Dry eye modifies the thermal and menthol responses in rat corneal primary afferent cool cells

Abstract

Dry eye syndrome is a painful condition caused by inadequate or altered tear film on the ocular surface. Primary afferent cool cells innervating the cornea regulate the ocular fluid status by increasing reflex tearing in response to evaporative cooling and hyperosmicity. It has been proposed that activation of corneal cool cells via a transient receptor potential melastatin 8 (TRPM8) channel agonist may represent a potential therapeutic intervention to treat dry eye. This study examined the effect of dry eye on the response properties of corneal cool cells and the ability of the TRPM8 agonist menthol to modify these properties. A unilateral dry eye condition was created in rats by removing the left lacrimal gland. Lacrimal gland removal reduced tears in the dry eye to 35% compared with the contralateral eye and increased the number of spontaneous blinks in the dry eye by over 300%. Extracellular single-unit recordings were performed 8-10 wk following surgery in the trigeminal ganglion of dry eye animals and age-matched controls. Responses of corneal cool cells to cooling were examined after the application of menthol (10 μM-1.0 mM) to the ocular surface. The peak frequency of discharge to cooling was higher and the cooling threshold was warmer in dry eye animals compared with controls. The dry condition also altered the neuronal sensitivity to menthol, causing desensitization to cold-evoked responses at concentrations that produced facilitation in control animals. The menthol-induced desensitization of corneal cool cells would likely result in reduced tearing, a deleterious effect in individuals with dry eye.

Keywords: cold cells; dry eye; rat; sensitization; trigeminal.

Fig. 1.

Effect of lacrimal gland removal on blinking, tearing, and corneal fluorescein staining. A: spontaneous blinking counted over a 5-min observation period was elevated in the eye ipsilateral to the side of gland removal (filled circles) compared with the contralateral eye (open circles) over an 8-wk period. B: tear measurements taken with a cotton thread in unanesthetized animals (see materials and methods) were lower on the side of lacrimal gland removal (filled circles) compared with the contralateral side (open circles). C: photomicrograph taken from a control animal demonstrating the absence of corneal fluorescein staining. D: corneal fluorescein staining in an animal 8 wk after lacrimal gland removal. In this case, the area of positive staining is >50% of the corneal surface, representing a score of 4 on the fluorescein rating scale. ***P < 0.001 vs. measurements taken from the contralateral side.

Fig. 2.

Effect of the dry eye condition on cold-evoked activity. Examples of cold-evoked discharge in a control (A) and dry eye animal (B) are shown. Similar temporal patterns of activity were found in both groups of animals. Activity was greatest during the change in temperature (phasic period) and decreased to a stable level at the temperature plateau (static period). C: average discharge evoked during the cooling stimulus, divided into 3 epochs: 0–30, 60–90, and 120–150 s. Cold-evoked activity was increased in dry eye animals (filled circles) compared with controls (open circles) only during the initial, phasic period of cooling. D: group average peak frequency of activation, which occurred during the phasic period of the cooling stimulus, in control and dry eye animals. E: average temperature threshold for activating cool cells in control and dry eye animals (n = 27 units in control animals and 22 units in dry eye animals). *P < 0.05; ***P < 0.001.

Fig. 3.

Responses to noxious heat in control and dry eye animals. A: example of an inhibitory response to noxious heat recorded in a control animal (top) and an excitatory response by the same stimulus in a dry eye animal (bottom). B: overall, mean discharge evoked during heating was greater in dry eye animals compared with control animals (n = 27 units in control animals and 22 units in dry eye animals). *P < 0.05.

Fig. 4.

Menthol-evoked discharge in control and dry eye animals. A: example of neuronal activity following application of vehicle, 100 μM menthol, and 1.0 mM menthol in a control animal. B: neuronal activity in a corneal unit following vehicle, 50 μM menthol, and 500 μM menthol in a dry eye animal. C: average response magnitude (Rmag) of units recorded in control animals calculated 0–30 s after the application of menthol. D: average Rmag 0–30 s after menthol application in dry eye animals. E: average Rmag recorded 30–120 s following menthol application in control animals. F: average Rmag calculated 30–120 s following menthol application in dry eye animals. In control animals, n = 27, 8, 11, 19, and 6 for vehicle, 10 μM, 50 μM, 100 μM, and 1 mM menthol, respectively. In dry eye animals, n = 22, 8, 17, 20, and 9 for vehicle, 10 μM, 50 μM, 100 μM, and 500 μM menthol, respectively. *P < 0.05 vs. vehicle.

Fig. 5.

Examples of the response to cooling applied 5 min after the application of vehicle (top) and 100 μM menthol (bottom). A: in a cool cell recorded from a control animal, menthol produced an increase in cold-evoked activity during the initial phasic period of cooling. B: in the dry eye animal, the same concentration of menthol inhibited the response to cooling.

Fig. 6.

Effect of menthol on the response to cooling in control (A, C, E) and dry eye animals (B, D, F). A: average discharge during the initial 30 s of cooling (phasic period) after vehicle and increasing concentrations of menthol in control animals. B: average discharge during the initial 30 s of cooling in dry eye animals. C: average peak frequency responses to cooling after vehicle and increasing concentrations of menthol in control animals. D: average peak frequency responses in dry eye animals. E: average cooling thresholds in control animals after vehicle or menthol application to the cornea. F: average cooling thresholds in dry eye animals after vehicle or menthol application. The number of neurons for each group is the same as in Fig. 4. *P < 0.05 vs. vehicle.