Volume 39 ·  Issue 08 ·  2003

To study trabecular meshwork induced glucocorticoid response protein (TIGR) gene mutation in primary open angle glaucoma (POAG), their relatives and normal controls in Chongqing.

(1) The coding sequence of TIGR was screened for sequence alterations using polymerase chain reaction (PCR) followed by single-strand conformation polymorphism (SSCP). (2) Samples corresponding to bands of altered mobility were sequenced. (3) The sequence alterations were analyzed by bioinformatics.

(1) In 15 POAG, TIGR gene mutation was found in 5 cases. In 10 POAG relatives, 2 TIGR gene mutations were found. No TIGR gene mutation was found in 20 normal persons in Chongqing. (2) The mutation sites Ser55Thr, Asp247Stop and in 49021 c-t alteration discovered in the present study have not been reported previously. The 63155 c-t alternation has been reported as a polymorphism site previously. (3) Bioinformatics analysis indicated that the mutations in structural domain could lead to the changes in the amino acid sequence coded, second structure of protein, isoelectric point and antigen binding site.

We found that the TIGR gene mutations is closely related to the occurrence of JOAG in Chongqing. The relatives of POAG patients may have a higher incidence of TIGR gene mutation as compared with the controls. Furthermore, after analyzed by bioinformatics, we found that the alteration of structure and biological activity of TIGR protein resulted from gene mutation may be one of the important changes that leading to the occurrence of POAG.

To study the anatomic background of non-penetrating trabecular surgery (NPTS) and to explore the resistant site of aqueous outflow in primary open-angle glaucoma.

NPTS was performed in 18 eyes of 12 cases (10 males, 2 females) with POAG. The age of these patients ranged from 42 to 65 years old. The deep scleral flap and external trabecular membrane were obtained during the surgical procedure and were examined by light microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM).

The beginning part of the collection canal in the outer wall of the Schlemm's canal were very narrow, and there was electron dense material (so-called plaques) around the outer wall of the Schlemm's canal. The external trabecular membrane consisted of adjacent trabecular meshwork and part of the corneoscleral trabecular meshwork. The number of the trabecular cells was decreased and a large amount of electron dense materials were deposited in the adjacent trabecular meshwork. The holes of the corneoscleral trabecular meshwork was small and irregular. In the corneoscleral trabecular meshwork, the trabecular lamellae was thicken, the amount of elastic-like fibers was increased and the trabecular cells, especially the nucleus, were enlarged. In the advanced stage of POAG, a great amount of electron dense material was deposited in the corneoscleral trabecular meshwork and the inter-trabecular spaces was blocked.

The deep scleral flap of NPTS contains the outer wall of Schlemm's canal and the external trabecular membrane, which consists of the inner wall of the Schlemm's canal, the adjacent trabecular meshwork and part of the corneoscleral trabecular meshwork. The main resistant site of aqueous outflow in POAG lies in the adjacent trabecular meshwork and in part of the corneoscleral trabecular meshwork. The outer wall of the Schlemm's canal is another important resistant site for aqueous outflow.

To study the effect of deep sclerectomy combined with laser trabecular puncture for the treatment of open angle glaucoma (OAG).

Deep sclerectomy was performed in 32 eyes of 32 patients with moderate to advanced stages of OAG. After the exposure of external wall of Schlemm's canal and the remaining trabecula, the intraocular optic fiber of diode laser photocoagulator was directed to the trabecula-limbus near the anterior edge of the scleral spur. Two or three punctures, approximately 0.5 mm in diameter, were performed with laser beam under direct visualization until aqueous humor was extravasated slowly. The scleral flap was closed with one or two sutures. Postoperative observation included intraocular pressure (IOP), intraocular tissue reaction, the appearance of filtering bleb, gonioscopy and ultrasound biomicroscopy. The follow-up period was 4 to 24 months (7.94±6.20) months.

The anterior chamber was recovered shortly after the operation and the visual acuity remained the same in all eyes. At the end of follow-up period, the postoperative IOP (14.86±4.15) mm Hg was lower than the preoperative IOP (29.68±5.76) mm Hg, the difference was statistically significant (t=7.415, P<0.001). Diffuse and obvious filtering blebs were found in all eyes after the operation, including 27 functional blebs and 5 blebs that disappeared 2 to 3 months later. Iris was burned slightly in 2 cases, pigment was released and appeared in the anterior chamber temporarily. No laser injury occurred in the cornea and the lens. High IOP was found in 2 cases after long-term follow-up and could be controlled by antiglaucoma medication.

Deep sclerectomy with laser trabecular puncture can effectively reduce the IOP, without the occurrence of serious complications that commonly seen after the trabeculectomy. It is an effective surgical method for the treatment of OAG.

To evaluate the interobserver and intraobserver agreement of parameters of optic nerve head by Heidelberg Retina Tomograph (HRT).

Optic nerve heads from 50 eyes (30 subjects) were imaged with the HRT. Three ophthalmologists majoring in glaucoma traced the disc margin with a contour line. One of these observers, repeatedly traced the disc margin with contour line in triplicate at three different times. Eleven topographic parameters of optic nerve head (including disc area, cup area, cup/disc area ratio, rim area, cup volume, rim volume, mean cup depth, maximum cup depth, cup shape measure, mean retinal nerve fiber layer thickness, sectional area of retinal nerve fiber layer) were analyzed with HRT software (version 2.01). Intraclass coefficients (ICC) and agreement of result estimation (kappa value) were calculated.

The ICCs results among three observers on 12 parameters of optic nerve head topograph were 0.976 ~ 0.999, which showed almost perfect agreement; and the kappa value were 0.649 ~ 0.767, which showed substantial agreement. The ICC of the same observer in different times were 0.983 ~ 0.999, which showed almost perfect agreement; and the Kappa values were 0.810 ~ 0.951, which showed substantial agreement.

Tracing the optic nerve head with contour line on HRT image is substantial to almost perfect within both interobserver and intraobserver in experienced ophthalmologist. This procedure is suitable for clinical diagnosis and follow-up observation of glaucoma.

To study and evaluate the outcome of trabeculectomy with amniotic membrane transplantation combining suture lysis of scleral flap in complicated glaucoma.

This operation was performed in 48 patients (67 eyes) with complicated glaucoma. Amniotic membrane was implanted under the scleral flap and the conjunctival flap and combined with suture lysis for scleral flap to control the outflow of the aqueous humor.

Follow-up period ranged from 5 to 38 months (mean 19.5 months). The intraocular pressure (IOP) was lowered from (43.86±7.12) mm Hg (preoperative IOP) to postoperative (19.63±2.57) mm Hg (postoperative IOP) in neovascular glaucoma (t=4.96, P<0.001); from (40.31±4.79) mm Hg to (18.09±2.21) mm Hg in glaucoma after IOL implant (t=3.54, P<0.05); from (37.94±5.63) mm Hg to (20.14±3.15) mm Hg in aphakic glaucoma (t=5.12, P<0.05); from (32.48±3.98) mm Hg to (16.54±1.84) mm Hg in juvenile glaucoma (t=4.23, P<0.01); from (36.01±4.13) mm Hg to (18.11±3.40) mm Hg in uveitic glaucoma (t=4.47, P<0.01); from (34.43±5.28) mm Hg to (18.31±1.52) mm Hg in glaucoma after failure of conventional filtering operation (t=2.05, P<0.05). Statistical significant difference was found in the IOP before and after the operation. The rate of formation of functional bulb was 80.6%. There were no reject reaction and severe complications.

Trabeculectomy with amniotic membrane transplantation combining suture lysis of scleral flap is a useful method in complicated glaucoma.

To investigate whether a Chinese herbal medicine, erigeron breviscapus (vant) hand-mazz (EBHM), can protect the retinal ganglion cells (RGC) damaged by calibrated optic nerve crush injury.

Forty-two Sprague-Dawley rats were randomly divided into two groups. Calibrated optic nerve crush injury model was induced in the right eyes by a special designed optic nerve clip. The left eyes served as a control. All 42 rats were randomly divided into 2 groups. Group A consisted of the rats with calibrated optic nerve crush injury and group B consisted of rats with calibrated optic nerve crush injury treated with EBHM. In group B, EBHM solution was given once after the crush injury. According to the time interval between the optic nerve crush and the sacrifice, both groups A and B were further divided into three subgroups (day 4, day 14 and day 21). Therefore, there were 7 rats in each subgroup. Three days before sacrifice, 3% fast blue was injected into superior colliculi bilaterally. The eyes were enucleated after the rat was sacrificed, and flat mounts of the retina from both eyes were prepared on a slide and observed under a fluorescence microscope. Four photos with 400× magnification were taken from each of the four quadrants of the retina 1 mm away from the optic disc. The labeled RGC were counted by a computerized image analyzer. The labeled RGC rate was used for statistical analysis (the labeled RGC rate = number of RGC in injured eye/control eye ×100%).

In group A, the labeled RGC rate was (77.79±7.11)%, (63.76±3.79)% and (54.66±4.75)% on day 4, day 14 and day 21, respectively. In group B, the labeled RGC rate was (80.13±12.03)%, (78.17±9.19)% and (83.59±12.61)% on day 4, day 14 and day 21, respectively. In group B, which was treated with EBHM after injury, the labeled RGC rate was significantly higher than that of group A on day 14 and day 21.

In the experimental optic nerve crush model in rats, EBHM therapy can increase the survival rate of the RGC and can rescue and/or restore the injured RGC.

To evaluate the efficiency and safety of indocyaine green (ICG) staining technique using in white cataract phacoemulsification.

In a prospective clinical study, 105 eyes with white senile cataract were divided into two groups: capsular-staining group using ICG (n=55) and noncapsular-staining group (n=50). The time for performing the circular continuous capsulorrhexis (CCC), the number of radial tears of anterior capsule, the number of the rupture of capsular bag and the incidence of IOL in-the-bag fixation during the operation were recorded. We also measured the intraocular pressure and observed the changes of corneal endothelial cells, the anterior chamber flare and the keratic precipitation before surgery, 1 day and 3 months after surgery, respectively. The results of these two groups were compared. The quantitative results were analyzed with two-tailed student's t-test, while the qualitative results were analyzed with chi-square test.

In operations performed with ICG staining technique, the CCC could be performed more quickly, no radial tears of the anterior capsule or rupture of capsular bag occurred and all IOL could be fixed in the bag. No statistically significant difference could be detected in the occurrence of these four aforementioned changes between these two groups. None of these patients had an elevation of intraocular pressure. The anterior chamber flare, the corneal endothelial cell density, the percentage of hexagonal cells and the corneal thickness showed no statistically significant difference between these two groups before and after surgery.

Indocyaine green dye staining is a safe and useful technique in the white cataract phacoemulsification. We suggested to use ICG staining technique in the white cataract phacoemulsification.

To assess the effect of bcl-X_L, an anti-apoptotic gene, on glutamate-induced apoptosis in cultured retinal photoreceptors.

Glutamate-induced apoptosis in cultured retinal photoreceptors was established. The experiment was divided into three groups: control, glutamate treatment and rAd-gfp-bcl-X_L + glutamate transfection group, the protection of bcl-X_L on retinal photoreceptors from apoptosis was evaluated. 6.5×10¹² pfu/L rAd-gfp-bcl-X_L were transfected into retinal photoreceptors in the rAd-gfp-bcl-X_L + glutamate group 48 h before Glutamate-induced apoptosis was established. The positive photoreceptors with green fluorescence were identified under fluorescence microscopy. The expression of Bcl-X₂ protein in rAd-gfp-bcl-X_L transfected and non-transfected neurons were assessed by immunohistochemistry assay. DNA fragment was detected by agarose gel electrophoresis. Nuclei were revealed by hoechst33258 staining.

rAd-mediated gene delivery can transfect retinal photoreceptors effectively. Increased expression of Bcl-X_L protein was demonstrated in the rAd-transfecting neurons. Transfection of bcl-X_L significantly decreased the number of apoptotic retinal photoreceptors in vitro.

Transfection of bcl-X_L protects cultured retinal photoreceptors from apoptosis induced by glutamate. Transfection the gene of bcl-X_L may be a potential gene therapy for retinal degenerative diseases.

To study the effects of iodine supplementation (in different kinds and doses) on the antioxidative ability of retina in iodine deficient rats.

One hundred and twenty eight iodine deficient Wistar rats were randomly divided into four groups, normal dose of iodate, normal dose of iodide, high dose of iodate and high dose of iodide. Concentration of serum thyroid hormones, including total TT₃ and total TT₄, were estimated by radioimmunoassay. GSH-Px, SOD, TAOC activities and MDA content in the retina were determined using biochemical methods in the 22nd week of iodine supplementation.

Statistical analysis showed that a significant difference in TT₃ level of serum was observed between animals treated with different doses. Serum TT₃ level in the groups treated with high doses was significantly higher than those with normal doses. However, no statistical difference could be detected at TT₄ level between animals treated with different doses. Different kind of iodine did not affect the level of thyroid hormones. Statistical analysis showed that a difference in SOD activity of retina was observed between animals treated with different doses. SOD activity in the groups with normal doses was significantly higher than that in groups with larger doses. Retina TAOC activity was significantly higher in groups treated with iodide than that in groups of iodate. Although there was no statistical difference in GSH-Px activity between different groups, it showed the same tendency as the SOD and TAOC activities, i. e. GSH-Px activity in the groups of normal doses was higher than that in the groups of high doses. GSH-Px activity in groups of iodide was higher than that in the groups of iodate. There was no significant difference in MDA content among these four groups.

Different iodine and doses have certain effects on the antioxidative ability of retina in iodine deficient rat. The rats supplemented potassium iodide at normal dose showed higher antioxidative ability of the retina than those of the others.