and water consumption were recorded on a daily basis. Surviving rats (20 in each group) were sacrificed 4 days after surgery and adhesions were evaluated. Each segment containing an anastomosis was removed and the bursting pressure was determined; the diameter and Lapace's law were used to calculate Bursting wall tension (BWT). RESULTS: The cause of death during the early postoperative period was dehiscence in 2 cases (1 in Group 2 and 1 in Group 3). A rat died in Group 3 due to non-specific side effects. Weight loss was significantly lower and water consumption significantly higher in Group 2 (Food) than in others groups. BWT was lower in Group 2 than in the other groups (53.61 x 10(3) dynes/cm +/- 24.51 x 10(3) dynes/cm in Group 1, 48.94 x 10(3) dynes/cm +/- 18.53 x 10(3) dynes/cm in Group 2 and 65.09 x 10(3) dynes/cm +/- 28.59 x 10(3) dynes/cm in Group 3). Nevertheless only comparison between Group 2 (Food) and Group 3 (Hyoscine) showed statistically significant difference (p = 0.03). In all cases leakage of dye was observed at the anastomotic line. General adhesion scores in Group 2 (Food Intake) were similar than Group 1 (Water only) and higher than in Group 3 (Hyoscine) (p = 0.036).