The primary objective of the study was to investigate the effects of experimental TFI on corneal responses to cool, mechanical, and chemical stimuli. The present study psychophysically demonstrates in humans that TFI induced by repeated trials of STARE produced bidirectional effects on corneal sensory processing, decreasing the responses to suprathreshold cold stimulation and enhancing irritation with mechanical and chemical stimuli. These novel results confirmed our working hypothesis that continuous ocular stimulation resulting from prolonged eye-opening (STARE) and associated TFI and ocular surface stress alters sensory processing/sensations. Although the STARE technique does not represent normal blinking as shown by the MBI results (
Table 4), the induced TFI may provide a model for understanding the neurosensory abnormalities that play an etiological role in dry eye.
5
Corneal sensations are mediated by different functional types of corneal sensory neurons through activation of modality-specific receptors.
33–35 The irritation/discomfort induced by mechanical stimuli increased (
Fig. 1C), with a similar trend for chemical stimuli (
Fig. 1D) in the study, suggesting that repeated STARE and TFI may result in promoting nociception of noxious stimuli. A few possible changes occurred in the ocular surface during STARE that may contribute to the altered neurosensory processing although the events during TFI remain a subject for speculation and are not well understood.
36 As the tear film thins and breaks up, increased evaporation should act to elevate tear film osmolarity, possibly as high as 800 mOsm/kg or higher.
37–39 It has been suggested that corneal polymodal neurons are excited when tear osmolarity is greater than 600 mOsm,
40 which has been postulated to occur during tear breakup.
38,39 In addition, it is possible that cell shrinkage may induced by hyperosmotic exposure to underlying corneal epithelial cells
41 during TBU. Deformation of surface cells secondary to drying, as suggested increased surface scatter with wavefront measurement,
42 may also stimulate corneal nociceptors. As corneal polymodal and mechanical neurons (and perhaps chemo-nociceptors
9,43) connect centrally to the second- and higher-order neurons that are responsible for nociception,
10,17,44 activation of these neurons is likely to increase sensory inputs to the nociceptive pathway evoking pain (including irritation).
5
Additionally, the hyperosmolarity that was likely to occur during STARE secondary to tear film evaporation during TFI
45 could have altered the activity of cold receptors as reported in animal studies.
16,40,46–48 These abnormal activities have been thought to underlie dry eye symptoms
49 and are perhaps involved in activation of high threshold cold-sensitive neurons and the connecting nociceptive pathway.
50,51 In the present study, besides the increased irritation with threshold and suprathreshold stimuli discussed above, symptoms of ocular discomfort increased after repeated STARE, as demonstrated by the shift in the CSQ scores to worse symptoms (
Fig. 2), similar to previous reports.
3,4 The abnormal sensory inputs from the ocular surface and the activation of the nociceptive pathway may contribute to this increased ocular irritation following STARE, supporting the hypothesis that TFI may induce neurosensory abnormalities,
1 which play an important role in dry eye development.
5
Presumably, activation of low threshold cold-sensitive neurons is in response to a small surface temperature reduction during normal blink cycle and elicits an innocuous cooling sensation,
5 while corneal high threshold cold receptors are activated by stronger stimulation evoking irritation.
52,53 The nonnoxious room-temperature pneumatic stimuli in the study elicited a cooling sensation similar to previous reports,
32,52,54 but the magnitude of coolness to pneumatic cool stimuli reduced after repeated STARE (
Figs. 1A,
1B), suggesting adaptation, inhibition, and/or masking of the neural mechanisms responsible for innocuous cold perception. The detection of cold was most likely through the opening of TRPM8 transducing channels of the corneal primary cold-sensitive neurons.
15,55 The sensory signals detected are transmitted by second-order neurons that respond to cooling and hyperosmolarity at the Vi/Vc transition region.
34,47 These second-order neurons appear to have distinct functional properties, with one group exclusively responding to innocuous cooling and another group having lower response to cooling and menthol but also responding to acid and noxious heat.
34 They likely receive input from different types of corneal primary afferent neurons, suggesting distinct pathways (“labeled-lines”) for processing sensory signals arising from the ocular surface involved in signaling cooling and drying
34 to regulate ocular homeostasis.
5 While the abnormal sensory inputs induced by TFI and associated ocular surface changes during STARE might lead to activation of the nociceptive pathway as discussed earlier, the reduction in coolness perception in the present study provides evidence that the neuronal pathway processing innocuous cold could also be affected, perhaps at the afferent and/or higher levels.
While there are many putative mechanisms that might account for these two seemingly paradoxical mechanical and cooling effects on corneal sensation following STARE, one possible explanation is related to the suppression of TRPM8-mediated responses to innocuous cooling in corneal cold receptors and sensitization of the nociceptors by inflammatory mediators as reported in animal models of allergic conjunctivitis and UV keratitis.
56,57 There are reports that inflammatory mediators inhibit the activation of TRPM8 channels
58,59 by the G protein subunit Gα
q.
59 In the present study, repeated STARE and TFI could potentially produce ocular surface stress resulting in local release of inflammatory mediators that may reduce the activity of cold receptors and enhance the activity of nociceptors. These effects on peripheral nerve activity may contribute to the changes in the magnitude of sensations evoked by cooling and mechanical stimulation.
Another possibility may be “labeled line” crosstalk or inhibitory and excitatory effects between corneal cooling and nociceptive pathways. Peripheral sensory neurons are presumably connected to specific neuronal pathways or labeled lines, evoking particular modality of sensation such as touch, itch, pain, and temperature sensitivity,
60–63 and the crosstalk among these labeled lines generates and shapes somatosensory perception, as has been reported in somatic sensation and pain reserch.
61,63 Human and animal studies have shown that innocuous cold could suppress nociception or pain and vice versa.
52,53,64,65 Using a spinal cord slice preparation, Zheng et al.
66 have shown that while two distinct populations of inhibitory interneurons in the superficial dorsal horn received specific inputs from TRPM8 (cold) and TRPV1 (heat-pain) expressing afferents, these interneurons converged and were reciprocally inhibitory, allowing interactions between specific afferent messages.
65,66 Like the spinal dorsal horn, the neurons within the trigeminal brainstem complex are extensively interconnected.
35 It is plausible, therefore, that differential engagement of modality specific primary corneal neurons using inhibitory and excitatory circuitries in the trigeminal brainstem complex and/or higher central processing pathway may underlie the crosstalk between coolness and irritation in the present study.
In this study, the sample size was relatively small, and it could be argued that STARE does not represent normal blinking conditions. However, although the induced TFI effects were short-lived and reversible, the present study reveals the complexity of sensory inputs arising from the ocular surface affecting human sensory processing and supports the notion that psychophysical channels
52 do not act independently. These results are important for a number of reasons. First, the experiment points to the utility of pneumatic stimuli for examining hypotheses that are more complex than simple sensitivity issues. We were only able to determine different cooling and discomfort effects because pneumatic esthesiometers provide us with the means to examine mechanical, chemical, and thermal effects. Secondly, the results highlight the usefulness of studying suprathreshold processing (that in the current context is much more experimentally tractable than threshold sensory processes). Finally, the dissociation of the effects on cold and pain sensing pathways reinforces the separability of these paths.
In conclusion, repeated STARE and TFI results in a reduction of innocuous cooling sensations and promotes irritation to noxious stimuli. While further study is needed to understand STARE induced TFI as a sensory stimulus, the present study provides physiological evidence for the first time that prolonged repeated periods of ocular surface stimulation by TFI lead to significant differences in suprathreshold scaling and appears to differently affect mechanical and cooling pathways. The bidirectional responses suggest complex interactions of neural mechanisms underlying the ocular surface sensations in normal and disease conditions such as dry eye.