Involvement Of Spinal GABA_B Receptor Subtypes And GABA Transporter-1 In The Neuropathic Pain On Rats

ObjectiveStudies on the transmitter pharmacology of the post - nerve injury have indicated that there existed changes of neurotransmitters and receptors in spinal cord induced by neuropathic pain. Changes of GABAB receptors and GABA transporters could affect the function of GABAergic system so to affect the transmission and modulation of nociceptive messages. Studies about the changes of GABAB receptors subtypes and GABA transporters help finding out more specific targets for pain treatment.The present study is to explore changes of GABA receptor subtypes ( GAB-AB1a,GABAB1b,GABAB2) and GABA transporter - 1 ( GAT - 1) in spinal cord of rats with chronic constriction injury induced neuropathic pain. Characteristic behavioral changes reflecting hyperalgesia of rats were observed, immunohisto-chemical method and western blot technique were applied to examine the expression of GABAB receptors and GAT - 1. Baclofen, a GABAB receptor agonist and NO -711, a GABA transporter - 1 inhibitor were respectively intrathecally injected to explore the anti - hyperalgesic effects and possible mechanism involved in, which lends further support to the analgesic effects of GABAergic drugs.Part OneExpression of GABAB Receptor Subtypes and GABA Transporter -1 on Rats with Chronic Constriction Injury Induced Neuropathic PainMaterials and Methods1. Chronic constriction injury ( CCI) model.1. 1 Animals. Experiments were performed on adult, male Sprague -Dawley rats weighing 230 ~ 300 g.1.2 Chronic constriction of sciatic nerve. The left common sciatic nerve of anesthetized rats was exposed, four silk ligatures (3 - 0) were tied loosely proximal to the sciaticas trifurcation. Sham surgery was done by exposing the left sciatic nerve without ligation.2. Experimental paradigm. Rats were divided into Sham group and CCI group. Hind paw thermal withdrawl latency (TWL) to thermal stimulus and mechanical withdrawl threshold ( MWT) of mechanical stimulus were recorded before ligation or on 1, 3, 5 , 7, 10, 14, 21, 28 day after ligation respectively ( n= 8). Expression of GABABreceptor subtypes and GAT - 1 were detected before ligation and on 1, 3, 7, 14, 28 day after ligation respectively using immunohis-tochemical assay (n=6) and Western blot technique ( n = 4).3. Data analysis and statistics. Data were expressed as mean ±SEM. The results were analyzed by analysis of variance ( ANOVA) with Student - Newman- Keuls as the post hoc test. Values of P <0.05 were considered as statistically significant.Results1. Effects of chronic constriction injury on the MWT and TWL of hind paws of ratsThere were no significant differences on the values of MWT and TWL before ligation between Sham group and CCI group. Compared with Sham group, the MWT and TWL of rats in CCI group significantly decreased after ligation ( P < 0. 01, P <0. 05 ) and continued to be decreased until 28 day after ligation.2. Effects of chronic constriction injury on the expression of GABAB1a, GABAB1b,GABAB2 in spinal cord of ratsBoth immunoreactive neurons and protein content of GABAB1a or GABAB2 subtypes increased on the 3rd day after ligation, then both decreased on the 7th day after ligation, and lasted for 28 days after ligation ( P <0. 01, P < 0. 05). There were no changes of GABAB1b after ligation ( P >0.05).3. Effects of chronic constriction injury on the expression of GAT - 1 in spinal cord of ratsThe GAT - 1 immunoreactive density in superficial spinal cord of rats in CCI group significantly increased after ligation ( P <0. 01) and continued to be increased until 7th day after ligation; the protein content of GAT - 1 also kept a higher level from the 3rd day to the 7th day after ligation.Part TwoEffects of Intrathecal Injection of GABAB Receptor Agonist Baclofen on Hyperalgesia and Spinal GAT -1 of Neuropathic RatsMaterials and MethodsR( ± ) baclofen was purchased from Sigma ( U. S. A. ) , the other drugs and reagents were the same as those in Part One.1. CCI rats. The same as that in Part One.2. Experimental paradigm. Behavioral experiment: After nerve ligation, 32 rats exhibiting neuropathic pain were divided into 4 groups (n=8): NS group, Bacl group, Bac2 group and Bac3 group, which were intrathecally injected with normal saline, 0. 1μg, 0. 3μg and 1. 0μg baclofen respectively. MWT and TWL were recorded before drug administration, 0.5h, 1h, 2h, 4h, 8h, 12h and 24h after drug administration respectively. Immunohistochemical and Western blot experiment: Neuropathic rats were divided into NS group and Bac group, which were intrathecally injected with normal saline or 0. 3μg baclofen respectively. Lumbar spinal cord of rats were removed before injection or on lh, 4h and 8h after intrathecal injection respectively to detect the GAT - 1 immunoreactive density(n=6) or were removed before injection or on 0.5h, 1h, 2h, 4h, 8h, 12h and 24h after intrathecal injection respectively to detect the protein contentof GAT-1 (n=4).3. Data analysis and statistics. The same as that in Part One.Results1. Effects of intrathecal injection of baclofen on the MWT and TWL of neuropathic ratsMWT and TWL of Bac1 group, Bac2 group and Bac3 group were significantly higher than those of NS group (P<0.01,P<0.05). Compared with NS group, the increasing of MWT and TWL in Bacl group, Bac2 group and Bac3 group lasted for 2h, 4h and 8h respectively ( P <0. 01 ,P <0. 05).2. Effects of intrathecal injection of baclofen on the expression of GAT - 1 in spinal cord of neuropathic ratsCompared with NS group and pre - drug values, both immunoreactive density and protein content of GAT - 1 remarkably decreased during 0. 5 ~4h after drug administration, on 8h after drug administration, there were no significant differences on the expression of GAT - 1 among the four groups ( P >0.05).Part ThreeMechanisms Underlying the Anti - hyperalgesic Effects of GABA Transporter -1 Inhibitor NO -711 on Neuropathic RatsMaterials and Methods*NO -711 was purchased from Sigma (U. S. A. ) , primary antibody to c -fos and primary antibody to pERK were purchased from Santa Cruz ( U. S. A. ) , the other drugs and reagents were the same as those in Part One.1. CCI rats. The same as that in Part One.2. Experimental paradigm.3.1 80 rats were divided into 10 groups (n=8): ANS, AN5, AN10, AN20, AN40, BNS, BN5, BN10, BN20 and BN40 group. Before nerve ligation, rats in BNS, BN5, BN10, BN20 and BN40 group were intrathecally injec-ted with normal saline, 5μg> 10μg, 20μg and 40μg NO -711 respectively, and TWL and MWT of rats were recorded before ligation and on 1, 2, 3, 5, 7, 10, 14 day after ligation. On the third day after nerve ligation, rats in ANS, AN5, AN10, AN20 and AN40 group were intrathecally injected with normal saline, 5μg, 10μg, 20μg and 40μg NO -711 respectively, TWL and MWT of rats were recorded before drug administration and on 15min, 0.5h, 1h, 2h, 4h, 8h and 24h after administration respectively.3.2 After nerve ligation, 64 neuropathic rats were divided into NO group and NS group, which were intrathecally injected with 20μg of NO -711 or normal saline respectively. Lumbar spinal cord of rats were removed before drug administration or on 15min, 0. 5h, lh, 2h, 4h, 8h and 24h after administration respectively to detect the protein level of GABAB1a, GABAB2 and GAT - 1.3.3 64 rats were divided into 4 groups ( n = 16) : Sham group, CCI group, NO group and NS group. Sciatic nerves were exposed but not ligated in Sham group, and rats were undergone nerve ligation in the other three groups. On the third day after operation, rats in NO group and NS group were intrathecally injected with 20μg of NO -711 or normal saline respectively, and then lumbar spinal cord of all the rats in the 4 groups were removed to detect Fos -like immunoreactive neurons, pERK immunoreactive neurons and protein contents of pERK.4. Data analysis and statistics. The same as that in Part One.Results1. Effects of intrathecal injection of NO - 711 on the TWL and MWT of neuropathic ratsCompared with ANS group and pre - drug values, TWL and MWT of rats in AN5, AN10, AN20 and AN40 group significantly increased (P <0. 05, P < 0.01) , and lasted for different time courses according to different doses of NO -711.2. Effects of intrathecal injection of NO -711 before sciatic nerve ligation on the TWL and MWT on rats with chronic constriction injuryCompared with those in BNS group, TWL in the BN5, BN10, BN20 and BN40 group did not decreased until 3-5 day after nerve ligation (P <0.05, P <0. 01) , but MWT in all the groups significantly decreased after operation.3. Effects of intrathecally injection of NO -711 on the expression of GAT -1, GABAB1a and GABAB2 of lumbar spinal cord on neuropathic ratsDuring 0. 5h ~ 8h after intrathecal injection with 20μg of NO - 711, expression of GAT - 1, GABAB1a and GABAB2 remarkably decreased in NO group (P<0.01,P <0. 05 ) , and then gradually increased. On 24h after injection, protein content of GABAB2 were significantly higher than those before intrathecal injection (P<0.05).4. Effects of intrathecally injection of NO -711 on the FOS -like immunoreactive neurons ( FLIN) and expression of pERK in lumbar spinal cord neurons on neuropathic ratsCompared with Sham group, the FLIN and pERK immunoreactive neurons in spinal cord significantly increased in CCI group ( P < 0. 01, P < 0. 05 ). Intrathecal injection of NO -711 decreased FLIN and pERK immunoreactive neurons in NO group. The expression of pERK on both nucleus and cytoplasm of lumbar spinal cord neurons increased in CCI group, nucleutic pERK but not cy-toplastic pERK significantly decreased in NO group.Conclusions1. There existed differences among expression of spinal GABAB receptor subtypes on rats with chronic constriction injury induced neuropathic pain. The expression of spinal GABAB1a and GABAB2 decreased while there were no changes of GABAB1b on neuropathic rats.2. Up - regulation of spinal GAT - 1 was involved in the forming of chronic constriction injury induced neuropathic pain on rats.3. Intrathecal injection of GABAB receptor agonist baclofen attenuated hyperalgesia of neuropathic rats with chronic constriction injury, in which down -regulation of spinal GAT - 1 may involved.4. Intrathecal injection of GAT - 1 inhibitor NO -711 significantly sup-...