Mechanisms For Inhibition Of Thyroid Hormone On Effects Of Propofol Anesthesia

Why is additional dose of propofol required during surgery for general anesthesia in the patients with hyperthyroidism? This problem has plagued the field of clinical Thyroid-tumor surgery and anesthesiology. The present study has investigated the related mechanisms involving thyroid hormone (TH) and substance P (SP) inhibition on GABA-activated currents, using patch-clamp technique, intracellular recording, MQAE chlorine ion fluorescent probe, immunofluorescence microscpy and western blot, which provided theoretical basis for rational use of anesthetic propofol and treatment of pain.Part 1:Influence of thyroid hormone on the anaesthetic effect of propofolObjective:In anaesthesia of propofol, usually larger propofol dose is needed during operation for patients with hyperthyroidism. In this study, we investigated the mechanism through which THs inhibit the effect of propofol anaesthesia.Methods:1. Hyperthyroidism model of rats was induced by daily injections of T3 (7 ?g/100 g BW in 0.01 mM NaOH, intraperitoneal for 14 days)2. Immunofluorescence microscopy was utilised to verify the difference in expression of GABAA receptors's subunit ?2 and ?2 in DRG of normal and hyperthyroid rats.3. Whole-cell patch clamp recordings were performed to detect the GABA-activated current in freshly isolated rat DRG neurons of normal and hyperthyroid rats.4. The change of the GABA-activated currents and the effects of propofol were evaluated after the pre-perfusion of propofol with and without T3 (0.1 ?mol/L?100?mol/L).5. After the pre-perfusion of GABAA receptor blocker bicuculline, observe whether GABAA receptor is the only factor to achieve the inhibition effect of T3 on the anaesthetic effect of propofol.Results:1. Additional propofol anaesthesia is required for rats with hyperthyroidism compared with control. Control group was 7.14 ± 0.51 (mg/100g BW), hyperthyroidism group was 11.02 ± 0.53 (mg/100g BW)2. Compared with normal rats, rats with hyperthyroidism shared the same subunit expression quantity of the GABAA receptors, ?2 and ?2, in DRG. No difference in GABA-activated currents in acutely isolated rat DRG neurons was observed.3. T3 can inhibit GABA-activated inward currents in DRG neurons. T3 of 10 ?mol/L can obviously suppress the GABA-activated current in DRG neurons. T3 reduced the currents in a dose-dependent manner. T3 alone could not induce any inward current or outward current.4. T3 inhibits or minimises the augmentation effect of propofol on the GABA-activated currents. T3 inhibits or minimises the effect of propofol on the augmentation of the GABA-activated currents (p< 0.05).5 ?mol/L propofol was pre-perfused followed by 100 ?mol/L GABA, which induced the augment of the inward current and proving that the induction is the effect of propofol on the augment of the GABA-activated currents, as well as the anaesthetic mechanism. The activated normalized current for GABAA receptor is 2.21 ± 0.36; the mixture of 10 ?mol/L T3 and 5?mol/L propofol was pre-perfused, followed by 100 ?mol/LGABA, which then evidently showed that effect of propofol on the augment of GABA-activated currents was significantly reduced. The activated normalized current for GABAA receptor is 1.24±0.21.5. The inhibition effect of T3 on propofol can be minimised by increasing the propofol concentration. The inhibition effect of T3 on propofol can be minimised by increasing the propofol concentration (P< 0.05). The propofol concentration increased, and detection of the 100 ?mol/L GABA-activated currents after the mixture of 10 ?mol/L T3 and 10 ?mol/L propofol was pre-perfused demonstrated that the inward current increased. This result indicated that an increased propofol concentration can partially offset the inhibition effect of T3 on GABA-activated currents. The activated normalized current for the GABAA receptor is 2.10± 0.31.6. Propofol enhance the GABA-activation current was blocked by bicuculline. GABA-activation current was blocked by bicuculline (100 ?mol/L). T3(10 ?mol/L) inhibit the GABA-activation current was blocked by bicuculline (100 umol/L). Propofol (5 ?mol/L) enhance the GABA-activation current was blocked by bicuculline (100 ?mol/L).7. Tetrac could not block the inhibitory effect of T3 on GABA-activated currents.Conclusions:The inhibition effect of T3 on the anaesthetic effect of propofol is achieved through the inhibition on the function of GABAA receptors through the non-genomic actions of the THs, instead of changing the number of GABAA receptors. This inhibition effect can be mitigated by increasing the propofol concentration.Part 2:Study on the Mechanism of thyroid hormone inhibit GAB A-activated currentObjective:GABAergic system is one of the important targets for regulating thyroid hormone. It is confirmed that the non-genomic pathways of thyroid hormone (instantaneous pathway) can inhibit GABA-activated current by suppressing the functions of GABAA receptor. But there is no report about whether the instantaneous pathway of thyroid hormone can affect the functions of chlorine ion transporter on cell membrane and the concentration of chloride ion in the neurons. GABA-activated current is caused by intracellular Cl- transmembrane flow, so change the chloride ion concentration can directly affect the size of the GABA-activated current. The concentration of chloride ions in the neurons can directly affect the intensity of GABA-activated Cl- current. Therefore, in this part of the present thesis Cl" concentration and expressions of chloride ions transporter in neurons were tested before and after incubation with thyroid hormone to explore whether there were changes in Cl- concentration and expressions of chloride ions transporter in the inhibition role of thyroid hormone on GABA-activated current.Methods:Freshly-isolated DRG and cerebral cortex neurons from adult SD rats were detected with MQAE probe for changes of chloride ion concentration in neurons before and after incubation with T3. Rat models of hyperthyroidism were established to detect the expressions of NKCC1 and KCC2 in the DRG and cerebral cortex.Results:1. T3 can inhibit GABA-activated inward currents in DRG neurons. T3 of 10 ?mol/L can obviously suppress the GABA-activated current in DRG neurons of SD rats, with an inhibition effect up to 41.2%±5.1%.2. T3 did not change and chloride ion concentration in the DRG neurons and cortical neurons. There were no significant differences in chloride ion concentration in both DRG and cerebral cortex neurons before and after incubation with T3 (10?mol/L), detected with MQAE probe.3. Compared with those of normal rats, there was no difference in chloride ion concentration in DRG and cortical neurons of hyperthyroid rats.4. There were no significant changes in the expression levels of NKCC1 and KCC2 in DRG and cortex between normal and hyperthyroid rats.Conclusion:The inhibitory effect of thyroid hormone on GABA activated inward current in both DRG and cortex neurons are not attributed to the changes in Cl- ion concentration and chloride ion transporter expression.Part 3:Mechanisms underlying SP and TH inhibition on GABA-activated currentsObjective:To investigate the mechanism underlying the modulatory effect of SP on GABA-activated response in rat dorsal root ganglion (DRG) neurons.Methods:SD rats, aged 8-10 weeks, weighing 250?280 g, were decapitated. The thoracic and lumbar segments of vertebrael column were dissected and longitudinally divided into two halves along the median sagittal plane. The DRGs together with dorsal and ventral roots, and spinal nerves were taken out and transferred into Dulbecco's Modified Eagle's Medium (DMEM) at pH=7.4, immediately. After the removal of attached nerves and surrounding connective tissues, the DRGs were minced with iridectomy scissors and incubated with enzymes including trypsin 0.25 mg/mL (type ?, Sigma, USA) and 0.5 mg/mL collagenase (type IA, Sigma, USA) in 5 mL DMEM at 37? in a shaking bath for 30 min. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique and intracellular recordings was used to record GABA-activated current.Results:1. GAB A can induce inward current in most DRG neuronsApplication of GAB A (1-1000 ?mol/L) induced a concentration-dependent inward current in most DRG neurons (89.8%,114/127). The selective GABAA receptor agonist muscimol (100 ?mol/L) mimicked GABA-activated response (n=11). Both GABA (100 ?mol/L) and muscimol (100 ?mol/L)-activated currents were completely and reversibly blocked by bicuculline (100 ?mol/L, n=9), indicating that the current was mediated by the GABAA receptor.2. NK1 receptor antagonist can completely block the SP inhibition of GABA-activated inward currents.During whole-cell patch clamp recordings, a pre-treatment of SP (0.001-1 ?mol/L) could significantly inhibit the GABA-activated inward current. The inhibitory effects could be blocked by the selective NK1 receptors antagonist, spantide (40 ?mol/L, n= 7), but not by the selective NK2 and NK3 antagonists L659187 (20 ?mol/L, n=7) and SR142801(20 ?mol/L, n=7). The inhibitory effect of spantide (5,20, and 40 ?mol/L) was concentration-dependent, while that of L659187 (1,10, and 20 ?mol/L) and SR142801 (1,10, and 20 ?mol/L) was not.3. The suppressing effect of SP on GABA-activated membrane depolarization is mediated by NK1 receptor.During intracellular recordings, application of GABA (1-1000 ?mol/L) evoked membrane depolarization in a concentration-dependent manner in 236 out of 257 DRG neurons (91.8%) examined. The average amplitude of membrane depolarization induced by 100 ?mol/L GABA was 12.3±3.6 mV (n=27). Application of SP (0.001?1 ?mol/L) suppressed the GABA-activated inward current and membrane depolarization. Consistent with the above patch-clamp results above, the inhibitory effect of SP (0.1 ?mol/L) on GABA-induced depolarization was abolished by the selective NK1 receptor antagonist spantide.4. PLC and PKC are involved in the SP inhibition of GABA-activated currents.The inhibitory effect of SP was strongly suppressed by pre-incubation of phospholipase C (PLC) inhibitor (U73122,1 ?mol/L, n=6) and the protein kinase C (PKC) inhibitor (chelerythrine,1 umol/L, n=9). The PKA inhibitor H-89 (1 ?mol/L, n=8) did not affect the SP effect. The inhibitory effect of U73122, chelerythrine was concentration-dependent.5. PKC? is involved in the SP inhibition of GABA-activated currents.The inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCE inhibitor epilon-V1-2 but not by PKCa inhibitor safingol and PKCp inhibitor LY333531.Conclusion:Our results suggest that NKi receptor mediates the inhibition of SP on GABA-activated current and membrane depolarization by activating intracellular PLC-PKC? cascade. Thus SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA.