Slit lamp

Male and female 10–12 week old mice underwent laser treatment as previously described^{23 (link)}. Briefly, mice were anesthetized with ketamine/xylazine (Akorn, Lake Forest, IL) and received 1 mg/kg subcutaneous injection of Meloxicam (Henry Schein Animal Health, Melville, NY). Eyes were anesthetized, dilated, and a cover slip was coupled to the cornea with Gonak (Akorn) for slit lamp biomicroscopy and laser. Four (immunofluorescence) or eight (flow cytometry, to increase inflammatory cell numbers) focal burns (75 μm, 100–120 mW, 100 ms) were administered in each eye using a 532 nm argon ophthalmic laser (IRIDEX, Mountain View, CA) via a slit lamp (Zeiss, Oberkochen, Germany).

The allergic condition was confirmed using a skin-prick test (SPT). Briefly, the outer epithelial layer of the forearm skin was scratched with a lancet without inducing bleeding to avoid false-positive results. Then, the allergen (Dermatophagoides pteronyssinus, Der p) or the control (histamine) were instilled over the skin. The SPT was read between 15–20 min after the allergen skin challenge; positive results were considered for Der p (wheal ≥ 3 mm diameter) compared with the histamine control. In addition, the serum total IgE (tIgE) was determined in all subjects included in the study.
The ophthalmological investigation was systematically documented by biomicroscopy, according to [39 (link)]. Schirmer test was performed without instillation of topical anesthetic, folding a Schirmer paper strip (AMCON Tear Flow Test Strips Nomax, Inc, MO, USA) over the temporal one-third of the lower lid margin inserted in the conjunctival sac for 5 min to measure the production of tears in millimeters. The tear break-up time (TBUT) was evaluated observing the cornea under a slit-lamp (Carl Zeiss, Meditec Inc. CA, USA) with a cobalt blue filter. TBUT was considered as the time required for the appearance of the first break in the tear film after blinking with fluorescein staining (Bio Glo Fluorescein Ophthalmic Strips USP, Gujarat, India), recording the mean value of three measurements.

On the 7 and 14th days after alkali injury, pictures of the corneas were taken by using a digital camera (ORCA ER; Hamamatsu, Japan), which was connected to the slit lamp (Zeiss, Jena, Germany). The CNV area was quantified using the following equation: area (mm²) = C/12 × 3.1416 × [R²-(R-L)²] (20 (link)), where C is the clock hour of CNV, R is the radius of the mouse cornea (R = 1.5 mm) and L is the average vessel length sprouting from the limbal vasculature. The percentage of CNV was the CNV area of each eye compared to the area of the entire cornea.

We examined participants on a slit lamp (Carl Zeiss, Oberkochen, Germany). Ophthalmodynamometry (Meditron GmbH, Poststrasse, Völklingen, Germany) was used to measure the force (ophthalmodynamometric force [ODF; Meditron Unit {mu}]) applied to the eye. The Meditron Ophthalmodynamometry is constructed of a conventional Goldmann three-mirror contact lens that enables direct visualization of the posterior pole, and a force transducer ring attachment made of flexible copper-beryllium trabeculae.² (link) Force applied to the eye was measured in Meditron Unit (mu), where 1 mu = 3.33 g of force. We calculated the induced IOP using our previously described formula: Induced IOP = 0.89 × ODF + baseline IOP.²⁵ (link)
The ophthalmodynamometer was calibrated by horizontal placement of the device on a flat surface before use. A range of ODF was applied to the eye from 0 to 50. Retinal video of the optic nerve and peripapillary region was simultaneously captured (Canon 5D Mark III, Japan), mounted on a slit lamp. The video also recorded the audible beeping from the pulse oximeter (Nellcor N65; Covidien, Mansfield, MA) applied to the participants’ index fingers, thereby allowing for synchronization of retinal vascular pulsation with cardiac cycles. This technique was used to measure PPG estimates of blood column pulse amplitude²⁸ (link) (Figs. 1A, 1B, Figs. 2A1–F1).

The CNV lesions were induced in rat eyes by laser photocoagulation as previously described [26 (link)]. Briefly, Brown Norway rats were anesthetized with an intramuscular injection of a mixture of 2% xylocaine (0.15 mL/kg body weight, Astra, Astra Sodertalje, Sweden) and ketamine (50 mg/kg body weight, Parke-Davis, Morris Plains, NJ, USA). Pupils were dilated with 1% tropicamide (Alcon Laboratories, Fort Worth, TX, USA). A piece of cover glass served as the contact lens to improve the visibility of the fundus. Argon laser (Novus Omni; Coherent, Palo Alto, CA, USA) irradiation was delivered through a slit lamp (Carl Zeiss, Oberkochen, Germany). Laser parameters: spot size of 50 µm, power of 400 mW, and exposure duration of 0.05 s. Disruption of Bruch’s membrane was detected by the emergence of a bubble at the center of photocoagulation in the laser spotted zone. Six lesions were generated in each eye at the 1, 3, 5, 7, 9, and 11 o’clock positions located at equidistance from the optic disk and between the major retinal vessels.