J Korean Med Sci.  2015 Dec;30(12):1902-1910. 10.3346/jkms.2015.30.12.1902.

Changes in the Expressions of Iba1 and Calcitonin Gene-Related Peptide in Adjacent Lumbar Spinal Segments after Lumbar Disc Herniation in a Rat Model

• Cho HK
• Ahn SH
• Kim SY
• Choi MJ
• Hwang SJ
• Cho YW

• KMID: 2359978
• DOI: http://doi.org/10.3346/jkms.2015.30.12.1902

Abstract

Lumbar disc herniation is commonly encountered in clinical practice and can induce sciatica due to mechanical and/or chemical irritation and the release of proinflammatory cytokines. However, symptoms are not confined to the affected spinal cord segment. The purpose of this study was to determine whether multisegmental molecular changes exist between adjacent lumbar spinal segments using a rat model of lumbar disc herniation. Twenty-nine male Sprague-Dawley rats were randomly assigned to either a sham-operated group (n=10) or a nucleus pulposus (NP)-exposed group (n=19). Rats in the NP-exposed group were further subdivided into a significant pain subgroup (n=12) and a no significant pain subgroup (n=7) using mechanical pain thresholds determined von Frey filaments. Immunohistochemical stainings of microglia (ionized calcium-binding adapter molecule 1; Iba1), astrocytes (glial fibrillary acidic protein; GFAP), calcitonin gene-related peptide (CGRP), and transient receptor potential vanilloid 1 (TRPV1) was performed in spinal dorsal horns and dorsal root ganglions (DRGs) at 10 days after surgery. It was found immunoreactivity for Iba1-positive microglia was higher in the L5 (P=0.004) dorsal horn and in the ipsilateral L4 (P=0.009), L6 (P=0.002), and S1 (P=0.002) dorsal horns in the NP-exposed group than in the sham-operated group. The expression of CGRP was also significantly higher in ipsilateral L3, L4, L6, and S1 segments and in L5 DRGs at 10 days after surgery in the NP-exposed group than in the sham-operated group (P<0.001). Our results indicate that lumbar disc herniation upregulates microglial activity and CGRP expression in many adjacent and ipsilateral lumbar spinal segments.

Keyword

Multisegmental; Neural Pathways; Lumbar Disc Herniation; Microglia; Calcitonin Gene-Related Peptide; Neuropathic Pain

MeSH Terms

Animals
Astrocytes/metabolism
Calcitonin Gene-Related Peptide/*metabolism
Calcium-Binding Proteins/*metabolism
Disease Models, Animal
Ganglia, Spinal/metabolism
Humans
Immunohistochemistry
Intervertebral Disc Displacement/*metabolism
Lumbar Vertebrae/*metabolism
Male
Microfilament Proteins/*metabolism
Microglia/metabolism
Neuralgia/metabolism
Rats
Rats, Sprague-Dawley
Spinal Cord Dorsal Horn/metabolism
Up-Regulation
Calcitonin Gene-Related Peptide
Calcium-Binding Proteins
Microfilament Proteins

Figure

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  Fig. 1 Segmental acquisition of spinal cord.

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  Fig. 2 Immunohistochemical staining of Iba1 in ipsilateral L3, L4, L5, L6, and S1 dorsal horns and relative area fractions of Iba1 immunoreactions compared to sham-operated controls (sham) at 10 days after surgery. (A) Iba1-positive microglia showed an increasing tendency in ipsilateral dorsal horns at multisegmental in the significant pain (Pain) and no significant pain subgroups (nPain). (B) In the NP-exposed group, increased immunoreactions of Iba1-positive microglia were noted in L5 dorsal horns (the NP implantation level) and in ipsilateral L4, L6, and S1 dorsal horns as compared with sham-operated controls. Results are presented as means±SEMs. *P<0.05 vs. sham-operated controls; bar = 100 µm.

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  Fig. 3 Immunohistochemical staining of CGRP in ipsilateral L3, L4, L5, L6, and S1 dorsal root ganglions (DRGs) and relative cell counts of CGRP-positive DRG cells compared to sham-operated controls (sham) at 10 days after surgery. (A) CGRP expression showed increasing tendencies in DRGs at several segments in the significant pain (Pain) and no significant pain subgroups (nPain). (B) In the NP-exposed group, increased CGRP expression was noted in L5 DRGs (the implantation level), and in ipsilateral L3, L4, L6, and S1 DRGs as compared with the sham-operated controls. Furthermore, significantly more CGRP expression was observed in L5 DRGs in the significant pain subgroup than in the no significant pain subgroup. Results are presented as means±SEMs. *P<0.05 vs. sham-operated group; †P<0.05 vs. the no significant pain subgroup. Bar=100 µm.

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  Fig. 4 Immunohistochemical staining of GFAP in ipsilateral L3, L4, L5, L6, and S1 dorsal horns and relative area fractions of GFAP immunoreactions compared to sham-operated controls (sham) at 10 days after surgery. (A) Red stained cells were more in the Pain subgroup, but not significant. (B) The relative area fractions of GFAP immunoreactions were similar in the significant pain subgroup (Pain), the no significant pain (nPain) subgroup, and sham-operated controls (Sham). Results are presented as means±SEMs. Bar = 100 µm.

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  Fig. 5 Immunohistochemical staining of TRPV1 in ipsilateral L3, L4, L5, L6, and S1 dorsal root ganglions (DRGs) and relative cell counts of TRPV1-positive DRG cells compared to sham operated controls (sham) at 10 days after surgery. (A) TRPV1 expressions were similar in the significant pain subgroup (Pain), the no significant pain subgroup (nPain), and sham-operated controls (Sham). (B) Results are presented as means±SEMs. Bar = 100 µm.

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