An in vivo study of gene expressions during collateral sprouting accelerated by electrical stimulation in rat dorsal root ganglia

Hao, Yawei (1998) An in vivo study of gene expressions during collateral sprouting accelerated by electrical stimulation in rat dorsal root ganglia. Masters thesis, Memorial University of Newfoundland.

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Abstract

Previous studies have demonstrated that collateral sprouting in sensory neurons is an NGF dependent process, and that the onset of this sprouting can be accelerated by electrical stimulation (depolarization) of nerves, producing a precocious collateral sprouting. However, the mechanism underlying this phenomenon is not clear. -- In the present study, the mechanisms underlying precocious collateral sprouting were studied in adult rats in vivo. Electrical stimulation was performed on the intact cutaneous nerves at 8V, 20 HZ, 1 min. These intact nerves were isolated from the adjacent nerves by dissecting the nearby nerves. The intact dorsal cutaneous nerves were treated under three paradigms: i) electrical stimulation (S); ii) isolation of intact nerves (I); iii) electrical stimulation plus isolation (S+I). After varying periods of time (lh, 4h, 8h, Id, 2d, 4d, 8d, and 14d), the dorsal root ganglia (DRGs) connected with these nerves were dissected and the possible factors related to precocious sprouting were investigated in the DRG neurons using in situ hybridization (ISH), immunocytochemistry (ICC) and Western blot assays. The parameters examined included immediate early genes (IEGs), such as CREB, egr-1, c-fos, c-jun, and Oct-2; NGF receptors (Trk A and p75); and potential members of the NGF – Trk A signal transduction pathways inducing downstream signaling (PI3-kinase, SHC, PLC-y, ERK1). -- The results showed that, among IEGs, CREB mRNA was quickly induced after 1h electrical stimulation, and this increase lasted to 4d. The effects of isolation started at Id, and the combination of isolation plus stimulation resulted in this occurring sooner. At the protein level, the expression of pCREB was only significantiy increased under stimulation at 8h (p<0.05). After 4h, electrical stimulation started to induce the elevation of egr-1 mRNA and this induction lasted until 2d, but the protein level was significant increased only at 8h. Isolation, which would result in increased NGF levels in the skin due to the adjacent cutaneous denervation, did not induce significant changes in Egr-1 protein during the experimental period. Isolation plus stimulation shortened the duration of Egr-1 increase (at 1d) and this increase lasted to 4d. Except for electrical stimulation alone and isolation alone, isolation and stimulation together induced significant increases of Fos in DRGs at 2d and 4d. Stimulation did not have significant effects on Jun protein, but after 8h, isolation plus stimulation, respectively, resulted in significant increases in Jun protein compared with control. Oct-2 was not affected by any of the treatments in these experiments. -- Under the treatments of electrical stimulation and isolation, expression of Trk A receptor mRNA and protein showed different patterns in these experiments. The mRNA level of Trk A did not significantly increase after electrical stimulation; however, isolation alone resulted in a significant increase of TrkA mRNA and this increase reached a peak at 4d. Combined with electrical stimulation, isolation induced a large increase at a very early time point (1h), but this gradually declined at later time points (2d and 4d). -- The protein level of Trk A was only increased at lh and 4h stimulation time points. There was, however, an increase induced by isolation plus stimulation at later time points (4d and 8d). The phosphorylated state of Trk A receptor did not appear to be increased except at the isolation treatment at 1h and longer time points of 4d and 8d. With respect to p75, mRNA levels were altered little by electrical stimulation. Isolation alone induced a peak change at 2d. The combination of electrical stimulation and isolation resulted in increased expression of p75 by 4h, peaking at Id, and then gradually decreasing. -- Among the proteins which propagate NGF signals, PLC-yl was slightly induced by stimulation and isolation at the short time periods (1h, 4h) and the very late time point (8d). PI-3 kinase was increased only at the latest time point (8d) following treatments. SHC and MAP kinase (ERK1) were not obviously affected by any of these treatments. -- The results addressed the hypotheses that, during precocious collateral sprouting, electrical stimulation induces some alterations in IEG expression and elevated Trk A receptor expression and/or activation, and acts in concert with the increased availability of NGF to result in an accelerated terminal sprouting response. This study provided information about the potential mechanisms associated with precocious sprouting at the molecular level.

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