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Intranasal trigeminal thresholds in healthy subjects
Environmental Toxicology and Pharmacology 19 (2005) 575–580
Intranasal trigeminal thresholds in healthy subjects Johannes Frasnelli, Thomas Hummel∗ Smell and Taste Clinic, Department of Otorhinolaryngology, University of Dresden Medical School, Fetscherstrasse 74, 01307 Dresden, Germany Available online 26 January 2005
Abstract The trigeminal chemosensory system responds to irritation of the nasal cavity. Despite its dominant role as a sentinel in protecting the respiratory tract from harmful substances and its involvement in the perception of odorous substances, it has received relatively little attention compared to the olfactory system. Aim of the present study was the comparison of two psychophysical techniques to assess intranasal trigeminal thresholds, namely (A) responses of subjects who focused on intranasal trigeminally mediated sensations, and (B) the ability of subjects to identify the side of the nose receiving unilaterally presented stimuli. Method A (0.81 > r > 0.56) was found to show a higher testretest reliability than Method B (0.48 > r > 0.40). Method A revealed thresholds that were approximately 32 times lower than those measured with method B. With method A women were found to have lower thresholds than men; no such difference could be detected between older and younger subjects. In conclusion, if the objective is to assess the level at which trigeminal sensations are detected with the utmost objectivity and unconfounded by smell, the obvious choice is B. If one’s purpose is to assess the level at which trigeminal sensations are detected and the quality perceived, in the context of an odor one might opt for Method A. Thus, preference of one method over the other may depend on the question being asked, provided a well-instructed/trained panel of subjects is used. © 2004 Elsevier B.V. All rights reserved. Keywords: Trigeminal nerve; Chemoreception; Threshold; Ratings; Lateralization
1. Introduction The trigeminal chemosensory system responds to irritation of the nasal cavity. Despite its dominant role as a sentinel in protecting the respiratory tract from harmful substances and its involvement in the perception of chemicals, it has received relatively little attention compared to the olfactory system. Today, we know that most odorants activate both the trigeminal and the olfactory systems (Doty et al., 1978; Elsberg et al., 1935; von Skramlik, 1924). For example, nicotine produces not only an odorous sensation, but also burning and stinging (Hummel and Kobal, 1992). Other trigeminally mediated sensations include cooling, prickling, freshness, and piquancy (see Kelly and Dodd, 1991; Laska et al., 1997). Different fiber types are involved in trigeminally mediated sensations: Activation of C-fibers produces dull and burning painful sensations, while sharp and stinging sensations are known to appear after excitation of A␦-fibers ∗
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(Hummel et al., 1994; Mackenzie et al., 1975; Torebj¨ork and Hallin, 1971). These sensations follow different time courses (Hummel et al., 1992) and may have a different impact on olfactory sensations. Apart from electrophysiological measures (e.g., eventrelated potentials (ERP) (Huttunen et al., 1986; Kobal and Hummel, 1988); negative mucosa potential (NMP) (Hummel et al., 1998b; Kobal, 1985; Thurauf et al., 2002)), a number of other techniques have been proposed to assess the trigeminal impact of odorants in humans. They include the assessment of the percentage of stimulus detection in anosmics and ratings from healthy subjects focusing on trigeminally mediated sensations (Doty et al., 1978), magnitude estimation procedures (Cain and Murphy, 1980), questionnaires (Hudnell et al., 1992), measures of respiratory reflexes (Warren et al., 1994) or reflexive changes in nasal blood flow (Th¨urauf et al., 1993), localization of the site of lateralized odor presentation (Berg et al., 1998; Kobal et al., 1989), assessment of ocular irritation (Cometto-Muniz et al., 1997), or pain thresholds after intranasal trigeminal activation with CO2 (L¨otsch et al., 1997). Unfortunately, almost no data are available concern-
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ing the reliability and validity of these methods. In addition, some of these measures seem to be only suited for the assessment of trigeminally mediated sensations at the suprathreshold level. The relation between results obtained by means of these different methods remains obscure. As threshold levels of trigeminal activation by odorants appear to be of high practical interest (Cain and Cometto-Muniz, 1995), studies are needed which systematically compare the relative value of methods to detect threshold levels of trigeminal stimulation. Aim of the present study was the comparison of two different psychophysical techniques to assess intranasal trigeminal thresholds: (a) responses of subjects who focused on intranasal trigeminally mediated sensations, and (b) the ability of subjects to identify the side of the nose receiving an unilaterally presented stimulus (compare Kobal et al., 1989). These techniques were chosen because of their high practicability, and their low apparative efforts. Comparisons between methods were based on test-retest reliability and absolute thresholds. These characteristics should also be used to judge the quality of the different measures. Further, it was investigated whether the two methods would reveal inter-individual differences in trigeminal sensitivity, which seems to be (a) greater in women than in men (Hummel et al., 1998a), and (b) which appears to decrease with age (Hummel et al., 1998a; Stevens et al., 1982).
2. Material and methods A total of 32 subjects participated in the study. The subjects’ health was ascertained through a thorough history and a detailed physical ENT-examination. Further, olfactory function was screened by means of the “Sniffin’ Sticks” (Hummel et al., 1997; Kobal et al., 1996). Half of the subjects (eight women and eight men) were between 18 and 35 years of age (mean age 25.5 years, standard deviation (S.D.): 0.7), the other half of the subjects (8w, 8m) were older than 45 years (mean age: 58.5 years, S.D.: 1.6). Thus, four groups were established (young women, young men, older women, and older men). The study was conducted according to the Declaration of Helsinki on biomedical research involving human subjects. During enrolment, subjects were given detailed information about all testing procedures. Written informed consent was obtained from all subjects prior to the study. This study was designed to systematically compare two methods to assess trigeminal thresholds. In Experiment A threshold concentrations were identified at which a “trigeminal” sensation appeared. In Experiment B threshold concentrations were determined at which the stimulated nostril can be determined after lateralized stimulus presentation. The order of methods was randomized across subjects. Both experiments were performed on different days. Subjects were retested after 6–25 days. During retesting the order of chemosensory testing methods was the same in individual
subjects. Two substances were investigated, namely linalool and menthol. Both substances are known to produce clear trigeminal sensations (Doty et al., 1978). For each substance 16 dilutions (numbered 1–16, #1 being the strongest and #16 being the weakest concentration) were prepared in geometric series (1:2) with propylene glycol as the solvent. The highest concentration (#1) was 97% for linalool and 50% g/g for menthol, #2 was 48.5% g/g and 25% g/g, respectively. The highest concentration produced a clear “supra-threshold” sensation in all subjects. The intensity of the highest concentrations was rated to be similar by a panel of experienced observers. Trigeminal thresholds were assessed with two different techniques. All measurements were performed in a quiet, well-ventilated room. In Experiment A odors were presented to both nostrils. The odors were tested sequentially and the order of testing was counterbalanced across subjects. Odors were presented in sniff bottles made from glass (brown color, volume: 250 ml, diameter of the bottle: 65 mm, opening diameter: 45 mm). Each bottle contained 40 ml of liquid. The mucosa of the eyes was protected from potential trigeminal stimulation by means of goggles. Threshold for each odor was determined using an initially ascending single staircase method (Ehrenstein and Ehrenstein, 1999). After presentation of each stimulus, subjects were asked whether the odor produced a sensation that was mediated via the trigeminal nerve or not (“have you felt any sensation like burning, stinging, cooling or tickling?”). Subjects were allowed to sniff more than once. The staircase started at the lowest concentration of the substance. If subjects indicated no “trigeminal” sensation, the next higher concentration was presented. This continued until subjects indicated the presence of a “trigeminal” sensation three times in a row for a given concentration. Then, the staircase was reversed, and the next lower concentration was applied. The next reversal was triggered when subjects did not indicate the presence of such a sensation, etc. This procedure was continued until seven reversals had occurred (Doty, 1991). The mean of the last four reversals was used as a threshold estimate. If the subject was not able to lateralize the highest concentration seven times in a row, a threshold of 1 was assigned as a substituted value. Higher numbers indicate lower thresholds. The interval between two stimulations was 30 s. The time required to test both substances was approximately one hour including a interval of 15 min between individual measurements. Experiment B was divided into two sessions during which the two odors were applied sequentially. Lateralization thresholds were obtained using a two-alternative, forcedchoice, staircase method (Ehrenstein and Ehrenstein, 1999). On each trial, the subject was presented with two polyethylene bottles (white color with a red spout, volume 250 ml, bottle diameter: 55 mm, opening diameter: 2 mm) mounted in a hand-held, manually operated holder, which allowed the simultaneous stimulation of both nostrils (Berg et al., 1998). Using this technique, the mean volume of applied substance
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containing air was 15 ml (Hummel et al., 2003). One bottle contained substance dissolved in propylene glycol, the other bottle contained solvent only. Subjects were asked to simultaneously insert the nosepiece from each pair of bottles snugly into their nostrils; then, the substance was presented to the left or the right nostril while the other received air only. The side of the presentation of the odor was randomized. Subjects indicated whether they believed the stimulus had been presented to the left or the right nostril. An incorrect detection on any trial resulted in the presentation of the next higher concentration, while three consecutive correct detections triggered the presentation of the next lower concentration. The interval between two stimulations was 30 s. Testing continued until seven reversals for each substance were reached; thresholds were calculated as the mean of the last four reversals. Again, higher results indicate lower thresholds. The time required to test the substances was approximately one hour including a 15-min break between assessment of individual thresholds. Statistical analyses were performed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The alpha level was 0.05. A repeated measures ANOVA (analysis of variance) was applied (repeated measurements design; between-subject factors “sex” and “age group”, within-subject factors “session”, “substance”, and “method”; Greenhouse-Geisser correction of degrees of freedom; post hoc testing using t-tests (calculated when interactions or main effects were significant)). Pearson’s coefficient of correlation was calculated for analysis of bivariate correlations.
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3. Results 3.1. Test-retest correlation The results of threshold determination were found to correlate between test and retest. In Experiment A higher degrees of test-retest correlation were found (A[Linalool]: r32 = 0.81, p < 0.001; A[Menthol]: r32 = 0.57, p = 0.001) than in Experiment B (B[Linalool]: r32 = 0.41, p = 0.022; B[Menthol]: r32 = 0.48, p = 0.006). 3.2. Repeated measures ANOVA Thresholds were found to differ between men and women (“sex”: F = 5.3; p = 0.028). When comparing the results of male and female subjects with post hoc tests, results were found to differ significantly for session 2 of Experiment A: women had lower trigeminal thresholds for linalool (women: A[Linalool 2] = 10.1; men: A[Linalool 2] = 6.8, p = 0.033) and menthol (women: A[Menthol 2] = 9.5; men: A[Menthol 2] = 5.7, p < 0.001). No significant differences between female and male subjects were found for the other methods. In addition, the results of the two methods were found to differ (“method”: F = 61.2; p < 0.001). The mean thresholds (see Fig. 1) obtained with method A ranged between 6.5 and 8.4, whereas mean thresholds assessed with method B ranged from 1.9 to 3.3 (all p < 0.001). This indicated that in Experiment A intranasal trigeminal thresholds were found at significantly lower concentrations.
Fig. 1. Results (means and standard errors of means) for intranasal trigeminal thresholds assessed in Experiment A (ratings) and Experiment B (lateralization) for two substances (linalool and menthol). Results of the first sessions are represented by filled black bars, results of the retest session are represented by filled gray bars. Data are presented separately for younger and older subjects in relation to the subjects’ sex (Y M: young male subjects; Y F: young female subjects; O M: old male subjects; O F: old female subjects). Higher values indicate higher sensitivity/lower thresholds.
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There was no significant main effect for within subject factors “substance”, “session”, and “age group”. However, a significant interaction was found for “substance” and “session” (F = 5.9; p = 0.021). This indicated that, when linalool was used as the stimulant, in seven out of eight cases lower thresholds were found in the second session compared to the first session (see upper part of Fig. 1). When menthol was used as the stimulant, in four cases results for the second sessions were lower than those in the first session (lower part of Fig. 1). Further, an interaction was found between factors “method” and “session” (F = 7.3; p = 0.011), indicating that especially with method A, higher scores were found in the second session, compared to the first session (left side of Fig. 1). As it were mostly female subjects showing higher scores in the second session, there was an additional interaction for “method”, and “session”, and “sex” (F = 7.9; p = 0.009). 3.3. Inter-method correlation The results of the two methods were not found to correlate significantly.
4. Discussion 4.1. Testing conditions (within-subject factors) Comparison of the two methods revealed clear and significant differences. The mean thresholds determined in Experiment A were found to be approximately five dilution steps higher than those obtained in Experiment B. This means that thresholds obtained with method A are approximately 32 (=25 ) times lower than those measured with method B. In addition, the methods were not correlated to each other. Although – with regard to test-retest correlation – both methods seem to be valid, they appear to measure different aspects of the trigeminal system. Thus, although awareness of trigeminal sensation seemed to occur at certain (lower) concentrations, which implies stimulation of the trigeminal system, the sensations produced at those concentrations appeared not to be strong enough to allow for localization. This finding is important, as results from lateralization testing may be used to set exposure standards or otherwise evaluate impact from exposures to volatile compounds. Such standards would be too high, as people are likely to perceive some trigeminal sensations at levels below these standards. The differences between the two methods could be explained in three ways: (1) in Experiment A subjects indicated the presence of trigeminal sensation at very low concentrations when in fact, they only had an olfactory sensation. In other words, the ratings of “trigeminal” sensations would have been confounded through olfactory mediated sensations. It could therefore be, that the trigeminal sensitivity was biased by different olfactory sensitivity, which, for example, is known to be lower in older subjects compared to
younger ones. This would implicate that the technique used in Experiment A would not be suitable to reliably measure intranasal trigeminal activation. However, in the present study, differences between trigeminal and olfactory activation were carefully explained to the subjects, so that the risk of confusion should have been minimized. Nevertheless, the possibility of confusion of the trigeminal ratings with olfactory stimulation cannot completely be ruled out. (2) Another possibility for the differences between the two methods is that lateralization of unilaterally presented trigeminal stimuli is only possible at supra-threshold levels. Thus, while the assessment of thresholds is possible with both techniques, two different thresholds are measured. Method A would assess the “subjective” trigeminal threshold, i.e., the lowest concentration, which produces trigeminal sensations. Method B, on the other hand, would measure the lateralization threshold, i.e., the lowest concentration, which allows to lateralize unilaterally presented trigeminal stimuli. This concentration would then be higher than the “subjective” trigeminal threshold. (3) A third explanation is that the concentration of substance in the nose, which is needed to successfully perform the task, is equal for both methods. In this scenario, the difference between thresholds would be due to the two different stimulation techniques. In both experiments the amount of substance, which gets in contact with the mucosa was determined by the concentration of the substance and the applied volume. Odor concentration was equal in both experiments. The applied volume, either passively or through sniffing, was different under the two conditions. While the mean applied volume was 15 ml in Experiment B, it was probably larger in Experiment A, where subjects were allowed to sniff as often as needed. In fact, during an odor threshold task, sniff volume was estimated to be in the range of 1l (Sobel et al., 2000). It has been shown that olfactory detection threshold depends on nasal flow rate and length of the sniff (Sobel et al., 2000). Thus, when subjects in a detection task are free to sniff as long and strong as they want, the volume flowing through their nose is much higher than the applied volume in Experiment B. It is known that stimulants of the trigeminal nerve show time-intensity trading (Cometto-Muniz and Cain, 1984; Frasnelli et al., 2003; Wise et al., 2003), i.e., the perceived intensity of a trigeminal stimulant is larger if the stimulus duration is longer. This indicates that trigeminal activation depends on the total time-integrated mass of the inhaled stimulant (Cometto-Muniz and Cain, 1984). Therefore, it is possible that the different results of the two methods are the effect of different volumes of substance containing air flowing through the nose: a small volume with a high concentration contains the same mass as a large volume with a low concentration. Therefore, the differing results of the two methods could reflect the same threshold. There was a significant interaction for “method” × “session”. In Experiment A, higher scores were found in the second session compared to the first session. No such difference was seen in Experiment B. Although no effect of the main factor “session” was found,
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the present results lead to the conclusion that there was a training effect for method A. Therefore, if this technique is applied, in addition to a detailed explanation of the difference between olfactory and trigeminally mediated sensations, in order to minimize sequence effects subjects should undergo training to become acquainted with the characteristics of intranasal trigeminal sensation. Menthol is typically described as cool and fresh (Laska, 2001), whereas linalool evokes a different, more burning sensation. However, there was no significant main effect of the factor “substance”. Thus, it seems that both stimulants were equally appropriate to be applied when investigating intranasal trigeminal activation.
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related loss of intranasal trigeminal function was also seen in ERP studies (Hummel et al., 1998a). However, in the present study, no significant differences between age groups could be detected. As there was a tendency to higher thresholds in the older subjects compared to the younger ones, it could be that the number of subjects in the presently investigated age groups was too small to detect significant age-related differences in trigeminal thresholds. However, it was large enough to detect the difference between age groups in olfactory testing: older subjects were found to have lower scores in the “Sniffin’ Sticks”-test, indicative of weaker olfactory function (p = 0.004). Thus, effects of age on the intranasal trigeminal system at threshold level may be less consistent than in the olfactory system.
4.2. Group differences (between-subject factors) Women were found to have lower intranasal trigeminal thresholds than men. This is in line with previous work: when sex differences are found in chemosensory tasks, female subjects exhibit higher sensitivity than male subjects (e.g., olfactory tests Doty et al., 1984; Kobal et al., 2000). Women also exhibit larger ERP amplitudes in response to intranasal trigeminal stimuli (e.g., Hummel et al., 1998a. The reason for this remains unclear. In a study investigating sex differences using magnetic resonance it was shown that women show higher activation following olfactory stimulation (Yousem et al., 1999). Specifically, in certain brain areas the women’s group-averaged activation maps showed up to eight times more activated voxels compared to those of the men. In the present study, post hoc tests showed significant sex differences only for the retest session of Experiment A. This indicates that method A is more sensitive to sex differences than method B. Another explanation may lie in the different tasks of both methods. Method B is an “objective” test; Method A is more “subjective”. It cannot be ruled out that there are sex differences in the attention to unpleasant sensations. Thus, it could be speculated that women would report such sensations more quickly. In this scenario female subjects would sooner report any trigeminal sensations. However, this inclination may not help them to reliably lateralize the stimulus. The conclusion could also be that females pay more attention to such sensations and thus report them at lower concentrations. In a sense, women might have a lower criterion for deciding that they are experiencing the intended sensation. However, as they are not able to lateralize the odor at lower concentrations, their sensitivity may not be different from that of males. A further hypothesis of the present study was that younger subjects would outperform older subjects. An age-related decrease in the perceived intensity of CO2 (Stevens et al., 1982) was reported as well as an age related elevation of the threshold for transitory apnea in response to CO2 (Stevens and Cain, 1986). Older subjects had less correct answers in lateralization (Hummel et al., 2003; Wysocki et al., 2003) or discrimination (Laska, 2001) tasks than young subjects, which is indicative of decreased trigeminal function. An age
5. Conclusions With regard to test-retest reliability, a higher correlation was found for method A than for method B. Although significantly differing threshold estimates were obtained with the two methods, it is possible that the absolute mass of stimulant needed to successfully perform the respective task is equal for both methods. As the intranasal trigeminal system is more sensitive to the absolute amount of stimulus than to stimulus concentrations, it appears possible that results obtained through both methods would reflect the same quantity of stimulant. Further, method A showed significant differences between male and female subjects. No significant difference between age groups could be detected with both methods. Therefore, method A seems the more sensitive method in terms of the effects of sex on trigeminally mediated sensations. Method B appears the more objective method, where it is the experimenter’s decision if the task was completed correctly or not. In contrast, with method A it is up to the subject to decide if there was a trigeminal component or not. Naturally, results obtained by means of this approach depend on the explanation of the differences between olfactory and trigeminal activation. If one’s objective is to assess the level at which trigeminal sensations are detected with the utmost objectivity and unconfounded by smell, the obvious choice is method B. If one’s purpose is to assess the level at which trigeminal sensations are detected and the quality perceived, in the context of an odor one might opt for Method A. The circumstances under which thresholds are assessed in A are more comparable to those with which people are confronted in everyday situations. Thus, preference of one method over the other may depend on the question being asked, provided a wellinstructed/trained panel of subjects is used. Acknowledgement This study was supported by the Environmental Sensory Research Institute, Baltimore, MD 21228, USA. It also received (partly) support through Philip Morris Inc., USA.
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Intranasal trigeminal thresholds in healthy subjects Johannes Frasnelli, Thomas Hummel∗ Smell and Taste Clinic, Department of Otorhinolaryngology, University of Dresden Medical School, Fetscherstrasse 74, 01307 Dresden, Germany Available online 26 January 2005
Abstract The trigeminal chemosensory system responds to irritation of the nasal cavity. Despite its dominant role as a sentinel in protecting the respiratory tract from harmful substances and its involvement in the perception of odorous substances, it has received relatively little attention compared to the olfactory system. Aim of the present study was the comparison of two psychophysical techniques to assess intranasal trigeminal thresholds, namely (A) responses of subjects who focused on intranasal trigeminally mediated sensations, and (B) the ability of subjects to identify the side of the nose receiving unilaterally presented stimuli. Method A (0.81 > r > 0.56) was found to show a higher testretest reliability than Method B (0.48 > r > 0.40). Method A revealed thresholds that were approximately 32 times lower than those measured with method B. With method A women were found to have lower thresholds than men; no such difference could be detected between older and younger subjects. In conclusion, if the objective is to assess the level at which trigeminal sensations are detected with the utmost objectivity and unconfounded by smell, the obvious choice is B. If one’s purpose is to assess the level at which trigeminal sensations are detected and the quality perceived, in the context of an odor one might opt for Method A. Thus, preference of one method over the other may depend on the question being asked, provided a well-instructed/trained panel of subjects is used. © 2004 Elsevier B.V. All rights reserved. Keywords: Trigeminal nerve; Chemoreception; Threshold; Ratings; Lateralization
1. Introduction The trigeminal chemosensory system responds to irritation of the nasal cavity. Despite its dominant role as a sentinel in protecting the respiratory tract from harmful substances and its involvement in the perception of chemicals, it has received relatively little attention compared to the olfactory system. Today, we know that most odorants activate both the trigeminal and the olfactory systems (Doty et al., 1978; Elsberg et al., 1935; von Skramlik, 1924). For example, nicotine produces not only an odorous sensation, but also burning and stinging (Hummel and Kobal, 1992). Other trigeminally mediated sensations include cooling, prickling, freshness, and piquancy (see Kelly and Dodd, 1991; Laska et al., 1997). Different fiber types are involved in trigeminally mediated sensations: Activation of C-fibers produces dull and burning painful sensations, while sharp and stinging sensations are known to appear after excitation of A␦-fibers ∗
Corresponding author. Tel.: +49 351 458 4189; fax: +49 351 458 4326. E-mail address: [email protected] (T. Hummel).
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(Hummel et al., 1994; Mackenzie et al., 1975; Torebj¨ork and Hallin, 1971). These sensations follow different time courses (Hummel et al., 1992) and may have a different impact on olfactory sensations. Apart from electrophysiological measures (e.g., eventrelated potentials (ERP) (Huttunen et al., 1986; Kobal and Hummel, 1988); negative mucosa potential (NMP) (Hummel et al., 1998b; Kobal, 1985; Thurauf et al., 2002)), a number of other techniques have been proposed to assess the trigeminal impact of odorants in humans. They include the assessment of the percentage of stimulus detection in anosmics and ratings from healthy subjects focusing on trigeminally mediated sensations (Doty et al., 1978), magnitude estimation procedures (Cain and Murphy, 1980), questionnaires (Hudnell et al., 1992), measures of respiratory reflexes (Warren et al., 1994) or reflexive changes in nasal blood flow (Th¨urauf et al., 1993), localization of the site of lateralized odor presentation (Berg et al., 1998; Kobal et al., 1989), assessment of ocular irritation (Cometto-Muniz et al., 1997), or pain thresholds after intranasal trigeminal activation with CO2 (L¨otsch et al., 1997). Unfortunately, almost no data are available concern-
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ing the reliability and validity of these methods. In addition, some of these measures seem to be only suited for the assessment of trigeminally mediated sensations at the suprathreshold level. The relation between results obtained by means of these different methods remains obscure. As threshold levels of trigeminal activation by odorants appear to be of high practical interest (Cain and Cometto-Muniz, 1995), studies are needed which systematically compare the relative value of methods to detect threshold levels of trigeminal stimulation. Aim of the present study was the comparison of two different psychophysical techniques to assess intranasal trigeminal thresholds: (a) responses of subjects who focused on intranasal trigeminally mediated sensations, and (b) the ability of subjects to identify the side of the nose receiving an unilaterally presented stimulus (compare Kobal et al., 1989). These techniques were chosen because of their high practicability, and their low apparative efforts. Comparisons between methods were based on test-retest reliability and absolute thresholds. These characteristics should also be used to judge the quality of the different measures. Further, it was investigated whether the two methods would reveal inter-individual differences in trigeminal sensitivity, which seems to be (a) greater in women than in men (Hummel et al., 1998a), and (b) which appears to decrease with age (Hummel et al., 1998a; Stevens et al., 1982).
2. Material and methods A total of 32 subjects participated in the study. The subjects’ health was ascertained through a thorough history and a detailed physical ENT-examination. Further, olfactory function was screened by means of the “Sniffin’ Sticks” (Hummel et al., 1997; Kobal et al., 1996). Half of the subjects (eight women and eight men) were between 18 and 35 years of age (mean age 25.5 years, standard deviation (S.D.): 0.7), the other half of the subjects (8w, 8m) were older than 45 years (mean age: 58.5 years, S.D.: 1.6). Thus, four groups were established (young women, young men, older women, and older men). The study was conducted according to the Declaration of Helsinki on biomedical research involving human subjects. During enrolment, subjects were given detailed information about all testing procedures. Written informed consent was obtained from all subjects prior to the study. This study was designed to systematically compare two methods to assess trigeminal thresholds. In Experiment A threshold concentrations were identified at which a “trigeminal” sensation appeared. In Experiment B threshold concentrations were determined at which the stimulated nostril can be determined after lateralized stimulus presentation. The order of methods was randomized across subjects. Both experiments were performed on different days. Subjects were retested after 6–25 days. During retesting the order of chemosensory testing methods was the same in individual
subjects. Two substances were investigated, namely linalool and menthol. Both substances are known to produce clear trigeminal sensations (Doty et al., 1978). For each substance 16 dilutions (numbered 1–16, #1 being the strongest and #16 being the weakest concentration) were prepared in geometric series (1:2) with propylene glycol as the solvent. The highest concentration (#1) was 97% for linalool and 50% g/g for menthol, #2 was 48.5% g/g and 25% g/g, respectively. The highest concentration produced a clear “supra-threshold” sensation in all subjects. The intensity of the highest concentrations was rated to be similar by a panel of experienced observers. Trigeminal thresholds were assessed with two different techniques. All measurements were performed in a quiet, well-ventilated room. In Experiment A odors were presented to both nostrils. The odors were tested sequentially and the order of testing was counterbalanced across subjects. Odors were presented in sniff bottles made from glass (brown color, volume: 250 ml, diameter of the bottle: 65 mm, opening diameter: 45 mm). Each bottle contained 40 ml of liquid. The mucosa of the eyes was protected from potential trigeminal stimulation by means of goggles. Threshold for each odor was determined using an initially ascending single staircase method (Ehrenstein and Ehrenstein, 1999). After presentation of each stimulus, subjects were asked whether the odor produced a sensation that was mediated via the trigeminal nerve or not (“have you felt any sensation like burning, stinging, cooling or tickling?”). Subjects were allowed to sniff more than once. The staircase started at the lowest concentration of the substance. If subjects indicated no “trigeminal” sensation, the next higher concentration was presented. This continued until subjects indicated the presence of a “trigeminal” sensation three times in a row for a given concentration. Then, the staircase was reversed, and the next lower concentration was applied. The next reversal was triggered when subjects did not indicate the presence of such a sensation, etc. This procedure was continued until seven reversals had occurred (Doty, 1991). The mean of the last four reversals was used as a threshold estimate. If the subject was not able to lateralize the highest concentration seven times in a row, a threshold of 1 was assigned as a substituted value. Higher numbers indicate lower thresholds. The interval between two stimulations was 30 s. The time required to test both substances was approximately one hour including a interval of 15 min between individual measurements. Experiment B was divided into two sessions during which the two odors were applied sequentially. Lateralization thresholds were obtained using a two-alternative, forcedchoice, staircase method (Ehrenstein and Ehrenstein, 1999). On each trial, the subject was presented with two polyethylene bottles (white color with a red spout, volume 250 ml, bottle diameter: 55 mm, opening diameter: 2 mm) mounted in a hand-held, manually operated holder, which allowed the simultaneous stimulation of both nostrils (Berg et al., 1998). Using this technique, the mean volume of applied substance
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containing air was 15 ml (Hummel et al., 2003). One bottle contained substance dissolved in propylene glycol, the other bottle contained solvent only. Subjects were asked to simultaneously insert the nosepiece from each pair of bottles snugly into their nostrils; then, the substance was presented to the left or the right nostril while the other received air only. The side of the presentation of the odor was randomized. Subjects indicated whether they believed the stimulus had been presented to the left or the right nostril. An incorrect detection on any trial resulted in the presentation of the next higher concentration, while three consecutive correct detections triggered the presentation of the next lower concentration. The interval between two stimulations was 30 s. Testing continued until seven reversals for each substance were reached; thresholds were calculated as the mean of the last four reversals. Again, higher results indicate lower thresholds. The time required to test the substances was approximately one hour including a 15-min break between assessment of individual thresholds. Statistical analyses were performed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The alpha level was 0.05. A repeated measures ANOVA (analysis of variance) was applied (repeated measurements design; between-subject factors “sex” and “age group”, within-subject factors “session”, “substance”, and “method”; Greenhouse-Geisser correction of degrees of freedom; post hoc testing using t-tests (calculated when interactions or main effects were significant)). Pearson’s coefficient of correlation was calculated for analysis of bivariate correlations.
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3. Results 3.1. Test-retest correlation The results of threshold determination were found to correlate between test and retest. In Experiment A higher degrees of test-retest correlation were found (A[Linalool]: r32 = 0.81, p < 0.001; A[Menthol]: r32 = 0.57, p = 0.001) than in Experiment B (B[Linalool]: r32 = 0.41, p = 0.022; B[Menthol]: r32 = 0.48, p = 0.006). 3.2. Repeated measures ANOVA Thresholds were found to differ between men and women (“sex”: F = 5.3; p = 0.028). When comparing the results of male and female subjects with post hoc tests, results were found to differ significantly for session 2 of Experiment A: women had lower trigeminal thresholds for linalool (women: A[Linalool 2] = 10.1; men: A[Linalool 2] = 6.8, p = 0.033) and menthol (women: A[Menthol 2] = 9.5; men: A[Menthol 2] = 5.7, p < 0.001). No significant differences between female and male subjects were found for the other methods. In addition, the results of the two methods were found to differ (“method”: F = 61.2; p < 0.001). The mean thresholds (see Fig. 1) obtained with method A ranged between 6.5 and 8.4, whereas mean thresholds assessed with method B ranged from 1.9 to 3.3 (all p < 0.001). This indicated that in Experiment A intranasal trigeminal thresholds were found at significantly lower concentrations.
Fig. 1. Results (means and standard errors of means) for intranasal trigeminal thresholds assessed in Experiment A (ratings) and Experiment B (lateralization) for two substances (linalool and menthol). Results of the first sessions are represented by filled black bars, results of the retest session are represented by filled gray bars. Data are presented separately for younger and older subjects in relation to the subjects’ sex (Y M: young male subjects; Y F: young female subjects; O M: old male subjects; O F: old female subjects). Higher values indicate higher sensitivity/lower thresholds.
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There was no significant main effect for within subject factors “substance”, “session”, and “age group”. However, a significant interaction was found for “substance” and “session” (F = 5.9; p = 0.021). This indicated that, when linalool was used as the stimulant, in seven out of eight cases lower thresholds were found in the second session compared to the first session (see upper part of Fig. 1). When menthol was used as the stimulant, in four cases results for the second sessions were lower than those in the first session (lower part of Fig. 1). Further, an interaction was found between factors “method” and “session” (F = 7.3; p = 0.011), indicating that especially with method A, higher scores were found in the second session, compared to the first session (left side of Fig. 1). As it were mostly female subjects showing higher scores in the second session, there was an additional interaction for “method”, and “session”, and “sex” (F = 7.9; p = 0.009). 3.3. Inter-method correlation The results of the two methods were not found to correlate significantly.
4. Discussion 4.1. Testing conditions (within-subject factors) Comparison of the two methods revealed clear and significant differences. The mean thresholds determined in Experiment A were found to be approximately five dilution steps higher than those obtained in Experiment B. This means that thresholds obtained with method A are approximately 32 (=25 ) times lower than those measured with method B. In addition, the methods were not correlated to each other. Although – with regard to test-retest correlation – both methods seem to be valid, they appear to measure different aspects of the trigeminal system. Thus, although awareness of trigeminal sensation seemed to occur at certain (lower) concentrations, which implies stimulation of the trigeminal system, the sensations produced at those concentrations appeared not to be strong enough to allow for localization. This finding is important, as results from lateralization testing may be used to set exposure standards or otherwise evaluate impact from exposures to volatile compounds. Such standards would be too high, as people are likely to perceive some trigeminal sensations at levels below these standards. The differences between the two methods could be explained in three ways: (1) in Experiment A subjects indicated the presence of trigeminal sensation at very low concentrations when in fact, they only had an olfactory sensation. In other words, the ratings of “trigeminal” sensations would have been confounded through olfactory mediated sensations. It could therefore be, that the trigeminal sensitivity was biased by different olfactory sensitivity, which, for example, is known to be lower in older subjects compared to
younger ones. This would implicate that the technique used in Experiment A would not be suitable to reliably measure intranasal trigeminal activation. However, in the present study, differences between trigeminal and olfactory activation were carefully explained to the subjects, so that the risk of confusion should have been minimized. Nevertheless, the possibility of confusion of the trigeminal ratings with olfactory stimulation cannot completely be ruled out. (2) Another possibility for the differences between the two methods is that lateralization of unilaterally presented trigeminal stimuli is only possible at supra-threshold levels. Thus, while the assessment of thresholds is possible with both techniques, two different thresholds are measured. Method A would assess the “subjective” trigeminal threshold, i.e., the lowest concentration, which produces trigeminal sensations. Method B, on the other hand, would measure the lateralization threshold, i.e., the lowest concentration, which allows to lateralize unilaterally presented trigeminal stimuli. This concentration would then be higher than the “subjective” trigeminal threshold. (3) A third explanation is that the concentration of substance in the nose, which is needed to successfully perform the task, is equal for both methods. In this scenario, the difference between thresholds would be due to the two different stimulation techniques. In both experiments the amount of substance, which gets in contact with the mucosa was determined by the concentration of the substance and the applied volume. Odor concentration was equal in both experiments. The applied volume, either passively or through sniffing, was different under the two conditions. While the mean applied volume was 15 ml in Experiment B, it was probably larger in Experiment A, where subjects were allowed to sniff as often as needed. In fact, during an odor threshold task, sniff volume was estimated to be in the range of 1l (Sobel et al., 2000). It has been shown that olfactory detection threshold depends on nasal flow rate and length of the sniff (Sobel et al., 2000). Thus, when subjects in a detection task are free to sniff as long and strong as they want, the volume flowing through their nose is much higher than the applied volume in Experiment B. It is known that stimulants of the trigeminal nerve show time-intensity trading (Cometto-Muniz and Cain, 1984; Frasnelli et al., 2003; Wise et al., 2003), i.e., the perceived intensity of a trigeminal stimulant is larger if the stimulus duration is longer. This indicates that trigeminal activation depends on the total time-integrated mass of the inhaled stimulant (Cometto-Muniz and Cain, 1984). Therefore, it is possible that the different results of the two methods are the effect of different volumes of substance containing air flowing through the nose: a small volume with a high concentration contains the same mass as a large volume with a low concentration. Therefore, the differing results of the two methods could reflect the same threshold. There was a significant interaction for “method” × “session”. In Experiment A, higher scores were found in the second session compared to the first session. No such difference was seen in Experiment B. Although no effect of the main factor “session” was found,
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the present results lead to the conclusion that there was a training effect for method A. Therefore, if this technique is applied, in addition to a detailed explanation of the difference between olfactory and trigeminally mediated sensations, in order to minimize sequence effects subjects should undergo training to become acquainted with the characteristics of intranasal trigeminal sensation. Menthol is typically described as cool and fresh (Laska, 2001), whereas linalool evokes a different, more burning sensation. However, there was no significant main effect of the factor “substance”. Thus, it seems that both stimulants were equally appropriate to be applied when investigating intranasal trigeminal activation.
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related loss of intranasal trigeminal function was also seen in ERP studies (Hummel et al., 1998a). However, in the present study, no significant differences between age groups could be detected. As there was a tendency to higher thresholds in the older subjects compared to the younger ones, it could be that the number of subjects in the presently investigated age groups was too small to detect significant age-related differences in trigeminal thresholds. However, it was large enough to detect the difference between age groups in olfactory testing: older subjects were found to have lower scores in the “Sniffin’ Sticks”-test, indicative of weaker olfactory function (p = 0.004). Thus, effects of age on the intranasal trigeminal system at threshold level may be less consistent than in the olfactory system.
4.2. Group differences (between-subject factors) Women were found to have lower intranasal trigeminal thresholds than men. This is in line with previous work: when sex differences are found in chemosensory tasks, female subjects exhibit higher sensitivity than male subjects (e.g., olfactory tests Doty et al., 1984; Kobal et al., 2000). Women also exhibit larger ERP amplitudes in response to intranasal trigeminal stimuli (e.g., Hummel et al., 1998a. The reason for this remains unclear. In a study investigating sex differences using magnetic resonance it was shown that women show higher activation following olfactory stimulation (Yousem et al., 1999). Specifically, in certain brain areas the women’s group-averaged activation maps showed up to eight times more activated voxels compared to those of the men. In the present study, post hoc tests showed significant sex differences only for the retest session of Experiment A. This indicates that method A is more sensitive to sex differences than method B. Another explanation may lie in the different tasks of both methods. Method B is an “objective” test; Method A is more “subjective”. It cannot be ruled out that there are sex differences in the attention to unpleasant sensations. Thus, it could be speculated that women would report such sensations more quickly. In this scenario female subjects would sooner report any trigeminal sensations. However, this inclination may not help them to reliably lateralize the stimulus. The conclusion could also be that females pay more attention to such sensations and thus report them at lower concentrations. In a sense, women might have a lower criterion for deciding that they are experiencing the intended sensation. However, as they are not able to lateralize the odor at lower concentrations, their sensitivity may not be different from that of males. A further hypothesis of the present study was that younger subjects would outperform older subjects. An age-related decrease in the perceived intensity of CO2 (Stevens et al., 1982) was reported as well as an age related elevation of the threshold for transitory apnea in response to CO2 (Stevens and Cain, 1986). Older subjects had less correct answers in lateralization (Hummel et al., 2003; Wysocki et al., 2003) or discrimination (Laska, 2001) tasks than young subjects, which is indicative of decreased trigeminal function. An age
5. Conclusions With regard to test-retest reliability, a higher correlation was found for method A than for method B. Although significantly differing threshold estimates were obtained with the two methods, it is possible that the absolute mass of stimulant needed to successfully perform the respective task is equal for both methods. As the intranasal trigeminal system is more sensitive to the absolute amount of stimulus than to stimulus concentrations, it appears possible that results obtained through both methods would reflect the same quantity of stimulant. Further, method A showed significant differences between male and female subjects. No significant difference between age groups could be detected with both methods. Therefore, method A seems the more sensitive method in terms of the effects of sex on trigeminally mediated sensations. Method B appears the more objective method, where it is the experimenter’s decision if the task was completed correctly or not. In contrast, with method A it is up to the subject to decide if there was a trigeminal component or not. Naturally, results obtained by means of this approach depend on the explanation of the differences between olfactory and trigeminal activation. If one’s objective is to assess the level at which trigeminal sensations are detected with the utmost objectivity and unconfounded by smell, the obvious choice is method B. If one’s purpose is to assess the level at which trigeminal sensations are detected and the quality perceived, in the context of an odor one might opt for Method A. The circumstances under which thresholds are assessed in A are more comparable to those with which people are confronted in everyday situations. Thus, preference of one method over the other may depend on the question being asked, provided a wellinstructed/trained panel of subjects is used. Acknowledgement This study was supported by the Environmental Sensory Research Institute, Baltimore, MD 21228, USA. It also received (partly) support through Philip Morris Inc., USA.
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