Study On Morphology And Distribution Of SP-mRNA In INR

The intestinal nerve of Remak (INR) is a unique avian autonomic ganglionatednerve trunk which runs in the mesentery dorsal to the intestine from the cloaca to theduodenum, sending many nerve branches to the intestine. Different from mammal,the avian enteric nervous system is composed of the intramural plexuses and INR,and INR has important regulative effects on the digestion and procreation of birds.Substance P (SP) is a kind of Neuropeptide, composed by 11 amino acids, and isvery important for maintaining the normal physiological functions of intestine. Inthis experiment, some histo-morphological researches were done on INR in chicken,the SP-mRNA probes were synthesized successfully, and then the SP-mRNA in INRwas detected by in situ hybridization histochemistry.â… Histo-morphology of the Intestinal nerve of Remak in chickenThe intestinal nerve of Remak (INR) is a unique avian ganglionated nerve trunk.In this experiment, the INR was frozen sectioned and then the slices were stained withhaematoxylin-eosin and toluidine blue separately. The morphology of the neurons inthe INR was observed by conventional optic microscopy, the number of the neuronswas counted, the areas of the neural somas were measured, and finally the data wasanalyzed by the Statistic software of SPSS. It was revealed that the ganglia of the INRcontained abundant neuronal bodies, the total number of neurons in the whole INRwas 31821, and the juxta- rectal portion of INR had the most neurons, amounted to27109. The average area of the neuronal bodies in the juxta-jejunal portion of INRreached 478.46μm² and was the largest in the whole INR, the proportion of the largerneuron cells was also the highest in this region; and in the juxta-rectal portion of INRthe average area of the neuronal bodies was 391.74μm² and was the smallest. It isspeculated that there are differences in the shape, size, and quantity of neurons amongdifferent regions of INR, and it's possible that a correlation exists between thisdifference and INR's role in the regulation of gut motility.â…¡Preparation of chicken SP-mRNA probeFirst of all, getting the SP-mRNA sequence from the GenBank, its serialnumber is XM₄18674, and then two couples of primers were designed according this sequence. The fragment of SP gene was obtained by RT-PCR through total RNAof chicken brains. The amplified cDNA fragments were subcloned into pGM-T easyvector, and the plasmids were transformed into E. coli top10 and chosen by"white-blue plaque selection". The recombinant plasmids were identified bysequencing. Then SP/pGM-T easy vectors were linearized with the restrictionenzyme of Nco I and Sal I respectively. The sense and anti-sense DIG labeled RNAprobes were producted by using SP6 and T7 RNA polymerase respectively and invitro transcription according to the protocol of "DIG RNA Labeling Kit (SP6/T7)".Certificated by dot blot hybridization, the sense and anti-sense RNA probes wereprepared successfully, and the concentration of the probes approximated 100 ng//μl,enough for the experiment of in situ hybridization histochemistry.â…¢The detection of SP-mRNA in INR by in situ hybridization histochemistryThe distribution of SP-mRNA in chicken INR was examined by using in situhybridization histochemistry (ISHH). Most of the positive cells had oval and otherpolygonal shapes, and the labeled neurons distributed in ganglion in layers or smallgroups and in nerve trunk between ganglions sporadically. The proportion of positivecells in total neurons was 82.98% in the juxta-jejunoileum portion and 98.01% in thejuxta-rectal portion of INR. In conclusion, by using the sense and anti-sense DIGlabeled SP-mRNA probes, the technique of ISHH provided an approach for thefurther study of the location of SP-mRNA in nerve tissue; the result of ISHHsuggested the existence of the SP-mRNA in the most neurons of INR.