Nutritional Effects Of Nerve Growth Factor On Interstitial Cell Of Cajal In Primary Culture

Disorders of gastrointestinal motility have the disorders of motor and/or sensory function for the damage of innervation. A new classification of disorders of gastrointestinal motility has been formulated in Bangkok based on neurogastroenterology. In some disorders, it has been reinforced by identification of underlying pathological change in enteric innervaton or musculature. Interstitial cell of cajals (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal tract and serve as electrical pacemakers, active propagation pathways for slow waves, and mediators of enteric motor neurotransmission. Abnormal distributions of ICC have been reported in a variety of GI motility disorders, such as chronic intestinal pseudoobstruction, Hirschsprung’s disease, idiopathic slow transit constipation, and diabetic gastroparesis. Alteration of ICC in the human GI tract may cause some gastrointestinal motility disorders. ICC reverts to smooth muscles when kit signaling pathway was blocked. However, the phenotype can be recovered by stem cell factor. As a result, the recovery of ICC in appearance and function may have a therapeutical effect in disorders of gastrointestinal motility. Recent studies have shown that IL-9, NO and 5-HT prompted the proliferation of ICC.Nerve growth factor (NGF) is the most thoroughly studied of the neurotrophins. NGF has an important role in maintenance and survival of central and peripheral nervous systems. Functional receptors for NGF are widely distributed among the diverse enteric phenotypes and argue for a novel NGF-mediated regulatory system within the enteric nervous system. So we put the hypothesis that:NGF may prompt the proliferation or regulate pacemaker current of ICC.Aims1. To obtain ICC by explant culture and identify it by immunofluorescence technique and methylene blue method.2. To investigate the effect of NGF on the quantity and network structure of ICC in primary culture.3. To investigate the effect of NGF on pacemaker current and chlorine current of ICC in primary culture.Methods1. ICC was isolated and cultured by explant culture. The jejumun was obtained from CD1 newborn mice. The musculature was cleaved along the deep muscular plexus and cut into millimetre-size pieces. These explants were gently placed on collagen-coated glass coverslips in dishes by using curved forceps, immersed in RMPI 1640 culture medium and incubated in 95%O2-5%CO2 at 37℃for 3 days. ICC was detected by immunohistochemistry and metylene blue methods.2. Longitudinal muscle with the attached myenteric plexus was obtained from CD1 adult mice. ICC was marked by metylene blue methods. The appearance and network structure of ICC and the relationship between ICC and enteric nerve ganglion were observed.3. ICC was isolated and cultured by explant culture, and divided into normal group and NGF group. NGF was added into culture medium after incubation for 12 hours in NGF group. The quantity and network structure of ICC were compared in two groups. The pacemaker current and outwardly rectifying chloride current of ICC were recorded. The amplitude and frequency of currents were compared.Results1. Different types of cells gradually grew from the explants after incubation of explants in culture medium for 3 days, Smooth muscle cells formed bundles, whereas ICC formed networks that contracted rhythmically. At the light microscopy level, the morphological characteristics of ICC included a triangular- or stellate-shaped cell body, large prominent nuclei with little perinuclear cytoplasm, and multiple, thin processes. Under a confocal microscope, ACK2 coupled to FITC stained ICC bright green, whereas smooth muscle was negative. ICC outside of the explants was dark blued by methylene blue stain, but living nerve cell and smooth muscle were negative. ICC stained by methylene blue could also be positive by ACK2.2. ICC was dark blue by methylene blue stain in longitudinal muscle with the attached myenteric plexus. The network structure was formed between ICCs. ICC was distributed among the stem of enteric nerve ganglion.3. The quantity of ICC in NGF group was increase by 85% compared to normal group by immunofluorescence (all n=6). The similar result was observed in methylene blue stain. The tighter network structure was formed between ICCs in NGF group. Rhythmical inward current was recorded in ICC by whole-cell patch clamp method. The pacemaker current appeared more strong rhythmicity and regular wave amplitude. The amplitude of pacemaker current in ICC was significantly enhanced (179.3±29 pA. n=6). Outwardly rectifying chloride current was recorded in ICC in two groups. The amplitude of current was evidently increase (850±140 pA, n=10). The obvious inwardly tail current appeared in NGF group.Conclusions1. We obtain ICC by explant culture and identify it by immunofluorescence and methylene blue methods. It is a good method for ICC labeling with methylene blue. It makes for the localization of ICC in situ by methylene blue stain.2. NGF can prompt the proliferation of ICC and make the network structure tighter.3. NGF can enhance the amplitude of pacemaker current in ICC. Outwardly rectifying chloride current may be the main component of pacemaker current. NGF can also increase the amplitude of chloride current.These results may provide new sight in the pathogenesis and treatment of disorders of gastrointestinal motility.