Secretion of immunomodulating neuropeptides (VIP, SP) and nitric oxide synthase in porcine small intestine during postnatal development

Abstract

Immunohistological identification/localization of immunomodulating neuropeptides [vasoactive intestinal polypeptide (VIP) and substance P (SP)] and enzyme nitric oxide synthase (NOS) as well as histomorphometric analyses of kinetics of their release and development of respective nerve fibers density during postnatal ontogenesis of porcine intestinal mucosal immune system (IMIS), were performed in order to assess the role of these molecules involved in maturation of the IMIS. The kinetcs of reactions to VIP, SP and NOS were demonstrated in the samples of jejunum and ileum from conventionally reared pigs. The samples were obtained at 0, 7, 14, 21, 28, 35, 42 and 49 days of age and processed for immunohistological staining. The VIP+ reaction was prevalently visible in the epithelial layer, lamina propria and Lieberkühn crypts (Lc) but also in the submucosa and lamina muscularis along blood and lymphatic vessels. The SP+ fibers were regularily distributed along enteric neurons in the muscular layer. The reaction to NOS was demonstrated in both mucosa and submucosa of ileum and jejunum and in the ileal Peyer's patches (PP). Intensity of the reaction was more pronounced in the epithelial layer and numerous NOS+ cells were observed around the Lc and inside the follicles of the PP. Also, we have noticed NOS+ blood vessels, particular neurons and nerve fibers in the submucosa and muscular layer of the small intestine. By analyzing quantitative patterns of SP+, VIP+ fibers and release of NOS we have concluded that intensity of their reactions gradually increases with age, except a short period of stagnation after weaning (at age of 28 days), reaching the highest values in the pigs aged between 42 and 49 days. The values obtained by Sperman rank order correlation test (rs) between days of age of pigs and intensity of the reactions in their jejunum/ileum to VIP (rs=0.97/0.95), SP (rs=0.97/0.97) and NOS (rs=0.98/0.95), respectively, showed positive correlations (P<0.05) according to Roemer Orphal scale. Current study showed that postnatal development of porcine IMIS was accompanied by a substantial increase in the secretion of neuropeptides/enzyme tested and that these molecules may participate in the functional maturation of immunoregulatory/bactericidal mechanisms of the local (intestinal) immune defense in young pigs.

Figure 1

Topographic patterns of VIP⁺ reaction in porcine small intestine in: epithelial layer, lamina propria and Lieberkühn crypts (Lc) (a), submucosal nerve fibers and along blood and lymphatic vessels (b), and nerve fibers of the muscular layer (c); intensity of VIP⁺ reaction in the ileum of 0-day-old pig (d) and 49-days-old pig (e). Scale bar: 50 µm.

Figure 2

Topographic patterns of SP⁺ nerve fibers along enteric neurons within muscular layer of the ileum of 0-day-old pig (a) or 49-days-old pig (b). Scale bar: 50 µm.

Figure 3

Topographic patterns of NOS⁺ reaction in porcine small intestine in: epithelial layer, lamina propria and Lieberkühn crypts (Lc) of jejunal mucosa (a), area of blood vessels within ileal submucosa (b), neurons and nerve fibers of the muscular layer within ileum (c); intensity of NOS+ reaction in the ileum of 0-day-old pig (d) and in the jejunum of 49-days-old pig (e); Scale bar: 50 µm.

Figure 4

The kinetics and intensity of reaction to neuropeptides VIP (a) and SP (b) and enzyme NOS (c) in the mucosa of jejunum (VIP r_s=0.97; SP r_s=0.97; NOS r_s=0.98) and ileum (VIP r_s=0.95; SP r_s=0.97; NOS rs=0.95) of pigs (N=5) aged from 0 to 49 days; the correlations between age of pigs (independent variable) and intensity of reactions to VIP, SP and NOS (dependent variable) in their small intestinal specimens were positive at P<0.05, respectively.