Backgound: To identify and compare specific protein levels between overacting inferior oblique (IO) muscles in superior oblique (SO) palsy patients and normal IO muscles. Methods: We obtained 20 IO muscle samples from SO palsy patients with IO overaction ≥ + 3 who underwent IO myectomies (IOOA group), and 20 IO samples from brain death donors whose IO had functioned normally, according to their ophthalmological chart review (control group). We used MyoD for identifying satellite cell activation, insulin-like growth factor binding protein 5 (IGFBP5) for IGF effects, thioredoxin for oxidative stress, and p27 for satellite cell activation or oxidative stress in both groups. Using immunohistochemistry and Western blot, we compared expression levels of the four proteins (MyoD, IGFBP5, thioredoxin, and p27). Results: Levels of thioredoxin and p27 were decreased significantly in the IOOA group. MyoD and IGFBP5 levels showed no significant difference between the groups. Conclusions: Based on these findings, the overacting IOs of patients with SO palsy had been under oxidative stress status versus normal IOs. Pathologically overacting extraocular muscles may have an increased risk of oxidative stress compared with normal extraocular muscles. Keywords: Oxidative stress, Inferior oblique muscle overaction, Superior oblique palsy, Thioredoxin, p27 Background circumference. However, little is known about the mo- Inferior oblique (IO) muscle overaction may occur as a lecular and microscopic differences between overacting primary condition or develop secondarily to specific and normal IOs. events such as superior oblique (SO) palsy. When myec- Extraocular muscles have different metabolic and tomies were performed to weaken the IOs, some over- structural components compared with other skeletal acting IOs were very bulky but others were of apparently muscles, both molecularly and microscopically . How- normal size. A previous study using magnetic resonance ever, both extraocular muscles and other skeletal mus- imaging (MRI) demonstrated that IO belly diameter in- cles generate free radicals with repetitive contraction, creased on upward gaze nearly equally between patients which can result in cellular oxidative damage in severe with and without inferior oblique overaction (IOOA) . or prolonged state [4–6]. Myocytes generally contain a Bagheri et al.  reported that there was no detectable network of antioxidant defense mechanisms to reduce correlation between IOOA and muscle position or the risk of oxidative damage against increased reactive oxygen species . Prolonged oxidative stress can result in reduced antioxidant capacity in extraocular muscles, * Correspondence: firstname.lastname@example.org and a previous report revealed that the medial rectus Department of Ophthalmology & Visual Science, College of Medicine, Seoul muscles (MRM) of patients with exotropia had a redox St. Mary’s Hospital, The Catholic University of Korea, Banpo-daero 222, Seocho-gu, Seoul 06591, Republic of Korea imbalance status compared with normal MRMs . Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chung et al. BMC Ophthalmology (2018) 18:128 Page 2 of 6 No previously reported study has compared extraocu- group were obtained from age-matched donor eyes of lar muscles under continuously contracting conditions, individuals within 12 h after the brain death. Ethics, as seen in IOOA due to SO palsy and normal extraocu- consent, permissions and approval were obtained with lar muscles. Thus, this study was undertaken to investi- written documents by their guardians based on each gate and identify any difference between pathologically donor’s intension of eyeball donation before brain overacting and normal IOs at the protein level using death. Any subject who had history of eyelid or MyoD, IGFBP5, thioredoxin, and p27 as example pro- extraocular muscle surgery or disease, orbital diseases, teins. The brief introductions of each protein are sum- and eyeball or periorbital trauma by their medical re- marized as follows. cords was excluded. MyoD, which is located in a specialized niche between All IOs were transferred to a portable tank filled with the myofiber sarcolemma and the the basal lamina, is an liquid nitrogen and immediately delivered from the op- essential protein for satellite cell differentiation. Satellite eration room to the laboratory. Before preservation at − cells represent 2–10% of total myonuclei [7, 8]. They are 80 °C, portions of secured IOs were separated and pro- known to be. Satellite cells become activated and express teins for analysis were extracted within 1 h from secur- the myogenic regulatory factors Myf5 and/or MyoD fol- ing IOs. lowing injury or growth stimulus, proliferate and gener- We selected four proteins for investigation; MyoD, ate the myogenic progenitors which are needed for insulin-like growth factor (IGF) binding protein 5 muscle regeneration [9–11] or become new muscle fi- (IGFBP5), thioredoxin, and p27. bers. Thus, we can identify in which group the satellite For immunohistochemistry, we first prepared cell is activated by measuring and comparing MyoD formalin-fixed, paraffin-embedded tissue sections. Then levels. the sections were deparaffinized with xylene and trans- IGF is known to induce hypertrophy in cultured neo- ferred to 100, 95, 70, and 50% alcohols in sequence, natal rat cardiomyocytes through a specific receptor twice each for 3 min. For blocking of non-specific bind- . IGFBP binds to IGF and regulates its half-life. ing activity, each slide was incubated in 1% bovine Thus, IGFBP5 detected in extraocular muscles can help serum albumin at room temperature for 10 min. After us to determine whether IGF has any influence on over- removing the blocking buffer, 100 μL of appropriately di- acting or normal IOs. luted primary antibodies (MyoD, IGFBP5 and p27; Santa Thioredoxin decreases, as an antioxidant, in an oxida- Cruz Biotechnology, Santa Cruz, CA, USA, thioredoxin; tive stress state [13, 14]. If IOOA is caused by oxidative Abcam, Cambridge, London, UK) were applied to the stress, thioredoxin would be expected to decrease sig- sections on the slides and the slides were incubated in a nificantly in the IOOA group versus the control group. humidified chamber at 4 °C for overnight. After washing P27 maintains or arrests the quiescent phase in the with PBS for 30 min, secondary antibody (rhodamine-- cell cycle, and decreases before cell division in stem cells conjugated, Santa Cruz Biotechnology, Santa Cruz, CA, like the satellite cells . Recently, p27 was shown to USA) was applied for 1 h. After washing, the sections play a role in an oxidative stress state [16, 17]. If IOOA were stained with Hoechest 33,258 (Sigma-Aldrich, St. is caused by hyperplasia or oxidative stress, a difference Louis, MO, USA). Then, a cover slip was mounted on in p27 levels between the groups, in conjunction with a the slide glass using mounting solution. The stained tis- difference in MyoD or thioredoxin level, would be sues were observed by fluorescence microscopy (Axio expected. imager 2, Carl Zeiss GmBH, Zena, Germany). Western blotting was performed to compare levels Methods of the protein, MyoD, IGFBP, thioredoxin, and p27 The IOOA group consisted of 20 IOs obtained from protein levels between the two groups. Total protein patients with secondary IOOA ≥ +3 from SO palsy. wasisolatedfromthe IOsusing RIPA buffer (25 mM A portion of the IO (8.0 mm from the insertion) was Tris-HCl, pH 7.4, 1% Tween 20, 0.1% SDS, 0.5% so- resected during IO myectomy surgery. Approval to dium deoxycholate, 10% glycerol, 150 mM NaCl, conduct this study was obtained from the Institu- 5mMEDTA, 1mMPMSF, 50 mMNaF, 1 mM tional Review Board of the Catholic Medical Center Na VO ,and 1 μg/mL of aprotinin, leupeptin, and 3 4 (#KC09TISI0365). Ethics, consent, permissions and pepstatin) and protein concentration was determined approval were obtained with written documents by all using a BCA protein assay kit (Pierce, Rockford, IL, participants prior to surgery. Approval to conduct USA). After separating 20 μg of protein using 10% and securing human tissue of this study was obtained sodium dodecyl sulfate polyacrylamide gel electro- from the institutional review board of the hospital phoresis, it was transferred onto polyvinylidene and the study protocol adhered to the tenets of the difluoride membranes (Amersham Life Science, Cleve- Declaration of Helsinki. 20 normal IOs as the control land, OH, USA) with approximately 5% skim milk in Chung et al. BMC Ophthalmology (2018) 18:128 Page 3 of 6 Table 1 Demographic data of the IOOA and control group IOOA Control P value* Number of patients (number of eyes) 20 (20) 10 (20) Age, years (SD) 52.35 (11.43) 55.25 (9.68) 0.438 Gender (M:F) 13:7 6:4 0.282 Deviation, prism diopters (SD) 26.2 (11.8) *Mann-Whitney test PBS-0.1% Tween 20 for blocking. Then, the primary value < 0.05 was considered to indicate statistical antibodies (MyoD, IGFBP5 and p27; Santa Cruz Bio- significance. technology, Santa Cruz, CA, USA, and thioredoxin; Abcam, Cambridge, London, UK) were reacted overnight in a 4 °C cold room, and the membrane was washed and Results reacted with horseradish peroxidase-conjugated goat Twenty overacting IOs from 20 patients with SO anti-rabbit antibody (1:1000; Santa Cruz Biotechnology, palsy were obtained as the IOOA group (13 males, 7 Santa Cruz, CA, USA) and was developed with an en- females; mean age = 55.25 years). 20 IOs were ob- hanced chemiluminescence solution (Santa Cruz Biotech- tained as the control group (6 males, 4 females; mean nology). Finally, bands on the X-ray film were quantitated age = 55.25 years). There was no significant difference by densitometry with β-actin as the protein loading in gender or age between the two groups (Table 1). control. In immunohistochemistry, few fluorescent dots Using densitometry (Image Master VDS 2.0; Pharma- representing MyoD and IGFBP5 were detected little cia Biotech Inc., San Francisco, CA, USA), we obtained in either groups (Fig. 1). However, many fluorescent the optical density of each protein band and divided dots for thioredoxin and p27 were detected in the them by the optical density of actin to obtain the relative control group but few dots were found out in the optical density of each protein. The relative optical dens- IOOA group (Fig. 2). ity of each protein was then compared between the On Western blotting and densitometry analysis, the IOOA and control groups. levels of MyoD and IGFBP5 did not show a statistically Statistical analyses were performed using SPSS statis- significant difference (P > 0.05). However, the levels of tical software for Windows (SPSS, version 20.0, Chicago, thioredoxin and p27 were significantly higher in the con- IL, USA). Student’s t-test and the Mann-Whitney U test trol group than in the IOOA group in optical density were used to evaluate differences between groups. A P (P < 0.001, respectively; Fig. 3). Fig. 1 Immunohistochemistry results. MyoD (a) inferior oblique overaction (IOOA) group, (b) control group, insulin-like growth factor binding protein 5 (IGFBP5) (c) IOOA group, (d) control group Chung et al. BMC Ophthalmology (2018) 18:128 Page 4 of 6 Fig. 2 Immunohistochemistry results. Thioredoxin (a) IOOA group, (b) control group, p27 (c) IOOA group, (d) control group Discussion extraocular muscles and there was a decrease in Oxidative stress was defined as “a disruption of redox sig- anti-oxidative capacity in the IOOA group. naling and control” by Jones . There are several A recent study showed another role for thioredoxin, in methods for measuring oxidative stress in muscles and that its downregulation resulted in the induction of thioredoxin is a known antioxidant and an oxidative stress apoptosis  and its overexpression inhibited tumor biomarker [14, 19]. To our knowledge, there is no previ- necrosis factor-alpha (TNF-α) induced apoptosis,  ous report about thioredoxin levels in extraocular muscles. indicating an anti-apoptotic role for thioredoxin. How- In the present study, thioredoxin was detected in the con- ever, in our study, the actin levels between the IOOA trol group and was decreased significantly in the IOOA and the control groups did not show a significant differ- group. This indicates that thioredoxin is an antioxidant in ence, indicating that there was no significant muscle Fig. 3 Western blot analyses of MyoD, IGFBP5, thioredoxin and p27. a Samples in the IOOA group are in lanes 1 to 2, and those in the control group are in lanes 3 to 4. b Levels of each protein in the IOOA group, compared with those in the control group. MyoD and IGFBP5 showed no statistical difference between the groups. However, thioredoxin and p27 were decreased significantly in the IOOA group versus the control group Chung et al. BMC Ophthalmology (2018) 18:128 Page 5 of 6 fiber loss in the IOOA group. Thus, we consider the de- Availability of data and materials The datasets used and analyzed during the current study are available from creased level of thioredoxin in the IOOA group to be the corresponding author on reasonable request. the result of the oxidative stress. The decrease in p27 in the IOOA group, in conjunction Authors’ contributions YWC: Analysis and interpretation of data, draft and modification of with the decrease in thioredoxin, may also be evidence of manuscript. JSC: Experiment and data collection. SYS: Conception and design oxidative stress. Sherr and Roberts reported that (selection of patients and proteins to be analyzed), Surgery and obtaining downregulation of p27 in a particular condition suggested parts of inferior oblique muscles, final approval of manuscript. All authors read and approved the final manuscript. the presence of other mechanisms in regulating p27 levels, distinguishing it from cellular apoptosis. Lesley et al.  Ethics approval and consent to participate revealed that hydrogen peroxide and the antioxidant del- Approval to conduct this study was obtained from the Institutional Review Board of the Catholic Medical Center (CMC) (#KC09TISI0365). Ethics, consent, phinidin seemed to regulate intracellular levels of p27 permissions and approval were obtained with written documents by all through regulating HIF-1 levels, which were, in turn, gov- participants prior to surgery. The methods for securing human tissue erned by its upstream regulators, involving the PI3K/Akt/ complied with the tenets of the Declaration of Helsinki. mTOR signaling pathway. These findings support the hy- Competing interests pothesis that oxidative stress and antioxidant regulate p27 The authors declare that they have no competing interests. by affecting PI3K-Akt signaling pathway. Satellite cell activation, and the effects of intrinsic Publisher’sNote IGF on the IOs, were investigated through the levels Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. of MyoD and IGFBP5 in the groups. However, they may have no or little effect in both the overacting Author details and normal IOs although further study are needed to Department of Ophthalmology, College of Medicine, St. Vincent’s Hospital, The Catholic University of Korea, Suwon, Republic of Korea. Department of confirm this. Ophthalmology & Visual Science, College of Medicine, Seoul St. Mary’s This study had some limitations. First, the IOs ob- Hospital, The Catholic University of Korea, Banpo-daero 222, Seocho-gu, tained from the IOOA group represented only a portion Seoul 06591, Republic of Korea. of the total IO length. Thus, the antioxidant capacity in Received: 22 December 2017 Accepted: 22 May 2018 the present study may not have represented the total capacity of IOs. To overcome this limitation, we in- References cluded a similar length of the IOs from donor eyes. Sec- 1. Kono R, Demer JL. 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Published: May 30, 2018
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