Quantitive Analysis Of Sensory And Motor Neurons Innervating The Gastrocnemius Muscle In Fetal Rats With Spina Bifida Occulta

PurposeNeural tube defects (NTDs) are among the most common congenital malformations and supervised by World Health Organization. Spina bifida is the most common type of NTDs which include spina bifida aperta and spina bifida occulta. Spina bifida aperta obviously affects the quality of life with severe urinary and fecal incontinence and limbs' sensorimotor functional disorder. Though the fetuses with spina bifida occulta may not have symptom when born, some individuals will present with the symptom of tethered cord syndrome with lapse of time, due to slight deformity of spinal cord and liparomphalus within neural canal. Even by operations, some functional disorders can't be cured. The spinal cord pathochanges of spina bifida occulta and the mechanism of clinical symptom are far from clear now. In this study, spina bifida was induced in rat fetuses with all trans-Retinoic acid (at-RA). Combined fetal microsurgery, microinjection techniques and retrograde nerve tracing, we investigated the development of sensory and motor neurons innervating gastrocnemius muscle of fetal rats with spina bifida occulta by injecting retrograde tracer fluorogold (FG) into the gastrocnemius muscle.Methods1. Animal models: Healthy virginal female rats weighting 240～300g were mated with males of the same strain. On day 10 of gestation, pregnant rats of experimental group were gavaged with at-RA(135mg/kg) through gastric tube; rats of control group were given olive oil through gastric tube. 2. Fetal microsurgery and microinjection: On day 20 of gestation, pregnant rats were anesthetized with 10％chloral hydrate intraperitoneally, then fetal microsurgery and microinjection techniques were performed to expose the gastrocnemius muscle of fetuses and 5％FG was injected with microinjector.3. etrograde tract-tracing technique and statistics: 24 hours later, the operated fetus was perfused transcardially with 0.9％saline followed by 4％paraformaldehyde in phosphate-buffered saline(PBS). We observed whether the fetus was malformed or not by microscope. Lumbosacral segment spinal cord of fetuses were dissected and taken out, and then cryoprotected in 20％sucrose in PBS for serial transverse cryosection after 24 hours, the FG-labelled sensory neurons and motoneurons were observed and counted by microscope. The image of the neurons was transported into computer. Compare sensory and motor neurons' number, shape and distribution among control group, normal appearance after dosage given group and spina bifida occulta group. Then results were statistically analyzed.ResultsControl group (groupⅠ): the neurons were located mainly in the posterior root ganglion of the lumbosacral segment spinal cord, which were regularly distributed. The neurons of normal appearance after dosage given group (groupⅡ) were similar with control group. Spina bifida occulta group (groupⅢ): the neurons were rare and irregularly distributed.The sensory neurons' number of groupⅡ(1152±379) is less than groupⅠ's (1357±393), but the difference between them isn't significant. The neurons' number of groupⅢ(481±188)is less than groupⅠ(1357±393) andⅡ's (1152±379), and the difference between them is significant (p＜0.05).The motor neurons' number of groupⅡ(180±53) is less than groupⅠ's (204±77), but the difference between them isn't significant. The neurons' number of groupⅢ(106±34) is less than groupⅠ(204±77) andⅡ's (180±53), and the difference between them is significant (p＜0.05). ConclusionsThe sensory and motor neurons innervating the gastrocnemius muscle in fetal rats with spina bifida occulta are fewer than that in the normal fetal rats, and distribute abnormally. The reduction and abnormal distribution of the sensory and motor neurons innervating the gastrocnemius muscle in fetal rats with spina bifida occulta are probably associated with sense and motor dysfunction.