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Mechanotransducers in Rat Pulpal Afferents
T.O. Hermanstyne1,
K. Markowitz2,
L. Fan3 and
M.S. Gold3,*
1 Program in Neuroscience, University of Maryland, Baltimore, MD 21201, USA;
2 Department of Oral Biology, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, 185 South Orange Ave, Newark, NJ 07103, USA; and
3 Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, 3500 Terrace Street, Rm E1440 BST, Pittsburgh, PA 15213, USA
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Figure 1. A modified Evans Blue Assay may be used to assess pulpal inflammation. Evans Blue was injected systemically as described in MATERIALS & METHODS. Ten min later, tissue was harvested, and Evans Blue was extracted and quantified with spectrophotometry. Naïve animals had intact teeth. Uninflamed animals received cavities in the 2nd and 3rd molars for labeling of pulpal afferents with DiI, and then the cavities were filled with Transbond, 14–17 days prior to tissue-harvesting. Inflamed animals were treated in a manner similar to that used for uninflamed animals, except that 3 days prior to tissue harvest, cavities were re-exposed. The Evans Blue content was significantly (p < 0.01) higher in inflamed bridges than in either uninflamed or naïve sections. Numbers in parentheses are the number of animals studied in each group. Data are plotted as a mean ± standard error of the mean (SEM).
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Figure 2. The majority of the known mechanotransducers are expressed in trigeminal ganglia. (top panel) An ethidium-bromide-stained agarose gel loaded with products of a nested PCR amplification of cDNA generated from mRNA extracted from rat trigeminal ganglia. To assess the sensitivity of the primer pairs, we diluted the cDNA 1:10,000. We used a nested PCR strategy to increase the fidelity of the PCR reaction as well as the specificity of the product amplified. The dominant bands in lanes with multiple bands correspond to the product of the expected molecular weight. In two cases, i.e., TREK1 and ENaC , products from both the first (i.e., larger PCR product) and second (i.e., smaller PCR product) amplifications can be seen. (bottom left) Results from a PCR analysis of the contents of a single pulpal afferent collected as described in MATERIALS & METHODS. Cyclophilin was used to assess recovery of cell contents, since this is an abundant mRNA species in most cells. The only mechanotransducer detected was ASIC3. (bottom right) Results from a PCR analysis of the contents of 4 pulpal neurons collected as described in MATERIALS & METHODS. cDNA generated from each afferent was probed for the presence of TRPA1 and ASIC3. Both genes were detected in the 1st and 4th afferents, while only one or the other was detected in the 2nd and 3rd neurons. In all three panels, lanes labeled BP were loaded with a DNA ladder used to estimate the size of PCR products.
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Figure 3. Frequency distribution of known mechanotransducers among pulpal neurons. Each bar represents the mean (± SEM) proportion of pulpal neurons observed in each rat. N = 5 for all except ASIC3, where n = 8, TRPA1, where n = 3, and TREK2, where n = 4. ASIC3 and TRPA1 were present in pulpal neurons at a higher frequency (p < 0.01) than any other transducer assessed.
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Journal of Dental Research, Vol. 87, No. 9,
834-838 (2008)
DOI: 10.1177/154405910808700910
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