Article Title: Abnormal activity of corneal cold thermoreceptors underlies the unpleasant sensations in dry eye disease
Figure Lengend Snippet: Intracellular calcium responses of trigeminal sensory neurons innervating the cornea of control and 4-week tear-deficient guinea pigs. (A) Trigeminal ganglion (TG) neurons innervating the cornea were retrogradely labeled with FM 1-43 applied on the corneal surface 6 days earlier and identified with fluorescence microscopy. Only one or 2 corneal neurons were found per culture plate. Photographs show (from left to right) bright field, FM 1-43 fluorescence when excited with 470-nm light and merged images. (B–E) Ratiometric fluorescence changes (expressed as the ratio of fluorescence of Fura-2 when excited at 340 and 380 nm, F 340 /F 380 ) recorded simultaneously in the same culture plate from several fresh cultured TG sensory neurons from control (B and D) and tear-deficient (C and E) animals, in response to a cooling ramp and to the addition to the bath solution of 100 μM menthol, 100 μM cinnamaldehyde, 1 μM capsaicin, and 30 mM K + ; the bath temperature was simultaneously recorded (lower channels). The blue lines correspond to recordings from TG neurons innervating the cornea. Neurons responding to cooling and menthol were classified as cold thermosensitive neurons (B and C); neurons not responding to cooling stimulus but responding to capsaicin and cinnamaldehyde were classified as putative polymodal nociceptive neurons (D and E). (F) Box plots showing [Ca 2+ ] i peak response to cooling ramps (peak value of the F 340 /F 380 ratio during the ramp) in cold sensitive corneal neurons of control (n = 36; median = 0.40, range = 0.21-1.87) and tear-deficient animals (29; median = 0.55, range = 0.21-2.10). Horizontal line, median; bar, 25th and 75th interquartile range; whiskers, 10th and 90th percentiles. (G) Box plots showing [Ca 2+ ] i threshold response to cooling ramps (temperature decrease required to evoke the [Ca 2+ ] i response) in the same population of corneal neurons of control (n = 36; median = −16.06, range = −4.79 to −20.59) and tear-deficient animals (n = 29; median = −17.23, range = −5.94 to −23.46).
Article Snippet: Labelling of trigeminal ganglion corneal neurons In guinea pigs anesthetized with 90 mg/kg ketamine and 5 mg/kg xylazine (i.p.), a 6 mm-diameter piece of Spongostan film (Ferrosan A/S, Soeborg, Denmark) saturated with the cell stain FM 1-43 (Molecular probes; Invitrogen) at 5 mM in saline solution was carefully placed in the center of the cornea to retrogradely label TG sensory cell bodies whose axons innervate the cornea.
Techniques: Labeling, Fluorescence, Microscopy, Cell Culture