GOLEBIOWSKI, BLANKA BOptom, PhD; PAPAS, ERIC B. PhD, FAAO; STAPLETON, FIONA MCOptom, PhD, FAAO
Measurement of sensitivity is an important tool in the investigation of the sensory function of the ocular surface. Whereas electrophysiological data illustrate corneal and conjunctival neural function in animals,1–4 such experiments can not be performed in humans. Consequently, sensory information about the human ocular surface in vivo has been gathered by evaluating subjective responses to controlled stimulation of the cornea and conjunctiva. Sensitivity measurement provides a means of examining the impact of ocular surface disease, refractive surgery, contact lens wear, and therapeutic agents on corneal and conjunctival physiology and neural function (see Millodot5 and Lawrenson6 for review).
To study the changes brought about by various induced or pathological conditions, it is important to first establish which factors cause ocular surface sensitivity fluctuations under normal circumstances. Although effects of factors such as age, gender, ocular location, and time of day have been investigated previously, much of the earlier work was carried out using traditional instrumentation and did not examine both corneal and conjunctival sensitivity. The results of previous studies are inconsistent regarding the influence of gender and age, and the impact of ambient environmental factors such as temperature and humidity has not yet been explored.
Ocular surface sensitivity has traditionally been measured using the Cochet-Bonnet esthesiometer, which mechanically stimulates the ocular surface with a nylon filament.7 The deficiencies of this instrument—the most crucial of which are its truncated intensity range and imprecise stimulus application8,9—have lead to development of newer instruments, most recently the CRCERT-Belmonte esthesiometer, or CBA,10 which uses a fine jet of gas as a stimulus. The flow, composition, and temperature of this gas can be altered to apply mechanical, chemical, and cooling stimuli to the ocular surface,8 thus enabling an investigation of the effects of these various stimuli. The CBA incorporates a number of key improvements over earlier air-jet esthesiometers, which allow a more precise application and characterization of the stimulus.8
Ambient temperature and relative humidity are known to affect the nylon filament of the Cochet-Bonnet esthesiometer, and alter the stimulus applied.11 As the CBA stimulus consists of a core of temperature controlled air and interacts with the tear film, sensitivity measurements with this instrument may also conceivably be affected by day-to-day fluctuations in ambient conditions.
Previous investigations have shown the cornea to be more sensitive than the conjunctiva to stimulation with both types of esthesiometer.10,12–19 However, the magnitude of the difference in the sensitivities of these two tissues and how this varies in the population has not yet been reported.
Studies examining gender differences in corneal sensitivity have varied in their conclusions. Increased,14,20,21 decreased,5,22 and more variable23 sensitivity in females compared to males has been reported, with most studies not noting a difference.24–27 Gender differences in conjunctival sensitivity have not been shown at all.
A number of studies report a decline in corneal sensitivity with age12,23,27 but there is uncertainty as to the age at which this becomes significant or whether it occurs at all. A recent study using the CBA found an age-related reduction of corneal sensitivity after approximately 50 years of age14 and investigations with the Cochet-Bonnet esthesiometer showed a similar reduction to occur after 50 years28 or later.25 However, when a cooled air-jet stimulus was used24 this decline occurred from the age of 20 years. Other investigators using air-jet stimuli have not shown age-related change in corneal sensitivity.20,21,26 Although investigated in less detail, conjunctival sensitivity has also been reported to decrease with age.12–14
Diurnal variation in corneal sensitivity has previously been shown by investigators using the Cochet-Bonnet as well as an air-jet esthesiometer. The consensus is that this manifests as a marked reduction in sensitivity overnight29,30 followed by a defined increase in the morning and a gradual rise through the day.10,29–31 The sole investigation into comparable changes in conjunctival sensitivity did not show a variation with time of day.10
Age, gender, time of day, and ambient environmental conditions may be important confounding factors in the measurement of ocular surface sensitivity. To gain a more complete understanding of the influence of these physiological and environmental fluctuations on corneal and conjunctival sensitivity measurement using the CBA, a retrospective analysis was carried out of data from multiple studies of normal subjects.
Corneal sensitivity measurements for 49 subjects and conjunctival measurements for 33 subjects were included in the analyses (Table 1). All subjects had normal corneas and were not habitual contact lens wearers. The study was carried out according to the guidelines set out in the Declaration of Helsinki and all procedures were approved by the University of New South Wales ethics committee. All subjects signed a statement of informed consent before commencement of the study.
The CRCERT-Belmonte esthesiometer10 (Fig. 1) was used for all sensitivity measurements. The CBA air stimulus in this study was intended to simulate a mechanical stimulus, and its characteristics were as follows: duration 1 s, flowrate 1 to 400 mL/min and temperature 34°C at the ocular surface (equivalent to the temperature of the corneal surface32).
Stimuli were delivered to one eye of each subject at the corneal apex and at an inferior conjunctival region 2 mm from the limbus. Thresholda to stimulation was determined using the Garcia-Perez staircase technique, as described previously.8,33 A refractory period of 30 s was allowed between stimulus presentations to minimize possible effects of summation or adaptation. Measurements were made between 9 am and 6 pm, at normal ranges of ambient temperature 20 to 26°C (mean 23 ± 2°C) and humidity 52 to 87% (mean 70 ± 7%). These measurement conditions were the same for male and female subjects. Because of the retrospective nature of the analysis, multiple measurements were available for some subjects whereas for others only single measurements were available.
Mixed model analysis of variance (ANOVA), Student’s t-test (paired and independent-samples, two-tailed) and Pearson’s correlation were used as appropriate to examine the effects of various factors on threshold. A 95% confidence level was considered to be significant. Interaction between factors was examined to inform subsequent subgroup analyses. As interactions were examined at an exploratory level, a 90% confidence level was used to make decisions for subsequent analysis.
Mean group corneal threshold measured 74.2 ± 23.9 mL/min and conjunctival threshold was significantly higher at 123.7 ± 49.3 mL/min (p < 0.001, paired t-test, Fig. 2a). Corneal and conjunctival thresholds were reasonably well correlated (p < 0.001, Fig. 2b) and neither gender nor age had a significant effect on this correlation.
Corneal and conjunctival thresholds of male subjects were found to be significantly higher than thresholds of female subjects (Fig. 2, Table 2).
When all subjects were considered together, a significant reduction with age was seen to occur in both corneal (r = −0.29, p = 0.046) and conjunctival (r = −0.37, p = 0.03) threshold. Mixed model ANOVA showed an interaction between age and gender for corneal threshold (p = 0.09) and consequently, the effect of age on threshold at each ocular site was evaluated separately for males and females. In females, a significant reduction in corneal threshold was found with age (Fig. 3c). Although a similar trend can be seen for conjunctival threshold, this association was not statistically significant (Fig. 3d). No effect of age on threshold at either site was evident for males (Figs. 3a, b).
As a significant correlation was found between the time of day of measurement and both ambient temperature and humidity (p < 0.03), the effect of each of these factors was examined separately. Mixed model ANOVA showed no significant effect on corneal or conjunctival threshold of time of day (cornea p = 0.31, conjunctiva p = 0.65), ambient humidity (cornea p = 0.45, conjunctiva p = 0.18) or temperature (cornea p = 0.65, conjunctiva p = 0.91).
This study found the central cornea to be more sensitive than the bulbar conjunctiva, which confirms previous findings.12,13,16,18 This difference in sensitivity is expected from the considerably higher innervation density of the cornea compared to the conjunctiva.12,34–36 In addition, whereby terminal receptors of corneal sensory neurons are located within the most superficial epithelial cell layers, conjunctival receptors lie more deeply in the subepithelial layers, which may render them less responsive to external stimulation.34,37–39 Corneal and conjunctival sensitivity are shown here to be positively correlated. This result is not surprising and reflects the overlapping nature of the receptive fields of mechanosensory and polymodal neurons innervating the two tissues which is seen in animal models.4
Male subjects were found to have reduced sensitivity at both the cornea and conjunctiva compared to females, in agreement with earlier findings using a similar esthesiometer.14,20,21 Comparable gender differences are known to occur in other sensory systems, such as the skin,40 the olfactory system41 and in the perception of pain. Differences in touch, smell, and pain have been ascribed to different levels of estrogens and adrenal hormones, although the exact mechanisms of action are still unknown.41,42 Previous investigators have also attributed gender differences in ocular surface sensitivity to hormonal changes.5,43 A recent finding of increased sensitivity in pre- but not postmenopausal women supports this hypothesis.14 Hormone mediated gender differences in sensitivity may be due to anatomical factors, differences in blood flow or due to a direct modulation of central nervous system activity.44 In terms of the ocular surface, gender-related differences in tear film structure and integrity may also play a role.
The increase of corneal sensitivity with age shown in female subjects has not previously been reported. A similar effect was not evident in men. Although this finding seems to be at odds with previous work showing a reduction in sensitivity with age, it must be noted that age-related changes in sensitivity have not previously been investigated with respect to gender. The causes of an age-related sensitivity increase in females could be genetic, hormonal, psychosocial or perhaps due to the confounding of CBA sensitivity measurements in older women by an increased prevalence of ocular surface disorders in this population. Corneal hypersensitivity to the same CBA stimulus has previously been shown in dry eye subjects and proposed to be a result of increased permeability of a compromised epithelial surface.26 A significant increase with age was not shown for conjunctival sensitivity, although this may have been obscured by the substantially greater intersubject variation seen in this tissue.
Measurement of corneal or conjunctival sensitivity was not found to be affected by time of day, in contrast to earlier investigations which showed a diurnal variation of corneal sensitivity.10,29–31 No effect of time has previously been reported for conjunctival sensitivity. There may be a number of reasons for the present findings. As the intersubject variation in ocular surface sensitivity is substantial, the effect of diurnal variation, which is relatively more subtle,10,29 is more effectively examined in a study designed specifically for this purpose. In addition, sensitivity in this study was not measured immediately upon waking, which is when the greatest changes in sensitivity are expected to occur,29,30 presumably as a consequence of hypoxic changes arising from lid closure.45 Future studies should encompass a greater time range than that available in this retrospective analysis.
Reassuringly, this study showed no effect of day-to-day fluctuations of ambient temperature and humidity on corneal or conjunctival sensitivity measurement. Ambient temperature and humidity were considered of potential consequence because both factors could influence detection of the CBA stimulus and perhaps affect the characteristics of the stimulus itself. It may be that relative differences in temperature between the air jet, the ocular surface, and the surrounding environment modulate stimulus detection. Ambient humidity may affect the interaction between the esthesiometer stimulus and the tear film, as the stability of the latter may be a factor of relative humidity.46,47 However, the present findings suggest that any such ambient environmental effects do not come into play within normal ranges of temperature or humidity.
From this analysis of normal subjects, it can be seen that ocular surface sensitivity varies as a function of gender and age and that the sensitivity levels of the two tissues are associated. Corneal and conjunctival sensitivity is higher in female subjects, who also show an increase in corneal sensitivity with age. In contrast, male ocular surface sensitivity is not dependant on age. Normal operating ranges of ambient factors such as temperature and humidity do not affect measurement of sensitivity with the CBA. A diurnal variation was not detected in this study, suggesting that this effect may be relatively minor during waking hours. However, it is important to bear in mind that results obtained with the CBA and those of earlier studies using the Cochet-Bonnet esthesiometer are not easily comparable. Dissimilarities between the dynamic and dispersed air jet of the CBA and the discrete punctate filament of the older instrument mean that the two stimuli will have a different effect on the tissues of the ocular surface.
Consideration should be given in the experimental design of sensitivity studies to demographic factors such as gender and age as these appear to have an influence on sensitivity measurements.
We thank Thomas Naduvilath for his advice on statistical analysis. This study was supported by the Australian Federal Government through the Cooperative Research Centres program with additional funding from the Contact Lens Society of Australia.
School of Optometry and Vision Science
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University of New South Wales
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aThreshold of detection of the esthesiometer stimulus was measured in this study, and the results are presented as such. However, for ease of comprehension, the results are discussed in terms of sensitivity, which is defined as the inverse of the measured threshold. Cited Here...