| Part1The Effect of Noradrenaline on GABAA Receptor Expression in Dorsal Root Ganglion Neurons of RatsObjective Noradrenaline (NA) has an important modulation on many receptors and channels via alpha-adrenoceptor (a-AR) in dorsal root ganglion (DRG) neurons, and participates in the nociceptive input. GABAA receptor is also an important modulator. In the development and maintenance of chronic neuropathic pain (NPP) the GABAergic systerm is downregulated. We applied NA to DRG neurons to detect the γ2subunit mRNA and protein expression. The aim of this experiment was to clarify whether NA modulats GABAA receptor expression to participate in the disinhibition of the GABAergic systerm.Methods After DRG neurons primary cultures of SD rat (160-220g), the distribution of γ2subunit in DRG neurons was detected by using immunofluorescence. The cells were cultured for48h and then were grouped. The first group:NA1μM, NA1μM+prazosin (PRZ, α1-AR blockers)10μM, NA1μM+yohimbine (YOH, α2-AR blockers)10μM and NA1μM+PRZ10μM+YOH10μM were added in the flasks, respectively. They were added for a second time at the next day, and then entered the RNA extraction procedure two days after that. Control groups were not treated with NA and α-AR blockers. The second group:NA0.01μM, NA0.1μM, NA1μM and NA10μM were added in the flasks, respectively. They were added for a second time at the next day, and then enter the subsequent protein extraction procedure two days after that. Control groups were not treated with NA. The third group:1μM NA were added in the flasks for5h,12h,24h, and48h, respectively, and then entered the protein extraction procedure. The fifth flask was added with1μM NA at the second day and the fourth day, and then entered the protein extraction procedure two days after that (total96h). Control groups were not treated with NA. Total RNA or protein was extracted from each group. The expression of γ2subunit under the different factors was observed by RT-PCR and Western blot technique.Results (1) Immunofluorescence staining in DRG neurons showed that yi subunite of GABAA receptor expressed in large, medium, and small size neurons. It mainly gathered on cell membrane, partly within the cytoplasm. The cores of the cells were lightly stained. The glial cells were’t stained;(2)1μM NA,1λM NA+10μM PRZ,1μM NA+10μM YOH and1μM NA+10μM PRZ+10μM YOH were applied to neuronal cultures, respectively. Accoding to the results of RT-PCR,γ2subunit mRNA expressions were not different beteen control group and treatment groups (P>0.05, n=3);(3)0.01μM NA,0.1μM NA,1μM NA and10μM NA were applied to neuronal cultures. The results from Western blot were that γ2subunit expressions were not different beteen control group and treatment groups (P>0.05, n=3);(4) The neuronal cultures were applied with1μM NA for5h,12h,24h,48h and96h. The results from Western blot were that γ2subunit expressions were not different beteen control group and treatment groups (P>0.05, n=3).Conclusion NA can not modulate GABAA receptor expression in DRG neurons.Part2Noradrenaline Inhibits GABA Current in Dorsal Root Ganglion Neurons in Rats with or without Chronic Constriction InjuryObjective In the process of NPP, the GABAergic system is down-regulated and many algogenic substances can modulate GABAA current. NA has an important modulation on nociceptive input in DRG neurons. We surveyed the effect of NA on GABAA current in order to clarify whether NA participates in the disinhibition of GABA in NPP.Methods The isolated DRG neurons were prepared through enzymatic digestion. The whole-cell patch-clamp technique was used to record GABA-activated currents (IGABA) and survey the effect of NA on IGABA-Further, phenylephrine (PHE, α1-AR agonist), PRZ (α1blockers), CLO (α2-AR agonist), YOH (α2-AR blockers), forskolin (FSK, PKA agonist), H-89(PKA inhibitor), PMA (PKC agonist), GF109203X (GF, PKC inhibitor) were applied to analyze the mechanism of the regulation of NA on IGABA. Preparation of CCI models was used to observe the action of NA under the pathological circumstance.Results (1) Application of GABA induced inward current which was mimicked by muscimol (Mus, a GABAA receptor agonist), and completely blocked by bicuculline (Bic, a GABAA receptor antagonist), indicating that GABAA receptor mediated IGABA in DRG neurons;(2) Pretreatment with NA suppressed IGABA.1μM NA has a more powerful effect. The effects of0.01-1μM NA gradually increased, but the inhibition reduced with the increase in NA concentrations at10μM-1000μM. The effects of NA was not significantly different among large, medium and small size neurons (P>0.05);(3) The inhibitory effect of NA was partly mimicked by the α1adrenergic receptor (α1-AR) agonist phenylephrine (PHE) and the α2-AR agonist clonidine (CLO), and partly depressed by the α1-AR antagonist PRZ and the α2-AR antagonist YOH, respectively. The inhibitory effect was also obtained with the protein kinase C (PKC) activator PMA and the protein kinase A (PKA) inhibitor H-89. The combination of PKC inhibitor GF109203X (GF) and the PKA activator forskolin (FSK) completely blocked the action of NA;(4) CCI induced a significant decrease of the paw withdrawal threshold of rats and IGBA in DRG neurons (P<0.01). The inhibitory effect of NA was enhanced after CCI (P<0.05).Conclusion NA modulates IGABA via both α1-AR/Gq/phospholipase C (PLC)/PKC pathway and α2-AR/Gi/cAMP/PKA pathway in DRG neurons. The nerve injury down-regulates IGABA and potentiates the action of NA. These mechanisms might participate in the process of nociceptive input.Part3Differential Expression of Alpha-Adrenoceptor Subtypes in Rat Dorsal Root Ganglion after Chronic Constriction InjuryObjective Accumulating studies demonstrate that mRNAs of the subtypes of α-AR are found in DRG neurons and the levels of α-AR mRNAs change after peripheral nerve injury. However, the datas from these studies are contradictory. To confirm the change of every a-AR subtype and clarify the mechanic of the increasing effect of NA after nerve injury, the expression of these subtype proteins in DRG neurons from normal rats and CCI rats was surveyed in this study.Methods Twenty-four male Sprague-Dawley rats (160-220g) were used in this study. They were randomly divided into three groups, normal control (n=3), shame control (n=3) and CCI group (n=18). To generate nerve injury, the rats in CCI group were anesthetized with sodium pentobarbital (60mg/kg) and their left sciatic nerves were exposed and tied. The sciatic nerves of shame control group were only exposed but not tied. The rats in normal control group have not been treated. By using the hot plate test, the pain thresholds on the injured paw were measured respectively on the pre-operative1day and post-operative14th day. At14th day after CCI, CCI rats and normal rats were perfused with4%paraformaldehyde and L5DRGs were dissociated. All tissue was mounted using OCT mounting media and sectioned on a cryostat to10μm. The sections were incubated with the antibodies by using immunofluorescence technique. The distribution of each a-AR subtype protein was studied through confocal microscopy imaging system.Results (1) CCI resulted in a reduction in withdrawal threshold to hot plate testing from23.0±1.0sec to11.5±1.0sec at14th day after surgery (P<0.01). The withdrawal thresholds of the shame control group before and after surgery weren’t significantly different (P>0.05);(2)α1A-AR and α1B-AR expressed in large, medium, and small size neurons in normal DRG (α1A-AR,49.4%; α1B-AR,38.2%, respectively). At14th day after CCI, they both significantly increased in all size neurons (α1A-AR,89.8%; α1B-AR,81.8%, respectively). α1A-AR gathered on cell membrane, within the cytoplasm, and occasionally in the nucleus.α1B-AR distributed throughout cell body, including the nucleus. α1D-AR moderately increased (control,48.8%; CCI,68.5%, respectively, P<0.01) and mainly in small size neurons, its protein was found on cell membrane, in the cytoplasm, and occasionally in the nucleus. α2A-AR protein was distributed on cell membrane, throughout the cytoplasm, and in many nuclei.22.8%of all neurons were α2A-AR positive neurons which increased significantly to57.7%(P<0.01) following the injury, especially in small size. However, the percent of α2C-AR positive cells decreased from control (44.0%) to CCI (32.2%, P<0.01), which mainly lied on the decrease of small positive cells. α2c-AR protein was predominantly distributed in the cytoplasm. α2B-AR immunoreaction (IR) wasn’t detected not only in normal DRG but also in CCI DRG;(3) Co-expressing of α1A-AR and a2A-AR was obvious in the same neuron of normal DRG; the double labeled neurons were mainly in the medium size ones and were upregulated post CCI (P<0.01).Conclusion There is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of α-AR subtype expressions change differently after CCI. The upregulation of α-AR subtype neurons may participate in the increase of the action of NA on IGABA and play an important role in the process of generating and transmitting NPP. |
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