Yonsei Med J.  2013 Mar;54(2):321-329. 10.3349/ymj.2013.54.2.321.

Intrathecal Lamotrigine Attenuates Mechanical Allodynia and Suppresses Microglial and Astrocytic Activation in a Rat Model of Spinal Nerve Ligation

• Choi YS
• Jun IG
• Kim SH
• Park JY

• KMID: 1503892
• DOI: http://doi.org/10.3349/ymj.2013.54.2.321

Abstract

PURPOSE
Lamotrigine, a novel anticonvulsant, is a sodium channel blocker that is efficacious in certain forms of neuropathic pain. Recently, microglial and astrocytic activation has been implicated in the development of nerve injury-induced neuropathic pain. We have assessed the effects of continuous intrathecal administration of lamotrigine on the development of neuropathic pain and glial activation induced by L5/6 spinal-nerve ligation in rats.
MATERIALS AND METHODS
Following left L5/6 spinal nerve ligation (SNL), Sprague-Dawley male rats were intrathecally administered lamotrigine (24, 72, or 240 microg/day) or saline continuously for 7 days. Mechanical allodynia of the left hind paw to von Frey filament stimuli was determined before surgery (baseline) and once daily for 7 days postoperatively. On day 7, spinal activation of microglia and astrocytes was evaluated immunohistochemically, using antibodies to the microglial marker OX-42 and the astrocyte marker glial fibrillary acidic protein (GFAP).
RESULTS
Spinal-nerve ligation induced mechanical allodynia in saline-treated rats, with OX-42 and GFAP immunoreactivity being significantly increased on the ipsilateral side of the spinal cord. Continuously administered intrathecal lamotrigine (240 microg/day) prevented the development of mechanical allodynia, and lower dose of lamotrigine (72 microg/day) ameliorated allodynia. Intrathecal lamotrigine (72 and 240 microg/day) inhibited nerve ligation-induced microglial and astrocytic activation, as evidenced by reduced numbers of cells positive for OX-42 and GFAP.
CONCLUSION
Continuously administered intrathecal lamotrigine blocked the development of mechanical allodynia induced by SNL with suppression of microglial and astrocytic activation. Continuous intrathecal administration of lamotrigine may be a promising therapeutic intervention to prevent neuropathy.

Keyword

Astrocytes; intrathecal; lamotrigine; microglia; neuropathic pain

MeSH Terms

Animals
Astrocytes/drug effects/*physiology
Disease Models, Animal
Hyperalgesia/*drug therapy
Infusions, Spinal
Ligation
Male
Microglia/drug effects/*physiology
Neuralgia/drug therapy
Rats
Rats, Sprague-Dawley
Spinal Nerves/*injuries
Triazines/administration & dosage/*therapeutic use
Voltage-Gated Sodium Channel Blockers/administration & dosage/*therapeutic use
Triazines
Voltage-Gated Sodium Channel Blockers

Figure

• 

  Fig. 1 Changes in mechanical sensitivity to von Frey filaments after left L5/6 spinal-nerve ligation and the effect of continuous intrathecal administration of lamotrigine (24, 72, and 240 µg per day) for 7 days after spinal nerve ligation. *p<0.05, †p<0.01 compared with control by one-way analysis of variance followed by Tukey's test (n=6 per group), ‡p<0.01 compared with baseline by one-way repeated-measures analysis of variance followed by Tukey's test (n=6 per group).

• 

  Fig. 2 The effect of continuous intrathecal administration of lamotrigine (24, 72, and 240 µg per day) for 7 days on rotarod performance time. Rotarod performance time was measured before (baseline) and after spinal nerve ligation. The results are expressed as mean±SEM. SEM, standard error of measurement.

• 

  Fig. 3 Effect of lamotrigine on spinal immunoreactivity to OX-42 after left L5/6 spinal-nerve ligation. Rats were continuously administered 24, 72, and 240 µg per day lamotrigine or intrathecal saline for 7 days after spinal nerve ligation. Prominent microglial activation was observed in the ipsilateral side spinal cord of rats in the control group. No group showed significant changes in the contralateral spinal cord. Nerve ligation-induced microglial activation was markedly suppressed by intrathecal administration of 72 and 240 µg lamotrigine per day. Scale bar: 100 µm. *p<0.05 compared with the sham group, †p<0.05 compared with the control group, ‡p<0.05 compared with the LTG72 group, by one-way analysis of variance followed by Tukey's test (n=6 per group).

• 

  Fig. 4 Effect of lamotrigine on spinal immunoreactivity to GFAP after left L5/6 spinal-nerve ligation. Rats were continuously administered 24, 72, and 240 µg per day lamotrigine or intrathecal saline for 7 days after spinal nerve ligation. Prominent astrocytic activation was observed in the ipsilateral side spinal cord of rats in the control group. No group showed significant changes in the contralateral side spinal cord. Nerve ligation-induced astrocytic activation was markedly suppressed by 72 and 240 µg intrathecal lamotrigine. Scale bar: 100 µm. *p<0.05 compared with the sham group, †p<0.05 compared with the control group, by one-way analysis of variance followed by Tukey's test (n=6 per group). GFAP, glial fibrillary acidic protein.

• 

  Fig. 5 (A) Immunohistochemical localization of microglia (OX-42; green), phospho-p38 (p-p38; red) and DAPI. Scale bars, 100 µm for the 100× and 200× images; 50 µm for the 400× images. p-p38: phosphorylated p38 mitogen-activated protein kinase. (B) Immunohistochemical localization of microglia (OX-42; green), phospho-p38 (p-p38; red) and phospho-p44/42 (p-ERK; red). Signals for microglia and p-p38 merged, whereas signals for microglia and p-p44/42 (p-ERK) did not. Scale bars, 100 µm for the 100× and 200× images, 50 µm for the 400× images. p-p38: phosphorylated p38 mitogen-activated protein kinase. p-ERK, phosphorylated extracellular signal regulated kinase; DAPI, 4',6-diamidino-2-phenylindole.

• 

  Fig. 6 (A) Immunohistochemical localization of astrocytes (GFAP; green), phospho-p38 (p-p38; red) and DAPI. Scale bars, 100 µm for the 100× and 200× images, 50 µm for the 400× images. (B) Immunohistochemical localization of astrocytes (GFAP; green), phospho-p38 (p-p38; red) and phospho-p44/42 (p-ERK; red). Signals for astrocytes and p-ERK merged, whereas signals for microglia and p-p38 did not. Scale bars, 100 µm for the 100× and 200× images, 50 µm for the 400× images. GFAP, glial fibrillary acidic protein; p-p38, phosphorylated p38 mitogen-activated protein kinase; p-ERK, phosphorylated extracellular signal regulated kinase; DAPI, 4',6-diamidino-2-phenylindole.

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