Safe use of retinal imaging with two-photon excitation in human eyes is crucial, as the effects of ultrashort pulsed lasers on the retina are relatively unknown. At the time of the study, the laser safety standards were inadequate due to the lack of biological data. This article addresses the feasibility of two-photon retinal imaging with respect to laser safety. In this study, rat retinas were evaluated at various laser exposure levels and with different laser parameters to determine the effects of laser-induced optical damage. The results were experimentally verified using confocal reflectance imaging, two-photon fluorescein angiography, immunohistochemistry, and correlated to the IEC 60825-1 laser safety standard.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Two-photon excited fluorescence (TPEF) imaging has been a mainstay in ophthalmology for exploring the structural and functional characteristics of the retina at a single-cell resolution [1–5]. The near-infrared (NIR) ultrashort pulsed lasers at high repetition rates are commonly employed for TPEF imaging since the pulsed laser beam enables efficient fluorophore excitation at higher scan rates, and the NIR light permits greater penetration depth, reduced scattering and less phototoxic effects than visible light [6,7]. Also, TPEF imaging can access the endogenous fluorophores like NAD (P) H and retinoids [8,9]; and excitation of these fluorophores by single-photon requires ultraviolet (UV) light which is blocked by the anterior segment of the human eye . Although TPEF imaging has been widely used to explore the retinal structures at a subcellular level in animal models, the TPEF imaging in human eyes is crucial since the tissue responses to short pulses are unknown. The laser safety standards such as the American National Standards Institute (ANSI) Z136.1, International Electrotechnical Commission (IEC) 60825-1 and International Commission on Nonionizing Radiation Protection (ICNIRP) provide guidance to protect the eye from hazardous laser exposures [11–13]. These standards are based on numerous experimental studies and determine the maximum permissible exposure (MPE) for the safe use of lasers. Despite that, the ultrashort pulsed laser safety in human eyes is still not well established due to insufficient biological data.
The pulsed laser generates peak powers that can cause potential mechanical, photochemical and thermal damage. The non-linear effects dominate with the ultrashort pulsed laser , and therefore, signals from fluorophore within the UV range can be generated within the retina by NIR pulsed laser. Thus, for the potential use of in vivo two-photon retinal imaging, the ultraviolet absorption (UVA) effects including the photodamage must be considered and evaluated. The effects of ultrashort pulsed laser on the retina with a stationary laser beam has been evaluated by numerous researchers to determine the safe use of these lasers for retinal imaging and treatment [14–18]. However, only a few studies have evaluated the retinal safety of two-photon laser scanning ophthalmoscopes [19,20]. Therefore, experimental studies on laser safety with respect to exposure levels and stationary vs scanning laser beams would help in a better understanding of the biological effects of the pulsed lasers and support refining the MPE for the safety standards.
We evaluated the effects of a pulsed laser (780 nm, 270 fs) on rat retinas for different power levels and exposure times. Although there are structural differences between rats and humans, this pilot study was carried out to determine the laser-tissue interactions in rodents since understanding the structural characteristics of the eye in different species is a key point. Also, rodents are widely used in ophthalmic research as retinal disease models which allow for studying disease mechanism and corresponding treatments.
2. Materials and methods
2.1 Animal preparation
Twelve brown Norway rats and twelve albino rats (Crl: CD (SD) IGS) with jugular vein catheterization were used for the experiment. Four brown Norway rats (pigmented) and four albino rats (non-pigmented) were exposed to the scanning laser beam, and eight brown Norway rats and eight albino rats to the stationary laser beam. Experiments were performed on one eye at a time and the fellow eye served as a control. The experimental rats were anesthetized using isoflurane 1-3% inhalant, and in addition, a drop of topical proparacaine anesthesia (proparacaine hydrochloride ophthalmic solution USP, 0.5% sterile) and a pupil dilator (0.5% tropicamide ophthalmic solution, USP) were applied to the eyes. Furthermore, sterile saline drops were applied to the experimental eyes every 30 seconds to keep the cornea moisturized. The Institutional Animal Care and Use Committee (IACUC) at Loma Linda University approved the animal experiments, and the experiments were carried out in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research.
2.2 Two-photon laser scanning ophthalmoscope
The two-photon prototype has been described in detail elsewhere . In brief, a compact femtosecond laser with 50 MHz repetition rate, 270 fs pulse width, 780 nm central wavelength, with tunable output power up to 500 mW was used as a light source. The confocal reflectance imaging and two-photon imaging can be performed using the same instrument in the high-resolution mode and high-speed mode with 30° x 30°, 20° x 20° and 15° x 15° transversal field of view. The scanning frequency per frame in the high-resolution mode is 5 Hz/7 Hz/9 Hz at 30°/20°/15° scan angle, whereas, in high-speed mode, the scan frequency is 9 Hz/12.5 Hz/16 Hz, respectively. The digital image readout for the high-resolution mode is 1536 × 1536, 1024 × 1024, 768 × 768; and for the high-speed mode 768 × 768, 512 × 512, and 384 × 384. In real time, the signal-to-noise ratio of the images was increased by a frame averaging. For this study, the standard objective lens (focal length, f = 30 mm) with an additional objective lens (f = 40/+25 diopters) from Heidelberg Engineering GmbH was used to collect the two-photon signals. The additional objective lens reduces the beam diameter by 70%, adapting the system to the shorter axial length of the rat eyes. The images were acquired in high-resolution mode with 30° scan angle for fundus overview and 20° and 15° scan angles for the detailed view of the retina.
2.3 Study design
Group 1: The retina was exposed to a scanning raster beam for two different time scales 100 seconds and 300 seconds at 160 mW for 30° and 15° scan angles. In total, four Brown Norway rats and four albino rats were exposed to the scanning laser beam as shown in Table 1.
Group 2: The retina was exposed to a stationary beam (spot size 5 µm) for different time scales, i.e., from 60 seconds to 600 seconds at 7 mW, 13 mW, 80 mW, and 160 mW laser power (Table 2). The threshold measurements for minimal visible lesions were determined using the stationary laser beam.
Prior to the experiment, a confocal reflectance image was acquired at a lower power level to determine the retinal location. The laser power levels were measured at the cornea since the power measurement at the retina has limitations. On rare incidents, the targeted eye drifts under the influence of anesthesia. At such instances, the exposure to the laser was blocked until the eye returned to its original position.
2.4 Two-photon fluorescein angiography
Fluorescein (10% AK-FLUOR, Akron) was administered through the jugular vein catheter for two-photon fluorescein angiography. The administered dose was in correlation with the animal’s body weight. The two-photon fluorescein angiography in animal models has been described in detail elsewhere . In brief, the fluorescein was injected to visualize the retinal vasculature, and in the two-photon mode, the flow of fluorescein through the blood vessels was captured. The two-photon fluorescein angiography was performed to assess the potential retinal damage, and to evaluate the efficacy of utilizing the two-photon fluorescein angiography in determining the light-induced damage.
The experimental rats were followed up for four days with confocal reflectance and two-photon fluorescein angiography and then euthanized humanely for histological analysis. The control and the experimental eye globes were enucleated and fixed in Davidson’s solution for 24 hours, and then embedded in paraffin. The retinal cross-sections of size 5 µm were cut from the paraffin-embedded eyes and transferred to the microscopic slides. The retinal sections were analyzed immediately under the light microscope, and after careful evaluation, some retinal sections were selected for hematoxylin and eosin (H&E) staining. In parallel to the H&E stained retinal sections, further sections from the same region were selected for the DeadEnd Fluorometric TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. The TUNEL assay was performed according to the manufacturer’s instructions (Promega Corporation, USA), and the retinal sections were mounted with VECTASHIELD + DAPI to allow for staining the nuclei. The TUNEL assay detects and quantifies the apoptotic cell death by measuring the nuclear DNA fragmentation, a vital biochemical hallmark of apoptosis in many cell types . The TUNEL assay is an established method to detect and quantify the apoptotic cells due to light damage [23–25].
The light microscopic (Aperio scan scope, Leica Biosystems) images of the H&E stained sections were captured to analyze the structural changes in the retina. The fluorescence microscopic (Olympus Corporation) images of the TUNEL stained retinal sections were captured to detect the apoptotic cells due to the laser exposure laser. Under a fluorescence microscope, the retinal sections were analyzed with 10 × , 20 × and 40 × objective lenses using a standard fluorescein filter at 520 nm to view the green fluorescence of fluorescein and at 490 nm for blue DAPI.
3.1 Scanning laser beam exposures
Figure 1 shows the confocal reflectance and two-photon fluorescein angiography images of brown Norway and albino rat exposed to the scanning laser beam exposure. The exposure settings were: scan angle 30°, exposure duration 300 seconds, laser power 160 mW. The confocal reflectance image [Fig. 1(a) and 1(d)] right after the laser exposure did not show any retinal abnormalities. The real-time two-photon fluorescein angiography from early phase [Fig. 1(b) and 1(e)] to late phase [Fig. 1(c) and 1(f)] showed neither hypofluorescence nor hyperfluorescence. Retinal capillaries were visible at the early phase of the fluorescein influx, and the fluorescein diminished slowly. The late phase demonstrated a graded elimination of dye from the retinal vasculature. The same results were observed for a scanning laser beam exposure at a 15° scan angle, 160 mW laser power exposures for 100 seconds and 300 seconds. Furthermore, the immunohistochemistry (TUNEL assay) analysis from the exposed retinal sections showed no cellular damages. The retinal sections of the control and the experimental eye were analyzed using the fluorescence microscope, and neither significant changes in cell loss nor disruption in the retina or retinal swelling were noticed. Figure 2 shows the fluorescence images of the control and the experimental retina of brown Norway and albino rats. The green fluorescence (arrow) noticed in Fig. 2(d) is an artifact of histology sections. And this was verified by one of the authors and a researcher from the department of pathology and human anatomy, Loma Linda University.
3.2 Stationary laser beam exposures
3.2.1 Brown Norway rats
Figure 3 shows the confocal reflectance and two-photon fluorescein angiography images of the brown Norway rat’s retina exposed to the stationary laser beam. The retina was exposed to 13 mW for 60 seconds (arrow 1), 100 seconds (arrow 2), 300 seconds (arrow 3) and 600 seconds (arrow 4) each on a single spot of the retina to determine the effects of the ultrashort pulsed laser (780 nm, 270 fs) to exposure durations. The stationary laser beam was the parked laser beam available in the service mode of the system. The confocal reflectance image of the retina was captured immediately after the exposure, and the visible retinal lesion induced by exposure to the stationary laser beam for different exposure times is shown in Fig. 3(b). With a longer exposure time, more radiation has been absorbed and hence there was an increase in the size of the lesion. After the exposure, two-photon fluorescein angiography was performed, and the early phase to late phase angiograms was recorded. High-contrast retinal microvasculature’s were clearly seen in the early phase two-photon fluorescein angiography [Fig. 3(c), yellow box], and at the late phase [Fig. 3(d), arrow] the hyperfluorescence was noticed on the laser-exposed retinal area. The irregularity in the nerve fiber layer and the absence of capillaries in the exposed area were noticed on day 4 [Fig. 3(e), 3(f)]. In Fig. 3(e), the confocal reflectance image of the retina shows high reflectivity and irregular nerve fiber layer at the laser-exposed area (yellow box), whereas the retina on the other side of the optic nerve (unexposed retina) did not show any structural changes or irregularities. Also, the early two-photon fluorescein angiography on day 4 [Fig. 3(f), yellow box] showed the absence of capillaries in the laser-exposed retina.
In addition, the H&E staining and TUNEL assays were performed on the retinal sections to evaluate the potential thermal damage to the retina. The retinal cross-sections were evaluated at different depths. The retinal sections close to the periphery of the eye globe did not show any structural damages on the retina [See Fig. 4(a), 4(d), 4(g)], whereas, the retinal sections closer to the exposed area showed disruption of the outer nuclear layer and the pigment epithelium [See Fig. 4(b), 4(c), 4(e), 4(f), 4(h), 4(i)]. The retinal swelling, a disorganized outer segment with no visible retinal pigment epithelium (RPE), and nuclear layer degeneration with a few apoptotic cells [Fig. 4(h) and 4(i), arrows] were noticed in the exposed area.
On the other hand, the stationary laser beam exposure at 80 mW induced the retinal burn and pigmentary change [Fig. 5(a), 5(b), 5(c)]. Large clear spaces were noticed in the subretinal area of the lesion disrupting the RPE. The inner and outer segments of photoreceptors severely disrupted, and the outer nuclear layer in the area of the lesion showed extensive loss of nuclei. The choroid was disrupted as well, and few apoptotic cells were noticed at the laser exposed area [Fig. 5(d), 5(e), 5(f)].
3.2.2 Albino rats
Conversely, in albino rats, no potential damages were noticed for the stationary laser beam exposure at 7 mW, 13 mW and 80 mW. Even at 160 mW laser power exposure for 100 seconds, no damages on the exposed retina were noticed. Only at 160 mW laser power exposure for 300 seconds, damages occurred [Fig. 6]. The confocal reflectance [Fig. 6(a), 6(b), 6(e)] and the two-photon fluorescein angiography images [Fig. 6(c), 6(d), 6(f)] of the albino rat were acquired immediately to monitor the retinal damages due to the laser exposure. The hyperfluorescence [Fig. 6(d), arrow] was minimal in the exposed area compared to the brown Norway rats [See Fig. 3(d)]. Likewise, in the follow-up examination on day 4, neither change in the nerve fiber layer nor the absence of capillaries were noticed as in the brown Norway rat [See Fig. 3(f) and 6(f)].
The TUNEL assay on the retinal sections of albino rats [Fig. 7(a) and 7(b)] did not show any apoptotic cells due to the laser exposure. Neither cell death nor disruption in the retina was noticed. Few green fluorescence cells were noticed in the outer nuclear layer (ONL) both on the control and the experimental retinal sections [Fig. 7(a) and 7(b), yellow box]. This could be due to the acute bright light exposure which can damage the RPE and photoreceptors even in control eyes. Within the control group, light-induced damages can be multifactorial including environmental conditions, genetic diseases, and aging. Animal environment, housing, and management are essential for maintaining the health and wellbeing of laboratory animals ensuring the reliable outcome of scientific investigations .
3.2.3 Retinal damage recovery
Brown Norway rat exposed to a stationary laser beam at 13 mW for a single exposure time was followed up for 4 days with no fluorescein injection to determine the recovery from light damage. Only confocal reflectance imaging and TUNEL assay were performed on this rat to monitor the significant effects of light damage and structure recovery. The retina exposed to a stationary laser beam of 13 mW for 300 seconds is shown in Fig. 8. The confocal reflectance image [Fig. 8(a)] immediately after the laser exposure and the follow-up retinal image on day 4 [Fig. 8(b)] visualize the retinal lesion [Fig. 8(a) and 8(b), arrows]. However, no irregularities in nerve fiber layers noticed on day 4. Also, the reflectivity from the laser-exposed area was not as severe compared to the retina exposed to the laser at multiple sessions [See Fig. 3(e)]. The retina with a single laser exposure at 13 mW recovered by itself without any medications and even the fluorescence image analysis did not show any irregularities or disruptions on the retina [See Fig. 4 and 8(c)].
Although no irregularity in the nerve fiber layer or apoptosis were noticed in the exposed retinas due to the laser exposure in Fig. 8(c), different approaches must be carried out to better locate the light damage on a cellular level. As for histology, it was difficult to locate the exact retinal lesion by a cross-section analysis. Therefore, the whole mount retinal analysis would be an efficient way to determine the effects of laser-induced damage for a single exposure. Optical coherence tomography (OCT) would be another efficient method to look at the retinal lesions for cross-section analysis.
3.3 Immunohistochemistry analyses of the retina for multiple exposures of the scanning laser beam
Fluorometric TUNEL analysis performed on the rats that were used for two-photon autofluorescence imaging (From our previous study ). The retinas exposed to the scanning laser beam multiple times at 160 mW laser power. The imaging procedure in these rats took approximately 45-60 minutes. The fluorescence microscopic analysis of these retinal sections reported no apoptosis or structural changes in the retina even after multiple exposures. The fluorescence images of the brown Norway rat’s retinas exposed to the scanning laser beam is shown in Fig. 9(a) and 9(b). Since no apoptosis was noticed in the exposed retinal sections, a positive control test was performed on retinal sections according to the manufacturer’s instructions . The apoptosis was clearly noticed [Fig. 9(c)] on the positive control retinal sections. This shows that the scanning laser beam even after multiple exposures produces no considerable damages.
4.1 Pigmented vs. non-pigmented retinal toxicity to laser exposures
A scanning laser beam returned no retinal toxicity in both animal models was verified by confocal reflectance imaging, two-photon fluorescein angiography, and TUNEL assay. However, for stationary laser beam exposure, the retinal lesions were noticed at 13 mW for 60 seconds in brown Norway rats; and only after exposure to 160 mW laser power for 300 seconds in albino rats. The number of TUNEL positive cells was significantly higher in brown Norway rats compared to albino rats. Retinal swelling, a disorganized outer segment with no visible RPE and nuclear layer degeneration were noticed in brown Norway retinal sections. In contrast, the albinos did not show any retinal damages at low laser power and only minimal damages at longer exposure to high laser power. The albino rats had a higher laser safety threshold than the pigmented brown Norway rats due to lack of melanin in the RPE layer. Absorption serves fundamentally in determining the potential toxicity of light on the retina.
Also, no potential thermal damages noticed on the brown Norway rat retina exposed to the stationary laser beam of 7 mW, while, the stationary laser beam exposure at 80 mW and 160 mW induced a retinal burn, pigmentary change, retinal detachment, and hemorrhage. These results infer the morphological changes related to tissue radiation interaction. In brown Norway rats, this was primarily due to the absorption of radiant energy by melanin within the RPE and the choroidal melanocytes. But in albino rats, the laser-induced effects occurred because of multiple scattering, together with absorption within hemoglobin and possibly also within tissue water .
4.2 Laser safety studies in animal models
Rodents have been predominantly used in ophthalmic research to study the disease progression and effect of therapies. Also, the rodents (rats 0.43) have larger numerical apertures compared to human eyes (0.20), and this possibly could resolve smaller retinal features . Two-photon retinal imaging has been studied in different animal models including rodents, however, for the laser safety study rodents may not be the appropriate animal models due to several factors. The laser safety standards determine the MPE values by the means of experimental studies in non-human primates (NHP) since NHP eyes are closer to human eyes. Also, the retinal damage depends on the transmission of the laser radiation (ocular transmission in rats is 0.9 or greater at 780 nm), absorption, and the diameter of the laser spot at the retina. Multiphoton absorption by ultrashort pulsed laser needs to be ensured for safe retinal imaging, and in rats, it might not be possible to evaluate the UVA since the rat’s cornea transmits UV radiation and supports its vision .
Laser safety guidelines specify that the diameter of the irradiance profile (D) at the retina determines the damaging potential of energy incident on the retina . The retinal irradiance diameter can be calculated as , where is the source angle and is the effective focal length of the eye (rats, , humans, ). The retinal irradiance diameter for the small source () in rats (5 µm) is 3 times lower than the human eyes (25 µm). Therefore, the energy required to create the damage must be lower in rats than humans since the irradiance diameter is smaller in rats. However, in this study, the visible retinal lesions were noticed in Brown Norway rats only at 13 mW laser power which is 2.3-fold higher than the safety standard (see Appendix). This could be due to the influence of the retinal-laser spot diameter, and differences in animal species. Also, rats have higher refractive power and higher-order aberrations, and these factors influences the spot diameter. Therefore, accurate analysis of the potential retinal damage in different animal species needs to be carefully evaluated and correlated for safe two-photon imaging in humans.
4.3 Future directions
From our previous study, the power threshold required for the two-photon autofluorescence imaging with our prototype was determined to be 160 mW  and is two orders of magnitude higher than the safety standards. Although no potential thermal damages noticed due to the scanning laser beam, further improvement in light delivery is needed for the two-photon ophthalmoscope in clinical applications. Implementing adaptive optics to the current two-photon prototype would improve light delivery by a factor of four. Also, adaptive optics has emerged as an empowering technology for retinal imaging enabling diffraction-limited and holds potential for non-invasive detection and diagnoses of eye diseases. However, the cost and complexity of adaptive optics ophthalmoscopes with a limited field of view currently impede its clinical use . The high-magnification objective (HMO) lens (Heidelberg Engineering GmbH, Germany) with phase plates has the potential to establish adaptive optics in a clinical application by simplifying its incorporation in the prevailing systems [32,33]. The combination of HMO with the phase plates could resolve ocular microstructures without the use of complex adaptive optics system for an 8-degree field of view. Furthermore, a femtosecond laser with a reduced pulse width would improve the two-photon efficiency, since the shortest pulse width coupled with group velocity dispersion compensation would greatly increase the two-photon excitation induced fluorescence . Thus, employing a shorter pulse width laser (~55 fs) with adaptive optics to the prototype will offer high-resolution retinal imaging sustaining the laser safety standards.
This study concludes that the use of two-photon scanning laser ophthalmoscope for in-vivo retinal imaging is safe in rats. No potential thermal damages observed due to the scanning laser beam at both high laser power and multiple exposures. The high contrast two-photon fluorescein angiography images were effective in evaluating the light-induced retinal damage. Furthermore, the immunohistochemistry analysis supported the study by analyzing the damages at a cellular level. Since no potential thermal damages were noticed even at high laser power, the physiological and biological process of the retina in rodents can be studied in vivo using the two-photon ophthalmoscope. However, for implementation of this technique in clinics, the appropriate light safety standards must be well established. The retinal toxicity evaluation at different operating wavelengths, and for shorter pulse widths (typically < 100 fs) in NHP could establish a broader sense of these safety standards. Shorter pulse widths have a direct relationship to the generated two-photon fluorescence, and therefore it is vital to establish the correlation of the shorter pulse widths to retinal phototoxicity.
A. Laser safety analysis according to IEC 60825-1:2014
A.1 Laser safety analysis of a stationary laser beam
Laser wavelength, repetition frequency, pulse duration,
for the spectral region 700 to 1050 nm; since the beam is emitted for a small source.
Pupil aperture with a diameter of 7 mm
For repetitively pulsed lasers the following conditions should be tested to determine the MPE.
Condition 1: The exposure from any single pulse shall not exceed the single-pulse MPE. Thus, the radiant exposure for the time period 270 fs is:
Condition 2: The average exposure for a pulse train of exposure duration T shall not exceed the MPE for a single pulse of duration T. A reasonable estimate of hazardous chance exposure time can be taken as 10 s.
Since there are pulses in 10 s period, the average irradiance criteria result in a single pulse radiant exposure:
Condition 3: The average exposure from pulses within a pulse train shall not exceed the MPE for a single pulse multiplied by the correction factor (where). The maximum exposure duration for 700 nm wavelength is
Since the laser is operating at high repetition the multiple pulses appearing within the period of are counted as a single pulse to determine N and the radiant exposure of the individual pulses are added to compare with the MPE of . Hence, the effective pulse repetition frequency is:
For NE pulses, each of durationin 10 s period the radiant exposure under this criterion would be:
Condition 1 and 2 are applicable to the pulse of energy, Q, while condition 3 is applicable to pulse of energy . Hence, dividing the by enables comparison of three MPEs.
Comparing the three MPE’s, the condition 2 yields the most restrictive and therefore the single pulse MPE for the two-photon prototype must be 562 µW. The MPE in laser safety guidelines is 10 times lower than the damage threshold.
A.2 Laser safety analyses for an extended source
Here the total illuminated field considered as an extended source. The repetition rate of the scan field equals the frame rate; for 30° and 15° scan angle.
The angular subtense of the scan field is 525 × 525 mrad and 262.5 × 262.5 mrad for 30° and 15° scan angle respectively. Duration of the field illumination,for 30° and 15° scan angle.
Condition 1: The exposure from any single pulse (frame) within a pulse train (frame rate) shall not exceed the MPE for a single pulse (frame).
Condition 2: The average power for a pulse train (frame rate) of emission duration T shall not exceed the power corresponding to the MPE for a single pulse. Here two different scan times considered for evaluation:
Since, , . Therefore, condition 3 will result in the same as condition 1. The thermal limits for the two-photon prototype can be interpolated from condition 2 and it is 21 mW and 16 mW for 100 and 300 s. For intentional long-term viewing, the output power must be 5 mW for the study prototype. The laser safety guidelines developed the standard for the human eye with a focal length of 17 mm and the pupil size of 7 mm. Therefore, the MPE for the rat’s retinal imaging can be obtained by scaling the obtained MPE using the square of the ratio of effective focal length of the rat and human eyes .
The authors thank Dr. Ubaldo Soto, Department of Microbiology, Loma Linda University for the fluorescence microscopy; and John Hough, Department of Anatomy, Loma Linda University for his assistance with the tissue embedding and sectioning. The authors also thank the animal care facility at Loma Linda University for their support and handling the animals.
The authors declare that there are no conflicts of interest related to this article.
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