Effects Of Low-dose Ketamine On The Expression Of Bradykinin And Substance P In Spinal Cord Of Morphine Tolerance Rats With Tibia Cancer

Objective:To investigate the analgesic effect of low dose ketamine or ketamine combined with morphine and the effects on the expression level of bradykinin and Substance P in spinal cord of morphine tolerance rats with tibia cancer pain,thus proposed the posssible mechanisms of analgesic of ketamine.It can bring experimental evidence for the clinical treatment of cancer.Methods:72 healthy female SD rats weighting 220-260g were randomly divided into 3 groups: normal control group(Group A,n=10), cancer group(Group B,n=52) and sham operation group(Group C,n=10).Group B was the model of tribial cancer pain and it was reproduced according to Medhurst method and the method was improved.The animals were anesthetized with ip 10% chloral hydrate 350mg/kg, MADB-106 rats mammary cancer cells3μl(4.8×106/ml) were injected into left tibia medullar cavity of rats in Group B.The same volume of PBS solution was used for rats in group C.No treatment was administered for rats in group A.X-ray imaging of left tibia was taken after 10 and 14 days for all the experimental rats, H-E straining after 8 and 14 days and the determination of pain behavior, to test tibia cancer pain model was succesfully established.Except the rats of modeling failure,42 rats in Group B which were successfully established an tibia cancer pain model were used for producing morphine tolerance model.Morphine sc 10mg/kg were injected for 42 rats (two times per day,12-hours interval,a total of 9 days). A catheter was inserted into subarachnoid space at L3-4 for 42 rats that was formed morphine tolerance.Both hindlimbs of rats occurred feeling or movement disorders within 3 days and catheter extrusion were rejected as the object of study.2% lidocaine was injected through the catheter after 3 days,both hindlimbs Paralyzed within 30s as the sign of operation success.Thirty two rats which had a success operation were divided into 4 groups:control group(Group B1),ketamine group(Group B2),morphine group(Group B3) and ketamine comined with morphine group (Group B4).Rats in Group B1 were treated with 10μl normal saline one times per day intrathecally;Rats in Group B2 were treated with 25μg ketamine through the catheter one times per day; Rats in Group B3 were treated with morphine sc10mg/kg two times per day;Rats in Group B4 were treated with morphine 10mg/kg sc two times per day and 25μg ketamine through the catheter after the first time injected ketamine. Intrathecal drugs were diluted into 10μl by normal saline,then injected the same volume of normal saline to ensure the drugs full access to the subarachnoid space.Drugs were administerd continuously for 7 days. The day before tibia cancer pain model surgery and 10th day,14th day ,20 th day after the surgery, 3th day ,5th day,7 th after used ketamine or ketamine combined with morphine, thermal withdrawal latency was tested as pain indicator to observe its trends of pain behavior.After 39th of the experiment 1 hour of last administerd drugs,the rats were killed and the lumbar spinal cord was taken immediately. Bradyknin in spinal cord was determinated by enzyme-linked immunosorbant assay method, Subtance P (SP) was determinated by Immuno-histo-chemical method.Results:1. monitoring indicators of tibia cancer pain model1.1 HE staining of the left tibia in rats Histological changes in left tibia of rats with optical microscope:in Group B,after 8 days of tibia cancer pain model operation,some big heteromorphic multicore cells were filled with medullar cavity,bone trabecula was not destroyed.After 14 days of the operation,medullar cavity was full of more big tumor cells,most of tumor cells were active, cortical bone get thin, a large number woven bone formed.bone trabecula was widely destroyed. All the rats were killed and taken the left tibia on the 37 day of the experiment, medullar cavity was completely filled with tumor cells, tumor cells necrosis in the central of medullar cavity and tumor cells were active on the edge of medullar cavity. Bone trabecula was widely destroyed.While only the normal bone marrow cells can be seen in medullar cavity of rats in group A and C and no tumor cell growth and bone destruction1.2 radiation researchLeft tibia of rats in Group B had a obvious damage, the time is longer,the more serious damage.On the 10th day of the tibia cancer pain model operation only to see the small radioactive lesions in X-ray film; on the 20th days, bone destruction got more severe , cortical bone completely missing and tibia of two rats had fractured. While there were no bone destruction in rats of group A and group C.1.3 Comparision on thermal withdrawal latencyThere were no significant differance among 3groups before operation of tibia cancer pain model (P>0.05). The thermal withdrawal latency of rats in group B was significantly lower than group A, also lower than group C on 10th day,14th day ,17th day,20 th day after the tibia cancer sugery(P<0.05). There were no significant differance between group A and C(P>0.05).3. The intervention of low-dose ketamineThere were no significant differance among Group B1, Group B2, Group B3 and Group B4 before ketamine or ketamine combined with morphine injection(P>0.05).Within 7 days in the period of ketamine or ketamine combined with morphine injection, the thermal withdrawal latency of rats in Group B4was significantly lower than Group B1 (P<0.05), also lower than Group B2 (P<0.05),and Group B3 (P<0.05).4. Comparation on bradyknin in spinal cord in ratsThe difference of bradyknin among group A, group B1, group B2, group B3and group B4 was significant (p<0.05). There was no significant difference between group B2 and group B3 (P>0.05), namely,the expression leval of bradykmin had no statistical difference between the two groups, the expression leval of bradyknin in group B4 was lower than group B2 (p<0.05),also lower than group B3 (p<0.05).5. Comparation of SP in spinal cord in rats SP-immunoreactive products were mainly distributed in the spinal dorsal hornⅠ,Ⅱu nder light microscope, espesially in the outer zone, brown,distributed in the cytoplasm,the background was not colored or pale yellow. Controlled trials of immunostaining were almost negative. Group B1 in five groups was the most positive, granules were brown and densely distributed.SP-immunoreactive products of group B4 were obviously sparse than Group B2,and also group B3.Analysis with IPP software showed that average optical density of SP of Group B4 was significantly lower than Group B1 ( P<0.05), also lower than Group B2 and Group B3 (P<0.05).Conlusion:1. Intrathecal low-dose ketamine can enhance the analgesic effect of morphine and the reverse morphine tolerance,alleviate pain hyperalgesia symptoms of morphine tolerance in rats.The effect was stronger than morphine alone,or ketamine.It is an effective way of treatment of intractable cancer pain.2. The mechanism of ketamine enhance the analgesic effect of morphine is probably by regulating content of bradykmin and SP in pain pathways.