The in vivo exposure system has been previously described in detail . Therefore, configuration of the exposure system is only briefly described here. Figure 1 depicts a block diagram of the system which comprises power sources, i.e., a signal generator (75 and 95 GHz) or a signal generator and amplifier (40 GHz), directional couplers, power sensors, and a power meter (E4417A, Agilent Technologies). Power sensors A and B, shown in Fig. 1, are used to measure incident power to the antenna and reflected power from the antenna, respectively, via directional couplers. A spot-focus-type lens antenna, i.e., a conical horn antenna with a ϕ-15-cm lens, was used to ensure localization to the eye. By using this equipment, exposure of the skin, i.e., upper and lower eyelids surrounding the eye tissue, to MMW can be avoided preventing burns; facial burns are a limitation in results of MMW exposure studies due to variation in individual values of ocular damage . The level of exposure was calculated as the spatially averaged incident power density over a circular region of 13 mm in diameter, the average size of the corneal region in Dutch-belted rabbits. The special profiles of incident power density radiating from the antenna aperture were measured using open-ended waveguide probes .
All animal experiments were conducted in accordance with the animal study guidelines of Kanazawa Medical University (Kahoku, Japan) and the ARVO (Association for Research in Vision and Ophthalmology) statement for the use of animals in ophthalmic and vision research .
One hundred and thirty male Dutch-belted pigmented rabbits (12–14 weeks old, 1.9–2.2 kg) were purchased from Sankyo Labo Service Co., Inc. (Toyama, Japan) and kept with unrestricted access to food and water. At baseline, all rabbit eyes were examined using a SL-130 slit-lamp microscope (Zeiss, Tokyo, Japan) to ensure absence of abnormalities in the anterior segment, and each eye was photographed.
Each rabbit was injected intramuscularly (IM) with a solution containing 0.8–1.0 mg/kg of medetomidine hydrochloride (Domitor, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) to induce general anesthesia, and immobilized in an acrylic rabbit restrainer, specially constructed for studies of exposure to MMW . Immediately before MMW exposure, 2% lidocaine hydrochloride topical anesthetic (Xylocaine 2%; AstraZeneca, Osaka, Japan) was administered to each eye. The upper and lower eyelids were held open with tape. Because anesthesia suppressed blinking, saline drops, pre-warmed to 35–37 °C, were administered to the eyes as necessary to prevent damage to corneal epithelial cells resulting from corneal desiccation.
Following each ocular examination, all rabbits were administered topical ofloxacin ointment (Tarivid; Santen Pharmaceutical Co., Ltd., Osaka, Japan) to prevent secondary infection. Temperature and humidity during exposure were maintained at 24 ± 2 °C and 60 ± 10%, respectively, using an air conditioner and dehumidifier. Anesthesia was reversed with 0.8–1.0 mg/kg IM atipamezole hydrochloride (Antisedan, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) to help the rabbits’ recovery.
The center of the corneal surface of each rabbit’s right eye was positioned 135 mm from the antenna aperture and on the line of maximum radiation of the antenna [7, 8]. The exposure point was set using red and green laser pointers on a target , and the right eye of 105 rabbits was exposed to continuous MMW of 10–600 mW/cm2 (40 GHz), 50–300 mW/cm2 (75 GHz), or 50–300 mW/cm2 (95 GHz) for 6 min. The left eye of each rabbit was unexposed and regarded as a control eye.
To assess the ocular effects of exposure to MMW for 30 min, the right eye of another set of 21 rabbits was exposed to 75 GHz (10–300 mW/cm2) MMW, and the left eye of each of these rabbits was treated as non-exposed control eyes. All other experimental conditions were identical to those in rabbit eyes exposed to MMW for 6 min.
In order to assess the effects of sham exposure for 30 min or exposure to very low intensity infrared (IR) irradiation, another set of four rabbits was used. The right eye of each of these was sham exposure which involved the power supply of all exposure systems set to the ON state, but the signal generator did not feed the system with the (MMW) signal. The left eye of each of these was exposed with a 60-W desk lamp, such that the corneal surface temperature reached about 38 °C (IR exposure). Ocular conditions were assessed prior to exposure and 10 min and 1 day after exposure. Other experimental conditions were identical to those in rabbit eyes exposed to MMW for 6 min.
Corneal Surface Temperature
Corneal surface temperatures were recorded in MMW and IR exposed eyes at 5 s prior to exposure and at 5 s before the end of MMW exposure using a thermography camera (R300, NEC Avio, Tokyo, Japan). The same measurements were taken in control and sham exposure eyes.
Examination of Ocular Injury
The anterior segment was evaluated before IR and MMW exposure, and at 10 min, and 1, 2, and 3 days after exposure. Corneal epithelial damage was observed by slit-lamp microscopy using a modified method involving fluorescein staining of damaged corneal epithelial cells . Briefly, rabbit eyes were gently washed with saline, and fluorescein solution (0.05%, 25 μl) was instilled into the cul-de-sac with a micropipette. After a single blink, excess fluorescein was washed out with saline, and images of the anterior segment were recorded with a slit-lamp microscope, following excitation with blue light and monitoring with green light using appropriate filters (excitation light cutting filter), corneal cross-sectional thickness was measured and recorded by optical coherence tomography (OCT, Zeiss model 5000, Tokyo, Japan), and corneal opacity was assessed using a slit-lamp microscope.
Categorization of Corneal Epithelial Injury
Corneal epithelial injury was defined as a round area of epithelial injury in the central pupillary zone of exposed eyes, with no similar injury observed in unexposed eyes. Other types of corneal epithelial injury, such as desiccation of the cornea (i.e., dry eye) and mechanical damage, were excluded.
The probability of corneal damage at 1 day after exposure depending on the power densities to different frequencies of MMW was evaluated by maximum likelihood estimation with probit analysis . Morphological changes in the cornea were assessed by slit-lamp microscopy, including fluorescein staining, and optical coherence tomography. Ocular disorders, including corneal epithelial disorders, corneal opacity, and corneal edema, were determined by observation at 1 day after exposure. The dose-response relationship between corneal injury and range of power density at each frequency was evaluated by fitting with a cumulative lognormal distribution function for probit analysis using R Ver 3.3.3 software . The MMW power density indicating the probability of eye damage was defined as damage dose (DD) and was derived from the best-fit probit function.