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Intrathecal Administration of Methylene Blue Reduces Pain Behavior in Neuropathic Rats
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IntrathecalAdministration of Evans Blue Reduces Pain Behavior in Neuropathic Rats

Ok HwaYoon, Jin PyoHong*, Dong WoonKim*, and Won HyungLee

Department of Anesthesiology and Pain Medicine, Anatomy*, ChungnamNational University Hospital, Daejeon, South Korea




Neuropathic pain induced by spinal or peripheral nerve injury is highly resistant to common pain killers, nerve block, and other pain management approaches. Recently, several new  therapeutic drug candidates are being developed to control neuropathic pain. In this study, we used the spinal nerve L5 ligation (SNL) model to investigate the ability of intrathecal  evans  blue to decrease pain behavior, and to study the relationship between  evans  blue and the neural structure of pain transmission




Neuropathic pain (allodynia) of the left hind paw was induced by unilateral SNL in Sprague-Dawley rats (n = 10) in each group. Evans blue (5, 15, 50 μg/10 μl) or phosphate buffer saline (PBS, 10 μl) was injected intrathecally at 7 days post-ligation. Mechanical sensitivity was assessed using Von Frey filaments at 7 days post-ligation, and 2 hours, on days 1, 2, 3, 5, 7 after intrathecal evans blue injection.  Microglia and glutaminergic neurons in the dorsal horn and VNUT (vesicular nucleotide transporter) in the dorsal root ganglia were tested to evaluate co-staining with evans blue. The experimental procedures were performed in accordance with the animal care guideline of the Korean Academy of Medical Science (Animal ethic committee of Chungnam National University Hospital: CNUH-014-A0005-1).





 Tight ligation of the L5 spinal nerve induced allodynia in the left hind paw on 7 days post-ligation. Intrathecal evans blue most significantly (P <0.001) alleviated allodynia at 2 days after intrathecal injection.  Glutaminergic neuron in the dorsal horn and VNUT in the dorsal root ganglia were co-stained with evans blue. On the other hand, microglia in the dorsal horn was partially co-stained with evans blue.  




We confirmed that  evans  blue may have an analgesic effect in neuropathic pain of the SNL animal model. These results suggest  evans blue may be a potential new drug for the treatment of chronic pain.


This research was supported by the National Research Foundation of Korea (NRF-2014R1A1A2056019) funded by the Ministry of Education. Patent No is 10-2016-0182639.


Fig1. The change of mechanical threshold  after intrathecalevans blue injection


Fig 2. Glutaminergicneurons(A: neurons, B: vGlut2) in the dorsal horn were co-stained with evansblue, but were not co-stained  in inhibitory neurons (C: GABA, D: calretinin).


Fig 3.Microglia (A: IBa1) in the dorsal horn was partially co-stained with evansblue, but were not co-stained in astrocytes (B: GFAP).


Fig 4.VNUT (vesicular nucleotide transporter)  in the dorsal root ganglia (A) and axon (B) were co-stained with evansblue.


Fig 5. The suspected targets of evans blue in the control of neuropathic pain.


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