Korean J Pain.  2011 Dec;24(4):185-190. 10.3344/kjp.2011.24.4.185.

Tumor Necrosis Factor-alpha and Apoptosis Following Spinal Nerve Ligation Injury in Rats

• Kim SH
• Nam JS
• Choi DK
• Koh WW
• Suh JH
• Song JG
• Shin JW
• Leem JG

• KMID: 2074012
• DOI: http://doi.org/10.3344/kjp.2011.24.4.185

Abstract

BACKGROUND
Spinal nerve ligation (SNL) injury in rats produces a pain syndrome that includes mechanical and thermal allodynia. Previous studies have indicated that proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) play an important role in peripheral mediation of neuropathic pain, and that altered dorsal root ganglion (DRG) function and degree of DRG neuronal apoptosis are associated with spinal nerve injury. The present study was conducted to evaluate the expression of TNF-alpha and the extent of apoptosis in the dorsal root ganglion after SNL in rats.
METHODS
Sprague-Dawley rats were subjected to SNL of the left L5 and L6 spinal nerves distal to the DRG and proximal to the formation of the sciatic nerve. At postoperative day 8, TNF-alpha protein levels in the L5-6 DRG were compared between SNL and naive groups using ELISA. In addition, we compared the percentage of neurons injured in the DRG using immunostaining for apoptosis and localization of activated caspase-3.
RESULTS
SNL injury produced significant mechanical and cold allodynia throughout the 7-day experimental period. TNF-alpha protein levels were increased in the DRG in rats that had undergone SNL (12.7 +/- 3.2 pg/100 microg, P < 0.001) when compared with naive rats (4.1 +/- 1.4 pg/100 microg). The percentage of neurons or satellite cells co-localized with activated caspase-3 were also significantly higher in rats with SNL than in naive rats (P < 0.001, P < 0.05, respectively).
CONCLUSIONS
SNL injury produces mechanical and cold allodynia, as well as TNF-alpha elevation and apoptosis in the DRG.

Keyword

apoptosis; dorsal root ganglion; neuropathic pain; tumor necrosis factor alpha

MeSH Terms

Animals
Apoptosis
Caspase 3
Cold Temperature
Cytokines
Diagnosis-Related Groups
Enzyme-Linked Immunosorbent Assay
Ganglia, Spinal
Hyperalgesia
Ligation
Negotiating
Neuralgia
Neurons
Rats
Rats, Sprague-Dawley
Sciatic Nerve
Spinal Nerves
Tumor Necrosis Factor-alpha
Caspase 3
Cytokines
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Behavioral tests for mechanical (left axis) and cold allodynia (right axis). Mechanical allodynia was tested by application of von Frey filaments, and cold allodynia by application of acetone. Data are mean ± SD and *P < 0.05 compared with baseline (before surgery).

• 

  Fig. 2 Double immunofluorescence staining of activated caspase-3 and cell markers in the rat dorsal root ganglion (DRG). Tissue samples from the naïve and spinal nerve ligation (SNL) groups were immunostained with caspase-3 (B, E, H, K), an indicator of apoptosis, and cell markers, such as NeuN (A, D) and GFAP (G, J), and the co-localization of caspase-3 was visualized in a merged image (C, F, I, L). (A-F) Immunostaining for caspase-3 with NeuN in naïve and SNL DRG; (G-L) Immunostaining for caspase-3 with GFAP in naïve and SNL DRG. Note that NeuN is a marker of neurons, and GFAP is a marker of activated astrocytes. Examples of apoptotic cells are marked with white arrows.

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  Fig. 3 Quantitative analysis of different phenotypic-specific markers in positive cells expressing caspase-3 (%) in naïve and SNL groups. The change in caspase-3 was striking in astrocytes and neurons. *,†Indicate significant difference at P < 0.05 compared with naïve group. Error bars represent SEM.

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