| The gastrointestinal(GI)tract takes in food,digests them,absorbs nutrients and eventually excretes the unwanted byproducts.The intrinsic movements are central of the GI tract's function,which facilitate proper anterograde propulsion of luminal contents during digestion and excretion.Under physiological conditions,the GI motility is tightly regulated by the enteric system(ENS)and involves coordinated cooperation of the pacemakers(interstitial cells of Cajal,ICC)and effectors(smooth muscle cell,SMC).The GI motility is a relatively autonomic event and accepts assistive regulations of extrinsic nerves from central nervous system(CNS)and various neuroendocrine mediators,such as 5-TH,acetylcholine,and cholecystokinin.The ENS provides the intrinsic innervation of GI tract and is divided into myenteric plexus and submucosal plexus by the location of neurons aggregation.The myenteric plexus,between longitudinal and the circular muscles,involves in regulating GI motility.As pacemaker,current researches suggest that ICC embedded between ENS and SMC can not only generate rhythmic slow waves spontaneously but also mediate signal transmission between ENS and SMC,thus regulating the generation and maintenance of GI motility.In short,the gut,richly innervated by ENS,senses food-induced stretch and chemical stimulation.This information is relayed by interneurons and causes ascending excitatory and descending inhibitory neurotransmissions from the myenteric plexus,which are integrated into slow wave by ICC predominantly the ICC-MY,resulting in a wave of coordinated contraction and relaxation of SMC and pushing luminal contents forward eventually.The ENS arises from the neural crest.Enteric neural crest-derived cells(ENCDC)migrate along the gut wall,proliferate,differentiate,until form a mature ENS finally,a complex and time-consuming process and regulated by multitudinous genes and intestinal micro-environmental factors.Failure in these processes and consequent abnormal ENS development result in neuronal intestinal malformations(NIM)eventually.Hirschsprung's disease(HSCR)and intestinal neuronal dysplasia(IND)represent common subtype of NIM with serious impaired colonic motility,and are considered to be different outcomes of defective ENS.At the same time,studies also reveal the coexisting of micro-environment anomalies and ICC abnormalities in the defective ENS.Howerer,a definitive diagnosis can be made only on histological analysis of rectal biopsies due to the unclear of pathogenesis.This study is divided into two parts.The first part is the expressions of neurexinl and neuroliginl in a mouse model of neuronal intestinal dysplasia and their effects on colonic motility.The second part is the expression of Laminin and its correlated significance to interstitial cells of Cajal in Hirschsprung's disease.Part IThe Expressions of Neurexinl and Neuroliginl in a MouseModel of Intestinal Neuronal Dysplasia and their Effects onColonic MotilityBackground and ObjectiveNeurexins represent a family of presynaptic neuronal cell-adhesion proteins.Interactions between neurexins and neuroligins,the most well-known postsynaptic ligand of neurexins,orchestrate the events of formation,differentiation,stabilization,and balance of excitatory glutamatergic and inhibitory y-GABAergic synapses in CNS.Previous studies found that neurexins and neuroligins also expressed in myenteric plexus of ENS and increased gradually as the growth of embryo.The temporal correlation with the development of ENS indicated that they may participate in the development of ENS.The expressions of neurexins and neuroligins were significantly reduced in HSCR accompanied by abnormalities of related neurotransmitters in serum,a decrease of serum concentration of glutamic acid(Glu)and an increase of ?-GABA,and may be involved in the pathogenesis of colonic motor dysfunction in HSCR.GI motility also depends on the coordination of excitatory and inhibitory impulses.So,a hypothesis was proposed that neurexins and neuroligins may affect the GI motility by controlling the formation of synapses.IND is a type of Hirschsprung's allied disease(HAD)and belongs to NIM together with HSCR.Although pathological pictures of IND,characterized by hyperplasia of the submucosal and myenteric plexuses,giant ganglia,ectopic ganglion cells,etc.,are different from HSCR showing absence of ganglion cells,IND are clinically indistinguishable from HSCR in most patients at presentation due to the similarities of clinical manifestations,both of which exist severe impaired colonic motility.IND can exist as an isolated disease or in combination with HSCR,immediately proximal to the segment of aganglionosis and often presenting persistent obstructive symptoms after surgical operations.Although investigators have raised doubts about the existence of IND as a distinct histopathologic entity,one strong piece of evidence that IND is a real entity stems from animal models.Tlx2 knockout(Tlx2-1-)mice show hyperplasia of myenteric plexus resembling IND,and is an important animal model for IND.Studies have confirmed that Neuroliginl is mainly expressed in glutamatergic synapses.Interaction between Neuroliginl and Neurexins can trigger the formation of glutamate synapses by recruiting synapse-related proteins and receptors.Based on the discussion and hypothesis above,the Tlx2-/-mice were chosen as the animal model and further studies were employed to investigate the expressions of neurexinl,neuroligin1,and markers of glutamatergic synapses in the mice model and to explore the effects of neurexinl and neuroliginl on colonic motility.Methods1.The Tlx2 gene was knocked out through CRISPR/Cas9 gene targeting technique and Tlx2+/-mice were generated.Tlx2+/-mice were interbred to obtain Tlx2-/-mice.The mutant offspring were numbered and genotyped by Southern-blot and gene sequencing.At age of 8 weeks,colonic motilities of wild type(WT),Tlx2+/-and Tlx2-/-male mice were measured by glass bead technique.A glass bead was push into the colon 2cm through anus and the time required to discharge the glass bead represented the colon emptying time.After anaesthetized via pentobarbital,blood samples were taken from the heart and full-thickness specimens of the distal colon,1.5cm from ileocecal junction,were harvested after dissection.2.Immunohistochemistry staining was employed to detect the histological appearance of neurexin1,neuroligin1,a presynaptic marker of glutamatergic synapses vesicular glutamate transporter-1(VGLUT1),and NR1 subunit of NMDA receptors(NR1).Western-blot and qRT-PCR analysis were applied for investigating the relative abundances of Neurexinl,Neuroligin1,VGLUT1 and NR1 in mice with different genotypes at protein and mRNA level respectively,and serum concentrations of Glu were measured by ELISA,3.The 8-week-old Tlx2-/-male mice were divided into 5 groups randomly.Huperzine A was given to the Tlx2-/-mice at a dose of 0.lmg/Kg for 8 weeks by intragastric gavage(group HIG)and retention-enema(group HRE).At the same time,a same dose of physiological saline was given also through intragastric gavage(group SIG)and retention-enema(group SRE).The Tlx2-/-mice without any intervention were CON group.4.One day after intervention,the colonic motilities the Tlx2-/-mice were measured,and the blood samples and full-thickness colon specimens were obtained after anesthesia.The relative expressions of Neurexinl,Neuroligin1,VGLUT1 and NR1 of the Tlx2-/-mice in different groups were detected by western-blot and qRT-PCR,and the serum concentrations of Glu were measured by ELISA.Results1.Southern-blot and gene sequencing confirmed that 173bp region of nucleotide sequence of the second exon of the Tlx2 gene was knocked out.Tlx2+/-mice showed normal and were indistinguishable from WT mice.Tlx2-/-" mice often exhibited retarded growth with a lower weight and distended abdomens.26%(33/127)of Tlx2-/-mice died up to 8 weeks after birth,and the dilated appendix,cecum,distal ileum,proximal colon and the constricted distal colon were found in gross anatomy.2.Hyperplasia of myenteric plexus was observed in immunohistochemistry staining.Positive stained cells of Neurexinl,Neuroligin1 and VGLUT1,NR1 were both concentrated mostly in ganglion cells of myenteric plexus.In Western-blot and qRT-PCR analysis,relative expressions of Neurexinl and Neuroliginl in Tlx2-/-mice were up-regulated compared to WT and Tlx2+/-mice(P<0.05).Expressions of VGLUT1 and NR1 in Tlx2-1-mice shown similar trends with Neurexinl and Neuroliginl(P<0.05),accompanied by an increase in serum concentration of Glu(P<0.05).At the same time,Tlx2-/-mice showed longer expelling time of glass bead in the colon motility test,which indicated the dysfunction of colon motility(P<0.05).Comparisons between Tlx2+/-and WT mice shown no statistical difference.3.After intervened by Huperzine A,expressions of Neurexin1 and Neuroligin1 in Tlx2-/-mice colon of group HIG and HRE decreased compared to group SIG,SRE and CON in Western-blot and qRT-PCR analysis(P<0.05).The expressions of markers of glutamatergic synapses,VGLUT1 and NR1,in group HIG and HRE also shown decreases(P<0.05),accompanied by a decrease in serum concentration of Glu(P<0.05).At the same time,a shorter expelling time of glass bead in Tlx2-/-mice of group HIG and HRE was detected in the colon motility measuring,which indicated a recovery of the impaired colon motility(P<0.05).Comparisons between group HIG and HRE mice shown no statistical difference.Conclusion1.Neurexinl and Neuroliginl are closely related to the colonic motility.The presence of glutamate synapses in the ENS of colon and the variations accompanied by the expressions of Neurexinl and Neuroliginl indicated that the effects of Neurexinl and Neuroligin1 on colon motility may be achieved through their regulations of colonic glutamate synapses.2.The variations of serum Glu concentrations are consistent with the variations of colonic glutamate synapses and colonic motility,and may be a potential auxiliary indicator reflecting the intestinal glutamate synapses and colon motility.3.Neurexinl and Neuroliginl genes may be potential targets for Huperzine A.Part IIThe Expression of Laminin and its Correlated Significances to Interstitial Cells of Cajal in Hirschsprung's DiseaseObjectiveHSCR,which occurs in 1/5000 human live births and is characterized by the absence of ganglion cells in the ENS of distal gut,is the second congenital GI malformation in children following congenital anorectal malformation.Patients with HSCR endure severe impaired colonic motility and clinically present delayed meconium,severe constipation,and intestinal peseudo-obstruction.Although a variety of surgical operations,such as the transanal endorectal pull-through procedure,the Soave procedure and the Duhamel procedure,have been widely performed in clinical practice for many years with good therapeutic effects,the pathogenesis of HSCR is still not fully elucidated.So far,there are two major pathogenesis theories of HSCR,the"genetic abnormalities theory" and the "micro-environment abnormalities theory",but neither of them can explain all HSCR cases,and the clear pathogenesis still needs further research.ICC is a special type of cells distributing throughout the musculature of GI tract and closely associating with nerve varicosities of ENS and forms a three-dimensional cellular network structure.A variety of gap junctions are expressed between ICC and SMC,which facilitate the communication and enable ICC electrical coupling to SMC to form a multicellular functional syncytium.As pacemaker of GI tract,ICC plays an important role in the generation and maintenance of GI motility.ICC not only can spontaneously generate rhythmic pacemaker activity,conduct to smooth muscle cells,and drive electrical slow waves and phasic contractions,but also can integrate excitatory and inhibitory neurotransmission from motor nerves of ENS with slow-wave activity to orchestrate peristaltic motor activity of the GI tract.Impairment of the function of ICC will cause severe GI motor disorders,such as gastroparesis,constipation,achalasia,etc.Some studies showed that the decrease and structural abnormalities of ICC were observed in affected colonic segments of HSCR and may have a certain relationship with their prognosis.However,the mechanism of this phenomenon is still unclear.Extracellular matrix(ECM)is one of the important micro-environment factor of GI tract,and laminin contribute to the structure of ECM and influence the behavior of associated cells such as cell adhesion,migration,differentiation,morphological stability,and resistance of apoptosis.Studies have revealed that functional abnormalities of laminin during the embryonic period may cause the differentiation and colonization of GNCDC prematurely before normal migration,thus failing to reach the distal colon and leading to HSCR consequently.However,the pelvic plexus migrated and differentiated from GNCDC and affected by ECM as well is normal.Whether there are other pathways affected by laminin still needs further research to confirm.So,the "micro-environment abnormalities theory" were combined with ICC in this study to investigate the expression of laminin in HSCR and its effect on ICC to further elaborate the pathogenesis of HSCR.Methods1.From June 2017 to June 2019,48 cases of serum and colon samples of HSCR were collected,and 48 cases of serum samples of inguinal hernia were collected as a control.All cases of patients were inpatients in department of pediatric surgery at Qilu Hospital of Shandong University,and all cases of patients with HSCR were confirmed by postoperative pathological diagnosis.2.Stenosed,transitional,dilated and normal segments of colon were harvested from surgically resected colon of children with HSCR.The mucosal layer of each colon specimens was stripped under a dissecting microscope to reduce the interferences from mast cells,and the retained musculature was divided into three parts.In the first part,double immunofluorescence staining for laminin and ICC was performed on paraffin-embedded sections after fixed by paraformaldehyde to observe their expressions and localizations in differen colon specimens.In the second part,immunofluorescence staining on whole mount preparations of colon and scan images by confocal microscopic were used to visualize the ICC network.In the third part,relative expressions of laminin and c-Kit in colon specimens by were detected by Western-blot and qRT-PCR.3.Serum samples were collected during routine hematological examinations after admission to hospital.After the blood was coagulated,serum was harvested by centrifugation.The concentrations of laminin and c-Kit in serum samples of HSCR and control group were detected by ELISA.4.The ICC were isolated from the gut of 16-days-old embryonic Wistar rat by enzymatic dissociation and enriched by immunomagnetic sorting(MACS).Cellular immunofluorescence staining was used to identify the sorted cells.ICC was intervened by siRNA transfection to silence the expression of laminin or adding with exogenous laminin protein.The effects of laminin to the expression of c-Kit were explored by Western-blot and qRT-PCR,and it's effects on the cell viabilities and apoptosis of ICC were investigated by MTT assay and TUNEL staining.Results1.Double immunofluorescence on paraffin-embedded section revealed that the expression of laminin in stenosed segments of HSCR was down-regulated significantly,and the ICC in myenteric plexus(ICC-MY)and intramuscular ICC(ICC-IM),labeled by c-Kit,were reduced significantly compared to normal segment.The expressions of laminin and ICCs were relatively reduced in transitional and dilated segments of HSCR.ICC cells were surrounded by laminin and overlapped in some parts,suggesting that there may be interaction between laminin and ICC.Immunofluorescence staining on whole mount preparations of colon showed that the ICC-MY had a network structure and ICC-IM had a parallel-like structure.ICC cells were significantly reduced in stenosed segments of HSCR,and relatively reduced in in transitional and dilated segments of HSCR.At the same time,the structures of ICC-MY and ICC-IM were damaged.The results of Western-blot and qRT-PCR shown similar expression trends with the immunofluorescence.The expressions of laminin and c-Kit were lowest in stenosed segments of HSCR stenosis,followed by transitional and dilated segments,and highest in normal segments(P<0.05).2.Regrettably,the serum concentrations of laminin and c-Kit in children with HSCR was not statistically different from that of the control group in ELISA assay.3.After enzymatic dissociation and enriched by MACS,the expression of ICC marker c-Kit in the extracted cells was positive,and the smooth muscle marker smooth muscle actin(SMA)was negative.Western-blot and qRT-PCR results showed that expression of c-Kit decreased after the expression of laminin was silenced by siRNA,and expression of c-Kit was increased after the addition of exogenous laminin(P<0.05).MTT results showed that the cell viabilities of ICC decreased after the expression of laminin was silenced by siRNA,and the cell viabilities of ICC was increased after the addition of exogenous laminin(P<0.05).TUNEL staining experiments showed that the apoptosis in ICC increased after the expression of laminin was silenced,and the addition of exogenous laminin can inhibit the apoptosis induced by TNF-?.Conclusion1.In the affected colon of children with HSCR,the expression laminin was down-regulated and the number of ICC was reduced with the damage of its normal structure.2.Laminin can sustain the cell viabilities of ICC and resist its apoptosis.3.The decrease ICC in the affected colon of children with HSCR may be induced by the decreased expression of laminin,thus leading to the abnormality of pacemaker activity in the affected colon and eventually causing the dysfunction of colonic motility in HSCR. |
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