Anti-scarring effect of sodium hyaluronate at filtration pathway after filtering surgery in rabbits

INTRODUCTION

Glaucoma is the first irreversible blindness disease,characterized by atrophy and depression of the optic disc, visual field defect, and vision decreased, and is often related to the level of intraocular pressure (IOP).Nowadays the glaucoma treatment often includes two aspects:reducing IOP and protecting the optic nerve. Unfortunately,the results of clinical trials about optic neuroprotective is disappointing. Treatments of reducing IOP is still the only effective method for glaucoma treatment, among which,the surgical treatment is easier to achieve the target of lowerlevel IOP compare to drug and laser. Trabeculectomy is the most effective surgical method but has many postoperative complications. For example, filtering bubble fiber wrapping is a common postoperative complication, which is also the primary reason leading to the failure of trabeculectomy.In order to solve this problem, researchers have carried out many explorations like using the anti-fiber preparations as adjuvant therapy during surgery. But unfortunately,these methods can induce other side effects, such as endophthalmitis.

Sodium hyaluronate (HA) is a natural component of the human eye, which has high water solubility and is easy to produce. It is widely used as a viscoelastic agent in ophthalmic surgery. Combined trabeculectomy, which applies detachable suture technology, antimetabolites, anterior chamber puncture technology or intraoperative pre-evaluation of postoperative filtration in trabeculectomy, is a currently common anti-glaucoma surgery. HA has been widely used in dermatology, joint surgery and many other medical filed.Previous studies showed HA has effects of anti-inflammatory,anti-scarring, and pain-relieving. Whether the HA play an anti-inflammatory effect, thereby reducing the incidence of fibrosis in the filtration channel was not clear. Unfortunately,there are few researches in this area, most clinical studies and animal experiments about the HA use in glaucoma filtering surgery always focus on its effect on maintaining the morphology and function of blebs, and avoid postoperative shallow chamber. However, there are some clinical trials reported that HA could not reduce postoperative complications,but may increase the postoperative intraocular. We found a small amount of HA injection at the end of combined trabeculectomy can effectively avoid early postoperative low IOP and shallow anterior chamber. Patients with HA assisted anterior chamber formation had better IOP, and their filtering blebs were more diffuse and more inclined to be type I filtering blebs(Figure 1). In this study we used a preservativefree medical HA gel in an animal experiment to clarify the relationship between HA and fibrosis of the filtration channel after filtration surgery and explore a new way to reduce the complications of filtration surgery in glaucoma patients.

MATERIALS AND METHODS

Fifteen healthy adult New Zealand white rabbits, weighing 1.8-2.2 kg, not limited to males and females are provided by the Department of Experimental Animals of Central South University. The animals were raised in the Department of Experimental Animals, bred for one week before the experiment. All experimental operations are in compliance with the Central South University Laboratory Animal Welfare Ethics Review Form.

综上所述，对孕妇进行饮食管理、妊娠期糖尿病孕妇进行饮食控制可对其血糖水平进行控制，可减少其出现不良妊娠结局的几率，减轻其经济负担，经济实惠、方法简单容易掌握，值得在以后临床中妊娠期糖尿病产妇治疗中进一步推广应用。

One day before the operation,IOP was measured three times. The rabbits were anesthetized by intravenous injection of 1% sodium pentobarbital solution(3 mL/kg). Oxybucaine hydrochloride eye drop and 1%lidocaine hydrochloride subconjunctival injection were used as local anesthesia. Make a conjunctival flap with the fornix as the base on the upper temporal part to expose the sclera. Make the scleral flap and excised the rectangular trabecular tissue.Cut off the peripheral iris tissue of the corresponding part, and the aqueous humor of the anterior chamber is released. The 0.1 mL of HA gel was injected in anterior chamber and under scleral flap in right eye and 0.1 mL of RS was injected in left eye (Figure 2).

Medical HA gel was purchased from Bausch &Lomb Company (composed of HA and physiological buffer without any preservatives). RS was purchased from Sichuan Kelun Pharmaceutical Corporation. Mouse anti alpha-smooth muscle actin (α-SMA) monoclonal antibody was purchased from Abcam, UK, rabbit anti transforming growth factor-beta(TGF-β) polyclonal antibody was purchased from Beijing Boaosen Biotechnology Corporation, FITC-labeled mouse anti rabbit IgG fluorescent secondary antibody was purchased from Santa Cruz, USA. SABC-POD(F) (mouse IgG) and DAB color development kit were purchased from Wuhan Boster Bioengineering Corporation.

Fifteen New Zealand white rabbits were numbered sequentially. Because different rabbits had significantly different baseline IOP and wound-healing reactions. Drawing lessons and experience from other studies about filtering surgery in rabbits, we performed the surgery on both rabbits' eyes to diminish the impact of individual difference. After the surgery, the right eye was used as HA group, and 0.1 mL medical HA was injected into the anterior chamber, while the left eye was used as the Ringer's solution(RS) group, and 0.1 mL sodium lactate RS was injected into the anterior chamber.

Three eyes (20.00%) and 5 eyes (33.33%) had anterior chamber hemorrhage in HA and RS group respectively(=0.682) 1d after the operation, which was faded within 7d.One eye (6.67%) in RS group had a shallow anterior chamber(degree I) and recovered after 4d without any treatment.

On the 7, 60, and 90day after the operation, 5 rabbits were randomly selected and sacrificed. Eyeball tissue was immersed in phosphate buffer saline (PBS). Cut off a small amount of corneal tissue. The specimens were immersed in 4% paraformaldehyde-PBS fixative solution at 4℃ for 24h, and made frozen sections for the Masson's trichrome staining, α-SMA immunohistochemical staining, and TGF-β immunofluorescence staining.

HA has effect of anti-scar and antiinflammation on filtration pathway after filtering surgery within 3mo by inhibiting fibroblast proliferation and collagen deposition.

SPSS23.0 statistical software (SPSS Inc.,Chicago, IL, USA) was used for data analysis. The difference between two groups was compared with independent sample-test. The difference in different timepoint within the same group was compared with one-way analysis of variance. Data of postoperative complications were analyzed using Chisquare test.＜0.05 was considered statistically significant.

RESULTS

There was no significant difference in IOP between HA and RS group before operation (=0.764;Table 1). The IOP of the two groups was significantly lower than that of the preoperative. The IOP on 1d after operation in RS group was significantly higher than 7d and 28d after operation(=0.018), while there were not significantly different at each time period after operation in HA group (＞0.05).

为助力中国制造接轨工业4.0，浩亭于2018年10月22日～24日联合德国电气电子行业协会（ZVEI）与佛山机器人学院、佛山中德工业服务区和互联网博览会等合作伙伴共同组织了参观访问团，并邀请了中日行业媒体代表及德国和亚洲的客户参观其珠海工厂，以加强交流与合作探讨。在井然有序的车间，可以看到，浩亭以精益制造的看板管理系统强化对品质的过程监控，还有实施反馈的监测系统和全面细致的检测系统也同样在无时不刻地为浩亭输出高品质产品做出坚定的技术保障。

On the first day after the operation,functional filtering blebs were formed in all the operated eyes, and the filtering blebs were highly bulged and diffuse,accompanied by conjunctival congestion and edema, and the congestion gradually subsided after 3d. On the 7day after operation, both groups of filtering blebs were diffused with thin walls. In the RS group, sparse and slender neovascularization were seen on the surface, while in the HA group, no new blood vessels were seen on the surface of the filter blebs. At 28d postoperatively, the filtering blebs in the RS group were flat and diffuse, with abundant surface new blood vessels; in the HA group, the filtering blebs were flat and diffuse, with a small amount of new blood vessels on the surface, which were formed by the branches and growth of peripheral conjunctival small blood vessels. At 60 and 90d postoperatively, there were no functional filtering blebs in both groups. The filtering blebs were scarred, and a dense vascular network was formed on the surface. Compared with the peripheral conjunctiva, the conjunctiva of the surgical area was rich in blood vessels and irregular in shape (Figure 4).

On 1, 7, 28, 60, and 90d after operation, the size, shape, height, and surface blood vessel distribution of the filtering blebs were observed under an operating microscope. Refer to Kronfield classification method: Type I (microcystic type): the filtering vesicles are microcystic or multi-cystic, with thin walls and pale ischemia on the surface; Type II (flat diffuse type): the bubble wall is slightly thicker, slightly raised, diffuse, and the surface is relatively ischemic; Type Ⅲ (scarring type): no filtering bleb or conjunctival hyperemia and micro bulging, subconjunctival scarring and adhesion on the scleral surface, multi-vascular appearance; type Ⅳ (wrap type): limited dome-shaped bulge,thick wall hyperemia, showing cystic hyperplasia. Among them, type I and type II are functional filtering vesicles, and type III and type IV are non-functional filtering vesicles.

（3）权力集中度较高。我国涉农企业整体而言，自然人控股且家族式企业较多，董事长和总经理兼任的现象普遍存在，造成权力过于集中，不能实行有效的授权，导致公司管理层独立性和积极性较差，严重制约了管理层职能的发挥，治理效率低下。在部分企业中高管总体素质不高，对企业发展中权利的集中与充分授权思想意识认识不够，特别在家族式的涉农企业中，只有权利的高度集中才能获取利润的思想存在，严重阻碍公司治理效率的提升。

At 7d after operation, the expression of α-SMA was obvious in both groups, and α-SMA positive cells showed a diffuse distribution in the conjunctiva and superficial sclera. There was no significant difference of average optical density (AOD) between the two groups (=0.501). At 60d postoperatively, α-SMA positive cells were mainly distributed at the junction of conjunctiva and sclera and gathered around blood vessels. After 90d, a small amount of α-SMA positive cells were under the conjunctiva in the RS group, and there was almost no positive staining in the HA group. The AOD in RS group was significant higher than of the HA group at 60 and 90d after operation (=0.032, 0.000; Figure 6, Table 3).

At 7d after operation, the conjunctiva,sclera and filtration channel of the HA group and the RS group showed significant expression of TGF-β, especially in RS group (=0.024). After 60d, the expression of TGF-β was significantly reduced, mainly concentrated in the subconjunctiva. After 90d, there was almost no expression of TGF-β in both groups, and occasionally scattered a small amount of positive staining in the subconjunctiva or deep sclera (Figure 7, Table 4).

The collagen fibers in the RS group are tightly arranged and bent and tortuous and disorder in HA group. At 7d after operation, there was no significant difference of collagen volume fraction (CVF) between the two groups(=0.389). It was statistically different at 60 and 90d after operation (=0.044, 0.036; Table 2, Figure 5).

DISCUSSION

Glaucoma is a type of ophthalmopathy characterized by atrophy and depression of the optic disc, visual field defect,and vision decreased. Trabeculectomy is currently an important method to reduce IOP and protect visual function.Postoperative filtering bleb scarring is often the primary cause of surgical failure. Previous studies have shown that HA had anti-inflammatory, biocompatibility and biodegradability,and is hopefully to be used to reduce the incidence of filtration channel fibrosis.

The IOP of RS group decrease significantly at 1d postoperatively,and then slowly reversed. IOP of HA group decreased slowly and remained at a relatively stable level (Figure 3). The IOP in HA group was higher at 1d (=0.005) and lower at 28d(=0.011) than that of the RS group after the operation.

It is preliminarily shown that HA has a certain inhibitory effect on the scarring of filtering blebs and is beneficial to maintain the functional morphology of filtering blebs. Meanwhile, it can reduce the incidence of complications, such as anterior chamber hemorrhage and shallow anterior chamber. The results showed that the collagen fibers in the HA group were bent and arranged disorderly, while the collagen fibers in the RS group were dense and tightly arranged. At the same time, the CVF of the RS group was significantly higher than that of the HA group at 60d and 90d after surgery. HA has an inhibitory effect on the deposition of collagen fibers at the filtration channel.

In the process of trabeculectomy wound healing and fibrotic scar formation, fibroblasts in Tenon's capsule are the main cells. It was found that the α-SMA positive cells were diffusely distributed 7d after the operation, but 90d after the operation, the HA group was almost no positive staining.A small amount of α-SMA positive cells were under the conjunctiva of the RS group. The measurement results of the AOD showed that there was no difference in the two groups at 7d after the operation, while the AOD of the RS group was greater than the HA group at 60 and 90d after the operation,and the difference was statistically significant. The AOD value measured by immunohistochemical staining and the CVF value measured by Masson staining have the same changing trend, suggesting that sodium hyaluronate can inhibit the proliferation and activation of fibroblasts, thereby inhibiting the formation and deposition of collagen fibers.

TGF-β can stimulate the proliferation and migration of human eyeball fascial sac fibroblasts, and play an important role in the process of scar repair after glaucoma trabeculectomy.At 7d postoperatively, TGF-β was significant expression in the conjunctiva, sclera, and filtration channel in both groups.At this time, the AOD value of the RS group was greater than that of the HA group. TGF-β is significantly reduced at 60 and 90d postoperatively. The results show that HA has an inhibitory effect on the expression of TGF-β, which can inhibit the proliferation and migration of fascial capsule fibroblasts,the aggregation of various inflammatory cells, the expression of various growth factors, and the new collagen contraction.Masson staining shows that the new collagen fibers in the HA group are slender and curved and arranged irregularly.In conclusion, HA has effect of anti-scar and anti-inflammation on filtration pathway after filtering surgery within 3mo by inhibiting fibroblast proliferation and collagen deposition.

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Supported by National Natural Science Fundation of China (No.81974132; No.81770927); National Key R&D Program of China (No.2021YFA1101202); Hunan Provincial Health Commission (No.202207022839).

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