Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation

Mutations in PITX2 cause Axenfeld–Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.


Introduction
Glaucoma is a multifactorial optic neuropathy characterized by retinal ganglion cell (RGC) loss, which accompanies optic nerve axonal injury. The disease leads to irreversible blindness if left untreated. Elevated intraocular pressure (IOP) is a major risk factor for glaucoma development and progression [1]. Congenital glaucoma, a major cause of childhood blindness, is a severe form of glaucoma that occurs in children from birth to the age of 3 years (infantile glaucoma) or after 3 years of age (juvenile glaucoma). According to the American Academy of Ophthalmology, congenital glaucoma is diagnosed only in 25% of babies who manifest the condition at birth and may occur in as many as 1 in 10,000 live births [2,3]. The increase in IOP in congenital glaucoma is due to developmental abnormalities in the anterior chamber angle, including trabeculodysgenesis, which leads to increased aqueous humor outflow resistance and thus elevated IOP [4,5]. Despite extensive research on congenital glaucoma, the molecular and cellular pathological events that occur are still unclear.
Genetic studies have been conducted to identify genes associated with congenital glaucoma. Research has shown that mutations in certain genes, such as PITX2, FOXC1, PAX6, and CYP1B1, are associated with congenital glaucoma [6][7][8][9][10][11]. Among these genes, extensive work has been carried out to identify the functions of PITX2. PITX2 is involved

Slit Lamp Examination
Anterior segments of mouse eyes were examined with a slit lamp (SL-D7; Topcon, Tokyo, Japan), and images were taken with a digital camera (D100; Nikon, Tokyo, Japan). Slit lamp examinations were performed on conscious animals. Six C57BL/6J and twentytwo egl1 mice were examined longitudinally at age 4 weeks to 12 months.

Anterior Segment Histologic Examination
For histologic examination, animals were euthanized by exposure to 10% to 30% cage volume/min carbon dioxide. After death was confirmed, eyes were enucleated and fixed in 10% neutral formalin (Electron Microscopy Sciences, Hatfield, PA, USA) overnight. Eyes were then dehydrated with ethanol and xylene and embedded in paraffin. Sagittal sections (5 µm) were prepared, mounted on glass microscope slides, and stained with hematoxylin and eosin (H&E) for structural evaluation. Three C57BL/6J and six egl1 mice were examined before and 4 weeks after intraocular pressure elevation.

Aqueous Humor (AH) Outflow Facility Measurement
The egl1 mice at ages 8 weeks and 1 year were used for aqueous humor outflow facility measurement, which was performed using a constant flow infusion method established previously [33][34][35][36]. In brief, mice were anesthetized by an intraperitoneal injection of a cocktail of ketamine/xylazine (100/10 mg/kg, respectively; maintenance: 1/2 × to 1/4 × induction dose). One drop of 0.5% proparacaine HCl was applied for corneal anesthesia (Alcaine, Alcon, Fort Worth, TX, USA). The anterior chamber of each eye was cannulated with a 32-gauge needle attached to tubing connected to a pressure transducer (BLPR2; World Precision Instruments (WPI), Sarasota, FL, USA) and a glass microsyringe (Hamilton Company, Reno, NV, USA) filled with sterile PBS and loaded onto a microdialysis infusion pump (SP101i; WPI). The eyes were infused at a flow rate of 0.1 µL/min initially for approximately 30 min to stabilize the pressure registered by the pressure transducer. On pressure stabilization, 3 pressure readings, spaced 5 min apart, were obtained over the following 10 min period. The flow rate was then increased to 0.2 µL/min, and following 5 min for stabilization, 3 pressure readings were obtained in a similar manner. The process was then repeated at flow rates of 0.3, 0.4, and 0.5 µL/min. Mean stabilized pressure flow rate curves were generated for each eye and fit using simple linear regression. The total aqueous humor outflow facility was calculated as the reciprocal of the slope of each respective curve. All measurements were conducted in a single masked manner. Seven egl1 mice (3 aged 2 months and 4 aged 12 months) were assessed.

Retinal Ganglion Cell Function Assessment
Pattern electroretinography (PERG) was performed in 6 egl1 mice using the JORVEC System (Intelligent Hearing Systems, Miami, FL, USA) as described previously [37]. The egl1 mice with severe corneal opacity that blocked central cornea were excluded from the PERG study. Briefly, mice were anesthetized with intraperitoneal injections of a mixture of ketamine and xylazine (100 and 10 mg/kg, respectively). The PERG responses were recorded from a stainless-steel needle (Grass, West Warwick, RI, USA) placed in the snout subcutaneously. Pattern stimuli consisting of contrast-reversing gratings with a spatial frequency of 0.05 cycles/deg and maximum contrast were displayed on two custom-made tablets. The contrast reversal frequency was 1 Hz. A total of 2232 responses were averaged. The amplitude was measured from the positive peak to the negative trough, and the latency was the time to the peak of the response.

Retinal Ganglion Cell Quantification
Quantification of retinal ganglion cells (RGCs) was performed using immunostained retinal whole mounts [38]. Briefly, mice (5 C57BL/6J and 21 egl1 mice) were euthanized by exposure to 100% CO 2(g) . Following the cessation of breathing and heartbeat, eyes were enucleated and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 2 h at 4 • C. Retinas were dissected from fixed eyes and blocked with 0.3% Triton X-100 in PBS containing 2% goat serum for 2 h. Retinas were incubated in rabbit polyclonal RBPMS antibody (1:200, diluted in 0.3% Triton X-100 in PBS, GeneTex GTX118619, Irvine, CA, USA) [39] overnight at 4 • C. Following washes in PBS, retinas were further incubated in AlexaFluor596 goat-anti-rabbit (1:1000, diluted in 0.1% Triton X-100 in PBS) overnight at 4 • C. After washes with PBS, the retinas were cut into four quadrants and mounted on glass slides (Fischer Scientific, Pittsburgh, PA, USA). Eight images were taken from the peripheral and mid-peripheral regions in four quadrants of each retina. The number of cells from each image (0.0867 mm 2 retina area) was counted using Adobe Photoshop software V22.2 (Adobe Systems, Inc., San Jose, CA, USA). The average of all counts from each retina was used as the number of RGCs presented in each eye. Cell counts were performed in a masked manner.

Statistical Analysis
One-way ANOVA followed by a Tukey post hoc test was performed to analyze intragroup differences. The unpaired Student's t-test was used for the comparison of differences between the two groups. The correlation between RGC counts and IOP exposure was assessed by Pearson's correlation coefficient. Data are presented as means ± SEM, and p < 0.05 was considered statistically significant.

Anterior Segment Morphology in egl1 Mice
We performed slit lamp examination on C57/BL/6J ( Figure 1A) and egl1 mice ( Figure 1B-I) aged 4 weeks to 12 months. The egl1 mice developed anterior segment abnormalities of various types and severities ( Figure 1C-I). Approximately 60% of eyes showed localized faint opacity in the paracentral zone of the cornea ( Figure 1C). The onset of the corneal lesion was as early as 4 weeks old and appeared independent of mechanical stimulation. Some corneas of aged egl1 mice remained clear, despite repeated IOP measurements ( Figure 1B), similar to those of C57BL/6J wildtype animals ( Figure 1A). The corneal lesion progressed to dense corneal opacity with neovascularization in some animals as they aged ( Figure 1D,E). In a few animals aged 12 months, we observed the diffuse opacity covering the entire cornea ( Figure 1F).
The egl1 mice also developed iris defects including anterior synechiae ( Figure 1G-I), atrophy ( Figure 1D), and pupil deviation ( Figure 1E). We further examined the anterior segment histology of 8-week-old young adult mice. In the egl1 mice with normal IOP, the iridocorneal angle remained open, and the ciliary body appeared normal ( Figure 1K) compared to C57BL/6J wildtype mice ( Figure 1J). In eyes with elevated IOP ( Figure 1L), there were anterior synechiae and ciliary body atrophy. Despite the ocular abnormalities, egl1 animals appeared similar in body size and coat color compared to C57BL/6J mice.

IOP Elevation in egl1 Mice
The egl1 mice were maintained on the C57BL/6J background; thus, C57BL/6J mice were used as control animals in this analysis. We monitored the conscious IOP longitudinally in a cohort of 22 egl1 mice and 6 C57BL/6J mice using the TonoLab tonometer ( Figure  2A-C). At 3 weeks of age, egl1 mice exhibited an IOP of 15.9 ± 0.5 mmHg (mean ± SEM; n = 44) which was similar to age-matched C57BL/6J control mice (14.0 ± 1.3 mmHg; mean ± SEM; n = 12). A significant elevation of IOP was observed in egl1 mice as early as 4 weeks old and remain elevated at all ages examined. The IOP values were 19.2 ± 0.6, 21.7 ± 0.8,
The IOP elevation in egl1 mice was variable and asymmetric. By age 8 weeks, 55% of mice examined exhibited IOPs under 20 mmHg ( Figure 2E). The elevated IOP values also demonstrated a wide range in mice of the same age. Some mice had IOPs greater than 35 mmHg at all time points examined. Furthermore, individual animals showed dramatic asymmetry in IOPs, which differed by more than 15 mmHg.

respectively.
The IOP elevation in egl1 mice was variable and asymmetric. By age 8 weeks, 55% of mice examined exhibited IOPs under 20 mmHg ( Figure 2E). The elevated IOP values also demonstrated a wide range in mice of the same age. Some mice had IOPs greater than 35 mmHg at all time points examined. Furthermore, individual animals showed dramatic asymmetry in IOPs, which differed by more than 15 mmHg.

Aqueous Humor Outflow Facility in egl1 Mice
The AH circulation plays a key role in IOP regulation. We measured total aqueous humor outflow facility in young (2 months) and aged (1 year) egl1 mice. The outflow facility in young animals was 30.25 ± 1.34 nL/min/mmHg (mean ± SEM; n = 5); The aged animals demonstrated significantly reduced outflow facility (17.07 ± 1.44 nL/min/mmHg, mean ± SEM; n = 7, p < 0.001), indicating a significantly higher outflow resistance in the aged animals ( Figure 3A). The outflow facility and IOP measurement showed a trend of negative correlation, but this did not achieve statistical significance ( Figure 3B).

Aqueous Humor Outflow Facility in egl1 Mice
The AH circulation plays a key role in IOP regulation. We measured total aqueous humor outflow facility in young (2 months) and aged (1 year) egl1 mice. The outflow facility in young animals was 30.25 ± 1.34 nL/min/mmHg (mean ± SEM; n = 5); The aged animals demonstrated significantly reduced outflow facility (17.07 ± 1.44 nL/min/mmHg, mean ± SEM; n = 7, p < 0.001), indicating a significantly higher outflow resistance in the aged animals ( Figure 3A). The outflow facility and IOP measurement showed a trend of negative correlation, but this did not achieve statistical significance ( Figure 3B).

RGC Death in egl1 Mice
The degeneration of RGCs is a key feature of glaucoma. We quantified the RGC loss in response to IOP elevation in egl1 mice to assess the glaucomatous neurodegeneration.

RGC Death in egl1 Mice
The degeneration of RGCs is a key feature of glaucoma. We quantified the RGC loss in response to IOP elevation in egl1 mice to assess the glaucomatous neurodegeneration. The egl1 mice with normal IOPs showed similar numbers of RGCs (263 ± 6/0.0867 mm 2 ; mean ± SEM; n = 17) in the retina, compared to C57BL/6J control mice (270 ± 8/0.0867 mm 2 ; mean ± SEM; n = 10). As expected, prolonged exposure to high IOP induced RGC loss in egl1 mice. RGC numbers (217 ± 14/0.0867 mm 2 ; mean ± SEM; n = 14) were significantly decreased in mice with 8 weeks of IOP elevation ( Figure 4A,B).

RGC Functional Loss in egl1 Mice
To further examine glaucomatous neurodegeneration in egl1 animals, we performed PERG recording in mice with normal and elevated IOP. The egl1 animals demonstrated a normal PERG waveform with a positive peak elicited at 89.6 ± 2.4 ms, followed by a negative trough ( Figure 5A). The PERG amplitude in the egl1 mice with normal IOP was 23.0 ± 3.2 µV (mean ± SEM; n = 5). After 2 weeks of IOP elevation, there was a significant decrease in PERG amplitude (9.2 ± 1.3 µV) ( Figure 5B) as well as an increase in latency (155.7 ± 21.6 ms, mean ± SEM; n = 6), which indicates damaged RGC function ( Figure 5C). To determine the correlation between RGC death and IOP elevation, we further plotted the RGC numbers against IOP exposure calculated as the area under the IOP-time curve. The RGC counts showed a moderate negative correlation (R square: 0.43, p < 0.001) with IOP exposure ( Figure 4C).

RGC Functional Loss in egl1 Mice
To further examine glaucomatous neurodegeneration in egl1 animals, we performed PERG recording in mice with normal and elevated IOP. The egl1 animals demonstrated a normal PERG waveform with a positive peak elicited at 89.6 ± 2.4 ms, followed by a negative trough ( Figure 5A). The PERG amplitude in the egl1 mice with normal IOP was 23.0 ± 3.2 µV (mean ± SEM; n = 5). After 2 weeks of IOP elevation, there was a significant decrease in PERG amplitude (9.2 ± 1.3 µV) ( Figure 5B) as well as an increase in latency (155.7 ± 21.6 ms, mean ± SEM; n = 6), which indicates damaged RGC function ( Figure 5C).

Glaucomatous ONH Changes in egl1 Mice
Elevated IOP induces glial activation in the ONH and optic nerve axonal degeneration, hallmarks of glaucomatous optic neuropathy. To evaluate these phenotypes in egl1 mice, we collected eyes from C57BL/6J and egl1 mice before and 2 and 8 weeks after IOP elevation and performed immunofluorescence staining using specific antibodies against glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba-1), and neurofilament (NF) (Figure 6). Eyes of C57BL/6J and egl1 mice with normal pressure exhibited similar expression levels and patterns of GFAP, Iba-1, and NF. Elevated IOP increased expression levels of GFAP and Iba-1, which peaked at 2 weeks, indicating astrocyte activation and microglia infiltration in the ONH in response to glaucomatous insult. Quantification of GFAP and Iba-1 fluorescence intensity showed a trend of increase at 2 weeks after IOP elevation without statistical significance (n = 3-6). The pattern of neurofilaments became disorganized after 2 weeks of IOP elevation. Eyes with extended pressure elevation further exhibited disruption of neurofilaments, suggesting optic nerve ax-

Glaucomatous ONH Changes in egl1 Mice
Elevated IOP induces glial activation in the ONH and optic nerve axonal degeneration, hallmarks of glaucomatous optic neuropathy. To evaluate these phenotypes in egl1 mice, we collected eyes from C57BL/6J and egl1 mice before and 2 and 8 weeks after IOP elevation and performed immunofluorescence staining using specific antibodies against glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba-1), and neurofilament (NF) (Figure 6). Eyes of C57BL/6J and egl1 mice with normal pressure exhibited similar expression levels and patterns of GFAP, Iba-1, and NF. Elevated IOP increased expression levels of GFAP and Iba-1, which peaked at 2 weeks, indicating astrocyte activation and microglia infiltration in the ONH in response to glaucomatous insult. Quantification of GFAP and Iba-1 fluorescence intensity showed a trend of increase at 2 weeks after IOP elevation without statistical significance (n = 3-6). The pattern of neurofilaments became disorganized after 2 weeks of IOP elevation. Eyes with extended pressure elevation further exhibited disruption of neurofilaments, suggesting optic nerve axonal damage in the egl1 mice.

Discussion
The present study characterized the glaucoma phenotype in a mouse strain carrying a missense mutation of Pitx2 named the egl1 mutation. The egl1 mutant mice developed glaucoma with a significant variation in the age of onset and phenotypic severity within and between animals. The mice with ocular hypertension demonstrated glaucomatous retinopathy and optic nerve neuropathy.
As a powerful tool, mouse models carrying the Pitx2 mutation or deletion have been developed to investigate the functions of PITX2 and the pathogenesis of glaucoma. The egl1 mice have a chemically induced Pitx2 mutation and develop early-onset glaucoma. The mouse strain is homozygous for the egl1 mutation, which was mapped to chromosome 3. High-throughput sequencing further identified the mutation as a single G to T transversion in Pitx2 exon 2, changing amino acid 115 from arginine to leucine [26,27]. In humans, PITX2 has been identified as a glaucoma-causing gene. Mutations in PITX2 are associated with Axenfeld-Rieger syndrome, which involves ocular malformations leading to congenital and childhood glaucoma in more than 50% of the affected patients [40]. Similarly, we found that the egl1 mouse strain developed anterior segment abnormalities, and by the age of 6 weeks, approximately 50% of the animals developed early-onset glaucoma despite having an identical genotype and genetic background. Gender difference in

Discussion
The present study characterized the glaucoma phenotype in a mouse strain carrying a missense mutation of Pitx2 named the egl1 mutation. The egl1 mutant mice developed glaucoma with a significant variation in the age of onset and phenotypic severity within and between animals. The mice with ocular hypertension demonstrated glaucomatous retinopathy and optic nerve neuropathy.
As a powerful tool, mouse models carrying the Pitx2 mutation or deletion have been developed to investigate the functions of PITX2 and the pathogenesis of glaucoma. The egl1 mice have a chemically induced Pitx2 mutation and develop early-onset glaucoma. The mouse strain is homozygous for the egl1 mutation, which was mapped to chromosome 3. High-throughput sequencing further identified the mutation as a single G to T transversion in Pitx2 exon 2, changing amino acid 115 from arginine to leucine [26,27]. In humans, PITX2 has been identified as a glaucoma-causing gene. Mutations in PITX2 are associated with Axenfeld-Rieger syndrome, which involves ocular malformations leading to congenital and childhood glaucoma in more than 50% of the affected patients [40]. Similarly, we found that the egl1 mouse strain developed anterior segment abnormalities, and by the age of 6 weeks, approximately 50% of the animals developed early-onset glaucoma despite having an identical genotype and genetic background. Gender difference in the risk for glaucoma and glaucoma blindness has been well documented [41,42]. In the egl1 mice, ocular hypertension developed in male and female mice at a similar percentage. Larger-scale studies are needed to reveal potential sex-associated differences in glaucoma development and severity in egl1 animals. The IOP elevation in egl1 animals exhibited significant variation and asymmetry. The onset of ocular hypertension was as early as 3 weeks old, and IOP was elevated to a maximum of 20 to 37 mmHg. Individual animals exhibited significant ocular hypertension in one eye and normal IOP in another. The variation and asymmetry in the development of ocular hypertension may require larger animal cohorts in the research design to ensure a sufficient sample size for statistical analysis. On the other hand, the wide range of IOP values offers a spectrum of disease severity and eyes with normal tension, providing an ideal strain-matched control.
Through our longitudinal IOP measurements, we also found that the IOP elevation in the egl1 mice demonstrated two patterns: sustained mild elevation until the age of 4 months or high elevation followed by normal pressure. Ciliary body atrophy has been described in the Pitx2 +/− mice as well as in human chronic cases when left untreated [25]. Similarly, the histologic examination showed ciliary body atrophy in the egl1 mice with prolonged ocular hypertension. The reduction of IOP following a high pressure elevation in the egl1 mice is likely due to ciliary body atrophy. Aqueous humor is produced by the epithelium of the ciliary body and drained primarily through the conventional outflow pathway consisting of the trabecular meshwork (TM), Schlemm's canal, the scleral collector channels, and aqueous veins, sequentially. It has been recognized that increased aqueous humor outflow resistance is associated with elevated IOP in glaucoma [43,44]. In addition, studies in humans and monkeys have shown that aqueous outflow facility (the reciprocal of aqueous humor outflow resistance) naturally declines with aging, even in healthy eyes [45,46]. Thus, we measured the AH outflow facility in young and aged animals and assessed the correlation between IOP and AH outflow facility. There was a negative trend of correlation between IOP and AH outflow facility, suggesting a role played by declined outflow facility in IOP elevation in the egl1 mice. However, we did not detect a statistically significant correlation between IOP and facility.
Pattern ERG is a well-accepted standard for assessing RGC function. It allows noninvasive and longitudinal evaluation of RGC function. It detects RGC functional loss prior to significant morphological damage [47]. Using PERG, we detected RGC functional loss at 2 weeks after IOP elevation when a significant RGC death was undetectable. The results further demonstrate the glaucomatous phenotype in egl1 animals and support the use of PERG as a sensitive tool to monitor the disease progression in the egl1 mouse strain.
The quantification of RGCs showed a mild correlation between RGC loss and IOP exposure. In addition, outer retinal dysfunction has been reported in egl1 mice. At 3 months of age, there is a decreased rod b-wave and a lower cone response, which indicates a possible outer retinal degeneration [27]. A separate study also showed that the deletion of the Pitx2 gene disrupts the development of the retinal pigment epithelium [23]. It is also possible that outer retinal degeneration may in turn affect RGC health within the inner retina and contribute to the decrease of RGC numbers.
Recent studies have shown that glia play an important role in the pathogenesis of glaucoma [48][49][50][51]. Astrocytes, the major glial cell population in the ONH, are considered the mediators of axonal injury in glaucoma. They respond to mechanical compression due to IOP elevation and undergo a number of cellular changes, such as upregulation of GFAP cytoskeleton and neurotrophic or neuroinflammatory factors [49,52]. Similarly, microglia in the ONH become activated and redistributed, express cytokines and other secreted factors, and exert neuroprotective or neurotoxic effects in glaucomatous eyes [51]. In the egl1 animals, we found upregulated GFAP, and Iba-1 expression also peaked at an early stage of the disease, suggesting the activation of astrocytes and infiltration of microglia following glaucomatous insults. This further validates the glaucoma phenotype in the mutant animals. The results also support the use of egl1 mice as an in vivo model to investigate mechanisms of glial activation in glaucoma.
In summary, this study demonstrates that the egl1 mouse strain exhibits key features of early-onset glaucoma and provides an important tool to study glaucomatous neurodegeneration. Additional studies are needed to further characterize glaucomatous neuropathy in egl1 animals.