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UMSC-derived exosomes promote retinal ganglion cells survival in a rat model of optic nerve crush
Journal of Chemical Neuroanatomy 96 (2019) 134–139
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UMSC-derived exosomes promote retinal ganglion cells survival in a rat model of optic nerve crush
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Dongyan Pana,c,d,g,1, Xin Changd,g,1, Mengqiao Xub,e,f,1, Mingke Zhangd, Shoumei Zhangc, ⁎ ⁎ ⁎ Yue Wangd,g, Xueting Luob,e,f, , Jiajun Xuc, , Xiangqun Yangc, , Xiaodong Sunb,e,f a
Department of Ophthalmology, Changhai Hospital, Second Military Medical University School of Medicine, Shanghai, China Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University School of Medicine, Shanghai, China c Department of Anatomy, Second Military Medical University School of Medicine, Shanghai, China d Department of Histology and Embryology, Second Military Medical University School of Medicine, Shanghai, China e Shanghai Key Laboratory of Fundus Diseases, Shanghai, China f Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, China g Shanghai Key Laboratory of Cell Engineering, Shanghai, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Mesenchymal cells Wharton’s jelly Exosomes Optic nerve crush Retinal ganglion cells Glia cells
Traumatic optic neuropathy or glaucoma lead to retinal ganglion cells loss and cause blindness, and there is no effective therapy strategy by far. Mesenchymal cells from the Wharton’s jelly of the umbilical cord (umbilical cord mesenchymal stem cells, UMSCs) and UMSC-derived exosomes (UMSC-Exos) are promising candidates for allogeneic therapy in regenerative medicine, but their effort on optic nerve injury and the underlying mechanism remains undefined. In the present study, we investigated the functions of UMSC-Exos in a rat optic nerve crush (ONC) model. After three times of treatments with an interval of one week, we found that the UMSC-Exos significantly promoted Brn3a+ retinal ganglion cells (RGCs) survival in retinal ganglion cell layer compared with PBS controls. UMSC-Exos also significantly promoted GFAP+ glia cells activation in retina and optic nerve. However, no increase of GAP43+ axon counts in the optic nerve was found after UMSC-Exos treatment. Thus, our results demonstrate that UMSC-derived exosomes may play a role in neuroprotection by promoting the RGCs survival and glia cells activation but not the axon regeneration.
1. Introduction Traumatic optic neuropathy or glaucoma lead to retinal ganglion cells (RGCs) loss and can cause inevitable blindness. Mesenchymal stem cells (MSCs) are multipotent stromal cells isolated from mesenchymal tissues including bone marrow(BM), (Campagnoli et al., 2001)adipose tissue (Rodriguez et al., 2005), circulating blood (Campagnoli et al., 2001), dental pulp (Pierdomenico et al., 2005), placenta (Fukuchi et al., 2004), amniotic fluid (You et al., 2008), umbilical cord blood (Gang et al., 2004), and umbilical cord Wharton’s jelly (Wang and Peng, 2004). MSCs have demonstrated therapeutic efficacy at protection and regeneration of central nervous system (CNS) neurons, including retinal ganglion cells (RGCs) (Chen et al., 2013; Chung et al., 2016; Cox et al., 2011; Johnson et al., 2010; Mead et al., 2013; Nichols et al., 2013). But the mechanism of MSCs’ neuroprotection is still poorly understood. MSCs transplanted into the vitreous showed no differentiation or
migration/integration into retinal tissue, implicating paracrine over cell replacement is the dominant mechanism (Johnson et al., 2010; Mead et al., 2016, 2013). Exosomes have been identified as a new kind of major paracrine factor released by various types of cells, including MSCs. Exosomes are a type of membrane vesicle with diameters of 40–150 nm containing proteins, mRNA and miRNA. They have been reported to be an important mediator of cell-to-cell communication (Lener et al., 2015). Newly studies demonstrate that MSCs-derived exosomes can reduce neuroinflammation, promote neurogenesis, and improve functional recovery in animal models (Kim et al., 2016; Zhang et al., 2015, 2017a). The neuroprotective and neurogenesis effects of MSCs-derived exosomes might be due to miRNA (Johnson et al., 2014; Mead and Tomarev, 2017). These reports indicate that MSCs-derived exosomes may promise to be a better therapy for neurodiseases than MSCs. Mesenchymal cells from the Wharton’s jelly of the umbilical cord
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Corresponding authors. E-mail addresses: [email protected] (X. Luo), [email protected] (J. Xu), [email protected] (X. Yang). 1 These authors contribute equally to this work. https://doi.org/10.1016/j.jchemneu.2019.01.006 Received 24 October 2018; Received in revised form 12 December 2018; Accepted 9 January 2019 Available online 10 January 2019 0891-0618/ © 2019 Elsevier B.V. All rights reserved.
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FL) to expose the optic nerve. The optic nerve was clamped with an aneurysm clip (mini clip, temporary, Aesculap, Germany) 2 mm posterior to the lamina cribosa for 8 s without damaging any small vessels. Rats were kept warm until fully awake. Deliver of exosomes was done as previously described (Mead and Tomarev, 2017). In the aid of a medical head-mounted microscope with cold light illuminator(x2.5, HAOLANG MEDICAL LIGHTING CO., LTD,Nanjing, China), a 5 μl volume of PBS loaded with 1 × 109 exosomes was injected slowly with a 33 g Hamilton syringe (Hamilton Company, Beltsville, MD, http://www.hamiltoncompany.com/) into the vitreous just posterior to the limbus. The needle was retracted after 2 min to minimize backflow.
(umbilical cord mesenchymal stem cells, UMSCs) possess stem cell properties and have broad differentiation potential, low immunogenicity, no ethical issues due to noninvasive collection (Batsali et al., 2013; Gang et al., 2004) and were efficient to obtain, suggesting that UMSCs exosomes are promising candidates for allogeneic therapy in regenerative medicine (Drela et al., 2016). In the present study, we investigated the function of UMSCs exosomes in a rat optic nerve crush (ONC) model. We found that UMSCderived exosomes could promote the RGCs survival but not the axon regeneration. 2. Materials and methods 2.1. Culture of human UMSCs and isolation of exosomes
2.5. Exosome tracking
Umbilical cords were obtained from the Changhai Hospital affiliated to the Second Military Medical University of China with medical informed consent. The collection and use of human biological specimens were approved by the Ethics Committee of the Changhai Hospital. Primary culture of UMSCs and exosome isolation were performed on standard procedures as previously reported (Fang et al., 2016; Zhang et al., 2017b). In briefly, umbilical cords were washed with 70% ethanol and subsequently excess blood was removed with sequential washes in DMEM (DMEM; Hyclone, Massachusetts, USA). Tissue were minced into small pieces (2 × 2 mm) and incubated with medium in small dishes at 37 °C. Only UMSCs in passages 2–5 were used. Collected cell culture suspension about 200 ml was transferred to conical tubes for centrifugation at 300 g for 10 min at 4 °C and was again centrifuged at 16,500 g for 20 min at 4 °C to further remove cell debris. After filtered through a 0.22-μm filter, the flow was transferred to new tubes and then ultracentrifuged again at 120,000 g for 70 min at 4 °C to pellet the exosomes. For maximal exosome retrieval, the exosome-enriched pellet was resuspended in a small volume (approximately 200 μl) with PBS. The presence of exosomes was confirmed by electron microscopy, and exosomes were characterized by using NANO Gold. (iZON® Science, New Zealand). Detection of exosomal surface markers CD81 and CD63 was performed by using Western blotting.
To track exosomes in vivo, the green lipophilic fluorescent dye PKH67 (System Biosciences) was used to label purified exosomes prior to intravitreal injection, according to the manufacturer’s instructions. Exosomes were labeled PKH67 for 5 min and the reaction was stopped by the addition of exosome-depleted FBS. Exosomes were then centrifugated for 70 min at 120,000 g, washed three times with PBS before being resuspended in 1 ml of PBS, and kept on ice until intravitreal injection on the same day. 24 h after PKH67 labeled exosomes were injected into the ONC rats’ vitreous body. Animals were euthanized by over dosage of 10% chloral hydrate and received extraction of retina. Fluorescent labeling was examined in frozen sections of retina under a confocal microscopy and images were captured at 1024 × 1024 pixel resolution.
2.6. Tissue preparation At day 21 post-ONC, animals were sacrificed and perfused intracardially with 4% paraformaldehyde (PFA) in PBS. Eyes and optic nerves were removed and immersion fixed in 4% PFA in PBS for 24 h at 4℃ then in 10, 20, and 30% sucrose solution in PBS for 24 h at 4℃. Eyes and optic nerves were then embedded using optimal cutting temperature embedding medium (SAKURA Tissue-Tek® O.C.T. Compound) by rapid freezing under crushed dry ice and were stored at -80℃. After embedding, eyes and optic nerves were sectioned on a CM1950 cryostat microtome (Leica Microsystems Inc, Bannockburn, IL, http://www. leica-microsystems.com) at −22 ℃ at a thickness of 20 μm and 15 μm. Longitudinal optic nerve and parasagittal eye sections were stored at 22℃ before immunocytochemistry. To ensure RGC counts were done in the same plane, eye sections were chosen with the optic nerve head visible (each eye six sections).
2.2. Animals Adult male Wistar rats weighing 160–200 g (Experimental animal center of the Second Military Medical University, Shanghai, China)were maintained in accordance with the Guide for the Care and Use of Laboratory Animals recommended by the National Institutes of Health and approved by the Ethics Committee for Animal Experimentation of the Second Military Medical University. Animals were kept at 21℃ and 55% humidity under a 12 h light and dark cycle, given food/water and were under constant supervision. Animals were euthanized by over dosage of 10% chloral hydrate before extraction of retina.
2.7. Immunofluorescence analysis Tissues were washed in PBS (3 x 5 min) and then incubated in blocking solution (5% donkey serum, 0.5% Triton X-100, and 1% bovine serum albumin in PBS) for 1 h at room temperature (RT), followed by incubation in primary antibodies either overnight at 4 °C. The primary antibodies used were: rabbit anti-GFAP (1:500; Abcam), mouse anti-Brn3a (1:50; Santa Cruz), rabbit anti–GAP43 (1:500; Abcam). The next day, tissues were washed in PBS (3 × 5 min) and incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:400; goat anti–mouse; Life technologies) or Alexa Fluor 594 (1:400; goat anti–rabbit; Life technologies) for 1 h at RT. Retinas were incubated with the nuclear dye DAPI for 20 min, washed in PBS (3 × 5 min), before microscopic analysis. All sections were observed under fluorescence microscope (Leica DFC 7000 T) and x 200 magnification photomicrograph images were captured. RGC survival and glia activation were assessed as previous described (Morgan-Warren et al., 2016).
2.3. In vivo experimental design 18 adult rats were divided in the following 3 groups: Group 1, uninjured/untreated; Group 2 ONC/UMSCs-derived exosomes; Group 3, ONC/PBS. Only 1 eye per animal was used. ONC was performed on day 0, treatment was given on day 0, 7 and 14, all the animals were sacrificed on day 21. 2.4. Optic nerve crush and intravitreal delivery of exosomes The rats were intraperitoneally anesthetized with 10% chloral hydrate (4 ml/kg). Intraorbital ONC was performed as previously described (Puyang et al., 2016). Briefly, a small incision was made in the superior and lateral conjunctiva, and then a gentle dissection was made with fine forceps (Dumont #5B, World Precision Instruments, Sarasota, 135
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staining. In retina of intact animals, GFAP + glia immunoreactivity was inhibited in the NFL and GFAP + glial processes were absent from the IPL. 21 d post ONC, GFAP + immunoreactivity increased in the NFL, and GFAP + glial processes traversed the IPL. The number of GFAP + glial processes in the IPL was significantly increased in retina treated with UMSCs-exosomes 16.6 ± 1.4/250 μm compared with PBS 10.7 ± 1.7/250 μm (Fig. 3).
2.8. Microscopy and analysis Brn3a + RGC nuclei were counted in 20 μm-thick radial sections of the retina, along a 250 μm linear region of the ganglion cell layer (GCL) either side of the optic nerve, imaged using a fluorescence microscope. Brn3a stained nuclei similar to the co-localised DAPI were counted as Brn3a + RGC nuclei. The survival of RGCs was measured as proportion of Brn3a + RGCs nuclei among DAPI + nuclei. Six sections per retina and 6 retinae (from 6 different animals) per treatment group were quantified. For immunohistochemistry of the optic nerve, GAP43+ axons were counted in 15 μm thick longitudinal sections, imaged using a fluorescence microscope. The number of axons was quantified at 200 μm distance intervals distal to the crush site, up to a maximum distance of 1.0 mm. Six sections per optic nerve and 6 optic nerves per treatment group were quantified. The number of axons/mm width was determined by measurement of axons and the optic nerve diameter as previously described (Mead and Tomarev, 2017).The diameter of the nerve was measured at each distance to determine the number of axons/mm width. This value was then used to derive ∑ad, The total number of axons extending distance d in an optic nerve with radius r using:
3.4. UMSCs-exosomes enhanced glia activity in optic nerve in vivo Because UMSCs-exosomes didn’t promote optic nerve axon regeneration of optic nerve, we speculated whether UMSCs-exosomes treatment also affected the glial scar formation. We therefore stained optic nerve sections for GFAP to investigate the responses of astrocytes after optic nerve crush. Exosomes- and PBS-treated animals revealed a GFAP-free area 21d after optic nerve crush. And the GFAP/astrocytefree gap at the lesion site of exosomes-treated animals was significantly smaller than that of PBS-treated controls (85.8 ± 1.4 μm vs.124 ± 1.3 μm) (Fig. 4). 4. Discussion
Average number of axons/mm width ∑ ad = πr 2 × 0.015mm(section thickness)
To our knowledge, the study is the first time UMSCs exosomes have been delivered into the eye to treat ONC. After intravenous injection, exosomes successfully integrate into GCL. Using the ONC model, we demonstrated neuroprotection effect by UMSCs exosomes, but axogenesis is not promoted after treatment of exosomes on 21 d post ONC. Thus, our results demonstrated that UMSCs-exo had limited effect of neuroprotection but no obvious effect of axon regeneration. In this study, we adopted proportion% in 250 μm length beyond optic disk in Brn3a stained radial retinal sections to estimate retinal ganglion cell loss. Ben Mead et al compared the methods for estimating retinal ganglion cell loss in retinal sections and wholemounts, and they found estimates of RGC loss were similar in Brn3a stained radial retinal sections using proportion% in 250 μm length beyond optic disk compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG (Mead et al., 2014). They believed sections had the added advantage of reducing experimental animal usage. RGCs are normally unable to regenerate injured axons after optic nerve damage or in glaucomatous optic neuropathies and can cause permanent visual loss in severe cases (Vidal-Sanz et al., 2012). In recently years, research has shown that, under certain circumstances, mature RGCs can be transformed into an active regenerative state, enabling these neurons to survive and to regenerate axons over long distances in the injured optic nerve (Park et al., 2008; Sengottuvel et al., 2011). It was reported BM-MSCs exosomes had neuroprotection and axogenic effect on RGCs. And the therapeutic effects of exosomes diminished after knockdown of Argonaute-2, a key miRNA effector molecule (Mead and Tomarev, 2017). This implicated that the mechanism might relate to miRNA in exosomes. In our study, we found that UMSCsexosomes promoted RGC survival after ONC, but couldn’t promote axogenesis in optic nerve. The reason why we had different result might attribute to miRNA compositions in different kinds of MSC exosomes. As previously reported, the five most abundant miRNAs in human UMSCs exosomes were miRNA-21-5p, miRNA-125b-5p, miRNA-23a-3p, miRNA-100-5p, and let-7f-5p, accounted for about 34% of the total miRNA; (Fang et al., 2016)whereas the five most abundant miRNAs in BM-MSCs exosomes were miR-143-3p, miR-10b-5p, miR-486-5p, miR22-3p, and miR-21-5p,accounted for 43–59% of the total miRNA (Baglio et al., 2015). Different contents in exosomes might led to different effects. Another explanation is the damage to the optic nerve was too severe for exosomes to exert their treatment effect. By labeling membrane of exosomes before intravitreal injection, we
2.9. Statistical analysis Data were presented as mean ± SEM. The normality test was used to ensure all data were normally distributed before applying t-test or a one-way ANOVA followed by a Tukey test. P values < 0.05 were considered to be statistically significant. 3. Results 3.1. UMSCs-derived exosomes successfully integrate to the cells of GCL in vivo UMSCs-derived exosomes were visualized using electron microscopy and showed the exosomes were cup-shaped and the diameters were around 100 nm (Fig. 1A). Sizes of exosomes were confirmed by NanoSight NS300. The particle size of purified UMSCs-exosomes ranged from 80 to 200 nm. (Fig.1B). Exosomal surface markers CD81 and CD63 were detected positive by performing Western blotting (Fig. 1C). UMSCs-derived exosomes were tracked by PKH67. Fluorescent labeling was seen around DAPI + neuclei of inner retinal cells. Exosomes targeted the cells in retinal ganglion cell layer with non-selectivity (Fig. 1D). 3.2. UMSCs-exosomes promoted RGC survival in retinal ganglion cell layer but didn’t promote optic nerve axon regeneration in vivo To evaluate the effects of UMSCs-exosomes on RGC survival in vivo, we injected 5 μl PBS or 1 × 109 UMSCs-exosomes into the vitreous. In intact GCL, the proportion of Brn3a + RGCs was 55.9 ± 3.5%. In PBS group, 3 weeks after injury, the Brn3a + RGCs dramatically decreased to 5.9 ± 1.3%. Injection with UMSCs-exosomes significantly promoted RGC survival with 12.6 ± 2.7% (Fig. 2). 21 d After ONC, the numbers of GAP43+ axons beyond crush site were decreased in both groups. There was no significantly difference between PBS group and UMSCs-exosomes group. (data not showed) 3.3. UMSCs-exosomes enhanced glia activity in retina To evaluate if glia activity was involved in the neuroprotection effect of UMSCs-exosomes, we observed glia activity in retinal by GFAP 136
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Fig. 1. Characteristics of UMSCs-derived exosomes. (A) Transmission Electron microscopy showed cup-shaped exosomes. Scale bar: 0.2 μm. (B) Size of exosomes was confirmed by NanoSight. The particle size of purified UMSCs-exosomes ranged from 80 to 200 nm. (C) Exosomal surface markers CD81 and CD63 using Western blot. UFS: UMSCs exosome-free supernatant. (D) PHK67 labeling was seen around DAPI + nuclei of inner retinal cells as shown by confocal microscopy. Exosomes targeted the cells in retinal ganglion cell layer with non-selectivity (green: PHK67, blue: DAPI) (Scale bar: 50 μm).GCL: ganglion cell layer of retina, INL: Inner nuclear layer of retina.
axon regeneration after optic nerve transection by activation of retinal glia and sequencely produced RGC-trophic glia-derived factors (Ahmed et al., 2010; Lorber et al., 2008). Besides synthesizing growth factors, astrocytes also synthesized other cellular modulators that threaten neuronal survival and contributed to an irreversible loss of neuronal function (Hernandez, 2000). Modulating astrocyte activation is thought to be an effective therapeutic strategy for improving neuronal survival and axon regeneration. Recently, H.-J. Li et al reported inhibition of miR-21 ameliorates excessive astrocyte activation and promotes axon regeneration after optic nerve crush. They suggested that inhibition of miR-21 contributes to maintaining moderate astrocyte activation. In our results, we found that the glia cells in retina were significantly more active in UMSCs-exo group than in PBS group. There are two explanations: one is that
were able to track the exosomes and identify where their target was. We found a strong staining in inner retina without cell selectivity. This was in accordance with other researchers’ finding. They found exosomes integrate into both neurons and astrocytes (Mead and Tomarev, 2017). Since RGCs were not the only target of UMSC exosomes, it is not clear if the therapeutic effect was via a direct effect on the RGC or through glia cells. In a normal state, astrocytes and Müller cells are inactive, but in response to injury or disease, they remodel and become reactive, express cytokines which are neuroprotective for retinal ganglion cells (Chun et al., 2000). in vitro, activated adult rat retinal glia cells supported adult rat retinal ganglion cell regeneration (Bahr, 1991).And in vivo, intravitreal inflammation, induced by lens injury, or intravitreal injection of zymosan, protected RGC from apoptosis and stimulates
Fig. 2. Brn3a+ RGCs in the GCL. (green: Brn3a, blue: DAPI) (A) Brn3a+ RGCs in intact GCL. (B) Brn3a+RGCs in PBS group at 21d after ONC. (C) Brn3a+ RGCs in UMSCs-exosomes group at 21d after ONC. (Scale bar: 100 μm). GCL: ganglion cell layer of retina. (D) Proportion of Brn3a + RGCs in three groups. (**P < 0.01, n = 6). 137
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Fig. 3. GFAP+ gila cells in the retina. (blue: DAPI, red: GFAP). (A) GFAP + gila cells in intact retina. (B) GFAP + gila cells in PBS group. (C) GFAP + gila cells in UMSCs-exosomes group. (Scale bar: 100 μm). GCL: ganglion cell layer of retina, INL: Inner nuclear layer of retina. (D) Numbers of GFAP + gila cells in three groups. (**P < 0.01, n = 6).
Fig. 4. Longitudinal sections of optic nerves stained for GFAP. (A) Intact optic nerve. (B) 21d after optic nerve crush in PBS group. (C) 21d after optic nerve crush in UMSCs-exosomes. (Scale bar: 100 μm) (D) Quantification of the average width of GFAP-free area at the lesion site of the optic nerve 21d after surgery. (**P < 0.01, n = 6). Department of Ophthalmology, Shanghai Hospital, Second Military Medical University School of Medicine, Shanghai, China
site of exosomes-treated animals was significantly smaller than that of PBS-treated controls. This suggested astrocyte activation in the optic nerve in UMSCs-exo group was excessive, and this might be the reason why axon regeneration was not promoted in exosome group in vivo.
UMSCs-exo might promote the survival of RGCs by promoting the activation of glia cells. Since glia cells can secret neurotrophins, glia activation in retina may partly contribute to the promoted survival of RGCs in a certain stage. But the GFAP/astrocyte-free gap at the lesion 138
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The other explanation is that miRNAs in UMSCs-exo, other than miR21, promoted survival of RGCs. Since miR-21was detected the most abundant miRNA in UMSCs-exo, it accounted 12.5% of the total miRNA, a bit more than the NO.2 miRNA, miRNA-125b-5p, which accounted 10.2% of the total miRNA. The over activation of astrocyte might just be the function presented by miR-21.Functions exerted by other miRNAs such as promoting the intrinsic ability of RGCs to survival and regenerate axons might be covered and mixed. Further study is needed to determine the function and mechanism of UMSCs-exo in neuroprotection.
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5. Conclusion Our results showed that intravitreal injection of UMSCs exosomes promoted retinal ganglion cells survival, but has no obvious effect of axon regeneration on 21 d post-treatment in a rat model of optic nerve crush. UMSCs exosomes has limited neuroprotection effect for optic nerve injury and may be further modificated and studied as a candidate clinical therapy strategy. Ethical statement None. Acknowledgments This work was supported by China National Key Research and Development Program Stem Cell and Translational Research Key Projects2018YFA0108301, National Natural Science Foundation of China81700828, Shanghai Rising-Star Program17QA1405400, Shanghai Outstanding Youth Medical Talent Training Program2017YQ028, China Postdoctoral Science Foundation, and China SMMU Starting Scientific Research Foundation for post doctors. References Ahmed, Z., Aslam, M., Lorber, B., Suggate, E.L., Berry, M., Logan, A., 2010. Optic nerve and vitreal inflammation are both RGC neuroprotective but only the latter is RGC axogenic. Neurobiol. Dis. 37, 441–454. Baglio, S.R., Rooijers, K., Koppers-Lalic, D., Verweij, F.J., Perez Lanzon, M., Zini, N., Naaijkens, B., Perut, F., Niessen, H.W., Baldini, N., Pegtel, D.M., 2015. Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. Stem Cell Res. Ther. 6, 127. Bahr, M., 1991. Adult rat retinal glia in vitro: effects of in vivo crush-activation on glia proliferation and permissiveness for regenerating retinal ganglion cell axons. Exp. Neurol. 111, 65–73. Batsali, A.K., Kastrinaki, M.C., Papadaki, H.A., Pontikoglou, C., 2013. Mesenchymal stem cells derived from Wharton’s Jelly of the umbilical cord: biological properties and emerging clinical applications. Curr. Stem Cell Res. Ther. 8, 144–155. Campagnoli, C., Roberts, I.A., Kumar, S., Bennett, P.R., Bellantuono, I., Fisk, N.M., 2001. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 98, 2396–2402. Chen, M., Xiang, Z., Cai, J., 2013. The anti-apoptotic and neuro-protective effects of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) on acute optic nerve injury is transient. Brain Res. 1532, 63–75. Chun, M.H., Ju, W.K., Kim, K.Y., Lee, M.Y., Hofmann, H.D., Kirsch, M., Oh, S.J., 2000. Upregulation of ciliary neurotrophic factor in reactive Muller cells in the rat retina following optic nerve transection. Brain Res. 868, 358–362. Chung, S., Rho, S., Kim, G., Kim, S.R., Baek, K.H., Kang, M., Lew, H., 2016. Human umbilical cord blood mononuclear cells and chorionic plate-derived mesenchymal stem cells promote axon survival in a rat model of optic nerve crush injury. Int. J. Mol. Med. 37, 1170–1180. Cox Jr., C.S., Baumgartner, J.E., Harting, M.T., Worth, L.L., Walker, P.A., Shah, S.K., Ewing-Cobbs, L., Hasan, K.M., Day, M.C., Lee, D., Jimenez, F., Gee, A., 2011. Autologous bone marrow mononuclear cell therapy for severe traumatic brain injury in children. Neurosurgery 68, 588–600. Drela, K., Lech, W., Figiel-Dabrowska, A., Zychowicz, M., Mikula, M., Sarnowska, A., Domanska-Janik, K., 2016. Enhanced neuro-therapeutic potential of Wharton’s Jellyderived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture. Cytotherapy 18, 497–509. Fang, S., Xu, C., Zhang, Y., Xue, C., Yang, C., Bi, H., Qian, X., Wu, M., Ji, K., Zhao, Y., Wang, Y., Liu, H., Xing, X., 2016. Umbilical cord-derived mesenchymal stem cellderived exosomal MicroRNAs suppress myofibroblast differentiation by inhibiting the transforming growth Factor-beta/SMAD2 pathway during wound healing. Stem Cells Transl. Med. 5, 1425–1439. Fukuchi, Y., Nakajima, H., Sugiyama, D., 2004. Human placentaderived cells have mesenchymal stem/progenitor cell potential. Stem Cells Transl. Med. 649–658.
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UMSC-derived exosomes promote retinal ganglion cells survival in a rat model of optic nerve crush
T
Dongyan Pana,c,d,g,1, Xin Changd,g,1, Mengqiao Xub,e,f,1, Mingke Zhangd, Shoumei Zhangc, ⁎ ⁎ ⁎ Yue Wangd,g, Xueting Luob,e,f, , Jiajun Xuc, , Xiangqun Yangc, , Xiaodong Sunb,e,f a
Department of Ophthalmology, Changhai Hospital, Second Military Medical University School of Medicine, Shanghai, China Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University School of Medicine, Shanghai, China c Department of Anatomy, Second Military Medical University School of Medicine, Shanghai, China d Department of Histology and Embryology, Second Military Medical University School of Medicine, Shanghai, China e Shanghai Key Laboratory of Fundus Diseases, Shanghai, China f Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, China g Shanghai Key Laboratory of Cell Engineering, Shanghai, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Mesenchymal cells Wharton’s jelly Exosomes Optic nerve crush Retinal ganglion cells Glia cells
Traumatic optic neuropathy or glaucoma lead to retinal ganglion cells loss and cause blindness, and there is no effective therapy strategy by far. Mesenchymal cells from the Wharton’s jelly of the umbilical cord (umbilical cord mesenchymal stem cells, UMSCs) and UMSC-derived exosomes (UMSC-Exos) are promising candidates for allogeneic therapy in regenerative medicine, but their effort on optic nerve injury and the underlying mechanism remains undefined. In the present study, we investigated the functions of UMSC-Exos in a rat optic nerve crush (ONC) model. After three times of treatments with an interval of one week, we found that the UMSC-Exos significantly promoted Brn3a+ retinal ganglion cells (RGCs) survival in retinal ganglion cell layer compared with PBS controls. UMSC-Exos also significantly promoted GFAP+ glia cells activation in retina and optic nerve. However, no increase of GAP43+ axon counts in the optic nerve was found after UMSC-Exos treatment. Thus, our results demonstrate that UMSC-derived exosomes may play a role in neuroprotection by promoting the RGCs survival and glia cells activation but not the axon regeneration.
1. Introduction Traumatic optic neuropathy or glaucoma lead to retinal ganglion cells (RGCs) loss and can cause inevitable blindness. Mesenchymal stem cells (MSCs) are multipotent stromal cells isolated from mesenchymal tissues including bone marrow(BM), (Campagnoli et al., 2001)adipose tissue (Rodriguez et al., 2005), circulating blood (Campagnoli et al., 2001), dental pulp (Pierdomenico et al., 2005), placenta (Fukuchi et al., 2004), amniotic fluid (You et al., 2008), umbilical cord blood (Gang et al., 2004), and umbilical cord Wharton’s jelly (Wang and Peng, 2004). MSCs have demonstrated therapeutic efficacy at protection and regeneration of central nervous system (CNS) neurons, including retinal ganglion cells (RGCs) (Chen et al., 2013; Chung et al., 2016; Cox et al., 2011; Johnson et al., 2010; Mead et al., 2013; Nichols et al., 2013). But the mechanism of MSCs’ neuroprotection is still poorly understood. MSCs transplanted into the vitreous showed no differentiation or
migration/integration into retinal tissue, implicating paracrine over cell replacement is the dominant mechanism (Johnson et al., 2010; Mead et al., 2016, 2013). Exosomes have been identified as a new kind of major paracrine factor released by various types of cells, including MSCs. Exosomes are a type of membrane vesicle with diameters of 40–150 nm containing proteins, mRNA and miRNA. They have been reported to be an important mediator of cell-to-cell communication (Lener et al., 2015). Newly studies demonstrate that MSCs-derived exosomes can reduce neuroinflammation, promote neurogenesis, and improve functional recovery in animal models (Kim et al., 2016; Zhang et al., 2015, 2017a). The neuroprotective and neurogenesis effects of MSCs-derived exosomes might be due to miRNA (Johnson et al., 2014; Mead and Tomarev, 2017). These reports indicate that MSCs-derived exosomes may promise to be a better therapy for neurodiseases than MSCs. Mesenchymal cells from the Wharton’s jelly of the umbilical cord
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Corresponding authors. E-mail addresses: [email protected] (X. Luo), [email protected] (J. Xu), [email protected] (X. Yang). 1 These authors contribute equally to this work. https://doi.org/10.1016/j.jchemneu.2019.01.006 Received 24 October 2018; Received in revised form 12 December 2018; Accepted 9 January 2019 Available online 10 January 2019 0891-0618/ © 2019 Elsevier B.V. All rights reserved.
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FL) to expose the optic nerve. The optic nerve was clamped with an aneurysm clip (mini clip, temporary, Aesculap, Germany) 2 mm posterior to the lamina cribosa for 8 s without damaging any small vessels. Rats were kept warm until fully awake. Deliver of exosomes was done as previously described (Mead and Tomarev, 2017). In the aid of a medical head-mounted microscope with cold light illuminator(x2.5, HAOLANG MEDICAL LIGHTING CO., LTD,Nanjing, China), a 5 μl volume of PBS loaded with 1 × 109 exosomes was injected slowly with a 33 g Hamilton syringe (Hamilton Company, Beltsville, MD, http://www.hamiltoncompany.com/) into the vitreous just posterior to the limbus. The needle was retracted after 2 min to minimize backflow.
(umbilical cord mesenchymal stem cells, UMSCs) possess stem cell properties and have broad differentiation potential, low immunogenicity, no ethical issues due to noninvasive collection (Batsali et al., 2013; Gang et al., 2004) and were efficient to obtain, suggesting that UMSCs exosomes are promising candidates for allogeneic therapy in regenerative medicine (Drela et al., 2016). In the present study, we investigated the function of UMSCs exosomes in a rat optic nerve crush (ONC) model. We found that UMSCderived exosomes could promote the RGCs survival but not the axon regeneration. 2. Materials and methods 2.1. Culture of human UMSCs and isolation of exosomes
2.5. Exosome tracking
Umbilical cords were obtained from the Changhai Hospital affiliated to the Second Military Medical University of China with medical informed consent. The collection and use of human biological specimens were approved by the Ethics Committee of the Changhai Hospital. Primary culture of UMSCs and exosome isolation were performed on standard procedures as previously reported (Fang et al., 2016; Zhang et al., 2017b). In briefly, umbilical cords were washed with 70% ethanol and subsequently excess blood was removed with sequential washes in DMEM (DMEM; Hyclone, Massachusetts, USA). Tissue were minced into small pieces (2 × 2 mm) and incubated with medium in small dishes at 37 °C. Only UMSCs in passages 2–5 were used. Collected cell culture suspension about 200 ml was transferred to conical tubes for centrifugation at 300 g for 10 min at 4 °C and was again centrifuged at 16,500 g for 20 min at 4 °C to further remove cell debris. After filtered through a 0.22-μm filter, the flow was transferred to new tubes and then ultracentrifuged again at 120,000 g for 70 min at 4 °C to pellet the exosomes. For maximal exosome retrieval, the exosome-enriched pellet was resuspended in a small volume (approximately 200 μl) with PBS. The presence of exosomes was confirmed by electron microscopy, and exosomes were characterized by using NANO Gold. (iZON® Science, New Zealand). Detection of exosomal surface markers CD81 and CD63 was performed by using Western blotting.
To track exosomes in vivo, the green lipophilic fluorescent dye PKH67 (System Biosciences) was used to label purified exosomes prior to intravitreal injection, according to the manufacturer’s instructions. Exosomes were labeled PKH67 for 5 min and the reaction was stopped by the addition of exosome-depleted FBS. Exosomes were then centrifugated for 70 min at 120,000 g, washed three times with PBS before being resuspended in 1 ml of PBS, and kept on ice until intravitreal injection on the same day. 24 h after PKH67 labeled exosomes were injected into the ONC rats’ vitreous body. Animals were euthanized by over dosage of 10% chloral hydrate and received extraction of retina. Fluorescent labeling was examined in frozen sections of retina under a confocal microscopy and images were captured at 1024 × 1024 pixel resolution.
2.6. Tissue preparation At day 21 post-ONC, animals were sacrificed and perfused intracardially with 4% paraformaldehyde (PFA) in PBS. Eyes and optic nerves were removed and immersion fixed in 4% PFA in PBS for 24 h at 4℃ then in 10, 20, and 30% sucrose solution in PBS for 24 h at 4℃. Eyes and optic nerves were then embedded using optimal cutting temperature embedding medium (SAKURA Tissue-Tek® O.C.T. Compound) by rapid freezing under crushed dry ice and were stored at -80℃. After embedding, eyes and optic nerves were sectioned on a CM1950 cryostat microtome (Leica Microsystems Inc, Bannockburn, IL, http://www. leica-microsystems.com) at −22 ℃ at a thickness of 20 μm and 15 μm. Longitudinal optic nerve and parasagittal eye sections were stored at 22℃ before immunocytochemistry. To ensure RGC counts were done in the same plane, eye sections were chosen with the optic nerve head visible (each eye six sections).
2.2. Animals Adult male Wistar rats weighing 160–200 g (Experimental animal center of the Second Military Medical University, Shanghai, China)were maintained in accordance with the Guide for the Care and Use of Laboratory Animals recommended by the National Institutes of Health and approved by the Ethics Committee for Animal Experimentation of the Second Military Medical University. Animals were kept at 21℃ and 55% humidity under a 12 h light and dark cycle, given food/water and were under constant supervision. Animals were euthanized by over dosage of 10% chloral hydrate before extraction of retina.
2.7. Immunofluorescence analysis Tissues were washed in PBS (3 x 5 min) and then incubated in blocking solution (5% donkey serum, 0.5% Triton X-100, and 1% bovine serum albumin in PBS) for 1 h at room temperature (RT), followed by incubation in primary antibodies either overnight at 4 °C. The primary antibodies used were: rabbit anti-GFAP (1:500; Abcam), mouse anti-Brn3a (1:50; Santa Cruz), rabbit anti–GAP43 (1:500; Abcam). The next day, tissues were washed in PBS (3 × 5 min) and incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:400; goat anti–mouse; Life technologies) or Alexa Fluor 594 (1:400; goat anti–rabbit; Life technologies) for 1 h at RT. Retinas were incubated with the nuclear dye DAPI for 20 min, washed in PBS (3 × 5 min), before microscopic analysis. All sections were observed under fluorescence microscope (Leica DFC 7000 T) and x 200 magnification photomicrograph images were captured. RGC survival and glia activation were assessed as previous described (Morgan-Warren et al., 2016).
2.3. In vivo experimental design 18 adult rats were divided in the following 3 groups: Group 1, uninjured/untreated; Group 2 ONC/UMSCs-derived exosomes; Group 3, ONC/PBS. Only 1 eye per animal was used. ONC was performed on day 0, treatment was given on day 0, 7 and 14, all the animals were sacrificed on day 21. 2.4. Optic nerve crush and intravitreal delivery of exosomes The rats were intraperitoneally anesthetized with 10% chloral hydrate (4 ml/kg). Intraorbital ONC was performed as previously described (Puyang et al., 2016). Briefly, a small incision was made in the superior and lateral conjunctiva, and then a gentle dissection was made with fine forceps (Dumont #5B, World Precision Instruments, Sarasota, 135
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staining. In retina of intact animals, GFAP + glia immunoreactivity was inhibited in the NFL and GFAP + glial processes were absent from the IPL. 21 d post ONC, GFAP + immunoreactivity increased in the NFL, and GFAP + glial processes traversed the IPL. The number of GFAP + glial processes in the IPL was significantly increased in retina treated with UMSCs-exosomes 16.6 ± 1.4/250 μm compared with PBS 10.7 ± 1.7/250 μm (Fig. 3).
2.8. Microscopy and analysis Brn3a + RGC nuclei were counted in 20 μm-thick radial sections of the retina, along a 250 μm linear region of the ganglion cell layer (GCL) either side of the optic nerve, imaged using a fluorescence microscope. Brn3a stained nuclei similar to the co-localised DAPI were counted as Brn3a + RGC nuclei. The survival of RGCs was measured as proportion of Brn3a + RGCs nuclei among DAPI + nuclei. Six sections per retina and 6 retinae (from 6 different animals) per treatment group were quantified. For immunohistochemistry of the optic nerve, GAP43+ axons were counted in 15 μm thick longitudinal sections, imaged using a fluorescence microscope. The number of axons was quantified at 200 μm distance intervals distal to the crush site, up to a maximum distance of 1.0 mm. Six sections per optic nerve and 6 optic nerves per treatment group were quantified. The number of axons/mm width was determined by measurement of axons and the optic nerve diameter as previously described (Mead and Tomarev, 2017).The diameter of the nerve was measured at each distance to determine the number of axons/mm width. This value was then used to derive ∑ad, The total number of axons extending distance d in an optic nerve with radius r using:
3.4. UMSCs-exosomes enhanced glia activity in optic nerve in vivo Because UMSCs-exosomes didn’t promote optic nerve axon regeneration of optic nerve, we speculated whether UMSCs-exosomes treatment also affected the glial scar formation. We therefore stained optic nerve sections for GFAP to investigate the responses of astrocytes after optic nerve crush. Exosomes- and PBS-treated animals revealed a GFAP-free area 21d after optic nerve crush. And the GFAP/astrocytefree gap at the lesion site of exosomes-treated animals was significantly smaller than that of PBS-treated controls (85.8 ± 1.4 μm vs.124 ± 1.3 μm) (Fig. 4). 4. Discussion
Average number of axons/mm width ∑ ad = πr 2 × 0.015mm(section thickness)
To our knowledge, the study is the first time UMSCs exosomes have been delivered into the eye to treat ONC. After intravenous injection, exosomes successfully integrate into GCL. Using the ONC model, we demonstrated neuroprotection effect by UMSCs exosomes, but axogenesis is not promoted after treatment of exosomes on 21 d post ONC. Thus, our results demonstrated that UMSCs-exo had limited effect of neuroprotection but no obvious effect of axon regeneration. In this study, we adopted proportion% in 250 μm length beyond optic disk in Brn3a stained radial retinal sections to estimate retinal ganglion cell loss. Ben Mead et al compared the methods for estimating retinal ganglion cell loss in retinal sections and wholemounts, and they found estimates of RGC loss were similar in Brn3a stained radial retinal sections using proportion% in 250 μm length beyond optic disk compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG (Mead et al., 2014). They believed sections had the added advantage of reducing experimental animal usage. RGCs are normally unable to regenerate injured axons after optic nerve damage or in glaucomatous optic neuropathies and can cause permanent visual loss in severe cases (Vidal-Sanz et al., 2012). In recently years, research has shown that, under certain circumstances, mature RGCs can be transformed into an active regenerative state, enabling these neurons to survive and to regenerate axons over long distances in the injured optic nerve (Park et al., 2008; Sengottuvel et al., 2011). It was reported BM-MSCs exosomes had neuroprotection and axogenic effect on RGCs. And the therapeutic effects of exosomes diminished after knockdown of Argonaute-2, a key miRNA effector molecule (Mead and Tomarev, 2017). This implicated that the mechanism might relate to miRNA in exosomes. In our study, we found that UMSCsexosomes promoted RGC survival after ONC, but couldn’t promote axogenesis in optic nerve. The reason why we had different result might attribute to miRNA compositions in different kinds of MSC exosomes. As previously reported, the five most abundant miRNAs in human UMSCs exosomes were miRNA-21-5p, miRNA-125b-5p, miRNA-23a-3p, miRNA-100-5p, and let-7f-5p, accounted for about 34% of the total miRNA; (Fang et al., 2016)whereas the five most abundant miRNAs in BM-MSCs exosomes were miR-143-3p, miR-10b-5p, miR-486-5p, miR22-3p, and miR-21-5p,accounted for 43–59% of the total miRNA (Baglio et al., 2015). Different contents in exosomes might led to different effects. Another explanation is the damage to the optic nerve was too severe for exosomes to exert their treatment effect. By labeling membrane of exosomes before intravitreal injection, we
2.9. Statistical analysis Data were presented as mean ± SEM. The normality test was used to ensure all data were normally distributed before applying t-test or a one-way ANOVA followed by a Tukey test. P values < 0.05 were considered to be statistically significant. 3. Results 3.1. UMSCs-derived exosomes successfully integrate to the cells of GCL in vivo UMSCs-derived exosomes were visualized using electron microscopy and showed the exosomes were cup-shaped and the diameters were around 100 nm (Fig. 1A). Sizes of exosomes were confirmed by NanoSight NS300. The particle size of purified UMSCs-exosomes ranged from 80 to 200 nm. (Fig.1B). Exosomal surface markers CD81 and CD63 were detected positive by performing Western blotting (Fig. 1C). UMSCs-derived exosomes were tracked by PKH67. Fluorescent labeling was seen around DAPI + neuclei of inner retinal cells. Exosomes targeted the cells in retinal ganglion cell layer with non-selectivity (Fig. 1D). 3.2. UMSCs-exosomes promoted RGC survival in retinal ganglion cell layer but didn’t promote optic nerve axon regeneration in vivo To evaluate the effects of UMSCs-exosomes on RGC survival in vivo, we injected 5 μl PBS or 1 × 109 UMSCs-exosomes into the vitreous. In intact GCL, the proportion of Brn3a + RGCs was 55.9 ± 3.5%. In PBS group, 3 weeks after injury, the Brn3a + RGCs dramatically decreased to 5.9 ± 1.3%. Injection with UMSCs-exosomes significantly promoted RGC survival with 12.6 ± 2.7% (Fig. 2). 21 d After ONC, the numbers of GAP43+ axons beyond crush site were decreased in both groups. There was no significantly difference between PBS group and UMSCs-exosomes group. (data not showed) 3.3. UMSCs-exosomes enhanced glia activity in retina To evaluate if glia activity was involved in the neuroprotection effect of UMSCs-exosomes, we observed glia activity in retinal by GFAP 136
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Fig. 1. Characteristics of UMSCs-derived exosomes. (A) Transmission Electron microscopy showed cup-shaped exosomes. Scale bar: 0.2 μm. (B) Size of exosomes was confirmed by NanoSight. The particle size of purified UMSCs-exosomes ranged from 80 to 200 nm. (C) Exosomal surface markers CD81 and CD63 using Western blot. UFS: UMSCs exosome-free supernatant. (D) PHK67 labeling was seen around DAPI + nuclei of inner retinal cells as shown by confocal microscopy. Exosomes targeted the cells in retinal ganglion cell layer with non-selectivity (green: PHK67, blue: DAPI) (Scale bar: 50 μm).GCL: ganglion cell layer of retina, INL: Inner nuclear layer of retina.
axon regeneration after optic nerve transection by activation of retinal glia and sequencely produced RGC-trophic glia-derived factors (Ahmed et al., 2010; Lorber et al., 2008). Besides synthesizing growth factors, astrocytes also synthesized other cellular modulators that threaten neuronal survival and contributed to an irreversible loss of neuronal function (Hernandez, 2000). Modulating astrocyte activation is thought to be an effective therapeutic strategy for improving neuronal survival and axon regeneration. Recently, H.-J. Li et al reported inhibition of miR-21 ameliorates excessive astrocyte activation and promotes axon regeneration after optic nerve crush. They suggested that inhibition of miR-21 contributes to maintaining moderate astrocyte activation. In our results, we found that the glia cells in retina were significantly more active in UMSCs-exo group than in PBS group. There are two explanations: one is that
were able to track the exosomes and identify where their target was. We found a strong staining in inner retina without cell selectivity. This was in accordance with other researchers’ finding. They found exosomes integrate into both neurons and astrocytes (Mead and Tomarev, 2017). Since RGCs were not the only target of UMSC exosomes, it is not clear if the therapeutic effect was via a direct effect on the RGC or through glia cells. In a normal state, astrocytes and Müller cells are inactive, but in response to injury or disease, they remodel and become reactive, express cytokines which are neuroprotective for retinal ganglion cells (Chun et al., 2000). in vitro, activated adult rat retinal glia cells supported adult rat retinal ganglion cell regeneration (Bahr, 1991).And in vivo, intravitreal inflammation, induced by lens injury, or intravitreal injection of zymosan, protected RGC from apoptosis and stimulates
Fig. 2. Brn3a+ RGCs in the GCL. (green: Brn3a, blue: DAPI) (A) Brn3a+ RGCs in intact GCL. (B) Brn3a+RGCs in PBS group at 21d after ONC. (C) Brn3a+ RGCs in UMSCs-exosomes group at 21d after ONC. (Scale bar: 100 μm). GCL: ganglion cell layer of retina. (D) Proportion of Brn3a + RGCs in three groups. (**P < 0.01, n = 6). 137
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Fig. 3. GFAP+ gila cells in the retina. (blue: DAPI, red: GFAP). (A) GFAP + gila cells in intact retina. (B) GFAP + gila cells in PBS group. (C) GFAP + gila cells in UMSCs-exosomes group. (Scale bar: 100 μm). GCL: ganglion cell layer of retina, INL: Inner nuclear layer of retina. (D) Numbers of GFAP + gila cells in three groups. (**P < 0.01, n = 6).
Fig. 4. Longitudinal sections of optic nerves stained for GFAP. (A) Intact optic nerve. (B) 21d after optic nerve crush in PBS group. (C) 21d after optic nerve crush in UMSCs-exosomes. (Scale bar: 100 μm) (D) Quantification of the average width of GFAP-free area at the lesion site of the optic nerve 21d after surgery. (**P < 0.01, n = 6). Department of Ophthalmology, Shanghai Hospital, Second Military Medical University School of Medicine, Shanghai, China
site of exosomes-treated animals was significantly smaller than that of PBS-treated controls. This suggested astrocyte activation in the optic nerve in UMSCs-exo group was excessive, and this might be the reason why axon regeneration was not promoted in exosome group in vivo.
UMSCs-exo might promote the survival of RGCs by promoting the activation of glia cells. Since glia cells can secret neurotrophins, glia activation in retina may partly contribute to the promoted survival of RGCs in a certain stage. But the GFAP/astrocyte-free gap at the lesion 138
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The other explanation is that miRNAs in UMSCs-exo, other than miR21, promoted survival of RGCs. Since miR-21was detected the most abundant miRNA in UMSCs-exo, it accounted 12.5% of the total miRNA, a bit more than the NO.2 miRNA, miRNA-125b-5p, which accounted 10.2% of the total miRNA. The over activation of astrocyte might just be the function presented by miR-21.Functions exerted by other miRNAs such as promoting the intrinsic ability of RGCs to survival and regenerate axons might be covered and mixed. Further study is needed to determine the function and mechanism of UMSCs-exo in neuroprotection.
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