Expression Of MAChR And VIP In Guinea Pigs Of A From Deprivation Myopia Model & Long-term Follow-up Study Of PRK And LASIK

Expression of mAChR and VIP in guinea pigs of a form deprivation myopia modelBackgroundApplication of the nonselective muscarinic acetylcholine receptor (mAChR) antagonist, atropine, has showed its effectiveness in slowing the progression of myopia in clinical trial. Another nonselective mAChR antagonist, oxyphenonium, can also prevent form deprivation myopia (FDM) in chicks. To date, five distinct mAChR subtypes have been characterized (M1-M5). Studies on selective mAChR antagonists showed that pirenzepine (an Ml selective antagonist), himbacine (M4/M2 selective), are effective in preventing the progression of myopia in both animal models. These drugs inhibit axial elongation of the eyeball, which is the structural cause of myopia. However, their mechanism of action is still unknown.Atropine, pirenzepine, and himbacine prevent FDM when administered intravitreously. So, their effectiveness was previously assumed to be worked via binding mAChR in retina. However, studies on retina of chick and visual cortex of monkey showed that both the density and affinity of Ml to M4 mAChRs have no significant difference between FDM and control groups. Furthermore, ablation of mAChR and the cholinergic amacrine cells in the retina does not affect the axial elongation of the eyebal. Studies on choroid also demonstrated that mAChRs have similar distribution during normal and myopic eye development. So, it is reasonable to speculate sclera as a potential site for the action of mAChR antagonists. Indeed, in vitro study has shown direct suppressive effect of atropine on scleral cells and a recent study has demonstrated all the mAChRs (M1-M5) expression in human sclera at both mRNA and protein levels. However, study to investigate whether mAChRs expression change in visually regulated eye growth has not been conducted.Vasoactive intestinal polypeptide (VIP) is a neurotransmitter and distributes widely in human body. It acts important physiological function of information-introduction and modulation. Studies had shown that, in FDM model of primate animals, VIP up-regulated in amacrine cells in IPL of retina. Recent investigation in DNA level also confirmed this result. A mainstream viewpoint believes that the abnormal visual input induces the enhancement of VIP release by a kind of amacrine cells and in turn, VIP excites the proliferation of progenitor cells lying in peripheral retina to promote the growth of retina and enlargement of eyeball. But this argument is needed to be further confirmed from various levels.ObjectiveThe aim of our study was to investigate mAChRs expression at retina and sclera in form-deprived eye in comparison with open eye in a guinea pig model. We used specific genetic probes targeting sequences of the M1 to M4 and VIP mRNA and further verifis the expression of the subtype proteins with immunohistochemistry and Western blot analysis.Material and Methods1. Animals and establishment of FDMThirty pigmented guinea pigs (Cavia porcellus) ranging in age from 12 to 14 days were maternally reared and housed in a 12 h light/12 h dark cycle. All procedures were approved by the Shandong University Institutional Animal Care and Use Committee and were in compliance with the NIH statement for the use of Animals in Research.To study the development of FDM, treated group of 15 guinea pigs were raised with a diffuser worn over one eye (randomly selected as left or right in different animals) for 14 days. These ages were selected to span the period of emmetropization in the guinea pig. Meanwhile, control group of the other 15 guinea pigs were raised with two eyes open.Diffusers were translucent hemispheres moulded from white plastic (diameter 12 mm, thickness 0.8 mm). At the beginning of the study, diffusers were fixed by sutured to skin around treated eye, while guinea pigs were lightly anaesthetised with halothane. At the end of study, the diffusers were removed under anesthesia.Refractive error and ocular dimensions were measured in both eyes before trial and immediately after the diffuser was removed at the end of the rearing period.Refractive error was measured by streak retinoscopy in hand-held, awake animals in which cycloplegia had been previously induced with 2 drops 1% cyclopentolate. Stable refractive errors were generally obtained after 15 min and no pupil response. All refractive error data presented refer to the spherical component refractive error, defined as the mean refractive error in the horizontal and vertical meridians and were not corrected for any possible artefact of retinoscopy.Axial dimensions of the eye were measured by high frequency ultrasonography (20 MHz), while animals were anaesthetised (1-2% Halothane in oxygen). Axial length of the eye was defined as the distance from the front of the cornea to the vitreous-retinal interfaces.2. Tissue collectionAfter last examination, animals were euthanized by overdose of pentobarbital sodium and the eyes were enucleated. The posterior segment was isolated with a cut through the ora serrata and immersed in abundant Ames medium (Sigma). Then, the eye cups were cut into two halves. One for immunohistochemistry (IHC), the other for Western blot analysis and Real Time RT-PCR.After rinse, the neural retina was separated from the pigment epithelium and either processed immediately for total RNA isolation or stored in RNAlater (Sigma). This entire procedure was operated on ice and lasted <30 min. The other half was fixed in 4% paraformaldehyde and processed through graded alcohols, embedded in paraffin and cut into 5μm thick slices.3. Real Time RT-PCR analysis of Ml to M4 and VIPThe total RNA was extracted from retina and sclera using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and then was treated with 2.5 U RNAse-free DNAase I (Invitrogen, Carlsbad, CA, USA) to remove contaminated genomic DNA. The reverse transcription of RNA to cDNA was performed using the M-MLV Kit (Promega, Madison, USA) at 37℃for 1 h. The mRNA sequences of the investigated genes were obtained from GenBank. cDNA were amplified on a Light Cycler (Roche Applied Science). The real-time PCR reactions were carried out following the manufacturer's protocol. The primers and amplification conditions are shown. The SYBR Green I kit (TaKaRa Biotechnology, Dalian, China) was used for amplification and detection of the expression of Ml to M4 and VIP mRNA. Quantitative values were obtained from the threshold cycle value (Ct), the point where a significant increase of fluorescence is first detected. Housekeeping geneβ-actin was quantified as an internal RNA control. The target bands were verified by gel electrophoresis. The data were analyzed with 2^-ΔCT method.4. ImmunohistochemistryEndogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min. The sections were then blocked by 5% bovine serum albumin to prevent non-specific staining by the secondary antibody. After incubation overnight at 4℃with primary antibodies (polyclonal antibodies to Ml to M4 and VIP, Santa Cruz Co), the sections were exposed to the secondary antibody conjugated with horseradish peroxidase. Bound antibody was visualized using a light microscopy with diaminobenzidine. Omission of primary antibodies and staining with non-immune IgG served as negative controls for each antibody in the present study.All slices were analyzed by using an Olympus-BHS microscope coupled to an MTI color video camera (DAGE-MTI; 162 Michigan City, IN) at a magnification of 400x.5. Western Blot AnalysisProtein was boiled for 5 min. Equal amounts of protein were separated with a 14% SDS-PAGE and transferred to nitrocellulose membrane (BioRad, Hercules, CA). Following blocking with 5% non-fat milk, the blots were washed with PBS containing 0.1% Tween 20 and incubated with an appropriate primary anti-body at 4℃overnight. The blots were probed with antibodies againstβ-actin (rabbit, 1:1000) IκBα(mouse, 1:100), phospho p38 MAPK(mouse, 1:100), phospho ERK1/2 (mouse, 1:300), phospho JNK1/2 (rabbit, 1:100). After overnight incubation, the blots were washed with TBST and incubated with secondary antibody conjugated to HRP (Santa Cruz. 1:2000), and then washed again. The blots were then visualized with enhanced chemiluminescence and relative intensities of protein bands were analyzed by MSF-300G Scanner.6. Statistical analysisData were presented as Mean±SD and independent t test analysis was performed by SPSS13.0 software to compare the difference between treated and control groups. A value of P <0.05 was considered statistically significant.Results1. Refractive Change After FDMThe refractive state and axial length of the eyes after 12 days of FDM: control eyes were slight hyperopic (mean 0.8D) whereas the deprived eyes became myopic (mean -5.8). Ultrasound measurements show that form-deprivation eyes have significantly longer axial eye length compared with nondeprived normal control eye (P<0.05).2. The mRNA Expression of Muscarinic Acetylcholine Receptors and VIPThe mRNA expression of the four mAChR subtypes (M1 to M4) and VIP was present in all retina and sclera samples. The predicted size of the products amplified by the PCR primers matched the size of the individual mAChR subtypes and VIP. There were no products generated during the amplifying process in the negative control samples, indicating that they were free of genomic DNA in the DNAse-treated samples. Therefore, the cDNA samples used in the experiment were free of contaminated genomic DNA. In either retina or sclera, there were no significant difference of Ml to M4 mAChR mRNA expression between treated group and control group (P>0.05). In retina, there was significant difference of VIP mRNA expression between treated group and control group (P=0.000). 3. Presence of mAChR and VIP ProteinsImmunohistochemistry for mAChRIn retina, immunohistochemical staining revealed that all mAChR subtypes (Ml to M4) expressions were visible both at deprivation and open groups. Anti-mAChRs (M1-M4) immunoreactivity were mainly localized in bipolar cells of the inner nuclear layer (INL) and sparsely distributed in amacrine cells with a few cells in the ganglion cell layer (GCL). Though M3 and M4 immunoreactivity were more intense than M1 and M2, no obvious differences between deprivation and control groups were found.In sclera, immunohistochemical staining also revealed that all mAChR subtypes (M1 to M4) expressions were visible both at deprivation and open groups. The scleral tissue treated with anti-M1, -M2, -M3 and -M4 primary antibodies demonstrated yellow stained spindle cells (fibroblast cells).Immunofluorescence for VIPImmunofluorescence revealed VIP was positive in retina of both control and FDM group and mainly lies in inner plexiform layer (IPL). The expression in FDM was stronger than control.Western blot analysisWestern blot analysis showed the presence of mAChR proteins (Ml to M4) in the retinal and scleral tissue where M1was detected as a 52kDa band, M2 as a 72kDa band, M3 as a 75kDa band and M4 as a 70kDa band. Additionally, the M3 and M4 bands were more intensive than the Ml and M2 bands. Each mAChR subtype demonstrated more expression in retina than in sclera. However, in either retina or sclera, all subtypes (m1-m4) had no significant difference between deprivation and control groups (P>0.05). VIP protein in the retinal tissue showed a significant higher expression in FDM group than control group (P<0.05).Conclutions1. Our study showed that there were no different distributions of mAChRs (M1 to M4) in scleral tissue between FDM and control groups at both mRNA and protein levels. The action of mAChR antagonists is through muscarinic receptors in extraretinal and extrascleral tissues, or nonmuscarinic mechanisms.2. VIP plays an important role on the growth of retina in a FDM model. Long-term follow-up study of PRK and LASIKBackgroundSince the first treatment on sighted human eye with excimer laser photorefractive keratectomy (PRK) in 1988, millions of PRK procedures have been performed worldwide by now. PRK, together with two later-developed procedures (laser subepithelial keratomileusis, LASEK and epipolis laser in situ keratomileusis, epi-LASIK), belong to surface ablation. In the early 1990s, a combination of lamellar keratotomy and excimer laser ablation of the cornea was independently described by two surgeons. This new procedure, named laser in situ keratomileusis (LASIK), is now the most popular surgical procedure for correcting myopia, for its virtues of less postoperative discomfort and faster visual rehabilitation over PRK and other surface ablation procedures.However, though 6 months and 1 year follow-up showed that PRK and LASEK had very similar outcomes with respect to visual acuity and refraction, whether the long-term outcomes of them remains unclear. Several studies had offered long-term follow-up of PRK, but even the longest 12 years follow-up which provided detailed results was based on an old ablation profile. Thus, further long-term follow-up is necessary to assess the refractive stability of surface ablation. In the mean while, whether the long-term outcomes of LASIK and surface ablation procedures actually differ also remains unclear. Long-term follow-up is of particular importance to assess the refractive stability and potential late-phase complications of LASIK which is an elective procedure. Particularly, as a result from the formation of corneal flap, LASIK has less residual stromal bed than surface ablation procedures. AimsTo evaluate long-term visual and refractive outcomes of PRK and LASIK. The main outcome measures were predictability, efficacy, stability and safety.MethodsFor PRK, a long-term (11 years) prospective follow-up study of visual and refractive outcomes of patients who underwent PRK surgery in 1994 and 1995 was performed. Forty-six patients (85 eyes) underwent detailed clinical assessment. The mean age at surgery of the 46 patients was 26.7 years (range, 21-46 years), 47% males and 53% females. Preoperative mean myopic refraction was 5.15D with a range of 1.25D to 9.75D. Stratification of patients into 3 groups (mild, moderate and severe group) was in terms of myopic level. Laser ablation was done with a Summit apex excimer laser machine. A 6 mm ablation zone was adopted for all eyes. The last 11-year follow-up was performed at February 2006. The mean length of follow-up was 135 months with a range of 121-143 months.For LASIK, another long-term (7 years) prospective follow-up study of visual and refractive outcomes of patients who had LASIK surgery performed in 1998 and 1999 was conducted. Fifty-nine patients (104 eyes) that include 58.6% males and 41.4% females underwent detailed clinical assessment at 1 year and 7 years postoperatively. Preoperative mean spherical equivalent (SE) was -6.28D with a range of -3.25D to 15.25D. Stratification of patients is in terms of SE. The flaps were cut using the MORIA CB microkeratome which created the flaps with a diameter of 8.0-9.5 mm and a desired depth of 160μm. Laser ablation was done with a Summit apex plus excimer laser. A 6 mm ablation zone was adopted for all eyes. The 7 years follow-up was performed at June 2006. The mean follow-up at the last examination was 89 months (range 84 to 93 months). The mean age at last visit of the 59 patients was 34.2 years (range, 29-48 years).All patients had a detailed preoperative and postoperatively examination including unconnected visual acuity (UCVA), best spectacle corrected visual acuity (BSCVA), manifest refraction, slit lamp examination and dilated fundus examination. Topography, pachymetry and postoperative complications were also recorded at last follow-ups. All patients received a questionnaire including 11 items at last visit to assess their satisfaction with the procedures.Descriptive statistics for visual outcomes and a paired 2-tailed t test (P<0.05 being significant) for difference in refraction between two postoperative follow-ups were also made. Statistical analysis was performed using SPSS 13.0 software.ResultsAfter PRK, 56.5% of eyes were within±0.5D of attempted correction with 81.2% within±1.0D at 11 years postoperatively. Eighty-seven percent had a vision of 0.5 or better and 51.7% of 1.0 or better. In all cases, there was myopic regression (26.43D) in refraction with time, which was most aggressive in the severe group (23.55D) and continued up to the 11 years follow up. Moderate and severe subsets showed a significant change between 3 and 11 years. BSCVA was unchanged or improved in 90.6%. Eight eyes lost their BSCVA. There were no intraoperative complications of all the 85 eyes. Postoperatively, 9% (4/46) of patients reported glare, halos, and other night vision problems at 11 years. Haze were found in 4 eyes in which 3 eyes under 0.5 scale and 1 eye 0.5 scale by subjective assess. Most patients (89%) were satisfactory with the outcomes.After LASIK, 89.4% of eyes were within±0.5D of attempted correction with 90.4% within±1.0D at 7 years postoperatively. One hundred percent had a vision of 0.5 or better and 94.2% of 1.0 or better. The mean manifest refraction spherical equivalent (MRSE) was 0.29D at 1 year and 0.25D at 7 years. There was no significant regression (P<0.05) between 1 year and 7 years postoperatively. BSCVA was unchanged or improved in 80.8% eyes. There were no intraoperative complications of all the 104 eyes. Postoperatively complications were also rare, only 2 eyes with debris in the interface. No other grave complications, such as diffuse lamellar keratitis, epithelial ingrowth, etc. Three point four percent (2/59) of patients reported glare and halo at 7 years. Ten point two percent (6/59) patients complained dry eye syndrome. Patient satisfaction with the surgery was high(91.5 %).Conclusions1. Myopic PRK surgery shows predictable, accurate and safe outcomes in terms of refractive and visual outcomes with slight regression in refraction which mainly lies in severe myopes. To some extent, these outcomes can provide references to LASEK and epi-LASIK.2. LASIK surgery also shows predictable, accurate and safe results in terms of refractive and visual outcomes. Refractive stability was maintained over 7 years. There was no evidence of progressive late-onset complications after LASIK.3. No progressive late-onset myopic or hyperopic shift occurs in both PRK and LASIK.