The Changes Of Motoneurons Innervating The Gastrocnemius In Rats With Congenital Clubfoot By Neuroanatomical Tracing

ObjectiveCongenital clubfoot (CCF) is one of the most common congenital foot malformations in pediatric orthopedics with the incidence of about 1 in every 1000 live births. It seriously influences the morphology and function of the feet. The basic components of clubfoot are equinus, varus, adduction of forefoot and internal tibial torsion. Some theories have been proposed regarding the cause of clubfoot, such as primary soft tissue abnormalities within the neuromuscular units, gene abnormality, soft tissue contracture and primary bone germ abnormality. Nowadays, many scholars support the neuromuscular abnormalities, they think that the primary pathological change is the imbalance of muscle strength which cause secondary bony changes of foot and soft tissue contracture. We established animal model of congenital clubfoot and study the quantity and morphology of motoneurons innervating the gastrocnemius in rats with congenital clubfoot by neuroanatomical tracing in order to provid some evidences to the etiological theory of neuromuscular abnormalities.MethodsThere are 29 female Wistar rats in our experiment, retinoic acid (RA) -administration group has 15 pregnant rats while the control group has 14 pregnant rats. Congenital clubfoot malformation is induced in fetuses by gavage feeding of all - trans Retinoic acid (120mg/kg body weight) to the pregnant Wistar rats at day 10 of gestation. The control group were gavage fed mineral oil of thesame volume only at the same time. At the 20th day of gestation, fetal surgery was performed to the hindlimb gastrocnemius of both the congenital clubfoot rats and normal rats and injected 5% Fluorogold ( FG) by micro - injection. Fetuses were collected 24 hours later, douched by 0. 9% sodium chloride and fixed by 4% paraformaldehyde. The spinal cords were removed under the microscope for frozen section. The quantities, morphology and distribution of the motoneurons innervating the gastrocnemius in rats were observed under fluorescent microscope. At last, we compared the results of the quantity of motoneurons innervating the gastrocnemius in rats with or without congenital clubfoot in RA - administration group and normal control group.ResultsMacro - observation: In RA - administration group, 71. 37% ( 187feet/ 262feet) of the fetuses feet displayed congenital clubfoot. Other associated external malformations we found out were neural tube defects, anorectal malformations, tail deformities, eye deformities, craneofacial abnormalities. In control group, there was no obvious malformations. Fluorescent microscope -observation : The FG - lablled motoneurons were located mainly in the ventro - lateral zone of the 4th segment of lumber and the 3 segment sacral cord . The number of FG - lablled motoneurons in the fetuses with congenital clubfoot and neural tube defects, with congenital clubfoot only and without congenital clubfoot in normal controls were none, 266 ±134 and 441 ±222, respectively. The number of FG - lablled motoneurons in RA - administration group was significantly fewer than those in normal controls (P < 0.05). At the same time the size of the cells is smaller and the synapses between neurons are fewer than those in normal controls.ConclusionFrom the results of the experiment, we hypothesized that the motoneuron development of lumber - sacral spinal cord could influence the muscles in the...