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2 Neurogastroenterol Motil (28) 2, doi: /j x Differential changes in brain-derived neurotrophic factor and extracellular signal-regulated kinase in rat primary afferent pathways with colitis L.-Y. QIAO, M. A. GULICK,,1 J. BOWERS,,1 J. F. KUEMMERLE, & J. R. GRIDER, Department of Physiology, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA Department of Internal Medicine, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA Address for correspondence Li-Ya Qiao, PhD, Department of Physiology, School of Medicine, Virginia Commonwealth University, PO Box 98551, Richmond, VA , USA. Tel: +1 (84) ; fax: +1 (84) ; Received: 19 September 27 Accepted for publication: 21 February 28 1 These authors contributed equally to this study. Abstract Brain-derived neurotrophic factor (BDNF) has been postulated to participate in inflammationinduced visceral hypersensitivity by modulating the sensitivity of visceral afferents through the activation of intracellular signalling pathways such as the extracellular signal-regulated kinase (ERK) pathway. In the current study, we assessed the expression levels of BDNF and phospho-erk in lumbosacral dorsal root ganglia (DRG) and spinal cord before and during trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats with real-time PCR, ELISA, western blot and immunohistochemical techniques. BDNF mrna and protein levels were increased in L1 and S1 but not L6 DRG when compared with control (L1: two- to fivefold increases, P <.5; S1: two- to three-fold increases, P <.5); however, BDNF protein but not mrna level was increased in L1 and S1 spinal cord when compared with control. In parallel, TNBS colitis significantly induced phospho-erk1/2 expression in L1 (four- to five-fold, P <.5) and S1 (two- to threefold, P <.5) but not in L6 spinal cord levels. Immunohistochemistry results showed that the increase in phospho-erk1/2 expression occurred at the region of the superficial dorsal horn and grey commisure of the spinal cord. In contrast, there was no change in phospho-erk5 in any level of the spinal cord examined during colitis. The regional and timespecific changes in the levels of BDNF mrna, protein and phospho-erk with colitis may be a result of increased transcription of BDNF in DRG and anterograde transport of BDNF from DRG to spinal cord where it activates intracellular signalling molecules such as ERK1/2. Keywords colitis, mitogen-activated protein kinase, neurotrophin, primary afferents, rat. INTRODUCTION Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family, has been postulated to participate in inflammation-induced visceral hypersensitivity by modulating the sensitivity of visceral afferents. 1 4 After peripheral inflammation, BDNF is synthesized in the primary sensory neurons where it facilitates intracellular signal transduction and gene expression in the dorsal horn of the spinal cord via anterograde transport. 5,6 Brain-derived neurotrophic factor in DRG and/or spinal cord likely binds to TrkB receptor and activates intracellular signalling molecules such as extracellular signal-regulated kinase (ERK), and induces long-term changes in central excitability. 9,1 Recent studies demonstrate that spinal intrathecal injection of BDNF/TrkB antisense or antiserum significantly attenuates inflammation-induced hypersensitivity, 7,8 suggesting a central modulatory role of BDNF in mediating inflammatory pain. Extracellular signal-regulated kinase, activated by neurotrophins or neuronal activity in the central or peripheral nervous system, plays an essential role in the generation and maintenance of inflammation-induced hyperalgaesia by regulating nociceptive activities in primary sensory pathways. 11 Increased phosphorylation of ERK1/2 has been observed in rat spinal cord dorsal 928 Journal compilation Ó 28 Blackwell Publishing Ltd

3 Volume 2, Number 8, August 28 ERK activation and BDNF expression in colitis horn neurons in response to noxious stimulation of the peripheral tissue or electrical stimulation to the peripheral nerve. 12,13 Moreover, intrathecal injection of MEK/ ERK inhibitor U126 or PD9859 alleviated pain behaviour induced by inflammation of the hind paw 13 or viscera, 12 suggesting a prominent role of ERK in the regulation of peripheral inflammation. Colonic inflammation induces considerable changes in neurochemical and electrophysiologic properties of the gut primary afferent pathways, 39,4 which play an important role in the regulation of gut function. Our previous studies 17 with neural retrograde tracing demonstrated the segmental distribution pattern of colonic primary afferents in lumbosacral DRG that were L1, L2, L6 and S1 DRG. In the present study, we characterized the changes in BDNF mrna and protein in lumbosacral DRG and spinal cord at 3 and 7 days of colitis. The resulting increased expression in BDNF mrna and/ or protein in DRG and spinal cord during colitis may lead to increased activation of intracellular signalling molecules, such as MEK/ERK pathway. Therefore, we further examined the activation (phosphorylation) level of ERK1/2 and ERK5 in lumbosacral spinal cord before and after colitis induction in order to determine the correlation between BDNF expression, ERK activation and TNBS-induced colitis. The resulting region-specific changes in BDNF mrna, BDNF protein and ERK activation in DRG and spinal cord suggest a role of BDNF as a central neuronal mediator during colonic inflammation. Part of the data has been reported in abstract form at national and international meetings. 22,23 MATERIALS AND METHODS Animals and reagents Adult male Sprague Dawley rats (15 2 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA) and divided into control (n = 15) and colitis groups (n = 2). Chemicals used in this experiment were purchased from Sigma ImmunoChemicals (St Louis, MO, USA). Brain-derived neurotrophic factor polyclonal antibody was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Antibodies against total ERK1/2, phospho-erk1/2 and phospho-erk5 were from Cell Signaling Technology Inc. (Danvers, MA, USA). Fluorescence-conjugated secondary antibody was from Molecular Probes (Eugene, OR, USA). Western blot secondary antibody was from the enhanced chemiluminescence kit purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). TNBS-induced colitis To induce inflammation in the distal colon, fasted rats were anaesthetized and TNBS was instilled into the lumen of the colon at a dose of 9 mg kg )1 (1.5 ml kg )1 of 6 mg ml )1 solution in 5% EtOH) through a syringe-attached polyethylene catheter via the rectum 6 cm proximal to the anus. Animals that received an equal volume of 5% EtOH enema served as controls throughout these studies. All colonic instillations were performed under isoflurane (2%) anaesthesia. All above experimental protocols involving animal use were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Animal care was in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. Perfusion and tissue harvesting Animals were deeply anaesthetized with isoflurane (3 4%) and then euthanized via intracardiac perfusion, first with oxygenated Krebs buffer (ph 7.4) (95% O 2, 5% CO 2 ) followed by 4% paraformaldehyde. Dorsal root ganglia and spinal cord from level L1, L6 and S1 were identified 4,17 and the DRG was sectioned parasagitally at a thickness of 2 lm. Spinal cord segments were sectioned transversely at a thickness of 3 lm. Tissues from control and experimental animals were handled in an identical manner. Immunohistochemistry Dorsal root ganglia sections were immunostained using an on-slide technique. Spinal cord sections were immunostained by free-floating method. Generally, sections were incubated with blocking solution containing 3% normal donkey serum in PBS-Triton [.3% Triton X-1 in.1 mol L )1 phosphate buffered saline (PBS), ph 7.4] for 3 min followed by rabbit anti-bdnf (1 : 5) or phospho-erk1/2 (1 : 4) primary antibody overnight at 4 C for DRG, or 1 h at room temperature for spinal cord. After rinsing (3 1 min with.1 mol L )1 PBS), tissues were incubated with fluorescence-conjugated species-specific secondary antibody Alexa 594 for 2 h at room temperature. Following washing, the slides were coverslipped with Citifluor (Citifluor Ltd., London, UK). Immunostaining in the absence of primary or secondary antibody was processed for background evaluation. The specificity of the antibodies was also evaluated with western blot. Journal compilation Ó 28 Blackwell Publishing Ltd 929

4 L.-Y. Qiao et al. Neurogastroenterology and Motility Dorsal root ganglia cells were counted with a Nikon fluorescent photomicroscope by investigators who were blinded to which treatment was administered. Brain-derived neurotrophic factor cell profiles were counted in 6 1 sections of each selected DRG (L1, L6 S1), and expressed as mean ± SE for n animals. Within the specific segment of DRG (such as L1 DRG), the sections of similar size (i.e. L1, 2 mm 2 ; L6, 2 mm 2 ; and S1, 1 mm 2 ) were chosen with the microscope built-in grids, and all the positive cells were counted in the sections and expressed as number of cells per section. Between different segments of the DRG (such as between L1 and L6 DRG), we did not normalize the results according to the size of the sections; therefore, we compared the difference between TNBS-treated and untreated animals for a specific segment of DRG. No comparison was made between different segments of the DRG due to the size discrepancy at different spinal levels. We have chosen every third section for examination with one specific antibody to avoid double counting. The fluorescent images of the spinal cord sections were converted to a grey scale that ranges in intensity from (black) to 255 (white) for the purpose of densitometry. For scanning, the same number of standard-sized rectangles was overlaid on the area of interest (i.e. superficial dorsal horn in this study) for each spinal section, with one rectangle on the background area for subtraction. Intensity measured within the rectangles was averaged as one point. Protein extraction Freshly dissected DRG and spinal cord segments were homogenized in solubilization buffer containing 5 mmol L )1 Tris HCl, 15 mmol L )1 NaCl, 1 mmol L )1 EDTA, 1% Triton X-1, 1 mm NaF supplemented with protease inhibitor cocktail (Sigma, P834) and phosphatase inhibitor cocktail 1 (Sigma, P285). The homogenate was centrifuged at 2 2 g for 1 min at 4 C, and the supernatant was removed to a fresh tube for further analysis. The protein concentration was determined using Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Enzyme-linked immunosorbent assay Brain-derived neurotrophic factor ELISA kit (Promega Corporation, Madison, WI, USA) was used for this examination. Briefly, polystyrene microtitre plates (96 well) were precoated according to manufactureõs instruction. The protein extracts were sequentially treated with 1 N HCl and then neutralized with 1 N NaOH. The treated samples and BDNF standard were incubated sequentially with chicken anti-human BDNF pab (1 : 5), anti-igy HRP conjugate (1 : 2) and TMB (Promega Corporation, Madison, WI, USA) one solution (3,3,5, 5 -tetramethylbenzidine). The absorbance at 45 nm was measured within 3 min of stopping the reaction and the BDNF levels were expressed as pg BDNF per mg total protein calculated against the standard curve. Western blot Proteins were separated on a % sodium dodecyl sulphate polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk in Tris-buffered saline for 1 h and then incubated with phospho-erk1/ 2 (1 : 1) or phospho-erk5 (1 : 1) or BDNF (1 : 5) antibody followed by horseradish peroxidaseconjugated secondary antibody. For internal loading control, the same membrane was striped and re-probed with antibody against the non-phosphorylation form of ERK1/2 (1 : 1) or b-actin (1 : 2, Sigma). The immunoreactive bands were detected by enhanced chemiluminescence, and the densitometric quantification of immunoreactive bands was performed using the software FluorChem 88 (Alpha Innotech, San Leabdro, CA, USA). The expression level of the target protein in control animal from each independent experiment was considered as 1, and the relative expression level of the target protein in experimental animals was adjusted as a ratio to its internal loading control in each independent experiment. For phospho- ERK1/2, the p42 and p44 bands were analysed together. RNA extraction and quantitative real-time PCR Total RNA was extracted using a RNA extraction kit RNAqueous (Ambion, Austin, TX, USA). RNA concentration was determined spectrophotometrically. cdna was synthesized using cloned AMV first-strand synthesis kit (Invitrogen, Carlsbad, CA, USA) with random hexamers. Following reverse transcription, quantitative real-time PCR was performed for BDNF with a Taqman probe mixed with PCR Master-Mix for 4 cycles (95 C for 15 sec, 6 C for 1 min) on a 73 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR of the same sample was performed for b-actin expression as internal control for normalization. The changes in mrna levels of the test gene were calculated with DDCt (change in the cycle threshold) method using 93 Journal compilation Ó 28 Blackwell Publishing Ltd

5 Volume 2, Number 8, August 28 ERK activation and BDNF expression in colitis b-actin level in the same sample as a normalizer. The expression level of target mrna in the control animal from each independent experiment was considered as 1, and the relative expression level of target mrna in experimental animals was adjusted as a ratio to its control in each independent experiment and expressed as fold changes (2 DDCt -fold). Culture of spinal cord slices Spinal cord segment S1 was dissected from naïve animals and transversely sectioned at a thickness of 25 lm with a tissue sectioner. The sections were randomly divided into several cell culture wells containing DulbeccoÕs modified EagleÕs medium supplemented with 2 units ml )1 penicillin, 2 mg ml )1 streptomycin and 1 mg ml )1 gentamycin, and cultured for 4 6 h. Brain-derived neurotrophic factor (5 nmol L )1 ) was added to the culture medium 5 min, 3 min or 14 h prior to the collection of all slices (BDNF-treated or untreated) for assay. All cultures were maintained in a 1% CO 2 environment at 37 C. Statistical analysis The results from each study were presented as mean ± SE for n animals. Comparison between control and experimental groups was made by using one-way ANOVA followed by DunnettÕs test. Differences between means at a level of P.5 were considered to be significant. The results were confirmed by statistical power calculation with statistical power 8%. RESULTS Colitis-induced changes in BDNF protein level in lumbosacral DRG We previously reported that BDNF immunoreactivity was significantly increased in L1 and S1 DRG but not in L6 DRG at 7 days of colitis. 17 In the present study, we examined the changes in BDNF immunoreactivity (Fig. 1A) in L1, L6 and S1 DRG at 3 days of colitis and compared them with 7 days of colitis demonstrated previously. 17 Results showed a significant increase in the number of cells expressing BDNF in L1 DRG at 3 days of colitis (2.5-fold, P <.5) when compared with L1 control DRG (Fig. 1B), and was sustained until 7 days of colitis as reported previously. 17 In L6 DRG, there was no change in the number of cells expressing BDNF at 3 days of colitis when compared with control (Fig. 1B). In S1 DRG, we previously showed that the number of cells expressing BDNF immunoreactivity was increased at 7 days of colitis; 17 however, there was Figure 1 Brain-derived neurotrophic factor (BDNF) protein expression in lumbosacral dorsal root ganglia (DRG) before and during colitis. Representative section (L1 DRG from 3 days of colitis) of BDNF immunostaining (A) shows that BDNF was expressed in different sizes of DRG neurons. Histogram B shows that the BDNF cell profiles were significantly increased in L1 but not S1 DRG at 3 days of colitis (n = 5) when compared with control (n = 6). ELISA examination (C) and western blot (D) also show increases in BDNF protein level in L1 and S1 but not L6 DRG when compared with segment-matched controls (n = 4 to 5 for each time point examined). Con: control; T7: TNBS 7 days. Calibration bar = 6 lm. P <.5. A C BDNF/protein (pg mg 1 ) BDNF Control TNBS-3d TNBS-7d L1-DRG L6-DRG S1-DRG BDNF-IR/section B D Fold Changes BDNF b-actin L1-DRG L6-DRG S1-DRG Control TNBS-7d Control TNBS-3d L1-DRG L6-DRG S1-DRG Con T7 Con T7 Con T7 Journal compilation Ó 28 Blackwell Publishing Ltd 931

6 L.-Y. Qiao et al. Neurogastroenterology and Motility no change in the number of cells expressing BDNF at 3 days of colitis when compared with control (Fig. 1B). To confirm the change in BDNF protein level in lumbosacral DRG with colitis, we examined BDNF content with ELISA in L1, L6 and S1 DRG at 3 and 7 days of colitis (Fig. 1C). The results showed time and segment-dependent changes in BDNF protein level in L1, L6 and S1 DRG during colitis, which is consistent with the change profile in BDNF cell numbers in these DRG. In L1 DRG, the levels of BDNF protein were increased at both 3 and 7 days of colitis (2.6- to 5.2-fold, P <.5); in L6 DRG, there were no significant changes in the BDNF protein level at both 3 and 7 days of colitis; and in S1 DRG, BDNF protein level was significantly increased at 7 days (2.9-fold, P <.5) but not 3 days of colitis when compared with control. Western blot results also showed increases in BDNF protein level in L1 and S1 but not L6 DRG at 7 days of colitis when compared with segment-matched control (Fig. 1D). Colitis-induced changes in BDNF mrna level in lumbosacral DRG The level of BDNF mrna was examined with quantitative real-time PCR. Results (Fig. 2) showed significant increases in the level of BDNF mrna in L1 DRG (2.3-fold, P <.5) at 3 days of colitis; however, no change in BDNF mrna level was detected in L1 DRG at 7 days of colitis when compared with control. In L6 DRG, there were no significant differences in the level of BDNF mrna at any time points examined. In S1 DRG, BDNF mrna level was increased at 3 days (2.1- Fold Changes in BDNF mrna Control TNBS-3d TNBS-7d L1-DRG L6-DRG S1-DRG Figure 2 Brain-derived neurotrophic factor (BDNF) mrna expression in lumbosacral dorsal root ganglia (DRG) before and during colitis. There were significant increases in the levels of BDNF mrna in L1 and S1 DRG at 3 days of colitis; however, no changes in BDNF mrna level were detected in L1 and S1 DRG at 7 days of colitis when compared with control. In L6 DRG, there were no significant differences in the level of BDNF mrna at all the time point examined (n = 3 to 5 for each time point). P <.5. fold, P <.5) but not 7 days of colitis when compared with control. BDNF mrna and protein in lumbosacral spinal cord before and during colitis Quantitative real-time PCR (Fig. 3A) showed no changes in the level of BDNF mrna in L1 and S1 spinal cord at either 3 or 7 days of colitis when compared with segment-matched control. However, the level of BDNF protein was significantly increased in L1 and S1 spinal cord during colitis (Fig. 3B,C). In L1 spinal cord from control animals, the level of BDNF protein was 48 ± 5 pg BDNF per mg protein, which was significantly increased by 2.1-fold (P <.5) at 3 days BDNF/protein (pg mg 1 ) Fold Changes in BDNF mrna A B L1-Spinal cord L1-Spinal cord C L1 spinal cord TNBS Con 3 d 7d Control TNBS-3d TNBS-7d S1-Spinal cord Control TNBS-3d TNBS-7d S1-Spinal cord S1 spinal cord TNBS Con 3 d 7d BDNF b-actin Figure 3 Tri-nitrobenzene sulfonic acid (TNBS) colitis-induced changes in BDNF mrna and protein in lumbosacral spinal cord. Quantitative real-time PCR (A) showed no changes in the level of BDNF mrna in L1 and S1 spinal cord at both 3 and 7 days of colitis when compared with control (n =3 to 5). However, the levels of BDNF protein were significantly increased in L1 and S1 spinal cord during colitis when examined with ELISA (B) and western blot (C) (n =3to4). P < Journal compilation Ó 28 Blackwell Publishing Ltd

7 Relative level of p-erk1/2 Volume 2, Number 8, August 28 ERK activation and BDNF expression in colitis of colitis and by 4.3-fold (P <.5) at 7 days of colitis. Similarly, the levels of BDNF protein were increased in S1 spinal cord at both 3 and 7 days of colitis (3.1 to 7.3-fold, P <.5) when compared with control. Western blot results (Fig. 3C) also showed increases in BDNF protein expression in L1 and S1 spinal cord at both 3 and 7 days of colitis when compared with control, which was consistent with the BDNF change profiles determined by ELISA (compare with Fig. 3B). Increased phosphorylation of ERK1/2 but not ERK5 in lumbosacral spinal cord during colitis The activation of MAPK by BDNF has been reported in several systems. 9,2,21 In the present study, we examined whether ERK1/2, a member of MAPK family, was activated (phosphorylated) in lumbosacral spinal cord at 7 days of colitis, because at this time point the level of BDNF expression in the spinal cord was increased when compared with control. Western blot results showed that the phosphorylation level of ERK1/2 was significantly increased in L1 and S1 but not L6 spinal cord at 7 days of colitis (two- to five-fold increases, P <.5) when compared with segment-matched controls (Fig. 4A,B). Immunohistochemistry results (Fig. 5) showed that the increase in the density of phospho-erk1/2 immunoreactivity occurred at the superficial dorsal horn of the spinal cord (Fig. 5A,B,E). There were also increases in the size of the area immunoreactive to phospho-erk1/2 in deeper regions of the dorsal horn (compare the circled regions in Fig. 5A,B). As well, there were increases in the density and number of neurons expressing phospho-erk1/2 in the region of grey commisure of the spinal cord (Fig. 5C,D,F). We previously showed increased activation of another ERK isoform, ERK5, in lumbosacral DRG after colitis. 3 To further examine whether colitis induces region-specific changes in neurotrophin signalling in primary afferent pathways, we examined the level of phospho-erk5 in the spinal cord after colitis. Results showed that colitis did not cause changes in the level of phospho-erk5 in either L1 (Fig. 4C) or S1 (data not shown) spinal cord when compared with control. Effects of BDNF on ERK1/2 phosphorylation in cultured spinal cord slices We have determined that BDNF and phospho-erk1/2 were both increased in L1 and S1 but not L6 spinal A Control SP L1 TNBS SP L1 Control SP L6 Control SP S1 TNBS SP L6 TNBS SP S1 p - ERK1/2 Figure 4 Expression of phospho-erk1/ 2 and phospho-erk5 in lumbosacral spinal cord before and during colitis. Lumbosacral spinal cord (SP) L1, L6 and S1 from control and animals treated with tri-nitrobenzene sulfonic acid (TNBS) were homogenized and phospho-erk1/2 or phospho-erk5 was measured by western blot (A, C). An increased expression of phospho-erk1/ 2 but not phospho-erk5 was observed in L1 and S1 spinal cord during colitis (n = 4 for each points). There was no change in the level of phospho-erk1/2 in L6 spinal cord with colitis. Histogram (B) shows relative levels of phospho-erk1/2 expression in each segments examined. P <.5. B 6 Control 5 TNBS L1 L6 S1 Spinal Cord C Control TNBS Positive ERK1/2 p - ERK5 ERK1/2 b-actin Journal compilation Ó 28 Blackwell Publishing Ltd 933

8 L.-Y. Qiao et al. Neurogastroenterology and Motility A B C E D F Figure 5 Phospho-ERK1/2 immunoreactivity in L1 spinal cord before and during colitis. Immunohistochemistry results showed increases in the size of the area immunoreactive to phospho- ERK1/2 in deeper regions of the dorsal horn [compare the circled regions in (A) and (B)] and increases in the density of phospho-erk1/2 immunoreactivity at the superficial dorsal horn of the spinal cord (A, B and E). There were also increases in the density and number of neurons expressing phospho-erk1/2 in the region of grey commisure of the spinal cord (C, D and F; arrows indicate phospho-erk1/2-immunoreactive neurons). Three animals for each treatment were used, and four sections from each animal were scanned for densitometry. Bar = 5 lm in (A) and (B); 25 lm in (C) and (D). P <.5. segments after colitis (compare Figs 1 4). As BDNF has been reported to activate ERK1/2 in several systems, 9,2,21 the parallel changes in BDNF expression and ERK1/2 phosphorylation suggest a possible cause and effect relationship of BDNF activating ERK1/2 in L1 and S1 spinal cord. In this study, we incubated spinal cord slices (25 lm) from S1 segment with BDNF (5 nmol L )1 ) for 5 min, 3 min or 14 h and determined the effects of BDNF on ERK1/2 phosphorylation. Results (Fig. 6) showed that exogenous BDNF increased ERK1/2 activation in the spinal cord within 3 min of incubation (P <.5), suggesting that the colitis-induced BDNF expression/release in the spinal cord was able to activate ERK1/2 rapidly. DISCUSSION Brain-derived neurotrophic factor has been extensively reported to be involved in peripheral inflammation and hypersensitivity. 1,3,4 Previous studies showed that intraperitoneal injection of BDNF antibody blocked colitis-induced hypersensitivity. 1 However, the central contribution of BDNF to the maintenance of the nociceptive property of sensory neurons during colitis is unknown. In the present study, we found that the levels of BDNF mrna and protein were increased in L1 and S1 but not L6 DRG with colitis in a timedependent manner. Brain-derived neurotrophic factor mrna was elevated at 3 days of colitis in both L1 and S1 DRG; BDNF protein was elevated at 3 days and 7 days in L1 DRG and at 7 days in S1 DRG with colitis, suggesting that colitis caused initial BDNF transcription in these DRG followed by protein production. In L1 and S1 spinal cord, only increases in BDNF protein were observed, whereas BDNF mrna demonstrated no changes in these spinal segments at any time point examined. These results suggest that BDNF was not transcribed in the spinal cord, but rather that BDNF was likely synthesized in primary sensory neurons in DRG, transported to the central terminals of the primary afferents, and released into the spinal dorsal horn. 5,6 Within the spinal cord, BDNF may bind to 934 Journal compilation Ó 28 Blackwell Publishing Ltd

9 Volume 2, Number 8, August 28 ERK activation and BDNF expression in colitis Relative level of p-erk1/ min 5 min 3 min 14 hr (BDNF) TrkB receptor on second-order sensory neurons, and then activate intracellular signalling molecules such as MAPK signalling cascades, and induce long-term changes in central excitability. 9,1 In the present study, we determined that ERK1/2 was activated in L1 and S1 but not L6 spinal cord after colitis. However, another isoform of ERK, ERK5, was not activated in these segments, suggesting a specific afferent-mediated rather than systemic activation of ERK1/2 in the dorsal horn after colitis. The segment-dependent parallel changes in BDNF expression and ERK1/2 activation in the primary afferent pathway during colitis suggests that ERK1/2 may be a downstream element in BDNF signalling pathways in the lumbosacral spinal cord. This notion is supported by our experiment that, in spinal cord slices from S1 segment, exogenous BDNF increased ERK1/2 phosphorylation within 3 min of incubation. The distal colon receives dual spinal innervation from the hypogastric/splanchnic (L1 L2) and pelvic afferents (L6 S1 in rats). 14,15 Spinal afferents arise from the DRG and project into the dorsal horn of the spinal cord, 16 where they are thought to play a major role in nociception. Studies from our laboratory 17 and others 18 using retrograde tracing with the conventional neuronal tracing dye fast blue or 1,1 -dioleyl-3,3,3 3 -tetramethylindocarbocyanine demonstrate that primary afferent neurons with projection to colon are located in lumbosacral DRG including L1, L2, L6 and S1, representing innervation from the hypogastric/ splanchnic and pelvic afferents 14 respectively. We previously showed that increased calcitonin gene-related peptide (CGRP) in L1 and S1 bladder afferent neurons during colitis may result from p-erk1/2 ERK1/2 Figure 6 Effects of brain-derived neurotrophic factor (BDNF) on ERK1/2 phosphorylation in cultured spinal cord slices. S1 spinal cord was transversely sectioned at a thickness of 25 lm and incubated with BDNF (5 nmol L )1 ) for a designated time period. Results showed that exogenous BDNF was able to increase ERK1/2 activation in the spinal cord slices within 3 min of incubation (n = 4). P <.5. increased BDNF in these DRG. 17 In the current study, we further examined the expression pattern of BDNF in these DRG and confirmed that the levels of BDNF mrna and protein were exclusively increased in L1 and S1 DRG but not in L6 DRG at the time point examined (3 or 7 days) during colitis. Although L6 DRG also receives afferent input from the distal colon, 17 there were no changes in either BDNF protein or BDNF mrna level in L6 DRG at 3 or 7 days of colitis. This does not preclude the changes in BDNF mrna and/or protein in L6 DRG at other time points (such as prior to 3 days or post to 7 days after colitis induction). It is noteworthy that the control value of BDNF protein determined by ELISA (Fig. 1C) and western blot (Fig. 1D) was significantly higher (P <.5) in L6 and S1 DRG than in L1 DRG whereas cells counts appear to be similar (Fig. 1B). One of the explanations for the discrepancy may be that BDNF expression in the nerve fibres or structures other than neuronal cells can be picked up by ELISA and western blot but not by cell counting, and the nature of the structure for L1, L6 and S1 DRG is different, which includes but is not limited to (i) the size of L1, L6 and S1 DRG are different, with the area containing cell bodies in S1 DRG relatively smaller than in L1 and L6; and (ii) the shape of L1, L6 and S1 are different, with L1 round-like and L6 and S1 elongated with an oval- or spindle-like shape. These characteristic features of L1, L6 and S1 may contribute to the different level of BDNF cell number and content in these DRG in control animals. Concomitant examination of BDNF transcription and BDNF protein levels in lumbosacral DRG revealed a time-dependent change in BDNF mrna production and BDNF protein accumulation. Brain-derived neurotrophic factor transcripts were elevated at 3 days of colitis and declined at 7 days of colitis. Brain-derived neurotrophic factor protein levels had small or no increase at 3 days of colitis but were increased significantly at 7 days of colitis in both L1 and S1 DRG (Fig. 1). 17 The time-dependent changes in BDNF mrna and protein in lumbosacral DRG may indicate an initial release followed by the accumulation of BDNF protein when more BDNF was transcribed during colitis. That BDNF protein but not mrna was still detectable at 7 days of colitis in both L1 and S1 DRG also indicates a prolonged stability of BDNF protein that allows BDNF to act as one of the neuronal mediators. In our earlier studies, we demonstrated that colitis induced ERK5 activation in the lumbosacral DRG, 3 where it might enhance gene expression through the activation of transcription factor CREB, a camp-responsive element binding protein. 28,29 Several Journal compilation Ó 28 Blackwell Publishing Ltd 935

10 L.-Y. Qiao et al. Neurogastroenterology and Motility studies have demonstrated that the signalling pathway regulating BDNF expression involves a CREB family transcription factor-dependent mechanism Consistent with this notion, we demonstrated that the expression levels of BDNF mrna and protein were upregulated in lumbosacral DRG where ERK5 was activated during colitis. 35 These results suggest a central role of BDNF in inflammatory bowel and associated diseases. The central production of BDNF and the central role of BDNF signalling in peripheral or visceral inflammation have been suggested previously. Significant increases in BDNF and receptor TrkB mrna in DRG and spinal cord after inflammation have been reported in many systems. 4,36,37 Previous studies with a cystitis rat model 4 suggest that increased expression of TrkB in bladder afferent neurons may contribute to cystitisinduced bladder overactivity. Similarly, the present study suggests that increased BDNF and signalling in lumbosacral DRG and spinal cord may play a role in colitis-induced hypersensitivity. Taken together, these results also support our recent findings that increased BDNF in DRG may contribute to increased CGRP in these neurons. 17 Previous studies suggested the anterograde transport property of BDNF from DRG neurons to the spinal cord dorsal horn after peripheral noxious stimulation. 9,1 To test whether BDNF was involved in spinal dorsal horn neuronal activation, we examined BDNF protein level in L1 and S1 spinal cord before and during colitis. We showed that BDNF protein level was significantly increased in L1 and S1 spinal cord at both 3 and 7 days of colitis. To further examine the mechanism underlying BDNF protein upregulation in L1 and S1 spinal cord following colitis, we examined BDNF mrna level in these spinal cord segments and showed that there were no changes in BDNF mrna level at any time point examined after colitis induction when compared with control. These results suggest that increased BDNF protein level in the spinal cord may be the result of anterograde transport of BDNF from DRG where both BDNF mrna and protein were increased during colitis when compared with control. Neurotrophins induce three major signalling pathways in neurons: the ERK pathway, the PI 3-kinase/ Akt pathway and the PLCc pathway. 28 Previous studies show that activation of ERK by inflammatory mediators in primary sensory and second-order dorsal horn neurons participates in the generation and maintenance of inflammatory pain. 19,31 In a murine model of visceral pain and hyperalgaesia, intracolonic instillation of either capsaicin or mustard oil significantly induced the activation of ERK1/2 in lumbosacral spinal cord. 12 Along with this, in recent studies with cyclophosphamide-induced cystitis, the level of phospho- ERK1/2 was increased in L1 spinal cord after bladder inflammation, 26 and phospho-erk1/2 immunoreactivity was exhibited in neurons of laminae I-II, dorsal commisure and the intermediolateral grey matter, areas that receive projections from primary afferent neurons and that show responsiveness to bladder stimulation. 27 In the current study, colitis induced increased expression of phospho-erk1/2 in L1 and S1 spinal cord at the laminae I II and dorsal commisure regions. The increased BDNF expression most likely contributed to the increased activation of ERK1/2 in lumbosacral spinal cord after colitis. However, this does not preclude the contribution of other factors such as glutamate in the activation of ERK1/2 in the dorsal horn neurons. 38 We previously reported that another MAPK isoform, ERK5, was activated in lumbosacral DRG during colitis. 3 To further examine whether colitis induces region-specific changes in neurotrophin signalling in primary afferent pathways, we examined the level of phospho-erk5 in the spinal cord and showed no activation of ERK5 in L1 and S1 spinal cord where ERK1/2 was activated with colitis. The stark contrast of region-dependent activation of ERK1/2 and ERK5, in which ERK1/2 was activated in spinal cord but not in DRG whereas ERK5 was activated in DRG but not in the spinal cord, provides strong evidence that the central activation of ERK is not a systemic, nonspecific event after colitis. In summary, the present study demonstrated that colitis-induced BDNF expression in primary afferent pathway was initiated with BDNF transcription in the DRG followed by BDNF protein production in primary sensory neurons. The increased BDNF was then likely transported to the spinal cord dorsal horn where the central nerve terminals of the primary sensory neurons are localized. Enhanced nociceptive input and increased presynaptic release of BDNF in the dorsal horn during colitis may facilitate excitatory synaptic responses by activating intracellular signalling pathways such as ERK1/2 as we determined in this study, leading to gene expression. 24,25 Taken together, the present studies demonstrated a central modulatory role of BDNF and signalling that may contribute to colitisassociated hypersensitivity. SUPPORTING GRANTS A.D. Williams Fund/Virginia Commonwealth University (LYQ); The AGA Foundation for Digestive Health 936 Journal compilation Ó 28 Blackwell Publishing Ltd

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