Different spatial and temporal protein expressions of repulsive guidance molecule a and neogenin in the rat optic nerve after optic nerve crush with and without lens injury

Abstract

The failure of lesioned mammalian CNS neurons to regenerate their axons remains a challenge. Evidence is emerging that repulsive proteins contribute to this failure. The repulsive guidance molecule A (RGMA) induces growth cone collapse in vitro, accumulates in the scar after spinal cord injury, and is up-regulated in glaucoma. In this study, we evaluated the spatial and temporal localization pattern of RGMA and its receptor neogenin in the optic nerve after optic nerve crush (ONC) without or with lens injury (LI) at up to nine time points (6 hr to 20 days) postsurgery by performing immunohistochemistry and Western blots. We found RGMA at the crush site (CS) and in the developing scar of ONC rats at every time point investigated, whereas it was absent in the CS of ONC + LI rats. Independent of the model, many cells were RGMA(+) in the ON: nerve fibers, blood vessels, astrocytes, oligodendrocytes, some microglia, some macrophages, and the sheath of the ON. Western blots showed a significantly lowered amount of RGMA in ONC + LI animals at 2, 4, and 6 days after crush compared with ONC animals. Furthermore, LI in sham-operated animals showed an increase of RGMA in six of eight time points compared with the sham-operated animals. Moreover, the effects of LI on the morphology of the ON were characterized at a level of detail never reported before. Our results show that RGMA is present and might contribute to the inhibitory environment in the ON, especially in and around the CS after ONC.

RGMA and neogenin protein expression are influenced by lens injury following optic nerve crush in the rat retina

“…We localized RGMA at the crush site immediately after ONC alone, and RGMA remained present from there on, whereas it was always absent in the ONC + LI animals [25]. In all investigated groups (including control and sham animals), many cells were found to be RGMA+ in the ON: nerve fibers, blood vessel endothelial cells, astrocytes, oligodendrocytes, some microglial cells, and the shaft of the optic nerve.…”

“…We recently showed that RGMA was present at the crush site after optic nerve crush (ONC) and absent after ONC + lens injury (LI). RGMA was co-localized with nerve fibers, blood vessels, shaft of the ON, astrocytes, oligodendrocytes, and some microglial cells [25]. Moreover, we showed a significantly lowered amount of RGMA in ONC plus LI animals at 2, 4, and 6 days after crush compared to ONC animals [25].…”

“…Surgical procedures were performed as described previously [25]. Briefly, adult male Wistar rats (8-10 weeks old) (Harlan Winkelmann, Germany) were deeply anesthetized by an intraperitoneal injection of a mixture of 100 mg/kg ketamine (CEVA, Germany) and 10 mg/kg xylazine (CEVA, Germany).…”

“…Frozen sections were longitudinally cut (10-12 μm) on a cryostat (Leica CM 1900, Germany), thaw-mounted onto glass slides (Superfrost plus, R.Langenbrinck Labor-und Medizintechnik, Germany) and stored at −30°C until further use. Immunostaining was performed using standard procedures and described previously [25]. In double-labeling experiments, sections were labeled with antibodies against RGMA or neogenin together with one of the following antibodies: CD68, CD11b, CD45RC, GFAP, neurofilament, CD31, and anti-laminin.…”

“…Always two retinae were pooled. Western blots and quantification protocols were performed as previously described [25,34]. Tissues were homogenized with a Dounce homogenizer (Carl Roth, Germany) in RIPA buffer (50 mM Tris-Hcl, pH 8; 150 mM NaCl; 1% NP-40; 0,1% SDS; 1:100 Protease inhibitor cocktail Set III, Calbiochem, Germany) and spun at 3,200 rpm for 2 min at 4°C.…”