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Responses of Rat Pulp Cells to Heat Stress in vitro

¹ Department of Pathology,
² Department of Dental Anesthesiology, and
³ Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihama-ku, Chiba, 261-8502, Japan

View larger version (29K): [in a new window]   Figure 1. HSP expression over time course by Western blot analysis. HSP70 was expressed in pulp cells at all time points. Heat shock induced production of HSP70 at 1 hr, although HSP70 expression showed no dramatic change between pre-heating and time point 0. HSP70 expression gradually increased between 1 hr and 6 hrs. In contrast, expression of HSP25 was clearly observed before heat treatment, changing slightly, but not dramatically, over time compared with HSP70. Fold change relative to the control (Fold ) was determined from densitometric data after normalization to actin for HSP25 and 70. Pre-heat value was arbitrarily set at 1.0.

View larger version (78K): [in a new window]   Figure 2. Expression of connexin43 over time course. (A) Western blot analysis showed rapid degradation of CX43 at time point 0. CX43 expression returned to normal levels within 3–6 hrs following heat stress. (B) The 43- and 45-kDa bands, which correspond to phosphorylated forms of CX43, decreased dramatically after heat treatment. However, the 41-kDa band, which corresponds to the non-phosphorylated form, changed only slightly. (C) CX43 was observed between cell bodies or cell processes in pulp cells prior to being heated (Pre). Immunohistochemical findings revealed that heat stress led to dramatic degradation of CX43 at time point 0, which agreed with Western blot findings. Heat-induced CX43 degradation was reversible, since CX43 expression increased after cultures were returned to 37°C for 3 hrs. Bars = 20 µm.

View larger version (43K): [in a new window]   Figure 3. Gap-junctional communication. (A) Micrograph of dye transfer. Prior to being heated (Pre), definite transfer of Lucifer yellow dye was detectable. Stimulating cells at 42°C for 30 min significantly decreased the extent of dye transfer, suggesting disruption of gap-junctional communication. Degradation of gap-junctional communication was reversible, since it increased after cultures were returned to 37°C for 3 hrs. Dashed lines = cutting edge; arrows = transferred cell. Bars = 50 µm. (B) Communication resumed after 1 hr (1.6 ± 0.2), and the number of coupled cells increased at 3 hrs (1.9 ± 0.2). Significant differences were confirmed between pre-heating and time point 0 groups, and between pre-heating and one-hour groups (p < 0.01, n = 20).

View larger version (14K): [in a new window]   Figure 4. ALP activity. At time point 0, values decreased dramatically (303 ± 24), and ALP activity increased after 1 hr (374 ± 24). Values increased further at 6 hrs (485 ± 13). Statistically significant differences were observed between pre-heating and zero-, three-, and six-hour groups (p < 0.01, n = 15).