August 2009
Volume 50, Issue 8
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Retina  |   August 2009
The Role of αA- and αB-Crystallins in the Survival of Retinal Ganglion Cells after Optic Nerve Axotomy
Author Affiliations
  • Yasunari Munemasa
    From the Jules Stein Eye Institute and
    St. Marianna University School of Medicine, Kawasaki, Japan.
  • Jacky M. K. Kwong
    From the Jules Stein Eye Institute and
  • Joseph Caprioli
    From the Jules Stein Eye Institute and
    Brain Research Institute, University of California at Los Angeles School of Medicine, Los Angeles, California; and the
  • Natik Piri
    From the Jules Stein Eye Institute and
    Brain Research Institute, University of California at Los Angeles School of Medicine, Los Angeles, California; and the
Investigative Ophthalmology & Visual Science August 2009, Vol.50, 3869-3875. doi:10.1167/iovs.08-3138
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      Yasunari Munemasa, Jacky M. K. Kwong, Joseph Caprioli, Natik Piri; The Role of αA- and αB-Crystallins in the Survival of Retinal Ganglion Cells after Optic Nerve Axotomy. Invest. Ophthalmol. Vis. Sci. 2009;50(8):3869-3875. doi: 10.1167/iovs.08-3138.

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

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Abstract

purpose. Stress-induced crystallin expression is commonly viewed as activation of the cell survival mechanism. The authors analyzed the expression of αA- and αB-crystallins in a rat optic nerve transection (ONT) model characterized by specific retinal ganglion cell (RGC) degeneration and determined their role in RGC survival.

methods. ONT was performed on adult Wistar rats. Quantitative and spatial expression were examined with Western blot analysis and immunohistochemistry, respectively. Electroporation was used to deliver αA and αB expression constructs to RGCs. Cell-protective effects of αA and αB overexpression after ONT were determined by RGC density analysis.

results. Expression of αA and αB in the retina was observed predominantly in the ganglion cell layer, where most crystallin-positive cells were colocalized with RGCs. Levels of αA and αB proteins after ONT were decreased 1.6-fold. The effect of αA and αB overexpression on RGC survival was evaluated 7 and 14 days after axotomy. At day 7 after ONT, 1426 ± 70 and 1418 ± 81 RGCs/mm2 were present in retinas electroporated with αA and αB expression constructs, respectively, compared with 1010 ± 121 RGCs/mm2 in sham-transfected or 1016 ± 88 RGCs/mm2 in nontransfected retinas. Numbers of surviving RGCs at 14 days were 389 ± 57 and 353.57 ± 60 cells/mm2 after αA and αB transfection, respectively, compared with 198 ± 29 cells/mm2 after transfection with the vector alone or 206 ± 60 cells/mm2 in nontransfected retinas.

conclusions. Increases of approximately 95% and 75% in RGC survival mediated by αA and αB overexpression, respectively, were observed 14 days after ONT. At day 7, the RGC protective effect of αA and αB overexpression was approximately 40%.

Three main families of mammalian crystallins are α-, β-, and γ-crystallins. α-Crystallins (αA and αB) are homologous to small heat shock proteins and are considered molecular chaperones 1 2 3 4 5 6 ; β- and γ-crystallins share similar sequences, structure, and domain topology and have therefore been grouped together to form a superfamily of βγ-crystallins. 7 Although α- and βγ-crystallins are known to be the major structural components of the lens, these proteins are also expressed in other ocular and nonocular tissues. The role of the α-crystallins outside the lens is primarily attributed to chaperone and cell-protective functions. The function of βγ-crystallins is not well understood, though these proteins were recently associated with axonal regeneration of retinal ganglion cells (RGCs). 8 9 A number of studies reported the upregulation of crystallin genes in various tissues in response to stress or pathologic conditions. An increased αB-crystallin level has been associated with neurologic diseases, including Alexander’s, Creutzfeldt-Jakob, and Parkinson’s diseases. 10 11 12 13 The upregulation of crystallin expression has been reported in damaged retinas, such as from ischemia-reperfusion injury, light injury, and retinal tears, and in retinas of experimental diabetic rats. 14 15 16 17 Elevation in crystallin expression was suggested to be a cellular response mechanism against environmental and metabolic stresses, primarily because αA and αB have been associated with promoting cell survival by increasing cell resistance to stress-inducible apoptosis. 18 19 20 21 22  
Contrary to these observations, retinal gene profiles obtained from animal models of glaucomatous neuropathy showed the downregulation of several members of the crystallin superfamily, including α- and β-crystallins. 23 24 25 26 Quantitative analyses showed the downregulation of crystallin (αA, αB, βA3/A1, βA2, and βA4) expression at the transcriptional (approximately 40%–50%) and the protein (approximately 40%–70%) levels in rat glaucoma model retinas, with approximately 8% ganglion cell loss 2 weeks after intraocular pressure (IOP) elevation. 26 Interestingly, in experimental glaucomatous retinas with more advanced RGC loss (approximately 20%), the levels of crystallin mRNAs were comparable to those of control retinas, but the levels of the corresponding proteins were still lower. These observations suggest dynamic and coordinated regulation of crystallin expression in retinas with induced degeneration of RGCs. 
Here we extend our study on the regulation of crystallin expression associated with RGC degeneration by analyzing the expression of αA- and αB-crystallins in retinas of the rat axotomy model characterized by specific and rapid RGC loss. Furthermore, we determined the effect of the overexpression of α-crystallins on RGC survival in this model. 
Methods
Optic Nerve Axotomy Animal Model
All procedures involving animals were approved by the Animal Research Committee of the University of California at Los Angeles and were performed in compliance with the National Institutes of Health Guide for the Care and Use of Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Fifty-six male Wistar rats weighing 250 to 300 g each were housed with standard chow and water provided ad libitum. The animals were maintained for at least 1 week before surgical procedures. To perform optic nerve transection (ONT), the right optic nerve of the anesthetized animals was exposed through a lateral conjunctival incision, and the optic nerve sheath was incised with a needle knife 2 mm longitudinally, starting 3 mm behind the eye. A cross-section of the optic nerve was made through the opening of the optic nerve sheath, with care taken not to damage the adjacent blood supply. The conjunctival incision was sutured and tobramycin ophthalmic ointment (Tobrex; Alcon, Fort Worth, TX) was applied topically. The ONT procedure was performed on the right eye of each rat, whereas the contralateral eye remained untreated. The animals were euthanatized 2 weeks after the procedure. Retinas dissected from the control and ONT eyes were used for cell counting, RNA and protein extraction, in situ hybridization, and immunohistochemistry. 
RGC Counting after Optic Nerve Transfection
To identify RGCs, retrograde labeling was performed by placing a piece of sterile compressed sponge (Gelfoam; Pfizer, New York, NY) soaked with 6% neuronal retrograde tracer (Fluoro-Gold [FG]; Fluorochrome, Denver, CO) to the proximal cut surface of the optic nerve after ONT. Eyes were enucleated and immersed in 4% paraformaldehyde in a 0.1 M phosphate buffer for 1 hour. Retinas were dissected, mounted on glass slides, and divided into superotemporal, inferotemporal, superonasal, and inferonasal quadrants. Three sampling fields (0.32 mm × 0.24 mm) were collected at each region 1, 2, and 3 mm from the center of the optic nerve in each retinal quadrant under a fluorescence microscope (LSM410; Carl Zeiss, Oberkochen, Germany) at 200× magnification. Numbers of RGCs in 36 sampling fields from each retina were counted and averaged. Morphologically distinguishable glial cells (bright and small cells) were not counted. Quantification was performed in a masked manner. 
In Situ Hybridization
cDNA fragments corresponding to the genes of interest were obtained by RT-PCR with sequence-specific primers and first-strand retinal cDNA. DNA fragments were purified and subcloned into vector (pCRII-Topo; Invitrogen, Carlsbad, CA). Digoxigenin (DIG)-labeled sense and antisense cRNA probes were synthesized by in vitro transcription with T7 or Sp6 RNA polymerase according to the manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). In situ hybridization (ISH) was performed according to the standard protocol. Briefly, 10-μm–thick frozen sections fixed in 4% paraformaldehyde were washed with PBS for 30 minutes and equilibrated for 15 minutes in 5× SSC (0.75 M NaCl, 0.075 M Na-Citrate). After prehybridization for 2 hours in a solution containing 50% formamide, 5× SSC, and 40 μg/mL salmon sperm DNA, the sections were hybridized for 12 to 24 hours in a humid chamber at 58°C in a prehybridization solution, with the addition of the DIG-labeled RNA probe at a concentration of 400 μg/mL. After two washes with 2× SSC and two with 0.1× SSC at 65°C for 1 hour, the sections were incubated with alkaline phosphatase (AP)-conjugated anti–DIG antibodies. After incubation with the DIG-AP antibody at 4°C for 12 to 14 hours, color staining was developed with NBT/BCIP (Roche). FG-labeled cells were detected after ISH with primary rabbit anti–FG polyclonal (1/400; Fluorochrome) and secondary Alexa Fluor 488-goat anti–rabbit IgG (1/1000) antibodies. 
Immunohistochemistry
Ten-micrometer–thick sections were obtained along the vertical meridian through the optic nerve head. Sections were washed with PBS in nonionic surfactant (Triton X [T-PBS; 0.01 M PBS/0.2% Triton X-100]; Roche) and were incubated with blocking buffer (T-PBS containing 5% bovine serum albumin) at room temperature for 30 minutes After blocking, the sections were incubated with primary mouse monoclonal anti–neurofilament (NF) antibody (Chemicon, Temecula, CA) diluted 1:500 or with primary polyclonal anti–αA or anti–αB antibodies (provided by Joseph Horwitz, JSEI, UCLA) diluted 1:300 overnight at 4°C. Sections were washed three times with T-PBS for 10 minutes and incubated with rhodamine-conjugated anti–mouse IgG antibody or fluorescein isothiocyanate (FITC)-conjugated anti–rabbit IgG antibody (Cappel Research Products, Durham, NC) for 1 hour at room temperature. After mounting, photomicrographs of the sections at 1.0 to 1.5 mm from the center of the optic nerve were taken with a fluorescence microscope. The specificity of the immunoreaction was controlled by exclusion of the primary antibody. 
Immunoblot Analysis
Western blot analysis of αA and αB was performed as previously described. 27 Briefly, aliquots of 2 to 10 μg detergent-soluble retinal protein were separated on a 12% SDS-polyacrylamide gel and transferred to the membrane (ImmobilonP; Millipore, Billerica, MA). After incubation with primary antibodies to αA or αB, the membranes were treated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour. Immunoreactive bands were detected by chemiluminescence (ECL Western Blotting Detection Reagents; Amersham, Piscataway, NJ). Equal protein loading of retinal lysates from different animals was confirmed by reprobing blots with a monoclonal antibody to housekeeping β-actin (1:10,000; Sigma, St. Louis, MO). Intensities of the immunoreactive bands were analyzed by densitometry. 
Expression Constructs
Enhanced green fluorescent protein (EGFP)-fused αA (pCryαA-EGFP) and αB (pCryαB-EGFP) expression constructs were prepared in pEGFP-N3 vector (Clontech, Mountain View, CA). For pCryαA-EGFP, αA coding region was obtained by RT-PCR with sequence-specific primers containing EcoRI and BamHI restriction sites integrated into forward (CryαA-1; 5′-CTAGAATTCCGGAACATGGACGTCACCAT) and reverse (CryαA-2; 5′-ATATGGATCCGGACGAGGGTGCCGAGCT) primers, respectively. First-strand retinal cDNA synthesized with oligo-dT primer, as described, was used as a template in PCR. The PCR product was digested with the EcoRI and BamHI and was cloned into pEGFP-N3 vector upstream of the EGFP open-reading frame (ORF). To construct pCryαB-EGFP, αB cDNA was obtained with forward (CryαB-1; 5′-CTAGAATTCCTCTACACTCATCTAGCCATC [contains EcoRI site]) and reverse (CryαB-2; 5′-CTAACGCGTCTTCTTAGGGGCTGCAGTGAC [contains MluI site]) primers, and EGFP was amplified with EGFP-4 (5′-CTAACGCGTATGGTGAGCAAGGGCGAGGAGC [contains MluI site]) and EGFP-5 (5′-TATGGCTGATTATGATCTAGAGTC [contains XbaI site]). These PCR products were digested with EcoRI and MluI (αB cDNA) or with MluI and XbaI (EGFP) and cloned into pEGFP-N3 cleaved with EcoRI and XbaI. The resultant pCryαA-EGFP and pCryαB-EGFP constructs were sequenced to confirm the integrity of the αA and αB cDNAs and their in-frame cloning upstream of EGFP ORF. 
Electroporation-Mediated RGC Transfection
As described previously, with minor modifications, αA- and αB-crystallin expressing plasmid DNAs were delivered to RGCs by electroporation (ELP). 28 29 Briefly, animals were anesthetized with intramuscular injection of 0.8 mL/kg of a cocktail containing 5 mL ketamine (100 mg/mL), 2.5 mL xylazine (20 mg/mL), 1.0 mL acepromazine (10 mg/mL), and 1.5 mL normal saline. Four microliters of plasmid DNA (5 μg/μL) was injected into the vitreous cavity 0.5 mm posterior to the limbus under stereomicroscopy with a 34-gauge needle. Ten minutes later, the cathodal electrode was placed on the cornea, and an 18-gauge needle anodal electrode was inserted subcutaneously at the middle of the forehead. A pulse generator (ECM 830; BTX, San Diego, CA) was used to generate electric pulses with the following parameters: electric field strength, 6 V/cm; pulse duration, 100 ms; stimulation pattern, five pulses at a frequency of 1/s. After a 10-minute pause, five more pulses with the same parameters were delivered. The efficiency of ELP-mediated RGC transfection was analyzed 7 days after ELP. The optic nerve was transected as described, and RGCs were retrogradely labeled by placement of a sterile compressed sponge (Gelfoam; Pfizer) soaked with FG to the proximal cut surface of the optic nerve. Transfected RGCs (EGFP positive) were counted 2 days after FG labeling. FG labeling was visualized with a wide-band ultraviolet (UV) excitation filter (330–390 nm excitation and 420–480 nm emission), whereas EGFP was detected with green spectral filter (460–490 nm excitation and 510–550 nm emission) under fluorescence microscopy. 
The effect of αA and αB overexpression on cell survival was evaluated 7 and 14 days after ONT, which led to rapid degeneration of RGCs. ONT was performed 3 days after ELP to allow transgene expression in transfected cells before injury. 28 At that time, RGCs were FG-labeled as described. 
Statistical Analysis
Data are presented as the mean ± SD. Differences among groups were analyzed by one-way ANOVA, followed by the Mann-Whitney U test. P < 0.05 was considered statistically significant. 
Results
Optic Nerve Transfection Animal Model
To evaluate the extent of RGC degeneration 7 and 14 days after ONT, the number of surviving cells was determined by counting retrogradely labeled cells in flatmount retinal preparations. A dramatic reduction in the RGC population in axotomized retinas was observed (Fig. 1A) . Quantitative analysis showed that RGC density in control retinas was 2044 ± 56 cells/mm2 (n = 4), whereas 7 and 14 days after ONT, RGC numbers were reduced to 1016 ± 88 (n = 5) and 206 ± 60 (n = 9) cells/mm2, respectively. This corresponds to approximately 50% and 90% of RGC degeneration by 7 and 14 days after ONT, respectively (Fig. 1B)
In Situ Analyses of αA- and αB-Crystallin mRNA in Normal and Axotomized Retinas
For spatial analysis of αA and αB expression, control and axotomized (14 days after ONT) retinal sections were hybridized with DIG-labeled sense (negative control) and antisense riboprobes corresponding to αA and αB. The crystallin-positive cells were present in the GCL of control retinal sections hybridized with antisense riboprobes (Figs. 2A-1 B-1) , and most of these crystallin-positive cells were clearly colocalized with FG-labeled RGCs (Figs. 2A-2 B-2) . In axotomized retinas, only a few cells were found to be positive for crystallin or FG in the GCL (Figs. 2A-2 B-2) . Moreover, it appeared that the remaining RGCs after ONT were not crystallin positive or were stained with crystallin riboprobes noticeably weaker than in control retinas. Crystallin expression was also present in the inner nuclear layer (INL) and, to a lesser degree, in the outer nuclear layer (ONL). No staining was observed with the sense crystallin riboprobes used as negative controls (data not shown). 
Immunoblot and Immunohistochemical Analyses of αA- and αB-Crystallin Proteins in Normal and Axotomized Retinas
Western blot analyses of crystallins were performed to determine changes in the levels of αA- and αB-crystallins 14 days after ONT. Proteins extracted from axotomized and untreated contralateral retinas of eight animals were analyzed. Distinct bands at the expected molecular weight of approximately 22 to 23 kDa, corresponding to αA and αB, were detected by antibodies specific to these proteins (Fig. 3A) ; αA was represented on Western blot by two bands corresponding to αA and αAins. Quantitative analysis showed that the expression levels of αA (both isoforms were included) and αB were reduced by approximately 1.6-fold after ONT compared with the contralateral untreated retinas (Fig. 3B) . β-Actin housekeeping protein was used to control the equal loading of proteins isolated from axotomized and contralateral untreated retinas. 
Immunohistochemistry with antibodies recognizing αA and αB showed a similar pattern of staining for these proteins, with strong expression in the GCL and INL (Figs. 4A 4B) . In the GCL, crystallin-positive cells were predominantly colocalized with NF-labeled RGCs. Two weeks after axotomy, significant reductions in αA and αB crystallin expression as well as NF expression were observed in the GCL. Immunohistochemistry results correlate with ISH. 
Effect of αA and αB Overexpression on RGC Survival after Optic Nerve Axotomy
Transfection of retinal cells with EGFP-tagged αA and αB expression plasmids was performed with ELP. Transfected cells were primarily localized in the GCL (Fig. 5A) . Given that the GCL, along with RGCs, contains displaced amacrine cells and astrocytes, we colocalized the αA- and αB-EGFP–positive cells with NF-labeled RGCs (Fig. 5A) . Among transfected cells in the GCL, RGCs were predominant. EGFP-fused crystallin protein expression in transfected retinas was also analyzed by immunoblot with anti–EGFP antibodies (Fig. 5B) . One band with a molecular weight of approximately 27 kDa, corresponding to EGFP, was detected in retinas transfected with pEGFP-N3 vector. In lanes containing proteins isolated from pCryαA-EGFP– or pCryαB-EGFP–transfected retinas, bands of approximately 50 kDa, corresponding to αA-EGFP and αB-EGFP, were present. CMV-driven GFP expression from pEGFP plasmid, introduced to RGCs by ELP-mediated transfection, has been shown to reach its maximum on day 7. 28 Therefore, the efficiency of RGC transfection in this study was evaluated in control retinas 7 days after ELP by counting EGFP-positive cells colocalized with FG-labeled RGCs and the total number of FG-labeled RGCs (Fig. 5C) . The total number of αA- and αB-transfected RGCs was determined to be 26.21% ± 1.24% and 26.90% ± 3.29%, respectively. The distribution of the transfected cells was not uniform. In well-transfected areas, the efficiency of RGC transfection with αA was 43.70% ± 15.17% and 40.83% ± 14.42% with αB. RGCs constituted 84.74% ± 5.06% and 79.98% ± 1.38% of all αA- and αB-EGFP–transfected cells in the GCL, respectively. 
The effect of αA and αB overexpression on RGC survival was evaluated 7 and 14 days after ONT. At day 7 after ONT, 1426 ± 70 and 1418 ± 81 RGCs/mm2 were present in retinas electroporated with αA and αB expression constructs, respectively, compared with 1010 ± 121 in sham-transfected (pEGFP-N3) and 1016 ± 88 RGCs/mm2 in nontransfected retinas (Fig. 6A) . This is equivalent to an approximately 40% increase in RGC survival. At 14 days after ONT, the numbers of surviving RGCs after αA and αB transfection were 389 ± 57 and 353.57 ± 60 cells/mm2, respectively, compared with 198 ± 29 cells/mm2 after transfection with the vector alone (pEGFP-N3) or 206 ± 60 cells/mm2 in nontransfected retinas (Figs. 6B 6C) . These numbers correspond to approximately 95% and 75% increases in RGC survival mediated by αA and αB overexpression, respectively. 
Discussion
Crystallins are major lens proteins that constitute 80% to 90% of the soluble protein fraction. In the human lens, α-, β-, and γ-crystallins represent approximately 40%, 35%, and 25% of the total amount of crystallin, respectively. 30 Crystallins in the lens primarily function as structural proteins, and their integrity and stability are essential to lens transparency. 31 32 Mutations in αA, 33 34 αB, 35 βA1, 36 βB1, 37 βB2, 38 39 40 γC, 41 and γD 42 43 44 crystallin genes are associated with autosomal dominant congenital cataracts. Outside the lens, including in the retina, α-crystallin functions are related to their chaperone and apoptosis inhibitory activities. Although the roles of β- and γ-crystallins are not fully understood, their expression in various tissues and dynamic regulation in response to different insults suggest their functional importance. Recently, intravitreally applied crystallins of the β/γ superfamily were shown to stimulate RGC axonal regeneration, whereas treatment with α-crystallins was reported to be ineffective. 9  
In the present study, we first determined changes in αA- and αB-crystallin expression in axotomized retinas characterized by rapid and specific RGC degeneration 45 46 47 48 and then analyzed their roles in the survival of injured RGCs. We localized the expression of αA and αB primarily to the GCL and, to a lesser degree, to the INL and in photoreceptors. The distribution of αA and αB in the rat retina correlates with the expression of these proteins in the mouse retina. 49 By the colocalization of crystallin-positive cells with retrogradely labeled RGCs, we determined that most crystallin-expressing cells in the GCL are RGCs. As expected, few cells expressing crystallin genes in the GCL of axotomized animals were detected. At the protein level, αA and αB were expressed 1.6-fold less than in contralateral control retinas. Interestingly, in experimental glaucomatous retinas 2 weeks after IOP elevation, αA and αB were also downregulated approximately 40% compared with control retinas. 26 Similar levels of crystallin proteins observed in RGC-deficient retinas of the axotomy model (>90% RGC loss) and in retinas with approximately 20% RGC loss of the ocular hypertension model suggest that IOP elevation by a yet unknown mechanism strongly suppresses the expression of crystallin genes in RGCs. The downregulation of crystallin genes in RGCs may, in turn, affect the survival mechanism of these cells and be responsible for their degeneration in glaucomatous retinas. To determine the cell-protective role of α-crystallins, we transfected retinas with αA- and αB-expression constructs and analyzed the survival rates of RGCs 1 and 2 weeks after optic nerve axotomy. These time points were chosen based on our earlier characterization of cell loss at 3, 5, 7, and 14 days after axotomy showing significant RGC degeneration starting at day 5, approximately 50% at day 7, and more than 90% at day 14 after ONT. 47 48 Overexpression of αA and αB increases the number of survived RGCs by approximately 40% 1 week after axotomy and by approximately 95% and 75%, respectively, 2 weeks after ONT. Similar GFP expression level from ELP-mediated transfected plasmid on day 7 and day 21 has been shown, 28 suggesting that the expression level of the crystallin proteins in transfected cells at 7 and 14 days after ONT was comparable and was not a reason for the cell protective differences observed at these time points. 
In summary, the expression of αA and αB crystallin genes, which are known to be involved in cell protection and are generally upregulated in response to various stresses, was investigated in a rat model with optic nerve axotomy–induced RGC degeneration. Expression of these genes at the mRNA and protein levels in the retina was predominantly localized in the GCL, and crystallin-positive cells were primarily colocalized with RGCs. Levels of αA- and αB-crystallins were decreased in RGC-deficient retinas. Transfection of RGCs with αA and αB expression constructs had a significant protective effect on these cells after optic nerve axotomy. The neuroprotective effect of α-crystallins observed in this study was achieved by the transfection of approximately 26% of RGCs. We believe that the cell-protective effect of αA- and αB-crystallins could be much stronger with the use of alternative methods yielding higher transfection efficiency. 
 
Figure 1.
 
RGC loss after ONT. (A) Dramatic loss of retrogradely labeled RGCs was observed in flatmounted retinas 2 weeks after ONT. (B) RGC counts in retinas 7 and 14 days after ONT were 1016 ± 88 (n = 5) and 206 ± 60 cells/mm2 (n = 9), respectively, compared with 2044 ± 56 cells/mm2 in contralateral control retinas (n = 4). *P = 0.001; **P = 0.005. Scale bar, 50 μm.
Figure 1.
 
RGC loss after ONT. (A) Dramatic loss of retrogradely labeled RGCs was observed in flatmounted retinas 2 weeks after ONT. (B) RGC counts in retinas 7 and 14 days after ONT were 1016 ± 88 (n = 5) and 206 ± 60 cells/mm2 (n = 9), respectively, compared with 2044 ± 56 cells/mm2 in contralateral control retinas (n = 4). *P = 0.001; **P = 0.005. Scale bar, 50 μm.
Figure 2.
 
In situ analysis of αA- and αB-crystallin mRNA expression in control and axotomized retinas. Expression of (A-1) αA- and (B-1) αB-crystallins was observed primarily in the GCL of control retinas. Few crystallin-positive cells were present in the GCL 14 days after ONT. (A-2, B-2) Colocalization of crystallin-positive cells with RGCs retrogradely labeled with FG. Arrows: cells in the GCL that are crystallin and FG positive. Scale bar, 10 μm.
Figure 2.
 
In situ analysis of αA- and αB-crystallin mRNA expression in control and axotomized retinas. Expression of (A-1) αA- and (B-1) αB-crystallins was observed primarily in the GCL of control retinas. Few crystallin-positive cells were present in the GCL 14 days after ONT. (A-2, B-2) Colocalization of crystallin-positive cells with RGCs retrogradely labeled with FG. Arrows: cells in the GCL that are crystallin and FG positive. Scale bar, 10 μm.
Figure 3.
 
Comparative analysis of αA- and αB-crystallin proteins expressed in control and axotomized retinas. (A) Representative immunoblot images of αA- and αB-crystallin in untreated contralateral control and 14-day axotomized retinas. Immunoblot for β-actin was used to ensure that the amounts of the protein in control and experimental lanes were equal. (B) Significant decreases in αA (Control vs. ONT; *P = 0.016, n = 8 [n represents number of control and experimental retinas]) and αB (Control vs. ONT; **P = 0.005, n = 8 [n represents number of control and experimental retinas]) were observed in retinas 14 days after ONT. Proteins from each retina were analyzed separately; each lane on the immunoblot contains proteins extracted from one retina.
Figure 3.
 
Comparative analysis of αA- and αB-crystallin proteins expressed in control and axotomized retinas. (A) Representative immunoblot images of αA- and αB-crystallin in untreated contralateral control and 14-day axotomized retinas. Immunoblot for β-actin was used to ensure that the amounts of the protein in control and experimental lanes were equal. (B) Significant decreases in αA (Control vs. ONT; *P = 0.016, n = 8 [n represents number of control and experimental retinas]) and αB (Control vs. ONT; **P = 0.005, n = 8 [n represents number of control and experimental retinas]) were observed in retinas 14 days after ONT. Proteins from each retina were analyzed separately; each lane on the immunoblot contains proteins extracted from one retina.
Figure 4.
 
Immunohistochemical analysis of αA (A) and αB (B) in control and ONT retinas. αA and αB were predominantly localized in the GCL and INL, correlating with in situ mRNA localization of αA and αB (Fig. 3) . Most αA- and αB-immunopositive cells in the GCL were colocalized with neurofilament (NF)-stained RGCs. The numbers of αA-, αB-, and NF-positive cells were decreased significantly 14 days after ONT. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical analysis of αA (A) and αB (B) in control and ONT retinas. αA and αB were predominantly localized in the GCL and INL, correlating with in situ mRNA localization of αA and αB (Fig. 3) . Most αA- and αB-immunopositive cells in the GCL were colocalized with neurofilament (NF)-stained RGCs. The numbers of αA-, αB-, and NF-positive cells were decreased significantly 14 days after ONT. Scale bar, 50 μm.
Figure 5.
 
Electroporation-mediated transfection of retinal cells with EGFP-fused αA and αB expression constructs. (A) Colocalization of transfected cells with NF-positive RGCs. (B) Immunoblot analysis of nontransfected, pEGFP-N3–, pCryαA-EGFP–, and pCryαB-EGFP–transfected retinas with anti–EGFP antibodies. (C) RGC transfection with pCryαA-EGFP and pCryαB-EGFP was evaluated 7 days after ELP by counting EGFP-positive cells colocalized with FG-labeled RGCs. Scale bar, 50 μm.
Figure 5.
 
Electroporation-mediated transfection of retinal cells with EGFP-fused αA and αB expression constructs. (A) Colocalization of transfected cells with NF-positive RGCs. (B) Immunoblot analysis of nontransfected, pEGFP-N3–, pCryαA-EGFP–, and pCryαB-EGFP–transfected retinas with anti–EGFP antibodies. (C) RGC transfection with pCryαA-EGFP and pCryαB-EGFP was evaluated 7 days after ELP by counting EGFP-positive cells colocalized with FG-labeled RGCs. Scale bar, 50 μm.
Figure 6.
 
Effect of αA and αB overexpression on RGC survival 7 (A) and 14 (B, C) days after ONT. (A) ONT (n = 5) vs. ONT+αA-EGFP (n = 7), *P = 0.004; ONT+EGFP (n = 6) vs. ONT+αA-EGFP (n = 7), **P = 0.003; ONT (n = 5) vs. ONT+αB-EGFP (n = 6), *P = 0.006; and ONT+EGFP (n = 6) vs. ONT+αB-EGFP (n = 6), **P = 0.004. (B) ONT (n = 9) vs. ONT+αA-EGFP (n = 8), *P = 0.001; ONT+EGFP (n = 5) vs. ONT+αA-EGFP (n = 8), **P = 0.003; ONT (n = 9) vs. ONT+αB-EGFP (n = 8), *P = 0.001; and ONT+EGFP (n = 5) vs. ONT+αB-EGFP (n = 8), **P = 0.003. (C) Representative fluorescence photographs of FG-labeled RGCs in flatmount nontransfected, pEGFP-N3–, pEGFP-CryαA–, and pEGFP-CryαB–transfected retinas 14 days after ONT. Scale bar, 50 μm.
Figure 6.
 
Effect of αA and αB overexpression on RGC survival 7 (A) and 14 (B, C) days after ONT. (A) ONT (n = 5) vs. ONT+αA-EGFP (n = 7), *P = 0.004; ONT+EGFP (n = 6) vs. ONT+αA-EGFP (n = 7), **P = 0.003; ONT (n = 5) vs. ONT+αB-EGFP (n = 6), *P = 0.006; and ONT+EGFP (n = 6) vs. ONT+αB-EGFP (n = 6), **P = 0.004. (B) ONT (n = 9) vs. ONT+αA-EGFP (n = 8), *P = 0.001; ONT+EGFP (n = 5) vs. ONT+αA-EGFP (n = 8), **P = 0.003; ONT (n = 9) vs. ONT+αB-EGFP (n = 8), *P = 0.001; and ONT+EGFP (n = 5) vs. ONT+αB-EGFP (n = 8), **P = 0.003. (C) Representative fluorescence photographs of FG-labeled RGCs in flatmount nontransfected, pEGFP-N3–, pEGFP-CryαA–, and pEGFP-CryαB–transfected retinas 14 days after ONT. Scale bar, 50 μm.
The authors thank Min Song and Grace Hsu for excellent technical assistance. 
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Figure 1.
 
RGC loss after ONT. (A) Dramatic loss of retrogradely labeled RGCs was observed in flatmounted retinas 2 weeks after ONT. (B) RGC counts in retinas 7 and 14 days after ONT were 1016 ± 88 (n = 5) and 206 ± 60 cells/mm2 (n = 9), respectively, compared with 2044 ± 56 cells/mm2 in contralateral control retinas (n = 4). *P = 0.001; **P = 0.005. Scale bar, 50 μm.
Figure 1.
 
RGC loss after ONT. (A) Dramatic loss of retrogradely labeled RGCs was observed in flatmounted retinas 2 weeks after ONT. (B) RGC counts in retinas 7 and 14 days after ONT were 1016 ± 88 (n = 5) and 206 ± 60 cells/mm2 (n = 9), respectively, compared with 2044 ± 56 cells/mm2 in contralateral control retinas (n = 4). *P = 0.001; **P = 0.005. Scale bar, 50 μm.
Figure 2.
 
In situ analysis of αA- and αB-crystallin mRNA expression in control and axotomized retinas. Expression of (A-1) αA- and (B-1) αB-crystallins was observed primarily in the GCL of control retinas. Few crystallin-positive cells were present in the GCL 14 days after ONT. (A-2, B-2) Colocalization of crystallin-positive cells with RGCs retrogradely labeled with FG. Arrows: cells in the GCL that are crystallin and FG positive. Scale bar, 10 μm.
Figure 2.
 
In situ analysis of αA- and αB-crystallin mRNA expression in control and axotomized retinas. Expression of (A-1) αA- and (B-1) αB-crystallins was observed primarily in the GCL of control retinas. Few crystallin-positive cells were present in the GCL 14 days after ONT. (A-2, B-2) Colocalization of crystallin-positive cells with RGCs retrogradely labeled with FG. Arrows: cells in the GCL that are crystallin and FG positive. Scale bar, 10 μm.
Figure 3.
 
Comparative analysis of αA- and αB-crystallin proteins expressed in control and axotomized retinas. (A) Representative immunoblot images of αA- and αB-crystallin in untreated contralateral control and 14-day axotomized retinas. Immunoblot for β-actin was used to ensure that the amounts of the protein in control and experimental lanes were equal. (B) Significant decreases in αA (Control vs. ONT; *P = 0.016, n = 8 [n represents number of control and experimental retinas]) and αB (Control vs. ONT; **P = 0.005, n = 8 [n represents number of control and experimental retinas]) were observed in retinas 14 days after ONT. Proteins from each retina were analyzed separately; each lane on the immunoblot contains proteins extracted from one retina.
Figure 3.
 
Comparative analysis of αA- and αB-crystallin proteins expressed in control and axotomized retinas. (A) Representative immunoblot images of αA- and αB-crystallin in untreated contralateral control and 14-day axotomized retinas. Immunoblot for β-actin was used to ensure that the amounts of the protein in control and experimental lanes were equal. (B) Significant decreases in αA (Control vs. ONT; *P = 0.016, n = 8 [n represents number of control and experimental retinas]) and αB (Control vs. ONT; **P = 0.005, n = 8 [n represents number of control and experimental retinas]) were observed in retinas 14 days after ONT. Proteins from each retina were analyzed separately; each lane on the immunoblot contains proteins extracted from one retina.
Figure 4.
 
Immunohistochemical analysis of αA (A) and αB (B) in control and ONT retinas. αA and αB were predominantly localized in the GCL and INL, correlating with in situ mRNA localization of αA and αB (Fig. 3) . Most αA- and αB-immunopositive cells in the GCL were colocalized with neurofilament (NF)-stained RGCs. The numbers of αA-, αB-, and NF-positive cells were decreased significantly 14 days after ONT. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical analysis of αA (A) and αB (B) in control and ONT retinas. αA and αB were predominantly localized in the GCL and INL, correlating with in situ mRNA localization of αA and αB (Fig. 3) . Most αA- and αB-immunopositive cells in the GCL were colocalized with neurofilament (NF)-stained RGCs. The numbers of αA-, αB-, and NF-positive cells were decreased significantly 14 days after ONT. Scale bar, 50 μm.
Figure 5.
 
Electroporation-mediated transfection of retinal cells with EGFP-fused αA and αB expression constructs. (A) Colocalization of transfected cells with NF-positive RGCs. (B) Immunoblot analysis of nontransfected, pEGFP-N3–, pCryαA-EGFP–, and pCryαB-EGFP–transfected retinas with anti–EGFP antibodies. (C) RGC transfection with pCryαA-EGFP and pCryαB-EGFP was evaluated 7 days after ELP by counting EGFP-positive cells colocalized with FG-labeled RGCs. Scale bar, 50 μm.
Figure 5.
 
Electroporation-mediated transfection of retinal cells with EGFP-fused αA and αB expression constructs. (A) Colocalization of transfected cells with NF-positive RGCs. (B) Immunoblot analysis of nontransfected, pEGFP-N3–, pCryαA-EGFP–, and pCryαB-EGFP–transfected retinas with anti–EGFP antibodies. (C) RGC transfection with pCryαA-EGFP and pCryαB-EGFP was evaluated 7 days after ELP by counting EGFP-positive cells colocalized with FG-labeled RGCs. Scale bar, 50 μm.
Figure 6.
 
Effect of αA and αB overexpression on RGC survival 7 (A) and 14 (B, C) days after ONT. (A) ONT (n = 5) vs. ONT+αA-EGFP (n = 7), *P = 0.004; ONT+EGFP (n = 6) vs. ONT+αA-EGFP (n = 7), **P = 0.003; ONT (n = 5) vs. ONT+αB-EGFP (n = 6), *P = 0.006; and ONT+EGFP (n = 6) vs. ONT+αB-EGFP (n = 6), **P = 0.004. (B) ONT (n = 9) vs. ONT+αA-EGFP (n = 8), *P = 0.001; ONT+EGFP (n = 5) vs. ONT+αA-EGFP (n = 8), **P = 0.003; ONT (n = 9) vs. ONT+αB-EGFP (n = 8), *P = 0.001; and ONT+EGFP (n = 5) vs. ONT+αB-EGFP (n = 8), **P = 0.003. (C) Representative fluorescence photographs of FG-labeled RGCs in flatmount nontransfected, pEGFP-N3–, pEGFP-CryαA–, and pEGFP-CryαB–transfected retinas 14 days after ONT. Scale bar, 50 μm.
Figure 6.
 
Effect of αA and αB overexpression on RGC survival 7 (A) and 14 (B, C) days after ONT. (A) ONT (n = 5) vs. ONT+αA-EGFP (n = 7), *P = 0.004; ONT+EGFP (n = 6) vs. ONT+αA-EGFP (n = 7), **P = 0.003; ONT (n = 5) vs. ONT+αB-EGFP (n = 6), *P = 0.006; and ONT+EGFP (n = 6) vs. ONT+αB-EGFP (n = 6), **P = 0.004. (B) ONT (n = 9) vs. ONT+αA-EGFP (n = 8), *P = 0.001; ONT+EGFP (n = 5) vs. ONT+αA-EGFP (n = 8), **P = 0.003; ONT (n = 9) vs. ONT+αB-EGFP (n = 8), *P = 0.001; and ONT+EGFP (n = 5) vs. ONT+αB-EGFP (n = 8), **P = 0.003. (C) Representative fluorescence photographs of FG-labeled RGCs in flatmount nontransfected, pEGFP-N3–, pEGFP-CryαA–, and pEGFP-CryαB–transfected retinas 14 days after ONT. Scale bar, 50 μm.
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