Toxicity Effects Of Intraperitoneal Injection Of Diethylnitrosamine On Liver And Lung And Visceral Pain Behavior In Rats

Objective:To observe the changes in the toxicity of liver and lung and the changes of visceral pain,during establishing hepatocarcinoma model by intraperitoneal injection of diethylnitrosamine in different doses.Methods:Sixty clean male Wistar rats were used in the experiment and were purchased from Hebei Experimental Animal Center with a weight of 210g±10g.Normalized light?12h light and 12h darkness alternating?,constant temperature and humidity,the rats freely ingest food,ordinary tap water,regular matting,cleaning squirrel cage,to prevent infection.One week into the environment,observe and evaluate the growth of rats.The general performance of lively,responsive,smooth fur,clean near the anus,appetite,drinking water as normal health,can be used for experiments.Rats in the experimental group were randomly divided into 3 groups,the experimental group T₁,T₂ and control group C,20 rats in each group.After the drug was diluted with 0.9%sodium chloride solution on the day of administration,the rats in group T₁ were intraperitoneally injected with 25mg/kg DEN,group T₂ rats were injected with 50mg/kg DEN,and group C was intraperitoneally injected with a volume of 0.9%sodium chloride solution.The dosing regimen was injected twice a week for the first 4weeks,then once a week for 4 weeks,and then stopped at 14 weeks.At the 12th,14th and 16th weeks,2 rats were sacrificed in each group.All remaining rats were sacrificed.The rats were fixed with a self-made rat fixator,the left lower abdomen of the rat was selected as the injection site,75%of the alcohol cotton ball was sterilized and the 1ml syringe with a 27G needle was injected.The resistance of the needle into the peritoneum was disappearing and the needle was fixed.If no blood or urine was returned,the dose of the rat could be calculated by the weight of the rat.The rats were injected with liquid medicine,and the abdominal circumference of rats was observed before each administration.After injection,slowly rotate and gently pull out the needle to prevent liquid leakage.The first administration was recorded as the first week,and the experiment was performed for a total of 20 weeks.Rat body weight was measured once a week during the experiment and the general state of the rats was observed.Rats were individually placed in an open environment and the extent and time of the back arches in the rats within 5 minutes were observed:total score=accumulative arch time within 5minutes×score.The possible range of scores is from 0?300 seconds x 0=0,no bowback occurs for normal animals?to 1200?300 seconds x 4=1200,for animals with continuous bow arches throughout the 300-second test period?.Rats'visceral pain behaviors were assessed at 6 weeks to 8 weeks,12 weeks to 14 weeks,and 16 weeks to20 weeks,or until death?accidental death or death?.In this study,the same rats were tested weekly.At the 12th,14th,and 16th weeks,2 rats were killed in each group,and the remaining rats were all sacrificed by 20 weeks.Rats sacrificed and accidentally killed were dissected,and pathological changes in the lungs and liver were visually observed,then fixed with 10%formalin,paraffin-embedded sections,hematoxylin-eosin?HE?staining,and alcohol-based treatment.Dehydration,sealing,observation of pathological changes under the light microscope,determine the time of liver cancer formation.Results:1.During the experiment,the rats of the control group C were normal in growth and development,and their body weights steadily increased without death.Compared with the control group,the body weight gain of the T₁ group at the 5th week was slower?P<0.05?;at the 20th week after the first dose,there were 3 accidental deaths?3/20?.)Anatomy of 20 rats showed no tumor tissue.Compared with the control group,weight gain in the T₂ group was slower at the 3rd week,and weight began to decrease at the 10th week?P<0.05?.At the 20th week after the first dose,total weight Accidental death occurred in 5?5/20?rats.No tumor tissue was found in the lungs of 20 rats.At the 14th week after administration,it was found that the liver had typical liver cancer changes.2.The pathological analysis of the accidental dead rats showed that the size of the lungs on both surfaces of the rats of the two groups showed gray and white necrosis.The lumens of the bronchioles were filled with purulent secretions,and some of the alveolar spaces were broken and fused.Consolidation,with large abscess formation,no tumor tissue.At the 6th week after the first dose,there were 5 accidental deaths in the two groups.The liver surface of the T₂ group was coarser than that of the T₁ group in the naked eye;in the T₁ group,small blood vessels were seen under light microscopy,expansion,congestion,and partial hepatocyte edema.In addition,chronic focal inflammatory cell infiltration was observed in the lesions.In addition to the above-mentioned performances,fatty degeneration was observed in the T₂ group.A total of 3 accidental deaths occurred 7 weeks after the first dose.At the 14th and 16th weeks after the first dose,one T₂ group died unexpectedly.The liver capsule was destroyed with a naked eye,the surface was rough,and necrosis was scattered.Microscopically,the structure of the hepatic lobule was destroyed and pathological mitotic figures were seen.At the 18th week after the first administration,one rat died unexpectedly in the T₁ group.The liver was intact in the naked eye and the surface was rough,scattered in vesicle-like structures of different sizes.Typical pseudolobule structures were observed under the light microscope.3.During the whole experiment,the rats in the control group C had no bowing.The rats in group T1 began to appear arbor behavior from 6 weeks.Group T2 started from 4weeks.The two groups had obvious dorsal behavior after 16 weeks,and there was no exploratory behavior in group T2 rats at the end of the 20 week.Conclusion:Rat liver and lung are target organs of DEN,but their sensitivities to DEN are different.The liver may be more sensitive to the exposure dose and the lung may be more sensitive to exposure frequency.By observing the hunching behavior of rats,the visceral pain of rat hepatocellular carcinoma can be safely and intuitively evaluated,which provides an experimental basis for the subsequent study of the mechanism of hepatocarcinogenic visceral pain.