Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

Purpose:To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells(buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line),Morphological features of undifferentiated EScells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic acid(RA)as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C)as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Results:ES-D3cells cultured by BRL-CMgrew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RAand the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Con clusions:Combined use of RA and Ara-Ccan induce ES cells to differentiate into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science2000;16:1-6.

Effect of High Dosage of Methylprednisolone on Rat Retinal Ganglion Cell Apoptosis after Optic Nerve Crush

Purpose: To investigate the effect of high dose methylprednisolone (MP) on retinal ganglion cells (RGC) apoptosis.Methods: One hundred and twenty six Wistar rats were divided into normal control, crush control and MP treatment groups. High dose MP and 0.9% sodium chloride were injected intravenously at 1 h after injury in the treatment group and crush control group, respectively. Apoptotic cells, Bcl-2 and Bax positive cells were located and calculated by terminal-deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL), immunohistochemical method and retinal whole-mount combined with section technique at day 4, 7, 14 after crush, respectively.Results: The TUNEL-labeling cells, Bcl-2 and Bax positive cells were detected in the ganglion cell layer of retina. Few apoptotic cells, a few of Bcl-2 and Bax positive cells were observed in normal control groups. The numbers of positive cells increased gradually after crush. The number of apoptotic cells and Bax positive cells in treatment groups was less than that in crush group significantly (P < 0.05), while the number of Bcl-2 positive cells in treatment group was more than that in crush group at day 4, 7 and 14 after crush significantly (P < 0.05) Conclusions: These results suggest that high dose MP can inhibit the apoptosis of RGC after optic nerve crush probably through up-regulation of Bcl-2 expression and down-regulation of Bax expression.

Differentiation of Embryonic Stem Cells into Neurons and Retina-like Structure in Nude Mice

Purpose:To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods:Murine embryonic stem cells(D3 cell line)were cultured and maintained in an undifferentiated state in vitro,then transplanted into the anterior chamber of nude mice.Mophological and immunohistochemical examinations were implemented.Results:Two to three days after transplantation,yellow-white floating granules,sheets and masses were seen inside the anterior chamber and vitreous cavity,and enlarged gradually,14-20days later,the mice were executed.Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity.The morphology of these differentiated cells were similar to that of the retina.The cells were highly positive in NSE staining.Conclusion:The tranplanted embryonic stem cells cold grow in the eyes of nude mice with tendency to differentiate into neurons and retina-like structure.

Photodynamic Therapy for Age-related Macular Degeneration

Purpose: To evaluate short-term effects of single photodynamic therapy (PDT) for age-related macular degeneration (AMD) accompanied with choroidal neovascularization (CNV).Methods: We analyzed retrospectively the effects of single PDT for 20 patients (20 eyes) with CNV caused by AMD. Corrected visual acuity, fluorescein angiography (FA) and optic coherence tomography (OCT) were examined before and after PDT. All patients were followed up at least 3 months. Results: At the end of 3-month follow-up, 5 eyes had vision progress, 15 eyes had vision stable and no eye had vision deterioration. Fluorescein angiography one week post PDT showed cessation of fluorescein leakage in 8 eyes with predominant classic CNV, and reduction of fluorescein leakage in 12 eyes with minimal classic CNV or occult CNV without classic component. At the 3-month following PDT fluorescein angiography showed fluorescein leakage reappeared in 4 of 8 eyes with predominant classic CNV. Among 12 eyes with minimal classic CNV or occult CNV without classic component, 9 eyes showed decreased or unchanged fluorescein leakage, 3 eyes had a progression of fluorescein leakage. Optic coherence tomography showed obvious recovery of serous sensory retinal detachment after PDT. Conclusion: PDT may occlude or inhibit CNV caused by AMD in short term. No obvious side effects were noticed.

Distribution of the Toxin following Medial Rectus Muscular Injection of Botulinum Toxin Gel in Rabbits

Purpose: To determine the distribution of the toxin among individual muscles after intramuscular injection of botulinum toxin gel.Methods: One eye of 7 New Zealand white rabbits was randomized into group A, and the contralateral eye was into group B. Eyes in group A received medial rectus intramuscular injection of 2.5 IU of 125I-BTX-A gel in 0.1 ml, and those in group B received equivalent amount of 125I-BTX-A solution by medial rectus intramuscular injection. Four rectus muscles and the levator palpebrae superioris were harvested and the radioactivity of muscles was measured 16 hours after the injection.Results: In group A, the radioactivity of per gram medial rectus was significantly higher than that of other muscles (P ＜ 0.01), and there was no statistically significant difference in the radioactivity of per gram muscles among other muscles (P ＞ 0.05). In group B, the radioactivity of per gram medial rectus and levator palpebrae superioris was significantly higher than that of other muscles respectively(P ＜ 0.05), and the difference in the radioactivity of per gram muscles between medial rectus and levator palpebrae superioris was not statistically significant (P ＞ 0.05). The radioactivity of per gram medial rectus in group A was higher than that in group B (t=3.731 ,P=0.01), and there was no significant difference in the radioactivity of per gram muscles among other homonymous muscles (P ＞ 0.05).Conclusion: The toxin dispersed principally in the injected muscle and the local concentration of the toxin was much high following intramuscular injection of botulinum toxin gel.

External Approach Microsurgery of Retinal Dialysis

Purpose: To explore the effect of external approach microsurgery in retinal dialysis.Methods: Consecutive 30 eyes of 28 patients with retinal dialysis were enrolled for this study. The progresses of the external approach microsurgery were following. Under the surgical microscopy, the preplacement of mattress sutures for buckling and/or encircling following retrobulbar anesthesia and scleral exposure, draining subretinal fluid, the cryotherapy of retinal breaks, checking the position of breaks on scleral buckle and gases injection were performed in turn.Results: After drainage of subretinal fluid, with scleral depression cryotherapy reaction around breaks could be observed clearly under the microscopy. All breaks were located on anterior slope of the buckle. Intraoperative complications were mild subretinal hemorrhage at drainage site and corneal epithelium exfoliation in 3 eyes, respectively.Postoperative complications were mainly secondary glaucoma and retinal redetachment.The one-operation reattachmentl rate was 96.7% (29 eyes), and the final reattachment rate was 100% after one eye had a second external approach microsurgery. The postoperative vision acuity (VA) was significantly better than the preoperative VA (X2=9.529, P＜ 0.01).Conclusion: External approach microsurgery has favourable effect on the surgery of retinal dialysis.

Characteristics of Optic Nerve Damage Induced by Chronic Intraocular Hypertension in Rat

Purpose:To set up the Sharma’s chronic intraocular hypertension model and investigate the intraocular pressure (IOP) as well as the optic nerve damage of this model in rat. Methods:The operations of the chronic intraocular hypertension model were performed as described by Sharma in 60 male Lewis albino rats. IOP was measured using the Tono-Pen XL immediately after surgery and then at 5 day, 2 week or 4 week intervals. Cresyl violet staining of whole-mounted retinas was used to label retinal ganglion cells (RGCs), then RGCs were counted. Paraphenylenediamine (PPD) staining was performed in the semi-thin cross sections of optic nerve of rat, in order to know whether the axons of optic nerve were degenerated or not. Results:There were 47 rats with higher IOP after the episcleral veins cauterized in 60 rats. The ratio of elevated IOP was 78.3%. The IOPs were stable in 4 weeks. After cresyl violet staining, the RGCs loss was 11.0% and 11.3% was found in the central and peripheral retina respectively after 2 weeks of increased IOP. After 4 weeks of increased IOP, the loss of RGCs was 17% for the central retina and 24.6% for the peripheral retina. In the retinas without higher IOP, there was no loss of RGCs. PPD staining showed that optic nerve of rat with about 5.3% damage of axons located at the superior temporal region. Region of affected optic nerve 1 mm posterior to the globe by light microscope showed evidence of damaged axons with axonal swelling and myelin debris. Conclusion:Sharma’s chronic intraocular hypertension model is a reproducible and effective glaucoma model, which mimics human glaucoma with chronically elevation IOP and induced RGCs loss and damage of optic nerve. Eye Science 2004;20:25-29.

Bilateral Central Retinal Vein Occlusions Combined with Artery Occlusions in A Patient with Acquired Immune Deficiency Syndrome

Purpose: This is the first report of a bilateral nonischemic central retinal vein occlusionscombined with artery occlusions in a patient with acquired immune deficiency syndrome(AIDS). Methods: Case report. Results: A 22-year-old Chinese(male) with a positive human immunodeficiency virus(HIV) infection developed bilateral nonischemic central retinal vein occlusions combinedwith artery occlusions and severe vision loss. The manifestations of the fundus andfluorescein angiography were similar in both eyes.Conclusion: This case report provides the evidences that central retinal vein and arteryocclusions are probably part of the spectrum of AIDS vascular diseases.

Clinical Features of Endophthalmitis after Vitreoretinal Surgery

Purpose: To investigate the rate, clinical features, treatment outcomes and prognosis ofpostvitrectomy endophthalmitis.Methods: Patients undergoing pars plana vitrectomy for vitreous opacity or complicatedretinal detachment during 1988 to 2000 were collected. Vitrectomies for recentpenetrating trauma or endophthalmitis were excluded. Patients suffered from clinical orculture-proven postvitrectomy endophthalmitis were selected.Results: Postvitrectomy endophthalmitis happened to 3 of 7 000 patients, resulting in anoverall frequency of 0. 04%. Enhanced systemic and local antibiotics were used assoon as diagnosis was made. Endophthamitis were controlled within 6 days, 8 days and10 days, respectively. Final visual acuities were light perception in 2 patients and0.02 in 1 patient.Conclusion: Postvitrectomy endophthalmitis was rare, but it deteriorate the visualacuity. Both ophthalmologist and patients should pay high attention to it.

The Study on the Expression of Keratin Proteins in Pterygial Epithelium

Purpose:To evaluate the expression patterns of several keratin proteins in pteygial epithelium.Methods:The proteins of K8,K10,K14,K16andAE3 in the epithelium of 8pterygia and 4normal conjunctiva were detected using immunofluorescent staining.Results:In pterygial epithelium,the staining patterns of 5keratin proteins were different with normal conjunctiva.No staining of K8,K10and K16was detected in normal conjunctival epithelium,but in pterygial epithelium,K8and K16were present in full thickness and K10was present in superficial cells.In normal conjunctiva.only base layer of epithelium showed positive staining with antibody of K14and AE3proteins,but they were present in the full thickness in pterygial epithelium and the antibody of AE3 protein stained the superficial cells heavily comparing with base cells.Conclusion:The Abnormal expression of keratin proteins(K8,K10,K14,K16andAE3)in pterygia indicates that the epithelial cells in pterygia are in the state of hyperproliferation and keratinization,which may,be the cause of the abnormal tear function in patients with pterygia,Eye Science2000;16:48-52.

Clinical Investigation of Radiation Retinopathy Fundus and Fluorescein Angiographic Features

Purpose:To investigate the fundus and fluorescein angiographic features in the patients with radiation retinopathy.Clinical Materials:Color fundus photography and/or fluorescein angiography from 13 patients with nasopharyngeal carcinomas received external beam radiation were retrospectively analyzed.Reslts:In this study,26 damaged eyes of 13 patients eveloped some degree of radiation retinopathy.The earliest and most common finding was macular microvascular changes (microaneurysms and/or telangiectasia),which was observed in 100%(26/26)of the eyes.Intraretinal hemorrhages,macular capillary nonperfusion,and macular edema were noted in 84%,50%,and 42% of the eyes,respectively.Conclusions:Radiation retinopathy is common after external beam radiation of nasopharyngeal carcinomas.The prominent changes include maular microvascular changes,intraretinal hemorrhages and macular capillary nonperfusion.

New Management of Angle-closure Glaucoma by Phacoemulsification with Foldable Posterior Chamber Intraocular Lens Implantation

Objective:To investigate the management of angle-closure glaucoma by phacoemulsification with foldable posterior chamber intraocular lens(PC-IOL)implantation.Design:Retrospective,noncontrolled interventional case series.Participants:In36eyes with angle-closure glaucoma(ACG),there were18eyes with primary acute angle-closure glaucoma(PACG),14eyes with primary chronic angle-closure glaucoma(PCCG),3eyes with secondary acute angle-closure glaucoma(SACG)and 1eye with secondary chronic angle-closure glaucoma(SCCG).Intervention:Phacoemulsification with posterior chamber intraocular lens implantation.Main Outcome Measures:Postoperative visual acuity,IOP,axial anterior chamber depth.Results:After a mean postoperative follow-up time of 8.81±7.45months,intraocular pressure was reduced from a preoperative mean of 23.81±17.84mmHg to a postoperative mean of 12.54±4.73mmHg(P=0.001).Mean anterior hamber depth was1.75±0.48mm preoperatively and2.29±0.38mm postoperatively(P=0.000).Best spectacle-corrected visual acuith in 36eyes ranged from0.01to0.7(20/200to20/30)postoperatively,which was better than preoperative VA ranging from hand movement to 0.4(20/50)(P=0.000).Conclusion:Phacoemulsification with posterior chamber foldable intraocular lens implantation can be a good alternative in treating angle-closure glaucoma,Eye Science 2000;16:22-28.

Primary Posterior Chamber Intraocular Lens Implantation in Traumatic Cataract With Posterior Capsule Breaks

Background:In patients with incomplete posterior capsule support,posterior chamber intraocular lenses(PC-IOLs)were implanted with both haptics transs-cleral fixation.This causes more damage to the eye and may result in more com-plications,In patients with small posterior breaks,non-fixation or single haptic fixation may be adequate.Methods:Thiry-two consecutive patients of traumatic cataract with posterior capsule breaks caused by penetrating eye trauma were retospected.Posterior chamber intraocular lenses were implanted in all these patients with three tech-niques,ie,without fixation,with single haptic fixation and with both haptics fixation .The selection of the technique was based on the position and size of the posterior capsule.The follow-up period was 21days to 28months(mean,15.2months).Results:Intra-operative problems included ciliary body bleeding(Two patents,6.25%)and enlargement of posterior capsule breaks(2patients,6.25).Postoperative visual acuity was0.5or better(Corrected)in28case(87.5%)and 0.1-0.4in four patients(12.5%),Postoperative complications included hyphema(6eyes,18.8%),transient intraocular pressure elevation(6eyes,18.8%),transient hypotention(7eyes,21.8%).Postoperative IOL position were good except one case of IOL tilt.No pupillary capture or endophthalmitis was found.Conclusions:Not all PC-IOLs have to be fixed by two haptics.In patients with small posterior capsule breaks,PC-IOLmay not be fixed or fixed by only one haptics.Eye Science1995;11:140-142.

The Influence of Central Corneal Thickness and Corneal Curvature and Axial Length on the Measurement of Intraocular Pressure

Purpose: To examine the influence of central corneal thickness (CCT), corneal curvature (CC), and axial length (AL) on intraocular pressure (IOP).Methods: Eighty-one clinically normal eyes were included in our study. The IOP, CCT, CC, AL were measured using a Goldmann applanation tonometer, optical pachymeter, keratometer and A-scan ultrasound biometer respectively in all subjects.Results: A highly significant positive correlation was identified between IOP and CCT. Linear regression analysis suggests that an increase in CCT of 0. 010 mm is associated with a 4. 946 mmHg increment in IOP. No significant positive correlation was identified between IOP and CC. P values are 0. 724 and 0.414 respectively for vertical and horizontal readings. A paradoxically reversed correlation was present between IOP and axial length.Conclusion: Corneal thickness is a very important confounding factor in the measurement of intraocular pressure, which warrants further attention in our clinical practice.

The Study of Influencing Factors on the Growth Characters of Sprague-Dawley Rat Retinal Neurons in Vitro

Purpose:To investigate the influencing factors in culturing Srague-Dawley(S-D) rats retinal neurons in order to lay foundation for further experimental research.Materials and Methods:Retinal cells were plated on plastic plates and coverslips coated with poly-lysine or ethylene imine polymer for primary culture.The cultured cells were divided into following groups:1.Culture medium changed every 2 tp 3 days vs changed only once;2.Cytosine arabinoside(Ara-C)added to the culture medium vs not added.The cells were observed and pictured under inverted phase contrast microscope.The cells were identified through immunocytochemistry.Results:The immunofluorescence showed that most of the cultured cells were neurons,among them were a few retinal ganglion cells.In the cultured group of which substrata coated with poly-l-lysine and culture medium added with Ara-c,the neurons intended to aggregate into clusters with relatively straight neurites.In the group of which substrata coated with ethylene imine polymer and medium added with Ara-c,the neurons grew dispersively with bent neurites.Both of them survived for 2 to 3 weeks.The cells which plated in the medium not added with Ara-c did not aggregate into clusters and survived longer than 4 weeks.In the group of which medium changed several times,the survival time of neurons was shorter than that in the medium changed only once.Conclusions:The retinal neurons plated on the substrata coated with ethylene imine polymer are easy to observe because of its dispersive growth.It is not favorable for the growth of the neurons by changing culture medium many times.Ara-c may possibly have side effect on the growth of retinal neurons.

Culture and Differentiation of Human Orbital Preadipocytes in Thyroid-associated Ophthalmopathy

Purpose: To identify whether there are preadipocytes in the orbits of thyroid-associated ophthalmopathy (TAO) patients and whether they possess the capacity to undergo adipocytic differentiation.Methods: Orbital fat tissue from TAO patients was placed in primary culture and proliferating cells were sub-passaged. The preadiocytes were identified with immunohistochemistry. Confluent preadipocytes were subjected to a differentiation protocol. And Peroxisome proliferators-associated receptorγ (PPARγ)transcript levels were determined before and after differentiation.Result: In primary culture, cells were spindly and showed positive immunostaining for preadipocyte factor-1 (pref-1 ) protein. During the process of differentiation, orbital cells showed evidence of adipogenesis and relatively greater PPARγ gene expression with differentiation.Conclusion: There are active preadipocytes in the orbital tissue of TAO patients which might be able to undergo adipocyte differentiation. These observations suggest that the preadipocytes differentiation contributed to excess orbital adipose tissue volume in TAO.

Experimental Study on the Prevention of Anterior Segment Ischemia by Preservation of Anterior Ciliary Vessels

Purpose: To observe the effect of preserving anterior ciliary vessels (ACVs) on anteriorsegments of rabbit eyes undergoing tenotomy of extraocular muscles.Methods: Thirty-two adult New Zealand white rabbits were divided into four groups.Same procedures were done in both eyes in each group except that left eyes underwentpreservation of ACVs. In the first group medial and lateral recti, in the second group,superior and inferior recti, in the third group, medial, lateral and superior or inferior rectiand in the fourth group, all four recti, underwent tenotomy. Slit-lamp examination,intraocular pressure (IOP) measurement, total protein and lactic acid quantification inaqueous humor were done in all eyes pre- and post-operatively. By four weeks afteroperation, the eyes were enucleated for histological examination and electron microscopy.All data were analyzed using SPSS version 10.Results: In the left eyes of both group 1 and group 2, no inflammatory response wasobserved. In the left eyes of group 3 and 4, we observed mild inflammatory response withslit-lamp examination, which disappeared in one wk. However, we did not findsignificant changes in IOP, total protein and lactic acid of aqueous humor, histology andelectron microscopic examination in these groups. In the right eyes in group 2, 3 and 4,we observed moderate to severe inflammatory changes, a few even developed anteriorsegment ischemia, appeared as decreased IOP, increased total protein and lactic acid inaqueous humor, along with pathological and electron-microscopic changes.Conclusion: Simultaneous tenotomy of three or four recti or two vertical recti on one eyemay decrease anterior segment blood flow even lead to ischemia. ACVs preservation mayprotect the blood circulation in anterior segment. Our study suggests that ACVspreservation in strabismus surgeries especially those involving multi-recti tenotomies mayprevent potential anterior segment ischemia.

Clinical Features and Surgical Treatment of A-pattern Exotropia

Purpose: To investigate the clinical characteristics and determine the effective surgical managements of A-pattern exotropia.Methods: Thirty-two patients with A-pattern exotropia underwent superior oblique muscle weakening procedures, medial rectus resection or (and) lateral rectus recession. Pre- and post-operative eye position, deviation angle, superior oblique function and binocular vision were examined and analyzed in the cases.Results: Overaction of the superior oblique muscles (31/32) and underaction of the medial rectus muscle (20/32) were presented in the cases. Postoperatively, a satisfactory ocular alignment was obtained in 28 cases (87.5%), and the A-pattern was corrected in 31 cases (96.9%). Four cases got binocular vision after surgery.Conclusions: As one of the most common forms of A and V patterns, A-pattern exotropia showed clinical characteristics of superior oblique muscle overaction and medial rectus muscle underacion, which should be the primary factors in the etiology of A-pattern exotropia, and superior oblique weakening procedures combined with horizontal surgery should be an effective approach to A-pattern exotropia treatment.

The Clinical Analysis of Endogenous Endophthalmitis

Purpose: To study the clinical characteristics, therapeutic efficacy and investigate pro-gnostic factors of endogenous endophthalmitis. Methods: Twenty-eight cases (28 eyes) of endogenous endophthalmitis were surveyed retrospectively. The clinical characteristics, primary infection foci, predisposing systemic disease, complications, pathogens examination, therapeutic options and efficacy were analysed. Results: The endogenous endophthalmitis occurred more frequently in the right eye than in the left one. The respiratory tract was the most common primary foci. The positive rate of pathogens culture was higher in vitreous sample than that in other tissues. Cataract and retinal detachment were the common complications. The visual improvement and infection control were achieved in 13 eyes (46.43%). These 13 patients received treatment (3.77±2.49)days after onset of endophthalmitis being much earlier than that of others [(10.13±4.98)days, P=0.002]. The prognosis was relevant to the type of the disease. The anterior segment inflammation type(anterior type) had better prognosis than posterior segment inflammation type(posterior type) and that of inflammation in both parts(mix type) (P < 0.05). There were no significant relation between the prognosis and the age, predisposing systemic disease, vitreous antibiotic injection and vitrectomy (P > 0.05).Conclusions: Endogenous endophthalmitis is a vital ocular emergency. Early diagnosis and effective treatment combination with systemic and local antibiotics are of significant value. The anterior type is prone to have better outcome than the others.