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Immobilization Stress Induces BDNF in Rat Submandibular Glands

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

View larger version (19K): [in a new window]   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.

View larger version (34K): [in a new window]   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.

View larger version (89K): [in a new window]   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).

View larger version (45K): [in a new window]   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/ß-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.