In vivo examinations of the dendritic cell dynamics in the human cornea

Purpose : Dendritic cells (DC) can be imaged in the cornea with in vivo confocal laser scanning microscopy. The activation state of the cells has so far been differentiated based on cell size and dendrite number. Nevertheless, the cell phenotype varies greatly, limiting this method's reliability. The cell migration velocity, however, also reflects the activation state of DCs. Mature DCs move considerably slower than immature ones. The differentiation of cell stages based on their migration velocity offers a new approach to describing the immunological status of the cornea.

Methods : In order to determine the velocity of the DCs, large-area SNP mosaics were generated using in vivo confocal laser scanning microscopy at different time intervals (1 - 60 min). Since subbasal nerve migration can be neglected in these short intervals, the mosaics could be aligned with respect to each other based on their nerve structures. The ImageJ plugin "TrackMate" was used to manually track cell movements within the image series and calculate the migration velocity of individual DCs. Further, the optimal time intervals for reliable cell tracking were investigated.
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Results : At 1 min intervals, the movement distance was in the order of the measurement and alignment, preventing reliable distance measurements. With a time interval of more than 30 min, an unambiguous tracking of the immature DCs between two mosaic images was no longer reliably possible. The optimal time interval for tracking immature DCs was between 10 and 30 min, with the exception of very slow mature DCs. Previously tested measurement intervals of up to 4 h did not allow a precise assessment of the migration velocity of these cells.

Conclusions : With the developed method, the dynamics of the DCs can be examined in detail. Movement patterns can be assigned to the degree of DC maturity in corresponding time scales between 10 min and 24 h. Exact velocity measurements of immature DCs should be carried out at 10 - 30 min intervals. Larger measurement intervals (e.g., 24 h) should be selected for mature DCs. However, the spatial overlay of the registered mosaic images would have to be changed to static structures such as pressure-induced corneal tissue folds or nerve entry points, as the subbasal nerves are no longer usable as registration reference points due to their own migration. Ideal time intervals for mature cells are the subject of current research.