Evaluation of nintedanib as a new postoperative antiscarring agent in experimental extraocular muscle surgery

INTRODUCTION

As a consequence of the immunostaining, it was found that TGF-β expression,the marker of cell proliferation and differentiation, in subconjunctival area was highest in sham group compared to control, 1, 5, and 10 μmol nintedanib and TA groups (

=0.004,0.016, 0.030, 0.004, and 0.004 respectively). TA group hadhigher H-scores compared to 5-10 μmol nintedanib groups(

=0.046, and 0.043 respectively). The 1 μmol nintedanib group had higher H-scores compared to 5-10 μmol nintedanib groups (

=0.036, and 0.016 respectively). There was no statistically significant difference between 1 μmol nintedanib group and TA group (

=0.517). Additionally, there was no statistically significant difference between 5 and 10 μmol nintedanib groups (

=0.146).

The most commonly used anti-inflammatory and antifibrotic agents in EOM surgery are steroids. Although some published studies have revealed that subconjunctival steroid injection has cytocidal effects on adjacent fibroblasts and causes breakdown of the collagen fibers

, steroids may induce significant side effects, such as cataract formation and increased intraocular pressure. Currently, the most suitable material or pharmacologic agent has not been yet identified. Therefore,there is a need for experimental researches that aim to search for new agents that are easily applicable and obtainable with negligible side effects.

It is often difficult to deal with adhesions, once formed.Today, the most common medical therapy used for antiinflammatory and antifibrotic effect is steroids. But their adequacy is questionable. Various treatment options, such as polyglactin 910 mesh

, silicone sheet

, mitomycin C

, 5-fluorouracil

, seprafilm (Genzyme, Cambridge,Massachusetts)

, bevacizumab

, amniotic membrane

,all-trans-retinoic acid

, and pirfenidon

have been used to provide the least amount of adhesion formation after EOM surgery. However, these methods have not been yet in use because of their unavailability and associated complications.Thus a new antiscar agent which leads minimal side effects is needed.

綜上所述，由于氧化石墨烯具有電子遷移率高、比表面積大、表面含氧官能團豐富等優異特質，使復合材料在有機染料的吸附光催化降解領域表現出非常優異的性能。

Nintedanib, a multiple tyrosine kinase inhibitor, selectively binds to and inhibits vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor(PDGFR) and fibroblast growth factor receptor (FGFR).Nintedanib also inhibits members of the Src family of tyrosine kinases including Lyn, Lck and Flt-3

. It is clinically used for idiopathic pulmonary fibrosis (IPF) treatment

. In pulmonary fibrosis animal model studies, nintedanib’s antifibrotic and anti-inflammatory activities by affecting fibroblast proliferation, migration, differentiation, extracellular matrix(ECM) protein secretion and fibrotic gene expression have been shown

. These data made us hypothesize that nintedanib may also show these antifibrotic effects in EOM surgery.

No ophthalmologic formula is available for nintedanib. We preferred to perform subconjunctival injection. Subconjunctival administration can provide better results because it allows direct access of the drug to the target site. However, in subconjunctival application, the drug can be rapidly eliminated due to the blood and lymphatic circulation in the conjunctiva.By preparing any colloidal form of the drug such as liposome,nanoparticle, microemulsion and nanoemulsion, the effectiveness of the drug can be achieved by applying the drug with less frequency by providing continuous and controlled release in the target area

.

MATERIALS AND METHODS

All animal experiments were handled according to the ARVO Statement guidelines for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Experiments Ethics Committee of the Kocaeli University Medical Faculty.

Thirty New Zealand rabbits, weighing 2500 to 4000 g and 15 to 20 weeks old, were purchased from Aykut Bolu Experimental Animal Production and Supply Center (Bolu, Turkey). All rabbits were acclimatized for 2wk before experiments. Rabbits were randomly allocated to one of five treatment groups: 0.9% normal saline (NS;Sham) group (

=6), TA treatment group (

=6) and three different concentrations of nintedanib-treatment groups (

=6).The right eyes were used for the experiments. As a control group, left eyes of rabbits were used (

=6). Left eyes that did not undergo any procedure were used to comparatively demonstrate the effect of surgery on histopathological and immunohistochemical parameters. Standard preoperative procedures and surgical method have been applied to all rabbits. Standard EOM surgery was performed by one surgeon.Rabbits in sham group were treated with 0.1 mL 0.9% NS by subconjunctival injection immediately after surgery and on postoperative day (POD) 1, 2, 3, 5, and 7. Rabbits in TA group were treated with 0.1 mL of 4 mg (40 mg/mL) triamcinolone(Kenacort-A, Deva, Turkey) by subconjunctival injection immediately after surgery. Rabbits in nintedanib groups received subconjunctival injection with 0.1 mL of 1, 5, or 10 micromolar (μmol) of nintedanib immediately after surgery and on POD 1, 2, 3, 5, and 7. Surgical wound appearance was recorded by photographs on the 3

day, 1

, 2

and 4

weeks after surgery.

Nintedanib 50 mg powder obtained from MedChemExpress local distributor company (Suarge, Turkey) was used. The required amount of nintedanib was weighed with precision balances (Shimadzu,Japan) and dissolved in DMSO (Dimethylsulfoxide;SantaCruz, USA) to prepare a 1 mmol stock nintedanib solution. Working solutions of 1, 5 and 10 μmol were prepared from the obtained stock solution and placed in sterile vials and carried to the surgical field.

因為關于生長痛與鈣、磷、堿性磷酸酶的關系，目前仍存在不同的看法。有人認為生長痛與血鈣、血磷的水平沒有關系，在對某學校2837 名中小學生的普查中，符合生長痛診斷的所有患兒血鈣、血磷水平都在正常范圍。

Both 5 mg/kg xylazine hydrochloride(Rompun, Bayer, Turkey) and 40 mg/kg ketamine hydrochloride (Keta Control, Doga Pharma, Turkey) were administered intramuscularly for general anesthesia. For topical anesthesia, 0.5% proparacaine hydrochloride drops were used (Alcaine, Novartis, Switzerland).

（二）主產區分布情況 出以巷口鎮、火爐鎮、白馬鎮和長壩鎮等鄉鎮為代表的部分鄉鎮雖然肉牛養殖的絕對數量不低，但是單位面積或者單位農業人口的產出量很少，而以文復鄉、桐梓鎮、接龍鄉、后坪鄉和雙河鄉等鄉鎮不僅絕對數量較高，其平均產出量同樣很高，究其原因有二：其一這些鄉鎮的大部分地區海拔高度高于800米，適宜肉牛生長，其二飼草飼料資源比較豐富。山羊的分布同樣如此。

Corticosteroids are a frequently used drug group because of their anti-inflammatory, antiallergic and immunosuppressive effects on almost every organ. Steroids inhibit angiogenesis,proliferation and migration and delay the inflammatory phase and wound healing accordingly. TA, steroid with a long half life, has a wide therapeutic range due to its anti-inflammatory and immunosuppressive properties. TA has been shown to inhibit fibroblast proliferation and collagen synthesis. In this way, it alters wound healing

.

All rabbits were euthanized on the postoperative 28

day. After the surgical sites were excised,SRM and surrounding tissue were put in formalin solution and fixed by immersion method for 48h. After washing process in running tap water, the tissues were passed throughgradually increasing series of ethyl alcohol (70%, 90%, 96%,100% respectively; Merck, Germany) and dehydration was performed. After 30min of transparency with toluene (Merck,Germany), the tissues were kept in pure paraffin for 2h and embedded in paraffin blocks at room temperature. Serial cuts of 4 μm thickness were taken from the paraffin blocks with a microtome (Leica SM 2000R, Germany). Staining of the sections was started from the insertion of SRM.Haematoxylin eosin (H&E) staining was used to assess the presence of conjunctival vascularity, scleral and conjunctival inflammation. Demonstration of collagen fibers and grading of SRM and perimuscular fibrosis as well as the amount of scar tissue formation between muscle and sclera (adhesion) were performed by Masson’s trichrome (MT) staining. Parameters for histopathologic evaluation are listed in Table 1.

The 4-μm sections taken from paraffin blocks on polylyzed slides were kept in a 56°C oven for 1 night to deparaffin, and then they were kept in toluene for 3 times for 5min and thoroughly cleared of paraffin.Then, it was kept in alcohol for 2×5min at 100

, 1×5min at 96

,1×5min at 90

, 1×5min at 70

alcohol and finally in distilled water for 2×5min. The sections kept in phosphate buffered saline (PBS) solution for 5min, placed in citric acid solution for antigen retrieval, boiled for 10min in the microwave and kept for 20min to cool. Then, protein block solution (ab64264,Abcam) was applied for 10min to block non-specific antibody binding to the sections washed in PBS. Matrix metalloproteinase-2 (MMP-2; ab2462, Abcam, 1:200 dilution ratio), transforming growth factor-β (TGF-β; ab190503,Abcam, 1:200) and alpha-smooth muscle actin (α-SMA;ab7817, Abcam, 1/50 dilution rate) primary antibodies were dropped and kept at +4°C for one night/overnight. Tissue sections washed with PBS were incubated with biotiny goat antipolyvalent solution (ab64264, Abcam) for 10min. After 10min of incubation, the sections with MMP-2, TGF-β and α-SMA expressions were observed in brown color with the chromogen called diaminobenzidine. Tissues that were counterstained with Mayer hematoxylin (ab128990, Abcam) were taken into toluene after dehydration (passing through the residual alcohol series) and covered with Entellan (Merck, Germany) with a coverslip. For immunohistochemical examination, MMP-2,TGF-β, and α-SMA expressions were evaluated

.

The data was statistically analyzed by using the software SPSS 26.0. For descriptive statistics of histopathologic examination data, ratio and frequency values were used. Chi-squared (

) test was performed to analyze qualitative independent data. When

test conditions were not met, Fischer test was used. The

test was used to compare the data of conjunctival vascularity, conjunctival and scleral inflammation, as well as of adhesion, SRM and perimuscular fibrosis. For descriptive statistics of immunohistochemical examination data, the lowest and highest values of mean,standard deviation and median, frequency and ratio values were used. Distribution of variables was measured with Kolmogorov-Simirnov test. For analysis of quantitative independent data, Kruskal-Wallis and Mann-Whitney

tests were used. Values of

＜0.05 indicated statistical significance.

RESULTS

All rabbits appeared to be healthy and ate normally. For early postoperative days, conjunctival hyperemia was observed in many surgical sites (Figure 1). There was no evidence of systemic toxicity in any animal.

Five treatment groups showed conjunctival inflammatory cell infiltration in the histological examination of H&E stained sections. Sham group had significantly higher conjunctival inflammation compared to 1, 5, and 10 μmol nintedanib and TA groups (

=0.015, 0.015,0.002, and 0.002 respectively). Scleral inflammation had statistically significant difference in sham group compared to control, 1, 5, and 10 μmol nintedanib and TA groups (

=0.015,0.015, 0.015, 0.015, and 0.015 respectively). Conjunctival vascularity was significantly increased in sham group compared to control and 10 μmol nintedanib groups (

=0.002 and 0.015 respectively). 10 μmol nintedanib group showed statistically significant difference in reducing conjunctival vascularity compared to sham group (

=0.015; Table 2 and Figure 2).

Five to ten μmol nintedanib and TA groups showed significantly less intense perimuscular collagen staining compared to sham group (

=0.015, 0.002, and 0.002 respectively).

Only 10 μmol nintedanib group had statistically significant difference in reducing rectus muscle fibrosis compared to sham group (

=0.015). Adhesion between SRM and sclera was significantly increased in sham group compared to control group (

=0.002). Neither 1, 5, and 10 μmol nintedanib groups nor TA group differed statistically from sham group with regard to adhesion (

=0.182, 0.061, 0.061, and 0.182 respectively; Table 3 and Figure 3).

Nintedanib appears to attenuate postoperative inflammation and fibrosis after extraocular muscle surgery. Nintedanib may be a safer and stronger alternative agent in extraocular muscle surgery when compared to steroids. Further investigation is needed to prove antiadhesive effect of nintedanib.

Main factors that affect success of extraocular muscle(EOM) surgery are inflammation, fibrosis and adhesion formation. Fibrosis, resulting from chronic inflammation, is the replacement of normal tissues by connective tissue and leads to contracture of muscle. Adhesion, on the other hand,is the attachment between muscle and surrounding tissues that leads to restriction of the muscle

. Therefore, wound repair with minimal scar formation is important for obtaining normal function and structure of injured tissues.

To assess the degree of transdifferentiation to myofibroblasts,we performed immunohistochemical staining for α-SMA.Sham group had significantly higher values compared to 5-10 μmol nintedanib groups (

=0.01 and 0.004 respectively).TA group had significantly higher values compared to 10 μmol nintedanib group (

=0.01). Additionally, 1-5 μmol nintedanib groups had significantly higher values compared to 10 μmol nintedanib group (

=0.008 and 0.090 respectively).

Expression of MMP-2, the marker of fibroblast cell density,was significantly higher in subconjunctival tissue of sham group compared to 5-10 μmol nintedanib and TA groups(

=0.008, 0.004, and 0.045 respectively). The 1 μmol nintedanib group had significantly higher values compared to 5-10μmol nintedanib groups (

=0.043 and 0.010 respectively).Additionally, 5 μmol nintedanib group had significantly higher values compared to 10 μmol nintedanib group (

=0.044).There was no statistically significant difference between 1 μmol nintedanib and TA groups (

=0.337).

The statistical analysis showed that the H-scores of TGF-β expression in SRM were significantly higher in sham group compared to 1, 5, and 10 μmol nintedanib and TA groups (

=0.016, 0.004, 0.004,and

=0.048 respectively). It was determined that 1, 5, and 10 μmol nintedanib and TA groups revealed similar TGF-β expressions within SRM (

＞0.05).

Expression of α-SMA was significantly higher in SRM of sham group compared to 1, 5, and 10 μmol nintedanib and TA groups (

=0.036, 0.010, 0.010, and 0.030 respectively). Three nintedanib and TA groups revealed similar α-SMA expressions within SRM (

＞0.05).

由于移植的脂肪組織術后會出現一定程度的吸收，因此單純自體脂肪注射隆乳可能需要多次操作才能達到理想的胸部外形。移植脂肪的體積大多移植到3～6個月時才能達到穩定，因此再次手術實際也常選擇在此期間[27]。而在移植脂肪時選擇加入ADSC或ADSC+PRP，一次填充有效率則會明顯上升，再次填充的概率及再次填充的次數也會相應降低[28]。

The results of the immunostaining for MMP-2 in SRM revealed that positivity was significantly higher in sham group compared to 1, 5, and 10 μmol nintedanib and TA groups (

=0.004, 0.020, 0.004, and 0.004 respectively). When compared to 5-10 μmol nintedanib groups, TA group had statistically increased MMP-2 expression in SRM (

=0.020 and 0.004 respectively). One μmol nintedanib group showed statistically increased expression compared to 10 μmol nintedanib group (

=0.036). There was no statistically significant difference between 1 μmol nintedanib and TA group (

=0.808).Additionally, there was no statistically significant difference between 5 and 10 μmol nintedanib groups (

=0.374).

DISCUSSION

It is possible to see some restrictions in ocular motility after EOM surgery. Various reasons may be responsible for surgical failure. The most challenging one is believed to be the postoperative scar tissue and adhesion formation involving EOM, Tenon’s capsule, intermuscular membrane and sometimes orbital fat tissue.

由表5可知，試驗段瀝青路面平整度指標檢測結果均能夠滿足規范要求的不大于3mm標準，試驗段瀝青路面施工完成時路面平整度良好。……