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Comparative Study
. 2013 Aug 13;54(8):5441-55.
doi: 10.1167/iovs.13-11651.

Development of a model of elevated intraocular pressure in rats by gene transfer of bone morphogenetic protein 2

Lakisha K Buie  1 , Md Zahidul Karim, Matthew H Smith, Teresa Borrás
Affiliations
Comparative Study

Development of a model of elevated intraocular pressure in rats by gene transfer of bone morphogenetic protein 2

Lakisha K Buie et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: To determine whether inducing calcification in the trabecular meshwork results in elevated IOP in living rats. To use this property to create an elevated IOP animal model by gene transfer of bone morphogenetic protein 2 (BMP2).

Methods: Calcification was assessed by alizarin red staining in primary human trabecular meshwork (HTM) cells and alkaline phosphatase (ALP) activity in the angle tissue. Brown Norway (BN) and Wistar rats were intracamerally injected with Ad5BMP2 (OS) and control Ad5.CMV-Null (OD). IOPs were taken twice a week and expressed as mean integral pressures. Morphology was assessed on fixed, paraffin-embedded anterior segments. Retinal ganglion cells (RGCs) were quantified on retrograde and Brn-3a-labeled flat mounts using MetaMorph software.

Results: BMP2-treated cells displayed marked increase in calcification. Trabecular meshwork tissue showed moderate ALP activity at 13 days postinjection. Fifty-four of 55 BN and 15 of 19 Wistar rats displayed significantly elevated IOP. In a representative 29-day experiment, the integral IOP difference between treated and control eyes was 367.7 ± 83 mm Hg-days (P = 0.007). Morphological evaluation revealed a well-organized trabecular meshwork tissue, exhibiting denser matrix in the treated eyes. The Ad5BMP2-treated eye showed 34.4% ± 4.8% (P = 0.00002) loss of peripheral RGC over controls.

Conclusions: Gene transfer of the calcification inducer BMP2 gene to the trabecular meshwork induces elevated IOP in living rats without altering the basic structure of the tissue. This strategy generates an elevated IOP model in rats that would be useful for evaluation of glaucoma drugs targeting the outflow pathway.

Keywords: adenoviral gene transfer; elevated IOP model; rat; trabecular meshwork.

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Figures

Figure 1
Figure 1
Effect of overexpressing BMP2 in primary HTM cells. Cells at passage 5 and 70% to 80% confluency were infected with Ad5BMP2 at moi between 5000 and 8000 VP/cell and harvested 3 to 5 days postinfection. (A) Cells stained with alizarin red. (1) corresponds to a representative infected well at 3 days postinfection, whereas (2) represents a parallel uninfected dish. A second HTM cell line was aged in culture for 2 months, infected with Ad5BMP2, and stained at 5 days postinfection (3). (4) represents a parallel dish of 2 months aged in culture uninfected cells. (B, C) TaqMan analysis of the RNA extracted from HTM-infected and untreated controls at 3 days postinfection, in two different cell lines. Results are expressed in FC of the infected versus the untreated and normalized to 18S. Human TaqMan probes were BMP2, COL1A1, OGN, RUNX2, IBSP, BGLAP, human 18S. *P ≤ 0.05. Overexpression of BMP2 induced calcification in HTM cells as well as expression of COL1A1 and IBSP markers.
Figure 2
Figure 2
Delivery of human BMP2 cDNA to the living rat iridocorneal angle by Ad5BMP2 intracameral injection: its effect on expression of calcification genes. Tissue strips from the angle region containing the trabecular meshwork were dissected at 12 to 13 days postinjection and their RNA extracted for TaqMan analyses. Results are expressed in FC of the Ad5BMP2-injected rats versus controls and normalized to 18S. (A) OS eyes of BN rats (n = 4) were injected with 4 × 109 VP of Ad5BMP2 and compared with their OD receiving the same VPs of Ad5.CMV-Null. (B) OS eyes from Wistar rats (n = 4) were injected with 2 × 1010 VP of Ad5BMP2 and compared with OD and OS of Wistar Naïve rats (n = 2). TaqMan reactions were performed in triplicate, each from 2 to 4 independent RT reactions. TaqMan probes were human BMP2, rat COL1A1, rat RUNX2, rat IBSP, rat BGLAP, human 18S. *P ≤ 0.05. Transgene human BMP2 was efficiently delivered to the rat iridocorneal angle. At higher concentrations, overexpression of human BMP2 induced rat COL1A1, RUNX2, and IBSP calcification markers in the living rat.
Figure 3
Figure 3
Rat elevated IOP induced by overexpression of human BMP2 cDNA. Two representative BN rat groups were injected with 5 × 109 to 9 × 107 VP Ad5BMP2 (OS) and 7 × 109 to 9 × 108 VP Ad5.CMV-Null (OD), respectively. IOP was measured using a tonopen (A, C) or a TonoLab rebound tonometer (B, D). (A) IOP absolute values of treated and control eyes from the tonopen group (n = 10) during 48 hours after injection. Mean baseline IOP of all eyes was 25.3 ± 0.3 mm Hg. (B) IOP absolute values of treated and control eyes from the TonoLab group (n = 6) during 29 days after injection. Mean baseline IOP of all eyes was 9.9 ± 0.1 mm Hg. *P ≤ 0.05 at the last time points (A, B). (C, D) AUC plots of the mean IOPs of treated (broken line) and control eyes (continuous line) from the tonopen (C) and TonoLab (D) groups. The integral IOP differences are depicted in gray. (E) Summary table of the numeric values of measurements and calculations of IOP differences between treated and controls in both groups, P ≤ 0.007. Delivery and overexpression of the human BMP2 transgene to the iridocorneal of the living rat induces significant elevation of IOP when compared with controls.
Figure 4
Figure 4
Morphology of the rat trabecular meshwork after gene transfer of BMP2. Representative images of the eye angle region from a group of Wistar rats injected with 5 × 109 VP of Ad5BMP2 (OS, left) and 1 × 109 Ad5.CMV-Null (OD, right) and killed at 77 days postinjections. Anterior segment wedges were fixed in 4% paraformaldehyde/2.5% glutaraldehyde, embedded in paraffin, and 5-μm sections stained with hematoxylin and eosin. Original magnification: ×100 (top), ×200 (bottom). TM, trabecular meshwork; SC, Schlemm's canal. Ad5BMP2-treated eye shows an open angle and a well-conserved trabecular meshwork structure with a denser ECM than the contralateral Ad5.CMV-Null control.
Figure 5
Figure 5
Effect of BMP2-induced elevated IOP on RGC survival. (A) BN rats (n = 3) injected with 7 × 107 VP of Ad5BMP2 (OS) and 7 × 108 VP of Ad5.CMV-Null (OD) were retrograde labeled with 3% Dil solution at 29 days postinjection. (B) Retinas of BN rats (n = 7) injected with 2.7 × 1010 VP of Ad5BMP2 (OS) and 7 × 108 VP of Ad5.CMV-Null (OD) were flat mounted and stained with Brn-3a at 28 days postinjection. Left: percentage of Dil- (A) or Brn-3a-labeled (B) RGC cell loss in the treated eyes normalized to untreated. Right: representative fluorescence photomicrographs of treated and untreated flat-mounted retinas taken from the peripheral retina. The number of positive cells was obtained from eight peripheral fields per each flat-mounted retina using MetaMorph software. The number of positive RGC cells was significantly reduced in the elevated IOP-BMP2 treated eyes. *P ≤ 0.006.
Figure 6
Figure 6
Representative experiment of the evaluation of the BMP2 model with a prostaglandin-derived glaucoma drug. Eyes of Wistar rats were injected with 2 × 1010 Ad5BMP2 VP (n = 9) or left uninjected (n = 3). At 8 days postinjection, eyes were (A) treated twice a day for 3.5 days with one drop of 0.01% lumigan (n = 4), or (B) left untreated (no drops, n = 5). (C) Uninjected eyes treated with PBS (n = 3). IPO measurements are expressed as ΔIOP from baseline (A, B) or absolute values (C). *P ≤ 0.05. Rats with BMP2-induced elevated IOP exhibit a significant IOP reduction on treatment with a conventional glaucoma drug.
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