Abstract
Purpose :
We have previously reported that depletion of corneal plasmacytoid dendritic cells (pDCs) leads to a robust loss of corneal innervation. Herein, we demonstrate neurotrophic properties of the pDC secretome by concurrent pDC depletion and topical treatment with pDC-derived supernatant. Furthermore, we characterize functional alterations of corneal nerves by ex vivo electrophysiology.
Methods :
For rescue studies, pDCs were sorted from adult C57BL/6J mice and cultured in serum-free media for 3 days, at which point supernatant was collected. Adult BDCA2-DTR mice were used for depletion of pDCs by subconjunctival injections of diphtheria toxin (DT; 30 ng/eye) at day 0 and repeated every 2 days and were compared to non-depleted controls In rescue studies, all BDCA2-DTR animals received DT injections every 2 days and were topically treated with pDC supernatant or serum-free media (control) 3 times/day for one week. Corneas were stained with βIII-tubulin, imaged by confocal microscopy, and nerve density quantified using NeuronJ. For electrophysiology studies, recordings were performed ex vivo on enucleated eyes using a glass recording micropipette at the ocular surface in a recording chamber perfused with 34°C physiologic solution. The activity of nerve terminal impulses (NTIs) was recorded at baseline and in response to cooling and warming ramps. Analyses were performed in Spike2 software.
Results :
pDC supernatant treatment resulted in a significant increase in central cornea innervation compared to controls (131.49 + 10.49 vs 67.77 + 6.80 mm/mm2; p<0.01). This effect was due to a rescue of the subbasal plexus (100.10 + 8.92 vs 30.34 + 7.40 mm/mm2; p<0.001), as the stromal nerve density did not differ between groups. NTI activity of high-threshold cold receptors (HT-CRs) was altered by pDC depletion. Although background activity and cooling responses did not differ between groups. Interestingly, the cooling threshold, the temperature at which HT-CRs respond, was reduced in the pDC-depleted group (22.53 + 0.54°C vs 27.97 + 0.46°C).
Conclusions :
These findings demonstrate that corneal pDCs are crucial for both the function and maintenance of corneal nerves. The structural and functional alterations following pDC depletion, as well as rescue of corneal innervation by pDC supernatant, may have relevant consequences for ocular diseases in which sensory abnormalities predominate.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.