Are Spinal GABAergic Elements Related to the Manifestation of Neuropathic Pain in Rat?

Jaehee Lee; Seung Keun Back; Eun Jeong Lim; Gyu Chong Cho; Myung Ah Kim; Hee Jin Kim; Min Hee Lee; Heung Sik Na

doi:10.4196/kjpp.2010.14.2.59

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Lee, Back, Lim, Cho, Kim, Kim, Lee, and Na: Are Spinal GABAergic Elements Related to the Manifestation of Neuropathic Pain in Rat?

Original Article

Korean J Physiol Pharmacol 2010;14(2):59-69.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.2.59

Are Spinal GABAergic Elements Related to the Manifestation of Neuropathic Pain in Rat?

Jaehee Lee^1,^∗, Seung Keun Back^1,^∗, Eun Jeong Lim¹, Gyu Chong Cho¹, Myung Ah Kim¹, Hee Jin Kim², Min Hee Lee¹, Heung Sik Na^1,

¹Medical Science Research Center and Department of Physiology, Korea University College of Medicine, Seoul 136-705, Korea

²Department of Life Science, Yonsei University Wonju College of Science, Wonju 220-701, Korea

Corresponding to: Heung Sik Na, Department of Physiology, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Korea. (Tel) 82-2-920-6188, (Fax) 82-2-925-5492, (E-mail) hsna@korea.ac.kr

^∗ Jaehee Lee and Seung Keun Back contributed equally to the present work.

Received 11 November 2009 Revised 4 December 2009 Accepted 8 December 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Impairment in spinal inhibition caused by quantitative alteration of GABAergic elements following peripheral nerve injury has been postulated to mediate neuropathic pain. In the present study, we tested whether neuropathic pain could be induced or reversed by pharmacologically modulating spinal GABAergic activity, and whether quantitative alteration of spinal GABAergic elements after peripheral nerve injury was related to the impairment of GABAergic inhibition or neuropathic pain. To these aims, we first analyzed the pain behaviors following the spinal administration of GABA antagonists (1 μg bicuculline/rat and 5 μg phaclofen/rat), agonists (1 μg muscimol/rat and 0.5 μg baclofen/rat) or GABA transporter (GAT) inhibitors (20 μg NNC-711/rat and 1 μg SNAP-5114/rat) into naïve or neuropathic animals. Then, using Western blotting, PCR or immunohistochemistry, we compared the quantities of spinal GABA, its synthesizing enzymes (GAD65, 67) and its receptors (GABA_A and GABA_B) and transporters (GAT-1, and –3) between two groups of rats with different severity of neuropathic pain following partial injury of tail-innervating nerves; the allodynic and non-allodynic groups. Intrathecal administration of GABA antagonists markedly lowered tail-withdrawal threshold in naïve animals, and GABA agonists or GAT inhibitors significantly attenuated neuropathic pain in nerve-injured animals. However, any quantitative changes in spinal GABAergic elements were not observed in both the allodynic and non-allodynic groups. These results suggest that although the impairment in spinal GABAergic inhibition may play a role in mediation of neuropathic pain, it is not accomplished by the quantitative change in spinal elements for GABAergic inhibition and therefore these elements are not related to the generation of neuropathic pain following peripheral nerve injury.

Keywords: GABA, GAD65, GAD67, GAT-1, GAT-3, Peripheral nerve injury, Neuropathic pain

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Fig. 1.

A schematic diagram illustrating how the inferior (black arrow) and superior (open arrow) caudal trunks are composed and the level of the transection (X) of nerve trunks. The curved arrow indicates the S1 spinal nerve.

Fig. 2.

Changes in pain behaviors by pharmacological regulation of spinal GABAergic activity. (A) Mechanical allodynia induced by intrathecal administration of GABA antagonists, bicuculline and phaclofen, in naïve rats. (B) Attenuation of nerve injury-induced mechanical allodynia by GABA agonists, muscimol and baclofen, applied spinally. (C) Reversal of nerve injury-induced mechanical allodynia by intrathecally administered GAT inhibitors, NNC 711 for GAT-1 and SNAP 5114 for GAT-3. ‘Pre’ and ‘N14’ indicate 1 day before and 14 days after neuropathic surgery, respectively. ^∗p≤0.05, ^∗∗p≤0.001 vs. pre-injection value (One-way repeated measures ANOVA followed by Bonferroni t-test).

Fig. 3.

Western blot analysis for spinal GAD65 and GAD67 among the naïve, allodynic and non-allodynic animals. (A) Difference in the manifestation of neuropathic pain between the allodynic and non-allodynic groups following the same nerve injury, ^∗p≤0.001 vs. the naïve group; ^#p≤0.001 vs. the non-allodynic group (One-way ANOVA followed by all pairwise multiple comparison, utilizing Holm-Sidak method). (B) Immunoblots of two isoforms of GAD extracted from the S1 spinal segments (injured level) 2 weeks following neuropathic surgery. (C) The temporal courses of immunoreactivities of GAD65 and GAD67 in the ipsilateral hemi-cords. Data are normalized to β-actin and expressed as a ratio to naïve. GAD65-and GAD67-ir in the ipsilateral hemicords of the allodynic and non-allodynic groups are not different from those of the naïve group on any postoperative day.

Fig. 4.

Reverse transcription-PCR analysis for GAD65 and GAD67 mRNA expression in the naïve, allodynic and non-allodynic groups 2 weeks following neuropathic surgery. Behavioral differences among the groups are illustrated in Fig. 3A. (A) GAD65 and GAD 67 mRNA expression in the S1 spinal segments excised from the naïve, allodynic and non-allodynic animals. ATF3 mRNA expression in the S1 DRG was adopted as a marker of nerve injury. (B) Ipsilateral expression of GAD65 and GAD67 mRNA. Data are normalized to GAPDH and expressed as a ratio to naïve. Expressions of GAD65- and GAD67 mRNA in the ipsilateral hemi-cords of the allodynic and non-allodynic groups are not different from those of the naïve group.

Fig. 5.

Immunohistochemial analysis for spinal GAD65 and GAD67 among the naïve, allodynic and non-allodynic animals 2 weeks following neuropathic surgery. (A) Difference in the manifestation of neuropathic pain between the allodynic and non- all-odynic groups, ^∗p<0.05 vs. Pre-surgical value (One-way repeated ANOVA followed by Bonferroni t-test); ^#p<0.05 vs. non-allodynic group (Unpaired t-test). (B) Microphotographs illustrating distribution of GAD65- and GAD67-ir in the S1 spinal segment ipsilateral to nerve injury. Scale bar=100 μm. (C) Relative staining intensities of GAD65 and GAD67, compared to the naïve group. GAD65- and GAD67-ir in both the allodynic and non-allodynic groups are not different from those of the naïve group.

Fig. 6.

Immunohistochemial analysis for spinal GABA and its receptors, GABAA and GABAB, among the naïve, allodynic and non-allodynic animals 2 weeks following neuropathic surgery. Behavioral differences among the groups are illustrated in Fig. 5A. (A) Microphotographs illustrating distribution of GABA-, GABA_A and GABA_B-ir in the S1 spinal segment ipsilateral to nerve injury. Scale bar=100 μm. (B) Relative staining intensities of GABA, GABA_A and GABA_B, compared to the naïve group. Immunoreactivities in both the all-odynic and non-allodynic groups are not different from those of the naïve group.

Fig. 7.

Immunohistochemial analysis for spinal GABA transporters, GAT-1 and GAT-3 among the naïve, all-odynic and non-allodynic animals 2 weeks following neuropathic surgery. Behavioral differences among the groups are illustrated in Fig. 5A. (A) Microphotographs illustrating distribution of GAT-1- and GAT-3-ir in the S1 spinal segment ipsilateral to nerve injury. Scale bar=100 μm. (B) Relative staining intensities of GAT-1-and GAT-3 as compared to the naïve animals. Immunoreactivities in both the allodynic and non-allodynic groups are not different from those of the naïve group.

Fig. 8.

Reverse transcription-PCR analysis for GABA transporters, GAT-1 and GAT-3, mRNA expression in the naïve, allodynic and non-allodynic groups 2 weeks following neuropathic surgery. Behavioral differences among the groups are illustrated in Fig. 3A. (A) GAT-1 and GAT-3 mRNA expressions in the S1 spinal segments excised from the naïve, allodynic and non-allodynic animals. ATF3 mRNA expression in the S1 DRG was adopted as a marker of nerve injury. (B) Ipsilateral expression of GAT-1 and GAT-3 mRNA. Data are normalized to GAPDH and expressed as a ratio to the naïve animals. Expressions of GAT-1 and GAT-3 mRNA in the ipsilateral hemi-cords of the allodynic and non-allodynic groups are not different from those of the naïve group.

Table 1.

Sequences of primers used for RT-PCR

Gene	Forward	Reverse
Rat GAPDH	5′-GAGTCTACTGGCCTCTTCAC-3′	5′-CCATCCACAGTCTTCTGAGT-3′
Rat GAD65	5′-CCTATGAGATCGCCCCTGTA-3′	5′-ATCAAAAGCCCCGTACACAG-3′
Rat GAD67	5′-GCGAGAGATTGGATGGT-3′	5′-TCTATGCCGCTGAGTTTGTG-3′
Rat GAT1	5′-GAGGCATCTACCTGCTCAGTG-3′	5′-ACCTCCTGCCTTCATCCTAAA-3′
Rat GAT3	5′-AACAACAAGGTGGAGTTCGTG-3′	5′-CATAGCCGATGCCTTCAAATA-3′
Rat ATF3	5′-GCAGGAGCATCCTTTGTCTC-3′	5′-CTGATGAAACTCCCGGAAAA-3′

Table 2.

Postoperative changes in spinal levels of GABA synthesizing enzymes, GAD65 and GAD67. Data are expressed as percentage of naïve animals. POD indicates postoperative day. When compared with naïve animals, significant loss of spinal GAD65 or GAD67 was observed in neither ipsilateral nor contralateral side of both of the allodynic and non-allodynic groups at any postoperative day

		POD 1		POD 7		POD 14
		Ipsilateral	Contralateral	Ipsilateral	Contralateral	Ipsilateral	Contralateral
GAD65	Allodynic	96.79±8.33	98.50±7.97	101.67±5.49	102.06±6.24	95.67±2.03	96.63±3.07
	Non-allodynic	97.71±4.90	95.83±7.05	98.47±3.35	93.22±6.13	98.65±3.58	102.92±2.45
GAD67	Allodynic	99.71±3.50	105.34±9.99	106.38±9.76	108.08±8.47	103.46±4.83	105.09±4.94
	Non-allodynic	107.90±4.49	106.38±6.01	107.94±2.42	105.49±3.46	103.38±5.34	107.21±5.75

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