September 2012
Volume 53, Issue 10
Free
Retina  |   September 2012
Effects of Erythropoietin-Dextran Microparticle-Based PLGA/PLA Microspheres on RGCs
Author Affiliations & Notes
  • Xianfang Rong
    From the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China; and the
  • Sixing Yang
    School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
  • Huamao Miao
    From the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China; and the
  • Tingting Guo
    From the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China; and the
  • Zhenyu Wang
    From the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China; and the
  • Wanru Shi
    From the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China; and the
  • Xiaofen Mo
    From the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China; and the
  • Weien Yuan
    School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
  • Tuo Jin
    School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
  • *Each of the following is a corresponding author: Xiaofen Mo, Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, No. 83 Fenyang Road, Shanghai 200031, China; xfmo@fudan.edu.cn
  • Tuo Jin, School of Pharmacy, Shanghai Jiao Tong University, No. 800 Dong Chuan Road, Shanghai 200240, China; tjin@sjtu.edu.cn
Investigative Ophthalmology & Visual Science September 2012, Vol.53, 6025-6034. doi:10.1167/iovs.12-9898
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Xianfang Rong, Sixing Yang, Huamao Miao, Tingting Guo, Zhenyu Wang, Wanru Shi, Xiaofen Mo, Weien Yuan, Tuo Jin; Effects of Erythropoietin-Dextran Microparticle-Based PLGA/PLA Microspheres on RGCs. Invest. Ophthalmol. Vis. Sci. 2012;53(10):6025-6034. doi: 10.1167/iovs.12-9898.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: We explored the neuroprotective effects of erythropoietin (EPO)-loaded dextran microparticle-based Poly(DL-lactide-co-glycolide)/Poly(DL-lactide) (PLGA/PLA) microspheres (EPO-dextran PLGA/PLA microspheres) on retinal ganglion cells (RGCs) in optic nerve crush rats for a prolonged period of time.

Methods.: EPO-dextran PLGA/PLA microspheres were prepared first by a novel solid-in-oil-in-water (S/O/W) technique. Then, the in vitro EPO release profile was assessed. Afterward, the bioactive effect of EPO released from EPO-dextran PLGA/PLA microspheres was explored in vitro on the retinal explants. Lastly, the neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs were evaluated in optic nerve crush rats with TUNEL staining for apoptotic RGCs. The level of glial fibrillary acidic protein (GFAP) expressed in retina was explored by immunohistochemistry staining. Survival RGCs were observed by DiI retrograde labeling using a DiI fluorescent tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate).

Results.: The results demonstrated that a sustained release of EPO from PLGA/PLA microspheres could last for at least 60 days. EPO released from the microspheres showed as efficaciously neuroregenerative as EPO protein solution on retinal explants (P = 0.2554 for neurite density, P = 0.1004 for neurite length). TUNEL staining revealed that EPO-dextran PLGA/PLA microspheres remarkably reduced RGCs death when compared to the control (untreated) group (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush). Increased GFAP expression in retina was reduced greatly in EPO-dextran PLGA/PLA microspheres-administrated rats two weeks post optic nerve crush. DiI retrograde labeling revealed that a single injection of EPO-dextran PLGA/PLA microspheres significantly promoted RGCs survival (P < 0.01 at four and eight weeks post-crush).

Conclusions.: A single intravitreal injection of EPO-dextran PLGA/PLA microspheres appeared to have a prolonged protective effect on RGCs in optic nerve crush rats. The PLGA/PLA microspheres may be a feasible protein delivery system, such as EPO, to intravitreal injection for retinal degeneration diseases.

Introduction
Retinal diseases, such as glaucoma, age-related macular degeneration, and ischemia, are major causes of blindness due to retinal neural cell degeneration. Neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) 1,2 and ciliary derived neurotrophic factor (CNTF), 3,4 have been shown to be neuroprotective effectively in these disease models. Erythropoietin (EPO), which historically was best known for its hematopoietic properties, recently has been proved to exhibit neuroprotective effects on neurons in the retina and central nervous system. It is known to prevent apoptosis and protect neuronal cells from acute damage, such as optic nerve injuries, 5 retinal ischemia, 6 and oxidative damage, 7 and chronic degeneration diseases, such as retinitis pigmentosa, 8 glaucoma, 9 and diabetic retinopathy. 10,11 Direct delivery of EPO into the eye protects retinal ganglion cells (RGCs), 5,9 photoreceptors, 8,12 retinal pigment epithelial cells, 7,13 and the retinal vascular system. 11 Furthermore, EPO has been estimated to be neuroregenerative. 14 It stimulates neurite regrowth of axotomized RGCs in vitro 15 and in vivo. 16 In addition, the safety and efficacy of intravitreal injection of EPO has been proved in animals. 12  
Intravitreal administration may be an effective means of delivering therapeutic levels of EPO quickly and with minimal systemic side effects, especially for a long-term, high-dose treatment. However, EPO has a half-life of 6 to 8 hours, and to maintain the vitreous concentration of EPO in the therapeutic range, frequent injections often are needed, which may increase complications due to the invasive procedure, such as cataract formation, vitreous hemorrhage, and endophthalmitis. 17  
Sustained drug delivery devices offer an excellent alternative to multiple intravitreal injections. The most popular device is the microsphere, made of Poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA), 18 which has the inherent advantages of biodegradability and biocompatibility; it degrades into metabolic lactic and glycolic acids that are eliminated readily from the body. 19 In vivo studies have demonstrated the biodegradability, safety, and feasibility of intravitreal injection of PLGA microspheres. 20,21  
However, there are problems in the PLGA or PLA microsphere technique for preparing a protein drug sustained release system, due to the highly fragile structure of protein. In preparing for the microsphere procedure, these macromolecules (protein drugs) must be protected from water–solvent or water–air interfacial tension and from the hydrophobic environment of the polymeric matrix, the known causes of protein denaturing, aggregating, and immunogenicity. 22 Formulating proteins into solid particles before encapsulation into polymeric systems (e.g., PLGA) has been reported as an effective approach to endow proteins with immobility and resistance to organic solvents used to dissolve polymers. 23,24 However, the particle-forming process itself often is associated with hazardous conditions to proteins. For examples, complexation with bivalent metal ions used successfully to prepare particles of human growth hormone (hGH) 24 resulted in aggregation of EPO. 25  
In our study, we addressed the protein stability issues discussed above using a unique system of stabilized aqueous–aqueous “emulsion,” invented by Jin laboratory 22,26,27 to formulate EPO-dextran particles, after which we encapsulated the particles into PLGA/PLA to form EPO-dextran PLGA/PLA microspheres for a sustained release of EPO by the solid-in-oil-in-water (S/O/W) method. We explored the effects of releasing EPO from the microspheres on regrowth neurites from retinal explants in vitro, and the prolonged neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs in optic nerve crush rats by a single intravitreal injection. 
Materials and Methods
Preparation of EPO-Dextran-PLGA/PLA Microspheres
The preparation of EPO-dextran-PLGA microspheres was divided into two steps. First, a temperature-induced phase separation method was used to prepare EPO-dextran particles. 26 Briefly, a co-solution containing EPO (0.003% wt/wt; Shanghai Clonbiotech Co. Ltd., Shanghai, China), dextran 76,000 (1% wt/wt; Sigma, St. Louis, MO), and polyethylene glycol (PEG 8000, 8% wt/wt) was prepared in a 4°C water bath. After freezing at −20°C for 8 to 12 hours, the mixture was lyophilized to powder under 5.25 × 10−3 Pa for 24 hours using a laboratory freeze-dryer (Christ ALPHA 1-2; Martin Christ, Lower Germany). The lyophilized powders were re-suspended in dichloromethane to dissolve the PEG continuous phase, followed by centrifugation at 12,000 revolutions per minute (rpm) for 5 minutes on a microcentrifuge (Eppendorf 5415D; Eppendorf, Hamburg Germany). to remove the dissolved PEG. This washing procedure was repeated five times, and the pellets were evaporated under 1.33 Pa for 24 hours using a vacuum dryer (Fuma DZF-3; Shanghai Fuma Co. Ltd., Shanghai, China). 
Second, EPO-dextran-PLGA/PLA microspheres were prepared by using a S/O/W method. 28 In brief, the prepared EPO-loaded dextran particles were suspended in a dichloromethane solution of PLGA/PLA (Lakeshore Biopolymers Inc., Cincinnati, OH) (PLGA:PLA = 4:6, 12.5% wt/wt). The ratio of dextran particles to PLGA/PLA was 1:20 (wt/wt). The resulting suspension then was added to 5 mL of a pre-cooled hydrophilic continuous phase containing 1% (wt/wt) PVA and 5% (wt/wt) NaCl (forming a S/O/W emulsion) and stirred at 2000 rpm for 30 seconds using a magnetic mixer (Sile 98-1; Shanghai Sile Co. Ltd., Shanghai, China) at 4°C to form embryonic composite microspheres. The microspheres sample was transferred immediately into 1 L of 10% (wt/vol) NaCl solution at 0°C under gentle stirring (100 rpm) by an electromotive stirrer (Xinhang JJ-1; Jintan Xinhang Co. Ltd., Jiangsu, China) to extract the organic solvent and harden the embryonic microspheres. The process lasted 4 hours, after which the hardened microspheres were rinsed with distilled water and lyophilized again before storage. Blank microspheres were prepared in the same manner, except that no EPO-loaded dextran particles were added. 
Morphology of EPO-Dextran-PLGA/PLA Microspheres
To observe the shape and size of the microspheres, scanning electron microscopic (SEM) images were taken, using an FEI SIRION 200 system (FEI Co., Hillsboro, OR) at 5KeV sputtering energy. Powder samples were attached on a metal stub using a double-sided adhesive and exposed to gold spray under argon atmosphere for 10 minutes. 
In Vitro Release Profile of EPO-Dextran-PLGA Microspheres
EPO standard solutions of different concentrations were prepared to test the absorbance concentration relationship. After the solutions were assayed using an ELISA kit (R&D DEP00; R&D Systems, Minneapolis, MN), the standard curve was obtained: C = 64.1 × A 0.167 (r2 = 0.9999; A, absorption; C, concentration; linear range 2.5 mIU/mL∼100 mIU/mL). 
The composite microspheres (20 mg), prepared as described above, were added to a 4 mL sample vial containing 1 mL of PBS buffer (pH 7.4) and shaken in an incubator (KYC 100C; Fuma Co. Ltd., Shanghai, China) at 110 rpm and 37°C. The release medium was collected after centrifugation and replaced with the same amount of fresh buffer in the same day. All the release media were assayed using the same procedure as the standard solution. The concentrations were calculated using the standard curve relation equation. 
Animals
The in vitro experiments were conducted with 3-day-old Sprague-Dawley rats, and the in vivo experiments were conducted with adult Sprague-Dawley rats weighing approximately 250 g. The adult animals were maintained under a 12-hour light/12-hour dark cycle (7:00 AM–7:00 PM), and standard rodent chow and water were provided ad libitum. All experiments and animal care procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines of the University of Fudan Institutional Animal Care and Use Committee. 
Three-Dimensional Collagen Gel Culture of Retinal Explants
The 3-day-old rats were euthanized humanely by exposure to a rising concentration of carbon dioxide followed by cervical dislocation. Immediately, both eyes were enucleated and immersed in ice-cold Hank's balanced salt solution (HBSS) containing penicillin (100 U/mL) and streptomycin (100 μg/mL). The retinas were dissected under sterile conditions. With sharp blades, 500 μm square retinal explants were dissected 1.5 mm from the optic nerve head and were placed in 100 μL liquid collagen solution on dishes pre-coated with poly-L-lysine (10 μg/mL), with the nerve fiber layer facing downward. The retinal explants were fixed in collagen gel (Chemicon Co. Ltd., Kyoungki-Do, Korea) and cultured at 37°C in a 5% CO2 atmosphere in serum-free MEM (Invitrogen, Carlsbad, CA) containing 2.7 mg/mL glucose, 5 μg/mL insulin, 16.1 μg/mL putrescine, 10% bovine serum albumin, 3.7 mg/mL NaHCO3, 5.2 mg/mL Na2SeO3, and 3.6 mg/mL HEPES. EPO protein (EPO group) or EPO-PLGA/PLA releasing solution (EPO-PLGA/PLA group) was added to the retinal explants, for a final concentration of 1 IU/mL in the culture medium. The same amount of blank PLGA/PLA microsphere-releasing solution (PLGA/PLA group) was added to the culture medium, and nothing was added as a control group. Each day, we examined the retinal explants in culture under a phase-contrast light microscope (Axiovert 40 CFL; Carl Zeiss, Thornwood, NY). The mean density and length of neurites growing out from retinal explants were calculated at six days. Briefly, the mean density was expressed by the ratio of total number of the outgrowing neurites longer than 50 μm to the circumference of the explant. The mean length was expressed by ( a 1 × n 1 + a 2 × n 2 + a 3 × n 3 + ... + a n × n n ) / ( n 1 + n 2 + n 3 + ... + n n ) , where a1 , a2 , a3 ... an were the length of neurites elongated outside the retinal explant that were longer than 50 μm, and n1 , n2 , i... nn were the numbers of the neurites. 
Optic Nerve Crush Procedure
All the animals under experimentation were anesthetized with a mixture of ketamine (40 mg/kg body weight [BW]) and xylazine (4 mg/kg BW) by an intramuscular injection, as well as topical 0.4% oxybuprocaine eye drops (Santen Co. Ltd., Osaka, Japan). Then, 0.3% ofloxacin ophthalmic ointment (Santen Co. Ltd.) was applied to the ocular surface to prevent infection and corneal desiccation after surgery. Optic nerve crush injury was conducted as described previously. 28 Briefly, the left optic nerve was exposed intraorbitally and subjected to a 60-second crush 2 mm behind the globe with a 70-g micro-clip (Shanghai Medical Instruments Co. Ltd., Shanghai, China). A sham operation (the left optic nerve was exposed intraorbitally without crush) was performed and served as normal control. We examined the rat fundus ophthalmoscopically immediately after the injury to confirm the patency of the central retinal artery. 
Intravitreal Injection
Immediately after the optic nerve crush was performed, the animals received a single intravitreal injection of EPO-dextran-PLGA microspheres 1 mg suspended in 0.01 M PBS (corresponding to 20 IU EPO [1 μg EPO = 160 IU EPO]; EPO-PLGA/PLA group), or a single injection of 1 mg blank PLGA/PLA microspheres suspended in 0.01 M PBS (PLGA/PLA group). The rats underwent intravitreal administration of 5 IU EPO (EPO group) or 0.01 M PBS (PBS group) immediately after the crush and every two weeks afterward. The optic nerve crushed animals that underwent no injection were regarded as the control (untreated) group. The sham operation animals did not receive any injection (normal control group). All injections were performed on the operation eyes. 
TUNEL Labeling
At each time point (five days, one week, and two weeks after optic nerve crush) for each group, animals were anesthetized and perfused transcardially with 200 mL 0.9% saline and 4% phosphate-buffered paraformaldehyde (PFA). The eyes were enucleated and fixed by immersion in 4% PFA for 2 hours after the removal of the cornea, lens, and vitreous body. After being dehydrated in graded sucrose solutions (20–30% in PBS) at 4°C overnight, the eyecups were embedded in an optimal cutting temperature compound (Tissue-Tek; Ted Pella, Inc., Redding, CA) at −20°C. Cryostat sections 10 μm thick were obtained. TUNEL reactions were performed using an in situ apoptosis detection kit (Calbiochem; Merk KGaA, Darmstadt, Germany), according to the manufacturer's protocol to detect RGC death. Mounting media containing 40,6-diamidino-2-phenylindole (DAPI; Roche Applied Science, Mannheim, Germany) was used on all the slides to image the nuclei. TUNEL-positive cells were imaged using a fluorescence microscope (Leica Microsystems, Bensheim, Germany). TUNEL-positive RGCs were counted in four areas of each section from seven different eyes. The number of apoptotic RGCs was expressed by the ratios of TUNEL-positive RGCs to the total number of RGCs nuclei stained with DAPI in the RGC layer. 
Glial Fibrillary Acidic Protein (GFAP) Immunohistochemical Staining
Cryostat sections were obtained as described above. Nine rats were used in each group at two weeks post-crush. GFAP immunohistochemistry staining was performed. Briefly, cryostat sections were put in 0.01% tween 20 at room temperature for 5 minutes, and incubated with 5% BSA and 1% goat serum mixture for 1 hour. Monoclonal mouse anti-rat primary antibody to GFAP (1:200; Invitrogen) was applied for 24 hours at room temperature. After three times in 0.01 M PBS, goat anti-mouse fluorescent secondary antibody (1:500; Invitrogen) was applied for 1 hour and followed by three washes in 0.01 M PBS. Cell nuclei were labeled by DAPI (1 μg/ml; Roche) for 5 minutes, followed by three washes in 0.01 M PBS, and mounted in 1:1 PBS and glycerol (by volume). All preparations were observed under a fluorescent microscope (MPS 30; Leica Microsystems). Quantification of GFAP expressed in the retina was performed as described previously. 29 Briefly, the ratio of the area of the GFAP-positive signal versus the total retinal area in each field was determined with IPP 6.0 and expressed as mean density of image arbitrary units (IAUs, mean ± SD). All images were analyzed with IPP 6.0 and evaluated by an experienced masked histopathologist. 
Retrograde Labeling of RGCs
Seven days before the rats were euthanized humanely, the RGCs were labeled retrogradely with a DiI fluorescent tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR) applied to both superior colliculi (SC), as described previously. 30 Briefly, under anesthesia, both sides of the SC were exposed and 5 μL of a DiI suspension (150 mg/mL in N,N-dimethylformamide) were injected on each side. 
Four and eight weeks after the optic nerve crush was performed, the animals (n = 7, at each time point in each group) were perfused as described above, and whole mount retinas were examined under a fluorescence microscope (Leica Microsystems). Three selected areas at 2, 3, and 4 mm from the optic disc were photographed in the four retinal quadrants at a final magnification of ×200. The DiI-labeled cells in the photographs were counted, and the density of labeled RGCs/mm2 was calculated by averaging the 12 counts in each retina. Data were expressed as the average density of survival RGCs per mm2 in each retina of seven rats in each group at each time point (mean ± SD). 
Statistical Analysis
All data were expressed as mean ± SD and analyzed with the Bonferroni tests after one-way ANOVA. Statistical significance was declared at P < 0.05. All calculations were performed using SPSS statistical software program. 
Results
Morphology and In Vitro Release of EPO-Dextran-PLGA/PLA Microspheres
EPO-dextran-PLGA/PLA microspheres were observed using SEM. As shown in Figure 1, the microspheres possessed a spherical shape, smooth surfaces, and diameters ranging from 40 to 100 μm. The microsphere sizes meet the criteria for formulating composite sustained-release polymer microspheres without causing severe burst release, in that the diameter of the inner particles should be less than 1/20 of diameter of the microspheres. 
Figure 1. 
 
EPO-dextran-PLGA/PLA microspheres analyzed by SEM.
Figure 1. 
 
EPO-dextran-PLGA/PLA microspheres analyzed by SEM.
The EPO sustained-release profile was achieved with an initial burst release of approximately 20% of the total EPO loadings released on the first day and followed with a smooth release within a month. Finally, more than 90% of loadings were released in 60 days (Fig. 2). Eventually, the total bounding efficacy of EPO in the EPO-dextran-PLGA/PLA microspheres was approximately 83%. 
Figure 2. 
 
In vitro release profile of EPO from EPO-dextran-PLGA/PLA microspheres (n = 3).
Figure 2. 
 
In vitro release profile of EPO from EPO-dextran-PLGA/PLA microspheres (n = 3).
Mean Density and Length of Neurite Outgrowth from Retinal Explants
To measure in vitro EPO activity, the ability of EPO released from EPO-dextran-PLGA/PLA microspheres to enhance neurite outgrowth from retinal explants was investigated. The culture results showed that some small neurites appeared on the edge of the retinal explants in collagen gel at 48 hours, and that grew longer by 72 hours to form the typical shape of neurites. The neurites could be distinguished from the extending processes of fibroblasts and other non-neuronal cells by the unique structure of the spread growth cones at the tips of the neurites. 31 Neurites growing out from retinal explants reach a peak at six days in culture. The average density and length of outgrowing neurites that were elongated outside the retinal explants were counted and analyzed on that day. There was a possibility that the counted number of neurites in the present experiments might have erroneously included the processes of non-neuronal cells, but their numbers were negligible. 
The results revealed that the EPO and EPO-PLGA/PLA groups had a remarkably higher number of neurites in density and length, compared to the control group (P < 0.01); the EPO group had an average density of 48.50 ± 8.57/mm2 and average length of 311.88 ± 12.69μm, and the EPO-PLGA/PLA group had an average density of 48.75 ± 4.65/mm2 and average length of 302.88 ± 11.15 μm (Figs. 3, 4). No significant differences were found between the EPO and EPO-PLGA/PLA groups (P = 0.2554) in neurite density, and the same results were found for the EPO and EPO-PLGA/PLA groups (P = 0.1004) in neurite length. The PLGA/PLA group had similar average density (29.25 ± 4.65/mm2) and length (148.88 ± 7.57 μm) of neurites as the control group (26.25 ± 2.71/mm2 in density and 158.75 ± 10.31 μm; P values were 0.1328 and 0.0728 for density and length, respectively; Figs. 3, 4). 
Figure 3. 
 
Average density of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 3. 
 
Average density of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 4. 
 
Average length of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 4. 
 
Average length of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
TUNEL Staining for Apoptotic RGCs
No TUNEL-positive cells were found in the RGC layer of the normal retina. In the untreated group, many apoptotic RGCs could be detected (Fig. 5) in the initial five days after crush, with an apoptotic rate of 11.24 ± 1.21%. The rate was 2.78 ± 1.04% in the EPO-PLGA/PLA group, 2.50 ± 1.29% in the EPO group, 10.77 ± 1.33% in the PLGA/PLA group, and 11.30 ± 1.34% in the PBS group (Table 1). The apoptotic rate reached a peak one week after crush in the untreated (30.30 ± 3.88%), PLGA/PLA (32.59 ± 3.36%), and PBS (31.86 ± 3.31%) groups. Higher apoptotic rates also were detected in the EPO-PLGA/PLA (11.42 ± 2.12%) and EPO (11.85 ± 2.06%) groups at one week than at five days. At two weeks, the apoptotic rate declined in the untreated (14.85.30 ± 2.93%), PLGA/PLA (15.81 ± 3.98%), and PBS (16.37 ± 3.71%) groups. However, the rate remained almost unchanged in the EPO-PLGA/PLA (11.91 ± 2.14%) and EPO (12.84 ± 2.55%) groups. The results revealed that the apoptotic rates in the EPO-PLGA/PLA and EPO groups were statistically significant (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush) when compared to the untreated, PLGA/PLA, and PBS groups (Table 1). At the same time, no differences were found between the EPO-PLGA/PLA and EPO groups, or between the untreated, PLGA/PLA, and PBS groups. 
Figure 5. 
 
TUNEL staining of apoptotic RGCs in RGC layer one week post-crush. (A) No apoptotic RGCs were found in the RGC layer of the normal retina. TUNEL-positive RGCs could be identified in all operation groups. In the untreated group (B), a substantial number of TUNEL-positive RGCs were detected. However, fewer apoptotic RGCs were found in the EPO-PLGA/PLA and EPO groups (C, D) compared to the untreated group. No differences were found between the EPO-PLGA/PLA and EPO groups. Almost the same number of TUNEL-positive RGCs was detected in the PLGA/PLA and PBS groups (E, F) when compared to the untreated group.
Figure 5. 
 
TUNEL staining of apoptotic RGCs in RGC layer one week post-crush. (A) No apoptotic RGCs were found in the RGC layer of the normal retina. TUNEL-positive RGCs could be identified in all operation groups. In the untreated group (B), a substantial number of TUNEL-positive RGCs were detected. However, fewer apoptotic RGCs were found in the EPO-PLGA/PLA and EPO groups (C, D) compared to the untreated group. No differences were found between the EPO-PLGA/PLA and EPO groups. Almost the same number of TUNEL-positive RGCs was detected in the PLGA/PLA and PBS groups (E, F) when compared to the untreated group.
Table 1. 
 
Apoptotic Rate (%) of RGCs of Each Group at Five Days, One Week, and Two Weeks Post-Crush
Table 1. 
 
Apoptotic Rate (%) of RGCs of Each Group at Five Days, One Week, and Two Weeks Post-Crush
Group N Time Post-Crush
5 Days 1 Week 2 Weeks
Untreated 7 11.24 ± 1.21 30.30 ± 3.88 14.85 ± 2.93
EPO-PLGA/PLA 7 2.78 ± 1.04* 11.42 ± 2.12* 11.91 ± 2.14†
EPO 7 2.50 ± 1.29* 11.85 ± 2.06* 12.84 ± 2.55†
PLGA/PLA 7 10.77 ± 1.33 32.59 ± 3.36 15.81 ± 3.98
PBS 7 11.30 ± 1.34 31.86 ± 3.31 16.37 ± 3.71
Immunohistochemistry Staining of GFAP Expressed in Retina
GFAP expression was localized mainly to the inner limiting membrane in normal retina. Optic nerve crush injury resulted in significantly increased GFAP expression in retina and reached a peak by two weeks (Fig. 6). Meanwhile, animals in the PLGA/PLA and PBS groups also displayed a significantly high level of GFAP expression in retina (Fig. 6), and no differences were found when compared to the untreated animals (P = 0.4180 in the PLGA/PLA group, P = 0.2474 in the PBS group). However, increased GFAP level was remarkably lower in the EPO-PLGA/PLA group (Figs. 6, 7; P < 0.01 when compared to the untreated group). Similar results were observed in the EPO group. 
Figure 6. 
 
Immunohistochemistry staining level of GFAP expressed in retina two weeks after optic nerve crush. (A) Limited amount of GFAP was expressed in the inner limiting membrane in the normal retina. (B) The level of GFAP reached a peak at two weeks in the untreated group. Increased GFAP expression was greatly lower in the EPO-PLGA/PLA (C) and EPO (D) groups compared to the untreated group (B). However, the level of GFAP expressed in the PLGA/PLA (E) and PBS (F) groups was almost the same as the level in the untreated group (B).
Figure 6. 
 
Immunohistochemistry staining level of GFAP expressed in retina two weeks after optic nerve crush. (A) Limited amount of GFAP was expressed in the inner limiting membrane in the normal retina. (B) The level of GFAP reached a peak at two weeks in the untreated group. Increased GFAP expression was greatly lower in the EPO-PLGA/PLA (C) and EPO (D) groups compared to the untreated group (B). However, the level of GFAP expressed in the PLGA/PLA (E) and PBS (F) groups was almost the same as the level in the untreated group (B).
Figure 7. 
 
Level of GFAP expressed in retina two weeks after optic nerve crush. **P < 0.01 when compared to the untreated group.
Figure 7. 
 
Level of GFAP expressed in retina two weeks after optic nerve crush. **P < 0.01 when compared to the untreated group.
Retrograde Labeling of RGCs
To determine the prolonged neuroprotective effect of EPO-dextran PLGA/PLA microspheres on RGC survival, we counted DiI-labeled RGCs in flat-mounted retinas four and eight weeks post-crush (Fig. 8 and Table 2). The mean number of labeled RGCs in the intact normal retinas was 2368.52 ± 161.98 cells/mm2 (mean ± SD). Four weeks after optic nerve crush, the mean number of RGCs had decreased significantly to 755.35 ± 53.23 cells/mm2 in the untreated group. The number was 1326.38 ± 67.37 cells/mm2 and 1428.51 ± 100.99 cells/mm2 in the EPO-PLGA/PLA and EPO groups, respectively. This is significantly higher than in the PLGA/PLA (816.01 ± 51.16 cells/mm2), PBS (808.83 ± 75.55 cells/mm2), and untreated groups (P < 0.01, Table 2). 
Figure 8
 
DiI labeling of survival RGCs in flat-mounted retinas at four weeks post-crush. RGCs was labeled in normal retina (A). A large amount of RGCs was lost four weeks after optic nerve crush (B). A higher number of survival cells remained in the EPO-PLGA/PLA and EPO groups (C, D) than in the untreated group. However, the survival RGCs in the PLGA/PLA and PBS groups (E, F) seemed similar to those in the untreated group.
Figure 8
 
DiI labeling of survival RGCs in flat-mounted retinas at four weeks post-crush. RGCs was labeled in normal retina (A). A large amount of RGCs was lost four weeks after optic nerve crush (B). A higher number of survival cells remained in the EPO-PLGA/PLA and EPO groups (C, D) than in the untreated group. However, the survival RGCs in the PLGA/PLA and PBS groups (E, F) seemed similar to those in the untreated group.
Table 2. 
 
Mean Density of Survival RGCs (cells/mm2) at Four Weeks and Eight Weeks Post-Crush
Table 2. 
 
Mean Density of Survival RGCs (cells/mm2) at Four Weeks and Eight Weeks Post-Crush
Group N Time Post-Crush
4 Weeks 8 Weeks
Untreated 7 755.35 ± 53.23 537.49 ± 37.05
EPO-PLGA/PLA 7 1326.38 ± 67.37* 911.91 ± 35.41*
EPO 7 1428.51 ± 100.99* 1118.80 ± 81.91*
PLGA/PLA 7 816.01 ± 51.16 581.50 ± 46.36
PBS 7 808.83 ± 75.55 570.70 ± 63.43
At eight weeks post-crush, the mean number of RGCs had decreased to 537.49 ± 37.05 cells/mm2 in the untreated group. The EPO-PLGA/PLA and EPO groups maintained a significantly higher number, that is 911.91 ± 35.41 cells/mm2 and 1118.80 ± 81.91 cells/mm2, respectively compared to the PLGA/PLA (581.50 ± 46.36 cells/mm2), PBS (570.70 ± 63.43 cells/mm2), and untreated groups (Table 2). However, no statistically significant differences were detected between the EPO-PLGA/PLA and EPO groups, or between the PLGA/PLA, PBS, and untreated groups at four and eight weeks after optic nerve crush. 
Discussion
In our study, we formulated EPO-dextran PLGA/PLA microspheres by loading EPO into dextran particles first, and then microencapsulating the particles in PLGA/PLA microspheres using the S/O/W method. The first step protected the protein from denature during the manufacturing and releasing process, and the S/O/W method showed stable sustained release of EPO for a prolonged time without aggregation. 27  
It is demonstrated that, despite the neuroprotective effects on the retina in the central nervous system (CNS), EPO induces neuroregeneration in vitro and in vivo. 14,15 EPO is capable of stimulating neurite outgrowth in neurons of rat retina explants directly through EPO-receptors on RGCs. 15 Therefore, we explored the bioactivity of EPO released from the microspheres by observing the effect of EPO on the neurite outgrowth from retinal explants in a three-dimensional collagen gel culture system. The regenerated neurites from the retinal explants were embedded in the stable condition of collagen gel, which protected retinal tissue and made it easy to observe neuronal regeneration over a long period of culture. 32 Our results revealed that EPO released from the EPO-dextran PLGA/PLA microspheres (EPO-PLGA/PLA group) had almost the same effects on neurite regeneration as the EPO protein (EPO group) in neurite density and length, indicating the remaining bioactivity of EPO through the complicated microsphere manufacturing and release process. No significant differences in neurite density or length were found between the PLGA/PLA and control groups, or between the EPO-PLGA/PLA and EPO groups. These results, to a certain degree, suggested that the degraded products of PLGA/PLA solution were biocompatible and biologically safe, and that the EPO-dextran PLGA/PLA microsphere-releasing solution was as efficacious and safe as EPO protein solution. 
In vivo, we investigated whether a single intravitreal injection of EPO-dextran PLGA/PLA microspheres had a prolonged neuroprotective effect on RGCs after optic nerve crush. We reported previously that EPO-loaded PLGA/PLA microspheres significantly promoted RGC survival for a prolonged period by an intraperitoneal injection in optic nerve crush rats. 28 It is good evidence for applying EPO sustained-release microspheres in CNS degeneration diseases. However, in the consideration of ocular application, the need for consistent availability of bioactive level of EPO in the retina will require a relatively high level of the protein systemically, which may increase systemic complications, such as excess viscosity and blood pressure. 33,34 Therefore, in our study, we applied EPO-dextran PLGA/PLA microspheres by intravitreal injection. 
In the optic nerve crush rats, apoptotic RGCs appeared at five days post-crush, accompanied with a very high number of TUNEL-positive cells. The apoptosis reached a peak at one week and was observable for up to six weeks, with most TUNEL-positive cells appearing in the first two weeks post-crush. No TUNEL-positive RGCs cells were seen ten weeks post-crush. This was consistent with the previous report. 35 This long time frame for possible therapeutic intervention was an attractive feature for in vivo investigations on the prevention of retinal degeneration diseases. In our study, our results revealed that at five days, one week, and two weeks after optic nerve crush, significantly fewer apoptotic RGCs were detected in the EPO-PLGA/PLA and EPO groups, and no statistical differences were observed between the two groups. In the PLGA/PLA and PBS groups, the RGCs were in the same apoptotic profile as the RGCs in the untreated group, but not those in the EPO-PLGA/PLA or EPO group. In the EPO-PLGA/PLA and EPO groups, the apoptotic RGCs decreased over the course of the post-crush monitoring. Unlike the untreated group, the apoptotic RGCs did not have an apoptotic peak, and a higher apoptotic rate was detected at one week than at five days after optic nerve crush. However, the rate remained stable two weeks post-crush compared to one week. Then, the apoptotic cells decreased four weeks post-crush. These results revealed that EPO-dextran PLGA/PLA microspheres and EPO might reduce RGC death and provide a neuroprotective effect by promoting RGC survival and delaying part of RGC apoptosis. It was demonstrated that, in retina injury, EPO promotes antiapoptotic proteins and suppresses apoptotic proteins by EPO/EPO receptor signaling through ERK and Akt pathways. 5,36 Further investigation may be needed to determine which part of RGC apoptosis was delayed by EPO. 
PLGA/PLA degrades into metabolic lactic and glycolic acids, 19 which may cause low degrade inflammation, and result in further RGCs death and retinal injury. Glial cell activation is found commonly after retinal injury, 37 and has been thought as an important factor contributing to RGCs death. 3840 EPO is neuroprotective by reducing reactive gliosis and stimulating neurotrophin expression in retina. 41 In our study, greatly increased level of GFAP was expressed in retina at two weeks post crush in the untreated group. PLGA/PLA microspheres administration did not cause further increased GFAP expression. The results demonstrated that PLGA/PLA and its degraded products caused negative effects on glial activation in retina. EPO-dextran PLGA/PLA microspheres reduced significantly the increased level of GFAP. No differences were found between the EPO-PLGA/PLA and EPO groups. It revealed that the neuroprotective effect of EPO-dextran PLGA/PLA microspheres was as efficacious as the protein EPO without further side effects. 
The number of survival RGCs was explored by retrogradely labeling four and eight weeks post-crush. Our results revealed that the mean density of survival RGCs in the EPO-PLGA/PLA group was not significantly different from that in the EPO group at the two time points. It indicated the prolonged neuroprotective effect of a single intravitreal injection of EPO-dextran PLGA/PLA microspheres. The efficacy was almost as effective as multiple injections of EPO protein solution, regardless of the side effects caused by repeated injections. In addition, there were no statistically significant differences in the number of survival RGCs of the apoptotic rate of the RGCs among the PLGA, PBS, and untreated groups, and no significant differences in RGC apoptosis were found between the EPO-PLGA/PLGA and EPO groups. All these results might suggest that the single intravitreal injection of EPO-dextran PLGA/PLA microspheres or blank PLGA/PLA microspheres did not cause further RGC death, other than a certain extent of optic injury. It might indicate the safety and feasibility 20,21 of the sustained-release microspheres by intravitreal injection for retinal protein drug delivery. However, further studies will be important for demonstrating the functional consequences of the treatments, and to explore the detailed mechanism of EPO on RGCs. Meanwhile, our work will be to extend the work to large animals and to explore new neurotrophic factors loaded in PLGA/PLA microspheres for ocular applications. 
In summary, EPO released from the PLGA/PLA microspheres was showed to be effectively bioactive, and it stimulated significantly the regeneration of neurites from retinal explants. A single intravitreal injection of EPO-dextran PLGA/PLA microspheres apparently promoted RGC survival in optic nerve crush rats for a prolonged period of time. As such, an effective and feasible drug delivery system might be to apply EPO or even other protein drugs ocularly to prevent retinal degenerative diseases. 
References
Chen H Weber AJ. BDNF enhances retinal ganglion cell survival in cats with optic nerve damage. Invest Ophthalmol Vis Sci . 2001;42:966–974. [PubMed]
Zhang CW Lu Q You SW CNTF and BDNF have similar effects on retinal ganglion cell survival but differential effects on nitric oxide synthase expression soon after optic nerve injury. Invest Ophthalmol Vis Sci . 2005;46:1497–1503. [CrossRef] [PubMed]
Cen LP Luo JM Zhang CW Chemotactic effect of ciliary neurotrophic factor on macrophages in retinal ganglion cell survival and axonal regeneration. Invest Ophthalmol Vis Sci . 2007;48:4257–4266. [CrossRef] [PubMed]
Lingor P Tönges L Pieper N ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells. Brain . 2008;131 (Pt 1):250–263. [PubMed]
Weishaupt JH Rohde G Pölking E Siren AL Ehrenreich H Bähr M. Effect of erythropoietin axotomy-induced apoptosis in rat retinal ganglion cells. Invest Ophthalmol Vis Sci . 2004;45:1514–1522. [CrossRef] [PubMed]
Junk AK Mammis A Savitz SI Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci U S A . 2002;99:10659–10664. [CrossRef] [PubMed]
Wang Z Shen L Tu LL Erythropoietin protects retinal pigment epithelial cells from oxidative damage. Free Radic Biol Med . 2009;46:1032–1041. [CrossRef] [PubMed]
Rex TS Wong Y Kodali K Merry S. Neuroprotection of photoreceptors by direct delivery of erythropoietin to the retina of the retinal degeneration slow mouse. Exp Eye Res . 2009;89:735–740. [CrossRef] [PubMed]
Zhong L Bradley J Schubert W Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice. Invest Ophthalmol Vis Sci . 2007;48:1212–1218. [CrossRef] [PubMed]
Hernández C Simó R. Erythropoietin produced by the retina: its role in physiology and diabetic retinopathy. Endocrine . 2012;41:220–226. [CrossRef] [PubMed]
Zhang J Wu Y Jin Y Intravitreal injection of erythropoietin protects both retinal vascular and neuronal cells in early diabetes. Invest Ophthalmol Vis Sci . 2008;49:732–742. [CrossRef] [PubMed]
Xie Z Chen F Wu X Safety and efficacy of intravitreal injection of recombinant erythropoietin for protection of photoreceptor cells in a rat model of retinal detachment. Eye (Lond.) . 2012;26:144–152. [CrossRef] [PubMed]
Gawad A Schlichting L Strauβ O Zeitz O. Antiapoptotic properties of erythropoietin: novel strategies for protection of retinal pigment epithelial cells. Eye (Lond) . 2009;23:2245–2250. [CrossRef] [PubMed]
King CE Rodger J Bartlett C Esmaili T Dunlop SA Beazley LD. Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection. Exp Neurol . 2007;205:48–55. [CrossRef] [PubMed]
Böcker-Meffert S Rosenstiel P Röhl C Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats. Invest Ophthalmol Vis Sci . 2002;43:2021–2026. [PubMed]
Kretz A Happold CJ Marticke JK Isenmann S. Erythropoietin promotes regeneration of adult CNS neurons via Jak2/Stat3 and PI3K/AKT pathway activation. Mol Cell Neurosci . 2005;29:569–579. [CrossRef] [PubMed]
Prasad AG Schadlu R Apte RS. Intravitreal pharmacotherapy: applications in retinal disease. Compr Ophthalmol Update . 2007;8:259–269. [PubMed]
Sinha V Trehan A. Biodegradable microspheres for protein delivery. J Control Release . 2003;90:261–280. [CrossRef] [PubMed]
Soppimath KS Aminabhavi TM Kulkarni AR Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release . 2001;70:1–20. [CrossRef] [PubMed]
Giordano GG Chevez-Barrios P Refojo MF Garcia CA. Biodegradation and tissue reaction to intravitreous biodegradable poly (D, L-lactic-co-glycolic) acid microspheres. Curr Eye Res . 1995;14:761–768. [CrossRef] [PubMed]
Veloso A Zhu Q Herrero-Vanrell R Refojo MF. Ganciclovir-loaded polymer microspheres in rabbit eyes inoculated with human cytomegalovirus. Invest Ophthalmol Vis Sci . 1997;38:665–675. [PubMed]
Jin T Zhu J Wu F Yuan W Geng LL Zhu H. Preparing polymer-based sustained-release systems without exposing proteins to water-oil or water-air interfaces and cross-linking reagents. J Control Release . 2008;128:50–59. [CrossRef] [PubMed]
Morita T Horikiri Y Yamahara H Suzuki T Yoshino H. Formation and isolation of spherical fine protein microparticles through lyophilization of protein-poly (ethylene glycol) aqueous mixture. Pharm Res . 2000;17:1367–1373. [CrossRef] [PubMed]
Johnson OFL Jaworowicz W Cleland JL The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm Res . 1997;14:730–735. [CrossRef] [PubMed]
Zale SE Burke PA Bernstein H Brickner A inventors. Composition for sustained release of non-aggregated erythropoietin. US Patent US5716644. February 10, 1998.
Yuan W Wu F Geng Y Xu S Jin T. Preparation of dextran glassy particles through freezing-induced phase separation. Int J Pharm . 2007;339:76–83. [CrossRef] [PubMed]
Geng Y Yuan W Wu F Chen J He M Jin T. Formulating erythropoietin-loaded sustained-release PLGA microspheres without protein aggregation. J Control Release . 2008;130:259–265. [CrossRef] [PubMed]
Rong X Mo X Ren T Neuroprotective effect of erythropoietin-loaded composite microspheres on retinal ganglion cells in rats. Eur J Pharm Sci . 2011;43:334–342. [CrossRef] [PubMed]
Zhang X Bao S Lai D Rapkins RW Gillies MC. Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas. Diabetes . 2008;57:1026–1033. [CrossRef] [PubMed]
Dezawa M Takano M Negishi H Mo X Oshitari T Sawada H. Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach. Micron . 2002;33:1–6. [CrossRef] [PubMed]
Takano M Horie H. Critical period for degradation of adult rat retinal ganglion cells and their regeneration capability after axotomy. Neurosci Lett . 1994;175:129–132. [CrossRef] [PubMed]
Oshitari T Dezawa M Okada S The role of c-fos in cell death and regeneration of retinal ganglion cells. Invest Ophthalmol Vis Sci . 2002;43:2442. [PubMed]
Duplay D. Physicians' Desk Reference . Montvale, NJ: Thomson Healthcare Inc.; 2004.
Drüeke TB Locatelli F Clyne N Normalization of hemoglobin level in patients with chronic kidney disease and anemia. New Engl J Med . 2006;355:2071–2084. [CrossRef] [PubMed]
Bien A Seidenbecher CI Böckers TM Sabel BA Kreutz MR. Apoptotic versus necrotic characteristics of retinal ganglion cell death after partial optic nerve injury. J Neurotrauma . 1999;16:153–163. [CrossRef] [PubMed]
Shen J Wu Y Xu JY ERK-and Akt-dependent neuroprotection by erythropoietin (EPO) against glyoxal-AGEs via modulation of Bcl-xL, Bax, and BAD. Invest Ophthalmol Vis Sci . 2010;51:35–46. [CrossRef] [PubMed]
Middeldorp J Hol EM. GFAP in health and disease. Prog Neurobiol . 2011;93:421–443. [CrossRef] [PubMed]
Wang X Tay SS Ng YK. An immunohistochemical study of neuronal and glial cell reactions in retinae of rats with experimental glaucoma. Exp Brain Res . 2000;132:476–484. [CrossRef] [PubMed]
Chen H Weber AJ. Expression of glial fibrillary acidic protein and glutamine synthetase by Müller cells after optic nerve damage and intravitreal application of brain-derived neurotrophic factor. Glia . 2002;38:115–125. [CrossRef] [PubMed]
Woldemussie E Wijono M Ruiz G. Müller cell response to laser-induced increase in intraocular pressure in rats. Glia . 2004;47:109–119. [CrossRef] [PubMed]
Hu LM Luo Y Zhang J EPO reduces reactive gliosis and stimulates neurotrophin expression in Muller cells. Front Biosci (Elite Ed) . 2011;3:1541–1555. [PubMed]
Footnotes
 Supported by the Shanghai Rising-Star Program (10QH1400400, 2006QA14011), the Key Program of Shanghai Science and Technology Commission (10JC1402302), the National Natural Science Foundation of China (NSFC81070764), the New Century Training Program Foundation for Talent by the State Education Commission (NCET-05-0370), the National Basic Research Program of China (No. 2007CB512204/No. 2007CB512205), and the Program of Eye and ENT Hospital of Fudan University (EENT-2010-09).
Footnotes
 Disclosure: X. Rong, None; S. Yang, None; H. Miao, None; T. Guo, None; Z. Wang, None; W. Shi, None; X. Mo, None; W. Yuan, None; T. Jin, None
Figure 1. 
 
EPO-dextran-PLGA/PLA microspheres analyzed by SEM.
Figure 1. 
 
EPO-dextran-PLGA/PLA microspheres analyzed by SEM.
Figure 2. 
 
In vitro release profile of EPO from EPO-dextran-PLGA/PLA microspheres (n = 3).
Figure 2. 
 
In vitro release profile of EPO from EPO-dextran-PLGA/PLA microspheres (n = 3).
Figure 3. 
 
Average density of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 3. 
 
Average density of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 4. 
 
Average length of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 4. 
 
Average length of neurites regenerated from retinal explants. **P < 0.01 when compared to the control group.
Figure 5. 
 
TUNEL staining of apoptotic RGCs in RGC layer one week post-crush. (A) No apoptotic RGCs were found in the RGC layer of the normal retina. TUNEL-positive RGCs could be identified in all operation groups. In the untreated group (B), a substantial number of TUNEL-positive RGCs were detected. However, fewer apoptotic RGCs were found in the EPO-PLGA/PLA and EPO groups (C, D) compared to the untreated group. No differences were found between the EPO-PLGA/PLA and EPO groups. Almost the same number of TUNEL-positive RGCs was detected in the PLGA/PLA and PBS groups (E, F) when compared to the untreated group.
Figure 5. 
 
TUNEL staining of apoptotic RGCs in RGC layer one week post-crush. (A) No apoptotic RGCs were found in the RGC layer of the normal retina. TUNEL-positive RGCs could be identified in all operation groups. In the untreated group (B), a substantial number of TUNEL-positive RGCs were detected. However, fewer apoptotic RGCs were found in the EPO-PLGA/PLA and EPO groups (C, D) compared to the untreated group. No differences were found between the EPO-PLGA/PLA and EPO groups. Almost the same number of TUNEL-positive RGCs was detected in the PLGA/PLA and PBS groups (E, F) when compared to the untreated group.
Figure 6. 
 
Immunohistochemistry staining level of GFAP expressed in retina two weeks after optic nerve crush. (A) Limited amount of GFAP was expressed in the inner limiting membrane in the normal retina. (B) The level of GFAP reached a peak at two weeks in the untreated group. Increased GFAP expression was greatly lower in the EPO-PLGA/PLA (C) and EPO (D) groups compared to the untreated group (B). However, the level of GFAP expressed in the PLGA/PLA (E) and PBS (F) groups was almost the same as the level in the untreated group (B).
Figure 6. 
 
Immunohistochemistry staining level of GFAP expressed in retina two weeks after optic nerve crush. (A) Limited amount of GFAP was expressed in the inner limiting membrane in the normal retina. (B) The level of GFAP reached a peak at two weeks in the untreated group. Increased GFAP expression was greatly lower in the EPO-PLGA/PLA (C) and EPO (D) groups compared to the untreated group (B). However, the level of GFAP expressed in the PLGA/PLA (E) and PBS (F) groups was almost the same as the level in the untreated group (B).
Figure 7. 
 
Level of GFAP expressed in retina two weeks after optic nerve crush. **P < 0.01 when compared to the untreated group.
Figure 7. 
 
Level of GFAP expressed in retina two weeks after optic nerve crush. **P < 0.01 when compared to the untreated group.
Figure 8
 
DiI labeling of survival RGCs in flat-mounted retinas at four weeks post-crush. RGCs was labeled in normal retina (A). A large amount of RGCs was lost four weeks after optic nerve crush (B). A higher number of survival cells remained in the EPO-PLGA/PLA and EPO groups (C, D) than in the untreated group. However, the survival RGCs in the PLGA/PLA and PBS groups (E, F) seemed similar to those in the untreated group.
Figure 8
 
DiI labeling of survival RGCs in flat-mounted retinas at four weeks post-crush. RGCs was labeled in normal retina (A). A large amount of RGCs was lost four weeks after optic nerve crush (B). A higher number of survival cells remained in the EPO-PLGA/PLA and EPO groups (C, D) than in the untreated group. However, the survival RGCs in the PLGA/PLA and PBS groups (E, F) seemed similar to those in the untreated group.
Table 1. 
 
Apoptotic Rate (%) of RGCs of Each Group at Five Days, One Week, and Two Weeks Post-Crush
Table 1. 
 
Apoptotic Rate (%) of RGCs of Each Group at Five Days, One Week, and Two Weeks Post-Crush
Group N Time Post-Crush
5 Days 1 Week 2 Weeks
Untreated 7 11.24 ± 1.21 30.30 ± 3.88 14.85 ± 2.93
EPO-PLGA/PLA 7 2.78 ± 1.04* 11.42 ± 2.12* 11.91 ± 2.14†
EPO 7 2.50 ± 1.29* 11.85 ± 2.06* 12.84 ± 2.55†
PLGA/PLA 7 10.77 ± 1.33 32.59 ± 3.36 15.81 ± 3.98
PBS 7 11.30 ± 1.34 31.86 ± 3.31 16.37 ± 3.71
Table 2. 
 
Mean Density of Survival RGCs (cells/mm2) at Four Weeks and Eight Weeks Post-Crush
Table 2. 
 
Mean Density of Survival RGCs (cells/mm2) at Four Weeks and Eight Weeks Post-Crush
Group N Time Post-Crush
4 Weeks 8 Weeks
Untreated 7 755.35 ± 53.23 537.49 ± 37.05
EPO-PLGA/PLA 7 1326.38 ± 67.37* 911.91 ± 35.41*
EPO 7 1428.51 ± 100.99* 1118.80 ± 81.91*
PLGA/PLA 7 816.01 ± 51.16 581.50 ± 46.36
PBS 7 808.83 ± 75.55 570.70 ± 63.43
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×