Study of reinnervation process in patients with 2 type of spinal muscular atrophy: Clinical experimental study

Abstract

OBJECTIVES: The aim of the research was to study the correlation between the level of NGF of blood serum and electroneuromyography data with impact of blood serum of patients with 2 type SMA on growth of dorsal root ganglion neurites of 10-12-day old chicken embryos in an organotypic tissue culture. METHODS: 10 patients with (2 type SMA) aged 8-12 years were examined. Electroneuromyography study was done to all of them. Study blood serum of the 10 patients was done in an organotypic tissue culture. Studies were performed on 500 explants sensory dorsal root ganglia of the chick embryo. The ganglia were cultured for three days on collagen supports in Petri dishes at 36.5 °C and 5% CO2. CO2 incubator (Sanyo, Japan) was used. In experimental dishes, blood serum of patients with 2 type SMA was added to the cultural medium. The growth of explants in tissue culture was controlled on vital preparations using a confocal laser scanning microscope LSM-710 (Carl Zeiss, Germany). The preparations were also visualized by a television microscope attachment Axiostar Plus (Carl Zeiss, Germany). Neurites outgrowth was quantified using the Image J program. The area index (AI) was used to estimate the neurite outgrowth. It was calculated in relative units as the ratio of total explant area to the central zone area. All values were taken as 100% in the control experiments. Level Beta-NGF in 10 patients (SMA 2 type) was determined in serum using Beta-NGF ELISA Kit (RayBiotech, Inc). RESULTS: According to electroneuromyography study in patients with 2nd type SMA reinnervation process was poor. It was found out that concentration NGF (nerve growth factor) in serum of patients with 2nd type SMA is considerably higher than in control group. We studied the influence of blood serum of patients with 2 type SMA on growth of neurites of dorsal root ganglia of 10-12 days old chicken embryos in organotypic culture. Blood serum of 12 patients with type 2 SMA was researched in a wide range of dilution (1:100-1:2). The plasma of patients in a dilution of 1:2-1:50 completely blocked the growth of dorsal root ganglion neurites. AIl of experimental explants cultured in the medium containing blood serum in dilution 1:70 was 25% less than the control value. CONCLUSIONS: For the first time it was shown that blood serum of patients with 2nd type SMA inhibits the growth of neurites of dorsal root ganglia in dose-dependent manner. Apparently, neurite-inhibitory effect is caused by high concentration of neurotrophins (NGF) in patient serum, and due to that the reinnervation in 2 type SMA patients is not efficient.