The combination of IGF1 and FGF2 and the induction of excessive ocular growth and extreme myopia

Abstract

Different growth factors have been shown to influence the development of form-deprivation myopia and lens-induced ametropias. However, growth factors have relatively little effect on the growth of eyes with unrestricted vision. We investigate whether the combination of insulin-like growth factor 1 (IGF1) and fibroblast growth factor 2 (FGF2) influence ocular growth in eyes with unrestricted vision. Different doses of IGF1 and FGF2 were injected into the vitreous chamber of postnatal chicks. Measurements of ocular dimensions and intraocular pressure (IOP) were made during and at the completion of different treatment paradigms. Histological and immunocytochemical analyses were performed to assess cell death, cellular proliferation and integrity of ocular tissues. Treated eyes had significant increases in equatorial diameter and vitreous chamber depth. With significant variability between individuals, IGF1/FGF2-treatment caused hypertrophy of lens and ciliary epithelia, lens thickness was increased, and anterior chamber depth was decreased. Treated eyes developed myopia, in excess of 15 diopters of refractive error. Shortly after treatment, eyes had increased intraocular pressure (IOP), which was increased in a dose-dependent manner. Seven days after treatment with IGF1 and FGF2 changes to anterior chamber depth, lens thickness and elevated IOP were reduced, whereas increases in the vitreous chamber were persistent. Some damage to ganglion cells was detected in peripheral regions of the retina at 7 days after treatment. We conclude that the extreme myopia in IGF1/FGF2-treated eyes results from increased vitreous chamber depth, decreased anterior chamber depth, and changes in the lens. We propose that factor-induced ocular enlargement and myopia result from changes to the sclera, lens and anterior chamber depth.

Figure 1

Receptors to insulin, IGF and FGF are widely expressed in different ocular tissues. RT-PCR was used to detect transcripts to the insulin receptor, IGF1 receptor, IGF2 receptor, FGFR1, FGFR2 and FGFR3. PCR was performed on cDNA pools obtained from retina, choroid+RPE, ciliary body+lens capsule, lens fiber cells and sclera. Abbreviations: FGFR – fibroblast growth factor receptor, IGF – insulin-like growth factor, GAPDH - Glyceraldehyde 3-phosphate dehydrogenase, RPE – retinal pigmented epithelium.

Figure 2

Three consecutive daily intraocular injections of 800ng IGF1 and 200ng FGF2 stimulate excessive ocular growth. Injections began at P5, ended at P7, and measurements were performed on enucleated eyes at P8 (paradigm 1). Measurements of enucleated eyes were made from digital images using ImagePro6.2. Measurements of eyes (n=6) were made on axis (a) and on profile (b). The histogram illustrates the mean (±SD) difference in ocular dimension (mm; c). Measurements included corneal circumference (corneal circ), corneal arc, equatorial circumference (eq circ), dorsal-ventral equatorial diameter (D–V eq diameter), and axial length. Statistical significance (*p<0.05, **p<0.001, n=6) was determined by using a paired, two-tailed student's t-test.

Figure 3

Treatment with 3 consecutive daily injections of 800ng IGF1 and 200ng FGF2 causes cell death, glial migration and proliferation within the retina. Retinas were harvested 1 day after the last of 3 consecutive daily injections of factors (paradigm 1). Vertical sections of central regions of the retina were labeled for fragmented DNA using the TUNEL method (a and b) or with antibodies to Sox9 (c and d), transitin (e and f), PCNA (g and h) or Brn3a (I and j). Arrows indicate dying cells (b), reactive Müller glia (d), proliferating Müller glia (h) or abnormal ganglion cell nuclei (j). Arrow-heads indicate putative NIRG cells that are PCNA-positive (h). The insets (i' and j') in panel j are 2-fold enlargements of the boxed-out regions in i and j. The calibration bar (50 μm) in panel j applies to all panels. Abbreviations: INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer, PCNA – proliferating cell nuclear antigen.

Figure 4

Four consecutive daily intraocular injections of 200ng IGF1 and 200ng FGF2 stimulate excessive ocular growth. Injections began at P5, ended at P8, and ocular measurements were performed at P9 (paradigm 2; a–d and g) or P16 (paradigm 3; e,f and h). Measurements were made by using ImagePro6.2 on digital images of enucleated eyes (a–f) and by using A-scan ultrasonography (g and h). Histograms represent the mean and standard deviation for data-sets. Measurements using ImagePro6.2 included nasal-temporal equatorial diameter (N–T diameter), dorsal-ventral equatorial diameter (D–V diameter), equatorial circumference, axial length, corneal circumference, and corneal arc. Measurements using A-scan ultrasound included axial length, vitreous chamber depth, anterior chamber depth and lens thickness. Statistical significance was determined by using a paired, two-tailed student's t-test.

Figure 5

Four consecutive daily intraocular injections of 200ng IGF1 and 200ng FGF2 do not have short-term effects upon cell death, glial reactivity or the integrity of ganglion cells, whereas the long-term survival of ganglion cells in the peripheral retina is compromised. Injections of IGF1 and FGF2 began at P5, ended at P8, and tissues were harvested at P9 (paradigm 2; a–f) or P15 (paradigm 3; g–l). Vertical sections of control (a,c,e,g,i and k) and treated retinas (b,d,f,h,j and l) were labeled for TUNEL (a,b,g and h), Sox9 (c,d and k–l), transitin (c and d), and Brn3a (e,f,i and j). Arrows indicate labeled nuclei. The calibration bar (50 μm) in panel n applies to all panels. Abbreviations: INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.

Figure 6

Four consecutive daily intraocular injections of 200ng IGF1 and 200ng FGF2 dramatically change the anterior segment of the eye. Sections were obtained from control eyes (a and c) and from eyes treated with IGF1 and FGF2 (b, d and e) and were stained with hematoxylin and eosin. Injections began at P5, ended at P8, and ocular measurements were performed at P9 (paradigm 2; a, b and f) or P16 (paradigm 3; c–e and g). The arrows indicate the zonules that attach to the lens, straight lines indicate the angle, and arrow-heads indicate delaminated iris epithelium. Histograms indicate the mean and standard deviation for the angle (degrees; f and g). Significance of difference (p-value) was determined using a two-tailed student t-test.

Figure 7

Intraocular injections of 200ng IGF1 and 200ng FGF2 stimulate the proliferation of cells in far peripheral regions of the retina, progenitors in the CMZ, equatorial regions of lens capsule, and non-pigmented epithelial cells in the zonules. Sections were labeled with DRAQ5 (nuclei; blue), PCNA (red) and BrdU (green). Sections were obtained from control eyes (paradigm 2; a, c and e) and eyes 1 day after the last of 4 consecutive daily injections of IGF1 and FGF2 (paradigm 2; b, d and f). The calibration bar (50 μm) in panel b applies to panels a and b, the bar in d applies to c and d, and the bar in f applies to e and f. Abbreviations: CMZ – circumferential marginal zone, PCNA – proliferating cell nuclear antigen.

Figure 8

Intraocular injections of the combination of IGF1 and FGF2 increase intraocular pressure (IOP) in a dose-dependent manner. Histograms indicate the mean and standard deviation for the IOP of eyes treated with 4 consecutive daily doses of 300ng IGF1 alone, 200ng FGF2 alone, 1μg insulin + 200ng FGF2, 50ng IGF1 + 200ng FGF2, 100ng IGF1 + 100ng FGF2, 200ng IGF1 + 200ng FGF2, or 300ng IGF1 + 200ng FGF2 (paradigm 6). IOP measurements were made at 1 or 7 days after the last injection. Significance of difference was performed using ANOVA (p<0.0001), two-tailed student's t-test to assess significance at each time or treatment, and a post-hoc two-way Bonferroni test was used to determine the significance of difference (p=0.002) between 1 and 7 days after treatment with 300ng IGF1 and 200ng FGF2.

Figure 9

Consecutive daily intraocular injections of 200ng IGF1 and 200ng FGF2 stimulate excessive ocular growth progressively over 4 days. Injections began at P5, ended at P8. Ocular measurements were performed at P5, P6, P7, P8 and P9 (paradigm 4; n=6). Measurements were made by using streak retinoscopy (a), a Tonolab tonometer (b), and A-scan ultrasonography (c–f). Ultrasound measurements include axial length (c), vitreous chamber depth (d), anterior chamber depth (e) and lens thickness (f). Histograms represent the mean and standard deviation for data-sets. Measurements using A-scan ultrasound included axial length, vitreous chamber depth, anterior chamber depth and lens thickness. Statistical significance (*p<0.05 and **p<0.005) was determined by using two-way ANOVA and was followed by a post-hoc, two-tailed student's t-test.

Figure 10

Four consecutive daily intraocular injections of 100ng IGF1 and 100ng FGF2 do not stimulate excessive ocular elongation. Injections began at P5, ended at P8, and ocular measurements were performed at P9 (paradigm 5; n=6). Measurements were made by using ImagePro6.2 on digital images of enucleated eyes (a), streak retinoscopy (b), and A-scan ultrasonography (c). Histograms represent the mean and standard deviation for data-sets. Measurements using ImagePro6.2 included equatorial circumference, corneal circumference, and corneal arc. Measurements using A-scan ultrasound included axial length, vitreous chamber depth, anterior chamber depth and lens thickness. Hematoxylin and Eosin-stained sections through anterior ocular structures included a control eye (e–g), a treated eye exhibiting no significant IGF1/FGF2-mediated effects (h–j), and a treated eye exhibiting significant hypertrophy of cells in the ciliary body, lens capsule and iris epithelium (k–m). The boxed-out areas in panels e, h and k are enlarged 2-fold in the panels immediately below. Statistical significance was determined by using a paired, two-tailed student's t-test.