Open Access
Glaucoma  |   November 2023
Comparison Between Two Types of Viral-Induced Anterior Uveitis In Vitro and In Vivo: A Stronger Response in Herpes Simplex Virus Type 1 Than in Murine Cytomegalovirus
Author Affiliations & Notes
  • Yuhang Li
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Weishaer Ke
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Xin Liu
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Qi Zhang
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Naiji Yu
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Kaijun Wang
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Min Chen
    Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
    Zhejiang Provincial Key Lab of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China
  • Correspondence: Min Chen, Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, No. 1 Xihu Boulevard, Hangzhou, Zhejiang Province 310009, China; [email protected]
  • Kaijun Wang, Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, No. 1 Xihu Boulevard, Hangzhou, Zhejiang Province 310009, China; [email protected]
  • Footnotes
     YL and WK contributed equally to this work and share first authorship.
Investigative Ophthalmology & Visual Science November 2023, Vol.64, 20. doi:https://doi.org/10.1167/iovs.64.14.20
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      Yuhang Li, Weishaer Ke, Xin Liu, Qi Zhang, Naiji Yu, Kaijun Wang, Min Chen; Comparison Between Two Types of Viral-Induced Anterior Uveitis In Vitro and In Vivo: A Stronger Response in Herpes Simplex Virus Type 1 Than in Murine Cytomegalovirus. Invest. Ophthalmol. Vis. Sci. 2023;64(14):20. https://doi.org/10.1167/iovs.64.14.20.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To observe the similarities and differences between herpes simplex virus type 1 (HSV-1) and murine cytomegalovirus (MCMV)–induced viral anterior uveitis (VAU), both in vitro and in vivo.

Methods: Primary rat trabecular meshwork cells (RTMCs) were infected by HSV-1 or MCMV to clarify the pattern of virus replication and the effect on cells. In vivo, intracameral injection of HSV-1 or MCMV was performed to establish the VAU rat models. The clinical manifestation, intraocular pressure (IOP), histological characteristics, ultrastructural changes, and the expression of inflammatory cytokines in the anterior segment were observed and compared between these two types of VAU models.

Results: Both viruses could infect the RTMCs but HSV-1 exhibited an earlier and greater cytopathic effect in vitro. In vivo, both VAU rats showed typical acute VAU signs, and the IOP elevation seemed to be correlated with the inflammatory progression. Histopathological findings and ultrastructural changes revealed tissue damage and cell infiltration in the anterior chamber angle. In both models, similar proinflammatory cytokines were upregulated. HSV-1 and MCMV viral particles were identified under transmission electron microscopy.

Conclusions: HSV-1 and MCMV infection share certain similarities but have significant differences both in vitro and in vivo. HSV-1 usually has a stronger anterior segment inflammation with a longer duration compared with MCMV in VAU models. Our results provided a valuable animal model for investigating pathogenesis and exploring therapeutic strategies for clinical VAU.

Human herpesvirus is a common cause of human infectious diseases, second only to the cold and influenza viruses.1 In the eye, it became a considerable source of ocular morbidity. Herpes simplex virus (HSV) and varicella-zoster virus have long been considered as the principal agents affecting all parts of the eyeball in immunocompetent patients, and cytomegalovirus (CMV) is reported as the most common infectious cause of uveitis in immunosuppressed patients.1,2 Recent advancements in diagnostic techniques have shifted the focus of herpetic ocular disease to the anterior segment, due to accumulated evidence showing the presence of herpesviruses in the aqueous humor during acute episodes of infectious anterior uveitis (AU).24 Studies showed controversial results regarding whether CMV and HSV-1 are linked with Posner-Schlossman syndrome and Fuchs’ uveitis syndrome during an acute virus attack.47 
Viral anterior uveitis (VAU), which is characterized by recurrent, unilateral anterior segment inflammation and raised intraocular pressure (IOP), is the most common form of infectious uveitis.3,5,8,9 Human herpesvirus, notably HSV-1 and CMV, have been most frequently reported as the causative agents in Asia and Europe VAU cases, regardless of the immune status of the host.2,4,6 An acute attack of VAU usually brings great discomfort that could last for a few hours to several weeks.10 Also, with the recurrent and chronic courses, these affected eyes may develop severe glaucomatous optic neuropathy or loss of vision.5 The prevalence of raised IOP in patients with VAU ranges from 50% to 90%, whereas approximately 13% of them lead to glaucoma.3,11 
The diagnosis of VAU is based mainly on clinical features, supplemented by aqueous analysis for viral nucleic acid.4,11 Although there is some limit to the categorization into HSV-AU and CMV-AU through clinical features since there are some overlapping manifestations, some differential points still exist. Numerous clinical researches revealed that hyperemia and pain of the eye, medium-to-large keratic precipitates (KPs), cells and flare in the anterior chamber (AC), and posterior synechia were observed more frequently in HSV-AU than in CMV-AU. In contrast, coin-shaped KPs, diffuse iris atrophy, elevated IOP, and the necessity of glaucoma surgery were more common in CMV-AU.5,11,12 
In this study, we simulated two types of VAU models in immunocompetent Sprague-Dawley (SD) rats through intracameral injection (IC) of HSV-1 or murine CMV (MCMV) with a suitable and relatively consistent viral titer. For the first time, we investigated and compared the clinical and pathological manifestations of VAU in rats caused by HSV-1 and CMV, expected to clarify the pathogenic mechanism of VAU and identify the relationship between viral infection and AC inflammation. 
Material and Methods
Cells and Viruses
Primary rat trabecular meshwork cells (RTMCs) were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China), which were cultured and propagated as described previously.13,14 In brief, trabecular meshwork (TM) tissue was micro-dissected from SD rats aged between three to four weeks old, and placed on plate to facilitate migration of RTMCs with the complete RTMCs medium (Procell, Wuhan, China). When the cells reached 80% confluence, they were passed sequentially in a 1:3 ratio and maintained in the same medium. Passage 3 to 4 RTMCs were used in the present study. 
HSV-1 (McKrae strain) and MCMV (labeled with an enhanced green fluorescent protein, MCMV-eGFP, K181strain) were both gifted by the Institute of Immunology, Zhejiang University. They were propagated, purified and titrated in cultured Vero cells and NIH/3T3 (Mouse Embryonic Fibroblast Cells), respectively, according to a previous study.15 The viral titer of cell culture infectious dose (TCID50) per 0.1 mL was quantified by endpoint dilution assay before the virus-containing supernatant was stored at −80°C and diluted with fresh Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) to the appropriate concentration for each experiment. 
Infection of RTMCs With HSV-1 or MCMV
Confluent RTMCs were seeded in 24-well plates and were divided into treatment and control groups. In the treatment groups, virus medium (HSV-1 or MCMV suspended in DMEM) was added at the same multiple of infection (MOI = 0.5) for two hours following a previously described method.16 The same volume of DMEM was added to the control group. Subsequently, the medium was removed and replaced with maintenance medium, and the cells were further cultured for 48 hours. 
Cell viability assays were performed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. The RTMCs were also subjected to immunofluorescence examination at five timepoints after infection. Specifically, after treatment, cells were fixed, washed, treated with 0.5% Triton, blocked with goat serum (Boster Biological Technology Co., Ltd., Wuhan, China), and incubated with primary antibodies, followed by secondary antibodies (antibodies see Supplementary Table S1). The cells were then mounted with DAPI (Invitrogen Corporation, Carlsbad, CA, USA), observed under a fluorescence microscope, and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). 
Animals and Grouping
Male SD rats (6–8 weeks old, 200–250 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., and housed in a specific-pathogen-free animal facility. One hundred twenty-three rats were randomly allocated into three groups: (i) HSV-1-induced VAU group (HSV-1 group), (ii) MCMV-induced VAU group (MCMV group), and (iii) normal control group (CTRL group). We kept all the animals strictly in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the Second Affiliated Hospital of Zhejiang University, School of Medicine (No. AIRB-2022-195). 
Viral-Induced Anterior Uveitis Animal Models
The rat VAU model was elicited by IC as previously described.15,17 In brief, rats were anesthetized, and the cornea was treated with a drop of topical anesthetic. AC paracentesis was performed using a 33-gauge needle Hamilton syringe (Hamilton, Reno, NV, USA) to penetrate the limbus into the AC of the right eye at the 3 o'clock position, avoiding injuries to the iris and lens. Rats in the HSV-1 group and MCMV group accepted 5 µL (1 × 105 TCID50/0.1 mL) virus stock of HSV-1 or MCMV, respectively. The same dose of DMEM was injected by IC for the control group. Chlortetracycline eye ointment was then applied to the ocular surface, and eyelids were closed for 30 seconds to allow the viral suspension to stain in AC. Rats were placed on an electric blanket for resuscitation. 
Clinical Evaluation of the VAU Models
Slit-Lamp and Optical Coherence Tomography Examination
Clinical manifestations of the rats in three groups were investigated by slit-lamp examination and anterior segment optical coherence tomography (ivue; Optovue, Fremont, CA, USA) at different time points. The severity of ocular inflammation was evaluated using a scoring system ranging from 0 (normal) to 4 (most severe) as previously described,18 based on the degree of corneal edema, KPs, AC flare, and iris hyperemia. The evaluation and scoring process was conducted by an independent ophthalmologist who was blinded to the treatment history. The specific criteria of the scoring rubric were provided in Supplementary Table S2 according to previous studies.1822 
IOP Measurement
Before and after injection with DMEM or virus, binocular IOPs of the rats were recorded every day until sacrificed. A handheld TonoLab rebound tonometer (iCare Laboratory, Espoo, Finland) was used to measure IOP in conscious rats according to the manufacturer's instructions. Six consecutive measurements were performed, and an average IOP value was automatically calculated by the software and recorded for analysis. IOP was measured between 2:00 PM and 4:00 PM by the same researcher to minimize interobserver viability and increase repeatability. All the investigators participating in clinical evaluations were independent, and the results were cross-masked during the measurement process. 
Histopathological Examination
For histopathologic analysis, rats were sacrificed at four specific time points. Intact eyes were enucleated, fixed in formalin, dehydrated, embedded in paraffin, and cut into 5 µm sections through the AC. Sections were stained with hematoxylin and eosin (H&E) and evaluated through a microscope. To assess inflammatory conditions, the paraffin sections were observed and scored by a single pathologist blinded to treatment history as described previously.23 The scoring criteria are shown in the Supplementary Material (Supplementary Table S3)
Electron Microscopy Examination
Rats were euthanized at specific points, and the central cornea, iris, lens, most of the ciliary body (CB), and vitreous were removed with forceps, then the corneoscleral rims tissue was isolated under a dissection microscope, leaving the scleral ring containing the TM. After fixing, samples were dehydrated, embedded, incubated, placed on copper grids, stained and examined under a 120 kV transmission electron microscopy (TEM; Tecnai G2 Spirit; FEI, Hillsboro, OR, USA). 
Evaluation of Inflammatory Associated Factors
At each time point, corneoscleral rims tissue was isolated for cytokine analysis. Total mRNA extraction and cDNA synthesis were carried out using FlaPure Animal Tissue DNA Extraction Kit (Genesand Biotech Co., Ltd., Beijing, China) and HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (Vazyme Biotech Co., Ltd., Nanjing, China), respectively. After that, quantitative real-time polymerase chain reaction (qRT-PCR) was performed in a ChamQ SYBR qPCR Master Mix Kit (Vazyme Biotech Co., Ltd.) using an ABI Fast 7500 RT-PCR system (Life Technologies, v2.0.6). Gene expression was normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase mRNA and assessed using 2−ΔΔCt quantification. Supplementary Table S4 lists all primers. 
Immunohistochemical staining was done to further validated the above results. The paraffin-embedded eyeballs sections were incubated with primary antibodies, with species-specific secondary antibodies (antibodies see Supplementary Table S1). The slides were stained with 3,3′-diaminobenzidine, counterstained with hematoxylin, dehydrated, and covered with neutral gum. The average optical density was counted using ImageJ software. 
Statistical Analysis
All experiments were repeated three times unless otherwise stated. Data were analyzed using GraphPad Prism (version 8) and SPSS (version 24). The data were presented as the mean ± standard error of the mean (SEM). One-way or two-way ANOVA followed by Bonferroni's post-hoc test was used for multiple groups. A P value < 0.05 was considered significant. 
Results
Changes of RTMCs After HSV-1 or MCMV Infection In Vitro
The results of light microscopy (LM) and fluorescence microscopy (FM) showed that both HSV-1 and MCMV could infect RTMCs in vitro (Fig. 1A). HSV-1 infected cells developed characteristic cytopathic effect (CPE) at 12 hours post-infection (hpi) (Fig. 1A a3), with most cells becoming round, shrunken, and loosely attached by 24 hpi (Fig. 1A a5). MCMV-infected cells showed CPE at 24 hpi (Fig. 1A b5). Green fluorescence-positive RTMCs were remarkable at 24 hpi in both groups, with a stronger fluorescence intensity in the HSV-1 group (Fig. 1A a6). RTMCs in the control group remained bright, fusiform, and firmly adherent throughout. 
Figure 1.
 
Changes of RTMCs after viral infection in vitro. Primary RTMCs were inoculated with HSV-1 or MCMV at a low multiple of infection. (A) Results of light and fluorescence microscopy of infected RTMCs in vitro. LM, light microscopy; FM, fluorescence microscopy. White arrows indicate typical CPE; the white dotted box indicates the enlarged image of the corresponding picture. Scale bar: 200 µm in LM and 50 µm in FM. These studies were each repeated six times with similar results. (B) Effects of HSV-1 or MCMV on RTMCs’ cell survival probability. Every six hours until 48 hpi after treatment, cell viability was measured by CCK8 assay. Data were compared with the control. The mean value of control samples defines as 100%. (C) Immunofluorescence analysis of HSV-gB and MCMV expression was detected respectively by Image J Semi-quantitative analysis of immunofluorescence. Data are presented as mean ± SEM (n = 6). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Figure 1.
 
Changes of RTMCs after viral infection in vitro. Primary RTMCs were inoculated with HSV-1 or MCMV at a low multiple of infection. (A) Results of light and fluorescence microscopy of infected RTMCs in vitro. LM, light microscopy; FM, fluorescence microscopy. White arrows indicate typical CPE; the white dotted box indicates the enlarged image of the corresponding picture. Scale bar: 200 µm in LM and 50 µm in FM. These studies were each repeated six times with similar results. (B) Effects of HSV-1 or MCMV on RTMCs’ cell survival probability. Every six hours until 48 hpi after treatment, cell viability was measured by CCK8 assay. Data were compared with the control. The mean value of control samples defines as 100%. (C) Immunofluorescence analysis of HSV-gB and MCMV expression was detected respectively by Image J Semi-quantitative analysis of immunofluorescence. Data are presented as mean ± SEM (n = 6). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Cell viability appeared in an obvious downtrend until 48 hpi in the HSV-1 group, and the green fluorescence signal was concomitantly attenuated (Fig. 1B). In the MCMV group, a slight decrease of cell activity occurred as early as 6 hpi, whereas recovery of cell viability was denoted from 24 hpi on. At 48 hpi, RTMCs showed fully restored cell viability, although poor cell morphology was observed at that time (Fig. 1A b10). 
Clinical Manifestations in HSV-1 or MCMV-Induced VAU Model
The clinical manifestations of different groups (n = 12 for each group) were shown in Figure 2. All the rat eyes in the control group retained a normal AC appearance, except for transient and localized corneal edema at the IC site at one day post-infection (dpi) (Fig. 2 c1). The rats in the VAU groups exhibited typical signs very similar to those that appeared in human uveitis, such as corneal edema, KPs, fibrin exudation, iris blood vessel dilatation, and pupil occlusion. However, there were some differences in AC inflammation between the HSV-1 and MCMV groups as measured by slit lamp examination and anterior segment optical coherence tomography . 
Figure 2.
 
Clinical manifestations of rat eyes after anterior chamber inoculation of HSV-1 (left), MCMV (middle), or DMEM (right) at one, three, seven, and 14 DPI, upper and lowercase letters labeled the same eye by slit-lamp examination and AS-OCT separately (n = 12, each). Representative images of anterior uveitis were observed in the VAU groups. Triangle, cornea edema; asterisk, anterior chamber fibrin exudation; square, KPs; dotted arrow, KNs; solid arrow, occlusion of pupil or discoria. Original magnification of slit-lamp images, × 25 magnification; AS-OCT, anterior segment optical coherence tomography; KNs, Koeppe nodules.
Figure 2.
 
Clinical manifestations of rat eyes after anterior chamber inoculation of HSV-1 (left), MCMV (middle), or DMEM (right) at one, three, seven, and 14 DPI, upper and lowercase letters labeled the same eye by slit-lamp examination and AS-OCT separately (n = 12, each). Representative images of anterior uveitis were observed in the VAU groups. Triangle, cornea edema; asterisk, anterior chamber fibrin exudation; square, KPs; dotted arrow, KNs; solid arrow, occlusion of pupil or discoria. Original magnification of slit-lamp images, × 25 magnification; AS-OCT, anterior segment optical coherence tomography; KNs, Koeppe nodules.
In the HSV-1 group, uveitis was aggravated in the initial three to seven days after infection and gradually alleviated within two weeks. Corneal edema persisted for three to five days in most rats, with three rats showing prolonged edema until seven dpi and shallow AC. Centrally located sporadic medium-to-large mutton fat KPs were frequently seen in this group (Fig. 2A3 a3). Inflammatory exudates were obvious and sustained longer in HSV-1 infected rats, some even formed a membrane covering the pupil on the 14 dpi (Fig. 2 a4). The Koeppe nodules were observed in only 2/12 of rats from three dpi on. Less than half of the rats showed miosis or discoria, but transient dilation of the pupil was observed in three rats in the early period, and four rats showed a dilated pupil at five dpi with an anterior iris bowing. Compared with the HSV-1 group, rats in the MCMV group developed milder inflammation during the first three days. Koeppe nodules were more common in this group, whereas KPs could only be found in two rats. 
Clinical Manifestation Scores and IOP Fluctuations
As shown in Figure 3A, the cumulative scores of clinical manifestations in both VAU groups increased immediately after IC, peaked at one dpi (P < 0.05, compared with the CTRL group), and then gradually subsided, but HSV-1-infected rats had higher overall rating than the MCMV group. 
Figure 3.
 
Clinical manifestations score (A) and IOP fluctuation (B) in rats. Data are presented as mean ± SEM, n = 12 in per group. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with the control group.
Figure 3.
 
Clinical manifestations score (A) and IOP fluctuation (B) in rats. Data are presented as mean ± SEM, n = 12 in per group. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with the control group.
Meanwhile, the IOP fluctuated with the changes of inflammatory reaction (Fig. 3B). Both VAU groups experienced significant IOP elevation in the first two days, with a peak IOP at one dpi (P < 0.001, compared with the CTRL group). In the MCMV group, the IOP values gradually returned to normal at three dpi. However, after a temporary drop, a second peak of IOP appeared around seven dpi (19.33 ± 1.82 mm Hg, P < 0.01) in the HSV-1 group. In the control group, both the clinical scores and the IOP values remained within the normal range. No obvious changes in IOP were observed in the left eye. 
Pathological Changes in the Anterior Chamber Angle
Histological analysis revealed obvious pathognomonic features in both VAU models. In the HSV-1 group, inflammatory damages to the TM, Schlemm's canal, and adjacent tissue were remarkable immediately after infection, which manifested as trabecular endothelial cell swelling, collapse of intertrabecular spaces, loss of trabecular endothelial cells (Fig. 4A a4; white arrows) and AC exudation (Figs. 4A a2, a4, a6; black arrows). For the MCMV group, the marked infiltration of inflammatory cells was noticed at one dpi, especially around the CB (Fig. 4A b2, black arrows), whereas the inflammatory exudation was obviously alleviated at three dpi and almost cleared at seven dpi. 
Figure 4.
 
Representative histopathological changes of anterior chamber after infection on light microscopy and the histopathological score in injected rats. (A) Light microscopic images of the anterior chamber angle in HSV-1 (left column), MCMV (middle column), and control group (right column) at each observing time. Square images on the right represent the amplification of the rectangle area of the left corresponding image. Asterisk indicates protein exudation; black arrow indicates inflammatory cell infiltration; white arrow indicates trabecular endothelial cell swelling and collapse of intertrabecular spaces. COR, cornea; AC, anterior chamber; S, Schlemm's canal; LEN, lens; RE, retina. Scale bar: 50 µm. The histopathologic score was assessed for severity of inflammation at one DPI (B), three DPI (C), seven DPI (D), and 14 DPI (E). Data are presented as mean ± SEM (n = 3 per group at each time point). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Figure 4.
 
Representative histopathological changes of anterior chamber after infection on light microscopy and the histopathological score in injected rats. (A) Light microscopic images of the anterior chamber angle in HSV-1 (left column), MCMV (middle column), and control group (right column) at each observing time. Square images on the right represent the amplification of the rectangle area of the left corresponding image. Asterisk indicates protein exudation; black arrow indicates inflammatory cell infiltration; white arrow indicates trabecular endothelial cell swelling and collapse of intertrabecular spaces. COR, cornea; AC, anterior chamber; S, Schlemm's canal; LEN, lens; RE, retina. Scale bar: 50 µm. The histopathologic score was assessed for severity of inflammation at one DPI (B), three DPI (C), seven DPI (D), and 14 DPI (E). Data are presented as mean ± SEM (n = 3 per group at each time point). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
The results of pathologic analysis (Figs. 4B–E) were consistent with the clinical manifestation, because the histopathologic scores of the VAU groups were notably higher than those of the control group in the first three timepoints, whereas the scores of the MCMV group at one and three dpi were significantly lower than those of the HSV-1 group (P < 0.05). In all observed samples, no inflammation extended to the posterior segment of the eye (Supplementary Fig. S1). Also, no obvious abnormalities were observed in the contralateral eyeball. 
Ultrastructural Changes in the Anterior Chamber Angle After Viral Infection
The viral particles were discovered in three types of cells—rat trabecular meshwork cells (TMCs), corneal endothelial cells (CECs), and nonpigmented ciliary epithelium cells (NPCECs), which confirmed the infection and replication of the virus. In the HSV-1 group, a large number of HSV-1 viral particles, about 100 nanometers (nm) in diameter, were found in the TMCs consistently from 3 to 14 dpi (Figs. 5A2–A4). In addition, the viral inclusion was observed in the CECs at 14 dpi (Fig. 5B2). In the MCMV group, 200 nm-sized MCMV particles were identified in NPCECs at one and three dpi (Figs. 5C1, 5C2) and appeared transiently in TMCs at three dpi (Fig. 5A6). 
Figure 5.
 
Representative electron micrographs of ocular ultrastructure in rat TMCs (A), CECs (B), and NPCECs (C) at different points after intracameral injection. Short red arrow indicates virus particles; long red arrow indicates inclusion body, and the upper right boxes indicate the magnified virus. Ultrastructure cytopathic changes in cellular morphology were observed. Yellow triangle indicates karyolysis; yellow star indicates mitochondrial swelling; long yellow arrow indicates autophagosome; short yellow arrow indicates autolysosome; red asterisk indicates ER dilation; red star indicates monocyte. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; PL, lamina elastic posterior.
Figure 5.
 
Representative electron micrographs of ocular ultrastructure in rat TMCs (A), CECs (B), and NPCECs (C) at different points after intracameral injection. Short red arrow indicates virus particles; long red arrow indicates inclusion body, and the upper right boxes indicate the magnified virus. Ultrastructure cytopathic changes in cellular morphology were observed. Yellow triangle indicates karyolysis; yellow star indicates mitochondrial swelling; long yellow arrow indicates autophagosome; short yellow arrow indicates autolysosome; red asterisk indicates ER dilation; red star indicates monocyte. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; PL, lamina elastic posterior.
Besides, ultrastructural changes were also found within the three types of cells. For the TMCs, control rats showed a clear morphological cellular structure whereas HSV-1-infected TMCs revealed obvious cytotoxic changes—loss of normal cellular structure began at 1 dpi; karyolysis, mitochondrial swelling, endoplasmic reticulum dilation along with vacuolated lesions and autophagosomes were presented under TEM from 1 to 14 dpi (Figs. 5A1–A4). Mitochondrial swelling and autophagosomes of TMCs were also noted in the MCMV group (Figs. 5A6, 5A7), but returned to normal morphology at 14 dpi. Importantly, an inclusion body was found at 3 dpi in the MCMV group (Fig. 5A6; red long arrows). An interesting finding in CECs was that a monocyte was observed near the corneal endothelium in the HSV-1 group at 1 dpi (Fig. 5B1). As for NPCECs, the TEM showed detailed cytopathic changes in the MCMV group in the early postinfection period—the cell became swollen, the number of mitochondria was reduced, and dilated cisterna of the endoplasmic reticulum and numerous autosomes were visible (Fig. 5C1). 
Inflammatory Associated Factors in HSV-1 or MCMV-Induced VAU
To map out the trajectory of the local immune responses generated after HSV-1 or MCMV infection, we evaluated the levels of cytokine and chemokine at the primary site of infection by qRT-PCR. As Figure 6 shows, the proinflammatory cytokines, namely IL-1α, IL-1β, IL-6, IFN-γ, and TNF-α were upregulated significantly in both VAU groups relative to the control group and gradually returned thereafter to baseline levels. 
Figure 6.
 
Comparison of local inflammatory cytokines and chemokines between HSV-AU and MCMV-AU. qRT-PCR analysis of the gene expression of cytokine and chemokine including IL-1α (A), IL-1β (B), IL-6 (C), IL-10 (D), MCP-1 (E), IFN-γ (F), and TNF-α (G) in the corneoscleral rims tissue of HSV-1-infected rats (solid line), MCMV-infected rats (dashed line), and normal control group rats (dotted line). The qRT-PCR with GAPDH primers was performed to serve as an internal control for input DNA. Data are the averages of three independent DNA samples from the infected cells. Values are the mean ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 versus untreated rats. Statistical analysis was performed according to ANOVA followed by Bonferroni tests. MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 6.
 
Comparison of local inflammatory cytokines and chemokines between HSV-AU and MCMV-AU. qRT-PCR analysis of the gene expression of cytokine and chemokine including IL-1α (A), IL-1β (B), IL-6 (C), IL-10 (D), MCP-1 (E), IFN-γ (F), and TNF-α (G) in the corneoscleral rims tissue of HSV-1-infected rats (solid line), MCMV-infected rats (dashed line), and normal control group rats (dotted line). The qRT-PCR with GAPDH primers was performed to serve as an internal control for input DNA. Data are the averages of three independent DNA samples from the infected cells. Values are the mean ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 versus untreated rats. Statistical analysis was performed according to ANOVA followed by Bonferroni tests. MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Specifically, the level of IL-1α showed a statistical increase in the MCMV group whereas the IL-1β mRNA level was generally higher in the HSV-1 group. As for monocyte chemoattractant protein–1, a chemokine capable of recruiting both macrophages and lymphocytes,24 we observed a monophasic peak in both VAU groups, and the magnitude of induction was significantly higher in the HSV-1 group, which was concordant with the inflammation manifestations detected by H&E staining (Fig. 6E). As an anti-inflammatory cytokine, the IL-10 was significantly higher in HSV-1 group, it secreted immediately, peaked at five dpi, and lasted to 14 dpi until recovery of uveitis (Fig. 6D). IFN-γ, together with TNF-α, is a regulatory type 1 cell (Th1) cytokine. In our study, TNF-α peaked at 12 hpi in the MCMV group, corresponding with the first peak of IFN-γ at 1 dpi (Figs. 6F, 6G). Meanwhile, a unique bimodal distribution of IL-1β, IL-6, and IFN-γ was exhibited in the HSV-1 group. There was no significant change in the levels of cytokines in the DMEM control group. The immunohistochemical findings were in accordance with the corresponding qRT-PCR results (Supplementary Fig. S2). 
Discussion
In this study, we successfully infected RTMCs with HSV-1 or MCMV, and observed the clinical characters and pathological features in these two types of VAU rat models. To our knowledge, this was the first comparative study of the similarities and differences between HSV-1 and MCMV-induced VAU models. 
The host's immune defense mechanism is critical for preventing and controlling of viral infection.2527 Here, we established an immunocompetent VAU model in SD rats by IC, which was different from the immunosuppressive model of Zhang et al.,17 who established a CMV-related keratouveitis rat model with only 1/100 of our viral load but elicited a comparable AC inflammation to our MCMV group. Also, because of a low and appropriate level of virus injection, the immune-privileged structure of the eyeball,2729 and various susceptibility and resistance of between rats and mice,30,31 the VAU models in our study differed from previously reported CMV-related retinitis,12,27 HSV-related herpetic contralateral retinitis or encephalitis,26,32 which was more suitable for simulating clinically AC viral infection. 
In our current study, there were some similarities between HSV-1 and MCMV-induced AU. In vitro, both viruses could infect the RTMCs and start replicating rapidly. They shared the same peak time of viral expression, and both developed CPE that would lead to decreased cell viability in the early infected period. In vivo, both groups’ eyes showed typical clinical signs of acute VAU and experienced a significant IOP elevation in the early stage, also, inflammatory cell infiltration and viral particle localization in the anterior chamber angle were both confirmed. Similar proinflammatory cytokines were upregulated in both groups. 
Meanwhile, there were notable differences in the manifestations and pathological characteristics between these two types of VAU models: (1) RTMCs were more vulnerable to HSV-1 than MCMV in vitro; (2) the overall inflammation presentation was severe in HSV-1-infected rats while mild in the MCMV group; (3) a bimodal IOP fluctuation was only seen in the HSV-1 group; (4) diverse cellular tropisms between these two viruses; and (5) different levels of inflammatory cytokine expression. We speculated that there might be two possible reasons accounting for the above differences. On the one hand, different virulence of the virus leads to distinct clinical presentations.12 HSV-1 belongs to the Alpha-Herpesviridae, which was characterized by the ability to replicate and spread quickly, then destroy infected host cells in a short time.33 In contrast, as a member of the Beta-subfamily,12 human CMV and its mouse counterpart MCMV generally replicate more slowly and display strict host species specificity,27 which was consistent with our in vitro results. The balance between virulence and the host immune response sways the outcome of viral infection. On the other hand, the unique tissue tropism of the virus resulted in different manifestations. As identified by the TEM results in our study, HSV-1 viral particles mainly presented in TM, occasionally in cornea endothelium, while MCMV preferred isolating in CB. Viral infection in the TM is not uncommon, because HSV,8,34 MCMV,10,15 and Zika virus35 had been reported to exist in the TM and result in TM damage and an elevated IOP. As the site for aqueous humor production,36 CB was also identified as the virus replication site in MCMV-infected mice,37,38 as well as in current study, which might give a reasonable explanation for the less IOP increase in our MCMV group. 
Several studies had already shown that the Th1 cytokine (like IL-1, IFN-γ, TNF-α), as well as the Th2 cytokines (like IL-6 and IL-10) were all implicated in the pathogenesis and persistence of uveitis, but had respective cytokine profiles in different types of ocular disease.2,3942 Our finding suggested that both viruses induced acute inflammation of the AC microenvironment, with a stronger response in the HSV-1 group compared with MCMV group (Fig. 6). The specific expression of immunoregulatory cytokine IL-10 in the HSV-1 group indicated its unique mechanism for relieving inflammation.39 The monocyte chemoattractant protein–1 was a key chemokine in the development of VAU, involved in recruiting neutrophils and monocytes,42 and upregulating the collagen mRNA expression.4345 Previous studies demonstrated different roles of IFN-γ in the fastigium of acute AU and TNF-α in the early stage of the inflammation46; hence, the upregulation of IFN-γ and TNF-α in our study suggested an earlier inflammatory response in the MCMV group. Interestingly, a bimodal distribution of IL-1β, IL-6 and IFN-γ mRNA expression was found in the HSV-1 group, which was in concordant with the IOP fluctuation in the HSV-AU rats. There might be two possible explanations: first, the bimodal expression of cytokines could be the results of the innate and adaptive immune responses built in the AC; and second, the release of one cytokine might influence the secretion of other cytokines.46 Nevertheless, MCMV-infected rats in our study developed mild AC inflammation but expressed obvious IOP elevation in the early inflammation stage, suggesting that other mechanisms might be involved, which needs our further exploration. 
There are certain limitations to our research. First, we only simulated the primary viral infection in vivo in the current study, yet VAU typically presented with latency and recurrence in clinical practice. Second, despite the fact that rebound tonometry is currently the most commonly used method for IOP measurement in animal models,15,17 the variability in IOP measurement due to corneal edema when severe uveitis occurs cannot be ignored. Third, only male rats were involved in our study. In terms of human studies on VAU, the incidence rate of CMV infection was higher in males than in females, although there was no gender difference in HSV-1 infection.4,9,12 Gender differences should be taken into account in our future studies. Despite these limitations, our study induced two types of VAU models in vitro and in vivo and highlighted the similarities and differences between HSV-1 and MCMV. For the first time, we directly observed the viral particles, tissue tropism, and ultrastructure changes of crucial organelles in the anterior chamber angle of these two VAU models. Focus on local immune state and inflammatory microenvironment is required for investigation on HSV-1 and CMV-associated eye disease. 
In summary, HSV-1 and MCMV infection share certain similarities but have significant differences both in vitro and in vivo. HSV-1 usually has a stronger AC inflammation with a longer duration compared with MCMV in VAU models. Our results provided a valuable animal model for investigating pathogenesis and exploring therapeutic strategies for clinical VAU. 
Acknowledgments
The authors thank Beibei Wang in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for her technical assistance on Transmission Electron Microscopy. 
Supported by the National Natural Science Foundation of China (Nos. 81700829 and 82171045) and the National Natural Science Foundation of Zhejiang Province (No. LZ23H120001). 
Disclosure: Y. Li, None; W. Ke, None; X. Liu, None; Q. Zhang, None; N. Yu, None; K. Wang, None; M. Chen, None 
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Figure 1.
 
Changes of RTMCs after viral infection in vitro. Primary RTMCs were inoculated with HSV-1 or MCMV at a low multiple of infection. (A) Results of light and fluorescence microscopy of infected RTMCs in vitro. LM, light microscopy; FM, fluorescence microscopy. White arrows indicate typical CPE; the white dotted box indicates the enlarged image of the corresponding picture. Scale bar: 200 µm in LM and 50 µm in FM. These studies were each repeated six times with similar results. (B) Effects of HSV-1 or MCMV on RTMCs’ cell survival probability. Every six hours until 48 hpi after treatment, cell viability was measured by CCK8 assay. Data were compared with the control. The mean value of control samples defines as 100%. (C) Immunofluorescence analysis of HSV-gB and MCMV expression was detected respectively by Image J Semi-quantitative analysis of immunofluorescence. Data are presented as mean ± SEM (n = 6). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Figure 1.
 
Changes of RTMCs after viral infection in vitro. Primary RTMCs were inoculated with HSV-1 or MCMV at a low multiple of infection. (A) Results of light and fluorescence microscopy of infected RTMCs in vitro. LM, light microscopy; FM, fluorescence microscopy. White arrows indicate typical CPE; the white dotted box indicates the enlarged image of the corresponding picture. Scale bar: 200 µm in LM and 50 µm in FM. These studies were each repeated six times with similar results. (B) Effects of HSV-1 or MCMV on RTMCs’ cell survival probability. Every six hours until 48 hpi after treatment, cell viability was measured by CCK8 assay. Data were compared with the control. The mean value of control samples defines as 100%. (C) Immunofluorescence analysis of HSV-gB and MCMV expression was detected respectively by Image J Semi-quantitative analysis of immunofluorescence. Data are presented as mean ± SEM (n = 6). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Figure 2.
 
Clinical manifestations of rat eyes after anterior chamber inoculation of HSV-1 (left), MCMV (middle), or DMEM (right) at one, three, seven, and 14 DPI, upper and lowercase letters labeled the same eye by slit-lamp examination and AS-OCT separately (n = 12, each). Representative images of anterior uveitis were observed in the VAU groups. Triangle, cornea edema; asterisk, anterior chamber fibrin exudation; square, KPs; dotted arrow, KNs; solid arrow, occlusion of pupil or discoria. Original magnification of slit-lamp images, × 25 magnification; AS-OCT, anterior segment optical coherence tomography; KNs, Koeppe nodules.
Figure 2.
 
Clinical manifestations of rat eyes after anterior chamber inoculation of HSV-1 (left), MCMV (middle), or DMEM (right) at one, three, seven, and 14 DPI, upper and lowercase letters labeled the same eye by slit-lamp examination and AS-OCT separately (n = 12, each). Representative images of anterior uveitis were observed in the VAU groups. Triangle, cornea edema; asterisk, anterior chamber fibrin exudation; square, KPs; dotted arrow, KNs; solid arrow, occlusion of pupil or discoria. Original magnification of slit-lamp images, × 25 magnification; AS-OCT, anterior segment optical coherence tomography; KNs, Koeppe nodules.
Figure 3.
 
Clinical manifestations score (A) and IOP fluctuation (B) in rats. Data are presented as mean ± SEM, n = 12 in per group. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with the control group.
Figure 3.
 
Clinical manifestations score (A) and IOP fluctuation (B) in rats. Data are presented as mean ± SEM, n = 12 in per group. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with the control group.
Figure 4.
 
Representative histopathological changes of anterior chamber after infection on light microscopy and the histopathological score in injected rats. (A) Light microscopic images of the anterior chamber angle in HSV-1 (left column), MCMV (middle column), and control group (right column) at each observing time. Square images on the right represent the amplification of the rectangle area of the left corresponding image. Asterisk indicates protein exudation; black arrow indicates inflammatory cell infiltration; white arrow indicates trabecular endothelial cell swelling and collapse of intertrabecular spaces. COR, cornea; AC, anterior chamber; S, Schlemm's canal; LEN, lens; RE, retina. Scale bar: 50 µm. The histopathologic score was assessed for severity of inflammation at one DPI (B), three DPI (C), seven DPI (D), and 14 DPI (E). Data are presented as mean ± SEM (n = 3 per group at each time point). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Figure 4.
 
Representative histopathological changes of anterior chamber after infection on light microscopy and the histopathological score in injected rats. (A) Light microscopic images of the anterior chamber angle in HSV-1 (left column), MCMV (middle column), and control group (right column) at each observing time. Square images on the right represent the amplification of the rectangle area of the left corresponding image. Asterisk indicates protein exudation; black arrow indicates inflammatory cell infiltration; white arrow indicates trabecular endothelial cell swelling and collapse of intertrabecular spaces. COR, cornea; AC, anterior chamber; S, Schlemm's canal; LEN, lens; RE, retina. Scale bar: 50 µm. The histopathologic score was assessed for severity of inflammation at one DPI (B), three DPI (C), seven DPI (D), and 14 DPI (E). Data are presented as mean ± SEM (n = 3 per group at each time point). *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001.
Figure 5.
 
Representative electron micrographs of ocular ultrastructure in rat TMCs (A), CECs (B), and NPCECs (C) at different points after intracameral injection. Short red arrow indicates virus particles; long red arrow indicates inclusion body, and the upper right boxes indicate the magnified virus. Ultrastructure cytopathic changes in cellular morphology were observed. Yellow triangle indicates karyolysis; yellow star indicates mitochondrial swelling; long yellow arrow indicates autophagosome; short yellow arrow indicates autolysosome; red asterisk indicates ER dilation; red star indicates monocyte. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; PL, lamina elastic posterior.
Figure 5.
 
Representative electron micrographs of ocular ultrastructure in rat TMCs (A), CECs (B), and NPCECs (C) at different points after intracameral injection. Short red arrow indicates virus particles; long red arrow indicates inclusion body, and the upper right boxes indicate the magnified virus. Ultrastructure cytopathic changes in cellular morphology were observed. Yellow triangle indicates karyolysis; yellow star indicates mitochondrial swelling; long yellow arrow indicates autophagosome; short yellow arrow indicates autolysosome; red asterisk indicates ER dilation; red star indicates monocyte. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; PL, lamina elastic posterior.
Figure 6.
 
Comparison of local inflammatory cytokines and chemokines between HSV-AU and MCMV-AU. qRT-PCR analysis of the gene expression of cytokine and chemokine including IL-1α (A), IL-1β (B), IL-6 (C), IL-10 (D), MCP-1 (E), IFN-γ (F), and TNF-α (G) in the corneoscleral rims tissue of HSV-1-infected rats (solid line), MCMV-infected rats (dashed line), and normal control group rats (dotted line). The qRT-PCR with GAPDH primers was performed to serve as an internal control for input DNA. Data are the averages of three independent DNA samples from the infected cells. Values are the mean ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 versus untreated rats. Statistical analysis was performed according to ANOVA followed by Bonferroni tests. MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 6.
 
Comparison of local inflammatory cytokines and chemokines between HSV-AU and MCMV-AU. qRT-PCR analysis of the gene expression of cytokine and chemokine including IL-1α (A), IL-1β (B), IL-6 (C), IL-10 (D), MCP-1 (E), IFN-γ (F), and TNF-α (G) in the corneoscleral rims tissue of HSV-1-infected rats (solid line), MCMV-infected rats (dashed line), and normal control group rats (dotted line). The qRT-PCR with GAPDH primers was performed to serve as an internal control for input DNA. Data are the averages of three independent DNA samples from the infected cells. Values are the mean ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 versus untreated rats. Statistical analysis was performed according to ANOVA followed by Bonferroni tests. MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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