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Submandibular Glands Contribute to Increases in Plasma BDNF Levels

¹ Department of Maxillofacial Diagnostic Science, Division of Pathology,
² Department of Craniofacial Growth and Development Dentistry, Division of Orthodontics, and
³ Department of Oral Medicine, Division of Operative Dentistry and Endodontics, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan

^* corresponding author, ktsukino{at}kdcnet.ac.jp

	ABSTRACT

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Brain-derived neurotrophic factor (BDNF) promotes survival and differentiation of neural cells in the central and peripheral nervous systems. BDNF has been detected in plasma, but its source has not yet been established. Expression of BDNF mRNA has been identified in the submandibular glands when male rats are exposed to acute immobilization stress. In the present study, we investigated whether plasma BDNF is influenced by the submandibular glands in this model. Acute immobilization stress for 60 min significantly increased the level of plasma BDNF. However, plasma BDNF elevation was markedly suppressed in bilaterally sialoadenectomized rats. There were no significant differences between stressed (60 min) and non-stressed rats with respect to the BDNF mRNA expression in the hippocampus, heart, lung, liver, pancreas, or spleen, as determined by real-time polymerase chain-reaction. These findings suggest that the submandibular glands may be the primary source of plasma BDNF in conditions of acute immobilization stress.

KEY WORDS: brain-derived neurotrophic factor (BDNF) • plasma • rat submandibular gland • acute immobilization stress

	INTRODUCTION

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Brain-derived neurotrophic factor (BDNF) is the most abundant neurotrophin in the central nervous system, and it is closely involved in survival, maintenance, and neural transmission in neural cells (Lewin and Barde, 1996). In the hippocampus in particular, expression of BDNF varies, depending on stress (Givalois et al., 2004), exercise (Adlard and Cotman, 2004), and learning (Egan et al., 2003), and BDNF plays an important role in facilitating the formation of neural networks and elevating neural activity. In contrast, in neuropathies such as depression (Karege et al., 2005), schizophrenia (Tan et al., 2005), Alzheimer’s disease (Michalski and Fahnestock, 2003), and Parkinson’s disease (Parain et al., 1999), decreased BDNF expression has been confirmed, and alteration of BDNF content in the central nervous system has been reported to play an important role in the pathogenesis of neural disease (Lommatzsch et al., 2005). In addition, levels of plasma and serum BDNF are decreased in persons with schizophrenia (Tan et al., 2005) or depression (Karege et al., 2005). Because plasma BDNF levels increase with the use of psychotropic agents (Shimizu et al., 2003), BDNF content in serum or plasma may have clinical significance as a diagnostic tool for mental disorders. However, the biological roles of BDNF in serum and plasma remain unknown.

Serum BDNF originates from platelets (Yamamoto and Gurney, 1990). Because large amounts of BDNF accumulate in platelets, BDNF in serum is at a relatively high level (Radka et al., 1996). When platelets are destroyed by coagulation, BDNF release is identified (Lommatzsch et al., 2005). However, levels of plasma BDNF are markedly lower than those of serum BDNF (Radka et al., 1996), and the origin of plasma BDNF has not been fully elucidated.

In recent years, BDNF expression has been reported in various organs outside the central nervous system, and there has been a high level of interest in identifying new functions of BDNF. In rat submandibular glands, acute immobilization stress increases BDNF expression (Tsukinoki et al., 2006). Furthermore, it has been suggested that the levels of BDNF in blood may fluctuate during acute immobilization stress. Interestingly, acute immobilization stress decreases BDNF content in the hippocampus (Ueyama et al., 1997). Stress is an important onset factor for mental disorders in humans, but few studies have measured the levels of plasma BDNF in conditions of acute immobilization stress.

Plasma is useful for investigating the role of BDNF as a hormone or humoral factor. Hence, in the present study, we determined whether plasma BDNF is altered by acute immobilization stress, and whether plasma BDNF is affected by the submandibular gland.

	MATERIALS & METHODS

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Animals
Nine-week-old male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) were used in this study. They were housed in groups of 4 animals per cage in a room maintained under standardized light (12:12 hr light-dark cycle) and temperature (22 ± 3°C) conditions. The animals had free access to food pellets and tap water.

Experimental Procedures
All experiments were performed with 4 rats per experimental group. On the first day of each experiment, the animals were immobilized to produce acute stress, in accordance with a well-established protocol (Hori et al., 2004, 2005). The rats were exposed to immobilization stress for 30 min, 60 min, or 180 min. Five-week-old rats in which the bilateral major salivary glands had been resected (sialoadenectomized rats) were also exposed to immobilization stress for 60 min, according to the same protocol. 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 accordance with the Guidelines for Animal Experimentation of Kanagawa Dental College.

Preparation of Plasma
Plasma samples were obtained from each rat’s heart and collected in Vacutainer^® tubes containing EDTA (TERUMO, Tokyo, Japan) or sodium citrate (Becton-Dickinson, Franklin Lakes, NJ, USA). The tubes were immediately placed on ice and then centrifuged at 2000 X g at 4°C. The supernatants were collected in tubes and stored at -80°C before use.

Measurement of ACTH and BDNF of Plasma
Adrenocorticotropic hormone (ACTH) was assayed by means of a radioimmunoassay (RIA) kit (ELISA-ACTH, CIS; Atomic Energy Laboratory of Biochemical Products, Gif sur Yvette, France), following the manufacturer’s instructions. Levels of ACTH were reported in pg/mL. We used the Promega BDNF Emax ImmunoAssay System to measure the amount of BDNF in each plasma sample (Promega, Co., Madison, WI, USA), in accordance with the manufacturer’s instructions. Absorbance was measured at 450 nm by an automated plate reader. Levels of BDNF were reported in pg/mL. The Mann-Whitney U-test was used for analysis, and P < 0.05 was considered statistically significant. In addition, statistical analysis regarding the association between ACTH and BDNF was performed with Pearson’s correlation coefficient.

RNA Extraction and cDNA Synthesis
Total RNA was isolated 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). Reverse transcription was performed by means of a single-strand cDNA synthesis kit (Roche Diagnostics Ltd., Lewes, UK), as outlined in the manufacturer’s instruction manual.

Real-time PCR for BDNF
We performed real-time polymerase chain-reaction (PCR) using a LightCycler system (Roche) as previously described (Tsukinoki et al., 2006). The primer sequences used were 5'-CAGGGG CATAGACAAAAG-3' (forward) and 5'-CTTCCCCTTTT AATGGTC-3' (reverse) (Nippon Gene Laboratory, Sendai, Japan). BDNF gene expression was expressed in terms of the ratio of BDNF copy number to that of ß-actin for each sample (expressed as mean ± SD). The Mann-Whitney U-test was used for analysis, and P < 0.05 was considered statistically significant.

	RESULTS

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ACTH Levels in Stress or Non-stress Conditions
When the rats were exposed to immobilization stress for 30, 60, or 180 min, ACTH levels were 2657.5 ± 668.64 pg/mL at 30 min, 2404.54 ± 679.74 pg/mL at 60 min, and 1306.66 ± 453.46 pg/mL at 180 min (Fig. 1). Non-stressed rats had ACTH levels of 775.833 ± 121.12 pg/mL. There were significant differences in ACTH levels between non-stressed rats and rats that experienced 30, 60, or 180 min of acute immobilization stress (p < 0.05 for all comparisons).

View larger version (18K): [in this window] [in a new window]   Figure 1. Stress levels following immobilization stress. For stressed rats, ACTH levels were 2657.5 ± 668.64 pg/mL at 30 min, 2404.54 ± 679.74 pg/mL at 60 min, and 1306.66 ± 453.46 pg/mL at 180 min (N = 4; error bars = SD). Non-stressed rats had ACTH levels of 775.833 ± 121.12 pg/mL. There were significant differences in the ACTH levels of non-stressed rats and rats that experienced 30, 60, or 180 min of acute immobilization stress (p < 0.05).

View larger version (15K): [in this window] [in a new window]   Figure 2. BDNF protein levels following immobilization stress. Plasma BDNF level was 6.15 ± 1.64 pg/mL for non-stressed rats (N = 4; error bars = SD). For stressed rats, plasma BDNF levels were 4.31 ± 0.33 pg/mL at 30 min, 65.5 ± 19.7 pg/mL at 60 min, and 15.64 ± 3.32 pg/mL at 180 min (N = 4; error bars = SD). Significant differences were found in plasma BDNF levels between non-stressed rats and those that experienced 60 min or 180 min of acute immobilization stress (p < 0.05). Significant differences were noted between rats that experienced 30 min and those that experienced 60 or 180 min of acute immobilization stress (p < 0.05), and also between rats that experienced 60 min and 180 min of stress. Acute immobilization stress for 60 min significantly increased the level of plasma BDNF.

Plasma BDNF Levels in Sialoadenectomized and Normal Rats
The plasma BDNF level was 8.01 ± 5.07 pg/mL for non-stressed sialoadenectomized rats. When the sialoadenectomized rats were exposed to acute immobilization stress for 60 min, plasma BDNF levels increased to 27.9 ± 14.8 pg/mL (Fig. 3). There were significant differences in plasma BDNF levels between stressed rats and sialoadenectomized rats stressed for 60 min (p < 0.05 for all comparisons). In non-sialoadenectomized rats, the BDNF level was 10.1-fold higher after acute immobilization stress, compared with levels in non-stressed rats. In sialoadenectomized rats, the BDNF level was 3.48-fold higher after acute immobilization stress, compared with levels in non-stressed rats.

View larger version (18K): [in this window] [in a new window]   Figure 3. BDNF protein levels in sialoadenectomized rats. (A) Plasma BDNF levels in non-stressed rats. Plasma BDNF levels were 6.15 ± 1.64 pg/mL in normal rats and 8.01 ± 5.07 pg/mL in sialoadenectomized rats (N = 4; error bars = SD). (B) Plasma BDNF levels in stressed rats. Plasma BDNF levels were 65.5 ± 19.7 pg/mL in normal rats and 27.9 ± 14.8 pg/mL in sialoadenectomized rats (N = 4; error bars = SD). There were significant differences in plasma BDNF levels between non-sialoadenectomized rats and sialoadenectomized rats (p < 0.05).

	DISCUSSION

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BDNF is abundant in adult human brain tissues, such as the hippocampus, and because its levels are low in the brain during the fetal period, it is considered to play an important role in neural differentiation and functional regulation, rather than in proliferation of neural cells (Lewin and Barde, 1996). When rats are chronically immobilized, BDNF levels have been reported to decrease in the hippocampus, after which memory disturbance is induced (Radecki et al., 2005). However, if exogenous recombinant BDNF is administered in the hippocampus, stress does not affect memory (Radecki et al., 2005). In addition, because BDNF is involved in the mechanism of infarct tolerance in the cerebrum, direct intra-cerebral infusion of exogenous recombinant BDNF protects neural cells in the infarct (Yanamoto et al., 2000). Hence, elevation of BDNF level by exogenous BDNF is thought to contribute to the function and the protection of neural cells.

In the present study, when rats were exposed to stress for 30, 60, or 180 min, plasma BDNF levels in the 60- and 180-minute groups were significantly higher than those in the control and 30-minute groups. Thus, we confirmed that acute immobilization stress markedly increased plasma BDNF levels. It has been reported that elevated plasma BDNF protects against neural damage from methamphetamine (Kim et al., 2005). However, because a decrease in plasma BDNF level is correlated with the severity of schizophrenia accompanied by tardive dyskinesia, tardive dyskinesia may be induced by the reduction of neural cell protection provided by BDNF (Tan et al., 2005). In addition, BDNF is able to pass through the blood-brain barrier (Pan et al., 1998). Free BDNF entering the plasma (endogenous BDNF) is likely to protect neural cells and maintain neural cell function. Therefore, in the early stages, an increase in plasma BDNF may contribute to the protection of neural cells against damage caused by acute stress conditions.

In the present study, levels of plasma BDNF were significantly higher in the 60-minute stress group than in all other groups. Sialoadenectomy suppressed the increase of BDNF in plasma when 60-minute stress was experienced. However, because suppression was not complete in sialoadenectomized rats, expression of BDNF was investigated in organs relating to peripheral BDNF, such as the heart, lung (Timmusk et al., 1993), liver (Cassiman et al., 2001), pancreas (Hanyu et al., 2003), and spleen (Schuhmann et al., 2005). Our results showed that expression of BDNF mRNA in these organs did not significantly increase with immobilization. In a previous report, BDNF mRNA and protein in the pituitary gland were found to increase in rats exposed to acute immobilization stress for 60 min (Givalois et al., 2004). Because the pituitary gland produces various hormones, pituitary BDNF may be released into the bloodstream. This suggests the possibility that not only the salivary gland but also the pituitary gland influences plasma BDNF in acute immobilization stress. However, we consider that the submandibular glands are the primary source of plasma BDNF in acute immobilization stress, because the increase in plasma BDNF concentration was greater in non-sialoadenectomized rats than in sialoadenectomized rats. In addition, in response to acute immobilization stress, salivary BDNF may enter the bloodstream from the submandibular gland.

In recent years, many studies have reported that BDNF is produced by various organs outside of the nervous system. BDNF is found in blood cells such as lymphocytes (Sobue et al., 1998), macrophages (Rost et al., 2005), and eosinophils (Raap et al., 2005). BDNF may be involved in the protection of neural cells in inflamed tissue. In allergic diseases, such as atopic dermatitis, BDNF released from eosinophils raises plasma BDNF levels (Raap et al., 2005). In asthma, enhanced local BDNF production in the lung contributes to neural hyperreactivity and pathological bronchoconstriction (Braun et al., 2004). In addition, alterations in plasma BDNF levels are also observed in acute coronary syndrome (Manni et al., 2005) and during the menstrual cycle (Lommatzsch et al., 2005). At the cellular level, vascular endothelial cells are considered to be an important source of BDNF (Nakahashi et al., 2000). Plasma BDNF may play different roles in various conditions and processes, such as inflammation, allergy, heart disease, and menstruation. Therefore, acute stress not only causes neural cell injury, but also damages the gastrointestinal tract (e.g., acute gastric ulcer). Thus, rapid increases in plasma BDNF may provide protection for the gastrointestinal organs.

Finally, using ELISA, we found no correlation between the time-courses of plasma ACTH and BDNF concentration. In the rat pituitary gland, no correlation has been reported between plasma ACTH level and BDNF mRNA (Givalois et al., 2001). Because ACTH is rapidly secreted into the bloodstream in response to acute stress, the highest level of ACTH was identified after 30-minute stress. In contrast, since a peak in BDNF mRNA was detected after 30-minute acute stress (Tsukinoki et al., 2006), it is likely that plasma BDNF would peak after 60-minute stress. In addition, the salivary glands are basically governed by the autonomic nervous system rather than by hormones. Therefore, ACTH is not likely to regulate production of salivary BDNF.

In conclusion, under conditions of acute immobilization stress, rat submandibular glands increased BDNF production, thereby contributing to the elevation of plasma BDNF levels. We believe that the salivary glands can influence the health of distant organs. In this stress model, it is necessary to determine the target organs of plasma BDNF.

	ACKNOWLEDGMENTS

 
This work was supported by a Grant-in-Aid for High-Tech Research Center Projects from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Received May 10, 2006; Last revision October 13, 2006; Accepted November 16, 2006

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