| IntroductionTramadol is chemically(±)- 2 -(dimethyl methyl ammonia)- 1-(intermethoxyphenyl)-cyclohexanol, and a typital analgesic which was initially approved in Germany in 1977 for pain treatment.In fact,tramadol is both an agonist of opioid receptors with selectivity for theμ-and less affinity for theδ-andκ-receptors,and inhibitor of the reuptake of noradrenaline and serotonine.Moreover,tramadol shows a lower abuse potential and respiratory inhibition in comparision with morphine.More and more adverse effects are increasingly emerged.Tramadol abuse are mainly associated with both widely clinical prescription and the possible effect of addiction and substitute of other illegal drugs.The problem of tramadol abuse was basically under control during the late 1990s.However,abuse and dependence of tramadol,which has produced untoward impact on social community,are increasingly realized.Drug dependence including psychological and physical dependence,is a kind of complex mental illnesses of the nervous system.The mental dependence is a special reward effect generated by central nervous system,characterized by continued craving for forced seeking behavior.Physical dependence refers to a series of physiological disorders after withdrawal or use of the antagonist.Research shows that drug addiction is a psychological adaptation with long-term drug intake,which may inflect gene transcription.Currently,cAMP response element binding protein(CREB)is the transcriptive factor involved in the mechanism of drug abuse.The level of CREB is found to be increased in the frontal cortex after morphine dependence and withdrawa1 without changes in hippocampus and nucleus accumbens,which may be attributed to different distribution of opioid receptors in the brain.It is reported that CREB is the first transcriptive factor found to be related closely with drug dependence.CREB mediates signaling pathway by regulating gene transcription.Glial fibrillary acidic protein(GFAP)is astrocyte(AS)specific marker.Its expression is reportedly increased in response to cell degeneration or death because of injury and drugs which activates of AS,for enhancing the clearance of excitatory neurotransmitters and ions including glutamate,H~+,K~+ and free radicals.GFAP is also responsible for glycogen storge,synthesis of cytokines and neurotrophic factors,contributing to the reconstruction of the integrity of neurons and neuronal repair.In the present study, pCREB and GFAP expression were studied for the first time in the frontal cortex in rats with tramadol dependence by immunohistochemical staining and Western blotting.Materials and MethodsA total of male 30 rats,weighing 90～110g,were randomly divided into three groups, one control group and two experiment groups which include tramado- dependent group and withdrawal group.Rats were supplied by the Animal Laboratory of China Medical University.Rats were bred for 5 days at 18-26℃to adapt to the environment.Under the permission of Ethical Committee for Animal Experiment of China Medical University. Rats were intragastrically given hydrochloride tramadol 60mg/kg per day for 115 days in tramadol-dependent group.Rats were intragastrically administrated hydrochloride tramadol 60mg/kg per day for 112 days and then given saline instead in withdrawal group. Rats were given saline per day for 115 days in control group.The rats got chow and water freely and housed individually in cage.The behaviors of the drug dependence and withdrawal were observed and recorded and body weight was measured.Two hours after the last administration,the rats were executed by intragastrically,administration of overdose of sodium pentobarbital.Frontal cortex was collected and fixed in 4% parafonmaldehyde/0.01MPBS(pH7.4)for 48h.and then washed,dehydrated,and embedded in paraffin.5μm-thick consecutive paraffin sections were prepared.For Western blotting,fontal cortex was sampled on a wax plate cooled with ice.The immunostaining sections were observed at 400 times magnification under microscope at randomly selected 10 fields in frontal cortex,pCREB- or GFAP-positive cells were counted by two staff members independent of the experiment.Gray density was analyzed by computerized imaging system for Western blotting results,bands that means expression of strength.Band color intensity was analyzed by Fluochem V2.0 software. The data were expressed as mean±deviation.All data were statistically analyzed by SPSS11.0 for Windows with a single factor analysis of variance.P＜0.05 was statistically significant.Results1.Behavior changes in tramadol dependent and withdrawal ratsAs compared with control rats,rats in tramadol-dependent group showed the symptoms of exciting,hyper-reactivity to sound and touching stimulation,running,tail uprighting as well as run in circle.But 36-48 hours after the withdrawal of tramadol, symptoms were characterized by agitating,restlessness,outraging,jumps,screaming, abnormal postures,teeth clicking,the wet dog-like shivering,flings,ptosis,and the diarrhea.2.Morphological changes of the brain(1)Gross examinationNo abnormal changes were found.(2)Microscopic examinationMild cerebral edema and gliosis were detected in the frontal cortext in the rats with tramadol dependence and withdrawal.3.Body weight measurements(1)Tramadol-dependent groupIn comparison with those in the control group,increase in body weight was greater than in the tramadol-dependent rats during the first 1-3 weeks.However,the increase in body weight became slower than in the control group since the 4th week after drug delivery,which was statistically significant(P＜0.01).(2)Tramadol-withdrawal groupBody weight of control rats gained continuosly from 380g to 401g with an increase of 19g in average within the 3 days.The weight of tramadol-dependent rats increased from 223g to 232g with an increase of 9g in average.The weight of tramadol- withdrawal rats decreased from 226 g to 212 g with a reduce 14g in average.The difference was statistically significant in body weight increase or decrease in tramadol-dependent or withdrawal group as compared with the control respectively(P＜0.01).4.pCREB and GFAP immunohistochemical stainingpCREB-positive cells were mainly detected in the nucli of the neurons,and in a small number of glial cells in the cortex in control rats.GFAP-immunoreactivity was found in the cytoplasm and dendrites of the astrocytes.An intensive immunoreactivity of pCREB and GFAP were detected in frontal cortex of rat in dependence and withdrawal rats.The number of pCREB-positive cells in tramadol-dependent rats(78.5±3.79)or withdrawal group(130.5±2.53)was increased in comparison with the control group (40.9±1.89),which was statistically significant(P＜0.05).Gray density of pCREB in positive cells in dependence group(43.02±3.12)and withdrawal group(34.09±3.53) was decreased in comparison with that in the control group(70.87±4.85),which was statistically significant(P＜0.05).Increase in number of GFAP-positive cells in dependence group(22.23±9.34)or withdrawal group(34.91±8.36)was noted in comparison with the control group(12.56±6.43),which was statistically significant(P＜0.05).Gray density of GFAP-positive cells in dependence group(40.09±3.56)or withdrawal group(30.02±3.11)was decreased in comparison with the control group(65.87±3.21),which was statistically significant(P＜0.05).5.Western blottingAverage optical density of pCREB bands in dependence group(60.36±2.14)or withdrawal group(105.24±2.04)was increased in comparison with the control group(40.26±3.01),which was statistically significant(P＜0.05).Average optical density of GFAP in dependence group(22.34±2.04)and withdrawal group(34.61±3.01)was increased in comparison with the control group(12.22±3.29),which was statistically significant(P＜0.05).Discussion and ConclusionsAs compared with the control group,pCREB expression was increased significantly in the frontal cortex in tramadol-dependent and withdrawal rats,suggesting that pCREB may take part in the development of tramadol psychlolgical and physical dependence. pCREB is considered to enhance the neuronal survival and protection.Recent studies indicate that pCREB is the most critical transcriptve factor responsible for development of drug dependence,pCREB may promote gene expressions which are associated with abnormal behaviors in drug addiction.GFAP expression was significantly enhanced in frontal cortex in tramadol -dependent and withdrawal rat.Up-regulated GFAP expression in AS indicates glial proliferation and development,indicates the neurons adaptation to damage.Enhanced GFAP expression indicates increased stability of AS skeleton in tramadol dependence and withdrawal. Activated AS may be helpful for meeting their the increased of metabolic activities and reducing drug-induced neuronal injury.Our study further implicates the protective roles of AS in the tramadol-induced neuronal damage.
|
PDF Full Text Request