Quantitative real time PCR analysis.

<p>(A) Standard curve to determine the copies of EBV DNA in specimens. EBV DNA (approximately 1x10⁶ copies/μl) was diluted in ten-fold serially. Each diluted DNA was amplified using real-time PCR simultaneously at the time of amplification for DNA extracted from periapical granulomas and healthy gingivae. (B) Detection of EBV DNA in periapical granulomas and healthy gingivae. The copy of EBV DNA in each specimen was calculated using the standard curve. The median of EBV DNA copies in periapical granulomas was approximately 8688.01 per 1μg of total DNA, as shown by a horizontal bar. * showed statistical difference using Mann-Whitney <i>U</i> test (<i>p</i> = 0.0001).</p

Histological evaluations of the specimens.

<p>Paraffin sections (n = 40) were stained using hematoxylin and eosin. Scale bar = 100μm. (A) Periapical granulomas (n = 32) showing a large number of inflammatory cells and microvessels. (B) Radicular cyst (n = 8) showing epithelial cell layer and cholesterol clefts. (C) Healthy gingival tissues (n = 10) showing epithelial cell layer and lower cell number of infiltrating cells in comparison with periapical granulomas.</p

Epstein-Barr Virus Infection in Chronically Inflamed Periapical Granulomas

<div><p>Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/μg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney <i>U</i> test (<i>p</i> = 0.0001). Paraffin sections were also analyzed by <i>in situ</i> hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.</p></div

Detection of EBV DNA using quantitative real-time PCR.

<p>Periapical granulomas and healthy gingival tissues were analyzed to detect EBV DNA by quantitative real-time PCR. EBV DNA were highly detected from periapical granulomas in comparison with healthy gingival tissues.</p><p>* Mann-Whitney <i>U</i> test.</p><p>Detection of EBV DNA using quantitative real-time PCR.</p

Detection of EBER using <i>in situ</i> hybridization.

<p>(A-F) <i>In situ</i> detection of EBER-expressing cells in periapical granulomas (6 out of 9) was shown in the cytoplasm and nuclei of B cells (arrows) and plasma cells (arrow heads). (G-I) Three periapical granulomas showed EBER-negative expression. (J) Healthy gingival tissues never showed positive expression. Scale bar = 50μm.</p

Specimens used in this study.

<p>(A) X-ray observation of periapical lesions caused at lower incisal teeth. Radiolucency around the apex showed alveolar bone resorption. (B) Periapical lesion surgically removed from a patient showing at (A).</p

LMP-1 immunohistochemistry using serial sections of periapical granulomas.

<p>Framed boxes in left panels (A, C, E) were magnified to right panels (B, D, F, respectively). (A, B) LMP-1 immunohistochemistry showed positive staining in B cells (arrows) and plasma cells (arrow heads). (C, D) EBER-expressing cells were present at the same area of LMP-1-expressing cells. (E, F) A negative control using normal mouse IgG antibody did not exhibit LMP-1 expression. Scale bar = 50μm.</p

Photomicrographs and percentage of double retrograde tracing from M1 and M2 afferents to TG neurons.

<p>A, B and C: Low magnification photomicrographs of FG(+) TG cells at 3 days after FG application to M1 (A) and DiI application to M2 (B), and the photomicrograph of A merged with B (C). D, E and F: High magnification photomicrographs of FG(+) TG cells 3 days after FG application to M1 (D) and DiI application to M2 (E), and the photomicrograph of E merged with F (F). Ga: Percentage of FG and DiI double-labeled TG cells indicated by the yellow pie. White arrow heads indicate FG and DiI double-labeled TG cells, the white arrows are FG(+) TG cells and open arrow heads are DiI(+) TG cells.</p

Effect of FC injection into TG on masseter muscle activity and GFAP and pERK-IR cell expression.

<p>A and B: Typical example of masseter muscle EMG activities following capsaicin application to M2 in M1 CFA-applied rats with Veh (A) or FC (B) injection to TG. C: The mean area under the curve of integrated EMG following capsaicin application to M2 in M1 CFA-applied rats with Veh or FC-injection to TG (C). D and E: Photomicrographs of GFAP-IR cells in TG Veh-injected rats (D) and TG FC-injected rats (E). F: The mean relative number (%) of TG cells encircled with GFAP-IR cells following Veh- (solid bur) or FC- (open bur) administration in M1 CFA rats. G and H: Photomicrographs of pERK-IR cells following M2 capsaicin application in TG Veh- (G) and TG FC-injected rats (H). I: The mean relative number (%) of pERK-IR cells in TG following M2 capsaicin application in TG Veh- and TG FC-injected rats which received CFA in M1. Solid arrow in A indicates the timing of capsaicin application. White arrow heads in D and E indicate GFAP-IR cells and those in G and H are pERK-IR cells, respectively. Veh: vehicle for FC, FC: Fluorocitrate. #: p<0.05, +++: p<0.001, *: p<0.05, **: p<0.01.</p

FG-labeled TG cells and pERK-IR TG cells following capsaicin application to M2 in M1 CFA- or saline-applied rats.

<p>Fluorescent photomicrographs of FG(+) cells (A and D), pERK-IR cells (B and E) and FG merged with pERK-IR (C and F) in TG (M1 saline-applied rats: A, B and C; M1CFA-inected rats: D, E and F ). G and H: The mean percentages of FG (+) pERK-IR cells/FG (+) cells (G) and mean percentage of FG(–) pERK-IR cells/FG (–) cells (H). I: The photomicrograph of FG-applied M1 sections. FG can be seen as blue-stained product in M1. The solid arrow heads indicate FG(+) pERK-IR cells in A-F. The open arrow heads indicate FG(+) cells in D and F. The arrows indicate pERK-IR cells in B, C, E and F. The large white arrow in I indicates the apex of M1. The white star in I indicates coronal pulp cavity. * : p<0.05.</p