Inhibitory effect of ILK-siRNA-AAV on scar formation after glaucoma filtering surgery in rats / 中华实验眼科杂志

Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.

Efficacy and Safety of Sodium Hyaluronate with 1,4-Butanediol Diglycidyl Ether Compared to Sodium Carboxymethylcellulose in Preventing Adhesion Formation after Lumbar Discectomy

OBJECTIVE: Epidural injection of hyaluronic acid may prevent adhesion formation after spine surgery, but the compounds used to stabilize hyaluronidase could interfere with its anti-adhesion effects. The present study was conducted as a clinical trial to evaluate the efficacy and safety of an experimental medical gel in preventing adhesion formation. METHODS: This study was designed as a multicenter, randomized, double-blind, and comparative controlled clinical trial with an observation period of 6 weeks. Subjects were randomly assigned into two groups: group A with sodium hyaluronate + 1,4-butanediol diglycidyl ether (BDDE) and group B with sodium hyaluronate + sodium carboxymethylcellulose (CMC). Visual analogue scale (VAS) of back and leg pain and the Oswestry disability index (ODI) and scar score ratings were assessed after surgery. RESULTS: Mean scar grade was 2.37+/-1.13 in group A and 2.75+/-0.97 in group B, a statistically significant difference (p=0.012). VAS of back and leg pain and ODI scores decreased significantly from baseline to 3 and 6 weeks postoperatively in both groups (p0.3). The number of adverse reactions related to the anti-adhesion gels was not statistically different (p=0.569), but subsequent analysis of nervous adverse reactions showed group B was superior with a statistically difference (p=0.027). CONCLUSION: Sodium hyaluronate with BDDE demonstrated similar anti-adhesion properties to sodium hyaluronate with CMC. But, care should be used to nervous adverse reactions by using sodium hyaluronate with BDDE.

Effect of Hyaluronic Acid-Carboxymethylcellulose Solution on Perineural Scar Formation after Sciatic Nerve Repair in Rats

BACKGROUND: Scar tissue formation is the major cause of failure in peripheral nerve surgery. Use of a hyaluronic acid-carboxymethylcellulose (HA-CMC) membrane (Seprafilm) as a solid anti-adhesion barrier agent is one of the therapeutic approaches to reduce postoperative scar tissue formation. However, a solid membrane may not be suitable for repair of a weak peripheral nerve site. This study examined the effect of HA-CMC solution on perineural scar formation after peripheral nerve repair in rats. METHODS: The sciatic nerves of 40 rats were transected and then immediately repaired using 10-0 nylon. The nerves were divided randomly into two groups. Saline and HA-CMC solution were applied topically to the nerve repair sites in the control and experimental groups, respectively. Reoperation was performed at 3, 6, 9, and 12 weeks to assess scar tissue formation. The assessment included the quality of wound healing, presence of perinueral adhesion, cellular components of the scar tissue, thickness of the scar tissue and histomorphological organization of the repair site. RESULTS: Topical application of the HA-CMC solution significantly decreased the macroscopic nerve adherence score and the numbers of the cellular components such as fibroblasts and inflammatory cells (p < 0.05, Mann-Whitney U-test). The scar tissue formation index was significantly lower in the experimental group at 12 weeks than that in the control group (p < 0.05, Mann-Whitney U-test). The grading scores of the histomorphological axonal organization at the repair site were significantly higher in the experimental group than those in the control group at 12 weeks (p < 0.05, Mann-Whitney U-test). No evidence of wound dehiscence or inflammatory reactions against the HA-CMC solution was noted. CONCLUSIONS: Topical application of a HA-CMC solution is effective in reducing the perineural scar formation and adhesion after sciatic nerve repair in rats, and is effective in promoting peripheral nerve regeneration at the repair site.

Spinal cord injury-induced astrocyte migration and glial scar formation: Effects of magnetic stimulation frequency.

The effects of magnetic stimulation on spinal cord injury-induced migration of white matter astrocytes were studied using an established animal model. Ethidium bromide was injected into the dorsal spinal cord funiculus of adult Sprague-Dawley rats on the left side at T10-11. Animals then received 1.52 Tesla-pulsed magnetic stimulation for 5 min at different frequencies (0-20 Hz) for 14 consecutive days. Selected animals received the non-competitive MEK1/2 inhibitor U0126 (10 μM), prior to stimulation at 10 Hz. Lesion volumes were measured in hematoxylin/eosin-stained sections. Expression of glial fibrillary acidic protein (GFAP), microtubule associated protein-2 (MAP-2) and extra-cellular signal-regulated kinase1/2 (ERK1/2) near the epicenter of injury was examined by Western blotting with quantification using an image analysis system. Lesion volumes decreased and GFAP and p-ERK1/2 expression increased with increasing magnetic stimulation frequency (0-10 Hz). MAP-2 expression was not affected at any frequency. Pretreatment with U0126 reduced GFAP and ERK1/2 expression and increased lesion volumes in response to stimulation at 10 Hz. It is concluded that magnetic stimulation increases the migration of astrocytes to spinal cord lesions. Activation of the ERK1/2 signaling pathway is proposed to mediate astrocyte migration and glial scar formation in response to spinal cord injury.

The Effect of the Transcriptional Regulation of Sp1 for TGF-beta1 and CTGF Expression in Scar Formation

This study is to examine the relationship between TGF-b1 expression and CTGF expression, and to evaluate the effect of Sp1 blockade on the expression of TGF-b1, CTGF and extracellular genes, clones of fibroblasts stably transfected with Sp1 decoy ODN. R-Sp1 decoy ODN was highly resistant to degradation by nucleases or serum, compared to the linear or phosphorothioated-Sp1 decoy ODN. Skin wounds were created on the back of 36 anesthetized rats. They were divided into four groups-the rats with normal skin, with wounded skin without decoy, with wounded skin injected with R-Sp1 decoy, and with wounded skin injected with mismatched R-Sp1 decoy, respectively. Skins were collected at 3rd, 5th, 7th, 14th day after wounding. Cellular RNA was extracted by RT-PCR analysis. TGF-beta1 and CTGF were deeply related with skin fibrosis during scar formation and it appeared that TGF-beta1 may cause the induction of CTGF expression. R-Sp1 decoy ODN inhibited TGF-beta1 and CTGF expression both in cultured fibroblasts and in the skin of rats. These results indicate that targeting Sp1 with R-type decoy efficiently blocks extracellular matrix gene expression, and suggest an important new therapeutic approach to control the scarring in normal wound healing and fibrotic disorders.

Preventive Effect of Anti-adhesion Barrier Gel for Peridural Fibrosis in Rat Laminectomy Model

OBJECTIVE: The authors compare peridural scar formation and adhesion with and without the use of Antiadhesion Barrier Gel(Adba) in an animal model of laminectomy. METHODS: Forty-five Sprague-Dawley rats underwent a two level lumbar laminectomy. The Adba was applied to randomly assigned 30 rats around the dura. Remaining 15 rats underwent same operation without the use of Adba. The rats were sacrificed 2, 4, 8 weeks after surgery by 15 numbers. A gross anatomic assessment of scar formation was done using microdissection by an observer blinded to treatment. Amount of scar formation and tenacity were compared between experimental and control group by a numerical rating system. The histological comparing was also performed. RESULTS: The amount of scar tissue and tenacity were reduced grossly and histologically at postoperative 2, 4, 8 weeks in animal model using Adba. Adba material was absorbed around 4 weeks of postoperative period in model. No special inflammatory reaction was observed, and the healing of wound was not affected by Adba. CONCLUSION: Adba significantly reduces the amount of scar formation and tenacity in rat laminectomy model without impacting the healing of operation wound and other complications.

The fetal wound healing: a review

Fetal wound healing has drawn the attention of many researchers from diverse background and specialties. Fetal wound healing is unique and differs from postnatal healing in that fetal skin wounds heal rapidly without scar formation. If the mechanism underlying such phenomenon can be elucidated, it will be serve as a significant milestone in the study of wound healing. Furthermore, the implications for therapeutic applications in wound management and in diseases where scarring is the basic pathogenetic mechanism would be immense. Rather than to list the results and conflicting data of numerous studies, this article hopes to provide a general overview of the recent developments.

Effects of decorin, TGF-beta antagonist on wound healing of rats

It is known that TGF-beta induces scar in fetal wound healing. The fact gives us that inhibition of TGF-beta can reduce scar formation. It has been reported that neutralizing antibody of TGF-beta reduced scar in rat incisional wounds. Meanwhile decorin, which is main proteoglycan of extracellular matrix, has been known as other antagonist against TGF-beta. However there has been no report about effects of decorin on scar formation. This study examined the histologic findings and width of incisional wound of rat, which was treated with decorin, compsring with non treated wound. We found that scar width was narrower in wounds 2 and 8 weeks after incision and the amount of collagen fiber is less in wounds treated with decorin than in control group. The collagen fibers, especially in wound 8 weeks after incision, were thick and regularly arranged and similar to no dermis in wounds treated with decorin. These results suggest that decorin reduces scar formation and facilitates maturation in wound healing. Even though this study cannot confirm its mechanism, the effect of decorin might be due to inhibition of TGF-beta.