Pain, 49 (1992) 99-104 0 1992 Elsevier Science

99 Publishers

B.V. All rights



PAIN 02003

Sensitizing effects of leukotriene Sandra ’ Departments


a, Eric A. Whitsel

B, on intradental

‘, Heberto


primary afferents

b and William Maixner


of Endodontics and b Pharmacology, and ’ the Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7450 (USA) (Received

22 May 1991, revision


12 September

1991, accepted

19 September


Summary Previous studies have established that leukotriene B, (LTB,) sensitizes cutaneous nociceptors. In this study the effects of LTB, on spontaneous and stimulus-evoked nerve activity from primary afferents innervating the dentin of canines in adult cats were examined. LTB, treatment (25 pgg/ml) significantly enhanced stimulusevoked intradental nerve activity (INA) for at least 20 min after the removal of the compound from the recording preparation. Teeth treated with LTB, demonstrated enhanced spontaneous nerve activity following the removal of hypertonic saline used to evoked INA. These findings provide additional evidence that LTB, is able to sensitize nociceptors and may be a long-lasting hyperalgesic factor which may contribute to pain of pulpal origin. Key words: Nociception;



Tooth pulp; Intradental

Introduction Spontaneous pain and hyperalgesia are common features of the inflammatory process which occur in response to tissue damage. Although not fully understood, it is thought that hyperalgesia and spontaneous pain result from the activation of peripheral nociceptors by mediators of inflammation (Chapman et al. 1961; Bessou and Per1 1969; Juan and Lembeck 1974; Juan 1978; Dubner and Bennett 1983; Levine et al. 1984) as well as by changes in the response properties of central nociceptive neurons (Menetrey and Besson 1982; Woolf 1983; Hylden et al. 1989; Dubner 1991). Inflammatory products such as bradykinin, E-type prostaglandins and prostacyclin sensitize nociceptors in injured or inflamed tissue and likely contribute to the underlying mechanisms which produce spontaneous pain and hyperalgesia following tissue damage (Handwerker 1975; Ahlquist et al. 1976; Ferreira et al. 1978; Juan 1978; Moncada et al. 1979; Davies et al. 1984).

Correspondence to: Dr. Sandra Madison, DDS, Department of Endodontics, University of North Carolina, Chapel Hill, NC 275997450, USA. Tel.: (919) 966-2707.

nerve activity; Dentin

In addition to the prostaglandins, prostacyclins and bradykinin, it has been demonstrated that leukotriene B, (LTB,), a product of the cyclooxygenase independent 5-lipoxygenase pathway of arachidonic acid metabolism, is a potent mediator of inflammation and pain. The intradermal administration of LTB, produces thermal hyperalgesia in man (Bisgaard and Kristensen 1985), lowers the mechanical threshold of nociceptive reflexes (Levine et al. 1984; Levine et al. 1986), and lowers the threshold of activation of cutaneous C-nociceptors and A-delta nociceptors (Martin et al. 1987, 1988; Martin 1990). In contrast to these relatively well characterized effects on afferents innervating cutaneous structure, the sensitizing action of LTB, on sensory afferents originating from encapsulated structures such as tooth pulp has not been investigated. In the present study, the effects of LTB, on spontaneous and stimulus-evoked intradental nerve activity (INA) produced by hypertonic sodium chloride (NaCI) was evaluated. Several studies have demonstrated that stimulation of exposed dentin with hypertonic NaCl solutions produces sharp pain which is correlated with the activation of A-delta primary afferents innervating dentin (Scott and Stewart 1965; Anderson et al. 1970; Horiuchi and Matthews 1974; Ahlquist et al. 1976, 1984, 1985; Lisney 1978; Hirvonen et al. 1984; Fors et


al. 1986). Our findings show that LTB, treatment enhances stimulus-evoked INA and has a tendency to enhance spontaneous nerve activity. Portions of this work have been previously reported (Madison et al. 1989).


Surgical procedure Nine young adult male cats weighing between 2.5 and 4.0 kg were anesthetized with pentobarbital (35 mg/kg,i.p.). After cannulation of the trachea, femoral artery and vein, the animals were artificially respirated and alpha-chloralose (60 mg/kg,i.v.) was administered. Deep anesthesia was maintained with a constant infusion of alphachloralose (3-6 mg/kg/h). Rectal temperature, expired CO, and arterial pressure were continuously monitored and maintained within normal physiological limits.

Recording procedures A procedure which allows for the recording of multiple-unit discharge activity from intradental A-delta fibers was used (Scott and Stewart 1965; Panopoulos et al. 1983; Narhi 1985). Briefly, two dentinal cavities were prepared in healthy maxillary and mandibular canines that were free of any visible signs of trauma. Both preparations were made on the facial surfaces of the teeth: one towards the incisal tip and the other in the cervical area. During preparation. enamel was removed with a high-speed inverted cone bur with irrigation. After reaching the dentino-enamel junction, the dentinal preparations were made with an end-cutting carbide bur in a handmanipulated holder under a continuous saline drip. The gingival preparations were terminated just pulpal to the dentino-enamel junction. A reference electrode was placed in the gingival preparations and secured with a light cured resin. Deep incisal cavities were prepared to a depth where the pulpal vasculature was visible. A deep preparation was placed to diminish the effect of debris (i.e., the smear layer) on INA evoked with hypertonic NaCl (Horiuchi and Matthews 1974; Hirvonen et al. 1984; Narhi 1985). The incisal preparations were washed and filled with isotonic 0.15 M NaCI. A low-impedance platinum recording electrode was positioned in the incisal cavity preparation adjacent to the underlying pulp horn. Multi-fiber activity from the intradental nerves was amplified (~OOOOX), displayed on an oscilloscope, and passed through a threshold window discriminator. Individual spikes were discriminated, counted and stored on an IBM-AT computer for on-line display and off-line analysis.

LTB, Treatment: N = 16 0.30 1.0 3.0 0.15 Pre TX-Response


Post TX-Response


Vehicle Treatment: N=8

Pre TX-Response Curve

Post TX-Response Curve H

I ml”

Fig. 1. Protocol

time line showing sequence of solutions recording preparation.


to the

Experimental protocol Fig. I displays a time line of the protocol used in this study. Five-minute recordings were made with each solution in the recording preparation. After each 5-min recording epoch, the recording electrode was removed and the test solution rinsed from the preparation. The next test solution was immediately placed in the preparation and the electrode was repositioned and recordings resumed. Spontaneous nerve activity associated with 0.15 M NaCl (isotonic saline) was recorded. Stimulation-evoked responses produced with graded concentrations of NaCl ranging from 0.3 M to 3.0 M were obtained to generate a pre-treatment dose-response curve. Treatment solutions of LTB, (25 pg/ml) or LTB, vehicle (20% ethanol in distilled water) were added to the recording preparation for 20 min. Following treatment with LTB, or vehicle, INA recordings in response to graded concentrations of NaCl were again obtained to create a post-treatment dose-response curve.

Statistical analysis of the data The percentage change in the response relative to the maximum evoked response (E,_) obtained during the initial pre-treatment dose-response curve was determined for each treatment group (LTB, and vehicle). To accomplish this, the maximum response produced during the generation of the pre-treatment dose-response curve was determined and assigned a value of 100%. All other responses produced by the other concentrations of NaCl were normalized relative to this response and converted to a percentage value. To evaluate the effect of a given treatment on stimulus-evoked nerve activity, the number of spikes recorded in response to 0.3, 1.0 or 3.0 M NaCl following LTB, or vehicle treatment was divided by the number of spikes recorded prior to test solution application. The effects of direct application of LTB, or vehicle on spontaneous INA was evaluated by comparing the number of spikes recorded in response to 0.15 M NaCl administered at the beginning of the first dose-response curve to the number of spikes recorded during the last 5-min application of LTB, or vehicle. The effect of LTB, or vehicle treatment on spontaneous INA was determined by comparing the number of spikes recorded in response to 0.15 M NaCl administered at the beginning of the first dose-response curve to the number of spikes recorded in response to 0.15 M NaCl after the end of the second dose-response curve. Mean data were subjected to non-parametric statistical analysis using Wilcoxon’s rank sum test for small sample sizes. All data are presented as mean f 1 S.E.M. Significance was assumed at alpha = 0.05.


Intradental nerve activity was recorded from 24 of the possible 36 preparations. Activity was not recorded from 12 teeth that showed signs of traumatization, that had pulp exposures during cavity preparation or were non-responsive to the application of NaCl. Sixteen of the 24 preparations were treated with LTB, and 8 with vehicle. Leukotriene B, enhanced stimulus-evoked activity in 11 of 16, 14 of 16, and 10 of 16 preparations in response to 0.3, 1.0 and 3.0 M NaCl, respectively. In contrast, vehicle treatment enhanced stimulus-evoked activity in 2 of 8, 3 of 8, and 6 of 8 preparations in response to 0.3, 1.0 and 3.0 M NaCl, respectively. One of our best examples of the sensitizing action of LTB, on evoked INA is shown in Fig. 2. The top panel displays evoked nerve activity produced by 1.0 M NaCl applied to the recording preparation before treatment



Pre-LTB, 1.w N.CI










Post -LTB, I.oMN&I

0.3 M NaCl

1.0 M NaCl

3.0 M NaCl

Fig. 4. The relative effects of vehicle (dotted bars) and LTB, (open bars) treatment on nerve activity evoked by graded concentrations of NaCI. Each value represents the mean of 1 S.E.M.




300 Time(sec)

bin = 20 WC

Fig. 2. Peristimulus time histograms of evoked nerve activity produced by adding 1.0 M NaCl to the recording preparation prior to LTB, (top panel) and after a 20 min exposure to LTB, (lower panel). Activity was recorded from a single isolated unit and summed in 2-set bins.

with LTB,. The lower panel displays evoked nerve activity produced by 1.0 M NaCl after treatment of the preparation with LTB,. Comparison of the two panels demonstrates that LTB, significantly enhances stimulus-evoked INA. The responses from the 14 teeth that exhibited enhanced responses to 1.0 M NaCl following LTB, treatment were used for subsequent population data analysis.

As shown in Fig. 3, hypertonic NaCl (0.3-3.0 M) produced equivalent dose-dependent increases in INA prior to LTB, or vehicle treatment. The stimulus-response curves for both groups closely paralleled and overlapped each other and were not statistically different. These data suggest that LTB, and vehicle-treated teeth responded similarly to graded concentrations of NaCl. Teeth treated with LTB, exhibited greater INA in response to 0.3 and 1.0 M NaCl solutions when compared to teeth treated with vehicle (Fig. 4). There was a strong tendency for the response to 3.0 M NaCl to be enhanced relative to vehicle-treated teeth but it failed to achieve statistical significance due to the large


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Sensitizing effects of leukotriene B4 on intradental primary afferents.

Previous studies have established that leukotriene B4 (LTB4) sensitizes cutaneous nociceptors. In this study the effects of LTB4 on spontaneous and st...
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