Kinetics of corneal leukocytes by intravital multiphoton microscopy

Abstract

Corneal immune privilege is integral in maintaining the clear avascular window to the foreign world. The presence of distinct populations of corneal leukocytes (CLs) in the normal cornea has been firmly established. However, their precise function and kinetics remain, as of yet, unclear. Through intravital multiphoton microscopy (IV-MPM), allowing the means to accumulate critical spatial and temporal cellular information, we provide details for long-term investigation of CL morphology and kinetics under steady state and following inflammation. Significant alterations in size and morphology of corneal CD11c dendritic cells (DCs) were noted following acute sterile inflammation, including cell volume (4364.4 ± 489.6 vs. 1787.6 ± 111.0 μm, P < 0.001) and sphericity (0.82 ± 0.01 vs. 0.42 ± 0.02, P < 0.001) compared with steady state. Furthermore, IV-MPM analyses revealed alterations in both the CD11c DC and major histocompatibility complex class II (MHC)-II mature antigen-presenting cell population kinetics during inflammation, including track displacement length (CD11c: 16.57 ± 1.41 vs. 4.64 ± 0.56 μm, P < 0.001; MHC-II: 9.03 ± 0.37 vs. 4.09 ± 0.39, P < 0.001) and velocity (CD11c: 1.91 ± 0.07 μm/min vs. 1.73 ± 0.1302 μm/min; MHC-II: 2.97 ± 0.07 vs. 1.62 ± 0.08, P < 0.001) compared with steady state. Our results reveal in vivo evidence of sessile CL populations exhibiting dendritic morphology under steady state and increased velocity of spherical leukocytes following inflammation. IV-MPM represents a powerful tool to study leukocytes in corneal diseases in context.-Seyed-Razavi, Y., Lopez, M. J., Mantopoulos, D., Zheng, L., Massberg, S., Sendra, V. G., Harris, D. L., Hamrah, P. Kinetics of corneal leukocytes by intravital multiphoton microscopy.

Intravital Multiphoton Microscopy of the Ocular Surface: Alterations in Conventional Dendritic Cell Morphology and Kinetics in Dry Eye Disease

“…Animals were anesthetized, and body and ocular surface temperatures were tightly regulated as previously described (41,42). For central corneal IV-MPM, following intraperitoneal injection of ketamine (100 mg/kg), xylazine (20 mg/kg), and acepromazine (3 mg/kg) mixture, mice were placed on a custom and tiltable stereotactic stage to focus on the cornea and limbus.…”

“…Topical proparacaine hydrochloride (0.5%) and Genteal ophthalmic lubricant gel (Alcon, Fort Worth, TX, United States) were applied to the examined eye and a 5-0 non-absorbable silk suture (Surgical Specialities Corporation, Reading, PA, United States) was placed around the eye to prevent eye closure, blinking, and nystagmus while allowing the normal flow of blood to the eye. To maintain ocular surface temperature and moisture, a sealed chamber was created around the exposed portion of the eye using high vacuum grease (Dow Corning, Midland, MI, United States) and plastics placed on the grease as previously described (41). Genteal gel was added to the chamber, and hot water circulated through the tubing and chamber assembly to maintain a temperature range within 2 • C of the physiological ocular surface temperature (34 • C; range, 33-35 • C).…”

“…An Ultima Multiphoton Microscope System (Bruker, Fitchburg, WI, United States) equipped with two MaiTai Ti/Sapphire DeepSee lasers (Newport Spectra-Physics, Irvine, CA, United States), allowing for simultaneous coaxial 850 and 900 nm illumination to achieve two-photon excitation and second harmonic generation, was utilized for all experiments. Using a 20 × −1.0 NA (Olympus XLUMPLFLN, Tokyo, Japan) water immersion objective, scans of the ocular surface were taken every 30-60 sec over a period of up to an hour with 512 × 512 resolution and twofold line averaging, as previously described (41).…”

Melanoblasts Populate the Mouse Choroid Earlier in Development Than Previously Described

“…An analysis of cell parameters were performed as previously described. 25 In short, after optical removal of the RPE, a surface was created using the surface tool within Imaris software, allowing for analysis of cell-surface area (mm 2 ) and volume (mm 3 ) for MB/melanocytes (TRP2 + ) and myeloid (Iba-1 + ) cell populations in the developing choroid.…”

Tunneling Nanotubes and the Eye: Intercellular Communication and Implications for Ocular Health and Disease

“…The transparent tissue environment of the corneal stroma, with its unique arrangement of collagenous lamellae and absence of pigmentation, likely enabled the first visualization of these elusive cellular structures in a mammalian tissue. Unlike other tissues such as the skin, where resident tissue macrophages and dendritic cells are abundant (approximately 1000 cells/mm 2 ) [41], the density of immune cells in the cornea is comparatively low (100-150 cells/mm 2 ) [42]. Thus, intercellular communication between widely spaced cells in the cornea may be facilitated by the presence of long cellular extensions.…”