In: Health9 Sep 2009
Among the 64 rats used in this experiment, only data obtained from 48 rats were deemed suitable for analysis. Twelve rats either responded to the injection of saline and the resultant increased EMG activity did not return to baseline levels prior to the injection of capsaicin/ve-hicle, or the application of the preload agent failed to induce a straight lateral movement of the edema measurement needle. In 2 rats, the application of 1% capsaicin to the bupivacaine pretreated TMJ region led to reflex jaw muscle activity, which indicated an unsuccessful nerve conduction blockade. Moreover, there was an absence of blue PE staining in the TMJ region of 2 other rats. Therefore, for the measurement of tissue expansion, each experimental group consisted of 8 rats each, and for the EMG recording of jaw muscle activity, groups 1 and 2 consisted of 6 rats, group 3 consisted of 10 rats, and groups 4-6 consisted of 8 rats each.
The dose-response curve demonstrating the relationship between tissue expansion and increasing capsaicin concentrations for the saline pretreated experimental groups (1, 2, and 3) is presented in Figure 1. Mean baseline expansion distances caused by the trauma induced by the insertion of the catheter were similar among the 3 saline-pretreated subgroups (ANOVA, P > .05). The capsaicin-induced edema development occurred in a dose-dependent manner; however, statistical analysis revealed that only 1% capsaicin-evoked tissue expansion was significantly different from the vehicle group at time28 and onward (Tukey test, P < .05). In addition, the comparison of tissue expansion induced by 1% with that elicited by 0.1% capsaicin indicated statistical significance at time48 and onward (Tukey test, P <.05). Therefore, in addition to the demonstration of a dose-dependent capsaicin-induced tissue expansion in the rat TMJ region, a difference in time point at which edema development was statistically significant between various concentrations of capsaicin was revealed.
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Figure 1. Dose-response curve: tissue expansion versus increasing capsaicin concentration. Mean changes in tissue expansion evoked by the injection of various concentrations of capsaicin or vehicle control into the left temporomandibular joint (TMJ) region. Each data point represents mean tissue expansion ± SE for a certain time point over the 180-minute time course in 8 rats. Asterisk (*) indicates tissue expansion of that 1% capsaicin data point time28 and onward was significantly higher in comparison with that evoked by the vehicle control (ANOVA, P < .05). Small dagger (t) indicates that tissue expansion of that 1% capsaicin data point time48 and onward was significantly higher in comparison with that evoked by the 0.1% solution (ANOVA, P < .05).
To evaluate the effect of 0.5% bupivacaine pretreat-ment of the TMJ region on capsaicin-evoked edema, the saline preload groups were compared with their respective bupivacaine preload groups. Regardless of the type of preload utilized, there were no statistically significant differences in the resultant tissue expansion (2-way ANOVA, P > .05), which was indicated by the close superimposition of the 2 plots for the respective pairs of experimental groups. Therefore, 0.5% bupivacaine was clearly ineffective in reducing the capsaicin-induced tissue expansion in the TMJ region. A summary plot of the cumulative edema development of all 6 experimental groups is provided in Figure 2.
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Figure 2. Capsaicin-induced cumulative tissue expansion. Mean cumulative tissue expansion evoked by injection of various concentrations of capsaicin or vehicle control into the left temporomandibular joint (TMJ) region pretreated with a saline preload or bupivacaine preload. Each data point represents the mean ± SE of normalized values relative to baseline tissue expansion in 8 rats. The horizontal dotted line indicates mean baseline cumulative tissue expansion distance. Asterisk (*) indicates that cumulative tissue expansion induced by 1% capsaicin was significantly higher in comparison with that evoked by the vehicle control (Tukey test, P < .05). Small dagger (t) indicates that tissue expansion of that data point was significantly higher in comparison with that induced by the 0.1% capsaicin solution (Tukey test, P < .05). No statistical difference was demonstrated between cumulative tissue expansion induced by various concentrations of capsaicin or vehicle control in saline pretreated and bupivacaine pretreated experimental groups.
Furthermore, the injection of capsaicin or vehicle solution into the saline pretreated TMJ region at time5 elicited a sustained and reversible reflex ipsilateral masseter and digastric EMG response. However, the injection of capsaicin or vehicle solution into bupivacaine pretreated TMJ regions failed to evoke an increase in reflex ipsilateral masseter and digastric EMG activity. Therefore, successful nerve conduction blockade by the pretreatment of TMJ tissues with bupivacaine was confirmed by the absence of reflex EMG activity in response to capsaicin.
Since the EMG data were similar for both the masseter and digastric muscles, only the summarized graph displaying the EMG activity for the digastric muscle is given in Figure 3. A 2-way ANOVA demonstrated significance between the increased EMG activity in both masseter and digastric muscles evoked by capsaicin in saline pretreated and bupivacaine pretreated rats (Fx 37 = 6.604, P < .05; F137 = 20.568, P < .001, respectively). Furthermore, an interaction between the concentration of capsaicin and the type of preload agent administered on the resultant reflex EMG activity in masseter and digastric muscles (F237 = 3.69, P < .05; ^2,37 = 9.401, P < .001, respectively) was revealed. Moreover, the reflex EMG activity of both muscles increased in a dose-dependent manner; however, only 1% capsaicin in the saline pretreated group evoked a significant increase in masseter and digastric EMG activity when compared with the vehicle control (Tukey test, P < .05).
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Figure 3. Capsaicin-induced cumulative digastric electromyographic (EMG) activity. Cumulative mean changes in EMG area in left digastric muscle evoked by injection of various concentrations of capsaicin or vehicle control into the ipsilateral temporomandibular joint (TMJ) region. Each data point represents mean ± SE of the normalized values relative to baseline EMG activity in each rat, and the horizontal dotted line indicates mean baseline activity. Asterisk (*) indicates cumulative EMG activity induced by 1% capsaicin was significantly higher in comparison with 0.1% capsaicin solution and vehicle control (Tukey test, P < .05). Small dagger (t) indicates statistical significance among EMG activity in digastric muscle evoked by capsaicin in saline pretreated and bupivacaine pretreated rats (2-way analysis of variance [ANOVA], P < .001). Also, the interaction between the concentration of capsaicin and type of preload agent administered on the resultant reflex EMG activity in the digastric muscle was statistically significant (2-way ANOVA, P < .001).
Qualitative analysis of the postmortem dissection by the naked eye revealed an Evan’s blue dye response in the ipsilateral temporomandibular articular and periarticular tissues in all experimental groups (data not shown). However, the 1% capsaicin demonstrated more intense staining compared with that produced by the 0.1% solution or vehicle control. In addition, examination of the contralateral rat TMJ revealed an absence of Evan’s blue dye staining of the temporomandibular articular or periarticular tissues.
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