BrdU Positive Cells Induced in a Genetic Mouse Model of Glaucoma

Research Article

J Ophthalmol & Vis Sci. 2021; 6(1): 1046.

BrdU Positive Cells Induced in a Genetic Mouse Model of Glaucoma

Paris JR¹, Sklar NC² and Linn CL¹*

¹Department of Biological Sciences, Western Michigan University, Kalamazoo MI, 49008, United States

²Western Michigan University Homer Stryker M.D. School of Medicine, Kalamazoo, MI, United States

*Corresponding author: Cindy Linn, Department of Biological Sciences, Western Michigan University, 1903 W. Michigan Ave, Kalamazoo, MI 49008, USA

Received: January 08, 2021; Accepted: March 01, 2021; Published: March 08, 2021

Abstract

Previous studies have shown that eye drop application of the selective a7 nicotinic acetylcholine receptor agonist, PNU-282987, induces neurogenesis of RGCs in adult wild-type rodents. This study was designed to test the hypothesis that PNU-282987 reverses the loss of RGCs associated with glaucoma. A DBA/2J mouse model that auto-induces a glaucoma-like condition in adulthood was used for these studies. Short-term effects using PNU-282987 and BrdU eye drop treatments were examined, as well as the effects of early treatment and the effects in a chronic early treatment group in DBA/2J mice aged 3, 6 and 10 months. With and without treatment, retinas were removed, fixed, immunostained and RGC counts were assessed. IOP measurements were obtained weekly using a Tonolab tonometer. Results showed an average typical loss of BrdU positive RGCs by 29% by 10 months of age in this DBA/2J colony corresponding with a significant increase in IOP. However, the two-week short term application of PNU-282987 and BrdU induced a significant 21% increase in RGCs for DBA/2J mice at all ages. Chronic early PNU-282987 treatment produced a similarly significant increase in RGCs, while acute early treatment had no effect on RGC numbers. IOP measurements were not affected with PNU-282987 treatment. These studies demonstrated that 2-week treatment with PNU-282987, as well as chronic long-term treatment, induced a significant increase in the number of RGCs in the DBA/2J retina, counteracting the effects of the DBA/2J genetic glaucoma-like condition. These results suggest a potential future treatment of degenerative retinal diseases with PNU-282987.

Keywords: DBA; Glaucoma; Regeneration; PNU-282987

Abbreviations

RGCs: Retinal Ganglion Cells; BrdU: 5-Bromo-2’-Deoxyuridine; IOP: Intraocular Pressure; ACh: Acetylcholine; GCL: Ganglion Cell Layer; alpha7nAChRs: Alpha7 Nicotinic Acetylcholine Receptors; RPE: Retinal Pigment Epithelium; MG: Muller Glia; MDPCs: Muller- Derived Progenitor Cells; mRNA: Message Ribonucleic Acid; RNA seq: Ribonucleic Acid Sequencing; HB-EGF/Ascl1/Lin28a: Genes Involved in Dedifferentiation; qRT-PCR: Real Time Polymerase Chain Reaction; OTX2: Orthodenticle Homeobox 2; VSX2: Visual System Homeobox 2; WMU: Western Michigan University; IACUC: Institutional Animal Care and Use Committee; PBS: Phosphate Buffered Saline; MG: Milligram; ML: Milliliter; C: Centigrade; DAPI: 4',6-Diamidino-2-Phenylindole; Thy 1.2: Antibody also known as CD90; ANOVA: Analysis of Variance; SE: Standard Error; DBA/2J: Strain of Mouse; SVJ-129: Strain of Mouse.

Introduction

Glaucoma is a degenerative retinal disease characterized by loss of vision due to progressive death of Retinal Ganglion Cells (RGCs) and is the second leading cause of irreversible vision loss worldwide [1]. One of the primary risk factors for glaucoma is increased Intraocular Pressure (IOP), which is associated with death of RGCs. Presently, there is no cure for glaucoma and the loss of RGCs and their axons in the optic nerve is irreversible in humans [2]. All current treatments are aimed at decreasing IOP to prevent RGC death [3]. Unfortunately, these treatments can prevent further progression of the disease but cannot reverse any loss of vision. However, neuroprotection or regeneration of RGCs could potentially reverse the effects of glaucoma. Here, results are presented to provide evidence of BrdU positive RGCs after PNU-282987 eye drop treatment in adult animals using a DBA/2J genetic mouse model of glaucoma.

Recent studies from this lab have shown that neuroprotection or robust regeneration can be induced in the retinas of adult mammals when treated with a specific a7 nicotinic acetylcholine receptor agonist, PNU-282987 depending on the type of treatment [4-8]. In neuroprotective studies, PNU-282987 was shown to provide RGC neuroprotection against induced glaucoma-like conditions when it was intraocularly injected [4-6]. PNU-28987 was found to bind to alpha7 nAChRs on RGCs and mimicked the physiological neuroprotective effect of ACh typically released from starburst amacrine cells [6]. When injected intraocularly, PNU-282987 provided neuroprotection against the significant loss of RGCs that occurred in the GCL after inducing glaucoma-like conditions [6].

Adult mammalian retinal cells do not typically regenerate, but regeneration does occur naturally in many non-mammals, such as zebrafish and chick, in response to injury [9,10]. Recent studies from this lab have demonstrated that if PNU-282987 is applied as eye drops, it induces robust regeneration of new neurons in all layers of the retina without inducing an injury. Specifically, when applied as eye drops, PNU-282987 acts on alpha7nAChRs on the RPE to induce release of signaling molecules that cause de-differentiation of Muller Glia (MG) cells in adult mice [7-8]. Specifically, PNU-282987 has the ability to cause MG to generate Muller-Derived Progenitor Cells (MDPCs) and generate multiple types of new differentiated neurons in adult mammalian retinas [7,8]. The neurogenic response of PNU- 282987 in the adult murine retina is robust and the division of the MG into progenitor cells follows a similar pattern to that seen in zebrafish regeneration [7,8]. In mRNA sequencing studies, RNAseq was performed on MG following contact with RPE cells treated with PNU-282987. Up- or down-regulated genes were compared with published literature of MG dedifferentiation that occurs in lower vertebrate regeneration or with transcript profiles during early mammalian development. These studies provided evidence that the HB-EGF/Ascl1/Lin28a signaling pathway was involved in MG dedifferentiation to retinal progenitor cells [11]. RNA-seq results were verified using qRT-PCR and using immunocytochemistry, the presence of retinal progenitor markers OTX2, Nestin and VSX2 in MG were identified 48 hours post treatment with PNU-282987 treated RPE supernatant [11]. However, it is unknown if PNU- 282987 can regenerate or provide neuroprotection to neurons lost to retinal damage due to genetic disease.

In this study, PNU-282987 was analyzed in an in vivo DBA/2J mouse model. DBA/2J mice are an inbred strain of mouse characterized by a degenerative glaucoma-like condition that develops around 6-9 months of age [12]. This degenerative disease closely mimics human pigmentary glaucoma and is similarly characterized by a progressive increase in intraocular pressure that leads to retinal ganglion cell loss [2,13]. Thus DBA/2J mice are an ideal model for studying genetic glaucoma in humans. DBA/2J mice are homozygous for mutations in two separate genes. One mutation is found on the b allele of tyrosine related protein (Tyrp1b), which encodes a melanosomal protein [14,15]. The second mutant gene encodes Gpnmb, a transmembrane glycoprotein located within many cellular structures [15,16]. A distinguishing feature of the disease is loss of pigment granules within the iris. These pigment granules accumulate in the trabecular meshwork of the anterior chamber and block the drainage structures, which results in an elevated intraocular pressure [17,18]. The rise in IOP in DBA/2J mice is often reported by 6-9 months of age [19-24] and DBA/2J mice experience axonal loss and optic nerve damage shortly after the onset of elevated IOP [25,26]. Thus far, most studies involving DBA/2J mice have focused on neuroprotection, not regeneration. For example, erythropoietin and neuroglobin have both been shown to have a neuroprotective effect against the onset of the glaucoma-like disease in DBA/2J mice [27,28]. However, in this study, PNU-282987 was applied as eye drops to examine its effect in a genetic model of glaucoma. Specifically, these studies will provide evidence that early treatment with PNU- 28297 can prevent or reverse the progressive loss of RGCs associated with the disease in adult DBA/2J mice.

Materials and Methods

Animals

Adult DBA/2J mice (both sexes; aged between 3 and 12 months) were used for these studies and were kept in Western Michigan University’s (WMU) animal facility. The mice were obtained from Jackson laboratories. SVJ-129 wildtype mice were used as a control in one experiment. All animals were cared for in accordance with the approved guidelines of the Institutional Animal Care and Use Committee (IACUC) of WMU.

Intraocular Pressure (IOP) measurement

Intraocular pressure measurements were taken using an Icare Tonolab tonometer. Because IOP measurements tend to fluctuate throughout the day, measurements were taken at the same time each evening. IOP measurements were obtained two times each week from awake mice. Briefly, animals were removed from their home cages and held gently. A handheld Tonolab. tonometer was then applied to the cornea of experimental eyes to obtain IOP measurements before and after various eye drop treatments with PNU-282987. On each measurement day, 3 IOP measurements were obtained from each experimental animal and averaged. This was performed for between 5 and 20 animals depending on the designated end time point specified by the experimental protocols.

Experimental design

Experiments were designed to demonstrate that the a7 nAChR agonist ‘PNU-282987’ produced proliferation in the genetic mouse model. Three different age groups of DBA/2J mice were treated daily for 2 weeks with PNU-282987: 3 months old, 6 months old, and 10 months old. These ages were chosen to get a sampling of the different stages of the disease. 3-month-old animals have reached adulthood but have yet to develop the disease and thus have lost minimal RGCs. At 6 months old, the disease is in its early stages and at 10 months, the disease is fully developed. Five animals from each age group receive 2 weeks of treatment with PNU-282987. Their retinas were then removed and RGC survival was assessed.

Additional experiments were designed to examine the effects of early treatment with PNU-282987. The prevention of the disease with early treatment of PNU-282987 could not be shown in the previous induced surgical model of glaucoma. The use of the DBA/2J genetic model allows a unique opportunity to treat the animal in anticipation of the onset of the disease. In this experiment, DBA/2J mice received treatment before they developed their characteristic glaucomalike condition, to determine if it would prevent the progression of the disease. There were two treatment conditions: chronic early treatment and acute early treatment. In the chronic early treatment studies, animals started receiving once weekly PNU treatment at an early age that continued until the animals reached 10 months of age. There were two time points within this group: 3 months old and 6 months old. The 3-month-old group began treatment at 3 months of age and the 6-month-old group began treatment at 6 months of age. Five mice at each time point were assessed. Each group continued receiving PNU once per week until they were 10 months old. At 10 months of age, their retinas were removed and RGC survival was assessed.

The acute early treatment animals received two weeks of PNU treatment before the onset of the disease. Following that two weeks, the animals received no further treatment and were left alone until they were 10 months old; at which point their retinas were removed and RGC survival was assessed. 3 and 6-month old mice were used for this study. Five animals at each time point received 2 weeks of PNU treatment at 3 or 6 months old and then left, with no further treatment, until they were 10 months old.

Eye drop treatment and retina preparation

Both eyes of each experimental animal were treated once daily with PBS eye drops containing 1 mg/mL BrdU and 1 mM PNU- 282987. The concentration of PNU-282987 and BrdU used in eye drop experiments in rodents was previously established in dose response studies and the dosage that produced maximal effects was used [7-8,29]. Animals in the short-term treatment groups received this treatment once daily for 2 weeks while the long-term treatment animals received treatment once weekly for 3-6 months. Control animals received drops containing only PBS and 1 mg/mL BrdU. Details of the eye drop treatment are described in Linn et al. [29]. At specific times following the start of treatment, mice were euthanized. The eyes were subsequently removed, and retinas were excised, flatmounted, and fixed in 4% paraformaldehyde overnight at 4°C [4-5].

Immunohistochemistry

Following fixation, retinas were labeled with primary antibodies including sheep anti-BrdU (7.5 μL/mL, Abcam ab1894, Cambridge, UK) and rat anti-Thy1.2 (1:200, Abcam ab 218775). Thy-1.2 is a cell surface protein used as a marker for RGCs in mice. BrdU is used as a marker for cell cycle reentry. For BrdU staining, antigen retrieval was done as in Webster et al. [8] Retinas were blocked in PBS containing 1% Triton X-100 and 1% bovine serum. Retinas were incubated in primary antibodies overnight at room temperature in PBS containing 1% bovine serum and 1% goat serum, rinsed in PBS and incubated overnight with appropriate Alexa Fluor conjugated secondary antibodies (1:300, Life Technologies) diluted in PBS without serum. Nuclei were stained with DAPI.

Cell counting and normalization

Stained retinas were then flat mounted on slides and visualized using a Nikon C2+ scanning laser confocal microscope. Four images of each flat-mounted retina were taken from each of four quadrants of the retina. Each image was taken 4 mm from the optic nerve head [4-5]. To quantify the flat mounts, a fixed 200×200 μm² grid was applied to all images and the Thy1.2-positive RGCs within the grid for each retinal quadrant were counted. The RGC counts from the four quadrants were averaged together according to the procedure outlined in Mata et al. [5] Webster et al. [7] and Cooley-Themm et al. [6] This represented an “N” of 1 for flat-mounted retinas and “N”s of 5 were obtained for each experimental condition.

Statistical analysis

Statistical analysis was performed on all normalized data using an analysis of variance (ANOVA) as well as Tukey post-hoc analysis. P<0.01 was considered statistically different.

Results and Discussion

Progression of glaucoma-like RGC loss in DBA/2J mice

Experiments were designed to quantify the loss of RGCs in DBA/2J mice. (Figure 1) shows confocal microscope images obtained from untreated DBA/2J retinas of different ages. The retinas were fixed overnight in 4% paraformaldehyde and flat mounted with the retinal ganglion cell layer facing up. After thorough rinsing, the retinas were immunostained using antibodies against Thy-1.2, a marker for RGCs. RGCs were counted and averaged from 5 different animals under different age groups. (Figure 1A) illustrates an image of a DBA/2J retina from a 3-month-old mouse (Figure 1B) is from a 6-month-old DBA/2J mouse, (Figure 1C) is from a 10-month-old DBA/2J mouse, and (Figure 1D) is from a 12-month-old DBA/2J mouse. There was significant loss of RGCs in the 6, 10, and 12-month-old animals compared to the 3-month-old animals. (Figure 1E) quantifies the average number of cells lost in the different age groups. By 6 months of age, there was an average loss of 47 (SE ± 6.6) RGCs compared to 3-month-old mice, representing a 16% loss. By 10 months, there was an average loss of 88 (SE ± 5.9) RGCs (a 29% loss) and by 12 months, there was an average loss of 114 (SE ± 6.0) RGCs (a 38% loss).