(-)-Epigallocatechin Gallate Attenuates Spinal Motoneuron Death Induced by Brachial Plexus Root Avulsion in Rats

Background EGCG bene�ts a variety of insults. Methods Rats were randomized into: EGCG, Avulsion, and Sham. Results P-c-Jun-positive motoneurons and the ratio of p-c-Jun/c-Jun were signi�cantly lower in EGCG group at 3d and 7d after injury. Conclusions EGCG protected motoneurons


Introduction
The survival of spinal motoneurons is the key to the recovery of motor function [1].we explored whether EGCG administration had any bene cial effects on the motoneurons.

Animal Preparation
Rats were randomized into: EGCG, Avulsion, and Sham .The animals in EGCG group received daily doses of 100 mg/kg EGCG (Hangzhou Gosun Biotechnologies Co., Ltd., China) from day 1 to day 7 as described in previous study [2].
At the end of each survival time, animals were deeply anesthetized with a lethal dose of ketamine and xylazine and perfused transcardially with normal saline followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4).Every fth section from each animal was used for Nissl staining and immunohistochemisty analysis.

Immunohistochemical staining
An avidin-biotin kit was used for the immunohistochemical IHC staining.Brie y, the sections were depara nized and treated with 3% H2O2 for 15 min to block the endogenous peroxidase.The sections were exposed to normal bovine serum for 30 min and then incubated with the primary antibodies overnight at 4°C.

Statistical Analyses
The data are presented as the mean ± SD and were analyzed using statistical software IBM SPSS version 20.0 (IBM Corp. Released 2011.IBM SPSS Statistics for Windows, Version 20.0.Armonk, NY: IBM Corp.).

Survival of the Injured Motoneurons
Signi cant decreases of motoneurons in the spinal cords were observed from days 14 to 28 after the injury.

Apoptosis in Motoneurons
The number of caspase-3-positive motoneurons was signi cantly increased in the Avulsion group compared to the Sham group at 3d, 7d, 14d and 28d after the injury.

Effects of EGCG on phospho-JNK expression
The numbers of p-JNK-positive motoneurons were signi cantly increased in the Avulsion group compared to the Sham group at 3d and 7d after injury.

Effect of EGCG on phospho-c-Jun expression
At 3d, 7d and 14d after injury, the numbers of p-c-Jun-positive motoneurons were signi cantly increased in the Avulsion group compared with the Sham group.

Discussion
The data on astrocyte and microglia activity after brachial plexus root avulsion is described in previous publications.Although roots repair is unable to completely hampers neurodegeneration reimplantation of avulsed roots is neuroprotective by itself [9].Motoneuron survival after root repair is based on neurotrophic factor production by glial cells at the CNS/PNS interface [9].This has also been connected to the breakage of the blood-brain barrier, allowing neurotrophic factor producing immune cells to enter spinal cord gray matter environment [10].
Clinically, patients with brachial plexus injuries suffer multiple injuries that delay treatments such as surgical repair or reimplantation [11].Therefore, available neuroprotective treatments should be offered as early as it is safe to do so and the search for neuroprotective treatments must be expedited to prevent permanent loss of function among patients.
Therefore, we chose a 100 mg/kg does of EGCG for the treatment of brachial plexus avulsion in the current study.In the present study, we found that the motoneurons were protected by EGCG against the death induced by brachial plexus root avulsion as evidenced by our present data.

Conclusions
We observed that the motoneurons were protected against death by EGCG.Western blot analyses of the JNK and phospho-JNK levels in the ipsilateral spinal segments following root avulsion of the left brachial plexuses of adult rats.(A) The samples were obtained from rats that were subjected to sham operations or root-avulsions at 3d, 7d, 14 d and 28d post-injury.The optical density(OD) of each protein was measured from the Western blot.(B,C) Semiquantitative changes in the level of phospho-JNK normalized to JNK expression after root-avulsion were determined by OD measurements.The data are presented as the means±the SDs (n=5 every group, *P<0.05 between the Sham and Avulsion groups; D3, 3 day after injury; D7, 7 days after injury; D14, 14 days after injury; D28, 28 days after injury).Western blot analyses of the c-jun and phospho-c-jun levels in the ipsilateral spinal segments following root avulsion of the left brachial plexuses of adult rats.(A) The samples were obtained from rats that were subjected to sham operations or root-avulsions at 3d, 7d, 14 d and 28d post-injury.The OD of each protein was measured from the Western blot.(B,C) Semiquantitative changes in the level of phospho-cjun normalized to c-jun expression after root-avulsion were determined by OD measurements.The data are presented as the means±the SDs (n=5 every group, *P<0.05 between the Sham and Avulsion groups, #P<0.05 between the Avulsion and EGCG groups; D3, 3 day after injury; D7, 7 days after injury; D14, 14 days after injury; D28, 28 days after injury)