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Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles
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Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles Guo-quan Zhang a,b , Shu Yang a , Xiao-shuang Li a , De-shan Zhou a,∗ a b
Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China Department of Histology and Embryology, Logistics University of Chinese People’s Armed Police Force, Tianjin 300162, PR China
a r t i c l e
i n f o
Article history: Received 27 November 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online xxx Keywords: Cholinergic neurons IGF-IR Interstitial cells of Cajal Mouse Myenteric plexus Stomach
a b s t r a c t Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have tremendous trophic effects on the central, peripheral and enteric neurons. The loss of IGF-IR contributes to the development of diabetic gastroparesis. However, the nature and the function of the IGF-IR+ cells in the gastric myenteric plexus remain unclear. In this study, anti-ChAT, anti-S100 or anti-c-KIT antibodies were used to co-label IGF-IR+ cells and neurons, glial cells or interstitial cells of Cajal (ICCs), respectively. We also generated type 1 diabetic mice (DM) to explore the influence of impaired IGF-I/IGF-IR in the myenteric neurons. Results showed that IGF-IR was expressed in the epithelium, smooth muscles and myenteric plexi of the mouse stomach. Most of the IGF-IR+ cells in the myenteric plexi were ChAT+ cholinergic neurons, but not enteric glial cells and there were more IGF-IR+ neurons and fibers in the gastric antrum than in the corpus. The IGF-IR+ /ChAT+ neurons and ICCs were closely juxtaposed, but distinctly distributed in the myenteric plexus, indicating a possible role for the IGF-IR+ /ChAT+ neurons in the mediation of gastric motility through ICCs. Moreover, the decrease of IGF-IR and cholinergic neurons in the myenteric plexi and smooth muscles of DM mice suggested that IGF-I/IGF-IR signaling might play a role in neuron survival and neurite outgrowth, as well as stem cell factor (SCF) production, which is required for the development of ICCs. Our results provide insights into the effects of IGF-I/IGF-IR signaling on the development of gastrointestinal motility disorders. © 2014 Elsevier GmbH. All rights reserved.
Introduction Insulin-like growth factor-I (IGF-I), which shares substantial amino acid sequence homology with pro-insulin, is produced by many tissues, primarily in the liver, as well as in the brain, kidney, heart and lung (Gualco et al., 2009). IGF-I elicits biological effects on cellular survival, proliferation and differentiation through its specific receptor (IGF-IR) (Kuemmerle, 2003; Sonntag et al., 2000; Pantaleo et al., 2010). IGF-I is also considered a type of neurotrophic factor, due to its general neurotrophic effects on the central and peripheral nervous systems. Several lines of evidence have shown that IGF-I is able to restrict neuronal and glial cell loss and promote the recovery process after ischemic insult in the rodent brain (Beilharz et al., 1998; Hwang et al., 2004). IGF-I also reduces spinal cord edema by inhibiting neuronal nitric oxide synthase (nNOS) activity (Sharma et al., 1998). In diabetic peripheral neuropathy patients, IGF-I levels are decreased, which contributes to the refractoriness of nerve regeneration (Yasuda et al., 2003). The neuropathy in diabetic rats could be ameliorated or prevented by IGF-I or insulin
∗ Corresponding author. E-mail address: [email protected] (D.-s. Zhou).
treatment (Ishii and Lupien, 1995; Tomlinson et al., 1997). It has been proposed that a decrease in the IGF-1 mRNA expression in diabetic rats might be responsible for the delayed nerve growth factor (NGF) mRNA response by affecting the induction of c-FOS and thus impairing nerve regeneration (Xu and Sima, 2001). Additionally, the neurotrophic effects of IGF-I or insulin potentially act through ERK1/2 and Akt signaling (Altar et al., 2008). The enteric nervous system (ENS) is composed of the submucosal plexus and the myenteric plexus, the latter of which plays a crucial role in the regulation of gastrointestinal (GI) peristalsis. Mulholland et al. (1992) reported that IGF-I is able to stimulate the neurite outgrowth of myenteric cells derived from neonatal guinea pigs in vitro, indicating that IGF-I also has a neurotrophic effect on myenteric neurons. The effects of IGF-I are primarily mediated through interactions with IGF-IRs, which are widely expressed in the brain, ganglia and nerve fibers (Craner et al., 2002; Gualco et al., 2009). In the GI tract, IGF-IRs are present in the epithelium, smooth muscle cells, and myenteric plexi and are considered to contribute to the development of diabetic gastroparesis in mice, rabbits and humans (Rouyer-Fessard et al., 1990; Termanini et al., 1990; Horváth et al., 2006). However, exactly which type(s) of IGF-IR+ cells exist in the gastric myenteric plexus has not been explored. The elucidation of this issue will help in understanding
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Please cite this article in press as: Zhang G-q, et al. Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.01.011
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2 Table 1 Antibodies used in immunofluorescence. Primary antibodies
Secondary antibodies
Rabbit anti-IGF-1R␣ (1:100)
sc-712, Santa Cruz
Rabbit anti-IGF-1R (1:600)
3027 S, cell signal technology BA0120, Boster ACK2, eBiosciecnce NBP1–30052, Novus biologicals
Rabbit anti-S100 (1:100) Rat anti-KIT (1:200) Goat anti-ChAT (1:100)
FITC conjugated goat anti-rabbit IgG (1:100) or Cy3 conjugated goat anti-rabbit IgG (1:200) AF 594 conjugated donkey anti-rabbit IgG (1:200)
A21207, Life Technologies
Cy3 conjugated goat anti-mouse IgG (1:200) FITC conjugated goat anti-rat IgG (1:200) AF 488 conjugated donkey anti-goat IgG (1:100)
A10521, Life Technologies 629511, Life Technologies A10521, Life Technologies
the functions of IGF-I/IGF-IR signaling in the gastric myenteric plexus, as well as of their possible role in GI motility disorders.
F2765, Life Technologies A10520, Life Technologies
Immunohistochemistry
Ten male BALB/c mice (6-weeks old, 22–26 g) were purchased from the Animal Center of Capital Medical University (Beijing, China). All mice were maintained in a temperature-controlled room (23 ± 1 ◦ C) with a constant 12 h light/dark cycle. Food and water were available ad libitum. After fasting for 14 h, five mice received a single intraperitoneal injection of Alloxan monohydrate (200 mg/kg, Sigma–Aldrich). The remaining five littermates receiving the same dose of normal sodium were used as controls. The fasting blood glucose was measured by the use of an Accu-chek Active Complete blood glucose monitor (Roche, Germany) 72 h after injection. The mice with a blood glucose ≥11.1 mmol/L were considered as DM mice and employed in this study. The blood glucose of these DM mice was monitored every week. The following experiments were performed 8 weeks after the onset of DM. All experimental procedures were approved by the Institutional Animal Care Committee from Capital Medical University, Beijing China.
Mature IGF-IR is a heterotetramer consisting of two extracellular ␣ subunits and two  subunits (Ward and Garrett, 2004); therefore, both anti-IGF-IR␣ and anti-IGF-IR antibodies were used in this study. Non-specific binding sites were blocked with 1% bovine serum albumin (BSA) and 1% normal goat serum for 30 min. The specimens were incubated with primary antibody to IGF-IR␣ or IGF-IR overnight at 4 ◦ C, followed by incubation with the corresponding secondary antibody for 1 h at 25 ◦ C (Table 1). After washing, the specimens were mounted in fluorescent mounting medium containing DAPI (Zhongshan Jinqiao Biotechnology, China) and observed under a fluorescence microscope (Nikon, 80i, Japan) or confocal laser scanning microscope (CLSM, Leica, TCS SP5). The preparations were incubated with an isotype control antibody or without a primary antibody as negative controls. To determine the nature of the IGF-IR-positive cells, the preparations were double labeled as follows: (1) anti-IGF-IR␣ and anti-ChAT (a marker for cholinergic nerves) primary antibodies with FITC-, Cy3-, AF 594- or AF 488-conjugated IgG secondary antibodies; (2) anti-IGF-IR␣ and anti-S100 (an antibody against enteric glial cells) primary antibodies with FITC-, Cy3- or AF 594-conjugated IgG secondary antibodies; or (3) anti-IGF-IR␣ and anti-c-KIT (a marker for ICCs) primary antibodies with FITC-, Cy3or AF 594-conjugated IgG secondary antibodies (Table 1).
Whole-mount preparations
Image analysis and statistics
The whole-mounts were prepared as described previously (Chan et al., 2010). In brief, the stomach was removed and fully washed with pre-cooled phosphate-buffered saline (PBS, 0.01 M, pH 7.4). Next, the stomach was inflated to maintain its shape with 4% paraformaldehyde for IGF-IR, choline acetyl transferase (ChAT) and S100 staining or 100% acetone for c-KIT staining for 2 h. Finally, each sample was immersed in the same fixative for 12 h. Three samples of 0.5 cm × 0.5 cm were randomly cut from each gastric corpus and antrum. The mucosa and submucosa were removed, and the longitudinal muscle layer containing the myenteric plexus was dissected. The prepared muscles were placed in 0.01 M PBS at 4 ◦ C for (double) immunostaining.
Ten photographs per whole-mount were randomly taken at a magnification of 20×. The target area of interest was outlined, and the areas of myenteric ganglia and primary nerve strands, as well as the lengths of the primary nerve strands, were measured in Image Pro-Plus software 6.0 (Media Cybernetics, Bethesda, MD, USA) based on their immunoreactive intensity. The area density of the myenteric ganglia and primary nerve strands and the length density of the primary nerve strands were derived by dividing the total areas of myenteric ganglia and primary nerve strands and the total length of primary nerve strands, respectively. The number density of cholinergic neurons was determined by dividing the total number of cholinergic neuron by the total area of the fields. Statistical analyses were performed with SPSS 13.0 software (SPSS Inc., IBM, Chicago, IL, USA). Data are expressed as the means ± SEM and compared using a Student’s t-test. A p-value of 0.05 was considered statistically significant.
Materials and methods Generation of type 1 DM mouse
Cryosection preparations The samples from the gastric corpus and antrum were embedded in an optimal cutting temperature compound (O.C.T., Sakura) and then frozen in liquid nitrogen. Cryosections (10 m thick) were cut with a cryostat (CM3050S Leica Microsystems, Wetzlar, Germany) and mounted on poly-L-lysine-coated glass slides. The sections were fixed with 4% paraformaldehyde for IGF-IR, ChAT and S100 staining or 100% acetone for c-KIT staining for 30 min.
Results Distribution of IGF-IR in the stomach The same staining patterns for IGF-IR␣ and IGF-IR were observed in the gastric wall; therefore, only IGF-IR␣ (green) was
Please cite this article in press as: Zhang G-q, et al. Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.01.011
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Fig. 1. This set of immunofluorescence images shows the wide distribution of IGF-IR in the stomach. (A) In cryosections, IGF-IR is intensely expressed in the tunica muscularis (including the circular and longitudinal muscle layers), myenteric plexus (arrow) and muscularis mucosa (arrowhead). (B) In whole-mount preparations, myenteric ganglia (high-magnification figures in frames) and primary nerve strands (arrow), secondary nerve fibers (double arrow) and tertiary nerve fibers (arrowhead) are strongly stained for IGF-IR. IGF-IR+ cells are round or ovoid with distinct nuclei and neurites, suggesting that they are neuron-like cells. Nuclei are stained with DAPI (blue). (C) The neuronal network is loosened and the nerve fibers in the myenteric plexi are slender in DM mice compared with controls. (D) The area densities of the IGF-1R+ myenteric ganglia and their primary fibers of DM mice were significantly decreased compared with controls. p < 0.05. CM: circular muscle layer, LM: longitudinal muscle layer. MU: mucosa; SM: submucosa. The figures and the alterations of IGF-1R+ myenteric structures of the gastric antrum and corpus are essentially identical; here, we show only those from the antrum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
shown to represent IGF-IR. In cryosections, IGF-IR mainly localized to the tunica muscularis (both the circular and longitudinal muscle layers), myenteric plexus and muscularis mucosa (Fig. 1A). In whole mounts, stronger immunoreactivity for IGF-IR was detected in the myenteric plexus, including the ganglion cells, primary nerve strands, secondary nerve fibers and fine tertiary fibers; the IGF-IR+ cells in the myenteric ganglia were round or ovoid with distinct round nuclei, similar to the neurons (Fig. 1B). The area density of the IGF-IR+ myenteric ganglia, the area density of the IGF-IR+ primary nerve strands and the length density of the IGF-IR+ primary nerve strands in the myenteric plexi of the gastric antrum were all significantly higher than those in the gastric corpus (Table 2). We investigated the alteration of the IGF-IR+ myenteric plexi in the gastric corpus and antrum in DM mice. Results showed that the connecting nerve fibers in myenteric plexi were more slender and the distance between adjacent ganglia was increased in DM
Table 2 Semi-quantification of IGF-1R-positive myenteric plexus.
Area density of IGF-1R-positive myenteric ganglia (mm2 /mm2 ) Area density of IGF-1R-positive nerve fibers (mm2 /mm2 ) Length density of IGF-1R-positive nerve fibers (mm/mm2 ) *
p < 0.05 compared with corpus.
Corpus (mean ± SEM)
Antrum (mean ± SEM)
0.245 ± 0.006
0.265 ± 0.006*
0.268 ± 0.016
0.304 ± 0.015*
0.366 ± 0.019
0.383 ± 0.017*
mice (Fig. 1C). The area densities of the IGF-IR+ myenteric ganglia and their primary fibers of DM mice were significantly decreased compared with controls (Fig. 1D). The alterations in the myenteric plexi of gastric corpus and antrum were nearly the same.
Identification of IGF-IR+ cells in the gastric myenteric plexus To determine the nature of the IGF-IR+ cells in the myenteric ganglia, double staining was performed. ChAT is the rate-limiting enzyme that is required for the acetylcholine (Ach) synthesis. CLSM indicated that the ChAT+ (green) ganglia and nerve fibers were mainly distributed in the gastric myenteric plexi (Fig. S1). Several distinct round or ovoid ChAT+ neurons were located within a single ganglion. Double staining with IGF-IR (red) and ChAT revealed that most IGF-IR+ cells also exhibited ChAT staining, indicating that these IGF-IR+ cells were cholinergic neurons (Fig. 2). However, several IGF-IR+ cells in the myenteric plexus were not labeled with ChAT (Fig. 2). In addition, the number density of cholinergic neurons both in gastric corpus and antrum was remarkably reduced in DM mice compared with controls (Table 2). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.acthis. 2014.01.011. Next, we performed double staining with IGF-IR (green) and S100ˇ (red). No co-localization areas were observed in the myenteric plexus. IGF-IR was only expressed in the neurons and nerve fibers, whereas the enteric glial cells stained by the anti-S100ˇ antibody did not exhibit IGF-IR immunoreactivity. These results
Please cite this article in press as: Zhang G-q, et al. Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.01.011
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Fig. 2. Double-labeling with ChAT and IGF-IR shows the nature of the IGF-IR-positive neurons. (A) ChAT staining (green). (B) IGF-IR staining (red). (C) Merged figure showing both ChAT and IGF-IR. Nuclei are stained with DAPI (blue). It can clearly be observed that most of the IGF-IR-positive cells are cholinergic neurons (arrow), while a few of the IGF-IR-positive cells are not labeled with ChAT, indicating that they may belong to other neuronal types (arrowhead). Similar results were observed in the gastric corpus (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. (A) IGF-IR staining (green). (B) S100 staining (red). (C) Merged figure for IGF-IR and S100. Double labeling shows that IGF-IR is not expressed in the glial cells around the neurons. Arrow: IGF-IR-positive neuron. Arrowhead: enteric glial cells. Similar results were observed in the gastric corpus (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
suggest that the IGF-IR+ cells in the myenteric plexus were not enteric glial cells (Fig. 3). Double-labeling with IGF-IR and c-KIT IGF-IR+ neurons (red) in the myenteric plexi were densely arranged in the myenteric ganglia (Fig. 4A). However, c-KIT+ ICCs (ICC-MY, green) with 2 or 3 long and fine processes formed a cellular network with one another and associated with the myenteric plexi (Fig. 4B). Double labeling experiments demonstrated that the ICCs did not co-immunostain for IGF-IR and instead formed a network independent from the myenteric plexus. However, the IGF-IR+ nerve fibers were in close proximity to the processes and bodies of the ICCs (Fig. 4C). c-KIT labeling was also observed in the mast cells in the gut; however, the mast cells could be distinguished from the ICCs by their distinct characteristics. Discussion The present study demonstrated that IGF-IR was widely expressed in the tunica muscularis, myenteric plexus and muscularis mucosa in the mouse stomach. The appearance of the soma and fiber bundles in the gastric myenteric plexus suggested that these cells were likely neurons. Double labeling further confirmed that most of the IGF-IR+ cells were cholinergic neurons, rather than enteric glial cells, in the gastric myenteric plexus. The decrease of IGF-IR in the myenteric plexi of DM mice diminished the number density of cholinergic neurons.
IGF-I/IGF-IR signaling plays an important role in neuronal survival, function and injury repair in response to environmental stimulation and injury (Neff et al., 1993; Bitar et al., 1997). Consistent with Horváth et al. (2006), we observed IGF-IR expression in the mouse gastric myenteric neurons, which indicated that IGFI/IGF-IR signaling might be involved in the survival and function of gastric myenteric neurons. In the population of myenteric neurons, most of the cholinergic neurons are classic excitatory neurons that release Ach to excite the M receptors in smooth muscle cells, thereby stimulating GI contraction and peristalsis based on slow waves (Uchiyama and Chess-Williams, 2004). The present study further confirmed that most of the IGF-IR+ neurons in the gastric myenteric plexus are cholinergic neurons. Bozyczko-Coyne et al. (1993) found that IGF-I promoted the survival of cholinergic neurons and increased the ChAT activity in embryonic rat spinal cord cultures. Exogenous IGF-I preferentially prevented the loss of cholinergic neurons in the striatum following ischemic brain injury in fetal sheep (Guan et al., 2000). In IGF-IR-immunolabeled cultured human central nervous system-derived neuronal cells, increased ChAT mRNA and protein expression levels were observed after the administration of IGF-I (Rivera et al., 2005). In this study, we also found that a decrease in IGF-IR was accompanied by a loss of myenteric cholinergic neurons in DM mice. We thus inferred that IGF-I/IGF-IR signaling could be responsible for the survival of cholinergic neurons in the murine ENS in vivo. Furthermore, IGF-IR could also be detected in the primary nerve strands, secondary nerve fibers and tertiary fibers. Therefore, we assume that IGF-I/IGF-IR signaling may be also related to neurite outgrowth.
Please cite this article in press as: Zhang G-q, et al. Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.01.011
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Fig. 4. (A) IGF-IR staining (red) in the myenteric plexus. (B) c-KIT staining (green) in the myenteric plexus. (C) Merged figure for IGF-IR and c-KIT in the myenteric plexus. Double labeling indicates that ICCs do not express IGF-IR. However, the IGF-IR-positive neurons are observed in close proximity to the ICCs. Similar results were observed in the gastric corpus (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ICCs are the pacemaker cells that generate and propagate spontaneous slow waves within the GI tract (Huizinga et al., 1995). ICCs are also known as mediators between neurons and smooth muscles. Although ICC depletion in diabetes is likely a consequence of reduced IGF-I signaling, mature ICCs are not direct targets of IGF-I because they lack IGF-IR (Horváth et al., 2006). The results from Horváth et al. (2006) raised the possibility that the effects of IGF-I on ICCs could be mediated in an indirect way. It is well known that smooth muscles and myenteric neurons are the main sources of stem cell factor (SCF), the natural ligand of c-KIT, which is fundamentally required for the survival and function of ICCs in the gut (Wu et al., 2000; Mei et al., 2006, 2009). In this study, we found that IGF-IR was also strongly expressed in the GI smooth muscles and myenteric plexi. Therefore, we presumed that IGF-I/IGF-IR signaling might play a role in maintaining ICC survival and function by stimulating SCF production in the GI muscles and myenteric plexus. Interestingly, IGF-IR+ neurons were found in close proximity to the ICCs. Because ICCs express the Ach receptor, it is reasonable that IGF-IR+ /ChAT+ neurons could also be involved in gastric motility mediation through ICCs. In addition, our results showed that several IGF-IR+ neurons in the myenteric plexi were not labeled with ChAT. These noncholinergic neurons might be related to inhibitory neurons, such as nNOS neurons and vasoactive intestinal peptide (VIP) neurons, or interneurons, such as peptidergic neurons and 5-HT neurons, which comprise the neuronal population in the myenteric plexus along with excitatory cholinergic neurons. To further confirm the cell types of these IGF-IR+ /ChAT− neurons, specific markers are needed to label each type of neurons specifically. For example, NADPHd is used as a marker for nitrergic neurons, NOS is used for NOS neurons, VIP is used for VIP neurons, and SP is used for SP neurons. It is known that nearly all myenteric neurons are either nitrergic or cholinergic, and the few remaining neurons have either the nitrergic− /cholinergic− or the nitrergic+ /cholinergic+ phenotype; these include peptidergic neurons. The literature suggests that, in adult rats, the myenteric neurons/cm2 in the antrum consist of 2723 nNOS+ neurons/cm2 and 6376 nNOS− neurons/cm2 (basically cholinergic); in the corpus, 2619 nNOS+ neurons/cm2 and 4622 nNOS− /cm2 are observed (Phillips et al., 2003). However, very little research has attempted to quantify the presence of peptideric neurons in the myenteric plexus of the mouse stomach. Hendershot et al. (2007) have demonstrated that 5.0 ± 0.02% of neural crestderived precursor cells immunoselected from an embryonic chick gut express VIP. In the antrum, diabetic mice show a decreased
number of VIP neurons in the myenteric ganglia, whereas the density of the NPY nerve fibers is constant (Spångéus and El-Salhy, 2001). To estimate the number of peptideric neurons (including subtypes), significant further work is required. Based on the general expression of IGF-IR in myenteric neurons, we propose that IGFI/IGF-IR signaling could play a general neurotrophic role in multiple myenteric neurons. The enteric glial cells, which surround and outnumber the enteric neurons, play an important role in the maintenance of gut homeostasis. The current evidence has suggested that enteric glial cells serve trophic and protective functions for enteric neurons through the secretion of multiple neurotrophic factors, e.g., NGF, glial cell-derived neurotrophic factor (GDNF), and transforming growth factor- (TGF-) (Liu et al., 2010; Chalazonitis et al., 2012; Wang et al., 2012). Notably, in contrast to the case for enteric neurons, enteric glial cell density was not reduced in diabetic rats (Kuemmerle, 2005; Pereira et al., 2011). It is probably due to that the decrease in serum IGF-I might have less of an effect on the enteric glial cells which were not labeled with anti-IGF-IR observed in this study. Glial preservation could exert a defense mechanism to maintain the viability of the enteric neurons after diabetic neuropathy (Pereira et al., 2011). It should be noted that in order to avoid the effects of preparation traction, the area density of IGF-IR+ myenteric ganglia, rather than the number of IGF-IR+ neurons, was analyzed in the current study because this measure was more constant during the preparations (Karaosmanoglu et al., 1996). Our results showed that, in the gastric antrum, the area density of the IGF-IR+ ganglia, the area density of the IGF-IR+ primary nerve strands and the length density of the IGF-IR+ primary nerve strands in the myenteric plexus were all higher than those in the gastric corpus, indicating a high neuron density in the gastric antrum. The frequency of slow waves in the stomach increased from proximal to distal locations; this is initiated by ICCs located in the myenteric plexus region that generate the action potential and corresponding gastric contraction. Moreover, the frequency of basal contraction was higher in the antrum than in the corpus (Cellini et al., 2011). The regional distributions of IGF-IR (antrum > corpus) are most likely associated with regional differences in slow wave and basal contractions and, accordingly, with the regional functions of the different gastric segments. In summary, IGF-IR is mainly expressed in smooth muscles and myenteric neurons in mouse stomachs. Most of the IGF-IR+ neurons are cholinergic neurons that are involved in the regulation of gastric motility. IGF-I/IGF-IR signaling might affect neuronal
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survival and neurite outgrowth, as well as SCF production, which is important for the survival, proliferation and function of ICCs in the GI tract. Furthermore, the different distributions of IGF-IR may be adapted for their functional requirements in the gastric antrum and corpus. Our results provide experimental evidence supporting the further investigation of IGF-I/IGF-IR signaling in the development of GI motility disorders. Acknowledgments This work was supported in part by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20111107110012 and 20121107120020), the National Science Foundation of China (81300285 and 31271290), the Scientific Research Key Program of Beijing Municipal Commission of Education (KZ201310025020) and the Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality (PHR201007113). References Altar CA, Hunt RA, Jurata LW, Webster MJ, Derby E, Gallagher P, et al. Insulin, IGF-1, and muscarinic agonists modulate schizophreniaassociated genes in human neuroblastoma cells. Biol Psychiatry 2008;64:1077–87. Beilharz EJ, Russo VC, Butler G, Baker NL, Connor B, Sirimanne ES, et al. Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic-ischemic in jury. Brain Res Mol Brain Res 1998;59: 119–34. Bitar MS, Pilcher CW, Khan I, Waldbillig RJ. Diabetes-induced suppression of IGF-I and its receptor mRNA levels in rat superior cervical ganglia. Diabetes Res Clin Pract 1997;38:73–80. Bozyczko-Coyne D, Glicksman MA, Prantner JE, McKenna B, Connors T, Friedman C, et al. IGF-I supports the survival and/or differentiation of multiple types of central nervous system neurons. Ann N Y Acad Sci 1993;692:311–3. Cellini J, DiNovo K, Harlow J, LePard KJ. Regional differences in neostigmine-induced contraction and relaxation of stomach from diabetic guinea pig. Auton Neurosci 2011;160:69–81. Chalazonitis A, Gershon MD, Greene LA. Cell death and the developing enteric nervous system. Neurochem Int 2012;61:839–47. Chan F, Liu Y, Sun H, Li X, Shang H, Fan D, et al. Distribution and possible role of PDGF-AA and PDGFR-␣ in the gastrointestinal tract of adult guinea pigs. Virchows Arch 2010;457:381–8. Craner MJ, Klein JP, Black JA, Waxman SG. Preferential expression of IGF-I in small DRG neurons and down-regulation following injury. Neuroreport 2002;13:1649–52. Gualco E, Wang JY, Del Valle L, Urbanska K, Peruzzi F, Khalili K, et al. IGF-IR in neuroprotection and brain tumors. Front Biosci 2009;14:352–75. Guan J, Bennet TL, George S, Waldvogel HJ, Faull RL, Gluckman PD, et al. Selective neuroprotective effects with insulin-like growth factor-1 in phenotypic striatal neurons following ischemic brain injury in fetal sheep. Neuroscience 2000;95:831–9. Hendershot TJ, Liu H, Sarkar AA, Giovannucci DR, Clouthier DE, Abe M, et al. Expression of Hand2 is sufficient for neurogenesis and cell type-specific gene expression in the enteric nervous system. Dev Dyn 2007;236:93–105. Horváth VJ, Vittal H, Lörincz A, Chen H, Almeida-Porada G, Redelman D, et al. Reduced stem cell factor links smooth myopathy and loss of interstitial cells of Cajal in murine diabetic gastroparesis. Gastroenterology 2006;130:759–70. Huizinga JD, Thuneberg L, Klüppel M, Malysz J, Mikkelsen HB, Bernstein A. W/kit gene required for interstitial cells of
Cajal and for intestinal pacemaker activity. Nature 1995;373: 347–9. Hwang IK, Yoo KY, Park SK, An SJ, Lee JY, Choi SY, et al. Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult. Neurochem Int 2004;45:149–56. Ishii DN, Lupien SB. Insulin-like growth factors protect against diabetic neuropathy: effects on sensory nerve regeneration in rats. J Neurosci Res 1995;40:138–44. Karaosmanoglu T, Aygun B, Wade PR, Gershon MD. Regional differences in the number of neurons in the myenteric plexus of the guinea pig small intestine and colon: an evaluation of markers used to count neurons. Anat Rec 1996;244:470–80. Kuemmerle JF. IGF-I elicits growth of human interstinal smooth muscle cells by activation of PI3K, PDK-1, and p70S6 kinase. Am J Physiol Gastrointest Liver Physiol 2003;284:G411–22. Kuemmerle JF. Endogenous IGF-1 protects human intestinal smooth muscle cells from apoptosis by regulation of GSK3 activity. Am J Physiol Gastrointest Liver Physiol 2005;288: G101–10. Liu W, Yue W, Wu R. Effects of diabetes on expression of glia fibrillary acidic protein and neurotrophins in rat colon. Auton Neurosci 2010;154:79–83. Mei F, Yu B, Ma H, Zhang HJ, Zhou DS. Interstitial cells of Cajal could regenerate and restore their normal distribution after disrupted by intestinal transection and anastomosis in the adult guinea pigs. Virchows Arch 2006;449:348–57. Mei F, Han J, Huang Y, Jiang ZY, Xiong CJ, Zhou DS. Plasticity of interstitial cells of Cajal: a study in the small intestine of adult Guinea pigs. Anat Rec 2009;292:985–93. Mulholland MW, Romanchuk G, Simeone DM, Flowe K. Stimulation of myenteric plexus neurite outgrowth by insulin and insulinlike growth factors I and II. Life Sci 1992;51:1789–96. Neff NT, Prevette D, Houenou LJ, Lewis ME, Glicksman MA, Yin QW, et al. Insulin-like growth factors: putative muscle-derived trophic agents that promote motoneuron survival. J Neurobiol 1993;24:1578–88. Pantaleo MA, Astolfi A, Nannini M, Biasco G. The emerging role of insulin-like growth factor 1 receptor (IGF1r) in gastrointestinal stromal tumors (GISTs). J Transl Med 2010;8:117. Pereira RV, Tronchini EA, Tashima CM, Alves EP, Lima MM, Zanoni JN. L-Glutamine supplementation prevents myenteric neuron loss and has gliatrophic effects in the ileum of diabetic rats. Dig Dis Sci 2011;56:3507–16. Phillips RJ, Kieffer EJ, Powley TL. Aging of the myenteric plexus: neuronal loss is specific to cholinergic neurons. Auton Neurosci 2003;106:69–83. Rivera EJ, Goldin A, Fulmer N, Tavares R, Wands JR, de la Monte SM. Insulin and insulin-like growth factor expression and function deteriorate with progression of Alzheimer’s disease: link to brain reductions in acetylcholine. J Alzheimers Dis 2005;8:247–68. Rouyer-Fessard C, Gammeltoft S, Laburthe M. Expression of two types of receptor for insulinlike growth factors in human colonic epithelium. Gastroenterology 1990;98:703–7. Sharma HS, Nyberg F, Westman J, Alm P, Gordh T, Lindholm D. Brain derived neurotrophic factor and insulin-like growth factor-1 attenuate upregulation of nitric oxide synthase and cell injury following trauma to the spinal cord—an immunohistochemical study in rat. Amino Acids 1998;14:121–9. Sonntag WE, Lynch C, Thornton P, Khan A, Bennett S, Ingram R. The effects of growth hormone and IGF-1 deficiency on cerebrovascular and brain ageing. J Anat 2000;197:575–85. Spångéus A, El-Salhy M. Myenteric plexus of obese diabetic mice, an animal model of human type 2 diabetes. Histol Histopathol 2001;16:159–65.
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Ward CW, Garrett TP. Structural relationships between the insulin receptor and epidermal growth factor receptor families and other proteins. Curr Opin Drug Discov Devel 2004;7:630–8. Wu JJ, Rothman TP, Gershon MD. Development of the interstitial cells of Cajal: origin, Kit dependence and neuronal and nonneuronal sources of Kit ligand. J Neurosci Res 2000;59:384– 401. Xu G, Sima AA. Altered immediate early gene expression in injured diabetic nerve: implications in regeneration. J Neuropathol Exp Neurol 2001;60:972–83. Yasuda H, Terada M, Maeda K, Kogawa S, Sanada M, Haneda M, et al. Diabetic neuropathy and nerve regeneration. Prog Neurobiol 2003;69:229.
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Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles Guo-quan Zhang a,b , Shu Yang a , Xiao-shuang Li a , De-shan Zhou a,∗ a b
Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China Department of Histology and Embryology, Logistics University of Chinese People’s Armed Police Force, Tianjin 300162, PR China
a r t i c l e
i n f o
Article history: Received 27 November 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online xxx Keywords: Cholinergic neurons IGF-IR Interstitial cells of Cajal Mouse Myenteric plexus Stomach
a b s t r a c t Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have tremendous trophic effects on the central, peripheral and enteric neurons. The loss of IGF-IR contributes to the development of diabetic gastroparesis. However, the nature and the function of the IGF-IR+ cells in the gastric myenteric plexus remain unclear. In this study, anti-ChAT, anti-S100 or anti-c-KIT antibodies were used to co-label IGF-IR+ cells and neurons, glial cells or interstitial cells of Cajal (ICCs), respectively. We also generated type 1 diabetic mice (DM) to explore the influence of impaired IGF-I/IGF-IR in the myenteric neurons. Results showed that IGF-IR was expressed in the epithelium, smooth muscles and myenteric plexi of the mouse stomach. Most of the IGF-IR+ cells in the myenteric plexi were ChAT+ cholinergic neurons, but not enteric glial cells and there were more IGF-IR+ neurons and fibers in the gastric antrum than in the corpus. The IGF-IR+ /ChAT+ neurons and ICCs were closely juxtaposed, but distinctly distributed in the myenteric plexus, indicating a possible role for the IGF-IR+ /ChAT+ neurons in the mediation of gastric motility through ICCs. Moreover, the decrease of IGF-IR and cholinergic neurons in the myenteric plexi and smooth muscles of DM mice suggested that IGF-I/IGF-IR signaling might play a role in neuron survival and neurite outgrowth, as well as stem cell factor (SCF) production, which is required for the development of ICCs. Our results provide insights into the effects of IGF-I/IGF-IR signaling on the development of gastrointestinal motility disorders. © 2014 Elsevier GmbH. All rights reserved.
Introduction Insulin-like growth factor-I (IGF-I), which shares substantial amino acid sequence homology with pro-insulin, is produced by many tissues, primarily in the liver, as well as in the brain, kidney, heart and lung (Gualco et al., 2009). IGF-I elicits biological effects on cellular survival, proliferation and differentiation through its specific receptor (IGF-IR) (Kuemmerle, 2003; Sonntag et al., 2000; Pantaleo et al., 2010). IGF-I is also considered a type of neurotrophic factor, due to its general neurotrophic effects on the central and peripheral nervous systems. Several lines of evidence have shown that IGF-I is able to restrict neuronal and glial cell loss and promote the recovery process after ischemic insult in the rodent brain (Beilharz et al., 1998; Hwang et al., 2004). IGF-I also reduces spinal cord edema by inhibiting neuronal nitric oxide synthase (nNOS) activity (Sharma et al., 1998). In diabetic peripheral neuropathy patients, IGF-I levels are decreased, which contributes to the refractoriness of nerve regeneration (Yasuda et al., 2003). The neuropathy in diabetic rats could be ameliorated or prevented by IGF-I or insulin
∗ Corresponding author. E-mail address: [email protected] (D.-s. Zhou).
treatment (Ishii and Lupien, 1995; Tomlinson et al., 1997). It has been proposed that a decrease in the IGF-1 mRNA expression in diabetic rats might be responsible for the delayed nerve growth factor (NGF) mRNA response by affecting the induction of c-FOS and thus impairing nerve regeneration (Xu and Sima, 2001). Additionally, the neurotrophic effects of IGF-I or insulin potentially act through ERK1/2 and Akt signaling (Altar et al., 2008). The enteric nervous system (ENS) is composed of the submucosal plexus and the myenteric plexus, the latter of which plays a crucial role in the regulation of gastrointestinal (GI) peristalsis. Mulholland et al. (1992) reported that IGF-I is able to stimulate the neurite outgrowth of myenteric cells derived from neonatal guinea pigs in vitro, indicating that IGF-I also has a neurotrophic effect on myenteric neurons. The effects of IGF-I are primarily mediated through interactions with IGF-IRs, which are widely expressed in the brain, ganglia and nerve fibers (Craner et al., 2002; Gualco et al., 2009). In the GI tract, IGF-IRs are present in the epithelium, smooth muscle cells, and myenteric plexi and are considered to contribute to the development of diabetic gastroparesis in mice, rabbits and humans (Rouyer-Fessard et al., 1990; Termanini et al., 1990; Horváth et al., 2006). However, exactly which type(s) of IGF-IR+ cells exist in the gastric myenteric plexus has not been explored. The elucidation of this issue will help in understanding
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2 Table 1 Antibodies used in immunofluorescence. Primary antibodies
Secondary antibodies
Rabbit anti-IGF-1R␣ (1:100)
sc-712, Santa Cruz
Rabbit anti-IGF-1R (1:600)
3027 S, cell signal technology BA0120, Boster ACK2, eBiosciecnce NBP1–30052, Novus biologicals
Rabbit anti-S100 (1:100) Rat anti-KIT (1:200) Goat anti-ChAT (1:100)
FITC conjugated goat anti-rabbit IgG (1:100) or Cy3 conjugated goat anti-rabbit IgG (1:200) AF 594 conjugated donkey anti-rabbit IgG (1:200)
A21207, Life Technologies
Cy3 conjugated goat anti-mouse IgG (1:200) FITC conjugated goat anti-rat IgG (1:200) AF 488 conjugated donkey anti-goat IgG (1:100)
A10521, Life Technologies 629511, Life Technologies A10521, Life Technologies
the functions of IGF-I/IGF-IR signaling in the gastric myenteric plexus, as well as of their possible role in GI motility disorders.
F2765, Life Technologies A10520, Life Technologies
Immunohistochemistry
Ten male BALB/c mice (6-weeks old, 22–26 g) were purchased from the Animal Center of Capital Medical University (Beijing, China). All mice were maintained in a temperature-controlled room (23 ± 1 ◦ C) with a constant 12 h light/dark cycle. Food and water were available ad libitum. After fasting for 14 h, five mice received a single intraperitoneal injection of Alloxan monohydrate (200 mg/kg, Sigma–Aldrich). The remaining five littermates receiving the same dose of normal sodium were used as controls. The fasting blood glucose was measured by the use of an Accu-chek Active Complete blood glucose monitor (Roche, Germany) 72 h after injection. The mice with a blood glucose ≥11.1 mmol/L were considered as DM mice and employed in this study. The blood glucose of these DM mice was monitored every week. The following experiments were performed 8 weeks after the onset of DM. All experimental procedures were approved by the Institutional Animal Care Committee from Capital Medical University, Beijing China.
Mature IGF-IR is a heterotetramer consisting of two extracellular ␣ subunits and two  subunits (Ward and Garrett, 2004); therefore, both anti-IGF-IR␣ and anti-IGF-IR antibodies were used in this study. Non-specific binding sites were blocked with 1% bovine serum albumin (BSA) and 1% normal goat serum for 30 min. The specimens were incubated with primary antibody to IGF-IR␣ or IGF-IR overnight at 4 ◦ C, followed by incubation with the corresponding secondary antibody for 1 h at 25 ◦ C (Table 1). After washing, the specimens were mounted in fluorescent mounting medium containing DAPI (Zhongshan Jinqiao Biotechnology, China) and observed under a fluorescence microscope (Nikon, 80i, Japan) or confocal laser scanning microscope (CLSM, Leica, TCS SP5). The preparations were incubated with an isotype control antibody or without a primary antibody as negative controls. To determine the nature of the IGF-IR-positive cells, the preparations were double labeled as follows: (1) anti-IGF-IR␣ and anti-ChAT (a marker for cholinergic nerves) primary antibodies with FITC-, Cy3-, AF 594- or AF 488-conjugated IgG secondary antibodies; (2) anti-IGF-IR␣ and anti-S100 (an antibody against enteric glial cells) primary antibodies with FITC-, Cy3- or AF 594-conjugated IgG secondary antibodies; or (3) anti-IGF-IR␣ and anti-c-KIT (a marker for ICCs) primary antibodies with FITC-, Cy3or AF 594-conjugated IgG secondary antibodies (Table 1).
Whole-mount preparations
Image analysis and statistics
The whole-mounts were prepared as described previously (Chan et al., 2010). In brief, the stomach was removed and fully washed with pre-cooled phosphate-buffered saline (PBS, 0.01 M, pH 7.4). Next, the stomach was inflated to maintain its shape with 4% paraformaldehyde for IGF-IR, choline acetyl transferase (ChAT) and S100 staining or 100% acetone for c-KIT staining for 2 h. Finally, each sample was immersed in the same fixative for 12 h. Three samples of 0.5 cm × 0.5 cm were randomly cut from each gastric corpus and antrum. The mucosa and submucosa were removed, and the longitudinal muscle layer containing the myenteric plexus was dissected. The prepared muscles were placed in 0.01 M PBS at 4 ◦ C for (double) immunostaining.
Ten photographs per whole-mount were randomly taken at a magnification of 20×. The target area of interest was outlined, and the areas of myenteric ganglia and primary nerve strands, as well as the lengths of the primary nerve strands, were measured in Image Pro-Plus software 6.0 (Media Cybernetics, Bethesda, MD, USA) based on their immunoreactive intensity. The area density of the myenteric ganglia and primary nerve strands and the length density of the primary nerve strands were derived by dividing the total areas of myenteric ganglia and primary nerve strands and the total length of primary nerve strands, respectively. The number density of cholinergic neurons was determined by dividing the total number of cholinergic neuron by the total area of the fields. Statistical analyses were performed with SPSS 13.0 software (SPSS Inc., IBM, Chicago, IL, USA). Data are expressed as the means ± SEM and compared using a Student’s t-test. A p-value of 0.05 was considered statistically significant.
Materials and methods Generation of type 1 DM mouse
Cryosection preparations The samples from the gastric corpus and antrum were embedded in an optimal cutting temperature compound (O.C.T., Sakura) and then frozen in liquid nitrogen. Cryosections (10 m thick) were cut with a cryostat (CM3050S Leica Microsystems, Wetzlar, Germany) and mounted on poly-L-lysine-coated glass slides. The sections were fixed with 4% paraformaldehyde for IGF-IR, ChAT and S100 staining or 100% acetone for c-KIT staining for 30 min.
Results Distribution of IGF-IR in the stomach The same staining patterns for IGF-IR␣ and IGF-IR were observed in the gastric wall; therefore, only IGF-IR␣ (green) was
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Fig. 1. This set of immunofluorescence images shows the wide distribution of IGF-IR in the stomach. (A) In cryosections, IGF-IR is intensely expressed in the tunica muscularis (including the circular and longitudinal muscle layers), myenteric plexus (arrow) and muscularis mucosa (arrowhead). (B) In whole-mount preparations, myenteric ganglia (high-magnification figures in frames) and primary nerve strands (arrow), secondary nerve fibers (double arrow) and tertiary nerve fibers (arrowhead) are strongly stained for IGF-IR. IGF-IR+ cells are round or ovoid with distinct nuclei and neurites, suggesting that they are neuron-like cells. Nuclei are stained with DAPI (blue). (C) The neuronal network is loosened and the nerve fibers in the myenteric plexi are slender in DM mice compared with controls. (D) The area densities of the IGF-1R+ myenteric ganglia and their primary fibers of DM mice were significantly decreased compared with controls. p < 0.05. CM: circular muscle layer, LM: longitudinal muscle layer. MU: mucosa; SM: submucosa. The figures and the alterations of IGF-1R+ myenteric structures of the gastric antrum and corpus are essentially identical; here, we show only those from the antrum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
shown to represent IGF-IR. In cryosections, IGF-IR mainly localized to the tunica muscularis (both the circular and longitudinal muscle layers), myenteric plexus and muscularis mucosa (Fig. 1A). In whole mounts, stronger immunoreactivity for IGF-IR was detected in the myenteric plexus, including the ganglion cells, primary nerve strands, secondary nerve fibers and fine tertiary fibers; the IGF-IR+ cells in the myenteric ganglia were round or ovoid with distinct round nuclei, similar to the neurons (Fig. 1B). The area density of the IGF-IR+ myenteric ganglia, the area density of the IGF-IR+ primary nerve strands and the length density of the IGF-IR+ primary nerve strands in the myenteric plexi of the gastric antrum were all significantly higher than those in the gastric corpus (Table 2). We investigated the alteration of the IGF-IR+ myenteric plexi in the gastric corpus and antrum in DM mice. Results showed that the connecting nerve fibers in myenteric plexi were more slender and the distance between adjacent ganglia was increased in DM
Table 2 Semi-quantification of IGF-1R-positive myenteric plexus.
Area density of IGF-1R-positive myenteric ganglia (mm2 /mm2 ) Area density of IGF-1R-positive nerve fibers (mm2 /mm2 ) Length density of IGF-1R-positive nerve fibers (mm/mm2 ) *
p < 0.05 compared with corpus.
Corpus (mean ± SEM)
Antrum (mean ± SEM)
0.245 ± 0.006
0.265 ± 0.006*
0.268 ± 0.016
0.304 ± 0.015*
0.366 ± 0.019
0.383 ± 0.017*
mice (Fig. 1C). The area densities of the IGF-IR+ myenteric ganglia and their primary fibers of DM mice were significantly decreased compared with controls (Fig. 1D). The alterations in the myenteric plexi of gastric corpus and antrum were nearly the same.
Identification of IGF-IR+ cells in the gastric myenteric plexus To determine the nature of the IGF-IR+ cells in the myenteric ganglia, double staining was performed. ChAT is the rate-limiting enzyme that is required for the acetylcholine (Ach) synthesis. CLSM indicated that the ChAT+ (green) ganglia and nerve fibers were mainly distributed in the gastric myenteric plexi (Fig. S1). Several distinct round or ovoid ChAT+ neurons were located within a single ganglion. Double staining with IGF-IR (red) and ChAT revealed that most IGF-IR+ cells also exhibited ChAT staining, indicating that these IGF-IR+ cells were cholinergic neurons (Fig. 2). However, several IGF-IR+ cells in the myenteric plexus were not labeled with ChAT (Fig. 2). In addition, the number density of cholinergic neurons both in gastric corpus and antrum was remarkably reduced in DM mice compared with controls (Table 2). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.acthis. 2014.01.011. Next, we performed double staining with IGF-IR (green) and S100ˇ (red). No co-localization areas were observed in the myenteric plexus. IGF-IR was only expressed in the neurons and nerve fibers, whereas the enteric glial cells stained by the anti-S100ˇ antibody did not exhibit IGF-IR immunoreactivity. These results
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Fig. 2. Double-labeling with ChAT and IGF-IR shows the nature of the IGF-IR-positive neurons. (A) ChAT staining (green). (B) IGF-IR staining (red). (C) Merged figure showing both ChAT and IGF-IR. Nuclei are stained with DAPI (blue). It can clearly be observed that most of the IGF-IR-positive cells are cholinergic neurons (arrow), while a few of the IGF-IR-positive cells are not labeled with ChAT, indicating that they may belong to other neuronal types (arrowhead). Similar results were observed in the gastric corpus (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. (A) IGF-IR staining (green). (B) S100 staining (red). (C) Merged figure for IGF-IR and S100. Double labeling shows that IGF-IR is not expressed in the glial cells around the neurons. Arrow: IGF-IR-positive neuron. Arrowhead: enteric glial cells. Similar results were observed in the gastric corpus (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
suggest that the IGF-IR+ cells in the myenteric plexus were not enteric glial cells (Fig. 3). Double-labeling with IGF-IR and c-KIT IGF-IR+ neurons (red) in the myenteric plexi were densely arranged in the myenteric ganglia (Fig. 4A). However, c-KIT+ ICCs (ICC-MY, green) with 2 or 3 long and fine processes formed a cellular network with one another and associated with the myenteric plexi (Fig. 4B). Double labeling experiments demonstrated that the ICCs did not co-immunostain for IGF-IR and instead formed a network independent from the myenteric plexus. However, the IGF-IR+ nerve fibers were in close proximity to the processes and bodies of the ICCs (Fig. 4C). c-KIT labeling was also observed in the mast cells in the gut; however, the mast cells could be distinguished from the ICCs by their distinct characteristics. Discussion The present study demonstrated that IGF-IR was widely expressed in the tunica muscularis, myenteric plexus and muscularis mucosa in the mouse stomach. The appearance of the soma and fiber bundles in the gastric myenteric plexus suggested that these cells were likely neurons. Double labeling further confirmed that most of the IGF-IR+ cells were cholinergic neurons, rather than enteric glial cells, in the gastric myenteric plexus. The decrease of IGF-IR in the myenteric plexi of DM mice diminished the number density of cholinergic neurons.
IGF-I/IGF-IR signaling plays an important role in neuronal survival, function and injury repair in response to environmental stimulation and injury (Neff et al., 1993; Bitar et al., 1997). Consistent with Horváth et al. (2006), we observed IGF-IR expression in the mouse gastric myenteric neurons, which indicated that IGFI/IGF-IR signaling might be involved in the survival and function of gastric myenteric neurons. In the population of myenteric neurons, most of the cholinergic neurons are classic excitatory neurons that release Ach to excite the M receptors in smooth muscle cells, thereby stimulating GI contraction and peristalsis based on slow waves (Uchiyama and Chess-Williams, 2004). The present study further confirmed that most of the IGF-IR+ neurons in the gastric myenteric plexus are cholinergic neurons. Bozyczko-Coyne et al. (1993) found that IGF-I promoted the survival of cholinergic neurons and increased the ChAT activity in embryonic rat spinal cord cultures. Exogenous IGF-I preferentially prevented the loss of cholinergic neurons in the striatum following ischemic brain injury in fetal sheep (Guan et al., 2000). In IGF-IR-immunolabeled cultured human central nervous system-derived neuronal cells, increased ChAT mRNA and protein expression levels were observed after the administration of IGF-I (Rivera et al., 2005). In this study, we also found that a decrease in IGF-IR was accompanied by a loss of myenteric cholinergic neurons in DM mice. We thus inferred that IGF-I/IGF-IR signaling could be responsible for the survival of cholinergic neurons in the murine ENS in vivo. Furthermore, IGF-IR could also be detected in the primary nerve strands, secondary nerve fibers and tertiary fibers. Therefore, we assume that IGF-I/IGF-IR signaling may be also related to neurite outgrowth.
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Fig. 4. (A) IGF-IR staining (red) in the myenteric plexus. (B) c-KIT staining (green) in the myenteric plexus. (C) Merged figure for IGF-IR and c-KIT in the myenteric plexus. Double labeling indicates that ICCs do not express IGF-IR. However, the IGF-IR-positive neurons are observed in close proximity to the ICCs. Similar results were observed in the gastric corpus (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ICCs are the pacemaker cells that generate and propagate spontaneous slow waves within the GI tract (Huizinga et al., 1995). ICCs are also known as mediators between neurons and smooth muscles. Although ICC depletion in diabetes is likely a consequence of reduced IGF-I signaling, mature ICCs are not direct targets of IGF-I because they lack IGF-IR (Horváth et al., 2006). The results from Horváth et al. (2006) raised the possibility that the effects of IGF-I on ICCs could be mediated in an indirect way. It is well known that smooth muscles and myenteric neurons are the main sources of stem cell factor (SCF), the natural ligand of c-KIT, which is fundamentally required for the survival and function of ICCs in the gut (Wu et al., 2000; Mei et al., 2006, 2009). In this study, we found that IGF-IR was also strongly expressed in the GI smooth muscles and myenteric plexi. Therefore, we presumed that IGF-I/IGF-IR signaling might play a role in maintaining ICC survival and function by stimulating SCF production in the GI muscles and myenteric plexus. Interestingly, IGF-IR+ neurons were found in close proximity to the ICCs. Because ICCs express the Ach receptor, it is reasonable that IGF-IR+ /ChAT+ neurons could also be involved in gastric motility mediation through ICCs. In addition, our results showed that several IGF-IR+ neurons in the myenteric plexi were not labeled with ChAT. These noncholinergic neurons might be related to inhibitory neurons, such as nNOS neurons and vasoactive intestinal peptide (VIP) neurons, or interneurons, such as peptidergic neurons and 5-HT neurons, which comprise the neuronal population in the myenteric plexus along with excitatory cholinergic neurons. To further confirm the cell types of these IGF-IR+ /ChAT− neurons, specific markers are needed to label each type of neurons specifically. For example, NADPHd is used as a marker for nitrergic neurons, NOS is used for NOS neurons, VIP is used for VIP neurons, and SP is used for SP neurons. It is known that nearly all myenteric neurons are either nitrergic or cholinergic, and the few remaining neurons have either the nitrergic− /cholinergic− or the nitrergic+ /cholinergic+ phenotype; these include peptidergic neurons. The literature suggests that, in adult rats, the myenteric neurons/cm2 in the antrum consist of 2723 nNOS+ neurons/cm2 and 6376 nNOS− neurons/cm2 (basically cholinergic); in the corpus, 2619 nNOS+ neurons/cm2 and 4622 nNOS− /cm2 are observed (Phillips et al., 2003). However, very little research has attempted to quantify the presence of peptideric neurons in the myenteric plexus of the mouse stomach. Hendershot et al. (2007) have demonstrated that 5.0 ± 0.02% of neural crestderived precursor cells immunoselected from an embryonic chick gut express VIP. In the antrum, diabetic mice show a decreased
number of VIP neurons in the myenteric ganglia, whereas the density of the NPY nerve fibers is constant (Spångéus and El-Salhy, 2001). To estimate the number of peptideric neurons (including subtypes), significant further work is required. Based on the general expression of IGF-IR in myenteric neurons, we propose that IGFI/IGF-IR signaling could play a general neurotrophic role in multiple myenteric neurons. The enteric glial cells, which surround and outnumber the enteric neurons, play an important role in the maintenance of gut homeostasis. The current evidence has suggested that enteric glial cells serve trophic and protective functions for enteric neurons through the secretion of multiple neurotrophic factors, e.g., NGF, glial cell-derived neurotrophic factor (GDNF), and transforming growth factor- (TGF-) (Liu et al., 2010; Chalazonitis et al., 2012; Wang et al., 2012). Notably, in contrast to the case for enteric neurons, enteric glial cell density was not reduced in diabetic rats (Kuemmerle, 2005; Pereira et al., 2011). It is probably due to that the decrease in serum IGF-I might have less of an effect on the enteric glial cells which were not labeled with anti-IGF-IR observed in this study. Glial preservation could exert a defense mechanism to maintain the viability of the enteric neurons after diabetic neuropathy (Pereira et al., 2011). It should be noted that in order to avoid the effects of preparation traction, the area density of IGF-IR+ myenteric ganglia, rather than the number of IGF-IR+ neurons, was analyzed in the current study because this measure was more constant during the preparations (Karaosmanoglu et al., 1996). Our results showed that, in the gastric antrum, the area density of the IGF-IR+ ganglia, the area density of the IGF-IR+ primary nerve strands and the length density of the IGF-IR+ primary nerve strands in the myenteric plexus were all higher than those in the gastric corpus, indicating a high neuron density in the gastric antrum. The frequency of slow waves in the stomach increased from proximal to distal locations; this is initiated by ICCs located in the myenteric plexus region that generate the action potential and corresponding gastric contraction. Moreover, the frequency of basal contraction was higher in the antrum than in the corpus (Cellini et al., 2011). The regional distributions of IGF-IR (antrum > corpus) are most likely associated with regional differences in slow wave and basal contractions and, accordingly, with the regional functions of the different gastric segments. In summary, IGF-IR is mainly expressed in smooth muscles and myenteric neurons in mouse stomachs. Most of the IGF-IR+ neurons are cholinergic neurons that are involved in the regulation of gastric motility. IGF-I/IGF-IR signaling might affect neuronal
Please cite this article in press as: Zhang G-q, et al. Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.01.011
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Please cite this article in press as: Zhang G-q, et al. Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.01.011