Dig Dis Sci DOI 10.1007/s10620-015-3695-8
ORIGINAL ARTICLE
Brain-Derived Neurotrophic Factor Contributes to Colonic Hypermotility in a Chronic Stress Rat Model Xiaojing Quan1 • Hesheng Luo1 • Han Fan1 • Qincai Tang1 • Wei Chen1 Ning Cui1 • Guang Yu1 • Hong Xia1,2
•
Received: 28 December 2014 / Accepted: 29 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Background Brain-derived neurotrophic factor (BDNF) has prokinetic effects on gut motility and is increased in the colonic mucosa of irritable bowel syndrome. Aims We aimed to investigate the possible involvement of BDNF in stress-induced colonic hypermotility. Methods Male Wistar rats were exposed to daily 1-h water avoidance stress (WAS) or sham WAS for 10 consecutive days. The presence of BDNF and substance P (SP) in the colonic mucosa was determined using enzyme immunoassay kits. Immunohistochemistry and western blotting were performed to assess the expression of BDNF and
& Hesheng Luo [email protected] Xiaojing Quan [email protected] Han Fan [email protected] Qincai Tang [email protected] Wei Chen [email protected]
its receptor, TrkB. The contractions of muscle strips were studied in an organ bath system. Results Repeated WAS increased the fecal pellet expulsion and spontaneous contractile activities of the colonic muscle strips. Both BDNF and SP in the colonic mucosa were elevated following WAS. Immunohistochemistry revealed the presence of BDNF and TrkB in the mucosa and myenteric plexus. BDNF and TrkB were both up-regulated in colon devoid of mucosa and submucosa from the stressed rats compared with the control. BDNF pretreatment caused an enhancement of the SP-induced contraction of the circular muscle (CM) strips. TrkB antibody significantly inhibited the contraction of the colonic muscle strips and attenuated the excitatory effects of SP on contractions of the CM strips. Repeated WAS increased the contractile activities of the CM strips induced by SP after BDNF pretreatment, and this effect was reversed by TrkB antibody. Conclusions The colonic hypermotility induced by repeated WAS may be associated with the increased expression of endogenous BDNF and TrkB. BDNF may have potential clinical therapeutic use in modulating gut motility. Keywords Rats
BDNF Muscle contraction Stress Colon
Ning Cui [email protected] Guang Yu [email protected]
Introduction
Hong Xia [email protected]
Irritable bowel syndrome (IBS) is a common functional bowel disorder characterized by recurrent abdominal pain and altered bowel habits [1]. Altered visceral perception and gut motility are the main pathophysiological factors associated with IBS [2–5]. It is known that IBS belongs to a group of stress-sensitive chronic disorders [6]. A variety of stressor types, including physical and psychosocial
1
Department of Gastroenterology, Renmin Hospital of Wuhan University, Number 238, Jiefang Road, Wuhan 430060, Hubei Province, China
2
Key Laboratory of Hubei Province for Digestive System Diseases, Wuhan, China
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stressors, play prominent roles in the development and modulation of IBS symptoms [6–8]. In both healthy humans and animals, it has been shown that stressors can result in a characteristic stress-induced increase in colonic motility and acceleration of intestinal transit [4, 9, 10]. Although the pathogenesis that underlines increased colonic motility is not fully understood, experimental studies have revealed some factors that are involved in colonic hypermotility induced by chronic stress, including the brain–gut axis, gastrointestinal (GI) hormones, gaseous molecules, and L-type calcium channels in smooth muscle cells [4, 5, 11]. Brain-derived neurotrophic factor (BDNF) is a type of neurotrophin that regulates the development, survival, and differentiation of central and peripheral neurons [12–14]. The majority of cellular responses of BDNF are mediated by its cognate tyrosine kinase receptor, TrkB, and/or the nonspecific neurotrophin receptor p75NTR [15]. Recently, much attention has been focused on the role of BDNF as a neuromodulator, especially in GI motility. Data on the effects of exogenous BDNF on GI motility remain inconclusive, but several studies have shown that BDNF has prokinetic effects. For example, recombinant BDNF has been shown to dose-dependently accelerate colonic transit and increase stool frequency in healthy subjects and in patients with constipation [16]. In rats, BDNF increases colonic myoelectric activities [17]. Endogenous BDNF released during mucosal stroking is involved in the peristaltic reflex in the rat colon. Recently, it has been reported that the mucosal BDNF level is much higher in patients with diarrhea-predominant IBS than in healthy controls [18]. Although these findings suggest excitatory effects of BDNF on gut motility, the interaction between stress-induced colonic hypermotility and the prokinetic effect of BDNF has not been studied. Given the role of BDNF in gut motility, we investigated the possibility that BDNF and its high-affinity receptor, TrkB, contribute to disturbances in colonic motility disorder in chronic stress. The present study investigated BDNF and substance P (SP) release in colonic mucosa over the course of repeated water avoidance stress (WAS) exposures. We also examined the expression of BDNF and TrkB in the rat proximal colon. Finally, we examined the effects of BDNF and TrkB-Ab on SP-induced contraction in both WAS and sham WAS (SWAS) rats.
1 week of acclimation, the rats were subjected to the experimental protocols. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Wuhan University (approval ID: WHU 20110312) and adhered to the ethical guidelines of the International Association for the Study of Pain. Water Avoidance Stress Protocol The test apparatus consisted of a Plexiglass cage (45 9 25 9 25 cm) with a block (8 9 8 9 10 cm) affixed to the center of the floor. The cage was filled with warm water (25 °C) to within 1 cm of the top of the block. The rats were weighed and then placed on the block for 1 h daily for 10 consecutive days in accordance with the WAS protocol. The SWAS rats were placed on the same block in a waterless container. The procedures were performed between 8:00 a.m. and 10:00 a.m. to minimize the effects of circadian rhythm. The total number of fecal pellets was counted at the end of each 1-h WAS or SWAS session. Colonic Motility Tests In Vitro
Materials and Methods
Rats were anesthetized with 10 % (w/v) chloral hydrate after the last stress session. A 2-cm segment of proximal colon was removed and immediately placed in a Petri dish filled with Tyrode’s buffer with the following composition (in mM): NaCl 147.0, KCl 4.0, CaCl2 2.0, NaH2PO4 0.42, Na2HPO4 2.0, MgCl2 1.05, and glucose 5.5. The segment was incised along the mesenteric border and pinned in the dish with the mucosa facing up. Circular muscle (CM) or longitudinal muscle (LM) strips (3 9 10 mm, width 9 length) were cut in the direction of the circular or longitudinal axis after removing the mucosa and submucosa. Each fresh smooth muscle strip was fixed in a tissue chamber containing 6 ml of Tyrode’s buffer that was bubbled with a mixture of 97 % O2 and 3 % CO2 and maintained at 37 °C using a circulating water jacket. One side of the strip was pinned to a hook at the bottom of the chamber, while the other side was attached to an isometric force transducer (JZJOIH, Chengdu, China) to record muscle contractions. The muscle strip was incubated for 60 min under a resting preload of 1.0 g and washed every 20 min with Tyrode’s buffer before the experimental procedures were started. The mean contractile amplitude of each strip was recorded with a RM6240 multichannel physiological signal system.
Animals
ELISA
Male Wistar rats (WKY, 180–200 g, Vital River, Beijing, China) were maintained under a normal 12:12 dark/light cycle and provided with food and water ad libitum. After
Immediately after collection, the colonic mucosa samples were frozen on dry ice and stored at -80 °C until they were processed. The samples were initially homogenized in
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ice-cold 100 mM Tris–HCl buffer, pH 7.0, containing a cocktail of protease inhibitors supplemented with 1 mM phenylmethanesulfonyl fluoride. The levels of BDNF and SP in the supernatants were measured with specific ELISA kits. The detection range of the BDNF assay was 7.8–500 pg/ml. The detection range of the SP assay was 9.76–10,000 pg/ml. Each sample was analyzed in duplicate. The final concentrations of BDNF and SP were normalized to the total protein concentration. Immunohistochemistry The localizations of the BDNF and TrkB proteins were examined in the proximal colon by immunohistochemistry. Formalin-fixed tissues were embedded in paraffin and cut into 4-lm-thick sections. Following antigen unmasking, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TrkB and anti-BDNF antibodies (1:250 and 1:500, respectively). They were then washed three times with phosphate-buffered saline (PBS) and incubated for 2 h at room temperature with an anti-rabbit secondary antibody in PBS/Triton and streptavidin–horseradish peroxidase. Diaminobenzidine was used as a chromogen, and hematoxylin was used for counterstaining. Western Blotting Immediately after collection, samples were frozen on dry ice and stored at -80 °C until they were processed. The proximal colon devoid of mucosa and submucosa was homogenized in ice-cold RIPA lysis buffer composed of 20 Mm Tris–HCl, 0.1 mM PMSF, and 5 ll/ml protease inhibitor cocktail. The protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were loaded onto 8 and 15 % gels, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and electrophoretically transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in 5 % nonfat dry milk in TBS-T (Tris-buffered saline, pH 7.6 plus 0.1 % Tween-20) and were incubated with rabbit anti-TrkB (1:1000) and anti-BDNF (1:500) primary antibodies overnight at 4 °C. The membranes were then washed and incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) and enhanced with a chemiluminescence substrate. The data were expressed as the band intensity ratios of the target proteins to GAPDH (1:1000). Chemicals BDNF was purchased from PeproTech (Rocky Hill, NJ, USA), and SP was purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Both the rabbit anti-BDNF and antiTrkB antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH was purchased from CST. A BDNF ELISA kit was purchased from Promega, and a SP ELISA kit was obtained from Assign Designs. All chemicals were dissolved in Tyrode’s buffer. Statistical Analysis The data were analyzed with SPSS 17.0 and GraphPad Prism 5.01 software. All data in the figures were expressed as mean ± SEM. Significant differences between groups were evaluated using paired or unpaired Student’s t tests. The mean value of the average tension for the 5-min period before treatment with SP, BDNF, or TrkBAb was taken as the baseline. In the groups pretreated with BDNF or TrkB-Ab, the baseline was the average tension after the administration of BDNF or TrkB-Ab. The average tension for 3 min after each SP treatment was normalized to a standardized ratio (R), the baseline of which was equal to one for each group. The significance level was set to P \ 0.05.
Results Effects of WAS on Fecal Pellet Expulsion and Body Weight Intestinal propulsive motor activity was increased by stress, and this effect was significantly greater in the WAS rats compared with the SWAS rats (Fig. 1a). The mean numbers of fecal pellets expelled daily over the 10-day period for the WAS and SWAS rats were 6.2 ± 0.2 and 2.2 ± 0.1 pellets/h, respectively (P \ 0.01, n = 10/group). The WAS-induced pellet output values consistently increased at each daily session over the 10-day period. Both the WAS and SWAS rats gained weight daily during the experimental period (Fig. 1b). Except for the weight on day 10 (WAS 278.9 ± 2.7 g vs. SWAS 288.3 ± 2.1 g, P \ 0.05, n = 10/group), there was no significant difference between the WAS and SWAS rats over the 10-day period. Repeated WAS Increased Spontaneous Contractile Activity of Proximal Colonic Strips In Vitro The spontaneous contractile activity of the proximal colon from the WAS rats was significantly increased compared with that of the SWAS rats (Fig. 2A). The mean active tension of the LM from the WAS rats was significantly higher than that from the SWAS rats (1.2 ± 0.1 vs.
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Fig. 1 Effects of repeated WAS on rat weight and defecation. a Fecal pellet expulsion during each of the ten stress sessions. b Changes in body weight during the 10-day study period. Values are expressed as
mean ± SEM (n = 10 rats/group). Stress induced a significantly greater rate of fecal pellet expulsion (#P \ 0.01 compared with SWAS, according to an unpaired Student’s t test)
0.9 ± 0.1 g, P \ 0.01; Fig. 2B-a). The mean active tension of the CM from the WAS rats was significantly higher than that from the SWAS rats (0.37 ± 0.02 vs. 0.25 ± 0.02 g, P \ 0.01; Fig. 2B-b).
Effects of Chronic WAS on BDNF and TrkB Expression in the Proximal Colon
Measurements of BDNF and SP in Colonic Mucosa The WAS rats showed a significant up-regulation of total free BDNF in the colonic mucosa compared with the SWAS rats (WAS 6.4 ± 0.3 ng/mg protein vs. SWAS 3.3 ± 0.2 ng/mg protein, P \ 0.01, n = 10/group; Fig. 3a). As shown in Fig. 3b, the level of SP in the colonic mucosa of the WAS rats was significantly higher than that in the SWAS rats (661.5 ± 20.6 pg/mg protein vs. 250.1 ± 12.1 pg/mg protein, P \ 0.01, n = 10/group).
Fig. 2 Effects of repeated WAS on contractile activities of proximal colonic muscle strips. a Representative traces of the spontaneous contraction of longitudinal muscle (LM) and circular muscle (CM) strips in the SWAS and WAS rats. b Summarized results of the LM (a) and CM (b) contractile activities in the SWAS and WAS rats. Values are expressed as mean ± SEM (n = 10 rats/group). WAS increased the contractile activities of both the LM and CM strips (#P \ 0.01 compared with SWAS, according to an unpaired Student’s t test)
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In both the WAS and SWAS rat colons, BDNF was strongly expressed in the cytosols of the circular and longitudinal smooth muscle cells and the myenteric plexus neurons. BDNF immunoreactivity (IR) was also detected in the mucosal and submucosal layers (Fig. 4a, b). As shown in Fig. 4c, d, densitometric analysis of the bands revealed that the levels of proBDNF and mature BDNF were significantly increased in the colonic tissue devoid of mucosa and submucosa compared with those of the SWAS rats (P \ 0.05). TrkB-IR was most intense in the neurons located in the myenteric plexus and in the mucosal epithelial cells in the
Dig Dis Sci Fig. 3 Expressions of BDNF and SP in the colonic mucosa were increased following repeated WAS. a, b Summary of the levels of BDNF and SP released from the colonic mucosa in the SWAS and WAS rats. Values are expressed as mean ± SEM (n = 10 rats/group). Significant differences between the SWAS and WAS rats were evaluated using an unpaired Student’s t test (#P \ 0.01)
samples obtained from both the control and stressed animals (Fig. 5a, b). Western blotting analysis showed that the WAS treatment resulted in the up-regulation of the expression of TrkB in the colonic tissue devoid of mucosa and submucosa (P \ 0.01; Fig. 5c, d).
Effects of SP, BDNF, and TrkB-Ab on Contractile Activity of Colonic Strips in SWAS Rats As shown in Fig. 6a, b, SP (1 lM) significantly increased the amplitudes of the contractile activities of the LM and CM strips in the SWAS rats (control 0.85 ± 0.06 g vs. SP 1.02 ± 0.05 g and control 0.22 ± 0.03 g vs. SP 0.81 ± 0.07 g, n = 10, P \ 0.01). Exogenous BDNF (10 nM) did not significantly change the spontaneous contractile activities of the LM and CM
strips in the SWAS rats following administration for 10 min (Fig. 6c, d). The spontaneous contractions of the LM and CM strips obtained from the SWAS rats were inhibited significantly after the application of TrkB-Ab (1:1000) (Fig. 6e, f). The mean amplitude of the LM strip before the addition of TrkB-Ab was 1.53 ± 0.07 g, and it was reduced to 1.26 ± 0.08 g after TrkB-Ab was added (P \ 0.01). The mean amplitude of the CM strip before the addition of TrkB-Ab was 0.53 ± 0.05 g, and it was reduced to 0.34 ± 0.06 g after its addition (P \ 0.01). Effects of BDNF and TrkB-Ab on Colonic Strip Contractions Induced by SP in SWAS Rats The contractions of the LM and CM strips from the SWAS rats induced by SP (1 lM) after BDNF (10 nM) and TrkB-
Fig. 4 Repeated WAS increased the expression of BDNF in the proximal colon. a, b Distribution of BDNF in the SWAS and WAS rats. c Representative western blotting for proBDNF and mature BDNF in extracts from the SWAS and WAS rats. d Summarized results showing that WAS increased the expression of BDNF in the colon devoid of mucosa and submucosa. Values are expressed as mean ± SEM (n = 10 rats/group). Significant differences between the SWAS and WAS rats were evaluated using an unpaired Student’s t test. *P \ 0.05 versus SWAS. Magnification 920
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Dig Dis Sci Fig. 5 Expression of TrkB in the proximal colon following repeated WAS. a, b Distribution of TrkB in the SWAS and WAS rats. c Representative western blotting for TrkB in the SWAS and WAS rats. d Summarized results showing that WAS increased the expression of TrkB in the colon devoid of mucosa and submucosa. Values are expressed as mean ± SEM (n = 10 rats/group). #P \ 0.01 compared with SWAS, according to an unpaired Student’s t test
Ab (1:1000) pretreatment for 10 min were compared with those induced by SP (1 lM) before incubation (Fig. 7). BDNF incubation for 10 min did not alter the mean amplitude of the LM strips induced by SP (P [ 0.05; Fig. 7a). In contrast to the effect of BDNF incubation on the LM strips, pretreatment of the CM strips with BDNF for 10 min significantly enhanced the mean amplitude of the SP-induced contractions and caused a 132 ± 4 % increase in the control peak contractions measured before the incubation period (P \ 0.01, n = 6; Fig. 7b). Pretreatment with TrkB-Ab for 10 min significantly decreased the excitatory effect of SP on the contraction of the CM strips (100 % vs. 66 ± 7.0 %, P \ 0.01, n = 6; Fig. 7b), but had no effect on the LM strips (P [ 0.05).
8.71 ± 0.90, respectively (P \ 0.01, n = 6). Moreover, the R values of the WAS group were significantly higher than those of the SWAS group (P \ 0.05; Fig. 8d). Following pretreatment with TrkB-Ab (1:1000) for 10 min, the R values of the LM strips after the application of SP (1 lM) in the SWAS and WAS groups increased from 1 (baseline) to 1.16 ± 0.03 and 1.15 ± 0.02, respectively (P \ 0.05, n = 7), while the R values between the two groups did not significantly differ (P [ 0.05; Fig. 9c). The R values of the CM strips for the SWAS and WAS groups increased from 1 (baseline) to 8.78 ± 2.36 and 2.83 ± 0.47, respectively (P \ 0.05, n = 7), and the R values of the SWAS group were significantly higher than those of the WAS group (P \ 0.05; Fig. 9d).
Comparison of the Effects of BDNF and TrkB-Ab on Colonic Strip Contractions Induced by SP in SWAS and WAS Rats
Discussion
In both the SWAS and WAS rats, SP (1 lM) caused an increase in the spontaneous contraction of the LM strips after BDNF (10 nM) pretreatment for 10 min. The R values of the LM strips from the SWAS and WAS groups increased from 1 (baseline) to 1.12 ± 0.05 and 1.08 ± 0.01, respectively (P \ 0.05, n = 6), while the R values between the two groups did not significantly differ (P [ 0.05; Fig. 8c). Pretreatment of the CM strips with BDNF (10 nM) for 10 min enhanced the amplitude of the SP-induced contractions in both the SWAS and WAS rats. The R values of the CM strips in the SWAS and WAS groups increased from 1 (baseline) to 5.38 ± 0.75 and
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Chronic stress plays an important role in the development and exacerbation of symptoms in functional gastrointestinal disorders. Repeated exposure to WAS in rats is an acceptable animal model for studying the possible mechanisms underlying stress-induced abnormal colonic motility [4, 11, 19]. In the present study, WAS increased fecal pellet expulsion and the spontaneous contractile activities of both the LM and CM strips. These results suggest that WAS induces colonic hypermotility, which is consistent with previous studies [4, 11]. Notably, the diminished weight gain of the WAS rats on day 10 may be a result of altered food intake and delayed gastric emptying which are responses to changes in the autonomic nervous system following chronic stress [8].
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Fig. 6 Effects of SP, BDNF, and TrkB-Ab on the contractile activities of the colonic strips in the SWAS rats. a, c, e Original traces of the spontaneous contractions of the longitudinal muscle (LM) and circular muscle (CM) in the SWAS rats after the strips were incubated with SP (1 lM), BDNF (10 nM), or TrkB-Ab (1:1000). b,
d, f Summary of the effects of SP, BDNF, and TrkB-Ab on the LM and CM strips. Values are expressed as mean ± SEM (n = 10 rats/group). #P \ 0.01 compared with SWAS, according to a paired Student’s t test
Fig. 7 Effects of BDNF and TrkB-Ab on the SP-induced contraction of the colonic muscle strips in the SWAS rats. a, b Summary of the effects of BDNF (10 nM) and TrkB-Ab (1:1000) on the SP-induced contraction of the longitudinal muscle (LM) strips and circular muscle
(CM) strips. Data normalized to preincubation SP contraction levels. Values are mean ± SE of the percent of SP-induced contraction (n = 6 rats/group, #P \ 0.01 vs. SWAS)
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Fig. 8 Repeated WAS increased the SP-induced contractile activities of the CM strips after BDNF pretreatment. a, b Original traces of the effects of BDNF (10 nM) pretreatment on the SP-induced contractions of the longitudinal muscle (LM) and circular muscle (CM) in the SWAS and WAS rats. c, d Summarized results showing that WAS
increased the SP-induced contractile activities of the CM strips after BDNF pretreatment. Values are expressed as mean ± SEM (n = 6 rats/group). *P \ 0.05 compared with SWAS, according to an unpaired Student’s t test
The roles of BDNF in development, survival, and pathological conditions of the enteric nervous system (ENS) are well documented [12, 13, 20, 21]. Recently, the prokinetic effects of exogenous BDNF on GI motility have drawn increasing attention [15, 16, 22, 23], and increased BDNF levels in the colonic mucosa have been found in patients with IBS [18]. However, little is known regarding the link between colonic hypermotility induced by chronic stress and the excitatory effect of BDNF on GI motility. It is known that BDNF coexists with certain neuropeptides including SP [24, 25]. Although the interactions between BDNF and SP have been investigated [21–23], there has been no study of their interactive involvement in colonic hypermotility induced by chronic stress. Therefore, the present study examined the possibility that colonic hypermotility caused by WAS is due to the increased expression of BDNF in the colon, combined with its modulatory role in SP-induced contraction. First, we observed that the releases of BDNF and SP in the colonic mucosa were significantly increased in the WAS rats. Second, the expression of BDNF and its high-affinity receptor, TrkB, in the colon devoid of mucosa and submucosa was both upregulated in the WAS rats. Third, exogenous BDNF had no obvious excitatory effects on the contractile activities of LM and CM strips in the colon, while the application of TrkB-Ab significantly inhibited the contraction of both the LM and CM strips. Fourth, the contraction of the CM strips
as induced by SP after BDNF incubation, but not that of the LM strips, was enhanced in the SWAS rats, while it was attenuated after TrkB-Ab incubation. Because several reports have indicated that BDNF exerts its biological effects in the muscle through TrkB receptor activation, we did not evaluate the participation of p75NTR, which is the lowaffinity receptor for BDNF [26, 27], in contractile activities in the SWAS rats. Considering these results, along with the localization of BDNF and its receptor, TrkB, in the gut, our findings suggest an important role of enhanced BDNF expression in chronic stress-induced colonic hypermotility. In the present study, the localizations of BDNF and TrkB were identified. In both the SWAS and WAS colons, high levels of BDNF expression were observed in the mucosal epithelium, smooth muscle cells of the lamina propria, and neurons of the myenteric plexus, which is consistent with previous reports [21, 27]. TrkB-like IR was intense in the nucleus of the myenteric plexus and mucosal epithelial cells but was absent in the smooth muscle cells, and a similar staining has been described in the human colon [28]. However, BDNF and TrkB expression in the colon shows marked differences. For example, BDNF-IR is absent in the smooth muscle of the fetal intestine [13], and TrkB-IR is not detected in the epithelium of the mouse colon [27]. It is possible that these major differences in the distributions of BDNF and TrkB are due to different technical approaches and species differences.
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Fig. 9 Repeated WAS reduced the SP-induced contractile activities of the CM strips after the application of TrkB-Ab. a, b Original traces of TrkB-Ab (1:1000) pretreatment on the SP-induced contractions of the longitudinal muscle (LM) and circular muscle (CM) in the SWAS and WAS rats. c, d Summarized results showing that WAS reduced
the SP-induced contractile activities of the CM strips after the TrkBAb pretreatment. Values are expressed as mean ± SEM (n = 7 rats/group). *P \ 0.05 compared with SWAS, according to an unpaired Student’s t test
Gut motility is mainly regulated by the ENS, and the contractile activities of smooth muscle are innervated by hundreds of excitatory and inhibitory motor neurons [29]. There is growing evidence that BDNF is a classical excitatory neurotransmitter [30, 31]. In the present study, both BDNF and TrkB were expressed in the neurons of the enteric plexus, suggesting a direct effect of BDNF on neuronal activity and excitability in the ENS. In addition to neurons, mucosal epithelial cells were found to be a potential source of BDNF. These findings together with the presence of TrkB-IR in the epithelium indicate that BDNF–TrkB plays a signaling role in the functioning of the colonic mucosa and that BDNF-containing mucosal cells in the colon may contribute to hypermotility in WAS rats by regulating the release of BDNF. It has been reported that BDNF-expressing epithelial cells are a major source of endogenous BDNF that is released during mucosal stroking [15]. The present study showed that the release of BDNF in the colonic mucosa was increased in the WAS rats. Furthermore, BDNF and TrkB expression in the colon devoid of mucosa and submucosa was upregulated in the WAS rats. Theoretically, increased BDNF release from either the colonic mucosa or enteric neurons
and smooth muscle cells may contribute to colonic motility disorder. To our knowledge, this is the first report of the stressinduced colonic up-regulation of BDNF, which may be responsible for the enhanced contraction of the colon in WAS rats. In the present study, the excitatory effect of SP on the rat colon was consistent with previous studies of humans, mice, and rabbits [22, 32, 33]. Moreover, the data obtained in this study provide further insight into the neuromodulatory action of BDNF on colonic motility. Consistent with this notion, the application of TrkB-Ab significantly inhibited the spontaneous contraction of both the LM and CM strips but not exogenous BDNF. This result indicates that the enhancement of gut motility caused by BDNF [15, 16, 22, 23], apart from possible regional and species-dependent differences, was due to a mechanism other than direct neuronal excitation. One possible mechanism, namely that BDNF enhanced the neurotransmitter-elicited contraction of the muscle strips, was detected by challenging the LM and CM strips with SP after BDNF incubation for 10 min. It is known that BDNF coexists with SP in both the central nervous system (CNS) and peripheral nervous system (PNS) [25, 34], and the interactions between them have been well
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documented. In the postnatal rat spinal cord, BDNF has been shown to enhance the release of SP in lamina II, and this activity is blocked by the Trk antagonist K252a or anti-TrkB antibody [35]. Furthermore, BDNF can cause the enhancement of Ca2? signaling responses induced by SP in cultured myenteric neurons of rats [21]. The present study showed that the levels of BDNF and SP in the colonic mucosa were both increased in the WAS rats. Furthermore, exogenous BDNF significantly enhanced the contraction of the CM strips induced by SP in both the SWAS and WAS rats. In addition, the SP-induced contraction of the CM strips was significantly attenuated after immunoneutralization of TrkB with TrkB antibody. This finding strongly suggested that the SP-induced contractile activities of the CM strips were augmented by BDNF as a result of interactions with TrkB receptors. However, similar effects of BDNF and TrkB-Ab on the CM strips were not detected in the LM strips of either the SWAS or WAS rats. In contrast to our findings, a recent report has shown that BDNF enhances the contractile activity of LM strips as elicited by SP from the distal colon of mice [22]. The reason for this discrepancy may be related to the different anatomical regions studied. Alternatively, it is possible that BDNF augmented the LM contraction elicited by other excitatory transmitters in the rat colon. Previous publications have shown that BDNF enhances the peristaltic reflex by augmenting the release of 5-HT [15] and causes an enhancement of Ca2? transients induced by 5-HT in cultured myenteric neurons [21]. There is recent evidence demonstrating that exogenous BDNF can augment the CCh-induced contraction of LM strips from the rabbit intestine [23]. Taken together, these results strongly support the notion that BDNF could enhance not only the effect of SP but also the effects of 5-HT and CCh on gut motility. In summary, this study provides evidence that the enhanced expression of BDNF and its high-affinity receptor, TrkB, in the colon may contribute to chronic stress-induced colonic hypermotility. Furthermore, BDNF, acting as a neuromodulator, may act on the TrkB receptor in enteric neurons and, consequently, facilitate the action of SP in promoting colonic motility. Due to the similar characteristics of the animal model used in the study of IBS, these findings can help to elucidate the pathogenesis of IBS and may offer interesting clinical therapeutic perspectives. Conflict of interest
None.
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29. Hansen MB. The enteric nervous system II: gastrointestinal functions. Pharmacol Toxicol. 2003;92:249–257. 30. Kafitz KW, Rose CR, Thoenen H, Konnerth A. Neurotrophinevoked rapid excitation through TrkB receptors. Nature. 1999;401:918–921. 31. Blum R, Kafitz KW, Konnerth A. Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9. Nature. 2002;419:687–693. 32. Liu L, Shang F, Morgan MJ, King DW, Lubowski DZ, Burcher E. Cyclooxygenase-dependent alterations in substance P-mediated contractility and tachykinin NK1 receptor expression in the colonic circular muscle of patients with slow transit constipation. J Pharmacol Exp Ther. 2009;329:282–289. 33. Mangel AW, Fitz JG, Taylor IL. Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y. Peptides. 1991;12:1063–1067. 34. Lever IJ, Bradbury EJ, Cunningham JR, et al. Brain-derived neurotrophic factor is released in the dorsal horn by distinctive patterns of afferent fiber stimulation. J Neurosci. 2001;21: 4469–4477. 35. Merighi A, Bardoni R, Salio C, et al. Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord. Dev Neurobiol.. 2008;68:457–475.
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ORIGINAL ARTICLE
Brain-Derived Neurotrophic Factor Contributes to Colonic Hypermotility in a Chronic Stress Rat Model Xiaojing Quan1 • Hesheng Luo1 • Han Fan1 • Qincai Tang1 • Wei Chen1 Ning Cui1 • Guang Yu1 • Hong Xia1,2
•
Received: 28 December 2014 / Accepted: 29 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Background Brain-derived neurotrophic factor (BDNF) has prokinetic effects on gut motility and is increased in the colonic mucosa of irritable bowel syndrome. Aims We aimed to investigate the possible involvement of BDNF in stress-induced colonic hypermotility. Methods Male Wistar rats were exposed to daily 1-h water avoidance stress (WAS) or sham WAS for 10 consecutive days. The presence of BDNF and substance P (SP) in the colonic mucosa was determined using enzyme immunoassay kits. Immunohistochemistry and western blotting were performed to assess the expression of BDNF and
& Hesheng Luo [email protected] Xiaojing Quan [email protected] Han Fan [email protected] Qincai Tang [email protected] Wei Chen [email protected]
its receptor, TrkB. The contractions of muscle strips were studied in an organ bath system. Results Repeated WAS increased the fecal pellet expulsion and spontaneous contractile activities of the colonic muscle strips. Both BDNF and SP in the colonic mucosa were elevated following WAS. Immunohistochemistry revealed the presence of BDNF and TrkB in the mucosa and myenteric plexus. BDNF and TrkB were both up-regulated in colon devoid of mucosa and submucosa from the stressed rats compared with the control. BDNF pretreatment caused an enhancement of the SP-induced contraction of the circular muscle (CM) strips. TrkB antibody significantly inhibited the contraction of the colonic muscle strips and attenuated the excitatory effects of SP on contractions of the CM strips. Repeated WAS increased the contractile activities of the CM strips induced by SP after BDNF pretreatment, and this effect was reversed by TrkB antibody. Conclusions The colonic hypermotility induced by repeated WAS may be associated with the increased expression of endogenous BDNF and TrkB. BDNF may have potential clinical therapeutic use in modulating gut motility. Keywords Rats
BDNF Muscle contraction Stress Colon
Ning Cui [email protected] Guang Yu [email protected]
Introduction
Hong Xia [email protected]
Irritable bowel syndrome (IBS) is a common functional bowel disorder characterized by recurrent abdominal pain and altered bowel habits [1]. Altered visceral perception and gut motility are the main pathophysiological factors associated with IBS [2–5]. It is known that IBS belongs to a group of stress-sensitive chronic disorders [6]. A variety of stressor types, including physical and psychosocial
1
Department of Gastroenterology, Renmin Hospital of Wuhan University, Number 238, Jiefang Road, Wuhan 430060, Hubei Province, China
2
Key Laboratory of Hubei Province for Digestive System Diseases, Wuhan, China
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stressors, play prominent roles in the development and modulation of IBS symptoms [6–8]. In both healthy humans and animals, it has been shown that stressors can result in a characteristic stress-induced increase in colonic motility and acceleration of intestinal transit [4, 9, 10]. Although the pathogenesis that underlines increased colonic motility is not fully understood, experimental studies have revealed some factors that are involved in colonic hypermotility induced by chronic stress, including the brain–gut axis, gastrointestinal (GI) hormones, gaseous molecules, and L-type calcium channels in smooth muscle cells [4, 5, 11]. Brain-derived neurotrophic factor (BDNF) is a type of neurotrophin that regulates the development, survival, and differentiation of central and peripheral neurons [12–14]. The majority of cellular responses of BDNF are mediated by its cognate tyrosine kinase receptor, TrkB, and/or the nonspecific neurotrophin receptor p75NTR [15]. Recently, much attention has been focused on the role of BDNF as a neuromodulator, especially in GI motility. Data on the effects of exogenous BDNF on GI motility remain inconclusive, but several studies have shown that BDNF has prokinetic effects. For example, recombinant BDNF has been shown to dose-dependently accelerate colonic transit and increase stool frequency in healthy subjects and in patients with constipation [16]. In rats, BDNF increases colonic myoelectric activities [17]. Endogenous BDNF released during mucosal stroking is involved in the peristaltic reflex in the rat colon. Recently, it has been reported that the mucosal BDNF level is much higher in patients with diarrhea-predominant IBS than in healthy controls [18]. Although these findings suggest excitatory effects of BDNF on gut motility, the interaction between stress-induced colonic hypermotility and the prokinetic effect of BDNF has not been studied. Given the role of BDNF in gut motility, we investigated the possibility that BDNF and its high-affinity receptor, TrkB, contribute to disturbances in colonic motility disorder in chronic stress. The present study investigated BDNF and substance P (SP) release in colonic mucosa over the course of repeated water avoidance stress (WAS) exposures. We also examined the expression of BDNF and TrkB in the rat proximal colon. Finally, we examined the effects of BDNF and TrkB-Ab on SP-induced contraction in both WAS and sham WAS (SWAS) rats.
1 week of acclimation, the rats were subjected to the experimental protocols. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Wuhan University (approval ID: WHU 20110312) and adhered to the ethical guidelines of the International Association for the Study of Pain. Water Avoidance Stress Protocol The test apparatus consisted of a Plexiglass cage (45 9 25 9 25 cm) with a block (8 9 8 9 10 cm) affixed to the center of the floor. The cage was filled with warm water (25 °C) to within 1 cm of the top of the block. The rats were weighed and then placed on the block for 1 h daily for 10 consecutive days in accordance with the WAS protocol. The SWAS rats were placed on the same block in a waterless container. The procedures were performed between 8:00 a.m. and 10:00 a.m. to minimize the effects of circadian rhythm. The total number of fecal pellets was counted at the end of each 1-h WAS or SWAS session. Colonic Motility Tests In Vitro
Materials and Methods
Rats were anesthetized with 10 % (w/v) chloral hydrate after the last stress session. A 2-cm segment of proximal colon was removed and immediately placed in a Petri dish filled with Tyrode’s buffer with the following composition (in mM): NaCl 147.0, KCl 4.0, CaCl2 2.0, NaH2PO4 0.42, Na2HPO4 2.0, MgCl2 1.05, and glucose 5.5. The segment was incised along the mesenteric border and pinned in the dish with the mucosa facing up. Circular muscle (CM) or longitudinal muscle (LM) strips (3 9 10 mm, width 9 length) were cut in the direction of the circular or longitudinal axis after removing the mucosa and submucosa. Each fresh smooth muscle strip was fixed in a tissue chamber containing 6 ml of Tyrode’s buffer that was bubbled with a mixture of 97 % O2 and 3 % CO2 and maintained at 37 °C using a circulating water jacket. One side of the strip was pinned to a hook at the bottom of the chamber, while the other side was attached to an isometric force transducer (JZJOIH, Chengdu, China) to record muscle contractions. The muscle strip was incubated for 60 min under a resting preload of 1.0 g and washed every 20 min with Tyrode’s buffer before the experimental procedures were started. The mean contractile amplitude of each strip was recorded with a RM6240 multichannel physiological signal system.
Animals
ELISA
Male Wistar rats (WKY, 180–200 g, Vital River, Beijing, China) were maintained under a normal 12:12 dark/light cycle and provided with food and water ad libitum. After
Immediately after collection, the colonic mucosa samples were frozen on dry ice and stored at -80 °C until they were processed. The samples were initially homogenized in
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ice-cold 100 mM Tris–HCl buffer, pH 7.0, containing a cocktail of protease inhibitors supplemented with 1 mM phenylmethanesulfonyl fluoride. The levels of BDNF and SP in the supernatants were measured with specific ELISA kits. The detection range of the BDNF assay was 7.8–500 pg/ml. The detection range of the SP assay was 9.76–10,000 pg/ml. Each sample was analyzed in duplicate. The final concentrations of BDNF and SP were normalized to the total protein concentration. Immunohistochemistry The localizations of the BDNF and TrkB proteins were examined in the proximal colon by immunohistochemistry. Formalin-fixed tissues were embedded in paraffin and cut into 4-lm-thick sections. Following antigen unmasking, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TrkB and anti-BDNF antibodies (1:250 and 1:500, respectively). They were then washed three times with phosphate-buffered saline (PBS) and incubated for 2 h at room temperature with an anti-rabbit secondary antibody in PBS/Triton and streptavidin–horseradish peroxidase. Diaminobenzidine was used as a chromogen, and hematoxylin was used for counterstaining. Western Blotting Immediately after collection, samples were frozen on dry ice and stored at -80 °C until they were processed. The proximal colon devoid of mucosa and submucosa was homogenized in ice-cold RIPA lysis buffer composed of 20 Mm Tris–HCl, 0.1 mM PMSF, and 5 ll/ml protease inhibitor cocktail. The protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were loaded onto 8 and 15 % gels, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and electrophoretically transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in 5 % nonfat dry milk in TBS-T (Tris-buffered saline, pH 7.6 plus 0.1 % Tween-20) and were incubated with rabbit anti-TrkB (1:1000) and anti-BDNF (1:500) primary antibodies overnight at 4 °C. The membranes were then washed and incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) and enhanced with a chemiluminescence substrate. The data were expressed as the band intensity ratios of the target proteins to GAPDH (1:1000). Chemicals BDNF was purchased from PeproTech (Rocky Hill, NJ, USA), and SP was purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Both the rabbit anti-BDNF and antiTrkB antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH was purchased from CST. A BDNF ELISA kit was purchased from Promega, and a SP ELISA kit was obtained from Assign Designs. All chemicals were dissolved in Tyrode’s buffer. Statistical Analysis The data were analyzed with SPSS 17.0 and GraphPad Prism 5.01 software. All data in the figures were expressed as mean ± SEM. Significant differences between groups were evaluated using paired or unpaired Student’s t tests. The mean value of the average tension for the 5-min period before treatment with SP, BDNF, or TrkBAb was taken as the baseline. In the groups pretreated with BDNF or TrkB-Ab, the baseline was the average tension after the administration of BDNF or TrkB-Ab. The average tension for 3 min after each SP treatment was normalized to a standardized ratio (R), the baseline of which was equal to one for each group. The significance level was set to P \ 0.05.
Results Effects of WAS on Fecal Pellet Expulsion and Body Weight Intestinal propulsive motor activity was increased by stress, and this effect was significantly greater in the WAS rats compared with the SWAS rats (Fig. 1a). The mean numbers of fecal pellets expelled daily over the 10-day period for the WAS and SWAS rats were 6.2 ± 0.2 and 2.2 ± 0.1 pellets/h, respectively (P \ 0.01, n = 10/group). The WAS-induced pellet output values consistently increased at each daily session over the 10-day period. Both the WAS and SWAS rats gained weight daily during the experimental period (Fig. 1b). Except for the weight on day 10 (WAS 278.9 ± 2.7 g vs. SWAS 288.3 ± 2.1 g, P \ 0.05, n = 10/group), there was no significant difference between the WAS and SWAS rats over the 10-day period. Repeated WAS Increased Spontaneous Contractile Activity of Proximal Colonic Strips In Vitro The spontaneous contractile activity of the proximal colon from the WAS rats was significantly increased compared with that of the SWAS rats (Fig. 2A). The mean active tension of the LM from the WAS rats was significantly higher than that from the SWAS rats (1.2 ± 0.1 vs.
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Fig. 1 Effects of repeated WAS on rat weight and defecation. a Fecal pellet expulsion during each of the ten stress sessions. b Changes in body weight during the 10-day study period. Values are expressed as
mean ± SEM (n = 10 rats/group). Stress induced a significantly greater rate of fecal pellet expulsion (#P \ 0.01 compared with SWAS, according to an unpaired Student’s t test)
0.9 ± 0.1 g, P \ 0.01; Fig. 2B-a). The mean active tension of the CM from the WAS rats was significantly higher than that from the SWAS rats (0.37 ± 0.02 vs. 0.25 ± 0.02 g, P \ 0.01; Fig. 2B-b).
Effects of Chronic WAS on BDNF and TrkB Expression in the Proximal Colon
Measurements of BDNF and SP in Colonic Mucosa The WAS rats showed a significant up-regulation of total free BDNF in the colonic mucosa compared with the SWAS rats (WAS 6.4 ± 0.3 ng/mg protein vs. SWAS 3.3 ± 0.2 ng/mg protein, P \ 0.01, n = 10/group; Fig. 3a). As shown in Fig. 3b, the level of SP in the colonic mucosa of the WAS rats was significantly higher than that in the SWAS rats (661.5 ± 20.6 pg/mg protein vs. 250.1 ± 12.1 pg/mg protein, P \ 0.01, n = 10/group).
Fig. 2 Effects of repeated WAS on contractile activities of proximal colonic muscle strips. a Representative traces of the spontaneous contraction of longitudinal muscle (LM) and circular muscle (CM) strips in the SWAS and WAS rats. b Summarized results of the LM (a) and CM (b) contractile activities in the SWAS and WAS rats. Values are expressed as mean ± SEM (n = 10 rats/group). WAS increased the contractile activities of both the LM and CM strips (#P \ 0.01 compared with SWAS, according to an unpaired Student’s t test)
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In both the WAS and SWAS rat colons, BDNF was strongly expressed in the cytosols of the circular and longitudinal smooth muscle cells and the myenteric plexus neurons. BDNF immunoreactivity (IR) was also detected in the mucosal and submucosal layers (Fig. 4a, b). As shown in Fig. 4c, d, densitometric analysis of the bands revealed that the levels of proBDNF and mature BDNF were significantly increased in the colonic tissue devoid of mucosa and submucosa compared with those of the SWAS rats (P \ 0.05). TrkB-IR was most intense in the neurons located in the myenteric plexus and in the mucosal epithelial cells in the
Dig Dis Sci Fig. 3 Expressions of BDNF and SP in the colonic mucosa were increased following repeated WAS. a, b Summary of the levels of BDNF and SP released from the colonic mucosa in the SWAS and WAS rats. Values are expressed as mean ± SEM (n = 10 rats/group). Significant differences between the SWAS and WAS rats were evaluated using an unpaired Student’s t test (#P \ 0.01)
samples obtained from both the control and stressed animals (Fig. 5a, b). Western blotting analysis showed that the WAS treatment resulted in the up-regulation of the expression of TrkB in the colonic tissue devoid of mucosa and submucosa (P \ 0.01; Fig. 5c, d).
Effects of SP, BDNF, and TrkB-Ab on Contractile Activity of Colonic Strips in SWAS Rats As shown in Fig. 6a, b, SP (1 lM) significantly increased the amplitudes of the contractile activities of the LM and CM strips in the SWAS rats (control 0.85 ± 0.06 g vs. SP 1.02 ± 0.05 g and control 0.22 ± 0.03 g vs. SP 0.81 ± 0.07 g, n = 10, P \ 0.01). Exogenous BDNF (10 nM) did not significantly change the spontaneous contractile activities of the LM and CM
strips in the SWAS rats following administration for 10 min (Fig. 6c, d). The spontaneous contractions of the LM and CM strips obtained from the SWAS rats were inhibited significantly after the application of TrkB-Ab (1:1000) (Fig. 6e, f). The mean amplitude of the LM strip before the addition of TrkB-Ab was 1.53 ± 0.07 g, and it was reduced to 1.26 ± 0.08 g after TrkB-Ab was added (P \ 0.01). The mean amplitude of the CM strip before the addition of TrkB-Ab was 0.53 ± 0.05 g, and it was reduced to 0.34 ± 0.06 g after its addition (P \ 0.01). Effects of BDNF and TrkB-Ab on Colonic Strip Contractions Induced by SP in SWAS Rats The contractions of the LM and CM strips from the SWAS rats induced by SP (1 lM) after BDNF (10 nM) and TrkB-
Fig. 4 Repeated WAS increased the expression of BDNF in the proximal colon. a, b Distribution of BDNF in the SWAS and WAS rats. c Representative western blotting for proBDNF and mature BDNF in extracts from the SWAS and WAS rats. d Summarized results showing that WAS increased the expression of BDNF in the colon devoid of mucosa and submucosa. Values are expressed as mean ± SEM (n = 10 rats/group). Significant differences between the SWAS and WAS rats were evaluated using an unpaired Student’s t test. *P \ 0.05 versus SWAS. Magnification 920
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Dig Dis Sci Fig. 5 Expression of TrkB in the proximal colon following repeated WAS. a, b Distribution of TrkB in the SWAS and WAS rats. c Representative western blotting for TrkB in the SWAS and WAS rats. d Summarized results showing that WAS increased the expression of TrkB in the colon devoid of mucosa and submucosa. Values are expressed as mean ± SEM (n = 10 rats/group). #P \ 0.01 compared with SWAS, according to an unpaired Student’s t test
Ab (1:1000) pretreatment for 10 min were compared with those induced by SP (1 lM) before incubation (Fig. 7). BDNF incubation for 10 min did not alter the mean amplitude of the LM strips induced by SP (P [ 0.05; Fig. 7a). In contrast to the effect of BDNF incubation on the LM strips, pretreatment of the CM strips with BDNF for 10 min significantly enhanced the mean amplitude of the SP-induced contractions and caused a 132 ± 4 % increase in the control peak contractions measured before the incubation period (P \ 0.01, n = 6; Fig. 7b). Pretreatment with TrkB-Ab for 10 min significantly decreased the excitatory effect of SP on the contraction of the CM strips (100 % vs. 66 ± 7.0 %, P \ 0.01, n = 6; Fig. 7b), but had no effect on the LM strips (P [ 0.05).
8.71 ± 0.90, respectively (P \ 0.01, n = 6). Moreover, the R values of the WAS group were significantly higher than those of the SWAS group (P \ 0.05; Fig. 8d). Following pretreatment with TrkB-Ab (1:1000) for 10 min, the R values of the LM strips after the application of SP (1 lM) in the SWAS and WAS groups increased from 1 (baseline) to 1.16 ± 0.03 and 1.15 ± 0.02, respectively (P \ 0.05, n = 7), while the R values between the two groups did not significantly differ (P [ 0.05; Fig. 9c). The R values of the CM strips for the SWAS and WAS groups increased from 1 (baseline) to 8.78 ± 2.36 and 2.83 ± 0.47, respectively (P \ 0.05, n = 7), and the R values of the SWAS group were significantly higher than those of the WAS group (P \ 0.05; Fig. 9d).
Comparison of the Effects of BDNF and TrkB-Ab on Colonic Strip Contractions Induced by SP in SWAS and WAS Rats
Discussion
In both the SWAS and WAS rats, SP (1 lM) caused an increase in the spontaneous contraction of the LM strips after BDNF (10 nM) pretreatment for 10 min. The R values of the LM strips from the SWAS and WAS groups increased from 1 (baseline) to 1.12 ± 0.05 and 1.08 ± 0.01, respectively (P \ 0.05, n = 6), while the R values between the two groups did not significantly differ (P [ 0.05; Fig. 8c). Pretreatment of the CM strips with BDNF (10 nM) for 10 min enhanced the amplitude of the SP-induced contractions in both the SWAS and WAS rats. The R values of the CM strips in the SWAS and WAS groups increased from 1 (baseline) to 5.38 ± 0.75 and
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Chronic stress plays an important role in the development and exacerbation of symptoms in functional gastrointestinal disorders. Repeated exposure to WAS in rats is an acceptable animal model for studying the possible mechanisms underlying stress-induced abnormal colonic motility [4, 11, 19]. In the present study, WAS increased fecal pellet expulsion and the spontaneous contractile activities of both the LM and CM strips. These results suggest that WAS induces colonic hypermotility, which is consistent with previous studies [4, 11]. Notably, the diminished weight gain of the WAS rats on day 10 may be a result of altered food intake and delayed gastric emptying which are responses to changes in the autonomic nervous system following chronic stress [8].
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Fig. 6 Effects of SP, BDNF, and TrkB-Ab on the contractile activities of the colonic strips in the SWAS rats. a, c, e Original traces of the spontaneous contractions of the longitudinal muscle (LM) and circular muscle (CM) in the SWAS rats after the strips were incubated with SP (1 lM), BDNF (10 nM), or TrkB-Ab (1:1000). b,
d, f Summary of the effects of SP, BDNF, and TrkB-Ab on the LM and CM strips. Values are expressed as mean ± SEM (n = 10 rats/group). #P \ 0.01 compared with SWAS, according to a paired Student’s t test
Fig. 7 Effects of BDNF and TrkB-Ab on the SP-induced contraction of the colonic muscle strips in the SWAS rats. a, b Summary of the effects of BDNF (10 nM) and TrkB-Ab (1:1000) on the SP-induced contraction of the longitudinal muscle (LM) strips and circular muscle
(CM) strips. Data normalized to preincubation SP contraction levels. Values are mean ± SE of the percent of SP-induced contraction (n = 6 rats/group, #P \ 0.01 vs. SWAS)
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Fig. 8 Repeated WAS increased the SP-induced contractile activities of the CM strips after BDNF pretreatment. a, b Original traces of the effects of BDNF (10 nM) pretreatment on the SP-induced contractions of the longitudinal muscle (LM) and circular muscle (CM) in the SWAS and WAS rats. c, d Summarized results showing that WAS
increased the SP-induced contractile activities of the CM strips after BDNF pretreatment. Values are expressed as mean ± SEM (n = 6 rats/group). *P \ 0.05 compared with SWAS, according to an unpaired Student’s t test
The roles of BDNF in development, survival, and pathological conditions of the enteric nervous system (ENS) are well documented [12, 13, 20, 21]. Recently, the prokinetic effects of exogenous BDNF on GI motility have drawn increasing attention [15, 16, 22, 23], and increased BDNF levels in the colonic mucosa have been found in patients with IBS [18]. However, little is known regarding the link between colonic hypermotility induced by chronic stress and the excitatory effect of BDNF on GI motility. It is known that BDNF coexists with certain neuropeptides including SP [24, 25]. Although the interactions between BDNF and SP have been investigated [21–23], there has been no study of their interactive involvement in colonic hypermotility induced by chronic stress. Therefore, the present study examined the possibility that colonic hypermotility caused by WAS is due to the increased expression of BDNF in the colon, combined with its modulatory role in SP-induced contraction. First, we observed that the releases of BDNF and SP in the colonic mucosa were significantly increased in the WAS rats. Second, the expression of BDNF and its high-affinity receptor, TrkB, in the colon devoid of mucosa and submucosa was both upregulated in the WAS rats. Third, exogenous BDNF had no obvious excitatory effects on the contractile activities of LM and CM strips in the colon, while the application of TrkB-Ab significantly inhibited the contraction of both the LM and CM strips. Fourth, the contraction of the CM strips
as induced by SP after BDNF incubation, but not that of the LM strips, was enhanced in the SWAS rats, while it was attenuated after TrkB-Ab incubation. Because several reports have indicated that BDNF exerts its biological effects in the muscle through TrkB receptor activation, we did not evaluate the participation of p75NTR, which is the lowaffinity receptor for BDNF [26, 27], in contractile activities in the SWAS rats. Considering these results, along with the localization of BDNF and its receptor, TrkB, in the gut, our findings suggest an important role of enhanced BDNF expression in chronic stress-induced colonic hypermotility. In the present study, the localizations of BDNF and TrkB were identified. In both the SWAS and WAS colons, high levels of BDNF expression were observed in the mucosal epithelium, smooth muscle cells of the lamina propria, and neurons of the myenteric plexus, which is consistent with previous reports [21, 27]. TrkB-like IR was intense in the nucleus of the myenteric plexus and mucosal epithelial cells but was absent in the smooth muscle cells, and a similar staining has been described in the human colon [28]. However, BDNF and TrkB expression in the colon shows marked differences. For example, BDNF-IR is absent in the smooth muscle of the fetal intestine [13], and TrkB-IR is not detected in the epithelium of the mouse colon [27]. It is possible that these major differences in the distributions of BDNF and TrkB are due to different technical approaches and species differences.
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Fig. 9 Repeated WAS reduced the SP-induced contractile activities of the CM strips after the application of TrkB-Ab. a, b Original traces of TrkB-Ab (1:1000) pretreatment on the SP-induced contractions of the longitudinal muscle (LM) and circular muscle (CM) in the SWAS and WAS rats. c, d Summarized results showing that WAS reduced
the SP-induced contractile activities of the CM strips after the TrkBAb pretreatment. Values are expressed as mean ± SEM (n = 7 rats/group). *P \ 0.05 compared with SWAS, according to an unpaired Student’s t test
Gut motility is mainly regulated by the ENS, and the contractile activities of smooth muscle are innervated by hundreds of excitatory and inhibitory motor neurons [29]. There is growing evidence that BDNF is a classical excitatory neurotransmitter [30, 31]. In the present study, both BDNF and TrkB were expressed in the neurons of the enteric plexus, suggesting a direct effect of BDNF on neuronal activity and excitability in the ENS. In addition to neurons, mucosal epithelial cells were found to be a potential source of BDNF. These findings together with the presence of TrkB-IR in the epithelium indicate that BDNF–TrkB plays a signaling role in the functioning of the colonic mucosa and that BDNF-containing mucosal cells in the colon may contribute to hypermotility in WAS rats by regulating the release of BDNF. It has been reported that BDNF-expressing epithelial cells are a major source of endogenous BDNF that is released during mucosal stroking [15]. The present study showed that the release of BDNF in the colonic mucosa was increased in the WAS rats. Furthermore, BDNF and TrkB expression in the colon devoid of mucosa and submucosa was upregulated in the WAS rats. Theoretically, increased BDNF release from either the colonic mucosa or enteric neurons
and smooth muscle cells may contribute to colonic motility disorder. To our knowledge, this is the first report of the stressinduced colonic up-regulation of BDNF, which may be responsible for the enhanced contraction of the colon in WAS rats. In the present study, the excitatory effect of SP on the rat colon was consistent with previous studies of humans, mice, and rabbits [22, 32, 33]. Moreover, the data obtained in this study provide further insight into the neuromodulatory action of BDNF on colonic motility. Consistent with this notion, the application of TrkB-Ab significantly inhibited the spontaneous contraction of both the LM and CM strips but not exogenous BDNF. This result indicates that the enhancement of gut motility caused by BDNF [15, 16, 22, 23], apart from possible regional and species-dependent differences, was due to a mechanism other than direct neuronal excitation. One possible mechanism, namely that BDNF enhanced the neurotransmitter-elicited contraction of the muscle strips, was detected by challenging the LM and CM strips with SP after BDNF incubation for 10 min. It is known that BDNF coexists with SP in both the central nervous system (CNS) and peripheral nervous system (PNS) [25, 34], and the interactions between them have been well
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documented. In the postnatal rat spinal cord, BDNF has been shown to enhance the release of SP in lamina II, and this activity is blocked by the Trk antagonist K252a or anti-TrkB antibody [35]. Furthermore, BDNF can cause the enhancement of Ca2? signaling responses induced by SP in cultured myenteric neurons of rats [21]. The present study showed that the levels of BDNF and SP in the colonic mucosa were both increased in the WAS rats. Furthermore, exogenous BDNF significantly enhanced the contraction of the CM strips induced by SP in both the SWAS and WAS rats. In addition, the SP-induced contraction of the CM strips was significantly attenuated after immunoneutralization of TrkB with TrkB antibody. This finding strongly suggested that the SP-induced contractile activities of the CM strips were augmented by BDNF as a result of interactions with TrkB receptors. However, similar effects of BDNF and TrkB-Ab on the CM strips were not detected in the LM strips of either the SWAS or WAS rats. In contrast to our findings, a recent report has shown that BDNF enhances the contractile activity of LM strips as elicited by SP from the distal colon of mice [22]. The reason for this discrepancy may be related to the different anatomical regions studied. Alternatively, it is possible that BDNF augmented the LM contraction elicited by other excitatory transmitters in the rat colon. Previous publications have shown that BDNF enhances the peristaltic reflex by augmenting the release of 5-HT [15] and causes an enhancement of Ca2? transients induced by 5-HT in cultured myenteric neurons [21]. There is recent evidence demonstrating that exogenous BDNF can augment the CCh-induced contraction of LM strips from the rabbit intestine [23]. Taken together, these results strongly support the notion that BDNF could enhance not only the effect of SP but also the effects of 5-HT and CCh on gut motility. In summary, this study provides evidence that the enhanced expression of BDNF and its high-affinity receptor, TrkB, in the colon may contribute to chronic stress-induced colonic hypermotility. Furthermore, BDNF, acting as a neuromodulator, may act on the TrkB receptor in enteric neurons and, consequently, facilitate the action of SP in promoting colonic motility. Due to the similar characteristics of the animal model used in the study of IBS, these findings can help to elucidate the pathogenesis of IBS and may offer interesting clinical therapeutic perspectives. Conflict of interest
None.
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