Loss of retinal ganglion cells in a new genetic mouse model for primary open‐angle glaucoma

Abstract Primary open‐angle glaucoma (POAG) is one of the most common causes for blindness worldwide. Although an elevated intraocular pressure (IOP) is the main risk factor, the exact pathology remained indistinguishable. Therefore, it is necessary to have appropriate models to investigate these mechanisms. Here, we analysed a transgenic glaucoma mouse model (βB1‐CTGF) to elucidate new possible mechanisms of the disease. Therefore, IOP was measured in βB1‐CTGF and wildtype mice at 5, 10 and 15 weeks of age. At 5 and 10 weeks, the IOP in both groups were comparable (P > 0.05). After 15 weeks, a significant elevated IOP was measured in βB1‐CTGF mice (P < 0.001). At 15 weeks, electroretinogram measurements were performed and both the a‐ and b‐wave amplitudes were significantly decreased in βB1‐CTGF retinae (both P < 0.01). Significantly fewer Brn‐3a+ retinal ganglion cells (RGCs) were observed in the βB1‐CTGF group on flatmounts (P = 0.02), cross‐sections (P < 0.001) and also via quantitative real‐time PCR (P = 0.02). Additionally, significantly more cleaved caspase 3+ RGCs were seen in the βB1‐CTGF group (P = 0.002). Furthermore, a decrease in recoverin+ cells was observable in the βB1‐CTGF animals (P = 0.004). Accordingly, a significant down‐regulation of Recoverin mRNA levels were noted (P < 0.001). Gfap expression, on the other hand, was higher in βB1‐CTGF retinae (P = 0.023). Additionally, more glutamine synthetase signal was noted (P = 0.04). Although no alterations were observed regarding photoreceptors via immunohistology, a significant decrease of Rhodopsin (P = 0.003) and Opsin mRNA (P = 0.03) was noted. We therefore assume that the βB1‐CTGF mouse could serve as an excellent model for better understanding the pathomechanisms in POAG.

mice were screened by isolating genomic DNA from tail biopsies and testing for transgenic sequenced by PCR, using the following primer sequences: 5'-GGAAGTGCCAGCTCATCAGT-3' and 5'-GTGCGGGACAGAAACCTG-3'. 15 weeks old female and male mice were included in the study. All procedures concerning animals adhered to the ARVO statement for the use of animals in ophthalmic and vision research. All experiments involving animals were approved by the animal care committee of North Rhine-Westphalia, Germany. Mice were kept under environmentally controlled conditions with free access to chow and water.

| Measurement of IOP
Intraocular pressure of both eyes and both groups was measured at 5, 10 and 15 weeks (n = 6-10 animals/group) using a rebound tonometer (TonoLab, Icare) as described previously. 14,15 For this procedure, mice were anaesthetized with a ketamine/xylazine cocktail (120/16 mg/kg). All measurements were performed by one examiner at the same time of the day. For each analysis, the mean of 10 measurements was calculated.

| Electroretinogram analyses
For electroretinogram (ERG) measurements, mice were dark adapted overnight. The retinal function was monitored in both eyes using full-field-flash electroretinography (HMsERG system, OcuScience LLC) after 15 weeks as described previously (n = 8 animals/group). 16,17 Mice were anaesthetized with a ketamine/xylazine cocktail (120/16 mg/kg) and eyes were dilated with tropicamide (5%) and topically anaesthetized using conjuncain. Body temperature was maintained at 37°C with a feedback temperature controller (TC-1000; CWE Inc). Reference electrodes were placed subcutaneously below the right and left ear and a ground electrode was placed in the base of the tail. Contact lenses with silver thread recording electrodes were placed in the center of the cornea after application of methocel (Omni Vision). Before measurement, the electroretinography equipment was covered with a faraday cage. Scotopic flash ERGs were recorded at 0.1, 0.3, 1, 3, 10 and 25 cd.s/m 2 . For the light intensities of 0.1-3 cd.s/ m 2 , four flashes were averaged and for 10 and 25 cd.s/m 2 , one flash was measured. The interstimulus interval was 10 seconds between flashes of the same light intensity. Signals obtained from the corneal surface were amplified, digitized, averaged and stored using commercial software (ERGView 4.380R; OcuScience LLC).
A 50 Hz filtering of the data was applied before evaluating the amplitude of the a-and b-wave.

| Histological examination
All photographs were taken using a fluorescence microscope (Axio Imager M1 or M2). Two photos of the peripheral and two of the central part of each section were captured. The images were transferred to Corel Paint Shop Pro (V13, Corel Corporation) and equal excerpts were cut out. 19 Afterwards, Brn-3a + , PKCα + and opsin + cells were counted using ImageJ software. Regarding GFAP, glutamine synthetase, vimentin, rhodopsin and recoverin, area analyses were performed using an ImageJ macro. 19,20 Briefly, images were transformed into grayscale. To minimize interference with background labeling, a defined rolling ball radius was subtracted (Table 2). Then, for each picture, a suitable lower threshold was set. The ideal threshold was obtained when the grayscale picture and the original one corresponded (Table 2). Afterwards, the mean value of the lower threshold was calculated, and this number was used for final analysis. The percentage of the labelled area was measured between these defined thresholds.

| Quantitative real-time PCR
Both retinae of each animal (five animals/group) were pooled for RNA preparation and cDNA synthesis as previously described. 18,21 The designed oligonucleotides for Quantitative real-time PCR (qRT-PCR) are shown in Table 3. Β-actin and Cyclophilin served as reference genes. The qRT-PCR was performed using DyNAmo Flash SYBR Green (Thermo Scientific) on the PikoReal qRT-PCR Cycler (Thermo Scientific). 17,22

| Increase of IOP
Intraocular pressure was measured after 5, 10 and 15 weeks ( Figure 1A). We could not measure significant changes in the IOP between βB1-CTGF and WT animals after 5 weeks (βB1-CTGF: 10

| Loss of retinal ganglion cells
To evaluate a possible alteration in the number of RGCs, flatmounts were stained with an antibody against Brn-3a 18 (Figure 2A).
Additionally, qRT-PCR analyses were performed regarding the TA B L E 4 Summary of electroretinogram results. For all light intensities, the mean a-and b-wave amplitudes of wildtype (WT) and βB1-CTGF animals and the respective p-values are shown   Figure 2B). Furthermore, a significant down-regulation was observed in Pou4f1 mRNA levels at this age (0.40-fold; P = 0.02).
To confirm the loss of RGCs, cross-sections were labeled with anti-Brn-3a. To detect a possible apoptosis of RGCs, co-staining with an antibody against cleaved caspase 3 was performed ( Figure 3A).

| Macroglia activation
A possible macrogliosis was investigated by labeling retinae against anti-GFAP, anti-vimentin and anti-glutamine synthetase (Müller glia and astrocytes; Figure 4A). Furthermore, the mRNA expression levels of Gfap was evaluated via qRT-PCR ( Figure 4C). The GFAP area analysis showed a slight trend to more GFAP signal in βB1-CTGF  Figure 4B). However, qRT-PCR analyses revealed a significant up-regulation of Gfap expression levels in the βB1-CTGF retinae compared to WT (2.2-fold; P = 0.02; Figure 4C).

| Decrease in cone bipolar cells
To evaluate the number of bipolar cells, retinae were labelled with anti-PKCα (rod bipolar cells) and anti-recoverin (cone bipolar cells; Figure 5A). Additionally, qRT-PCR analyses were performed regarding the mRNA levels of Pkcα and Recoverin ( Figure 5C,E). We  Figure 5D). Also, we noted no  Figure 5E).

| D ISCUSS I ON
The pathomechanisms leading to glaucoma are still not known precisely. However, an elevated IOP has been consistently associated with the prevalence 25-28 and incidence [29][30][31] of open-angle glaucoma. 1 In most western countries, approximately half of the patients with manifest glaucoma are not diagnosed. 1,32,33 To improve both the treatment and the diagnosis of glaucoma patients, it is necessary to have appropriate models. The βB1-CTGF mouse seems to be a promising tool to analyze mechanisms in POAG. In 2012, Junglas et al reported that the overexpression of CTGF led to an increased IOP accompanied with a progressive loss of optic nerve axons. 12 In this study, the transgenic βB1-CTGF mice also developed an elevated IOP after 15 weeks. This is diverging to previous results, where an increased IOP was noted already after 1 month. 12  The majority of retinal diseases are associated with reactive gliosis. [37][38][39] The astrocytes and Müller glia cells become reactive during the pathogenesis of glaucoma, characterized by morphologic alterations and expression changes. [40][41][42] We found that the observed loss of RGC cells is accompanied by changes of the macroglia cells in the retinae of the βB1-CTGF mice. The immunohistological analysis showed a slight increase of astrocytic markers like GFAP and vimentin, but we found a profound increase of GFAP mRNA in the βB1-CTGF mice. We assume that the changes could be the start of a F I G U R E 6 (A), Rhodopsin (rods, green) and opsin (L-cones, red) staining of retinal sections was performed to evaluate photoreceptors. DAPI was added to visualize cell nuclei (blue). (B), Comparable rhodopsin + areas were detected in both groups (P > 0.05). (C), However, qRT-PCR analyses revealed a significant down-regulation of Rhodopsin mRNA expression levels in βB1-CTGF retinae (P = 0.003). (D), The number of opsin + cells remained unaltered in βB1-CTGF animals compared to wildtype (WT) (P > 0.05).
(E), The mRNA expression levels of Opsin were significantly down-regulated in in βB1-CTGF retinae (P = 0.03). ONL, outer plexiform layer; OS, outer segment. Values are mean ± SEM for immunohistology and median±quartile+maximum/minimum for qRT-PCR. Scale bar: 20 μm. *P < 0.05, **P < 0.01. Immunohistology: n = 7 eyes/ group; qRT-PCR: n = 5 animals/group remodelling process in the retinal astrocytes based on the IOP and the RGC loss. It is known that astrocytes of the optic nerve head respond strongly to glaucomatous damage. 43 In retinal diseases, the induction of GFAP in Müller cells is an early and very sensitive marker for reactive Müller cells, which is often accompanied by an increased expression of glutamine synthetase. 44 Accordingly, in the eyes of human donors suffering from glaucoma, an increased expression of GFAP in Müller cells has been detected. 45 In our study, the Müller glial cells showed a GFAP positive signal in the βB1-CTGF mice in comparison to the WT littermates. As the Müller glial signal of the GFAP staining had an overlap with the signal of the astrocytes it is rather difficult to quantify the differences. To overcome this problem, we stained against glutamine synthetase in both groups.
The Müller glial cells had a significant enhanced signal for glutamine synthetase, which emphasize a reactive state of the Müller glial cells.
The enhanced reactivity of Müller glial cells could be a response to the IOP induced RGC loss and can mediate both protective as well as detrimental effects on retinal neurons. 44 To characterize the βB1-CTGF model more precisely, we additionally investigated whether an increased IOP could affect also other retinal layers. In previous studies, the induction of OHT in mice of the albino Swiss strain caused a reduced recoverin immunoreactivity and the number of PKCα + rod bipolar cells was diminished. 46 Similar results were observed in a rat model after IOP elevation, were a decrease of rod bipolar cells was reported 5 weeks after IOP elevation. 47 In our study, we could only observe a diminished cone bipolar cell signal at 15 weeks of age in the retinae of the βB1-CTGF in comparison to their WT littermates. The different results regarding the rod bipolar cells could be explained by the different experimentally and genetically mechanisms causing the increased IOP. In the βB1-CTGF mouse, cone bipolar cells seem to be more sensitive to IOP changes. Nevertheless, ERG data from our study suggest a functional loss of inner nuclear cells and photoreceptors in βB1-CTGF animals. In glaucoma patients, ERG measurements revealed a degeneration of photoreceptor cells in late stages of glaucoma. 48,49 Also after laser coagulation in rats, a secondary loss of cones could be observed. 50 In aged DBA2/J mice, the a-wave amplitude was decreased, pointing towards an impairment of photoreceptors. 51 In summary, elevated IOP is accompanied by apoptotic RGC loss and reactive gliosis in the βB1-CTGF mice. We therefore conclude that the βB1-CTGF mouse model can be used to study pathomechanisms occurring in POAG.

ACK N OWLED G EM ENTS
This study was supported by FoRUM (Ruhr-University Bochum), Deutsche Forschungsgemeinschaft (FU734/4-1), and Ernst und Berta Grimmke foundation (Germany). We acknowledge support by the DFG Open Access Publication Funds of the Ruhr-University Bochum.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflict of interest.

AUTH O R CO NTR I B UTI O N S
SR, DK, FF, MW and CV performed the research; SR, DK, and MW analysed the data; RF and SCJ designed the research study; SR wrote the paper; HBD, RF, and SCJ reviewed the manuscript.