Glaucoma is a major cause of blindness worldwide. It has been estimated that there will be 60.5 million people worldwide suffering from glaucoma in 2010, among whom 47% would be Asian, and the total number is expected to increase to 79.6 million by 2020.1 Glaucoma is mainly characterized by the loss of retinal ganglion cells (RGCs), which is directly responsible for the visual loss. Thus, neuroprotection may be an attractive strategy for treating glaucoma.
Ciliary neurotrophic factor (CNTF) was originally described as a factor that supported the in vitro survival of parasympathetic neurons from the chick ciliary ganglia.2 Nowadays CNTF is believed to promote the growth and development and stimulate the regeneration of axons of RGCs and has consequently obtained a great deal of attention in the field of ophthalmology.
The objective of our study was to detect the expression of CNTF in the retina of a rat glaucoma model with increased intraocular pressure (IOP), which provides the basis for further investigations of the neuroprotective effects of CNTF on RGCs in a rat glaucoma model.
Adult male Sprague-Dawley rats with an approximate starting weight of 200 to 250 g were obtained from the Laboratory Animal Unit of Shanghai No. 6 People’s Hospital. A total of 72 rats were randomly divided into one normal control group and five experimental groups (1, 3, 7, 14, and 28-day post-surgery groups, 12 rats each group), and were maintained in good health for 1 week prior to the surgical procedures. This investigation adhered to the tenets of the Association of Research in Vision and Ophthalmology (ARVO) Statements for the Use of Animals in Ophthalmic and Vision Research.
Rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g weight). Experimental glaucoma was induced by electro-coagulating at least three episcleral and limbal veins after incising the conjunctiva. After ocular surgery, the eyes were treated topically with Ocustilla eye drops and an antibiotic ointment during recovery. The IOP was measured by a Perkins tonometer (Clement Clarke International Ltd, UK) before surgery and weekly after surgery. To evaluate the effectiveness of the model, the cell morphology was observed by transmission electron microscopy. The retinas were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer for at least 2 hours. The retinas were subsequently post-fixed, dehydrated, embedded, sectioned, and stained for electron microscopy. Ultrathin sections were examined under a transmission electron microscope (Philips, CM-120, the Netherlands).
The rats were sacrificed using an overdose injection of anesthetic. Immediately after death, the eyes were enucleated, immersion-fixed in 4% paraformaldehyde overnight at 4°C, and washed in 0.1 mol/L phosphate-buffered saline (PBS, pH 7.4). The eyes were then processed for paraffin sectioning. Each eye was oriented so that the sections (5-μm thickness) were cut from the superior to the inferior edge. The sections were collected on gelatin- and poly-L-lysine-coated slides and incubated with the primary anti-CNTF antibody (rat CNTF-specific goat IgG, 1:20, R&D Systems, USA) overnight at 4°C. Washes were performed three times for 5 minutes each in 0.1 mol/L PBS at room temperature (RT). To block nonspecific binding, 3% bovine serum albumin (BSA) was used. After washing in PBS, the secondary antibody (1:500, Santa Cruz, USA) was applied for 70 minutes at 37°C, and diaminobenzidine (DAB, Sigma, USA) was used as the chromogen.
Reverse-transcription polymerase chain reaction (RT-PCR)
The expression of CNTF mRNA was examined using RT-PCR, with the amplification of β-actin as a control. The eyes were removed, and the retinas were dissected in a shallow bath of cold PBS. Total RNA was extracted. The absorbance at 260 nm was used to determine the RNA yield. Each total RNA (1 μg) was reverse transcribed to cDNA, and 1 μl of cDNA was used as the template for PCR amplification in a 50 μl reaction volume using the PCR System (MJ Research, USA) under the following conditions: initial denaturation at 94°C for 5 minutes and 34 cycles of denaturation at 94°C for 50 seconds, annealing at 57°C for 40 seconds, and extension at 72°C for 1 minute, followed by extension at 72°C for 7 minutes. The sequences of the primer pairs were: CNTF: forward 5′-GGGACAGTTGATTTAGG-GG-3′ and reverse 5′-GGCAGAAACTTGAGCATA-3′ and β-actin: forward 5′-AACGAGCGGTTCCGATGCC-CTGAG-3′ and reverse 5′-TGTCGCCTTCACCGTTC-CAGTT-3′. The expected sizes for the CNTF and β-actin PCR products were 373 bp and 500 bp, respectively. The PCR products were evaluated by 1% agarose gel electrophoresis and ethidium bromide staining. The gel was photographed using short-wavelength ultraviolet. Each PCR product was compared with that obtained by amplifying β-actin cDNA in the same sample, and band intensities were determined by densitometry.
Fresh retinas were harvested immediately after the animals were sacrificed. Retina total protein was extracted in RIPA lysis buffer. After centrifugation at 15 000 × g for 30 minutes at 4°C, the supernatant was collected, boiled for 5 minutes, and stored at -80°C until all samples had been collected. Equal amounts of protein, determined using the bicinchoninic acid (BCA) protocol, were electrophoresed in an 18% SDS- polyacrylamide gel and transferred to a nitrocellulose membrane using transfer buffer. The protein blot was blocked with 5% nonfat dried milk at 37°C for 1 hour and then incubated overnight at 4°C with primary antibody diluted 1:200 in blocking buffer. The membrane was washed with TBS-T, and the immunoreactive bands were visualized using secondary antibody (1:4000) for 2 hours at RT. Bands were observed at an optimal time point and quantified by using chemiluminescence. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control.
The statistical analysis was performed using SAS 6.12. The experimental results are expressed as the mean ± standard deviation (SD). Student’s t-test was used to compare differences among the groups, and P<0.01 was considered statistically significant.
IOP in the rats
The IOP in the eyes of the normal control group was about 15 mm Hg and remained approximately at the same level. The IOP in the eyes of the experimental group was elevated gradually after surgery and stabilized at 30 to 35 mm Hg for up to 4 weeks. The pooled means of the IOP were 29.80±3.58, 33.00±4.14, 35.00±3.43, 35.20±3.16, 34.00±3.89 mm Hg for the 1, 3, 7, 14, and 28-day post-surgery groups, respectively. The IOP of all the experimental groups increased significantly after surgery (t=9.19, 13.85, 17.93, 24.82, 15.34, P<0.01).
Ultrastructure research using electron microscopy
Retinal samples, prepared as described above under fixation, were examined by transmission electron microscopy to observe cell morphology before and after surgery. Compared with the normal control cells, the cells of the experimental retinas exhibited marked changes (Fig. 1). The mitochondria in the RGCs and Müller cells of the experimental retinas were swollen, with shortening and disappearance of some cristae. The experimental RGCs showed fewer ribosomes, dispersion of the nuclear chromatin, and swollen axons. The experimental Müller cells had higher electron-dense cytoplasm and shorter processes. In addition, bipolar cells of the experimental retinas had become necrotic.
Immunostaining for CNTF
Immunohistochemistry was performed to detect changes in protein expression between the normal control and experimental retinas (Fig. 2). CNTF immunostaining in the normal rat retina was confined to the ganglion cell layer (GCL). At 7 days after surgery, CNTF immunoreactivity was detected not only in the GCL but also in the inner nuclear layer (INL) and outer nuclear layer (ONL). The CNTF staining intensity was stronger in the experimental retinas than in the normal control retinas. There was no significant change between the 1-day post-surgery and 3-day post-surgery retinas.
RT-PCR analysis of CNTF
To investigate the change of CNTF mRNA expression following ocular hypertension, we sampled retinas at various time points after surgery (Fig. 3). The CNTF mRNA signal was weak in the normal control retina. After surgery, an obvious fluctuation in the amount of endogenous CNTF mRNA was detected. CNTF mRNA was upregulated, gradually rising to a high level, peaking at 1 to 2 weeks, and then decreasing to the normal control level.
Immunoblot analysis for CNTF
An antibody that recognized CNTF as a single band with an apparent molecular mass of 24 kD in homogenized normal and experimental retinas was used for immunoblotting. The immunoblot analysis revealed a fluctuation in the level of endogenous CNTF in the experimental retinas (Fig. 4). The CNTF protein level was low in the normal control retina, and an increase was detected after surgery. The upregulated protein level reached a peak at 7 to 14 days after surgery and then decreased, suggesting that endogenous CNTF is elevated transiently following ocular hypertension.
The ratios of CNTF mRNA to β-actin mRNA and CNTF protein to GAPDH protein were determined from densi-tometric analysis of the bands (Figs. 3 and 4). Both ratios increased significantly after surgery (P<0.01).
Glaucoma is a leading cause of irreversible visual impairment and blindness. As IOP is a major risk factor for the progression of optic neuropathy, pharmacological and surgical treatments for glaucoma have been aimed at lowering IOP. These treatments, however, neglect the importance of the optic nerve and RGCs, whose dysfunction and death are directly responsible for the visual loss. The IOP of some glaucoma patients can be under control, yet their visual field defect is still aggravated. Other glaucoma patients may have optic nerve damage without elevated IOP. Thus, neuroprotection presents a novel approach to the treatment of glaucoma.
CNTF was first identified and partially purified from embryonic chick eye tissues. It was named for its survival-promoting effects on parasympathetic neurons from the ciliary ganglion of chicken embryo.2,3 CNTF supports the survival and/or differentiation of a variety of neuronal cell types, including sensory, sympathetic, and motor neurons. Many reports have discussed the neuroprotective actions of CNTF on RGCs.4-10 For example, Cui et al4 reported that intraocular injection of CNTF increased the regeneration of adult RGC axons into peripheral nerve grafts in vivo, and CNTF was at least one of the trophic factors that could promote axonal regeneration of axotomized RGCs. Intraocular injection of CNTF before or after a single toxic dose of N-methyl-D-aspartate (NMDA) greatly reduced the number of cells destroyed by toxin treatment, indicating that this factor protected retinal neurons from severe excitotoxic insult.5,6 Zhang et al7 showed that CNTF significantly increased the survival of RGCs 1 week after optic nerve injury. Maier et al8 demonstrated that CNTF has a neuroprotective effect on affected RGCs during acute optic neuritis. In addition, CNTF injection not only transiently ameliorated the degenerative changes in retinal ischemia induced by increasing IOP to 160 mm Hg for 90 minutes9 but also attenuated the significant and progressive loss of RGCs in a rat glaucoma model with chronic, moderately elevated IOP induced by laser photocoagulation of the episcleral and limbal veins.10 Some studies have shown that the retina reacts to nerve damage by upregulating endogenous CNTF, thus protecting RGCs.6,11-15 In one study, after a single mechanical lesion to the subretinal space and retina, there was a substantial increase in CNTF expression that persisted for the entire 10-day study period.11 In response to NMDA- and KA-induced neuronal death, the expression of CNTF in Müller cells was upregulated.6 CNTF upregulation was also detected 1 week after optic nerve transection; CNTF expression peaked at 2 weeks and fell to control levels at 4 weeks.12 The expression and cellular localization of CNTF in the rat retina following retinal lesion have also been investigated. In the normal retina, CNTF immunoreactivity was restricted to profiles in the GCL.13,14 Following optic nerve transection12,13,15 or retina ischemia induced by transiently increasing IOP,14 CNTF immunoreactivity appeared in Müller cell somata and processes, and its intensity increased. These findings all suggest that CNTF plays an important role in the endogenous neuroprotective system. The upregulation of endogenous CNTF after retinal lesion is responsible for RGC survival and axonal regeneration.
The present study documents the impact of glaucoma on the location and expression of CNTF, using immuno-histochemistry, RT-PCR, and immunoblot analysis. CNTF immunostaining in the normal rat retina was confined to the GCL. On day 7 after surgery, CNTF immunoreactivity was detectable in the INL and ONL. RT-PCR and immunoblot analysis respectively showed a minimum expression of CNTF mRNA and CNTF protein in the control retina; the expression of each was significantly increased after surgery, reached a peak on days 7 to 14 post-surgery, and then declined. The current study demonstrated that the retina reacts to the nerve damage associated with glaucoma by upregulating this neuroprotective cytokine, thus protecting RGCs. We suggest that the upregulated CNTF is responsible for RGC survival and axonal regeneration in glaucoma.
In conclusion, the retina reacts to the conditions of glaucoma by upregulating endogenous CNTF, thus protecting RGCs. These results raise intriguing possibilities for the management of glaucoma. Increasing endogenous CNTF or adding exogenous CNTF might provide a new treatment methodology for glaucoma; however, many issues remain to be resolved, such as the effective dose and an effective sustained delivery system. It has been suggested that intraocular injection of adeno-associated viral (AVV),16 lentiviral (LV),17 or adenoviral (AdV)18 vectors expressing CNTF might successfully increase RGC survival after axotomy. Additional investigations are needed on neuroprotection in glaucoma.
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