Aging-dependent decrease in the numbers of enteric neurons, interstitial cells of Cajal and expression of connexin43 in various regions of gastrointestinal tract

Intestinal transit in mice is significantly delayed with time

To evaluate the GI motility function during aging in vivo, we used intestinal transit rate as a concernful parameter. The transit of the carbon marker 30 minutes after gavage was restricted to the small intestine in different groups (Fig. 1A). The ink propulsion distance of 20- and 24-mo-old mice was markedly shorter than that of younger groups, but no remarkable difference was observed between mice from 2 mo to 16 mo. We also found that the length of the entire small intestine was added from 16 mo (Fig. 1B). Hence the transit rate was reduced from 88.25% in 12-mo-old mice to 42.13% in 24-mo-old mice (Fig. 1C).

ICCs in the mouse GI tract decrease with increasing age

ICCs express the proto-oncogene c-kit related to the development and maintenance of ICCs [24]. The immunofluorescence staining (Fig. 2A-D) and western blot analysis (Fig. 2F-H) of c-kit showed that the proportions of ICCs in the MP region of the mouse GI tract including stomach, jejunum and colon all tended to decrease during aging. In the case of stomach, in 2-mo-old mice, the cell body of ICCs was big, their processes were thick and there were many secondary and tertiary branches, which interlaced and formed a complete network (Fig. 2D). As age increased from 16 mo, not only the number of ICCs in stomach was decreased, but also the processes presented fewer branches and cellular network was sparse. Like those in stomach, the similar tendency was also observed in either jejunum or colon, however, the alterations in ICCs appeared later considerably: it was at 20 mo in jejunum, and at 24 mo in colon (Fig. 2E). And age-related damage to cellular network was clearly observed in the colon of the oldest individuals (24-mo-old).

The number of ENS neurons in the mouse GI tract decreases with aging

Immunofluorescence staining and NADPH-diaphorase (NADPH-d) histochemistry were used to label corresponding ENS neurons, respectively, in whole-mount preparations according to specific biomarkers as follows: (1) choline acetyltransferase (ChAT) for excitatory cholinergic neurons; and (2) nitric oxide synthase (NOS) for inhibitory nitrergic neurons.

ChAT is the rate-limiting enzyme that is required for the acetylcholine synthesis. Strong immunoreactivity for ChAT (green) was obviously seen in the MP of GI tract, including neurons with their primary strands, secondary bundles and fine tertiary fibers. In young mice, several distinct round or ovoid ChAT-positive neurons were located within each ganglion, and the granular positive reactant was clearly scattered around the cell body and also within interganglionic nerve bundles. The neural networks became sparse in old mice indicating the senescence phenomenon also appeared in the ENS of the mouse GI tract (Fig. 3A-C). According to statistical analysis, compared with young (2-mo-old) and middle-aged (12-mo-old) mice, ChAT-positive area of ganglia and nerve fibers per field was gradually decreased in the stomach from 16 mo; however, reduction of ChAT(+) area was started at 20 mo of age in the intestine (Fig. 3D). The similar results were obtained by western blotting (Fig. 3E-G).

NADPH-d histochemistry is believed to be a good method for quantifying numbers of nitrergic neurons, which represented one of subpopulation of enteric neurons. Intriguingly, the changes of NOS(+) neurons in different organs of GI tract were shown in coincidence with ChAT-positive neurons. NOS(+) neurons were small and very intensely stained, with clearly visible unstained nuclei; meanwhile, there were a few labelled cell bodies scattered within nerve fibers (Fig. 4A-D). Alterations of total numbers of NOS(+) neurons per unit area (mm²) with age were shown in Fig. 4A-C and Fig. 4E, respectively. A significant reduction in NOS(+) neuron number per mm² was shown in stomach between 12- and 16-mo-old mice, but no reduction in jejunum and colon until 20 mo. Furthermore, age-related expression of neuronal nitric oxide synthase (nNOS) protein, measured by western blotting, was markedly decreased in aging mice, which was consistent with the alteration of NOS(+) neurons (Fig. 4F-H).

Furthermore, the total enteric neurons were evaluated by pan-neuronal markers protein gene product 9.5 (PGP9.5) and human neuronal protein HuC/HuD (HuC/D) immunostaining and western blotting, as observed in young (2-mo-old) and old (16-, 20-mo-old) mouse colon. PGP9.5-positive MP including neurons and processes, as well as nerve fibers were well exhibited via immunofluorescence staining, and quantification of PGP9.5(+) area per field did not show significant reduction until 20 mo, matching results obtained from the test of ChAT- and NOS-positive MP (Fig. 5A-B). Western blot analysis on PGP9.5 (Fig. 5C) and HuC/D (Fig. 5D) protein in colonic muscle layer containing MP showed the similar results.

Cx43 expression in the mouse GI tract is reduced during aging

Gap junctions are important structures for cellular communication and electrical transmission. To clarify whether GI motility dysfunction in aged mice is related to the defect of gap junction structures, Cx43, as the main gap junction protein, was examined by western blotting and immunofluorescence staining in mice of different ages, respectively. Importantly, expressions of Cx43 in stomach (Fig. 6A), jejunum (Fig. 6B) and colon (Fig. 6C) showed a clear tendency of decrease in elderly mice (16-, 20- and 24-mo-old). Moreover, a double‐immunofluorescence labelling of c-kit and Cx43 was presented in Fig. 6D. The Cx43 immunopositive product (green) appeared as plaques, and c-kit-positive staining (red) included cell bodies of ICCs and their processes. The high density of Cx43-puncta distributed throughout colonic MP of 2-mo-old mice, and some co-localized with ICCs (arrows). Cx43-puncta was sparse in 16- and 24-mo-old mice, whereas c-kit-positive area was not significantly diminished until 24 mo (Fig. 6D-F), suggesting Cx43 decline prior to ICCs loss.

ICCs, enteric neurons and Cx43 are lost in the geriatric colon

To clarify differences of ICCs, enteric neurons and Cx43 between young and old cohorts, we collected 8 samples of normal colonic muscle layer, four each from young group (27-36 years old) and old group (72-82 years old). Age-related changes in protein expressions were measured by western blotting (Fig. 7). It was clearly seen that c-kit and ChAT were significantly reduced in old people. Simultaneously, nNOS and Cx43 were also shown to be down-expressed in elderly samples.

Increased inflammation and apoptosis are present in the aged colon

In order to explore mechanisms involved in aging that cause the loss of ICCs and enteric neurons, we performed mRNA expression analysis of classic pro-inflammatory cytokines including tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. A statistically significant rise in overall pro-inflammatory cytokines was found both in aged mice (Fig. 8A) and geriatric people (Fig. 8B) compared with young. We further investigated the expression of key proteins related apoptosis in the colonic muscle layer. As shown in Fig. 8C-D, decreased B-cell lymphoma-2 (Bcl-2) and increased cleaved caspase-3 were observed in old compared to young group.

Animals

A total of 60 C57BL/6 mice of either gender were purchased from Capital Medical University and divided into five groups according to age: 2-, 12-, 16-, 20- and 24-mo-old, to simulate life phases at approximately 20, 40, 50, 60 and 70 years of age in human [39]. Mice were housed in a controlled environment under a 12 h light/dark cycle at 22 ± 2 °C, 55 ± 5% humidity with access to food and water ad libitum. All mice were sacrificed by appropriate anesthesia and cervical dislocation prior to sample collection. The gastric body, the middle part of the jejunum and colon were surgically removed from mice and used for further experiments. All animal procedures were carried out strictly under protocols approved by the Animal Care and Use Committee of Capital Medical University (Permit Number AEEI-2015-143, 17 October 2015). Every effort was made to minimize the number of animals used as well as their suffering.

Human tissue samples

The clinical tissue samples, including 8 normal colonic muscle layers from different colorectal cancer patients, were collected immediately after surgical resection prior to any other therapeutic intervention at Beijing Friendship Hospital, Capital Medical University (Beijing, China). All patients were chemotherapy and radiation therapy naive. The young group (n=4) was 27 to 36 years old, and the elderly group (n=4) was 72 to 82 years old. The study protocol was approved by the Clinical Research Ethics Committee of Beijing Friendship Hospital, Capital Medical University (Permit Number 2015SY12, 9 March 2015). Tissue samples were stored at -80 °C immediately after collection.

Intestinal transit measurement

Mice were fasted for 16 h prior to experiments, and then received an intragastric administration of 0.2 mL carbonic ink to evaluate the intestinal transit rate. Thirty minutes later, mice were sacrificed, and the whole intestinal tract, from the cardia to the terminal rectum, was removed. Without applying tension, length of the small intestine as well as ink propulsion distance were measured. The intestinal transit rate was calculated by using the following formula:

ink propulsion rate (%) = migration distance of ink (cm) / small intestine length (cm) × 100%.

Whole-mount preparation

The gastric body, jejunum and colon were fully washed with pre-cooled phosphate buffered saline (PBS, 0.01 M, pH 7.4) at 4 °C, then inflated with 100% acetone for c-kit, Cx43 and NADPH-d staining or 4% paraformaldehyde for ChAT and PGP9.5 staining, and immersed in the same fixative at 4 °C for 24 h. After washing with PBS, mucosa and submucosa were removed, the longitudinal muscle with adherent MP was carefully dissected with the aid of a dissecting microscope (Leica S6E, Germany), then the whole-mount preparation with an area of 0.25 cm² (0.5 × 0.5 cm) was obtained.

Immunofluorescence staining

Whole-mount preparations were washed in PBS containing 0.3% Triton X-100 3 times for 10 min each. Non-specific binding was blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, USA) for 1 h, followed by primary (overnight at 4 °C or 24 h at 25 °C) and secondary antibodies (1 hour at 25 °C) as specified in Table 1. Negative-control sections were incubated in solutions lacking the primary antibody.

NADPH-d staining

NADPH-d activity was detected histochemically to show the neurons containing NOS in the GI tract. Whole-mount preparations were washed in PBS, and then incubated in phosphate buffer (0.1 M, pH 7.4) containing 0.3% Triton X-100, 0.5 mg/mL nitroblue tetrazolium (Cat.No.N6876, Sigma-Aldrich, USA) and 1.0 mg/mL β-NADPH (Cat.No.N7505, Sigma-Aldrich, USA) for 1 h at 37 °C. The reaction was halted by placing the sample in PBS.

Western blot analysis

The entire muscle layer of alimentary tract was detached from mucosa and submucosa using a dissecting microscope and stored in liquid nitrogen for western blot analysis. Total proteins were extracted from tissues using the RIPA lysis buffer (Cat.No.C1053, Applygen, China) containing protease inhibitor cocktail (Cat.No.P8340, Sigma-Aldrich, USA) and phosphatase inhibitor cocktail (Cat.No.P5726, Sigma-Aldrich, USA). The protein concentrations were determined by NanoDrop 2000c spectrophotometer (Thermo Scientific, USA) using BCA protein assay kit (Cat.No.P1511, Applygen, China). Equal amounts of total protein from each sample were electrophoresed on SDS–PAGE and transferred to a nitrocellulose membrane. After blocking with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk or 5% BSA for 1 h, membranes were then orderly incubated with primary and secondary antibodies as specified in Table 1. The protein bands were detected using enhanced chemiluminescence (Thermo Scientific, USA) and viewed in Fusion FX Vilber Lourmat (France). The housekeeping gene β-actin was used as an internal control.

RNA isolation and quantitative real-time PCR

Total RNA was extracted from tissues with TRIzol reagent (Cat.No.15596026, Life Technologies, USA) and converted to cDNA using All-In-One RT MasterMix (Cat. No. G486, ABM, Canada). Twenty microliter reactions were incubated in a Veriti 96-well Thermal Cycler (Applied Biosystems, USA) for 30 min at 42 °C and 5 min at 85 °C. Quantitative real-time PCR was performed on an ABI 7500 real-time PCR system (Life Technologies, USA) using Maxima SYBR Green/ROX qPCR Master Mix (Cat.No.K0222, Thermo Scientific, USA). For analysis, each gene was normalized to the housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following primers are listed in Table 2. Twenty-five microliter reactions were incubated at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 40 s. Relative fold change of gene was calculated using the 2^−ΔΔCt method.

Quantification of cell numbers and data analysis

Images were acquired with a fluorescence microscope (Nikon 90i, Japan), confocal microscope (Leica DM6000 CS, Germany) equipped with confocal spectral scanning system (Leica TCS SP8) or light microscope (Leica DM LB2) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics Inc., USA). Data analysis was assessed in randomly chosen non-overlapping fields-of-view. Four fields in each whole-mount preparation were randomly taken, 5 preparations of each mouse and 5 mice per group were used. Quantifications of ICCs and nitrergic neurons were performed by counting the number of positive cells of all fields from each whole-mount preparation, and the cell density was determined by the total area of the fields. The cell morphology of inclusion criteria, including fusiform or stellate-shaped ICCs with slender processes and nitrergic neurons colored purple-blue, was used for identification during counting. The positive areas of cholinergic neurons, PGP9.5(+), c-kit(+) neurons and their nerve fibers as well as Cx43-puncta were conducted derived by dividing the total areas of the fields.

Statistics

Obtained data were analyzed using one-way analysis of variance for multiple groups and Student’s t-test for two groups with SPSS 23.0 software (IBM Corporation, USA). A P value < 0.05 was considered to be statistically significant.