【摘要】 目的：研究HeNe激光对兔眼小梁切除术后滤过道瘢痕化的影响及其机制，探讨HeNe激光在青光眼小梁切除术中的应用价值。 方法：于小梁切除手术前采用功率密度200mW/cm2 HeNe激光照射兔眼(右眼)手术区，照射部位选择鼻上象限角膜缘紧邻上直肌处，光斑直径4mm，照射时间10min，1次/d，连续照射3d。最后一次照射后24h实施双眼小梁切除术。手术后第7,14,28d检测手术区组织结缔组织生长因子(connective tissue growth factor, CTGF);第14和第28d检测胶原密度(masson染色)。左眼设为单纯手术对照组。每时间点各7只兔。结果：手术后第7,14d，HeNe激光组CTGF表达较对照组减少(P=0.01;P=0.005)。手术后第14,28d HeNe激光组胶原密度显著低于对照组(P=0.013;P=0.01)。结论：功率密度200mW/cm2 HeNe激光预照射小梁切除术手术区域，可下调术后该区域成纤维细胞CTGF表达并减少胶原合成，可能有助于减少兔眼小梁切除术后滤过道瘢痕形成。HeNe激光有可能为预防青光眼滤过手术后瘢痕化提供新的方法。
Scar formation of the filtration channel is one of the most important causes to the failure of trabeculectomy. It is also the most intractable problem of the antiglaucoma surgery. How to make a permanent channel for the outflow of aqueous humor is the challenge all ophthalmologist confronted with.
Mitomycin and 5 Fluorouracil are widespread used in clinical nowadays, and to some extent they can inhibit the excessive proliferation of fibroblast hypoconjunctiva after the filtration surgery. However, these drugs also can cause some severe complications such as long time post surgery hypotension, filtration bubble leakage, endophthalmitis and etc. So the discovery of new antiscarring methods is very important to filtration surgery.
HeNe laser can inhibit the collagen formation of fibroblast in
vitro, reduce the dermatic scar effectively and prevent the scar formation of skin. It is widely used in cosmetology, and it is safe in effect dosage[5,6]. HeNe laser may have great clinical significance in prevention of scar formation after the filtration surgery. We use HeNe laser on rabbit ocular, observe the scar formation after the filtration surgery, and discover the mechanism of HeNe laser impact on the wound healing and scar formation.
MATERIALS AND METHODS
Experimental animal Twentyone healthy sanitation grade New Zealand albino rabbits, weight about 2kg, no gender limit, provided by the Experimental Animal Center of Tongji Medical College.
Instruments and reagents The HeNe laser machine used in this study was manufactured by Wuhan Nation Optoelectricity Laboratory max power density is 300mW/cm2, light spot diameter is 4mm. Rabbit antiCTGF multiclone antibody (Wuhan Boster); SABC kit (Wuhan Boster); DAB kit (Wuhan Boster) and Masson staining kit (Wuhan Guge).
Laser irradiation Site: the upper nasal limbus area next to the upper rectus muscle; diameter: 4mm; time: 10 minutes×3 days.
Surgery method Rabbits were intravenous anesthetized with 30g/L Pentobarbital, conjunctival sac were washed by 3g/L FPA. Trabeculectomy: made a conjunctival flap based on fornix zone just next to the upper rectus muscle, and a 3mm×3mm scleral flap (2/3 sclera thickness). Excised 1.5mm×1mm trabecula tissue in the corneoscleral limbus. Cut a 1.5mm×1.5mm iris periphery hole. Reset the sclera flap, sutured the sclera flap and bulbar conjunctiva.
Animal grouping Twentyone rabbits were randomly divided into three groups, representing the 7th, 14th and 28th day after surgery. The right eyes received laser irradiation, the left eyes served as control. Seven eyes each group at each time point.
Observation and sample preparation We observed the filtration bubble after surgery. Rabbits were executed on the 7th, 14th and 28th day after surgery, took the tissues of surgery site (the whole layer), fixed them with 40g/L paraformaldehyde solution for 24 hours, then paraffin imbedded and cut into slices.
Histopathological and Immunohistochemistry Examination
Massons staining We observed the proliferation of collagen in the filtration canal. Result criterion: the collagen was blue stained while the muscle fiber and cellulose were red stained. The slice was put under the microscope in the same light intensity to observe the blue stained area and image analysis software was used to calculate average luminosity.
CTGF immunohistochemistry staining We used the SABC kit to mark the antigen (CTGF) and observe after DAB staining. Result criterion: the CTGF staining positive cell were those with brown or dark brown kytoplasm. Five high power lens(×400) visual field were chosen randomly in every slice, we counted the positive and whole number of fibroblast, and calculated the average positive cell percentage.
Statistical Analysis The SPSS (16.0) software was used. Ttest was used to compare the expression of CTGF and collagen density groups. P<0.05 was assumed the sign for diversity had statistical significance.
When observed on the 1st, 7th and 14th day after surgery, hyperemia was much lesser and filtration bubble was much more obvious in laser group than those of control group.
The expression of connective tissue growth factor (CTGF) in the filtration site was notable on the 7th day, lesser on the 14th day, and a very small amount on the 28th day (Figure 1). The expression of laser group was much lesser than that of control group on the 7th and 14th day (t=3.078, 3.458; P=0.01, 0.005 ), and the expression diversity between the two groups on the 28th day had no statistical significance (t=0.110, P=0.915,Table 1).
Massons staining and image analysis showed that collagen expression was obviously lesser in laser group on the 14th and 28th day (Figure 2) (t=2.914, 3.032; P=0.013, 0.01,Table 2).
How to sustain a functional filtration bubble after surgery is very important, suppress the scar formation of filtration canal
Table 1 HeNe laser impact on the CTGF expression (%) of fibroblast in filtration site
effectively is the key point for the surgery. The scar formation of filtration canal is induced by the inflammatory factors result in tissue damage during the surgery. The factors stimulate the inflammatory reactions and the proliferation and shifting of fibroblast. Fibroblast is functional active fibro cell, can synthesis and secrete collagen, induce the massive proliferation of extracellular matrix as collagen fiber and elastic fiber, cause the scar formation of filtration canal and subconjunctiva tissue fibration and finally result in stenosis even shut down of the filtration canal. So the research is focused on the fibroblast.
HeNe laser machine is the first manufactured continuous emission gas laser, and is the most reliable one. It is widely used because of its portability. The working substances are helium and neon, and neon is the working gas. The laser is produced by neon atom, and mainly stimulates three wavelength, 632.8nm, 1.15μm and 3.39μm. The output power of laser is low, 0.1100mW generally. Organism exposed into HeNe laser will not result in nonreversible tissue damage, so it is safe.
According to the research, HeNe laser impact on organism is related to its power and energy density. Low dose HeNe laser radiation promotes the fibroblast proliferation and collagen synthesis, and cause wound healing. High dose HeNe laser radiation inhibits the fibroblast proliferation and promotes apoptosis. This research uses high level dose of HeNe laser to study its influence on the scar formation of the filtration canal.
CTGF is new discovered fibrosis factor in the 1990s, is a polypeptide rich in cysteine, and a member of CNN family. CTGF is widely expressed in many human tissues and cells, such as heart, brain, lung, kidney, placenta, pancreas and connective tissue. Its main functions are: promote cell proliferation and collagen synthesis, induce cell adhesion and chemotaxis, and facilitate blood vessel and granulation tissue formation. CTGF is none or extremely low expression in normal tissue. But CTGF is closely related to some hyperplastic and fibrosis diseases in pathologic status. Many researches approved that CTGF expression is positive correlation to the degree of fibrosis. As the important substance
Figure 1 CTGF expression of fibroblast in the filtration site (×400, arrow pointed are positive cells) A: Laser group on the 7th day after surgery: a small amount of CTGF positive cells in filtration site; B: Control group on the 7th day after surgery: a lot of positive cells in filtration site, and they are stained darker; C: Laser group on the 14th day after surgery: few CTGF positive cells in filtration site and stained lighter compare to the 7th day group; D: Control group on the 14th day after surgery: a small amount of CTGF positive cells in filtration site, and stained lighter compare to the 7th day group; E and F are laser and control group on the 28th day after surgery: a extremely small amount of CTGF positive cells in filtration site.
Figure 2 Arrow pointed is the filtration site, and blue stained tissue is collagen fiber (×200) A and B are laser and control group on the 14th day; C and D are laser and control group on the 28th day, and the collagen expression is higher in laser group.
of ECM, collagen is secreted by fibroblast, and its degree of proliferation can reveal the degree of scar formation.
The research shows that the fibroblast proliferation and collagen synthesis are most active on the 7th day after surgery, the activity went down on the 14th day, and went normal on the 28th day. All above are accord with the post surgery inflammatory process, is the same as the literature report. The research result points out that the pretreatment with the HeNe laser can improve the success rate of the filtration surgery significantly, represented by the good condition of the filtration bubble. This could be the result of HeNe laser downgrade the fibroblast CTGF expression and the activity of fibroblast proliferation and collagen synthesis, reduce the inflammatory reaction, and scar formation of filtration canal.
The mechanism of HeNe laser inhibit the proliferation of cell is not completely clear. Its impact on the proliferation and apoptosis of cell, and the safety of using HeNe laser on ocular need more study.
In conclusion, the HeNe laser with 200mW/cm2 power density can reduce the scar formation of rabbit ocular after the filtration surgery; it could be related to the downgrading of fibroblast CTGF expression and collagen synthesis. It is possible the HeNe laser could be a brandnew way to improve the success rate of filtration surgery.
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