Quantitative changes of nitrergic neurons during postnatal development of chicken myenteric plexus

Quantitative changes of nitrergic neurons during postnatal development of chicken myenteric plexus 1 first-author 1 2 1 1 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chenqsh305@yahoo.com.cn †E-mail:chenqsh305@yahoo.com.cn chenqsh305@yahoo.com.cn Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China Department of Anatomy and Histology, Sindh Agriculture University, Tandojam 70060, Pakistan Department of Anatomy and Histology, Sindh Agriculture University, Tandojam 70060, Pakistan Objective: 2 Information regarding the development of the enteric nervous system (ENS) is important for understanding the functional abnormalities of the gut. Because fertilized chicken eggs provide easy access to embryos, chicken models have been widely used to study embryonic development of myenteric plexus; however, no study has been focused on the postnatal period. The aim of this study was to perform a qualitative and quantitative analysis of the nitrergic neurons in the myenteric plexus of developing chickens in the postnatal period. Methods: 2 Whole-mount preparations of the myenteric plexus were made in 7-d, 15-d, and 40-d old (adult) chickens of either sex ( n =15). The myenteric plexus was studied after nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry using light microscopy, digital photography, and Image-Pro Plus 6.0 software. The numbers of positively stained neurons and ganglia were counted in the duodenum, jejunum, ileum, caecum, and colon in the different age groups. Data were expressed as mean±standard deviation (SD), and statistical analysis was performed using a one-way analysis of variance (ANOVA) test. Results: 2 The positively stained neurons showed various morphologies and staining intensities, and formed bead-shaped and U-shaped arrangements in the myenteric plexus. The densities of neurons and ganglia increased with age. However, the number of positive neurons per ganglion increased. The number of NADPH-d-positive neurons was highest in the colon, followed by the ileum, the jejunum, the duodenum, and the caeca in all age groups. Conclusions: 2 Developmental changes in the myenteric plexus of chickens continue in the postnatal period, indicating that the maturation process of the gastrointestinal function is gradual. In addition, no significant difference is happening among different intestinal segments during postnatal development, suggesting that the function of different intestinal segments had been determined after birth. NADPH-d histochemistry Enteric nervous system (ENS) Development Myenteric plexus Chicken Introduction 1 The enteric nervous system (ENS), the largest and most complex division of the autonomic nervous system, regulates the motility of the intestine. Abnormalities of the ENS, especially deficiency of the inhibitory innervation, may lead to motility disorders, impaired peristalsis or functional bowel obstruction, such as congenital hypertrophic pyloric stenosis, Hirschsprung’s disease, hypoganglionosis, intestinal neuronal dysplasia, and internal anal sphincter achalasia (Vanderwinden et al., 1992 1992 ; Hanani et al., 1995 1995 ; Hirakawa et al., 1995 1995 ). Studying the development of ENS is important for understanding functional abnormalities. The enteric neurons can be classified by their neurotransmitters. The three largest groups are the cholinergic, catecholamninergic (or adrenergic), and nonadrenergic-noncholinergic (NANC) enteric neurons (Sri Paran et al., 2009 2009 ). Identifying one of these neurotransmitter groups is a feasible technique to follow developmental changes in the myenteric plexus. Bult et al. (1990) reported that nitric oxide (NO) is released on stimulation of NANC nerves. Since then, it has been postulated that NO acts as an NANC neurotransmitter and mediates relaxation of the smooth muscle of the gastrointestinal tract (Boeckxstaens et al., 1990 1990 ; Burleigh, 1992 1992 ; Stark et al., 1993 1993 ). NO regulates smooth muscle cell tone, platelet aggregation and adhesion, cell growth, apoptosis, neurotransmission, and injury, as well as infection-induced immune reactions (Rosselli et al., 1998 1998 ). NO is produced by the neuronal NO synthase (NOS) in nitrergic neurons. Nicotinamide adenine dinucleotide phosphate (NADPH) and calcium are required for this synthesis. Two techniques are used to identify and quantify nitrergic neurons: NADPH diaphorase (NADPH-d) histochemistry and NOS immunohistochemistry. Dawson et al. ( 1991 1991 ) has previously reported that NOS staining is identical to NADPH-d staining. NADPH-d staining has been used in recent studies to qualify and quantify nitrergic neurons in the gut in different species: mice (Azzena and Mancinelli, 1999 1999 ), rats (Sandgren et al., 2003 2003 ), guinea-pigs (Wade et al., 2003 2003 ), sheep (Lalatta-Costerbosa et al., 2007 2007 ), rabbits (Junquera et al., 1998 1998 ), dogs (Ward et al., 1992 1992 ), pigs (Cserni et al., 2009b 2009b ), cats (Feher and Montagnese, 1994 1994 ), monkeys and humans (Degiorgio et al., 1994 1994 ; Wittmeyer et al., 2010 2010 ), pigs, guinea-pigs, and rats (Bodi et al., 2009 2009 ), and chicks (O′Donnell et al., 2006 2006 ). The whole-mount preparation technique allows the observation of the 3D meshwork of the myenteric plexus and quantitative and qualitative analyses of the myenteric neurons (Wester et al., 1999 1999 ). Fertilized chicken eggs provide an easy access to embryos and therefore chicken models have been widely used to study age-related changes in the myenteric plexus of the prenatal period (Bagyanszki et al., 2000 2000 ; O′Donnell et al., 2006 2006 ; Sri Paran et al., 2009 2009 ). Furthermore, such studies may provide us with some clues towards the periodic maturation of the human ENS. In previous literature, no studies have focused on the postnatal period of development of the myenteric nerves in chickens. Our aim was to perform qualitative and quantitative analyses of the nitrergic neurons in the myenteric plexus of developing chickens in the postnatal period using whole-mount preparations and NADPH-d histochemistry. Materials and methods 1 All normal young and adult broiler chickens of either sex were obtained from a commercial poultry farm. They were housed in temperature-controlled rooms (20 °C) with natural light, and had free access to food and tap water. The chickens were sacrificed by cervical dislocation under ether anesthesia. All the experiments abide by the regulations of the Chinese Committee for Animal Use for Research and Education. Segments of the duodenum, jejunum, ileum, caecum, and colon were removed from 7-d old (90–100 g), 15-d old (400–420 g), and 40-d old (adult; around 1 800 g) broilers (5 chickens of each age group). Then these segments were washed with 0.1 mol/L phosphate buffered saline (PBS) (pH 7.3), ligated at the two ends, and fixed with 4% paraformaldehyde in 0.1 mol/L PBS for 1.5–2.5 h at 4 °C. After being fixed, the segments were cleared with two rinses in 0.1 mol/L of PBS for 10 min, and stored overnight in PBS at 4 °C. Lastly, the segments were opened along the mesenteric border, sheared into 10-cm long segments, and the mucous membrane, submucosa, and circular muscle were peeled back to produce a whole-mount preparation of gastrointestinal myenteric plexus and longitudinal muscle using fine forceps. The preparations were then put in PBS to prepare for staining. According to the method of Cserni et al. ( 2009a 2009a ), the preparations were incubated in 0.5 mg/ml nitroblue tetrozolium (Sigma), 0.3% Triton X-100 (Sino-American), and 1 mg/ml NADPH (Sigma) in 0.1 mol/L PBS at 37 °C for 20–30 min; the reaction was terminated by washing in 0.1 mol/L PBS. Subsequently, the preparations were dehydrated and mounted on slides and examined with an Olympus microscope. Control experiments were performed in the same manner in the absence of NADPH. The intensely blue-labeled cells and weakly positive cells were regarded as positive, while the cells of which the bodies had the same color as the background, and where the cell outlines were seen dimly although the nucleus was obvious, were regarded as negative cells. However, data were presented only for the positive cells. A standard line was calibrated to scale on the glass slide, and the pictures were taken at different magnifications (total length 1 mm; the smallest scale 10 μm). These pictures were then imported to the Image-Pro Plus (IPP) 6.0 software. This was used as a standard to measure the different size parameters, such as the cell body diameter and microscopic field. All statistical data were obtained by the software of IPP. NADPH-d-positive neurons were counted in randomly selected microscopic fields (1 microscopic field was 1.3222 mm 2 ) and ganglia were counted at 100× magnification. In these ganglia, the area, length, and size of positive neurons were measured from randomly selected areas (1 microscopic field was 0.0846 mm 2 ) at 400× magnification. The diameter of the neuron was measured as the shortest diagonal line that made the shortest straight line, which passed across the center of the nucleus between the membranes at 400× magnification. Data from 10 measured areas per intestinal segment or sample per age group, per animal (5 chickens of each age group), were included in this study. Statistical analysis was performed using the one-way analysis of variance (ANOVA) test on SPSS 18.0 software and the Student-Newman-Keuls (SNK) test to evaluate the effect of age on the quantitative distribution of NADPH-d-positive neurons of the same segment. A probability of P < 0.05 was set as the level of significance in all analyses. The ganglionic and neuronal densities were calculated as the numbers of positive ganglia and stained cells per mm 2 of the whole-mount area, respectively. Data were expressed as mean±standard deviation (SD). The number of NADPH-d-positive neurons per ganglion and the size of NADPH-d-positive neurons were also calculated in relation to the developmental stage and location. Diagrams and tables were prepared with Microsoft Excel 7.0 computer program. Results 1 The NADPH-d histochemistry stained the NADPH-d-positive neurons blue with varying intensities. The length and thickness of the nerve fiber bundles connecting the ganglia increased with age, so that the meshwork appeared denser in the adult group. The ganglia appeared fusiform, showed a tendency of parallel arrangement, and were vertical to longitudinal smooth muscle (Fig. 1 1 ). The ganglia contained both positive and negative neurons (Fig. 2 2 ), and most of the positive ones were present within ganglia, but occasionally a single or a few neuron bodies could be found within the nerve fiber (axon) bundles (Fig. 2d 2d ). In the 7-d-old group, the diameters of positive cell bodies varied from 10 to 22 μm and most of them were between 12 and 18 μm. Their shape was mainly oval at the low magnification and the surface of cell bodies showed short processes at higher magnification (Fig. 2a 2a ). In the 15-d-old group, the diameters of positive cell bodies varied from 12 to 28 μm and most of them were between 13 to 19 μm. They were in oval, circular, fusiform, triangular or irregular shape and had many surface protrusions and small plate long processes (Fig. 2b 2b ). In the adult group, the diameters of positive cell bodies varied from 13 to 32 μm and most of them measured 15–24 μm and were oval, circular, fusiform, triangular or irregular. Single, bipolar and multipolar neurons have been identified (Fig. 2c 2c ). All the nuclei were plain and were situated on one side of the cytoplasm, which showed variable staining intensity. (a) Jejunal myenteric plexus in 7-d-old chicken; (b) Jejunal myenteric plexus in 15-d-old chicken; (c) Ileal myenteric plexus in adult chicken; (d) Cecal myenteric plexus in adult chicken. The meshwork of nerve bundles with ganglia containing NADPH-d-positive neurons is clearly seen. The myenteric ganglia with different sizes (arrows) are interconnected by short nerve fiber strands Note that NADPH-d-positive neurons increase their sizes during development. (a) The ganglia contain both NADPH-d-positive neurons (thick big arrows) and NADPH-d-negative neurons (thin big arrows) in the duodenum of the 7-d-old chicken. The positive neurons show variant sizes and shapes. (b) The ganglia contain both positive neurons (thick big arrows) and negative neurons (thin big arrows) in the jejunum of 15-d-old chickens. Most NADPH-d-positive neurons (big thick arrows) have many surface protrusions and thin long processes (thin short arrows). (c) NADPH-d-positive neurons (thick big arrows) with different shapes could be distinguished in the jejunum of the adult chicken. (d) Occasionally, a limited number of positive neuron cells are seen in the nerve bundles connecting different ganglia The positive neurons extended both long processes and short processes towards other negative or positive neurons (Figs. 2b and 2c 2b and 2c ). Some neurons that were linked together formed bead-shaped (Fig. 3a 3a ) and U-shaped ganglia (Fig. 3b 3b ). The positively stained nerve fibers arose from the positive cell bodies and branched to the secondary and tertiary bundles forming a complex network. The trend of fibers coincided with that of the longitudinal muscle (Fig. 3c 3c ). Most of the nerve fibers had varicosities (Fig. 3d 3d ). (a) Neurons connected with each other form a bead shape (boxed area) (bar=20 μm); (b) Neurons connected with each other form a U shape (boxed area) (bar=20 μm); (c) The primary NADPH-d-positive nerve fibers (PS) branch to secondary (SS) and tertiary (TS) bundles (bar=80 μm); (d) Most of the NADPH-d-positive nerve fibers have varicosities (hollow arrows) (bar=20 μm) In each group, there was little regional variation along the gastrointestinal axis in the morphological type, sizes of ganglia and labeled cells, as well as the branched form of nerve fibers. However, the spatial densities of ganglia and positive neurons in each segment were variable so that the form of meshwork was different (Figs. 1c and 1d 1c and 1d ). The sizes of NADPH-d-positive neurons were similar in different segments in the same age group. The growth of NADPH-d-positive neurons was evident with age (Table 1 1 ). The size of the cytoplasm increased markedly, while the sizes of nuclei only increased slightly in different segments throughout the development (Figs. 2a, 2b 2a, 2b , and 4 4 ). Data are expressed as mean±SD ( n =5) 2 Group 2 Size (μm 2 ) Body Nuclei Duodenum 7 d 171.27±49.25 72.43±16.21 15 d 203.74±62.21 79.29±17.82 Adult 267.45±74.61 85.48±30.36 Jejunum 7 d 164.65±35.29 76.35±19.42 15 d 215.42±45.42 78.43±21.25 Adult 258.57±80.54 88.26±22.98 Ileum 7 d 188.32±51.77 75.39±12.56 15 d 213.29±38.76 82.87±20.56 Adult 260.92±55.65 84.73±21.76 Caeca 7 d 155.79±26.16 70.71±11.52 15 d 195.49±24.52 76.48±19.23 Adult 247.68±28.35 80.29±18.22 Colon 7 d 198.32±38.74 69.89±19.22 15 d 227.32±43.37 80.29±16.72 Adult 271.49±56.36 87.34±19.52 Data are expressed as mean±SD ( n =5). The increase in size of NADPH-d-positive neurons was evident with age. However, this increase is predominantly because of the growth of the cytoplasm The number of NADPH-d-positive neurons per area was highest in the colon, followed by the ileum, jejunum, duodenum, and caecum (Fig. 5 5 ). The density of ganglia followed the same pattern (Fig. 6 6 ). However, the number of neurons per ganglion in the 7-d-old group was higher in the ileum than in the duodenum, followed by the colon, caecum, and jejunum. In the 15-d-old and adult groups, a higher number of neurons per ganglion was found in the duodenum than in the ileum, followed by the colon, caecum, and jejunum (Fig. 7 7 ). Table 2 2 shows the number of NADPH-d-positive ganglia and neurons per unit area, and the neurons per ganglion in small and large intestines of chickens. Data are expressed as mean±SD ( n =5). Different superscripts above the bars represent the significant differences among the number of NADPH-d-positive neurons per mm 2 in the same segment of different age groups ( P < 0.05) Data are expressed as mean±SD ( n =5). Superscripts above the bars represent the comparison between the numbers of NADPH-d-positive neurons per mm 2 in same segment of different age groups. Different letters mean the difference was significant ( P < 0.05) Data are expressed as mean±SD ( n =5) Data are expressed as mean±SD ( n =5). Different superscripts in the same column between age groups of each part of intestine are significantly different ( P < 0.05) 2 Group 2 Number per mm 2 2 Neuron/ganglion Ganglia Neuron 4 Duodenum 7 d 1.89±0.17 A 63.64±7.18 a 26.79±4.29 15 d 1.55±0.16 A 52.94±2.55 b 29.67±4.29 Adult 0.80±0.22 B 38.79±14.91 c 36.90±5.78 4 Jejunum 7 d 2.48±0.18 A 73.47±16.99 a 20.03±3.83 15 d 1.94±0.23 AB 58.88±3.16 b 20.07±2.82 Adult 1.24±0.18 B 45.92±17.51 c 23.72±4.38 4 Ileum 7 d 2.89±0.49 A 98.32±11.22 a 27.77±4.68 15 d 2.41±0.44 AB 89.62±11.36 a 28.09±3.67 Adult 1.79±0.26 B 77.52±9.03 b 31.84±4.76 4 Caeca 7 d 1.33±0.45 A 59.75±9.30 a 20.87±3.24 15 day 1.08±0.22 AB 45.38±5.86 ab 25.45±3.09 Adult 0.70±0.10 B 36.18±12.37 b 29.89±3.29 4 Colon 7 d 3.47±0.40 A 119.49±17.19 a 21.96±3.11 15 d 2.92±0.49 A 109.66±11.34 a 22.85±4.28 Adult 1.99±0.30 A 96.81±14.96 b 27.10±6.24 Comparing the three age groups, the densities of NADPH-d-positive neurons and ganglia were highest in the 7-d-old group, following by the 15-d-old and the adult groups. Discussion 1 Our qualitative and quantitative analyses of NADPH-d-positive neurons in the myenteric plexus of developing chickens show significant age-related changes in the postnatal period. These changes are similar to those previously reported in chicken embryos (Balaskas et al., 1995 1995 ; Bagyanszki et al., 2000 2000 ; O′Donnell et al., 2006 2006 ), and other species like guinea pigs (Nichols et al., 1992 1992 ), rabbits (Junquera et al., 1998 1998 ), rats (Cracco and Filogamo, 1994 1994 ), pigs (Sri Paran et al., 2009 2009 ), and monkeys and humans (Degiorgio et al., 1994 1994 ; Román et al., 2004 2004 ). Variance in staining intensity, shape and size of the NADPH-d-positive neurons observed in our study suggests the existence of different subpopulations of nitrergic neurons. The most well known functional subgroup of nitrergic neurons is the group of inhibitory motor neurons that mediate relaxation of the smooth muscle of the gastrointestinal tract (Vanden Berghe et al., 1999 1999 ). Some elements of the sensory innervation of the circular musculature may derive from nitrergic neurons as well. The rest of the nitrergic neurons may serve as inhibitory interneurons and control other (probably excitatory) motor neurons in the myenteric plexus (Cserni et al., 2009a 2009a ). The staining intensity of the positive neurons may be related to their NADPH-d content, activity, and isoenzymes (Cracco and Filogamo, 1994 1994 ). Cserni et al. ( 2009a 2009a ) hypothesized that neurons containing the higher amount of NOS/NADPH-d and stained darker are most likely to be inhibitory motor neurons. The cells stained weaker may be interneurons, sensory neurons, or glial cells (Cserni et al., 2009a 2009a ). The positive neurons stretched processes towards other positive or negative neurons (Llewellyn-Smith et al., 1992 1992 ). The bead-shaped and U-shaped neuronal arrangements suggest the existence of nitrergic neural circuits; however, the synaptic contacts need further exploration. Most of the NADPH-d-positive nerve fibers showed varicosities in our specimens. It would be easy to hypothesize that these varicosities are for storage of NO or NOS. NO is known as a gaseous messenger and it is released immediately as soon as it is synthesized, and is not stored in specific organelles or vesicles. However, NOS may be stored in these varicosities. Xiao et al. ( 1996 1996 ) demonstrated that NADPH-d either covers the surface of synaptic vesicles or exists in their insides, instead of being their specific contents. Gabella ( 1971 1971 ) observed in developing rats that the neuronal density decreased with age. Similar observations have been made in guinea-pigs, sheep, horses, pigs, and chick embryos (Gabella, 1987 1987 ; Young et al., 1993 1993 ; Doxey et al., 1995 1995 ; Zhang et al., 2004 2004 ; O′Donnell et al., 2006 2006 ). Decreasing NADPH-d-positive neuron density in the developing pig model has been used to explain why primary intussusception does not occur in adults (Cserni et al., 2007 2007 ). Our study showed similar changes in the spatial density of NADPH-d-positive neurons in the myenteric plexus of chickens as has been shown in other species as mentioned above. This may explain the occurrence of intussusception in young fowls (Okoye, 1985 1985 ). The decrease in the neuron density may be caused by the rapid growth of the bowel. Neuronal cell migration into ganglia or fusion of ganglia may be an explanation for ganglion size growth, but cell division or proliferation cannot be ruled out. The significant regional variation in the density of NADPH-d-positive neurons along the intestine may represent functional differences. The highest proportion of NADPH-d-positive cells has been found in the colon in our study. This is in accordance with the literature and may be responsible for long-term relaxation of the organ in the avian species (An et al., 2003 2003 ). In mammals, however, the caecum contains more relaxing neurons (Belai et al., 1992 1992 ). This may be explained by different motility patterns of the cecum in different tribes, or it is possible that the thinner caecal wall in the chicken may require fewer neurons to achieve relaxation. In the small intestine, we found nitrergic hyperinnervation of the ileum. This is similar in mammals (Nichols et al., 1992 1992 ; Pearson, 1994 1994 ). In the chicken, the muscle wall of the terminal ileum is relatively thick compared to other segments. This, along with the nitrergic hyperinnervation, suggests that the ileum plays a crucial role in digestion and absorption in the chicken. The food of the poultry is usually dry and eaten in large amounts. The intestine is able to adapt to these conditions. Nitrergic innervation regulates this adaptation by regulating motility and expanding capacity (Rand, 1992 1992 ). The density of NADPH-d-positive neurons of the chicken small intestine is significantly higher than that of the goat or pig (Liu et al., 2007 2007 ), but lower than that of the rat (An et al., 2003 2003 ; Bodi et al., 2009 2009 ). This suggests that smaller animals have more neuronal density in the myenteric plexus. The significance of this, however, is not clear. Balaskas et al. ( 1995 1995 ) and Bagyanszki et al. ( 2000 2000 ) working on chicken embryos reported that the overall density of NADPH-d-positive neurons was higher in embryos along the gastrointestinal tract, especially in the small intestine and colon, than that in the postnatal period revealed in the present study. In addition, the density of positive neurons in the colon was higher on gestational Day 14 than that in the ileum, but this trend changed to the opposite situation by Day 19 of development. In our study, we observed regional differences in relation to increasing age. However, the regional differences remain constant in the postnatal development of the chicken. Conclusions 1 Developmental changes in the myenteric plexus of chickens continue in the postnatal period, indicating that the maturation process of the gastrointestinal function is gradual. 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All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1300005 Project supported by the National Natural Science Foundation of China (Nos. 31172282 and 31272521), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Natural Science Foundation of Jiangsu Province for Youths (No. BK20130681), China 2013-01-04 2013-09-24 2013-05-28 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 14