| BackgroundThe general anesthesia status, induced forgotten, unconsciousness, analgesia, inhibit noxious stimulation-response, braking and other ingredients, has its own the corresponding regions in the central nervous system. The spinal cord might restrain the critical response in the noxious stimulation site. Propofol is a relatively new short-acting intravenous anesthetic with advantage of the quick effect,short duration,prompt revival,easy control and less side effects, which has been widely used in general anesthesia induction and maintenance. Previous studies had shown that propofol played the anti-peripheral role in the reaction of noxious stimulation, the target site of which might located in the spinal cord, but its mechanism had not been understood today. As we known, spinal cord is the effective organ of propofol, and the action of propofol in the spinal cord was discordant in the various region of spinal cord. Therefore, the research of propofol's uptake and distribution in spinal cord is favourable and meaningful to investigate explore the mechanism of general anesthesia.In the past decade, along with the improvement of theories and techniques of neural and molecular biology, many researches have been done to investigate the anesthetic mechanism of propofol. But its mechanism of general anesthesia was still not able to be explained. The anatomic structure and the distribution in the central nervous system have a certain degree of specificity that the anesthetics have its selective parts to play its narcotic pharmacological effect too. There are dozens of biologically active substances exist in spinal cord neurons mainly involving in the transmission of messages which promote the nociceptive transmission of glutamate and substance P, and inhibit the nociceptive transmission of opioid peptides, GABA and glycine. The GABA receptors in the central nervous system are the major inhibitory receptors, it widespreadly distributed, but had their own regions and the different pharmacological and electrophysiological characteristics to nerves. Synaptic doctrine is the most important doctrine in mechanism of general anesthesia, and it said that the role of anesthesia drugs and their impact was relevant to the function of synaptic transmission, and propofol may be mainly acting on GABA receptors. The GABA neurons are rich in spinal cord substantia gelatinosa, in particular dorsal horn layerⅡⅡ,Ⅲboundary between layers, and axons and primary afferent terminals and synapses ball structure formed by the deep projection neuron dendrites. After GABA receptors were activated, conductance increased as resulting from spinal cord neurons depolarization, and the resulting in general anesthesia effect. But as we all know that our understanding of the nervous system is still inadequate, a number of research methods have its limitations and there are many intervention factors. Many studies suggestted that surgical operations itself can affect the sensitivity of the spinal cord neurons to the anesthetics. Therefore, uptake and regional distribution of propofol in spinal cord must be investigated for understanding its'true anesthetic mechanism.In 1988, Upton proposed the concept of cerebral uptake and the fundamental research method of mass balancing principles. The process that the drug is filled into the brain with blood and goes into the brain parenchyma from the intravascular which is caused by drug distribution and elimination is known as cerebral uptake.The research about cerebral uptake of propofol was usually baseds on the mass balance principles. By measuring the propofol concentrations in arterial and venous blood of cerebral circulation, we can calculate the cerebral concentrations and evaluate the cerebral uptake of propofol. Based on this rule, many scholars had started the research on the brain uptake of propofol since the late 90s of the last century. In 1995, Shyr reported that propofol concentration in the rat spinal cord and brain tissue increased with the time increased when rats were anesthetized by propofol infusion at a constant rate of 60mg·kg-1·h-1. The recent research had found that when propofol was intravenously infused at a rate of 70mg·kg-1·h-1 to the brain intake balance, the propofol had the same distribution in all regional organizations of the dog brain. The experimental studies about the propofol brain uptaking and the distribution have have been fairly mature. Are the propofol in different regions of spinal cord tissue's uptake and distribution the same? Can the noxious stimulation response impact the distribution of propofol in different regions of the spinal cord? Previous experiments had not been answerd, so it is necessary to do some study.The aim of this study is to measure propofol concentration of spinal cord by high-pressure liquid chromatography ultra-violet spectroscopy (HPLC-UV) and to investigate the regional distribution of propofol in spinal cord under the circumstandce of noxious stimulation when the cerebral uptake being equilibrium in dogs.Material and methods12 healthy dogs aged 12-18 months (male and female) were divided randomly into two groups (group C and group S). All the experiment were scheduled during the day (9:00-11:00) and raised in diet for 12-hour prior to experiment. The venous channel was established in the great saphenous vein of the right posterior limb. Propofol was intravenously injected respectively at a single bolus 7mg·kg-1 in group S and group C in 15 sec. after their eyelid reflex and pedal reflex disappeared, animals were fixed supinely on the platform. The constant intravenous infusion of propofol was taken at a rate of 70 mg·kg-1·h-1 to maintain anesthesia.When the infusion of propofol was at the 45th min, animals of group S were given stimulation to the end of its tail by hemostat for 5min. Animals of group C were given no stimulation. The blood samples were taken from the right internal carotid and internal jugular vein at the 50th min in group S and at the 50th min in group C. Then the animal was scarificed immediately by decapitation. The frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus of spinal cord were further dissected for determination the concentrations of propofol.Propofol concentration was determined by HPLC-UV. External standard was a control article of propofol. The analysis was performed with a Shim-pack VP-ODS, 250x4.6mmID, Shim-pack GVP-ODS,10x4.6mmID and a 2996 Waters ultraviolet detector (270nm). The solvent system was purified water-methanol at flow rate of 1ml·min-1.The brain samples were extracted with acetonitrile (2ml·g-1) and homogenized and the blood samples were extracted with acetonitrile (di-volume). After being centrifuged, the supernatant was submitted to HPLC analysis. The sample volume is 20μl.Measurement data were expressed as mean±standard deviation. All data were analysed with the Statistics Package for Social Sciences (SPSS, version 13.0 for WINDOWS; SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant when P was less than 0.05. We used the Independent-Samples T Test, Paired-Samples T Test and Repeated Measure to test for differences. Multiple comparisons were analyzed by LSD test. Differences were considered statistically significant when P was less than 0.05.Results1. All experimental animals reached safely and quickly the condition of anesthesia and maintained a stable status. In the group C, PETCO2,PR and MAP were 34.83±1.47mmHg,80.50±1.38bpt and 87.83±1.17mmHg respectively. In group S, PETCO2 were 34.83±1.33mmHg and 35.17±1.47mmHg, MAP were 88.50±1.05mmHg and 101.83±1.72mmHg and PR were 81.67±1.86bpt and 94.17±1.94 bpt respectively before and after stimulation, the MAP and PR after stimulation were significantly higher than that before stimulation.2. The propfol concentration in blood plasma:The concentration of propofol in internal carotid artery and internal jugular vein blood plasma were 5.09±0.03μg·ml-1,5.07±0.23μg·ml-1 in group C and 5.08±0.03μg·ml-1,5.03±0.10μg·ml-1 in group S respectively and no significant differences.3. The propofol concentration in spinal cord tissues:In group S, the differences of propofol concentrations among every region of spinal cord were considered statistically significant (F=41.384, P=0.000),the concentration of propofol in the frontal horn (7.65±0.47)μg·kg-1 and the posterior funiculus (7.60±0.82)μg·kg-1 were higher than that in other region of spinal cord(P<0.05), the differences of propofol concentrations among other region were not considered statistically significant differences(P>0.05). In group C, the differences of propofol concentrations among every region of spinal cord were not considered statistically significant(F= 0.469, P=0.798).The propofol concentrations of the frontal horn and the posterior funiculus in group S were significantly higher than that in group C (F=1.099,P=0.319).The concentration of propofol in dorsal thalamus and subthalamus were highest under the condition of balance of the brain uptake and noxious stimulation.Conclusions1 The HPLC-ultra-violet spectroscopy combining with precipitation method can be used appropriately to determinate the propofol concentration of blood plasma, brain and spinal cord tissues.2 At 50 min after constant intravenous propofol injection at a rate of 70 mg·kg-1·h-1, plasma propofol concentration reaches equilibrium between internal carotid artery and internal jugular vein, and propofol is distributed evenly among regional spinal cord tissues (frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus) in dogs.3 Under the circumstance of noxious stimulation, the propofol concentration in frontal horn and posterior funiculus are higher than other region of spinal cord.
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