Yonsei Med J.  2015 Sep;56(5):1307-1315. 10.3349/ymj.2015.56.5.1307.

TWIK-Related Spinal Cord K+ Channel Expression Is Increased in the Spinal Dorsal Horn after Spinal Nerve Ligation

• Hwang HY
• Zhang E
• Park S
• Chung W
• Lee S
• Kim DW
• Ko Y
• Lee W

• KMID: 2163622
• DOI: http://doi.org/10.3349/ymj.2015.56.5.1307

Abstract

PURPOSE
The TWIK-related spinal cord K+ channel (TRESK) has recently been discovered and plays an important role in nociceptor excitability in the pain pathway. Because there have been no reports on the TRESK expression or its function in the dorsal horn of the spinal cord in neuropathic pain, we analyzed TRESK expression in the spinal dorsal horn in a spinal nerve ligation (SNL) model.
MATERIALS AND METHODS
We established a SNL mouse model by using the L5-6 spinal nerves ligation. We used real-time polymerase chain reaction and immunohistochemistry to investigate TRESK expression in the dorsal horn and L5 dorsal rot ganglion (DRG).
RESULTS
The SNL group showed significantly higher expression of TRESK in the ipsilateral dorsal horn under pain, but low expression in L5 DRG. Double immunofluorescence staining revealed that immunoreactivity of TRESK was mostly restricted in neuronal cells, and that synapse markers GAD67 and VGlut2 appeared to be associated with TRESK expression. We were unable to find a significant association between TRESK and calcineurin by double immunofluorescence.
CONCLUSION
TRESK in spinal cord neurons may contribute to the development of neuropathic pain following injury.

Keyword

TRESK protein; rat; neuralgia; pain

MeSH Terms

Animals
Disease Models, Animal
Hyperalgesia
Ligation
Male
Neuralgia/*metabolism/physiopathology
Neurons/metabolism
Nociceptors
Pain/metabolism/*physiopathology
Potassium Channels/*metabolism
Rats
Rats, Sprague-Dawley
Real-Time Polymerase Chain Reaction
Spinal Cord Dorsal Horn/*metabolism
Spinal Nerves/*injuries
Potassium Channels

Figure

• 

  Fig. 1 Mechanical allodynia after spinal nerve ligation (SNL). Paw withdrawal threshold in response to von Frey filament stimulation decreased from 6.67±0.94 g before surgery to 0.57±0.07 g 10 days after surgery. This mechanical allodynia persisted at least for 14 days (0.65±0.13 g). Sham-operated rats showed no signs of hypersensitivity. *p<0.001 vs. the corresponding contralateral side. co, contralateral to the injured hind paw; ips, ipsilateral to the injured hind paw.

• 

  Fig. 2-1 TRESK immunoreactivity was similar in the spinal dorsal horn of the sham group. The bilateral region of the sham group showed similar TRESK expression. Scale bars=100 µm in A; 50 µm in B and C. TRESK, TWIK-related spinal cord K+.

• 

  Fig. 2-2 TRESK immunoreactivity (IR) increased in the spinal dorsal horn of spinal nerve ligation (SNL) rats. Fourteen days following SNL, TRESK IR dramatically increased in the ipsilateral dorsal horn (C) vs. the contralateral side (B). Quantitative measurement of TRESK IR cells in the L5 spinal dorsal horn (D). The mean number of TRESK IR cells in a fixed area occupied in ipsilateral superficial laminae of the dorsal horn (dot-line) was significantly higher than their contralateral counterparts (*p<0.05) (mean±SEM, n=8). Scale bars=100 µm in A; 50 µm in B and C. TRESK, TWIK-related spinal cord K+.

• 

  Fig. 3 TRESK immunoreactivity was similar in the both DRG of sham group (A and B). 14 days after spinal nerve ligation (SNL), TRESK expression was dramatically reduced in the ipsilateral DRG (D) vs. the contralateral side (C). Scale bar=20 µm. TRESK, TWIK-related spinal cord K+; DRG, dorsal root ganglion.

• 

  Fig. 4 TRESK is expressed exclusively in neurons in the spinal dorsal horn 14 days after spinal nerve ligation (SNL). Double immunofluorescence staining in the ipsilateral lumbar spinal dorsal horn of TRESK (red; A and E) and NeuN, a neuronal marker (green; B) or GFAP, an astrocyte marker (green; F); TRESK was mainly located in neurons, but not astrocytes 14 days after SNL (C, D, G, H). Scale bar=20 µm. TRESK, TWIK-related spinal cord K+; GFAP, glial fibrillary acidic protein; DAPI, 40,6-diamidino-2-phenylindole.

• 

  Fig. 5 TRESK transcript expression increased in the dorsal horn of spinal cord of SNL rats. Quantitative real-time PCR of TRESK gene expression in the ipsilateral L5-6 spinal cord at 0, 7, 14, 21, and 28 days after SNL. Data are presented as the fold change from the control (naïve) mean±SEM, which represented normalized averages derived from the threshold cycles in 6 to 8 ipsilateral samples. *p<0.05 vs. control, †p<0.01 vs. control. TRESK, TWIK-related spinal cord K+; SNL, spinal nerve ligation; PCR, polymerase chain reaction.

• 

  Fig. 6-1 Immunofluorescence staining of TRESK (red) and GAD67 (green) in the spinal dorsal horn of SNL rats. TRESK expression is associated with inhibitory synapses. Scale bars=50 µm in A, B, and C; 20 µm in D, E, and F. TRESK, TWIK-related spinal cord K+; SNL, spinal nerve ligation.

• 

  Fig. 6-2 Immunofluorescence staining of TRESK (red) and VGlut2 (green) in the spinal dorsal horn of SNL rats. TRESK expression is associated with excitatory synapses. Scale bars=50 µm in A, B, and C, 20 µm in D, E, and F. TRESK, TWIK-related spinal cord K+; SNL, spinal nerve ligation.

• 

  Fig. 7 Immunofluorescence staining of TRESK (red) and calcineurin α (green) in the spinal dorsal horn of SNL rats. TRESK expression was not regulated exclusively by calcineurin in the ipsilateral L5-6 region of SNL rats. Scale bars=50 µm in A-D; 20 µm in E-H. TRESK, TWIK-related spinal cord K+; SNL, spinal nerve ligation.

• 

  Fig. 8 qPCR of calcineurin gene expression in the L5-6 ipsilateral lumbar enlargement of spinal cord at 0, 7, 14, 21, and 28 days after spinal nerve ligation. Data are presented as fold change from control mean±SEM, which represented normalized averages derived from the threshold cycles in 6 to 8 ipsilateral samples.

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