Expression of insulin-like growth factor 1 receptor in rat retina following optic nerve injury

Abstract

Purpose: To investigate the apoptosis in retinal ganglion cells (RGCs) and insulin-like growth factor 1 receptor (IGF-1R) in the retina following optic nerve crush. Methods: Healthy Wistar rats (N = 70) were divided into two groups: a normal control group and an optic nerve injury group. Immunohistochemistry and flow cytometry were performed to detect the expression of IGF-1R and to measure the apoptosis of RGCs, respectively. Results: Immunohistochemistry revealed that at 1 hr after optic nerve injury, IGF-1R immunoreactivity began to increase and reached a maximal level at 24 hr (p < 0.05), where it remained elevated up to 14 days after injury. RGC apoptosis in the normal control group was 0.53%, while the apoptosis rate in the optic nerve injury group increased over time. The apoptosis rate in the optic nerve injury group was 1.4% at 1 hr, 4.4% at 6 hr, 5.2% at 12 hr and reached a maximal level (8.5%) at 24 hr. Subsequently, the rate declined to 1.9% 7 days after injury and 0.9% 2 weeks after injury. Conclusion: The IGF-1R immunereactivity in the retina increased after optic nerve injury. IGF-1R may regulate the apoptosis and regeneration of RGCs at different stages after optic nerve injury. The optic nerve is similar to other nerves of the central nervous system in that regeneration does not occur following damage, and optic trauma often results in irreversible injury (Chen et al. 2000; Wong et al. 2003). Insulin-like growth factor 1 (IGF-1) is a specific neurotrophin that is involved in a wide variety of biological processes and has been shown to promote the postinjury regeneration of neurons and can block cell apoptosis (D’Mello et al. 1993; Biswas et al. 2008; Joseph D’Ercole & Ye 2008). IGF-1 and IGF receptor type-I (IGF-1R) are expressed in the retina and play a role in development and neurogenesis (de la Rosa et al. 1994; Burren et al. 1996; Lambooij et al. 2003). A number of animal studies have illustrated the importance of IGF-1 and IGF-1R in the eye. Koriyama et al. (2007) have reported upregulation of IGF-1 in goldfish retinal ganglion cells (RGCs) during the early stage of optic nerve regeneration after injury. Another study (Tagami et al. 2009) reported that axonal regeneration after a crush injury was mediated by IGF-1. An association between downregulation of IGF-1 and apoptosis of rat RGCs has been reported (Homma et al. 2007). Other studies have reported the influences of the IGF-1/IGF-1R system in age-related macular degeneration (Lambooij et al. 2003). Blunting IGF-1R expression has been shown to increase neuronal apoptosis after hypoxic/ischaemic injury (Liu et al. 2011). Thus, in the present study, we aimed to determine the temporal profiles of IGF-1R immunoreactivity and IGF-1R protein expression in a rat model of optic nerve injury and to determine the RGCs apoptosis rate ex vivo. Healthy male Wistar rats (N = 70; specific pathogen free) aged 2–3 months and weighing 200–250 g were obtained from and housed at the Experimental Animal Center of Suzhou University. This study was approved by the Institutional Animal Care and Use Committee of Suzhou University. Animals were given ad libitum access to food and water in an environment with a 12-hr/12-hr light/dark cycle and sound level of 40 ± 10 dB. Routine outer eye and eye fundus examinations were normal. The animals were randomly assigned into two groups: normal control group (n = 10) and optic nerve injury group (n = 60). Clamping of the optic nerve surgery was performed according to the method of Ohlsson and Svensson (2007) with modifications. Clamping of the optic nerve was performed in one eye of each rat, and animals were killed, using an overdose of 30% chloral hydrate, at 6 time points (1 hr, 6 hr, 12 hr, 24 hr, 1 and 6 weeks) after optic nerve injury. The treatment of all animals was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were anesthetized with ketamine at 50 mg/kg intraperitoneally, and areas surrounding the eyes were sterilized. The bulbar conjunctiva was opened, and the optic nerve was exposed. The optic nerve was clamped 2 mm from the globe for 30 s to introduce optic nerve injury, and then the clamp was removed followed by wound closure. The relative afferent pupillary defect (RAPD) of the eye with the clamped optic nerve, lens and blood supply to the retina was observed. Animals that developed the pathological conditions of interest were used for further study. At the designated time points, the eyes were collected and fixed in paraformaldehyde for 48 hr followed by dehydration in alcohol and embedding in paraffin. The eyes were cut into 5-μm sections and mounted with Elmer’s Glue-All™ onto slides. The contralateral eyes were also collected, and the retina was isolated and ground to prepare a retinal cell suspension. Immunohistochemistry for the identification of IGF-1R was performed using the ABC kit (Sigma, St Louis, MO, USA) according to the manufacturer’s instructions. The affinity-isolated antibody was a polyclonal Anti-IGF-1R (Ab-1346) antibody produced in rabbit. The retinal sections were hydrated in alcohol and treated with mouse anti-rat IGF-1R (1:50; Sigma) and then with biotin-conjugated goat anti-mouse IgG (Sigma). Visualization was done with 3,3′-diaminobenzidine (DAB) under light microscope. The positive cells were identified if brown granules were in the cytoplasm. Meanwhile, all tissue samples were stained with haematoxylin and eosin. An Image Pro Plus image analysis system (Version 6.0; Media Cybernetics, Silver Spring, MD, USA) was used to capture the representative images using the colour cube-based mode with H = 60, I = 255 and S = 255. The integrated optic density (IOD) was calculated. At 6 time points after optic nerve injury, animals were killed, and a RGC suspension was prepared in 1640 medium with or without a supplement of BMS-7548077 (0.5 μg/mL, IGF-1R inhibitor; Bristol-Myers Squibb Co., Ltd, New York, NY, USA). Rat RGC suspensions were obtained according to the previously published method (Kretz et al. 2004) with modifications. Briefly, the animals were killed using CO2 followed by cervical dislocation. After sterilizing the head with 70% ethanol, the eye was removed, immersed in 70% ethanol to reduce micro-organisms and then transferred to 37 °C culture medium. Under a laminar flow hood, using sterile instruments, an incision was made in the cornea around the ora serrata. The lens and vitreous body were removed, and the retina was detached. The retinas were dissociated mechanically to make cell suspension. Retinal ganglion cells were incubated at 37 °C in an atmosphere with 5% CO2 for 24 hr. The retinal cell suspension was mixed in 500 l of binding buffer, and the mixture was divided into four tubes, which were then divided into normal control group, annexin V (AV; Invitrogen, Carlsbad, CA, USA) group, propidium iodide (PI; Invitrogen) group and AV+PI group. The mixtures were then subjected to flow cytometry (Epics XL flow cytometer; Beckman-Coulter, Brea, CA, USA) to detect the apoptotic retinal cells. Flow cytometry was performed to dynamically measure the apoptosis rate. Retinal ganglion cells were lyzed in a solution containing 50 mm Tris (pH 8.1), 1% SDS, PMSF, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA and leupeptin for 30 min on ice. The cell lysate was centrifuged, and the supernatant was collected. A total of 20 μg of protein was separated by 10% SDS-PAGE, transferred to membranes and probed with mouse anti-rat IGF-1R (Sigma) diluted 1:200 in 5% nonfat dry milk in TBS for 16 hr at 4 °C. IGF-1R has a different molecular size (∼155 kDa) from that of insulin receptor (∼95 kDa). In this study, the Western blot data were from IGF-1R. An antibody against beta actin (Sigma) served as the internal control. The filters were incubated with horseradish peroxidase-conjugated secondary antibody (Boster; Wuhan, China) diluted 1:400 for 2 hr at room temperature. Normally distributed continuous variables were compared using a one-way analysis of variance (anova). When a significant difference between groups was apparent, multiple comparisons of means were performed using the Bonferroni procedure with a type-I error adjustment. Moreover, the comparison of apoptosis rates at each time point with BMS treatment and without BMS treatment was determined using an independent two-sample t-test. Data are presented as means ± standard deviation (SD). All statistical assessments were two-sided and evaluated at the 0.05 level of significance. Statistical analyses were performed using SPSS 15.0 statistics software (SPSS Inc, Chicago, IL, USA). No IGF-1R immunereactivity was detected in the retina of the control group (Fig. 1A). In contrast, IGF-1R immunereactivity appeared to increase 1 hr after optic nerve injury (there was no significant difference between the IGF-1R expression levels at 1 hr compared with 6 hr). IGF-1R immunereactivity increased significantly 12 hr after injury and reached a maximal level 24 hr after injury (Fig. 1B, C; p < 0.05). The IGF-1R immunoreactivity remained stable 48 hr after optic nerve injury and maintained at a relatively high level at 3–7 days after injury (p < 0.05). The IGF-1R immunoreactivity in the optic nerve injury group was significantly higher than that in the control group (p < 0.01). Immunohistochemistry of IGF-1R in the retina. (A) Normal retina; no IGF-1R immunoreactivity was found in RGCs (arrow) (×400). (B) Retina at 24 hr after optic nerve injury. The RGCs (arrow) had patchy IGF-1 immunoreactivity (bluish brown in the cytoplasm; ×400). Both (A and B) were counterstained using haematoxylin and eosin. (C) Retinal IGF-1R immunoreactivity, expressed as integrated optic density (IOD), at different time points after optic nerve injury. (D) The protein expression of IGF-1R in RGCs prepared at various times after optic nerve injury, quantified by Western blot. β-actin was used as internal control. * indicates p < 0.05 versus control group. Western blots were used to quantify IGF-1R protein expression in RGCs, which were prepared at various times after optic nerve injury. IGF-1R protein expression reached a maximal level at 24 hr (p < 0.05, Fig. 1D). Results of the analysis of RGC ex vivo apoptosis after nerve injury are shown in Fig. 2. Rats were killed at different time points after nerve injury; RGCs were immediately isolated and then subjected to the apoptosis assay. In the control group, the apoptosis rate was 0.53%. After optic nerve injury, the apoptosis rate increased over time: the apoptosis rate was 1.4% at 1 hr, 4.4% at 6 hr, 5.2% at 12 hr and 8.5% at 24 hr, respectively. However, the apoptosis rate decreased to 1.9% at 7 day after injury and remained at 0.9% at 1–2 weeks. When compared with the control group, the apoptosis rate in the optic nerve injury group was markedly increased at the different time points (all, p < 0.05). After treatment with the IGF-1R antagonist BMS, the apoptosis rate of RGCs increased over time after optic nerve injury. The apoptosis rate was 2.5% at 1 hr, 10.6% at 6 hr, 13.68% at 12 hr and reached a maximal level (20.2%) at 24 hr after optic nerve injury. The apoptosis rate decreased to 2.5% at 7 days after injury and remained at 1.2% at 1–2 weeks after optic nerve injury. Notably, compared with control group, the apoptosis rate in the optic nerve injury group was markedly increased at the different time points (all, p < 0.05). This result indicates that blockage of IGF-1 signalling enhances the apoptosis of RCGs ex vivo. In addition, the apoptosis rate at each time point was larger in the BMS treatment group than in the without BMS treatment group (all, p < 0.05, Fig. 2). Ex vivo apoptosis rates of RGCs in the presence or absence of IGF-1R antagonist BMS. Rats were killed at different time points after nerve injury; RGCs were immediately isolated and then were subjected to the apoptosis assay. *Indicates p < 0.05 vs. control group (n = 10). aIndicates p < 0.05 versus without BMS treatment. Following optic nerve injury, IGF-1R expression in the retina was markedly increased in this rat model. IGF-1R receptor expression coincided with an increase in apoptosis at 24 hrs after optic nerve crush. After treatment with the IGF-1R antagonist BMS, the RGC apoptosis rate further increases. These results suggest that blockage of the IGF signalling pathway induces RGC apoptosis after optic nerve injury. These findings indicate that changes in IGF-1R expression in the retina occur following optic nerve injury and suggest a protective effect of the IGF-1 signalling pathway on the optic nerve and retina. Whether the increase in IGF-1R expression translates into increased functional IGF-1R needs to be confirmed in future studies. At the early and middle stages after optic nerve injury caused by optic nerve clamping, numerous viable glial cells in the stump of optic nerve may be the source of neurotrophic molecules, which promote the increase in viable RGCs following optic nerve injury (Wong et al. 2003; Biswas et al. 2008). Clamping of the optic nerve results in a disorder of electrical conduction which is attributable to changes in ultrastructure of the optic nerve such as loose myelin structure, lamellar separation and blockage of axoplasmic transport (Tagami et al. 2009). This partial impairment of the optic nerve results in the fracture of axons, nerve oedema and ischaemia/hypoxia, which may lead to a disruption in axoplasmic transport, which influences the supply of neurotropin to the retina resulting in apoptosis of RGCs (Homma et al. 2007). Not surprisingly, intraperitoneal injections of the free-radical scavenger, ginkgo biloba, have been shown to increase the survival rate of RGCs in a rat optic nerve crush model (Ma et al. 2010). Experiments from isolated human optic nerves have shown that this tissue has a relatively limited capacity for retraction following exposure to elevated pressures (Hansen et al. 2011). In these studies, the optic nerve sheath diameter did not return to baseline values after exposure to subarachnoid space pressures greater than 35 mmHg. IGF-1R is a ligand-dependent receptor that is activated once ligand binding occurs (Joseph D’Ercole & Ye 2008). IGF-1 binds to the IGF-1R to promote cell growth, and the molecule affects signalling pathways involving other growth factors and their receptors (such as platelet-derived growth factor, epidermal growth factor, insulin-like receptor and oestrogen receptors) (de la Rosa et al. 1994; Liu et al. 2011). IGF-1 can act as a specific neurotrophin, which promotes the growth and development of neurons and blocks the apoptosis and death of cells leading to the postinjury repair of neurons (D’Mello et al. 1993; Joseph D’Ercole & Ye 2008). Recent studies have also shown that IGF-1R is related to the p53/Mdm2 signal transduction pathway, and disruption of the p53/Mdm2 system may lead to upregulation of IGF-1R (Girnita et al. 2000, 2003). However, wild-type p53 can induce cell apoptosis; wild-type p53 inhibits the transcription of the IGF-1R gene, and p53/Mdm2 can downregulate IGF-1R expression (Ohlsson & Svensson 2007). In the case of an optic nerve injury, an increase in p53 may protect IGF-1R against degradation by sequestering MDM2 (a ubiquitin ligase for both p53 and IGF-1R). Subsequently, the activated p53 could inhibit IGF-1R at the transcriptional level causing a downregulation of IGF-1R. Insulin-like growth factor receptors can also interact with the anti-apoptotic protein Bcl-2 (Chen et al. 2000; Biswas et al. 2008). Interestingly, a model of optic nerve crush showed that intravitreally administered glucagon-like peptide 1-producing cells can significantly increase Bc1-2 gene expression (Zhang et al. 2012). That study demonstrated the therapeutic potential of intravitreal implantation of genetically engineered cells into the eye that can be used to alter gene expression at a specified target gene. The growth hormone/insulin-like growth factor 1 axis has been shown to play an important role in the regulation of retinal cell survival in vivo (Seigel et al. 2006; Sanders et al. 2011). Intravitreous cGH siRNA causes a reduction in both GH and IGF-1 mRNA levels in the chick embryonic retina, concomitantly with an increase in the numbers of apoptotic cells in that tissue (Sanders et al. 2011). These effects are detected within 6 hr after treatment and can last for at least 24 hrs. In vivo studies with diabetic rats have shown that systemic treatment with IGF-1 can reduce the TUNEL-immunoreactive cells in the photoreceptor layer, inner nuclear layer and ganglion cell layers in the retina to the numbers seen in nondiabetic eyes (Seigel et al. 2006). Systemic IGF-1 also significantly reduced the number of p-Akt immunoreactive cells in the ganglion cell layer and the inner nuclear layers of diabetic rat retinas. Following optic nerve injury, nogo-A protein and IGF receptors exert distinct biological effects; nogo-A can activate neuron regeneration, but IGF receptors promote it (Chen et al. 2000). In addition, IGF-1 may activate the phosphatidyl inositol-3 kinase (PI3k/Ark) system at an early stage after optic nerve injury to regulate the apoptosis and regeneration of RGCs at different stages (Biswas et al. 2008; Liu et al. 2011). In certain circumstances, IGF-1 may have a detrimental effect on retinal neuron survival. A new cell type was discovered in a newly-hatched chick model that was termed the Nonastrocytic Inner Retinal Glia-like (NIRG) cell (Fischer et al. 2010). These cells respond to IGF-1 by proliferating, migrating distally within the retina and causing an upregulation of transitin. Microglia stimulated by IGF-1 upregulate CD45 and lysosomal membrane glycoprotein. The overall result of the IGF-1 stimulation of the NIRG and microglia was shown to be higher levels of cell death accompanied by focal detachments across the retina. IGF-1 acts in concert with other molecules, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF-1), to modulate retinal angiogenesis (Sengupta et al. 2010). In a mouse model of choroidal neovascularization (CNV), Sengupta et al. 2010 found that after laser-induced injury, mRNA levels for IGF-1, VEGF, SDF-1 and their receptors were elevated. Both IGF-1 and VEGF produced an additive stimulation of SDF-1-induced angiogenesis. Subretinal injections of antibodies to CXCR4, the primary receptor for SDF-1 (Son et al. 2006), reduced choroidal neovascularization in this model (Sengupta et al. 2010). In a laser-induced model of CNV, mice that received oral or intraperitoneal cyclolignan picropodophyllin (an inhibitor of IGF-1R activity) experienced significant reductions in CNV area and VEGF concentrations in the choroid (Economou et al. 2008). Whether the addition of exogenous IGF-1 can inhibit RGC apoptosis thereby treating the injury of optic nerve is unclear. If so, IGF-1 may be an effective treatment for injury of the optic nerve. Much of the current information comes from in vitro and in vivo studies using animal models. Further study in human cells and tissues is needed to verify these results. The IGF-1R expression in the retina increases after optic nerve injury, and IGF-1R may regulate the apoptosis and regeneration of RGCs at different stages after optic nerve injury.