Assessment of the general clinical condition and functional properties of the eyes of rabbits after THz irradiation

Abstract

THz radiation is increasingly used for diagnostics in medicine. As technology utilizing THz radiation continues to develop rapidly, it is becoming increasingly important to consider its biological effects and establish safe exposure standards and parameters. The paper presents data on the clinical status and functional properties of the anterior and posterior structures of the eyes of rabbits after THz irradiation at the frequency of 2.3 THz. Terahertz radiation was generated at Novosibirsk Free Electron Laser (NovoFEL) at “Siberian Synchrotron and Terahertz Radiation Centre” (Budker Institute of Nuclear Physics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia). The exposure durations used were 15 and 30 minutes. Intensity ranges were I1=0.012 mW/cm², I2=0.018 mW/cm², and I3=0.024 mW/cm². The study investigated the effects of various time and power irradiation protocols on the California rabbit’s eyes and after a period of one month, but no significant clinical or functional alterations were observed in response to the established intensity protocols. However, the study identified statistically significant changes in corneal hydration and endothelial cell density over time, particularly under protocols with 15- and 30-minute exposures. A negative correlation was found between endothelial cell density and corneal thickness (r=-0.36, p=0.042), suggesting that a reduction in the endothelial cell pool may be associated with increased corneal thickness. These changes were subclinical and did not lead to clinically significant pathological changes in the cornea. There were no signs of ASOCT (anterior segment-optical coherence tomography) hyperreflectivity. THz radiation with parameters listed above of 2.3 THz and an intensities of 0.012-0.024 mW/cm² for 30 minutes has been shown to be conditionally safe for the structures of the rabbit eye. However, the detected subclinical corneal changes require further study to determine safe exposure limits.

1. Introduction

The THz region of the electromagnetic spectrum is currently one of the most promising areas for the study of physical and biological phenomena. This region is located between the infrared and microwave ranges [1]. THz radiation exhibits high sensitivity to water vapor content in the air, which can interfere with THz spectroscopy, and to liquid water (free and bound) in biological samples [2], making it a major source of contrast in THz imaging and diagnostics [3,4]. The frequency of THz waves spans from 0.1 to 10 THz, with wavelengths ranging from 3 mm to 30 µm [5]. These waves can penetrate certain materials, such as biological tissues and non-metallic substances, enabling the detection and analysis of structural and compositional information. Due to their high frequency, they are applied in engineering communication [6], security systems [7], and medical imaging, such as terahertz imaging and spectroscopy [8]. They are also utilized in space measurement systems [9]. In medicine, particularly in ophthalmology, THz radiation is primarily explored in research settings for its potential diagnostic applications, although its clinical use has not yet been widely established [10,11]. Specifically, the use of different THz frequency ranges in medical spectroscopy has not been fully explored. The safety of terahertz spectroscopy, including its application for measuring corneal thickness, remains insufficiently understood. This method also enhances the ability to assess and comprehend the dynamics of tear film stability in patients with dry eye syndrome [12–16].

However, as THz radiation technology continues to advance rapidly, it becomes increasingly important to establish safe standards and exposure parameters [17–19]. THz radiation is non-ionizing, and the energy of intermolecular bonds in higher order biopolymer structures falls within the range of photon energies in the THz region. Consequently, THz radiation has the potential to influence both the entire body and intracellular processes [20]. The widespread applications of THz waves necessitate a careful evaluation of their biological effects. It is essential to standardize safe parameters for THz exposure duration and power, as well as to implement protective measures against potential harmful effects. When assessing the possible adverse effects of electromagnetic radiation at any wavelength, special attention is directed towards its impact on the skin and the cornea [21]. The cornea is therefore a critical target for studying the biosafety of THz radiation. Its properties, including transparency, high water content, and precise morphological structure, make it an effective model for research [22–24]. THz radiation can be used to influence corneal tissue and evaluate changes in its functional properties, such as hydration levels, endothelial barrier integrity, epithelialization rates, and other parameters [25]. A study conducted at ITMO University demonstrated that THz radiation with power levels ranging from 26 to 60.8 nW and frequencies between 0.1 and 1.8 THz accelerated epithelialization of rabbit corneas following iatrogenic erosion, though higher radiation powers were associated with a deceleration of this process [26]. Conversely, Safonova et al. investigated the effects of THz radiation under different conditions, utilizing frequencies of 0.3 to 0.4 THz and power densities ≤ 30 nW/cm² to evaluate changes in corneal hydration and tissue safety. Their findings revealed reversible morphological alterations confined to the anterior corneal epithelium. Unlike the ITMO study, which focused on epithelialization rates post-erosion, Safonova et al. examined intact corneal tissues using laser confocal microscopy and histological methods, highlighting the distinct aims and experimental parameters of the two studies [27].

It has been demonstrated that nine hours of exposure to radiation at a frequency of 10 THz causes alterations in the gene expression of mesenchymal stem cells in mice [28]. This may be attributed to the high sensitivity of these cells to external stimuli. The findings of this study highlight the necessity for further research into the effects of THz radiation on biological systems [29]. Other studies report the absence of significant biological effects from THz radiation. For example, the experimental work by S. Koyama et al. showed that prolonged 24-hour exposure to THz radiation at a frequency of 0.12 THz and an intensity of 5 mW/cm² did not induce a genotoxic response or pathological changes in isolated corneal epithelial tissue [30]. Y. Liu and colleagues confirmed the safety of THz eye irradiation, which was administered for four hours to 16 rabbit eyes at a power of 40 mW and a frequency of 0.3 THz, with no significant morphofunctional, biochemical, or immunohistochemical changes observed [31].

In vitro studies have demonstrated the biosafety of THz radiation under specific time and power parameters. For example, Wilmink et al. reported no significant effects on cell cycle kinetics, chromosomal DNA damage, or DNA repair activation in human dermal fibroblasts exposed to THz radiation at an intensity of up to 84.8 mW/cm², with only minor increases in heat shock protein expression attributed to photothermal effects [32]. A study on blood samples from nine healthy donors after 20 minutes of THz-radiation with an average intensity of 1 mW/cm² in the frequency range of 120 to 140 GHz found no direct chromosomal damage or alterations in the cell cycle [33]. Additionally, no genomic damage was detected in human skin cells exposed to THz radiation at frequencies up to 2.52 THz for two to eight hours, with intensities ranging from 0.03 to 0.4 mW/cm² [34].

Moreover, the research group led by Dr. Sergey E. Peltek reported that a significant response of living systems occurs at a frequency of 2.3 THz [35–37]. Their findings indicated activation of stress response systems, disturbances in transcription regulation, and repair mechanisms, underscoring the importance of studying this frequency in humans. The potential benefits of using THz radiation are supported by ongoing research and technological advancements in THz wave technology. However, there remain unanswered scientific questions regarding the safety of this form of radiation. A review of the literature reveals a lack of studies on the in vivo biosafety of THz radiation on the eyes, particularly at frequencies above 1 THz. The aim of this study is to investigate the general clinical and ophthalmological effects of 2.3 THz at various intensity levels and exposure protocols on rabbit corneas. To assess the biosafety of THz radiation, an experiment was conducted using controlled radiation intensity levels and exposure protocols This study focuses on establishing the maximum permissible standards for the safe application of THz radiation in medicine and other fields, evaluating its effects on the cornea at varying power levels and exposure durations.

2. Materials and methods

2.1. Animals

We used eight male California rabbits, weighing between two and three kilograms and aged three months, for this experiment. These rabbits were sourced from A.V. Evdishchenko's farm in Russia and housed in the N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry of the Siberian Branch of the Russian Academy of Sciences vivarium at 20 ± 3°С, with a complete diet and air conditioning, in compliance with GOST 33216-2014, “Interstate Standard: Guidelines for the Maintenance and Care of Laboratory Animals”. All procedures adhered to the European Community Directive 86/609/EEC to ensure humane treatment of the animals. The protocol, dated December 5, 2023, was approved by the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences Ethical Committee (No. 160).

2.2. Preparation for irradiation

The rabbits underwent a 14-day quarantine period after being received. During this time, they were given routine clinical examinations, and no reduction in body weight or deviations from their normal health status were observed. The animals were then randomly assigned to study groups. The rabbits were transported in ventilated cages to the Novosibirsk Free Electron Laser (NovoFEL) at “Siberian Synchrotron and Terahertz Radiation Centre” (Budker Institute of Nuclear Physics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia). To anesthetize and sedate the animals before irradiation, propofol was administered intravenously at a dose of 2.5 mg/kg, allowing for the placement of a laryngeal mask. Isoflurane was then administered at a concentration of 4-5% vol., which was reduced to 1% vol. after ten minutes.

2.3. Study design and group selection

The source of THz radiation used in this study was the Novosibirsk Free Electron Laser (NovoFEL), located at the Siberian Center for Terahertz and Synchrotron Radiation. The experiments were conducted at the “Chemical, Physical, and Biological Research Station at the FEL” workstation [38].

A single-pass accelerator-recuperator with a 12 MeV electron energy was used for the generation THz radiation. THz irradiation was applied at a wavelength of 130 µm (2.3 THz). The distribution of intensities within the THz beam followed a 2D Gaussian profile with σ₁ = 0.666 cm, and σ₂ = 1.5 cm define the beam's width along these axes, as described by Eq. (1). The THz beam has an ellipsoidal shape (x = 1.88 cm; y = 4.5 cm, S = 6.64 cm²) at the irradiation point. The eye temperatures for the rabbits were 38, 40, and 42°С, using three laser Gaussian maximum intensities: I₁ = 0.012 mW/cm², I₂ = 0.018 mW/cm², and I₃ = 0.024 mW/cm² respectively. All temperatures remained stable during exposure. The initial temperature of both eyes before irradiation was 33–34 °C. Corneal heating was detected within three to five minutes, and temperature control was performed using a high-precision thermal imaging system, TKVr-SVIT101 (Rzhanov Institute of Semiconductor Physics SB RAS, Novosibirsk, Russia), with an accuracy of up to 0.03°C. The NovoFEL optic schematic is presented in Fig. 1, while the irradiation station for rabbits is depicted in Fig. 2. NovoFEL parameters are shown in Table 1.

Fig. 1.

Scheme of the Novosibirsk Free Electron Laser (NovoFEL) at the “Siberian Synchrotron and Terahertz Radiation Centre” (Budker Institute of Nuclear Physics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia).

Fig. 2.

Irradiation of a rabbit on a laser installation on the “Chemical, physical and biological research Station at the NovoFEL”.

Table 1. The main parameters of the Novosibirsk Free Electron Laser [38].

Parameter	Value
Frequency, THz; wavelength, µm	3.3–0.75 (90-400)
Pulse repetition frequency, MHz	5.6–22.4
Pulse duration, ps	40–100
Average power, W	≤ 500
Peak power, MW	≤ 0.8
Minimum relative line width, %	0.2–2

The rabbits were divided into four groups, with two rabbits in each group, corresponding to 16 eyes in total. Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4). The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes. Each group thus included two eyes exposed to the same intensity but with different durations.

$$I=I_o\ast e^{\frac{-x^2}{2{{\sigma }_1}^2}}\ast e^{\frac{-y^2}{2{{\sigma }_2}^2}}$$ (1)

2.4. Analysis of the effect of THz-irradiation exposure and the effectiveness of recovery after irradiation

To evaluate functional changes in the cornea, several instrumental measurements were performed, including a general examination using a slit lamp and fluorescein dye, Schirmer's test, eye tonometry, ophthalmoscopy, and endothelial microscopy. Additionally, computed optical tomography was conducted. To monitor the dynamics of the anterior segment of the eye following radiation exposure, four time points were selected: before irradiation (time point 0), the first day after exposure (time point 1), one week after exposure (time point 2), and four weeks after exposure (time point 3). The study design is illustrated in Fig. 3.

Fig. 3.

Experimental study design.

2.5. Clinical evaluation with slit lamp biomicroscopy and fundus photography

A general ophthalmological examination (biomicroscopy) was conducted on the rabbits using a Kowa SL-17 slit lamp (Kowa Company, Japan). Two drops of 1% fluorescein solution were instilled into the conjunctival sac to detect corneal defects. The eye was then rinsed thoroughly with a large volume of 0.9% isotonic sodium chloride solution. Using a cobalt filter, the anterior segment was illuminated both directly and laterally during the assessment. A portable fundus camera, the MiiS Horus Scope DSC 200 (Miis, Taiwan), was used to capture images of the fundus in the rabbits.

2.6. Schirmer's test

To evaluate overall tear production, Schirmer test strips (MSD Animal Health) were used. The strip was placed in the conjunctival sac of the lower eyelid for 60 seconds, the standard duration for assessing tear production. After 60 seconds, the length of the wetted section of the test strip was immediately measured in millimeters and recorded. Testing was conducted prior to the ophthalmological examination at all time points.

2.7. Tonometry

Intraocular pressure (IOP) was measured using a rebound tonometer (Tonovet Plus; Icare, Finland). Measurements were taken the day before radiation exposure, the day following radiation, and at all other standard time points. The tonometer was positioned to measure strictly the central portion of the rabbit's cornea, held vertically in relation to the corneal surface. To assess IOP, a small probe was rapidly moved from a fixed distance towards the cornea, made brief contact, and then rebounded back to the device. Eyes with higher IOP caused the probe to return more quickly than eyes with lower IOP, as evaluated by the device based on the probe’s deceleration characteristics.

2.8. Endothelial microscopy

Corneal endothelial microscopy was conducted using a Topcon SP-1P endothelial microscope (Topcon, Japan) to calculate the endothelial cell density per unit area and assess the endothelial function of the cornea at all four time points.

2.9. ASOCT evaluation (anterior segment-optical coherence tomography)

Optical coherence tomography (OCT) of the anterior segment of the eye was performed using an RTVue XR Avanti optical tomograph (Optovue, USA) to measure corneal thickness (pachymetry) at all four time points.

2.10. Examination of the complete blood count and bacteriological seeding of conjunctival flushes

Complete blood count was conducted for all rabbits both before and immediately after following radiation exposure. Conjunctival flushes were bacteriologically analyzed before radiation, immediately after, on the first day following, and one week after radiation exposure. Following a full clinical examination, all selected rabbits were confirmed to be in good health. Sampling was performed under sterile conditions. For each rabbit, two ocular-conjunctival smears (one from each eye) were obtained before irradiation, immediately after, and one week later, totaling 48 samples. The smears were directly applied to plates containing various media: blood agar, yolk-salt agar, Sabouraud agar, chromogenic agar, and incubated at 37 °C under aerobic conditions for the first 24 hours, then for an additional 2 to 5 days at 22 °C. Chocolate agar was also used; the smears were incubated for the first 24 hours in a 5% CO₂-enriched atmosphere, then for 2 to 5 days under aerobic conditions at 22 °C. All cultures were processed at the bacteriological laboratory of the Novosibirsk Scientific Research Institute of Tuberculosis, under the Ministry of Health of the Russian Federation.

2.11. Statistical analysis

Software tools used for data collection and analysis included IBM SPSS Released 2007 for Windows, Version 16.0 (SPSS Inc., Chicago, USA), Jamovi Version 2.5 for Windows (The jamovi project, Sydney, Australia), and Excel 2016 (Microsoft, Washington, USA). The Shapiro-Wilk test was applied to assess the normality of the distribution of the non-categorical data, revealing that most of the data did not follow a normal distribution. Consequently, nonparametric tests were used for comparative analysis. Data for quantitative indicators are presented as median and interquartile range (IQR), mean and standard deviation, minimum and maximum values, and the 95% confidence interval for the mean. The exact p-value, indicating the probability of a type I error, was reported. Quantitative variables in related groups were analyzed using either the Wilcoxon or Friedman tests. Post-hoc test results for the Friedman test were adjusted using the Durbin-Conover method. For independent samples, the Mann-Whitney and Kruskal-Wallis tests were applied, with paired comparisons and the least significant difference method used for further analysis. Results were considered statistically significant when the p-value was less than 0.05.

3. Results

3.1. Clinical evaluation with slit lamp biomicroscopy and fundus photography

Ophthalmoscopy of the anterior segment using a slit lamp showed that the cornea and lens remained transparent in animals across all experimental and control groups. There were no clinical signs of vitreitis, retinitis, or optic nerve neuropathy observed before irradiation, as well as on the first day, one week, and four weeks after irradiation. Biomicroscopy with fluorescein staining the ocular surface of rabbits (n = 16) demonstrated stable baseline conditions of the ocular surface, tear film, and epithelium before and after irradiation at all designated time points (Fig. 4(A)).

Fig. 4.

(A) - Biomicroscopy of the anterior segment of the eyes of rabbits using a Kowa SL-17 slit lamp (Kowa Company, Japan) with fluorescein staining in groups 1, 2, and 3. (B) - Fundus imaging of the posterior segment of the eyes of rabbits in groups 1, 2, and 3 using a portable fundus camera, MiiS Horus Scope DSC 200 (Miis, Taiwan), showing ophthalmoscopy results at 4 weeks. Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4). The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes.

Ophthalmoscopy of the rabbits’ fundus, performed at all standard time intervals, revealed no pathological changes in either the early or delayed post-irradiation periods. All animals exhibited the merangiotic type of fundus. The optic nerve disc was horizontally oval and located in the dorsal part of the fundus, with myelin extending laterally and medially from the disc. Physiological excavation was noted in the center, and normal-caliber arterioles and venules extended from the optic nerve disc laterally and medially, filling with blood. The choroidal vessels were of normal size and ran parallel to each other (Fig. 4(B)).

3.2. Schirmer's test

Stable tear production levels, consistent with normal values for this group of animals, were recorded for all subjects in both the experimental and control groups before irradiation and at all time points. The analysis results indicate that no statistically significant differences were found between the experimental and control groups (p > 0.05). Neither the intensity of radiation nor the duration of exposure (15 minutes and 30 minutes) influenced the results. The Schirmer test values did not vary significantly depending on the intensity level or exposure duration across groups (p > 0.05). See Dataset 1 ^{(12.4KB, xlsx)} [50] (Sheet 1) for supporting content.

3.3. Tonometry

Stable markers of intraocular pressure (ophthalmotonus) were observed in all rabbits across the experimental and control groups before irradiation and at all specified time points, consistent with normal values for this group of animals. The analysis indicated no statistically significant differences (p > 0.05) between the experimental and control groups. The findings showed no significant differences in ophthalmotonus values between groups based on exposure duration and irradiation intensity (15 minutes and 30 minutes) (p > 0.05). See Dataset 1 ^{(12.4KB, xlsx)} [50] (Sheet 2) for supporting content.

3.4. ASOCT evaluation

The assessment of corneal hydration was carried out using high-precision optical coherence tomography of the anterior segment of the eye (ASOCT) [39]. Based on the pachymetry (corneal thickness) measurements obtained from this device, we evaluated the dynamics of corneal hydration. ASOCT evaluation revealed no abnormal hyperreflectivity in the stroma (Figs. 5 and 6).

Fig. 5.

Optical coherence tomography (OCT) of the anterior segment of the eye performed using an RTVue XR Avanti optical tomograph (Optovue, USA) to measure corneal thickness (pachymetry) and detect hyperreflectivity in three experimental groups exposed to THz irradiation intensities of I₁ = 0.012 mW/cm², I₂ = 0.018 mW/cm², and I₃ = 0.024 mW/cm² over a 4-week observation period. Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4). The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes.

Fig. 6.

Optical coherence tomography (OCT) of the anterior segment of the eye performed using an RTVue XR Avanti optical tomograph (Optovue, USA) to measure corneal thickness (pachymetry) and detect hyperreflectivity in groups with 15-minute and 30-minute exposures across a 4-week observation period. The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes. All right eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2) were included in the group with 15-minute exposure. The group with 30-minute exposure included all left eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2). Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4).

A statistical analysis of the central corneal thickness measurements was conducted based on the degree and duration of THz radiation exposure. The dynamics of corneal thickness changes (measured in µm) for the two groups exposed to varying durations of THz radiation are illustrated in Fig. 7. There is a tendency for a gradual increase in corneal thickness in the group exposed to THz radiation for a shorter duration (OD, 15 minutes) (p > 0.05). A consistent, statistically significant increase in corneal thickness was observed in the group exposed for a longer duration (OS, 30 minutes). This increase becomes apparent during the first week of observation and continues progressively throughout the 4-week follow-up period.

Fig. 7.

Dynamics of changes in corneal thickness for groups with 15-minute and 30-minute exposure to THz radiation. Arrows indicate statistically significant differences within the 30-minute exposure group (p₁ = 0.047, p₂ = 0.006). Data are presented as mean and standard deviation (Durbin-Conover criterion). The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes. All right eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2) were included in the group with 15-minute exposure. The group with 30-minute exposure included all left eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2). Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4).

In conducting multiple pairwise comparisons, statistically significant changes in corneal thickness were found for two-time intervals in the 30-minute exposure group: before irradiation versus one week after irradiation, and before irradiation versus the fourth week after irradiation. The corneal thickness was 352.5 ± 35.1, 355.0 ± 35.4, and 361.8 ± 35.5 microns at the time points “0” (before irradiation), one week, and four weeks, respectively (p = 0.047, p = 0.006).

These findings suggest that exposure duration has a differential impact on the cornea; longer exposure to THz radiation likely induces a cumulative effect in corneal tissue, leading to a persistent and statistically significant increase in corneal thickness. This is in contrast to the short-term exposure group, where only a tendency for such an increase was observed.

Statistical analysis of corneal thickness changes across the three experimental groups showed no statistically significant differences (p > 0.05) in corneal thickness in any of the groups exposed to different THz radiation intensities. This outcome is likely due to the small sample size in each study group (4 eyes) and the inclusion of 50% of eyes in each group with short-term 15-minute exposure (2 eyes). The dynamics of corneal thickness changes across the three experimental groups are depicted in Fig. 8.

Fig. 8.

Dynamics of corneal thickness changes for three experimental groups with I₁ = 0.012 mW/cm², I₂ = 0.018 mW/cm², I₃ = 0.024 mW/cm² of THz exposure intensity. Data are presented as mean and standard deviation. The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes. All right eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2) were included in the group with 15-minute exposure. The group with 30-minute exposure included all left eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2). Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4).

3.5. Endothelial microscopy

Figure 9 illustrates the dynamics of changes in the density of corneal endothelial cells (cells/mm²) for the two groups with different exposure durations to THz radiation. A gradual, persistent, and statistically significant decrease in endothelial cell density was observed in both the 15-minute and 30-minute exposure groups.

Fig. 9.

Dynamics of changes in the density of corneal endothelial cells for groups with 15-minute and 30-minute exposure to THz radiation. Gray arrows indicate statistically significant differences within the 15-minute exposure group (p₁ = 0.017, p₂ = 0.003, p₃ = 0.004), black arrows indicate statistically significant differences within the 30-minute exposure group (p₁ = 0.021, p₂ < 0.001, p₃ = 0.008). Data are presented as mean and standard deviation (Durbin-Conover criterion). The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes. All right eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2) were included in the group with 15-minute exposure. The group with 30-minute exposure included all left eyes of Group 1 (n = 2), Group 2 (n = 2) and Group 3 (n = 2). Group 1 was irradiated with an intensity of I₁ = 0.012 mW/cm² (n = 4). Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4).

A statistically significant decrease in cell density (p < 0.05) was observed in the 15-minute exposure group (OD). This decrease began on the first day after exposure and persisted until the fourth week of follow-up. The initial density of endothelial cells was higher in the 30-minute exposure group (OS), and a statistically significant increase in corneal thickness was also noted, starting from the first day of observation and continuing steadily over the 4-week period.

Statistically significant changes in endothelial cell density were observed for all three-time intervals in both exposure groups (15 and 30 minutes): before irradiation versus the first day after irradiation, the first day after irradiation versus one week after, and one week after irradiation versus four weeks after. In the 15-minute exposure group, endothelial cell density was 2663.3 ± 236.6, 2485.1 ± 211.9, 2428.6 ± 154.8, and 2452.5 ± 162.5 cells/mm² at time points “0” (before exposure), first day, first week, and fourth week, respectively (p1 = 0.017, p2 = 0.003, p3 = 0.004). In the 30-minute exposure group, cell density was 2896.6 ± 288.6, 2618.6 ± 154.7, 2559 ± 114.3, and 2591 ± 124.1 cells/mm² at the same time points (p₁ = 0.021, p₂ < 0.001, p₃ = 0.008).

The statistical analysis indicates that all exposure duration groups experienced a significant and lasting reduction in corneal endothelial cell density when exposed to radiation at a frequency of 2.3 THz.

Figure 10 shows the results of the statistical analysis of changes in corneal endothelial cell density for the three experimental groups. No statistically significant changes (p > 0.05) were observed across the groups with varying THz radiation intensities, likely due to the small sample size in each group (4 eyes) and the inclusion of 50% of eyes with only short-term (15-minute) exposure (2 eyes).

Fig. 10.

Dynamics of changes in the density of corneal endothelial cells for groups with 15-minute and 30-minute exposure to THz radiation. Data are presented as mean and standard deviation. Group 1 received an intensity of I₁ = 0.012 mW/cm² (n = 4) Group 2 received an intensity of I₂ = 0.018 mW/cm² (n = 4). Group 3 was exposed to I₃ = 0.024 mW/cm² (n = 4), while the control group (Group 4) received no irradiation (n = 4). The right eye of each rabbit in each group was irradiated at the respective intensity for 15 minutes, while the left eye was irradiated for 30 minutes.

3.6. Complete blood count

The results obtained before irradiation showed that initial blood parameters in the experimental group were within the reference range (Table 2). Only an initial increase in eosinophil levels by 56.5% relative to the upper limit of the normal range was recorded, likely due to stress associated with the blood collection procedure. Eosinophil levels for some rabbit breeds range from 0 to 9.5%, so in this case, the values did not exceed the reference norms [40].

Table 2. Hematological parameters of rabbits (n = 8). The data is presented in the form of an average value and a standard error of the arithmetic mean (M ± m). Reference come from [40].

Measure	Before irradiation	Immediately after irradiation	Reference
White blood cells (WBC), 103/µl	6.16 ± 0.44	5.89 ± 0.77	4.1-10.8
Neutrophils (neu), 103/µl	1.76 ± 0.23	2.89 ± 0.60	1.1-7.4
Lymphocytes (lym), 103/µl	3.89 ± 0.26	2.34 ± 0.39	0.5-6.5
Monocytes (mon), 103/µl	0.11 ± 0.03	0.15 ± 0.03	0-3.7
Eosinophils (eos), 103/µl	0.39 ± 0.08	0.51 ± 0.10	0-0.03
Basophils (bas), 103/µl	0	0	0-0.4
Neutrophils neu%	28.02 ± 2.44	48.58 ± 7.46	20-80
Lymphocytes lym%	63.92 ± 3.49	40.26 ± 6.09	30-85
Monocytes mon%	1.8 ± 0.37	2.52 ± 0.25	0-4
Eosinophils eos%	6.25 ± 1.07	8.65 ± 1.93	0-4
Basophils bas%	0.02 ± 0.02	0.02 ± 0.017	0-7
Red blood cells (RBC), 106/µl	6.71 ± 0.10	5.98 ± 0.25	4.5–6.9
Hemoglobin (HGB), g/L	146.33 ± 1.63	133.67 ± 4.20	80-150
Hematocrit HCT, %	41.28 ± 0.39	37.2 ± 1.33	31.3-43.3

Results from blood tests conducted before and immediately after irradiation showed that the observation group experienced a 39.8% decrease in lymphocytes, a 4.5% decrease in leukocytes, and a 10.9% decrease in erythrocytes, along with increases in hematocrit, hemoglobin, and platelets of 9.9%, 8.6%, and 10%, respectively. The mean red blood cell volume and the mean concentration of hemoglobin in red blood cells increased by 1% and 1.36%, respectively. Meanwhile, the average values of neutrophils, monocytes, and eosinophils increased by 64.2%, 36.4%, and 30.8%, respectively. We are calculated NLR (neutrophil to lymphocyte ratio), ELR (eosinophil to lymphocyte ratio), LMR (lymphocyte to monocyte ratio) to evaluate the systemic inflammatory response. Thus, NLR from 0.45 became 1.24, ELR from 0.10 became 0.22, and LMR from 35.6 became 15.62, indicating an acute inflammatory response. These values and percentage changes align with the physiological standards for this species, and their variations are not clinically significant.

The number of isolated microorganisms was calculated using the formula: CFU/ml = K × 10 × n, where K is the number of colonies grown, n is the dilution of the suspension, and 10 is the conversion factor per 1 cm³ of the suspension when plating 0.1 cm³ (0.1 cm³ is 1/10 of a cm³). The qualitative and quantitative analysis of the microflora in the conjunctival sac revealed that microorganisms characteristic of the normal biota of the conjunctiva in warm-blooded animals predominated in all experimental subjects (Table 3).

Table 3. Results of bacteriological analysis of conjunctival flushes from rabbits before irradiation, on the 1st day after exposure, and one week after radiation exposure (n = 8).

Group №1 (Rabbit №3)	OS	OD
Before irradiation exposure	Neisseria subflava 101 CFU/ml	Neisseria subflava 101 CFU/ml
1st day	Not detected	Not detected
1 week after exposure	Not detected	Pseudomonas oryzihabitans 105 CFU/mL, Staphylococcus aureus 101 CFU/mL
Group №1 (Rabbit №4)	OS	OD
Before irradiation exposure	Moraxella_sg_Branhamella catarrhalis <101 CFU/mL	Moraxella_sg_Branhamella catarrhalis <101 CFU/mL
1st day	Not detected	Not detected
1 week after exposure	Not detected	Not detected
Group №2 (Rabbit №5)	OS	OD
Before irradiation exposure	Not detected	Not detected
1st day	Not detected	Not detected
1 week after exposure	Not detected	Not detected
Group №2 (Rabbit №6)	OS	OD
Before irradiation exposure	Not detected	Not detected
1st day	Not detected	Moraxella_sg_Branhamella catarrhalis <101 CFU/mL
1 week after exposure	Not detected	Not detected
Group №3 (Rabbit №7)	OS	OD
Before irradiation exposure	Staphylococcus aureus <101 CFU/ml	Staphylococcus aureus <101 CFU/ml
1st day	Not detected	Not detected
1 week after exposure	Staphylococcus aureus 103 CFU/ml	Moraxella_sg_Moraxella osloensis 102 CFU/ml
Group №3 (Rabbit №8)	OS	OD
Before irradiation exposure	Not detected	Not detected
1st day	Not detected	Not detected
1 week after exposure	Not detected	Not detected
Group №4 (Rabbit №1)	OS	OD
Before irradiation exposure	Rothia nasimurium 103 CFU/ml, Staphylococcus aureus 105 CFU/ml, Streptococcus thoraltensis 103 CFU/ml	Rothia nasimurium 103 CFU/ml, Staphylococcus aureus 105 CFU/ml, Streptococcus thoraltensis 103 CFU/ml
1st day	Not detected	Not detected
1 week after exposure	Pantoea agglomerans 106 CFU/ml	Staphylococcus aureus 103 CFU/ml
Group №4 (Rabbit №2)	OS	OD
Before irradiation exposure	Not detected	Not detected
1st day	Staphylococcus aureus <101 CFU/ml	Not detected
1 week after exposure	Not detected	Not detected

Before the start of the study, it was observed that in the experimental rabbits, bacteria from the genera Rothia, Staphylococcus, and Streptococcus, which are typical inhabitants of the rabbit conjunctiva, were dominant in terms of their number and frequency of occurrence [34]. Immediately after irradiation, bacteria from the genus Staphylococcus and the genus Moraxella (Branhamella) were detected. The occurrence frequency of Moraxella catarrhalis_sg_Branhamella and Staphylococcus aureus decreased by 50% and 92%, respectively, from “before” to immediately after irradiation. These changes in microflora composition may be attributed to the potential bactericidal effect of THz radiation and the sensitivity of these microorganisms to it. Seven days post-irradiation, bacteria from the genera Pseudomonas, Pantoea, and Staphylococcus were predominant in both frequency and quantity. Based on the qualitative and quantitative analysis of the conjunctival sac microbiota in the experimental group, it can be concluded that THz radiation influences the composition of the microbiota by altering the ratio of different bacterial species.

In the experimental animals, the bacteria isolated before irradiation, immediately after, and seven days after irradiation belonged to the phyla Actinobacteria, Firmicutes, and Pseudomonadota, consistent with literature data indicating these as typical representatives of the rabbit conjunctiva [41]. Therefore, it can be concluded that the bactericidal effect of THz radiation may temporarily alter the bacterial composition of the conjunctiva. However, by the seventh day, the microflora of the conjunctival sac re-turned to its normobiota state in both quantitative and qualitative aspects.

3.7. Associations among test parameters

The observed differences and trends suggest that prolonged and more intense exposure may be linked to functional changes in the properties and composition of corneal cells. To investigate this hypothesis, pachymetry and endothelial cell density parameters were analyzed to identify potential associations between these variables. In rabbits with 30-minute exposure, a statistically significant negative correlation was found between endothelial cell density and pachymetry, with a correlation coefficient of r = -0.36 (p = 0.042). This correlation indicates that a reduction in the endothelial cell pool is associated with an increase in corneal thickness. No other statistically significant correlations were observed (p > 0.05).

4. Discussion

The use of THz radiation in ophthalmology is a rapidly developing field [15,16]. THz waves are sensitive to water content in tissues, making them suitable for diagnosing diseases and conditions associated with changes in hydration [12]. However, the safety profiles of THz exposure to eye tissues have not been fully explored and must be established before widespread use in ophthalmological practice. Therefore, this study aimed to compare two THz radiation protocols on the functional properties of rabbit eye structures, assessing exposure times of 15 and 30 minutes and intensities of I₁ = 0.012 mW/cm² (38 °C), I₂ = 0.018 mW/cm² (40 °C), and I₃ = 0.024 mW/cm² (42 °C) to evaluate the biosafety of THz radiation in medical and technical applications. THz radiation at a frequency of 2.3 THz (130 µm) and varying intensities was used [38].

The selected power range (not exceeding sunlight intensity) and exposure times were chosen based on permissible thermal limits for corneal and anterior segment structures, maintaining temperatures between 38–42 °C [42,43], to evaluate the radiation’s biological effect in vivo while preventing thermal denaturation. Biological effects of electromagnetic radiation can be immediate or delayed; therefore, a protocol was developed to monitor changes on the first day, one week, and four weeks post-exposure.

This study is the first to demonstrate the effect of THz irradiation at 2.3 THz (130 µm wavelength) with 15- and 30-minute protocols and intensity levels of I₁ = 0.012 mW/cm², I₂ = 0.018 mW/cm², and I₃ = 0.024 mW/cm² on eye structures. Results showed that the intensity parameter did not have a statistically significant effect. However, the 30-minute exposure protocol showed a consistent and statistically significant increase in corneal thickness (p < 0.05) within 10 µm, indicating a slight subclinical increase in hydration. Since corneal edema can arise from various causes, including endothelial dystrophy [29,44], a correlation analysis was conducted to investigate the relationship between these parameters. An increase in corneal thickness was found to be associated with a decrease in endothelial cell density (p < 0.05), consistent with findings from [45]. The 15-minute exposure protocol also showed a tendency for a gradual increase in corneal thickness (p > 0.05), likely linked to the statistically significant decrease in endothelial cell density in this group (p < 0.05), suggesting a time-sensitive effect that warrants further investigation to develop safe parameters for high-frequency THz exposure.

Analysis of tear film stability showed consistent results across all groups, with no effect of THz radiation on the corneal surface layers. Ongoing research [31] discusses the protective properties of the tear film, aqueous humor, and vitreous body against radiation exposure. Intraocular pressure measurements using a rebound tonometer indicated stable ophthalmotonus, showing no variations. The THz range of electromagnetic radiation did not affect the overall blood count of the experimental animals.

The conducted study revealed changes in the complete blood count of rabbits following THz irradiation, indicating the development of an acute inflammatory response. Firstly, the NLR (neutrophil-to-lymphocyte ratio) significantly increased immediately after irradiation. This reflects the activation of neutrophils and a simultaneous decrease in lymphocyte levels, which is characteristic of a systemic inflammatory response and stress reaction triggered by a damaging factor, such as radiation. The increase in NLR aligns with findings from other studies that describe the activation of the immune system and cellular stress responses under the influence of THz radiation. Specifically, in the study by Echchgadda et al., the effects of THz radiation at 2.52 THz were investigated on Jurkat cells (human T-lymphocytes) [46]. The authors demonstrated that the radiation induced changes in gene expression related to stress response activation and affected signaling pathways, including apoptosis and DNA repair mechanisms. These findings are consistent with the observed increase in neutrophils in our study.

Secondly, the increase in ELR (eosinophil-to-lymphocyte ratio) suggests potential tissue damage. Eosinophils, in addition to their involvement in allergic reactions, are activated in response to tissue injury [47], which is consistent with subclinical changes induced by THz radiation. Zhang et al. studied the effects of THz radiation at 0.1 THz in mice with induced arthritis and found a reduction in pro-inflammatory cytokines such as IL-6 and TNF-\u03b1, along with the restoration of the Th₁₇/Tr_eg cell balance [48]. These findings highlight the potential for both pro-inflammatory and restorative effects, depending on irradiation conditions and experimental models.

Finally, a decrease in the LMR (lymphocyte-to-monocyte ratio) was observed due to a reduction in lymphocyte levels and a relative increase in monocyte levels. This change may be associated with lymphocyte migration to the site of injury or their destruction under stress conditions. Simultaneously, an increase in monocyte levels indicates the activation of innate immunity and tissue repair mechanisms. These changes are consistent with the findings of Zhang et al., who demonstrated the immunomodulatory effects of THz radiation, including a reduction in pro-inflammatory cytokines and the restoration of cellular balance [49].

The obtained data confirm the presence of a systemic inflammatory response to irradiation, despite the absence of clinically significant changes, highlighting the need for further research. Additionally, this may represent an adaptive response of the animal organism rather than solely a manifestation of inflammation.

Analysis of the effect of THz radiation on bacteriological cultures of conjunctival flushes showed short-term changes in the natural microflora, but by the seventh day, the conjunctival microflora returned to its normobiota state in both quantitative and qualitative terms.

The absence of THz irradiation effects on the eye lenses suggests that the penetration depth was limited to the cornea and possibly the aqueous humor of the anterior chamber. This is likely due to the high sensitivity of THz waves to water barriers [3], with the cornea, tear film, and aqueous humor acting as such barriers.

Our study confirmed the safety of THz irradiation at a frequency of 2.3 THz with in intensity ranges of I₁ = 0.012 mW/cm², I₂ = 0.018 mW/cm², and I₃ = 0.024 mW/cm², using 15- and 30-minute protocols, as evidenced by the absence of significant clinical reactions in eye structures. Specifically, no increase in corneal hyperreflectivity was observed in ASOCT data, tear production remained stable (Schirmer's test), ocular surface parameters were intact (negative fluorescein test), and ophthalmotonus was consistent. Additionally, there were no local or systemic reactions based on blood tests and bacteriological analysis of conjunctival flushes.

However, subclinical reactions in the corneas were observed in response to THz exposure, manifested as a slight statistically significant increase in corneal thickness (within 10 µm) correlated with a decrease in endothelial cell density. These effects were not associated with thermal damage to eye tissues, as the temperature increase during irradiation was minimal and within the standards for corneal tissues — the primary biological barrier to THz radiation. Further research is needed to explore the effects of THz radiation on biological tissues under various parameters to develop safe and effective applications in medicine and other fields. The study of THz radiation’s biological effects is advancing, contributing to the understanding of THz impacts on living systems [2].

5. Conclusions

Our study investigated the effects of 2.3 THz radiation on rabbit eyes. The findings indicate a slight, statistically significant increase in corneal thickness as a result of THz exposure, which varies with exposure duration. The data highlight the need for a comprehensive investigation into the biosafety of THz radiation, especially for extended exposures (30 minutes or longer). As high-frequency terahertz systems move toward clinical application in ophthalmology, further research will be necessary to establish safety guidelines.

Funding

Ministry of Science and Higher Education of the Russian Federation10.13039/501100012190 (БЧ-2020-0039-1).

Disclosures

The authors declare no conflicts of interest.

Data availability

Data underlying the results presented in this paper are available in Dataset 1 ^{(12.4KB, xlsx)} [50].