Quantification of neural protein in extirpated tooth pulp

Abstract

Because the pulp tissue extirpated during root canal procedures might serve as a valuable resource with which to assess underlying mechanisms of persistent pain, we sought to determine whether standard Western blotting techniques could be used to quantify neural proteins in pulp extirpated from teeth with irreversible pulpitis. Pulp harvested from healthy intact teeth extracted for orthodontic reasons was used for comparison. The neural marker PGP9.5 was detectable in all samples tested. A membrane enrichment protocol enabled detection of even low abundance, high molecular weight proteins such as the sodium channel alpha-subunit NaV1.8. Importantly, it was possible to quantify a approximately 6-fold increase in the relative density of NaV1.8 in inflamed pulp compared with control pulp. Our results suggest that it should be possible to use extirpated tooth pulp to validate mechanisms of persistent pain implicated in preclinical studies as well as evaluate the therapeutic efficacy of novel antinociceptive interventions.

Figures

Figure 1

The neural marker PGP9.5 was detectable in pulp tissue obtained from control subjects (Control) and from subjects undergoing root canal therapy (Inflamed) (A). Each lane was loaded with tissue obtained from a different subject. The blot was first probed with an antibody against PGP9.5, stripped and then re-probed with an antibody against β-actin. The expected molecular weight for PGP9.5 is indicated by the arrow. The intensity of the PGP9.5 band was normalized to that of β-actin and data for control and inflamed pulps were pooled and plotted as a mean ± SEM (B). There was no statistically significant difference in the relative amount of PGP9.5 pulps from these two groups.

Figure 2

The membrane bound α-subunit of the Na+/K+ ATPase (ATPase) was used to monitor the effectiveness of a protocol for the enrichment of membrane bound proteins. A. Pulp tissue from 3 control subjects was divided and subjected to either the membrane enrichment protocol (Enriched) or processed for total protein (Total). Thus, protein loaded in lanes 1 and 4, 2 and 5, and 3 and 6 were from the same subjects. The blot was first probed with an antibody against ATPase, stripped and then re-probed with an antibody against β-actin. B. Pooled data suggests the enrichment protocol resulted in a ~3-fold increase in the relative amount of ATPase detected.

Figure 3

Pulpal inflammation is associated with a dramatic increase in NaV1.8. A. pulp tissue obtained from control subjects (Control) and from subjects undergoing root canal therapy (Inflamed) was subject to the membrane enrichment protocol. Blots were first probed with an antibody against NaV1.8 (expected molecular weight is indicated by the arrow), stripped and re-proved for the α-subunit of the Na+/K+-ATPase (ATPase). B. Pooled data from control and inflamed pulp indicates that there is a ~6-fold increase in the relative abundance of NaV1.8 in inflamed pulp (p = 0.03).