Histological Analysis Of The Amount Of Neuronal Nitric Oxide Synthase, Lipid And Nissl Body In The Spina Bifida Occulta Spinal Cord

ObjectiveSpina bifida occulta(SBO),is the malformation including a defect of spinal vertebrae with the vertebral arches open and neural tube intact,and the skin covered is usually normal.It is mostly found in S1 and S2 region.SBO has various clinical symptoms,including cutaneous abnormalities,incontinence of urine,urinary tract infection,constipation,increased deep tendon reflexes in the lower limbs,progressive scoliosis,asymmetric development of the lower limbs,and so on.The most frequent cause of the above symptoms is tethered cord syndrome,in which process the fixed spinal cord cannot freely move during the normal flexion and extension of the vertebral column.Repeated microtrauma of the spinal cord can result in a dysfunction of the corticospinal tract.In addition,there is the possibility of associated malformations, such as congenital scoliosis or apparent shortening of a limb,terminal syringohydromyelia and diastematomyelia.Extra-axial malformations are most frequently seen in the anorectal and urogenital region(kidney defects).In spite of the high incidence(20%or so)and various manifestations of SBO,it has been treated without much attention.Relative studies have only covered drug/knocked out animal model manufacture,basic HE staining of spinal cord,apoptosis and retrograde tracer fluorogold based investigations.By now,the pathogenesy and pathological transformations of SBO remains unclear.So,there is still a broad space for further study concerning the topic.And the result would benefit our understanding of SBO,a relatively common defect,and further direct antepartal prevention,as well as early treatment.Nitric oxide(NO)is described as a nonadrenergic non cholinergic(NANC) inhibitory neurotransmitter at various sites in the nervous system,and is known to have important function in the information transmission process.Among the three types of NO synthases,neuronal nitric oxide synthase(NOS1)broadly lies in the nervous pathway of various tissues,including central nervous system,peripheral nervous system,cardiovascular system,digestive system and genitourinary system,and associates with their function.NOS1 has recently attracted a lot of academic interest, and was deeply studied in many conditions,including brain injury,incontinence of urine,reperfusion and stress.According to the reports,NOS1 regulates the function of smooth muscle in the intestinal tract as well as the sphincter in the urinal tract.In some occasions,SBO is associated with lipoma,which can cause tethered cord syndrome. Close study can disclose its influence to the spinal cord.Finally,Nissl body is classically called ribosome,where the proteins mainly synthesize.It is very sensitive to various stimuli,so can act as an indicator of neuron attack.Here,we focus on the data concerning the amount of NOS1,lipid and Nissl body to detect the pathological transformations of spinal cord in valproic acid(VPA)-induced SBO fetal rats,and approach its pathogenesy.MethodsOn day 9 of gestation,rats were treated with either 0.5 ml physiologic saline or 350 mg/kg VPA given once at 9am and once at 4pm(7 h between doses)for a total dose of 700 mg/kg.On the 21st day of gestation,the fetuses were taken out,after. recording their sizes and weight,the specimens from fetal spines were stained with Alcian blue.SBO was diagnosed using the distance parameter of the two cartilaginous ends of the vertebra arch from L4 to S3 vertebrate,which was measured with image-analyser.A part of the specimens were later made into serial transverse cryosection.The expression of NOS1 was determined by immunofluorescence staining, and the amount and distribution of lipid in spinal cord was determined by Oil Red O staining.Others of the specimens were made into paraffin sections and then stained with Toluidine Blue,which showed Nissl bodies in neurons.ResultsThe distances between the two cartilaginous ends of the vertebra arch of all 60 fetus in the control group fell in the normal range,while 87 of the 92 VPA treated fetus showed wider distances,which means the incidence of SBO was 95%.In the normal group,the NOS1 positive neurons were deeply stained with regular size and shape. While in the malformation spinal cord,the NOS1 positive motoneurons were significantly more than that of normal spinal cord(p＜0.01).The plasma of some neurons was lightly stained,and their size and shape differs a lot.No positively stained neutral fat tissue was observed within the spinal cord in either normal group or malformation group.The color of Toluidine Blue stained neurons thinned(p＜0.01)and the number also decreased(p＜0.01)in the malformation specimens.ConclusionsThe data showed in our study suggest that it may be one of the important pathological bases of the emergence of SBO clinical symptoms that the amount of NOS1 in spinal cord with SBO is higher than that in the normal ones,whereas the amount of Nissl body decreases.Our study observed some features of the pathological transformations in the SBO spinal cord,and initially discussed the mechanism of its clinical symptoms.Our data may benefit further studies of SBO,and offer some useful clues for clinical treatment.