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Immobilization Stress Induces BDNF in Rat Submandibular Glands
K. Tsukinoki1,*,
J. Saruta2,
K. Sasaguri2,
Y. Miyoshi1,
Y. Jinbu3,
M. Kusama3,
S. Sato2 and
Y. Watanabe1
1 Department of Diagnostic Science, Division of Pathology and
2 Craniofacial Growth and Development Dentistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan; and
3 Department of Dentistry, Oral and Maxillofacial Surgery, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Kawachi-gun, Tochigi, 329-0498, Japan
Correspondence: * corresponding author, ktsukino{at}kdcnet.ac.jp
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ABSTRACT
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Brain-derived neurotrophic factor (BDNF) promotes survival and differentiation of the cells of the central and peripheral nervous systems. BDNF has been identified in non-neural tissue, including the heart, lung, platelets, lymphocytes, and lacrimal glands. Immobilization stress modifies BDNF mRNA expression in some organs. The present study examines the effect of immobilization stress on BDNF, and its receptor TrkB, in male rat submandibular glands. Increased BDNF mRNA and protein expression were observed in duct cells as a result of immobilization stress, as demonstrated by real-time PCR, Western blot, immunohistochemistry, and analysis by microdissection. TrkB mRNA was not detected in salivary gland tissue, or oral or esophageal mucosa, by RT-PCR. Rat submandibular gland was thus identified as an organ which expresses BDNF. Furthermore, the results of this study suggest that increased salivary BDNF expression occurs following immobilization stress.
Key Words: brain-derived neurotrophic factor (BDNF) • TrkB • rat submandibular gland • stress
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INTRODUCTION
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Salivary glands produce several cell growth factors and play an important role in human health (Tsukinoki et al., 2005). Mouse salivary gland tissue expresses a high level of nerve growth factor (NGF) (Aloe et al., 1986). The NGF family consists of NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4/5, -6, and -7, all of which are referred to as neurotrophins (Lewin and Barde, 1996). However, few reports describe the expression of neurotrophins other than NGF in the salivary gland.
Neurotrophins interact with Trk family high-affinity protein kinase receptors. Specifically, BDNF interacts with the TrkB receptor (Lewin and Barde, 1996). This BDNF-TrkB interaction promotes the survival and differentiation of neurons, and is involved in modification of neurotransmission and synaptic plasticity of the central and peripheral nervous systems (Leibrock et al., 1989). BDNF is predominantly found in the hippocampus and is associated with episodic memory (Egan et al., 2003). Immobilization stress reduces mRNA levels for neurotrophins such as NGF, BDNF, and NT-3 in the rat brain, especially in the hippocampus (Ueyama et al., 1997). In contrast, NGF expression is increased in response to stress in the mouse salivary gland (Aloe et al., 1986). The productions of various cell growth factors are often increased during episodes of stress, to maintain homeostasis in the salivary gland (Aloe et al., 1986; Konturek et al., 1991). Thus, we sought to examine whether there is a relationship between immobilization stress and BDNF expression within the salivary gland. The aim of the present study was, thus, to examine the effect of immobilization stress on BDNF and TrkB expression in male rat submandibular glands.
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MATERIALS & METHODS
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Animals
Sprague-Dawley male rats, aged 7–9 wks (Japan SLC, Shizuoka, Japan), were used in this study. They were housed in groups of 3 animals per cage in a room maintained under standardized conditions of light (12:12 hrs light-dark cycle) and temperature (22 ± 3°C). Animals had free access to food pellets and tap water.
Experimental Procedure
All experiments were performed with 6 rats per group. On the first day of each experiment, the animals were immobilized, to produce acute stress according to a well-established protocol (Hori et al., 2004, 2005). Rats were fixed on a wooden board (18 x 25 cm) in the supine position by means of a leather belt, after which each of their legs was fixed at an angle of 45 degrees to the body midline with adhesive tape. The rats were exposed to immobilization stress for 30 min, 60 min, or 180 min, respectively (immobilization stress group). In a separate group, the rats exposed to 30 min of immobilization stress were immediately anesthetized at 30, 60, or 180 min after the termination of restraint (post-immobilization stress group). During the post-stress period, all animals were returned to their home cages without food or water. The experimental protocol used in this study was reviewed and approved by the Committee of Ethics on Animal Experiments of Kanagawa Dental College, and was carried out in adherence with the Guidelines for Animal Experimentation of Kanagawa Dental College.
RNA Extraction and cDNA Synthesis
Total RNA isolation was performed with the ISOGEN reagent (Nippon Gene, Toyama, Japan), in accordance with the manufacturer’s instructions. RNA concentrations were determined by absorbance readings at 260 nm with a SmartSpec Plus spectrophotometer (BIO-RAD, Tokyo, Japan). Total RNA prepared from the rat brain was used as a positive control. Reverse transcription was performed as outlined in the instruction manual, with a 1st-strand cDNA synthesis kit (Roche Diagnostics Ltd, Lewes, UK).
Real-time PCR for BDNF
Real-time PCR was performed with a LightCycler (Roche), according to the manufacturer’s instructions. Reactions were performed in a 20-µL volume containing 0.3 mM of each primer and 4 mM MgCl2. Nucleotides, Taq DNA polymerase, and buffer were included in the LightCycler-DNA Master SYBR Green I mix (Roche). Oligonucleotide primers were designed to amplify rat BDNF and were specific for the coding region of exon V. The primer sequences were 5'-CAGGGGCATAGACAAAAG-3' and 5'-CTTCCCCTTTTAATGGTC-3', as designed and synthesized by Nippon Gene Laboratory. Denaturation was performed at 95°C for 10 min, after which a PCR step preceded Segment 1 (95°C for 10 sec), Segment 2 (60°C for 10 sec), and Segment 3 (72°C for 10 sec), with 40 cycles. We performed melting analysis and agarose gel electrophoresis to confirm the specificity of the PCR products obtained using each primer pair. Real-time PCR for amplification of the rat β-actin housekeeping gene was performed with a LightCycler Primer/Probe set, according to the manufacturer’s instructions (Nippon Gene Laboratory, Sendai, Japan). BDNF gene expression was expressed in terms of the relative copy number ratio of BNDF to β-actin for each sample. Mean ± SD was calculated. The Mann-Whitney U test was used for analysis. P < 0.05 was considered statistically significant.
PCR Analysis of TrkB Expression
Oligonucleotide primers, the primer sequences were 5'-TGACGCAGTCGCAGATGCTG-3' and 5'-TTTCCTGTACATG ATGCTCTCTGG-3' (Tokuyama et al., 1998). The primers used to detect the internal control marker, GAPDH, were 5'-TCCCAGAGCTGAACGGGAAGCTCATG-3' and 5'-TGGA GGCCATGTAGGCCATGAGGTCCA-3'. PCR was performed according to established procedures (Tokuyama et al., 1998).
Western Blot Analysis
Total protein was obtained from the rat brain positive control sample, as well as the submandibular gland tissue samples, after which protein concentrations were determined by the Bradford method using a SmartSpec Plus spectrophotometer (BIO-RAD). Each sample was separated by 12.5% SDS-PAGE and blotted. Each membrane was then incubated with anti-BDNF rabbit polyclonal antibody (sc-546, 1:100, Santa Cruz Biochemistry, Santa Cruz, CA, USA) for 1 hr. Membrane was incubated in Tris-PBS with anti-rabbit IgG conjugated with HRP (1:2000) (DAKO Cytomation, Glostrup, Denmark). Signals were detected on Hyperfilm ECL with an ECL system (GE, Healthcare Bio-Sciences Corp, Piscataway, NJ, USA).
Immunohistochemistry
Resected rat submandibular gland tissue samples were fixed in 10% formalin for 24 hrs. Immunohistochemical analysis was performed with the Simple stain MAX-PO kit (Nichirei, Tokyo, Japan). Sections were incubated with anti-BDNF rabbit polyclonal antibody (sc-546, 1:2000, Santa Cruz Biochemistry) or anti-human BDNF monoclonal antibody (1:30, TECHNE, Minneapolis, MN, USA) for 1 hr at room temperature. To provide negative controls, we used non-immunized rabbit or mouse IgG instead of primary antibody. Competitive assay was also attempted with the recombinant BDNF (R&D Systems, Minneapolis, MN, USA).
In situ Protein Expression and mRNA Analysis after Laser Capture Micro-dissection
Frozen sections of 8 µm were mounted onto non-coated slides and fixed in cold acetone for 5 min. Sections were then dried in cool air for 30 min. After this, they were immersed in phosphate-buffered saline (PBS) and incubated in 10% rabbit serum for 1 min. The primary antibody, BDNF monoclonal antibody (1:5, TECHNE), was applied for 5 min. The secondary antibody, FITC-labeled anti-mouse IgG antibody, was also applied for 5 min. Immunofluorescent-positive or -negative cells were dissected from stained sections with a laser capture microdissection system (ARCTURUS, Sunnyvale, CA, USA). Total RNA was extracted from these cells (approximately 1000 cells per sample) with the use of a PicopureTM RNA isolation kit (Roche).
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RESULTS
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Quantitative Analysis of BDNF mRNA
Melting curve analysis demonstrated a single fluorescent peak representing the melting temperature (Tm) of BDNF mRNA in all samples, except for the negative sample (data not shown). In addition, a single band was observed following agarose gel electrophoresis (data not shown). These findings confirmed that the PCR product was BDNF mRNA. BDNF/β-actin ratios were 0.066341 for brain as a positive control and 0.000367 ± 0.000205 for non-stressed rats. When the rats were exposed to immobilization stress for 30, 60, or 180 min, BDNF/β-actin ratios were 0.009028 ± 0.001 at 30 min, 0.001021 ± 0.000474 at 60 min, or 0.001463 ± 0.000662 at 180 min (Fig. 1A ). There were significant differences between non-stress and 30, 60, or 180 min of immobilization stress in the BDNF/β-actin ratios (p < 0.01 for all comparisons). BDNF/β-actin ratios were 0.001181 ± 0.000137 for 30 min, 0.000973 ± 0.000075 for 60 min, or 0.000135 ± 0.000031 for 180 min of post-immobilization stress (rats exposed to 30 min of immobilization stress were immediately killed at 30, 60, or 180 min after the termination of restraint) (Fig. 1B). There were significant differences between non-stress and 30 or 60 min of post-immobilization stress in the BDNF/β-actin ratios (p < 0.01 for both comparisons).
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Figure 1. BDNF mRNA quantification in rat submandibular gland tissue. (A) Graph showing BDNF mRNA of immobilization-stressed rats. BDNF mRNA level was 0.000367 ± 0.000205 for non-stress, 0.009028 ± 0.001 for 30 min, 0.001021 ± 0.000474 for 60 min, or 0.001463 ± 0.000662 for 180 min of immobilization stress (n = 6, bar = SD). There were significant differences between non-stress and 30, 60, or 180 min (*p < 0.01). (B) Graph showing BDNF mRNA levels in the post-immobilization stress (rats exposed to 30 min of immobilization stress were immediately killed at 30, 60, or 180 min after the termination of restraint). BDNF mRNA level was 0.001181 ± 0.000137 at 30 min, 0.000973 ± 0.000075 at 60 min, or 0.000135 ± 0.000031 at 180 min (n = 6, bar = SD). There was a significant difference between non-stress and 30 or 60 min of post-immobilization stress (*p < 0.01). BDNF mRNA levels approached those of non-stressed rats at 180 min of post-immobilization stress.
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RT-PCR Analysis of TrkB Expression
Amplified products corresponding to TrkB transcripts were detected in RT-PCR samples derived from the rat brain positive control sample. Each amplified TrkB fragment was 245 bp. TrkB mRNA was not detected in any salivary gland samples (Figs. 2A, 2B). GAPDH mRNA, an amplified fragment of 306 bp, was detected in all samples (Figs. 2A, 2B).
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Figure 2. Effect of immobilization stress on TrkB mRNA expression in rats. (A) Submandibular gland tissue. (B) Oral and esophageal mucosa. TrkB mRNA was not detected in any sample, except for brain tissue. P, Positive control (brain tissue); N, Negative control; 0, non-stress; 30, immobilization stress for 30 min; 60, immobilization stress for 60 min; 180, immobilization stress for 180 min.
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Immunohistochemistry
Two antibodies were used in this study. These antibodies demonstrated similar patterns of immunoreactivity; however, the polyclonal antibody was more intensely immunoreactive than the monoclonal antibody. Rat brain as a positive control showed high levels of BDNF expression in neural cells (data not shown). Non-stress submandibular gland tissue revealed little to no expression of BDNF in various duct-type cells (Fig. 3A). No apparent BDNF expression was consistently observed in acinar cells or myoepithelial cells in each of the immobilization stress groups. However, weak BDNF expression was observed in duct cells after 30 min of immobilization stress, while intense BDNF expression was observed in various duct-type cells after 60 or 180 min of immobilization stress (Fig. 3A). BDNF expression was not detected in the competitive assay (data not shown).
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Figure 3. BDNF protein levels following immobilization stress. (A) Photomicrographs showing the immunohistochemical localization of BDNF protein in paraffin-embedded tissues from rats immobilization-stressed with the anti-BDNF monoclonal antibody (n = 6). BDNF protein expression was observed in duct cells. Scale bar = 10 µm. (B) Western blotting demonstrated bands of 14 kDa in brain tissue and submandibular gland tissues of the immobilization-stress group (n = 6). P, Positive control (brain tissue).
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Western Blot Analysis
Bands of 14 kDa were clearly detected in brain tissue (as positive control), and in submandibular gland tissues at 30, 60, and 180 min of immobilization stress (Fig. 3B ).
Determination of BDNF mRNA and Protein Expression following Microdissection
The BDNF-positive cells were duct cells, while acinar cells were negative for BDNF after immobilization stress at 60 min (Fig. 4A). Laser-capture micro-dissection specifically dissected duct cells (Figs. 4B, 4C) or acinar cells (Figs. 4B, 4D). Dissected duct cells expressed BDNF mRNA, but there was no expression in acinar cells. β-actin mRNA was detected in all samples. The BDNF/β-actin ratio was 0.0001 for duct cells, and 0 for acinar cells (Fig. 4E).
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Figure 4. Results of microdissection analysis of BDNF mRNA and protein expression in rats immobilization-stressed for 60 min. (A) BDNF-reactivity was found within duct cells, but not acinar cells, by immunofluorescence staining. Scale bar = 10 µm. (B) Duct cells and acinar cells dissected from tissue sections. Scale bar = 10 µm. (C) Cap image showing dissected duct cells. Scale bar = 20 µm. (D) Cap image showing dissected acinar cells. Scale bar = 20 µm. (E) Graph showing BDNF/β-actin ratios of the dissected samples. BDNF mRNA level was 0.0001 for duct cells, and 0 for acinar cells. Duct cells showed the presence of BDNF mRNA, but acinar cells did not.
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DISCUSSION
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Using multiple techniques, we demonstrated increased expression of BDNF mRNA and protein in rat submandibular gland tissue following stress. Localization of BDNF protein and mRNA to ductal epithelium was observed in samples of rat submandibular gland tissue in a novel detection system, which combined microdissection of BDNF immunofluorescent-positive cells and quantitative RT-PCR. Ernfors et al. were the first to describe BDNF expression in rat submandibular gland tissue in the absence of stress, but BDNF mRNA was not detected by in situ hybridization with an oligonucleotide probe (Ernfors et al., 1990). The present findings were consistent with these results in the non-stress condition. In general, a high level of BDNF expression has been observed in the central and peripheral nervous systems, since BDNF mediates cell survival, and differentiation in neurons (Lewin and Barde, 1996). However, BDNF has also been reported to be present in non-neural tissue, such as the heart (Timmusk et al., 1993), lung (Timmusk et al., 1993), platelets (Radka et al., 1996), lymphocytes (Sobue et al., 1998), and lacrimal glands (Ghinelli et al., 2003) of rats. The present study identified the rat submandibular gland as a BDNF-expressing organ in immobilization stress.
Single or repeated immobilization stress markedly reduces BDNF mRNA and protein expression in the rat hippocampus (Adlard and Cotman, 2004). However, increased BDNF mRNA and protein levels occur in the pituitary glands of rats stressed for 60 min, while decreased levels occur following stress for 180 or 300 min (Givalois et al., 2001). In the present study, significant increases in BDNF mRNA and protein in the submandibular gland were observed in immobilization-stressed, compared with non-stressed, rats. Immobilization stress was performed according to a well-established protocol (Hori et al., 2004, 2005). This protocol is known to rapidly induce adrenocorticotropichormone (ACTH) and corticosterone (Adlard and Cotman, 2004). A sustained elevation of BDNF expression was observed in immobilization-stressed, compared with non-stressed, rats. Of note, a marked increase in BDNF mRNA was observed in rats immobilization-stressed for 30 min. Moreover, BDNF levels were decreased after 180 min of post-immobilization stress, compared with levels in non-stressed rats. These findings suggest that the salivary gland is sensitive to stress. Specifically, they demonstrate that BDNF expression increases within submandibular gland tissue in response to stress. BDNF expression is not observed in human or murine submandibular gland tissue (De Vicente et al., 1998). Although BDNF expression is not observed in human or murine submandibular gland tissue in the non-stress condition (De Vicente et al., 1998), alteration of BDNF expression may be induced in stress conditions.
Interestingly, in the non-stress and time-course of stress, TrkB mRNA was not detected in submandibular gland tissue or oral or esophageal mucosa by RT-PCR, despite the fact that increased levels of BDNF mRNA and protein were observed. Previous reports failed to demonstrate TrkB expression in the human salivary gland (De Vicente et al., 1998), or esophageal mucosa (Shibayama and Koizumi, 1996), in the absence of stress. BDNF derived from the submandibular gland might act at distant sites following secretion into the bloodstream. NGF is released from salivary glands into the bloodstream following stress induced by fighting (Aloe et al., 1986). There is a positive correlation between serum and brain BDNF protein levels (Karege et al., 2002). However, serum BDNF is unlikely to have an effect on the central nervous system, since serum BDNF is derived from platelets (Yamamoto and Gurney, 1990). Low levels of free BDNF exist in rat plasma (Radka et al., 1996). Since BDNF is able to cross the blood-brain barrier (Pan et al., 1998), levels of free BDNF in plasma might play a more significant role than serum levels of BDNF with regard to effects on the central nervous system. Although it is generally thought that trauma-induced alterations in neurotrophins and neurotrophin receptors within the central nervous system might protect against neuronal damage (Givalois et al., 2004), free BDNF in plasma might contribute to recovery against a decrease of BDNF. However, the source and role of plasma BDNF remain poorly understood. The results of the present study indicate that the rat submandibular gland may be an important source of plasma BDNF.
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ACKNOWLEDGMENTS
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This work was supported by a Grant-in Aid for High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Received for publication June 2, 2005.
Revision received April 14, 2006.
Accepted for publication May 5, 2006.
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Journal of Dental Research, Vol. 85, No. 9,
844-848 (2006)
DOI: 10.1177/154405910608500913
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ABSTRACT
INTRODUCTION