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Transplantation with retinal progenitor cells repairs visual function in rats with retinal ischemia–reperfusion injury
Neuroscience Letters 558 (2014) 8–13
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Transplantation with retinal progenitor cells repairs visual function in rats with retinal ischemia–reperfusion injury Xueying Li a , Qianyan Kang a,∗,1 , Shan Gao a , Ting Wei a , Yong Liu b , Xinlin Chen b , Haixia Lv b a b
Department of Ophthalmology, The First Affiliated Hospital, Xi’an Jiaotong University School of Medicine, Xi’an, China Institute of Neurobiology, National Key Academic Subject of Physiology, Xi’an Jiaotong University School of Medicine, Xi’an, China
h i g h l i g h t s • • • •
Retinal progenitor cells (RPCs) transplantation is effective for RIR therapy. Subretinal space and the superior colliculus are suitable sites for grafting. Subretinal space transplantation could significantly improve the ERG response. Superior colliculus transplantation could significantly improve the VEP response.
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Article history: Received 7 June 2013 Received in revised form 20 August 2013 Accepted 7 September 2013 Keywords: Electrophysiology Retinal ischemia–reperfusion injury Retinal progenitor cells Superior colliculus Subretinal space
a b s t r a c t The retinal ischemia–reperfusion injury (RIR) is a common pathological process that leads to progressive visual loss and blindness in many retinal diseases such as retinal vascular occlusion disease, diabetic retinopathy, and acute glaucoma. Currently, there has been no effective therapy. The purpose of this study was to investigate the effects of transplantation of retinal progenitor cells (RPCs) into the subretinal space (SRS) and the superior colliculus (SC) in a rat model of RIR injury. We used cultured postnatal day 1 rat RPCs transfected with adeno-associated virus containing the cDNA encoding enhanced green fluorescence protein (EGFP) for transplantation. RIR injury was induced by increases in the intraocular pressure to 110 mmHg for 60 min. The effects of transplantation were evaluated by immunohistochemistry, electroretinography (ERG), and visual evoked potentials (VEP). We found that in rats with RIR injury, RPCs transplanted into the SRS and the SC survived for at least 8 weeks, migrated into surrounding tissues, and improved the ERG and VEP responses. Cells transplanted into the SC improved the VEP response more than those transplanted into the SRS. Our data suggest that transplantation of RPCs into the SRS and the SC may be a possible method for cell replacement therapy for retinal diseases. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Retinal diseases such as glaucoma, diabetic retinopathy, agerelated macular degeneration, and vascular occlusion disease affect millions of people. Loss of retinal cells is regarded to be the irreversible cause of blindness in these retinal diseases. Currently, there are no effective treatments available for these retinal diseases. Recent studies have indicated that stem cells transplanted into the retina can survive and integrate into the host [25], suggesting that the development of stem cell therapy to replace missing retinal cells for the treatment of retinal diseases.
∗ Corresponding author. Tel.: +86 15991622903. E-mail addresses: [email protected] (X. Li), [email protected] (Q. Kang). 1 Present address. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.024
Retinal ischemia is a common cause of visual loss in humans [14]. An animal model of retinal ischemia–reperfusion (RIR) injury, which mimics clinical situations such as retinal vascular occlusion disease and acute glaucoma, is widely used to study retinal neuronal cell damage after ischemic insult [19]. RIR injury mainly causes retinal ganglion cell (RGC) loss or dysfunction. Replacement of missing RGCs via stem cell therapy is a promising treatment to restore vision loss in these retinal diseases. Several studies have shown that stem cells transplanted into the retina integrate into the host retina and restore visual function in RIR models [2,3], suggesting that stem cell therapy may be useful for the treatment of RIR. For the past few decades, several types of cells have been used for the treatment of retinal diseases, including embryonic stem cells [23], brain-derived precursor cells [22], bone marrowderived hematopoietic stem cells [12], and retinal progenitor cells (RPCs) [11]. Compared with poor integration of embryonic
X. Li et al. / Neuroscience Letters 558 (2014) 8–13
cells into the host retina, retinal precursors derived from postnatal day 1–7 (P1–P7) retinas show robust integration into the host retina [11], suggesting that retinal precursors derived from P1–P7 retinas may be suitable for transplantation. Because allogeneic transplants appear to be tolerated in the SRS without need for immunosuppression [1], many investigators choose the SRS as the site of transplantation [9,10]. Superior colliculus (SC), receiving approximately 65% of the contralateral RGC projections, represents the primary retinal projection area in rats [21]. RGCs can be retrogradely labeled by FluoroGold injection into the SC [15], and retinal injury can induce early onset and long-lasting neuronal damage in the SC [4,13]. Retinal precursors transplanted into the SC may result in migration of retinal precursors into the retina, and subsequent differentiation into RGCs. In this study, we aimed to explore the role of transplantation of RPCs derived from P1 retinas in repair of retinal function in a rat model of RIR injury. SRS and SC were selected as the sites for transplantation.
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intraocular pressure. Bacitracin ointment was applied to the eye after surgery. 2.4. Subretinal and superior colliculus Transplantation
2. Materials and methods
RPCs were transplanted into the SRS and SC immediately after RIR injury. After the superior temporal pole of the sclera was exposed, the sclera and choroid were penetrated by a 30-gauge syringe needle. A 1 l cell suspension (approximately 1 × 104 cells) was slowly injected into the SRS with a microinjector. The needle was allowed to remain in place for 30 s and was then very slowly withdrawn to minimize efflux of the transplanted cell suspension. For SC transplantation, the guide cannula was tied in the SC according to the following coordinates: 6 mm posterior to the bregma, 1.5 mm right to the midline, and 6 mm ventral to the skull surface. 1 l of the cell suspension was injected into the right SC. For the control RIR + R-PBS and RIR + SC-PBS groups, 1 l PBS was injected into the SRS and SC, respectively. No evidence of damage to the SC was found from histologic examination of SC sections performed at eight weeks after transplantation.
2.1. Animals
2.5. ERG and VEP recording
Seventy adult male Sprague-Dawley rats (weighing 200–250 g) and 3 P1 rats were used in this study. Rats were supplied by the Center of Experimental Animals, Xi’an Jiaotong University School of Medicine. Principles of laboratory animal care were followed and all procedures were conducted according to guidelines established by the National Institutes of Health. Rats were randomly assigned into 7 groups: control group (rats without any treatment), sham group (sham-operated group), RIR group (retinal ischemia–reperfusion injury group), RIR + SC-C group (RIR rats with transplantation of RPCs into the superior colliculus), RIR + SC-PBS group (RIR rats with transplantation of PBS into the superior colliculus), RIR + R-C group (RIR rats with transplantation of RPCs into the SRS), and RIR + R-PBS group (RIR with transplantation of RPCs into the SRS). Animals were sacrificed at eight weeks after the transplantation.
Full-field ERGs and VEP were recorded on the left eye for each rat eight weeks after the cell therapy. Animals were dark-adapted overnight and prepared for recording under dim red light. The responses were analyzed using RetiScan RetiPort electrophysiology equipment (Roland Consult, Germany). A silver wire loop was placed directly on the center of the rat cornea, and subcutaneous needle electrodes in the forehead and tail served as the reference and ground electrodes, respectively. For VEP, the recording electrode was inserted into the skin over the visual cortex. Bright-flash stimuli were given by a contact LED stimulator at a distance of 15 cm. VEP response was averaged from 100 sweeps.
2.2. Rat retinal progenitor cell preparation Retinal progenitor cell from P1 neural retinas were isolated and cultured in serum-free medium containing N2, B27, EGF and bFGF. Neurospheres were formed after 2–3 days of culture, and were then passaged every 7–10 days by mechanical dissociation. Rat retinal progenitor cells (RPCs) were passaged 2 times before transplantation. For adeno-associated virus (AAV) transfection of stem cells, rat RPC spheres were dissociated, and the cells were seeded at a density of 1.0 × 105 cells per well in 1 ml of fresh serum-free culture media. Cells were transfected with AAV containing the cDNA encoding enhanced green fluorescent protein (AAV-EGFP) at a multiplicity of infection of 100 for 72 h at 37 ◦ C in 5% CO2 . The spheres that arose were visualized with a fluorescent microscope, and only green spheres were used for the transplantation experiment. 2.3. RIR injury model Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5–4 ml/kg). Pupils were dilated with1% tropicamide eye drops. The middle of the anterior chamber of the left eye was cannulated with a steel 30-gauge infusion needle connected to a pressure device. The intraocular pressure was elevated to 110 mmHg for 60 min. In the sham-operated control group, the middle of the anterior chamber of the left eye was cannulated with a steel 30-gauge infusion needle without the elevation of the
2.6. Immunohistochemistry Animals were sacrificed at eight weeks after the transplantation. Enucleated and fixed the left eyes and brain then cut in 12-m-thick sections using a cryostat microtome. After blocking with 10% goat serum for 30 minutes, sections were incubated with primary antibodies anti-rhodopsin antibody (mouse IgG, 1:200; Abcam, MA, USA), anti-Pax6 antibody (rabbit IgG,1:200; Abcam, MA, USA), anti-Thy-1 antibody (H-110, rabbit IgG,1:200; santa cruz biotechnology, CA, USA), anti-protein kinas C alpha (C-term) (rabbit IgG,1:200; Epitomics, CA, USA), and anti-nestin antibody (mouse IgG,1:200; Abcam, MA, USA) at 4 ◦ C overnight, followed by incubation with fluorescent-labeled secondary antibody FITC (anti-mouse IgG, 1:200; Abcam, UK) or Cy3 (Goat Anti-Rabbit IgG or Rabbit Antimouse IgG, 1:200; Beijing CoWin Biosciences Co., Beijing, China). Sections were examined under a fluorescence microscope. 2.7. Statistical analysis The data are presented as mean ± SD. One-way ANOVA with subsequent post hoc tests was used to compare differences among groups. A value of P < 0.05 was considered significant. 3. Results 3.1. Characterization of cultured cells for transplantation The cultured cells, expressing pax6 (Fig. 1A) and Nestin (Fig. 1B), also expressed antigens specific to RGCs (Thy1.1; Fig. 1C), photoreceptors (rhodopsin; Fig. 1D), and bipolar cells (PKC␣; Fig. 1E). These results suggested that the colony-forming cells from the neural
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X. Li et al. / Neuroscience Letters 558 (2014) 8–13
Fig. 1. The expression of pax6 (A), nestin (B), Thy1.1 (C), rhodopsin (D), and protein kinase C alpha (PKC␣; E) in cultured cells. (A) Nuclear staining of pax6 (red), and DAPI (blue). (B) Cytoplasmic staining of nestin (red) and nuclear staining of DAPI (blue). (C) Plasma membrane staining of Thy1.1 (red), and nuclear staining of DAPI (blue). (D) Cytoplasmic staining of rhodopsin (red) and nuclear staining of DAPI (blue). (E) cytoplasmic staining of PKC␣ (red) and nuclear staining of DAPI (blue); Scalebar: 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
retinas were RPCs, which differentiated into specific retinal cells in vitro.
mature retinal cells. No EGFP-positive cells were observed in the retina (Fig. 2D).
3.2. Grafted retinal progenitor cells in retina and superior colliculus
3.3. Visual evoked potentials response
Under fluorescence microscopy, survived retinal progenitor cells exhibited blue intact nuclei and green EGFP fluorescence (Fig. 2A3, B3). We found that retinal progenitor cells survived for at least 8 weeks when they were transplanted into the SRS or SC after RIR injury. Cells integrated into the outer nuclear layer and differentiated into new photoreceptors (Fig. 2B3) after SRS transplantation. No EGFP-positive cells were found in the inner nuclear layer, the RGC layer (Fig. 2B3) and the SC (Fig. 2C). EGFP-positive cells were observed in the SC eight weeks after SC transplantation, but they did not express any markers (thy-1, PKC, rhodopsin) of
Representative VEP waveforms in each group are shown in (Fig. 3). The amplitudes of the p-wave were significantly different among these 7 groups (ANOVA: P < 0.0001). At 8 week after RIR injury, the RIR injury significantly reduced the amplitudes of the pwave by 71% compared with the control group (P < 0.0001; Fig. 4). The amplitude of the p-wave was significantly increased in the RIR + SC-C group compared with that in the RIR and RIR + R-R groups (P < 0.0001; Fig. 4). The amplitude of the p-wave in the RIR + R-C group was did not significantly differ from that in the RIR + R-PBS group (P = 0.091). There were no significant differences among RIR,
Fig. 2. The grafted cells in the retina and the superior colliculus. Retinal progenitor cells were labeled with EGFP (green). The triple labeling of DAPI (blue), EGFP (green) and rhodopsin (red) showed the differentiated state of the grafted retinal progenitor cells (arrow). (A1–A3) Representative images showing survived retinal progenitor cells (arrow) in the superior colliculus. The cells did not express any markers (thy-1, PKC, rhodopsin) of mature retinal cells. (B1–B3) Representative images showing survived retinal progenitor cells (arrow) in the retina. The cells integrated into the outer nuclear layer, and some of them differentiated into new photoreceptors (B3, red). (C) Representative image showing the SC of rat 8 weeks after subretinal transplantation of retinal progenitor cells. No EGFP-positive cells were observed. (D) Representative image showing the retina of rat 8 weeks after transplantation of retinal progenitor cells into the SC. No EGFP-positive cells were observed in the retina. Scale bar: 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
X. Li et al. / Neuroscience Letters 558 (2014) 8–13
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Fig. 3. Representative waveforms of VEPs, RRs, CRs, MRs, Ops, FRs in the Control, Sham, RIR, RIR + SC-C, RIR + SC-PBS, RIR + R-C, RIR + R-PBS groups.
Fig. 4. VEP amplitudes of the p-wave (VEP), ERG amplitudes of the RR b-wave (RR), MR b-wave (MR), and OP1 (OPS), CR b-wave (CR), and FR b-wave (FR). Values represent the mean ± SD (n = 8). RR: rod response; MR: maximal response; OP: oscillatory potential; CR: cone response; FR: flicker response; *P < 0.05; **P < 0.01; ***P < 0.001.
RIR + SC-PBS and RIR + R-PBS groups. No significant differences in the implicit time were found among these groups. 3.4. Electroretinography Representative waveforms are shown in Fig. 3. The amplitudes of the rod response (RR) b-wave, maximal response (MR) b-wave,
oscillatory potential (OP) 1, cone response (CR) b-wave and flicker responses (FR) second b-wave were significantly different among the 7 groups (ANOVA: P < 0.0001). RIR injury significantly reduced the amplitudes of the RR b-wave by 95%, the MR b-wave by 90%, the OP1 by 88%, the CR b-wave by 88%, and the FR second b-wave by 91%, compared with the control group at 8 week after RIR injury (P < 0.0001, Fig. 4). In the RIR + R-C group, the amplitudes of the
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RR b-wave, MR b-wave, OP1, CR b-wave and FR second b-wave were significantly increased compared with those in the RIR-R-PBS group (P < 0.0001, Fig. 4). The amplitudes of the RR b-wave, MR b-wave, OP1 (P = 0.002), CR b-wave and FR second b-wave were significantly increased in the RIR + R-C group compared with those in the RIR + SC-R group (P <0.0001, Fig. 4). There were no significant differences in the amplitude of these ERG responses among RIR, RIR + SC-PBS, and RIR + R-PBS groups. No significant differences in the implicit time of these ERG Reponses were found among the 7 groups. 4. Discussion This study found that injection of retinal progenitor cells (RPCs) into the SRS and SC improved substantial visual function in a rat model of RIR injury. ERG responses were significantly improved by transplantation of RPCs into the SRS, and VEP responses were significantly improved by transplantation of RPCs into SC. Our data suggest that the SRS and the SC are suitable for transplantation of RPCs. 4.1. Subretinal space transplantation The challenge for retinal progenitor cell therapy lies in the generation of numerous mature retinal cells, identification of the ideal developmental stage to integrate, and no immunogenicity. For the past decade, investigators have demonstrated that some cells could integrate into the host retina and make synaptic connections [11]. In the present study, we found that cultured P1 RPCs proliferated and expressed nestin and pax6. These RPCs differentiated and expressed many mature retinal cell markers, thy1, PKC, and rhodopsin. After transplantation into the SRS, these donor cells integrated into the outer layer of the host retina, expressed the photoreceoptor marker rhodopin, and improved retinal function. Our data shows that success has been achieved in the incorporation of donor cells into host retinal tissues, but specific and appropriate differentiation of donor cells is not always accomplished. 4.2. Superior colliculus transplantation SC represents the primary retinal projection area in rats. Retinal injury induces early onset and long-lasting damage in the retinal neurons, and their terminals in the brain [5,18]. Investigators observed that a decrease in the crosssectional area of neurons in the contralateral dLGN and SC was concomitantly associated with the RGC loss at 3 days post-operation [24]. Nuclear factor kappa-B is activated in the contralateral SC from 1 h to day 15 after retinal injury [4], and the expression of brain-derived neurotrophic factors (BDNF) and other neurotrophic factors in the LGN and SC are increased after retinal injury [6,20]. In the present study, we investigated the function and structural protective role of RPC transplantation into the SC in a rat model of RIR injury, and found that retinal progenitor cells survived in the SC for at least 8 weeks. 4.3. Visual function Visual electrophysiology is a useful tool to assess visual function and treatment efficacy even when only a minimal degree of function remains [17]. In this study, we used ERG and VEP to assess visual function and found that SC transplantation of RPCs significantly increased the amplitude of the VEP p-wave, and SRS transplantation of RPCs significantly improved ERG responses in the eyes of rats with RIR injury. In addition, SC transplantation significantly improved VEP responses more than SRS transplantation. A combination of SC and SRS transplantation of RPCs is expected to be more beneficial for the improvement of VEP and ERG response in
the eyes of rats with RIR injury compared with SC or SRS transplantation alone. However, in the present study, we did not transplant RPCs in the naïve rats, because it has been reported that stem cells do not migrate, integrate, and differentiate in normal rats [8]. We speculate that RPC transplantation may not improve the visual function in naïve rats. Although we aimed to find the differentiation and integration of transplanted cells into RGCs, the transplanted cells were integrated into the outer nuclear layer, but not the RGCs. The mechanism for improved visual function after RPC transplantation remains unclear. We found that RPC transplantation resulted in a decrease in RGCs, and an increase in retinal thickness in RIR rats (unpublished data), suggesting that RPC transplantation may improve the survival of RGCs in RIR rats. In addition, previous studies have shown that environmental enrichment can improve the visual system via upregulation of trophic factors, such as BDNF and insulin-like growth factor-1 (IGF-1) [7,16]. It has been reported that transplanted BMSCs continue to release the trophic factors such as CNTF, bFGF and BDNF for at least four weeks [8]. Secretion of trophic factor from transplanted cells may contribute to the improved visual function by RPC transplantation in the present study. The underlying mechanisms are under investigation. In summary, we find that retinal progenitor cells replacement therapy has potential for functional and structural retinal protection in eyes with RIR injury. Most importantly, we find that SC transplantation significantly improves the VEP in rats with RIR injury. This study, for the first time, reports the SC transplantation of RPCs in the RIR injured rat. Though the underlying mechanisms remain unknown, the new approach provides different treatment strategies for the treatment of RIR disease. Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) (No. 30772373) and Project of Science and Technology Development of Shaanxi Province (No. 2012K1611(04). The author thanks Zuoming Zhang and Feng Xia (Department of Aviation Clinical Medicine, School of Aviation & Aerospace Medicine, Fourth Military Medical University, Xi’an, Shhanxi Province, China) for excellent technical assistance in these studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2013.09.024. References [1] R.B. Aramant, M.J. Seiler, Transplanted sheets of human retina and retinal pigment epithelium develop normally in nude rats, Exp. Eye Res. 75 (2002) 115–125. [2] I.M. Fang, C.M. Yang, C.H. Yang, S.H. Chiou, M.S. Chen, Transplantation of induced pluripotent stem cells without C-Myc attenuates retinal ischemia and reperfusion injury in rats, Exp. Eye Res. 113C (2013) 49–59. [3] S.D. Grozdanic, A.M. Ast, T. Lazic, Y.H. Kwon, R.H. Kardon, I.M. Sonea, D.S. Sakaguchi, Morphological integration and functional assessment of transplanted neural progenitor cells in healthy and acute ischemic rat eyes, Exp. Eye Res. 82 (2006) 597–607. [4] M.S. Hernandes, L.S. Lima, C. Scavone, L.R. Lopes, L.R. Britto, Eye enucleation activates the transcription nuclear factor kappa-B in the rat superior colliculus, Neurosci. Lett. 521 (2012) 104–108. [5] Y. Ito, M. Shimazawa, Y.N. Chen, K. Tsuruma, T. Yamashima, M. Araie, H. Hara, Morphological changes in the visual pathway induced by experimental glaucoma in Japanese monkeys, Exp. Eye Res. 89 (2009) 246–255. [6] Y. Ito, M. Shimazawa, Y. Inokuchi, H. Fukumitsu, S. Furukawa, M. Araie, H. Hara, Degenerative alterations in the visual pathway after NMDA-induced retinal damage in mice, Brain Res. 1212 (2008) 89–101. [7] S. Landi, A. Sale, N. Berardi, A. Viegi, L. Maffei, M.C. Cenni, Retinal functional development is sensitive to environmental enrichment: a role for BDNF, FASEB J. 21 (2007) 130–139.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Transplantation with retinal progenitor cells repairs visual function in rats with retinal ischemia–reperfusion injury Xueying Li a , Qianyan Kang a,∗,1 , Shan Gao a , Ting Wei a , Yong Liu b , Xinlin Chen b , Haixia Lv b a b
Department of Ophthalmology, The First Affiliated Hospital, Xi’an Jiaotong University School of Medicine, Xi’an, China Institute of Neurobiology, National Key Academic Subject of Physiology, Xi’an Jiaotong University School of Medicine, Xi’an, China
h i g h l i g h t s • • • •
Retinal progenitor cells (RPCs) transplantation is effective for RIR therapy. Subretinal space and the superior colliculus are suitable sites for grafting. Subretinal space transplantation could significantly improve the ERG response. Superior colliculus transplantation could significantly improve the VEP response.
a r t i c l e
i n f o
Article history: Received 7 June 2013 Received in revised form 20 August 2013 Accepted 7 September 2013 Keywords: Electrophysiology Retinal ischemia–reperfusion injury Retinal progenitor cells Superior colliculus Subretinal space
a b s t r a c t The retinal ischemia–reperfusion injury (RIR) is a common pathological process that leads to progressive visual loss and blindness in many retinal diseases such as retinal vascular occlusion disease, diabetic retinopathy, and acute glaucoma. Currently, there has been no effective therapy. The purpose of this study was to investigate the effects of transplantation of retinal progenitor cells (RPCs) into the subretinal space (SRS) and the superior colliculus (SC) in a rat model of RIR injury. We used cultured postnatal day 1 rat RPCs transfected with adeno-associated virus containing the cDNA encoding enhanced green fluorescence protein (EGFP) for transplantation. RIR injury was induced by increases in the intraocular pressure to 110 mmHg for 60 min. The effects of transplantation were evaluated by immunohistochemistry, electroretinography (ERG), and visual evoked potentials (VEP). We found that in rats with RIR injury, RPCs transplanted into the SRS and the SC survived for at least 8 weeks, migrated into surrounding tissues, and improved the ERG and VEP responses. Cells transplanted into the SC improved the VEP response more than those transplanted into the SRS. Our data suggest that transplantation of RPCs into the SRS and the SC may be a possible method for cell replacement therapy for retinal diseases. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Retinal diseases such as glaucoma, diabetic retinopathy, agerelated macular degeneration, and vascular occlusion disease affect millions of people. Loss of retinal cells is regarded to be the irreversible cause of blindness in these retinal diseases. Currently, there are no effective treatments available for these retinal diseases. Recent studies have indicated that stem cells transplanted into the retina can survive and integrate into the host [25], suggesting that the development of stem cell therapy to replace missing retinal cells for the treatment of retinal diseases.
∗ Corresponding author. Tel.: +86 15991622903. E-mail addresses: [email protected] (X. Li), [email protected] (Q. Kang). 1 Present address. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.024
Retinal ischemia is a common cause of visual loss in humans [14]. An animal model of retinal ischemia–reperfusion (RIR) injury, which mimics clinical situations such as retinal vascular occlusion disease and acute glaucoma, is widely used to study retinal neuronal cell damage after ischemic insult [19]. RIR injury mainly causes retinal ganglion cell (RGC) loss or dysfunction. Replacement of missing RGCs via stem cell therapy is a promising treatment to restore vision loss in these retinal diseases. Several studies have shown that stem cells transplanted into the retina integrate into the host retina and restore visual function in RIR models [2,3], suggesting that stem cell therapy may be useful for the treatment of RIR. For the past few decades, several types of cells have been used for the treatment of retinal diseases, including embryonic stem cells [23], brain-derived precursor cells [22], bone marrowderived hematopoietic stem cells [12], and retinal progenitor cells (RPCs) [11]. Compared with poor integration of embryonic
X. Li et al. / Neuroscience Letters 558 (2014) 8–13
cells into the host retina, retinal precursors derived from postnatal day 1–7 (P1–P7) retinas show robust integration into the host retina [11], suggesting that retinal precursors derived from P1–P7 retinas may be suitable for transplantation. Because allogeneic transplants appear to be tolerated in the SRS without need for immunosuppression [1], many investigators choose the SRS as the site of transplantation [9,10]. Superior colliculus (SC), receiving approximately 65% of the contralateral RGC projections, represents the primary retinal projection area in rats [21]. RGCs can be retrogradely labeled by FluoroGold injection into the SC [15], and retinal injury can induce early onset and long-lasting neuronal damage in the SC [4,13]. Retinal precursors transplanted into the SC may result in migration of retinal precursors into the retina, and subsequent differentiation into RGCs. In this study, we aimed to explore the role of transplantation of RPCs derived from P1 retinas in repair of retinal function in a rat model of RIR injury. SRS and SC were selected as the sites for transplantation.
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intraocular pressure. Bacitracin ointment was applied to the eye after surgery. 2.4. Subretinal and superior colliculus Transplantation
2. Materials and methods
RPCs were transplanted into the SRS and SC immediately after RIR injury. After the superior temporal pole of the sclera was exposed, the sclera and choroid were penetrated by a 30-gauge syringe needle. A 1 l cell suspension (approximately 1 × 104 cells) was slowly injected into the SRS with a microinjector. The needle was allowed to remain in place for 30 s and was then very slowly withdrawn to minimize efflux of the transplanted cell suspension. For SC transplantation, the guide cannula was tied in the SC according to the following coordinates: 6 mm posterior to the bregma, 1.5 mm right to the midline, and 6 mm ventral to the skull surface. 1 l of the cell suspension was injected into the right SC. For the control RIR + R-PBS and RIR + SC-PBS groups, 1 l PBS was injected into the SRS and SC, respectively. No evidence of damage to the SC was found from histologic examination of SC sections performed at eight weeks after transplantation.
2.1. Animals
2.5. ERG and VEP recording
Seventy adult male Sprague-Dawley rats (weighing 200–250 g) and 3 P1 rats were used in this study. Rats were supplied by the Center of Experimental Animals, Xi’an Jiaotong University School of Medicine. Principles of laboratory animal care were followed and all procedures were conducted according to guidelines established by the National Institutes of Health. Rats were randomly assigned into 7 groups: control group (rats without any treatment), sham group (sham-operated group), RIR group (retinal ischemia–reperfusion injury group), RIR + SC-C group (RIR rats with transplantation of RPCs into the superior colliculus), RIR + SC-PBS group (RIR rats with transplantation of PBS into the superior colliculus), RIR + R-C group (RIR rats with transplantation of RPCs into the SRS), and RIR + R-PBS group (RIR with transplantation of RPCs into the SRS). Animals were sacrificed at eight weeks after the transplantation.
Full-field ERGs and VEP were recorded on the left eye for each rat eight weeks after the cell therapy. Animals were dark-adapted overnight and prepared for recording under dim red light. The responses were analyzed using RetiScan RetiPort electrophysiology equipment (Roland Consult, Germany). A silver wire loop was placed directly on the center of the rat cornea, and subcutaneous needle electrodes in the forehead and tail served as the reference and ground electrodes, respectively. For VEP, the recording electrode was inserted into the skin over the visual cortex. Bright-flash stimuli were given by a contact LED stimulator at a distance of 15 cm. VEP response was averaged from 100 sweeps.
2.2. Rat retinal progenitor cell preparation Retinal progenitor cell from P1 neural retinas were isolated and cultured in serum-free medium containing N2, B27, EGF and bFGF. Neurospheres were formed after 2–3 days of culture, and were then passaged every 7–10 days by mechanical dissociation. Rat retinal progenitor cells (RPCs) were passaged 2 times before transplantation. For adeno-associated virus (AAV) transfection of stem cells, rat RPC spheres were dissociated, and the cells were seeded at a density of 1.0 × 105 cells per well in 1 ml of fresh serum-free culture media. Cells were transfected with AAV containing the cDNA encoding enhanced green fluorescent protein (AAV-EGFP) at a multiplicity of infection of 100 for 72 h at 37 ◦ C in 5% CO2 . The spheres that arose were visualized with a fluorescent microscope, and only green spheres were used for the transplantation experiment. 2.3. RIR injury model Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5–4 ml/kg). Pupils were dilated with1% tropicamide eye drops. The middle of the anterior chamber of the left eye was cannulated with a steel 30-gauge infusion needle connected to a pressure device. The intraocular pressure was elevated to 110 mmHg for 60 min. In the sham-operated control group, the middle of the anterior chamber of the left eye was cannulated with a steel 30-gauge infusion needle without the elevation of the
2.6. Immunohistochemistry Animals were sacrificed at eight weeks after the transplantation. Enucleated and fixed the left eyes and brain then cut in 12-m-thick sections using a cryostat microtome. After blocking with 10% goat serum for 30 minutes, sections were incubated with primary antibodies anti-rhodopsin antibody (mouse IgG, 1:200; Abcam, MA, USA), anti-Pax6 antibody (rabbit IgG,1:200; Abcam, MA, USA), anti-Thy-1 antibody (H-110, rabbit IgG,1:200; santa cruz biotechnology, CA, USA), anti-protein kinas C alpha (C-term) (rabbit IgG,1:200; Epitomics, CA, USA), and anti-nestin antibody (mouse IgG,1:200; Abcam, MA, USA) at 4 ◦ C overnight, followed by incubation with fluorescent-labeled secondary antibody FITC (anti-mouse IgG, 1:200; Abcam, UK) or Cy3 (Goat Anti-Rabbit IgG or Rabbit Antimouse IgG, 1:200; Beijing CoWin Biosciences Co., Beijing, China). Sections were examined under a fluorescence microscope. 2.7. Statistical analysis The data are presented as mean ± SD. One-way ANOVA with subsequent post hoc tests was used to compare differences among groups. A value of P < 0.05 was considered significant. 3. Results 3.1. Characterization of cultured cells for transplantation The cultured cells, expressing pax6 (Fig. 1A) and Nestin (Fig. 1B), also expressed antigens specific to RGCs (Thy1.1; Fig. 1C), photoreceptors (rhodopsin; Fig. 1D), and bipolar cells (PKC␣; Fig. 1E). These results suggested that the colony-forming cells from the neural
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Fig. 1. The expression of pax6 (A), nestin (B), Thy1.1 (C), rhodopsin (D), and protein kinase C alpha (PKC␣; E) in cultured cells. (A) Nuclear staining of pax6 (red), and DAPI (blue). (B) Cytoplasmic staining of nestin (red) and nuclear staining of DAPI (blue). (C) Plasma membrane staining of Thy1.1 (red), and nuclear staining of DAPI (blue). (D) Cytoplasmic staining of rhodopsin (red) and nuclear staining of DAPI (blue). (E) cytoplasmic staining of PKC␣ (red) and nuclear staining of DAPI (blue); Scalebar: 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
retinas were RPCs, which differentiated into specific retinal cells in vitro.
mature retinal cells. No EGFP-positive cells were observed in the retina (Fig. 2D).
3.2. Grafted retinal progenitor cells in retina and superior colliculus
3.3. Visual evoked potentials response
Under fluorescence microscopy, survived retinal progenitor cells exhibited blue intact nuclei and green EGFP fluorescence (Fig. 2A3, B3). We found that retinal progenitor cells survived for at least 8 weeks when they were transplanted into the SRS or SC after RIR injury. Cells integrated into the outer nuclear layer and differentiated into new photoreceptors (Fig. 2B3) after SRS transplantation. No EGFP-positive cells were found in the inner nuclear layer, the RGC layer (Fig. 2B3) and the SC (Fig. 2C). EGFP-positive cells were observed in the SC eight weeks after SC transplantation, but they did not express any markers (thy-1, PKC, rhodopsin) of
Representative VEP waveforms in each group are shown in (Fig. 3). The amplitudes of the p-wave were significantly different among these 7 groups (ANOVA: P < 0.0001). At 8 week after RIR injury, the RIR injury significantly reduced the amplitudes of the pwave by 71% compared with the control group (P < 0.0001; Fig. 4). The amplitude of the p-wave was significantly increased in the RIR + SC-C group compared with that in the RIR and RIR + R-R groups (P < 0.0001; Fig. 4). The amplitude of the p-wave in the RIR + R-C group was did not significantly differ from that in the RIR + R-PBS group (P = 0.091). There were no significant differences among RIR,
Fig. 2. The grafted cells in the retina and the superior colliculus. Retinal progenitor cells were labeled with EGFP (green). The triple labeling of DAPI (blue), EGFP (green) and rhodopsin (red) showed the differentiated state of the grafted retinal progenitor cells (arrow). (A1–A3) Representative images showing survived retinal progenitor cells (arrow) in the superior colliculus. The cells did not express any markers (thy-1, PKC, rhodopsin) of mature retinal cells. (B1–B3) Representative images showing survived retinal progenitor cells (arrow) in the retina. The cells integrated into the outer nuclear layer, and some of them differentiated into new photoreceptors (B3, red). (C) Representative image showing the SC of rat 8 weeks after subretinal transplantation of retinal progenitor cells. No EGFP-positive cells were observed. (D) Representative image showing the retina of rat 8 weeks after transplantation of retinal progenitor cells into the SC. No EGFP-positive cells were observed in the retina. Scale bar: 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. Representative waveforms of VEPs, RRs, CRs, MRs, Ops, FRs in the Control, Sham, RIR, RIR + SC-C, RIR + SC-PBS, RIR + R-C, RIR + R-PBS groups.
Fig. 4. VEP amplitudes of the p-wave (VEP), ERG amplitudes of the RR b-wave (RR), MR b-wave (MR), and OP1 (OPS), CR b-wave (CR), and FR b-wave (FR). Values represent the mean ± SD (n = 8). RR: rod response; MR: maximal response; OP: oscillatory potential; CR: cone response; FR: flicker response; *P < 0.05; **P < 0.01; ***P < 0.001.
RIR + SC-PBS and RIR + R-PBS groups. No significant differences in the implicit time were found among these groups. 3.4. Electroretinography Representative waveforms are shown in Fig. 3. The amplitudes of the rod response (RR) b-wave, maximal response (MR) b-wave,
oscillatory potential (OP) 1, cone response (CR) b-wave and flicker responses (FR) second b-wave were significantly different among the 7 groups (ANOVA: P < 0.0001). RIR injury significantly reduced the amplitudes of the RR b-wave by 95%, the MR b-wave by 90%, the OP1 by 88%, the CR b-wave by 88%, and the FR second b-wave by 91%, compared with the control group at 8 week after RIR injury (P < 0.0001, Fig. 4). In the RIR + R-C group, the amplitudes of the
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RR b-wave, MR b-wave, OP1, CR b-wave and FR second b-wave were significantly increased compared with those in the RIR-R-PBS group (P < 0.0001, Fig. 4). The amplitudes of the RR b-wave, MR b-wave, OP1 (P = 0.002), CR b-wave and FR second b-wave were significantly increased in the RIR + R-C group compared with those in the RIR + SC-R group (P <0.0001, Fig. 4). There were no significant differences in the amplitude of these ERG responses among RIR, RIR + SC-PBS, and RIR + R-PBS groups. No significant differences in the implicit time of these ERG Reponses were found among the 7 groups. 4. Discussion This study found that injection of retinal progenitor cells (RPCs) into the SRS and SC improved substantial visual function in a rat model of RIR injury. ERG responses were significantly improved by transplantation of RPCs into the SRS, and VEP responses were significantly improved by transplantation of RPCs into SC. Our data suggest that the SRS and the SC are suitable for transplantation of RPCs. 4.1. Subretinal space transplantation The challenge for retinal progenitor cell therapy lies in the generation of numerous mature retinal cells, identification of the ideal developmental stage to integrate, and no immunogenicity. For the past decade, investigators have demonstrated that some cells could integrate into the host retina and make synaptic connections [11]. In the present study, we found that cultured P1 RPCs proliferated and expressed nestin and pax6. These RPCs differentiated and expressed many mature retinal cell markers, thy1, PKC, and rhodopsin. After transplantation into the SRS, these donor cells integrated into the outer layer of the host retina, expressed the photoreceoptor marker rhodopin, and improved retinal function. Our data shows that success has been achieved in the incorporation of donor cells into host retinal tissues, but specific and appropriate differentiation of donor cells is not always accomplished. 4.2. Superior colliculus transplantation SC represents the primary retinal projection area in rats. Retinal injury induces early onset and long-lasting damage in the retinal neurons, and their terminals in the brain [5,18]. Investigators observed that a decrease in the crosssectional area of neurons in the contralateral dLGN and SC was concomitantly associated with the RGC loss at 3 days post-operation [24]. Nuclear factor kappa-B is activated in the contralateral SC from 1 h to day 15 after retinal injury [4], and the expression of brain-derived neurotrophic factors (BDNF) and other neurotrophic factors in the LGN and SC are increased after retinal injury [6,20]. In the present study, we investigated the function and structural protective role of RPC transplantation into the SC in a rat model of RIR injury, and found that retinal progenitor cells survived in the SC for at least 8 weeks. 4.3. Visual function Visual electrophysiology is a useful tool to assess visual function and treatment efficacy even when only a minimal degree of function remains [17]. In this study, we used ERG and VEP to assess visual function and found that SC transplantation of RPCs significantly increased the amplitude of the VEP p-wave, and SRS transplantation of RPCs significantly improved ERG responses in the eyes of rats with RIR injury. In addition, SC transplantation significantly improved VEP responses more than SRS transplantation. A combination of SC and SRS transplantation of RPCs is expected to be more beneficial for the improvement of VEP and ERG response in
the eyes of rats with RIR injury compared with SC or SRS transplantation alone. However, in the present study, we did not transplant RPCs in the naïve rats, because it has been reported that stem cells do not migrate, integrate, and differentiate in normal rats [8]. We speculate that RPC transplantation may not improve the visual function in naïve rats. Although we aimed to find the differentiation and integration of transplanted cells into RGCs, the transplanted cells were integrated into the outer nuclear layer, but not the RGCs. The mechanism for improved visual function after RPC transplantation remains unclear. We found that RPC transplantation resulted in a decrease in RGCs, and an increase in retinal thickness in RIR rats (unpublished data), suggesting that RPC transplantation may improve the survival of RGCs in RIR rats. In addition, previous studies have shown that environmental enrichment can improve the visual system via upregulation of trophic factors, such as BDNF and insulin-like growth factor-1 (IGF-1) [7,16]. It has been reported that transplanted BMSCs continue to release the trophic factors such as CNTF, bFGF and BDNF for at least four weeks [8]. Secretion of trophic factor from transplanted cells may contribute to the improved visual function by RPC transplantation in the present study. The underlying mechanisms are under investigation. In summary, we find that retinal progenitor cells replacement therapy has potential for functional and structural retinal protection in eyes with RIR injury. Most importantly, we find that SC transplantation significantly improves the VEP in rats with RIR injury. This study, for the first time, reports the SC transplantation of RPCs in the RIR injured rat. Though the underlying mechanisms remain unknown, the new approach provides different treatment strategies for the treatment of RIR disease. Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) (No. 30772373) and Project of Science and Technology Development of Shaanxi Province (No. 2012K1611(04). The author thanks Zuoming Zhang and Feng Xia (Department of Aviation Clinical Medicine, School of Aviation & Aerospace Medicine, Fourth Military Medical University, Xi’an, Shhanxi Province, China) for excellent technical assistance in these studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2013.09.024. References [1] R.B. Aramant, M.J. Seiler, Transplanted sheets of human retina and retinal pigment epithelium develop normally in nude rats, Exp. Eye Res. 75 (2002) 115–125. [2] I.M. Fang, C.M. Yang, C.H. Yang, S.H. Chiou, M.S. Chen, Transplantation of induced pluripotent stem cells without C-Myc attenuates retinal ischemia and reperfusion injury in rats, Exp. Eye Res. 113C (2013) 49–59. [3] S.D. Grozdanic, A.M. Ast, T. Lazic, Y.H. Kwon, R.H. Kardon, I.M. Sonea, D.S. Sakaguchi, Morphological integration and functional assessment of transplanted neural progenitor cells in healthy and acute ischemic rat eyes, Exp. Eye Res. 82 (2006) 597–607. [4] M.S. Hernandes, L.S. Lima, C. Scavone, L.R. Lopes, L.R. Britto, Eye enucleation activates the transcription nuclear factor kappa-B in the rat superior colliculus, Neurosci. Lett. 521 (2012) 104–108. [5] Y. Ito, M. Shimazawa, Y.N. Chen, K. Tsuruma, T. Yamashima, M. Araie, H. Hara, Morphological changes in the visual pathway induced by experimental glaucoma in Japanese monkeys, Exp. Eye Res. 89 (2009) 246–255. [6] Y. Ito, M. Shimazawa, Y. Inokuchi, H. Fukumitsu, S. Furukawa, M. Araie, H. Hara, Degenerative alterations in the visual pathway after NMDA-induced retinal damage in mice, Brain Res. 1212 (2008) 89–101. [7] S. Landi, A. Sale, N. Berardi, A. Viegi, L. Maffei, M.C. Cenni, Retinal functional development is sensitive to environmental enrichment: a role for BDNF, FASEB J. 21 (2007) 130–139.
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