Experimental Study On The Developmental Abnormalities Of Enteric Nervous System In Rats Induced By All-trans Retinoic Acid

Objective We utilize the SD rat animal model of ARMs (Anoreetal Malformations)induced by ATRA (All-trans retinoic acid). Used PGP9.5(protein gene product9.5), SYP(synaptophysin) and TEM (Transmission electron microscopy) for evaluating the entericnervous system and neuromuscular development of intestinal tract, providing experimentalbasis for the prevention and treatment of ENS abnormalities disease.Methods1. Experiment oneunpregnant mature SD rats,40, weighing250-300g, provided by the ExperimentalAnimal Center of Ningxia Medical. Male and female ratio of4:1in the cage together aftermidnight,8:00morning vaginal smear, under a microscope to see sperm or vaginalsuppository pregnancy counted as0d, randomized feeding the confirmed pregnancy mice.30pregnant rats has been randomly divided into2groups (n=15). Experimental group by10days during pregnancy dosage of100mg/kg, will be all-trans retinoic acid in accordance withthe olive oil40mg/ml suspension formed after the first injection tube. The control group inonly10days of pregnancy for the dosage of2.5ml/kg of olive oil. Experimental group andcontrol group were Cesarean Section in sterile conditions by intraperitoneal anesthesia with6.5%Chloral Hydrate during pregnancy on14days,16days,18days,20days. Remove allembryos, check and determine the deformity and records the number of embryo survival.Specimens derived from embryonic rat by4%paraformaldehyde fixation, dehydration madeof wax block, the sagittal serial sections, continuous dynamic observation of gestational age14-20days and normal anorectal malformations in rat embryos anal Morphological changes inthe course of the rectum. Median sagittal election for paraffin section HE staining and SPimmunohistochemistry. Known positive slice as a positive control, phosphate buffered saline instead of primary antibody as blank control. Image analysis, data recording and analyzing.2. Experiment two8pregnant rats has been randomly divided into2groups (n=4). Experimental group by10days during pregnancy dosage of100mg/kg, will be all-trans retinoic acid in accordancewith the olive oil40mg/ml suspension formed after the first injection tube. The control groupin only10days of pregnancy for the dosage of2.5ml/kg of olive oil. Two groups wereCesarean Section in sterile conditions by intraperitoneal anesthesia with6.5%ChloralHydrate on21days. Remove all embryos, check and determine the deformity and records thenumber of embryo survival. Made into the1-2mm3specimens, double fixed, dehydrated,soaked, embedded, curing, pre-sliced positioning, ultra-thin sections and transmission electronmicroscopy, recording and analyzing.Result:1. Immunohistochemistry(1) Control group: On E16, neuxal erest cells already colonized in the whole intestineand scattered positive cells were seen in the rectum. No SYP fluorescence were shown in themajority of colorectal. From E16to E18, PGP9.5-positive cells appeared in the myentericplexus sites poradically. The positive expression of SYP can be seen in the intestinal wallsubserosa position, straighted into a cord-like. On E20, it can be observed that theyellowish-brown mass of PGP9.5-positive ganglion cells, showing cluster-like. The positivecells also can be seen in the submucosa. While at the same sections observed in the sameperiod in the intestinal wall, the development of ENS grew earlierly. On E20, the positiveexpression of SYP gradually increased, more clear in the upper colon than the rectum.(2) Experimental group: On E16-18, neural crest cells at the intestinal wall could also beseen and there was no significant difference between the two groups. The distribution ofpositive cells were sparse. No SYP were shown in the majority of colorectal. Only a veryweak could be found in the uppercolon. On E18, the development of SYP in the upper colon became more sophisticated. PGP9.5positive expression in proximal rectum and SYP browncord-like positive expression in colorectal. Compared with the upper segment of the controlgroup the intensity deereased, the difference is more obvious. On E20, compared with thenormal and upper colon, the density of positive cells reduced in the rectal termimus.2. TEM(1) Control group: Plexus are mainly distributed between the circular muscle andlongitudinal muscle, the structure arranged in neat rows. Microtubules, microfilaments,mitochondria, and dense ribosomes, unmyelinated nerve fiber in he stromal cells, the axonterminals aresynaptic contacts with the typical structure of the synapse.(2) Experimental group: Myometrial muscle structural disorder, microtubules, actinfilaments show is unclear, fuzzy structure of a variety of organelles, mitochondrial swelling.The distribution of immature glial cells, owning nucleolus or not, nuclear condensation,chromatin aggregation, cell body is relatively small, less cytoplasm.Conclusion1. All-trans retinoic acid is a good anorectal malformations induced animal model ofteratogenic agents.2. On E16, there was no significant difference between the two groups. From E18,ARMs group compared with the normal group, the development of ENS has lagged:the older,the greater difference between the two groups.3. On E21, electron microscopy results of the experimental group was different from thenormal tissue structure.