Safety profiles of terahertz scanning in ophthalmology

Abstract

Terahertz (THz) technology has emerged recently as a potential novel imaging modality in biomedical fields, including ophthalmology. However, the ocular biological responses after THz electromagnetic exposure have not been investigated. We conducted a rabbit study to evaluate the safety profiles of THz scanning on eyes, at a tissue, cellular, structural and functional level. Eight animals (16 eyes) were analysed after excessive THz exposure (control, 1 h, 4 h, and 1 week after continuous 4-h exposure; THz frequency = 0.3 THz with continuous pulse generated at 40 µW). We found that at all the time points, the corneas and lens remained clear with no corneal haze or lens opacity formation clinically and histopathologically. No thermal effect, assessed by thermographer, was observed. The rod and cone cell-mediated electroretinography responses were not significantly altered, and the corneal keratocytes activity as well as endothelial viability, assessed by in-vivo confocal microscopy, was not affected. Post-exposed corneas, lens and retinas exhibited no significant changes in the mRNA expression of heat shock protein (HSP)90AB1), DNA damage inducible transcript 3 (DDIT3), and early growth response (EGR)1. These tissues were also negative for the inflammatory (CD11b), fibrotic (fibronectin and α-smooth muscle actin), stress (HSP-47) and apoptotic (TUNEL assay) responses on the immunohistochemical analyses. The optical transmittance of corneas did not change significantly, and the inter-fibrillar distances of the corneal stroma evaluated with transmission electron microscopy were not significantly altered after THz exposure. These results provide the basis for future research work on the development of THz imaging system for its application in ophthalmology.

Figure 1

Clinical observation with slit lamp biomicroscopy (A,B) and fundus photography (C) for the control, 1 h, 4 h and 1 week after 4 h-exposure groups, respectively. Spectrum-wide transmittance trends of the corneas in different groups were also shown (D). The corneas remained clear without corneal haze formation (A1–A4), the lens were clear with no opacity (B1–B4), and there were no signs of vitritis, retinitis or optic neuropathy (C1–C4).

Figure 2

Representative IVCM micrographs at anterior stroma, posterior stroma and endothelial layer, after different periods of THz exposure (A). There were no significant changes in the mean intensity of stromal keratocytes reflectivity (B) and corneal endothelial density (C) when comparing to controls, at different time points. Error bars represent standard deviations.

Figure 3

Representative ASCOT pictures for the control (A), 1 h (B), 4 h (C) and 1 week after 4 h-exposure groups (D). The bar graph showed that the CCT was at a consistent level over time after exposure (E). Error bars represent standard deviations.

Figure 4

Representative thermographic maps showing the ocular surface temperature for the control (A), 1 h (B), 4 h (C) and 1 week after 4 h-exposure groups (D). The temperature was lower in the central area than that in the peripheral cornea, but no significant change was observed after different periods of exposure (E).

Figure 5

ERG responses after different duration of THz exposure. There were no significant changes in the scotopic a-wave (A) and b-wave (B) amplitudes after exposure compared to controls.

Figure 6

Histological sections with H&E staining presented that no inflammatory cell infiltrates or fibrotic reaction in the corneal stroma (A1–A4), and no retinal pathology such as gliosis, inflammation or degeneration of photoreceptors, were seen in the retinas (B1–B4), for the control, 1 h, 4 h and 1 week after 4 h-exposure groups, respectively. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL; outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. After THz exposure, cataractogenesis was not detected. The crystalline lens fibers remained concentric and intact without disruption or liquefaction thus demonstrating no cortical cataract formation (note: artificial disruptions were seen due to processing artefacts). No increased coloration of the nuclear lens was seen thus excluding nuclear sclerosis (C1: control, C2: 1 week after 4-h exposure). Scale bar: 100 μm.

Figure 7

There was no expression of CD11b (A) and HSP-47 (B) in corneas, lens epithelium and retinas at different time points. IPL: inner plexiform layer; ONL: outer nuclear layer. Nuclei were counterstained with DAPI (blue). Scale bar 100 μm.

Figure 8

Immunohistochemical analysis on fibronectin showed negative staining in corneas, lens epithelium and retinas at all time points (A). Staining for α-SMA was present at Bruch’s membrane and sclera with a comparable extent in all eyes (B). IPL: inner plexiform layer; ONL: outer nuclear layer. Nuclei were counterstained with DAPI (blue). Scale bar 100 μm.

Figure 9

There were no TUNEL-positive staining cells at all time points, in corneas, lens epithelium and retinal layers. IPL: inner plexiform layer; ONL: outer nuclear layer. Nuclei were counterstained with DAPI (blue). Scale bar 100 μm.

Figure 10

Transmission electron micrographs of the corneas showing transverse section of collagen fibrils (A, scale bar 200 nm) and keratocytes (B, scale bar 1 μm) for the control , 1 h , 4 h and 1 week after 4 h-exposure groups (A1–4 and B1–4, respectively).

Figure 11

Gene expression in corneas (A), lens (B) and sclero-retinal tissues (C) measured for different groups using qRT-PCR. The mRNA expression fold values (2^−ΔΔCT) were measured and normalized to that of the control group. There were no significant changes in the expression of HSP90AB1, DDIT3 and EGR1 in all the exposure groups.