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Nantotechnology and Regenerative Medicine  |   July 2012
Cell Therapy Modulates Expression of Tax1-Binding Protein 1 and Synaptotagmin IV in a Model of Optic Nerve Lesion
Author Affiliations & Notes
  • Louise A. Mesentier-Louro
    Programa de Terapia Celular and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
    Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens (INBEB), Rio de Janeiro, Brazil; and the
  • Juliana Coronel
    Programa de Terapia Celular and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
    Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens (INBEB), Rio de Janeiro, Brazil; and the
  • Camila Zaverucha-do-Valle
    Programa de Terapia Celular and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
    Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens (INBEB), Rio de Janeiro, Brazil; and the
  • Andre Mencalha
    Laboratório de Células-Tronco, Centro Nacional de Transplante de Medula Óssea, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.
  • Bruno D. Paredes
    Programa de Terapia Celular and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
  • Eliana Abdelhay
    Laboratório de Células-Tronco, Centro Nacional de Transplante de Medula Óssea, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.
  • Rosalia Mendez-Otero
    Programa de Terapia Celular and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
    Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens (INBEB), Rio de Janeiro, Brazil; and the
  • Marcelo F. Santiago
    Programa de Terapia Celular and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
    Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens (INBEB), Rio de Janeiro, Brazil; and the
  • Corresponding author: Louise A. Mesentier-Louro, Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Sala G028, Cidade Universitária, RJ 21941‐590, Rio de Janeiro, Brazil; lmesentier@biof.ufrj.br
Investigative Ophthalmology & Visual Science July 2012, Vol.53, 4720-4729. doi:10.1167/iovs.11-8198
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      Louise A. Mesentier-Louro, Juliana Coronel, Camila Zaverucha-do-Valle, Andre Mencalha, Bruno D. Paredes, Eliana Abdelhay, Rosalia Mendez-Otero, Marcelo F. Santiago; Cell Therapy Modulates Expression of Tax1-Binding Protein 1 and Synaptotagmin IV in a Model of Optic Nerve Lesion. Invest. Ophthalmol. Vis. Sci. 2012;53(8):4720-4729. doi: 10.1167/iovs.11-8198.

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

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Abstract

Purpose.: Bone marrow mononuclear cells (BMMCs) have been used with considerable success to improve regeneration and/or functional recovery in animal models of neurologic diseases. Injected into the host, they migrate to the damaged areas and release cytokines and/or trophic factors, which are capable of altering the genetic program of the injured tissue cells. In this study, there was a search for genes with altered expression in a model of optic nerve crush and cell therapy.

Methods.: Optic nerve crush was followed by an intravitreous injection of BMMCs or vehicle in adult rats. After 14 days, we obtained a transcriptome screening of the retinas using differential display and automatic sequencing, followed by q-PCR, Western blot, and immunohistochemistry of selected genes and proteins.

Results.: Among the differentially displayed genes, transcription of the antiapoptotic Tax1-binding protein 1 (Tax1BP1) and Synaptotagmin IV (Syt IV), an immediate early gene, is increased in the treated group. Tax1BP1 expression is robust in the ganglion cell layer and is significantly increased by cell therapy. Syt IV is expressed by activated Müller cells and astrocytes in the retina and optic nerve, without changes in protein levels among the groups.

Conclusions.: Tax1BP1 and Syt IV transcription and/or expression are differently modulated by optic nerve crush and BMMC treatment, and might be related to neuronal damage and cell-therapy effects in the retina. The increased expression of Tax1BP1 in the treated eyes could be involved in the neuroprotective effects of BMMCs that were described previously by our group.

Introduction
Injury to adult mammalian central neurons makes them unable to regrow functional axons and reestablish dendritic connections, with damaging consequences to communication even between unaffected neurons. 1 The section or crush of the optic nerve kills most retinal ganglion cells (RGCs), mainly by apoptosis. Cell-death pathways are believed to be triggered by signals relayed retrogradely from the lesion site, and by loss of trophic support from glia and central targets. 2 Intrinsic changes in the cell genetic program include activation of proapoptotic and prosurvival genes in the retina after glaucoma induction. 3 In addition, genes related to cell signaling, cell death, immune response, cytoskeleton, connectivity, vesicle-mediated transport, and basic metabolism are regulated at different time points after optic nerve crush or transaction. 4,5  
Among the strategies developed to enhance survival and regeneration of RGCs are the inhibition of myelin-derived proteins and blockage of rho kinase, 69 deletion of PTEN 10 and/or SOCS-3, 11,12 macrophage activation and delivery of oncomodulin, 1318 delivery and stimulation of ciliary neurotrophic factor, 9,1921 regulation of KLF family members, 22 and combinations of multiple approaches. 18 However, at present there are no clinically and currently applicable therapies to efficiently protect RGCs and enhance long-distance optic nerve axon regeneration. 
Recently, bone marrow–derived cells have been used to improve regeneration and/or functional recovery in animal models of neurologic pathologies. 2326 In the visual system, mesenchymal stem cells were able to increase RGC survival in a glaucoma model, 27 after optic nerve transaction. 28 Our group demonstrated that bone marrow mononuclear cells (BMMCs) increase neuronal survival and regeneration following optic nerve crush. 29 Bone marrow cells have the ability to migrate to damaged areas of the nervous system 30 and release cytokines and trophic factors, 31 which might alter the genetic program of the injured tissue cells, preventing their death and enhancing axon regeneration. 
In this study, we searched for genes with altered expression in a model of optic nerve crush and therapy with BMMCs. For this purpose, we used a modification of the differential display technique, 32 taking advantage of the possibility to unravel genes involved in RGC survival and optic nerve regeneration that were not described previously in this system. 
Materials and Methods
Animals
A total of 59 adult (3- to 5-month-old) Lister Hooded rats were used in this study. Animals were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, in protocols approved by the Committee for the Use of Experimental Animals of our Institution. Eight rats were used for extraction of bone marrow cells and 51 rats for optic nerve crush. Different sets of animals submitted to the surgery procedure were used for each protocol. For the flow cytometry analysis, we used 6 animals, 3 per time point after nerve crush and BMMC transplant. For messenger RNA (mRNA) differential display, 6 animals were used. For quantitative polymerase chain reaction (q-PCR), a total of 19 animals were used. For immunohistochemistry we used 12 animals, of which 7 were also used for anterograde tracing. For Western blot, we used 8 animals. 
BMMC Extraction
Adult Lister Hooded rats were deeply anesthetized by inhalation of isoflurane and euthanized by cervical dislocation. Bone-marrow cells were extruded from femurs and tibias as previously described by our group. 29 A total of 8 rats were used for BMMC extraction over all the experimental procedures. 
Optic Nerve Crush and Intraocular Injections
A total of 51 animals were submitted to optic nerve crush, performed as previously described. 29 Briefly, adult male Lister Hooded rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (15 mg/kg), and under a stereoscopic microscope the nerve was crushed 1 mm behind the eye with tweezers for 15 seconds, with care to avoid damage to the blood vessels (see Supplementary Material and Supplementary Fig. S1). Immediately after the crush, a 5-μL suspension of 5 × 106 BMMCs (treated group) or vehicle (0.9% saline, untreated group) was injected into the vitreous. In additional experiments, we injected BMMCs killed by heating at 80–90°C for 15 minutes. Cell counts and viability analysis were then performed (Countess Automated Cell Counter; Invitrogen, Carlsbad, CA). 
BMMC Labeling and Flow Cytometry Analysis
BMMCs were incubated with a commercial cell proliferation reagent for cell trace studies (CellTrace Far Red DDAO-SE, 2.5 μg/mL; Invitrogen) for 40 minutes at 37°C in a 5% CO2 incubator. Cells were washed three times in PBS, counted (Countess Automated Cell Counter; Invitrogen), and suspended in PBS, for flow cytometry analysis, or in saline, for transplant after optic nerve crush. Labeled BMMCs were visualized by confocal microscopy (see Supplementary Material and Supplementary Fig. S2) and analyzed by flow cytometry before injection to obtain the percentage of CellTrace+ cells. Retinas from crushed-nerve animals were dissected and enzymatically dissociated prior to flow cytometry analysis, at 1 (n = 3) or 14 days (n = 3) after the injury. Briefly, both retinas of each animal were incubated separately in a solution of papain (1 U/mL; Worthington Biochemical Corp., Lakewood, NJ), l-cystein (0.3 mg/mL; Sigma Chemical, St. Louis, MO), DNAse (0.06 U/mL; Worthington), and bovine serum albumin (BSA, 0.3 mg/mL; Sigma) in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM-F12) culture medium (Invitrogen) for 30 minutes at 37°C in a 5% CO2 incubator. The solution was replaced by DMEM-F12 and changed twice, after decantation of the dissociated tissue. The cell suspension was recovered by centrifugation. The enzymatically digested retina and the vitreous body were suspended in PBS 0.5% BSA and analyzed separately (data were acquired by BD FACS Aria IIu flow cytometer; Becton Dickinson, Franklin Lakes, NJ). Acquired data (analyzed by FlowJo v.7.6.4 software; FlowJo, Cincinnati, OH) from retina and vitreous were plotted on the same graph. 
RNA Extraction
Fourteen days after optic nerve crush, total RNA was extracted from retinas using a commercial reagent (TRIzol reagent; Invitrogen) according to the manufacturer's protocol. A total of 25 animals were used for RNA extraction, 6 of them for mRNA differential display and 19 for RT-PCR and q-PCR. RNA was purified from residual genomic DNA by treatment with DNAse I (1 U/μL; Invitrogen) for 20 minutes at room temperature, and inactivated by incubation at 65°C for 10 minutes. 
mRNA Differential Display
Alterations in mRNA levels were analyzed by the differential display method, with a modification in the original protocol. 32 Samples containing 3 μg of RNA were homogenized, generating a pool of RNA for each experimental group (control, n = 3; treated, n = 3; and untreated, n = 3). mRNAs were converted into separate cDNA populations using anchored primers with one varying nucleotide at their 3′ end. cDNA synthesis was carried out using 200 U/μL reverse transcriptase enzyme (Superscript II; Invitrogen) with 500 ng of total RNA from each pool, in the presence of 0.1 μM of each 3′-anchored oligo primer (HT11N, N = A, C, and G, equal mole; GenHunter, Nashville, TN) in 5× RT-PCR buffer, 0.5 mM dNTP Mix, 10 mM DTT, and 40 U/μL recombinant ribonuclease inhibitor (RNaseOUT; Invitrogen). Reverse transcription was validated by a PCR for β-actin (performed with Platinum Taq DNA Polymerase; Invitrogen), following the manufacturer's instructions. Briefly, PCR was conducted with a free radioactive nucleotide (α-[32P]dCTP 10 mCi/mL; Amersham Biosciences/GE Healthcare, Buckinghamshire, UK). Each reaction also included 4 μL of cDNA primed with anchored primers (HT11N, N = A, C, and G, equal mole; GenHunter) and a 12-mer arbitrary primer (AP2 to AP8; GenHunter). Twenty-one primer combinations were used. The cycle parameters were 1 cycle at 94°C for 2 minutes, 40 cycles at 94°C for 30 seconds, 40°C for 2 minutes, and 72°C for 30 seconds. A final extension was carried out at 72°C for 5 minutes. The resulting products were electrophoresed on 6% denaturing polyacrylamide gel and analyzed by autoradiogram exposure (O-MAT V film; Kodak, Carestream Health Inc., Rochester, NY). For cDNA recovery, bands differentially displayed between control, treated, and untreated groups were excised from the gel, hydrated in 200 μL of water, boiled for 15 minutes, then precipitated, eluted in 10 μL sterile distilled water, and reamplified using the same primer combination that generated them. Amplification PCR product sizes after reamplifications were evaluated by agarose gel. Products were sequenced using a commercial sequencing kit (BigDye Terminator Sequencing Kit; Invitrogen) and analyzed with the BLAST (Basic Location Alignment Search Tool, www.ncbi.nlm.nih.gov/Blast) program, in Rattus norvegicus genome databases using the blastN algorithm. 
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Samples containing 2 μg of total RNA were treated with DNAseI. cDNA synthesis was carried out using transcriptase reverse enzyme (Superscript II; Invitrogen) and OligodT18, according to the manufacturer's instructions. RT-PCR was performed by incubation at 42°C for 40 minutes and inactivation at 70°C for 15 minutes in a thermocycler (MyCycler; Bio-Rad, Hercules, CA). 
Quantitative PCR (q-PCR)
Quantitative determination of mRNA levels was performed using 10 ng of total cDNA, 0.2 μM of each primer, and 1× Power SYBR-Green (PCR Master Mix; Applied Biosystems, Foster City, CA). GAPDH mRNA levels were used for PCR normalizations. Reactions were performed in a commercial thermocycler (ABI Prism 7000; Applied Biosystems), with an initial incubation at 50°C for 2 minutes, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. All primers demonstrated equal amplification efficiency and specific PCR product through dissociation curve analysis. Fold-change mRNA levels were calculated using the 2(–ΔΔC T) method. 33 Primers were designed according to mRNA sequence of TAX1BP1 (NM_0010041) and SYT4 (NM_031693). Primer sequences follow: TAX1BP1 sense 5′-GTC TGT AGC CAG CCT GCT CG and antisense 5′-GCT GGA TCA GGA GGA ATG GGC; SYT4 sense 5′-GGC TCC TAT CAC CAC CAGC and antisense 5′-GAC ACA GTG AAG ACC AGG CC; GAPDH (NM_017008) sense 5′-ATG ATT CTA CCC ACG GCA AG and antisense 5′-CTG GAA GAT GCT GAT GGG TT. 
Immunohistochemistry
Fourteen days after optic nerve crush, the animals were euthanized with an overdose of anesthetics and perfused through the heart with ice-cold saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Eyes and attached nerve segments were dissected to the level of the optic chiasm, and transferred to increasing sucrose solutions until 30%. Tissue was embedded in optimal cutting temperature (Tissue-Tek Automated Embedding System; Sakura Finetek USA, Inc., Torrance, CA), cut longitudinally on a cryostat (Leica Microsystems GmbH, Wetzlar, Germany) at 14- to 20-μm thickness. Tissue sections were rinsed with 0.3% Triton X-100 in PBS (PBST) and incubated in 5% normal goat serum (Sigma) for 1 hour at room temperature, followed by incubation with primary antibodies (anti-Tax1-binding protein 1 [TRAF6BP], 1:50; Abcam, Cambridge, UK; anti-GFAP, 1:100; Biomedical Technologies, Inc., Stoughton, MA; Synaptotagmin IV, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA; TuJ1, 1:500; Covance Inc., Princeton, NJ) overnight at 4°C. Sections were then washed in PBST and then incubated with the appropriate secondary antibodies (Alexa488-anti-rabbit; Invitrogen; Cy3-anti-mouse; Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 hours at room temperature. Slides were rinsed in PBS, counterstained with DAPI (4′,6-diamidino-2-phenylindole), and mounted with commercial mounting media (VectaShield; Vector Laboratories, Burlingame, CA). A carbocyanine monomer nucleic acid stain (TO-PRO-3, 1:1000; Invitrogen) was also used for nuclear staining, and was incubated together with the secondary antibodies. Fluorescent samples were analyzed under an inverted-light microscope (Zeiss Axio Vert 200M microscope equipped with an Apotome slide; Carl Zeiss Microscopy GmbH, Jena, Germany) for structural illumination microscopy, or under a confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss Microscopy). 
Fluorescence Intensity Quantification
Retinas from the control, untreated, and treated groups were immunostained with Tax1BP1-specific antibody, and a nucleic acid stain (TO-PRO-3) was used to stain nuclei, as described above. Z-stack images of the retinas, in both the central and peripheral regions, were acquired with a confocal microscope (Zeiss LSM 510 Meta, ZEN 2009 software; Carl Zeiss Microscopy) under identical parameters for all slides. Individual slices of each stack were analyzed using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). The area of the ganglion cell layer (GCL) and of the total retina, excluding photoreceptor segments, was outlined in each image, in the Tax1BP1 channel; the monomeric fluorescent dye (TO-PRO-3) signal was used only for localization of the retinal layers. The average mean gray value per stack of the treated and untreated groups was normalized to the control group, and this ratio was used for statistical analysis. 
Western Blot
Retinas from nerve-crushed eyes from treated (n = 4), untreated (n = 4), and control (fellow eyes, n = 8) groups were dissected and transferred to a solution of lysis buffer (10 mM Tris-HCl, pH 6.8, 1% Triton X-100) and a protease inhibitor cocktail (EMD Biosciences [including Calbiochem], Gibbstown, NJ; and Merck KGaA, Darmstadt, Germany). The total lysates were centrifuged for 15 minutes at 15,000g and 25 μg was loaded onto SDS/PAGE and transferred to nitrocellulose membranes. Blots were blocked with 2% nonfat milk in 0.05% Tween-20 Tris-buffered saline (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 1 hour and then incubated with antibodies to Syt IV (1:1000, in TBS-T, 4°C, overnight; Synaptic Systems, Göttingen, Germany). Alpha-tubulin (1:70,000; Sigma) was used as a loading control after stripping for 20 minutes in a solution of 0.2 M glycine, pH 2.2. Horseradish peroxidase (HRP)–linked secondary antibodies (1:5000; Sigma) were incubated for 1 hour in TBS-T. Chemiluminescent detection was performed using a commercial reagent for Western blotting (Immobilon Western Chemiluminescent HRP substrate; Millipore, Billerica, MA), following exposure to a commercial multipurpose film (Hyperfilm ECL; GE Healthcare Life Sciences, Cleveland, OH). Films were scanned at 600 dpi resolution, and band densitometry was analyzed with ImageJ software. The final amount was expressed as the ratio of integrated density values of the bands corresponding to Syt IV to that of alpha-tubulin. 
RGC Axon Labeling
Cholera Toxin B subunit (conjugated to Alexa488, CTB-488; Invitrogen) was used as an anterograde tracer of RGC axons. Briefly, 4 μL of CTB-488 (0.2% diluted in PBS with 1% DMSO) was injected in the vitreous body. Two days after injection, the animals were perfused and the tissues analyzed by histologic procedures similar to those described above. 
Statistical Analysis
Comparisons among three groups were done by the one-way ANOVA with Tukey's multiple comparison posttest. Analysis of two groups was performed with the unpaired t-test. Results were normalized for the control group and displayed as the mean ± SEM. A commercial analytical software program (GraphPad Prism software; GraphPad Software, San Diego, CA) was used for all statistical analyses. 
Results
Transplant of CellTrace-Labeled BMMCs
BMMCs were labeled with CellTrace (99.8 ± 0.10% positive cells) prior to transplant (Fig. 1A). Flow cytometry analysis was performed to detect labeled cells in the retina and vitreous 1 and 14 days after optic nerve crush and intravitreous CellTrace–BMMC transplant. The fellow eye was used as a control for detection, showing a very low signal (0.008 ± 0.01%; n = 6) (Fig. 1B). Injected CellTrace–BMMCs were detected in the sample from the injured eye (6.84 ± 2.3%; n = 3) (Fig. 1C) 1 day after optic nerve crush and infusion of cells, but not after 2 weeks (0.009 ± 0.002%; n = 3) (Fig. 1D). 
Figure 1. 
 
Labeling and tracking of transplanted BMMC. BMMCs were labeled with CellTrace and analyzed by flow cytometry prior to the injection and after transplant in the vitreous body and retina. (A) Representative dotplot of the labeled BMMCs before injection. Cells were 99.8 ± 0.10% CellTrace+. (B) Representative dotplot of the noninjured and noninjected eye. The detected signal was very low (0.008 ± 0.01%). (C) Representative dotplot of the labeled cells in the injured eye 1 day after injection. The number of CellTrace+ cells was significantly higher than that in the control (6.84 ± 2.3%). (D) Representative dotplot of labeled cells in the injured eye 14 days after transplantation. The presence of CellTrace+ cells was minimal (0.007 ± 0.01%) and comparable to the fellow eye (not shown) and to the noninjured eye (B). The percentage numbers indicated in the plots refer to the rectangular gates.
Figure 1. 
 
Labeling and tracking of transplanted BMMC. BMMCs were labeled with CellTrace and analyzed by flow cytometry prior to the injection and after transplant in the vitreous body and retina. (A) Representative dotplot of the labeled BMMCs before injection. Cells were 99.8 ± 0.10% CellTrace+. (B) Representative dotplot of the noninjured and noninjected eye. The detected signal was very low (0.008 ± 0.01%). (C) Representative dotplot of the labeled cells in the injured eye 1 day after injection. The number of CellTrace+ cells was significantly higher than that in the control (6.84 ± 2.3%). (D) Representative dotplot of labeled cells in the injured eye 14 days after transplantation. The presence of CellTrace+ cells was minimal (0.007 ± 0.01%) and comparable to the fellow eye (not shown) and to the noninjured eye (B). The percentage numbers indicated in the plots refer to the rectangular gates.
Analysis of mRNA Differential Display
We screened for differences in mRNA levels in retinas of the control (n = 3), treated (n = 3), and untreated (n = 3) groups, using the differential display (DD) technique. Several differentially displayed cDNA fragments were identified in the autoradiogram showing the retina transcriptome. cDNA sequencing and BLAST (www.ncbi.nlm.nih.gov) search of selected fragments revealed four with a high homology to known genes (the Table). 
Table. 
 
Genes Identified after Automatic Sequencing
Table. 
 
Genes Identified after Automatic Sequencing
GenBank Homology Accession Number Homology (%) e-Value Primers
Tax1 (human T-cell leukemia virus type I) binding protein 1 (Tax1BP1) NM_001004199.1 99% 0.0 A7
Rattus norvegicus synaptotagmin IV (Syt IV) NM_031693.1 98% 2e-24 A7
Rattus norvegicus ribosomal protein S19 (Rps19) NM_001037346.2 97% 2e-113 A6
Rattus norvegicus PDZ domain containing 1 (Pdzk1) NM_031712.1 89% 2e-12 A3
Two genes, Tax1BP1 and Syt IV, displayed a tendency for upregulation in animals treated with BMMCs (Fig. 2A). Tax1BP1 is an antiapoptotic protein 34 that interacts with GABAc receptor in the retina. 35 Syt IV is an immediate early gene induced after seizures 36 and codifies a membrane-trafficking protein, with a controversial cellular localization and role in synaptic function. 3739 Thus, we considered that these genes were important to study in our model, since their transcripts are upregulated in treated but not in untreated animals. We also identified Rps19 and PDZk1 as differentially displayed genes, the former being a component of the small subunit of ribosomes 40 and the latter involved in the control of high-density lipoprotein (HDL) cholesterol metabolism. 41 Differential display analysis suggested that Rps19 was upregulated in the untreated animals and returned to control levels in the treated group (Fig. 2A), whereas Pdzk1 was downregulated by the lesion in both the untreated and treated groups (Fig. 2A). 
Figure 2. 
 
Sequenced genes with mRNA variations among experimental groups. (A) Differentially displayed fragments of retina cDNA in the experimental conditions. The panels show the genes identified after automatic sequencing. (B, C) qRT-PCR analysis of Tax1BP1 (B) and Syt IV (C). mRNA levels were normalized for GAPDH and for the control group. (B) Differences between Tax1BP1 mRNA levels in the retinas of the control (n = 8), untreated (Crush+Saline, n = 4), and treated (Crush+BMMC, n = 4) groups. **P < 0.01, Tukey's Multiple Comparison Test. (C) Differences between Syt IV mRNA levels in the retinas of the control (n = 5), untreated (n = 3), and treated (n = 5) groups. *P < 0.05, Tukey's Multiple Comparison Test.
Figure 2. 
 
Sequenced genes with mRNA variations among experimental groups. (A) Differentially displayed fragments of retina cDNA in the experimental conditions. The panels show the genes identified after automatic sequencing. (B, C) qRT-PCR analysis of Tax1BP1 (B) and Syt IV (C). mRNA levels were normalized for GAPDH and for the control group. (B) Differences between Tax1BP1 mRNA levels in the retinas of the control (n = 8), untreated (Crush+Saline, n = 4), and treated (Crush+BMMC, n = 4) groups. **P < 0.01, Tukey's Multiple Comparison Test. (C) Differences between Syt IV mRNA levels in the retinas of the control (n = 5), untreated (n = 3), and treated (n = 5) groups. *P < 0.05, Tukey's Multiple Comparison Test.
Tax1BP1 and Syt IV mRNAs in the Retina Are Upregulated by Cell Therapy
Fourteen days after crush, Tax1BP1 mRNA levels in the retina were significantly (P < 0.01) increased to 1.97 ± 0.32 in the treated group (ratio to control; n = 4) compared with the control (0.99 ± 0.08; n = 8) and untreated (0.67 ± 0.23, n = 4) groups (Fig. 2B). Similarly, the levels of Syt IV mRNA were significantly (P < 0.05) upregulated to 2.31 ± 0.41 in the treated group (n = 5) compared with the untreated (0.50 ± 0.20, n = 3) group, but no statistical differences (P > 0.05) were detected between the control (1.00 ± 0.29) and treated groups (Fig. 2C). To check for a possible unspecific effect of the transplant, we added a group that received dead BMMCs after the optic nerve crush, and there were no significant differences for both transcripts compared with the untreated group (see Supplementary Material and Supplementary Fig. S3). 
Tax1BP1 Is Expressed by RGCs
To describe the Tax1BP1 expression profile in the retina of control and experimental groups, we used antibodies specific for Tax1BP1, GFAP, and β-III tubulin (TUJ-1) in retinal sections. Tax1BP1 labeling was strongly expressed in the GCL of both the control (Figs. 3A–C) and treated (Figs. 3D–F) retinas, and did not colocalize with GFAP expressed by astrocytes and Müller cell endfeet (Figs. 3A–C). However, we observed Tax1BP1 in GCL cell bodies of RGCs identified by TUJ-1 staining (Figs. 3D–F), in both the central and peripheral retina (see Supplementary Material and Supplementary Fig. S4). Tax1BP1 is expressed around the nuclei of RGC and was also detected in the other retinal layers (Figs. 3G–I), which is consistent with previous studies. 35  
Figure 3. 
 
Tax1BP1 expression in the retina. (AC) Structural illumination microscopy images show that Tax1BP1 (green) is strongly expressed in the GCL, and does not colocalize with GFAP from astrocytes and Müller cell endfeet. (B, C) Structural microscopy Z-stack merged images show Tax1BP1 expression by TUJ1+ cells in the GCL. (DF) Confocal images of Tax1BP1 expression in retinas from control (G), untreated (H), and treated (I) animals. The intensity of labeling at GCL is higher in the control, decreases in the untreated retinas, and increases in the treated retinas. DAPI (AF, blue) or TO-PRO (GI, red) was used for nuclei staining. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm. (J, K) Graphs show the quantification of fluorescence intensity in GCL (J) and total retina (K), expressed by the average mean gray value of Z-stack images. Means of untreated (n = 4) and treated (n = 4) groups were normalized to the control group (n = 4). *P < 0.05, unpaired t-test. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 3. 
 
Tax1BP1 expression in the retina. (AC) Structural illumination microscopy images show that Tax1BP1 (green) is strongly expressed in the GCL, and does not colocalize with GFAP from astrocytes and Müller cell endfeet. (B, C) Structural microscopy Z-stack merged images show Tax1BP1 expression by TUJ1+ cells in the GCL. (DF) Confocal images of Tax1BP1 expression in retinas from control (G), untreated (H), and treated (I) animals. The intensity of labeling at GCL is higher in the control, decreases in the untreated retinas, and increases in the treated retinas. DAPI (AF, blue) or TO-PRO (GI, red) was used for nuclei staining. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm. (J, K) Graphs show the quantification of fluorescence intensity in GCL (J) and total retina (K), expressed by the average mean gray value of Z-stack images. Means of untreated (n = 4) and treated (n = 4) groups were normalized to the control group (n = 4). *P < 0.05, unpaired t-test. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.
BMMC Therapy Upregulates Tax1BP1 Expression Specifically in the GCL
To analyze the protein expression of Tax1BP1 in the RGC, we quantified the fluorescence intensity of the GCL and of the total retina (Figs. 3G–K) in retinas of the control (n = 4), untreated (n = 4), and treated (n = 4) groups after immunostaining with Tax1BP1-specific antibody (Figs. 3G–I). Fluorescence intensity in the GCL increased significantly (P < 0.05, Fig. 3J) in the treated group (0.75 ± 0.10, ratio to the control) in relation to the untreated group (0.35 ± 0.06). However, when the total retina was analyzed, we found no significant difference between the treated (0.55 ± 0.06) and untreated (0.78 ± 0.19) groups (P > 0.05, Fig. 3K). 
Syt IV Is Expressed in the Retina and Optic Nerve
Syt IV is strongly expressed in the GCL. Staining was also observed in the plexiform and nuclear layers (Figs. 4A–C). Syt IV expression colocalizes with GFAP expressed by astrocytes and Müller cells activated by optic nerve injury (Figs. 4D–F). After BMMC treatment, Syt IV remains expressed by the same glial cells (Fig. 4C). 
Figure 4. 
 
Syt IV expression in the retina (AF) and optic nerve (GN). Confocal images of the retina and optic nerve. Syt IV is strongly expressed in the GCL, without notable variation among control (A), untreated (B), and treated (C) groups. (DF) Syt IV expression colocalizes with GFAP (green) from astrocytes and/or Müller cells, which are activated by optic nerve injury (DF). Syt IV is expressed along the intraorbital extension of the optic nerve in all groups, but does not colocalize with CTB-488+ axons (green). (G). Syt IV is expressed by GFAP+ astrocytes (green) in the optic nerve (HJ). Arrows in (J) indicate regions of colocalization of Syt IV and GFAP. After injury to the optic nerve, Syt IV is strongly expressed around the crush site (K, M) and is expressed at levels similar to the control at sites distal from the lesion (L, N). Arrows in (M) indicate regions of contact between Syt IV+ astrocytes and CTB-488+ axons (green). TO-PRO was used to stain nuclei (blue) in all panels. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm.
Figure 4. 
 
Syt IV expression in the retina (AF) and optic nerve (GN). Confocal images of the retina and optic nerve. Syt IV is strongly expressed in the GCL, without notable variation among control (A), untreated (B), and treated (C) groups. (DF) Syt IV expression colocalizes with GFAP (green) from astrocytes and/or Müller cells, which are activated by optic nerve injury (DF). Syt IV is expressed along the intraorbital extension of the optic nerve in all groups, but does not colocalize with CTB-488+ axons (green). (G). Syt IV is expressed by GFAP+ astrocytes (green) in the optic nerve (HJ). Arrows in (J) indicate regions of colocalization of Syt IV and GFAP. After injury to the optic nerve, Syt IV is strongly expressed around the crush site (K, M) and is expressed at levels similar to the control at sites distal from the lesion (L, N). Arrows in (M) indicate regions of contact between Syt IV+ astrocytes and CTB-488+ axons (green). TO-PRO was used to stain nuclei (blue) in all panels. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm.
Syt IV expression was seen along the intraorbital extension of the optic nerve (Figs. 4G–N). Crushed nerves strongly express Syt IV at the lesion site (Figs. 4K, 4M), and also distal to the injury site, in close proximity to the optic chiasm (Figs. 4L, 4N), suggesting that the protein is expressed by glial cells rather than by axons. Indeed, we observed that at the nerve, Syt IV does not colocalize with RGC axons labeled with CTB-488 (Fig. 4G), but rather is expressed by GFAP+ astrocytes (Figs. 4H–J). At the crush site, it is possible to observe Syt IV+ astrocytes in close proximity to CTB-488+ axons (Fig. 4M, arrows). 
Expression of Syt IV Protein in the Total Retina Was Not Altered 14 Days after Nerve Crush and BMMC Therapy
We used Western blot to investigate protein variations in total retina lysates from the control, treated, and untreated groups. Although transcript levels differed among the untreated and treated groups 14 days after nerve crush, we did not observe differences in Syt IV (Fig. 5) protein levels in the total retina among the groups. 
Figure 5. 
 
Western blot analysis. Western blot of retinal lysates from the control (n = 8), untreated (n = 4), and treated (n = 4) groups, using specific antibodies against Syt IV. Alpha-tubulin was used as a loading control. Data represent the ratios of densitometric values of the Syt IV band for alpha-tubulin, normalized to the control group. Error bars show the SEM for each experiment. Representative images of the respective bands for Syt IV (top panels) and alpha-tubulin (bottom panels).
Figure 5. 
 
Western blot analysis. Western blot of retinal lysates from the control (n = 8), untreated (n = 4), and treated (n = 4) groups, using specific antibodies against Syt IV. Alpha-tubulin was used as a loading control. Data represent the ratios of densitometric values of the Syt IV band for alpha-tubulin, normalized to the control group. Error bars show the SEM for each experiment. Representative images of the respective bands for Syt IV (top panels) and alpha-tubulin (bottom panels).
Discussion
In previous studies we have shown that BMMC treatment decreases RGC death and increases nerve regeneration 14 days after optic nerve crush. 29 Here we applied the differential display method to search for specific genes with altered transcription in the retina after optic nerve crush and cell therapy. Considering that 2 weeks after the transplant, virtually no BMMCs remain in the retina (Fig. 1), the observed changes are likely to be derived from retinal cells, rather than the transplanted cells. 
We identified an increase in Tax1BP1 gene transcription in the retina after optic nerve crush and cell therapy. Although a Western blot of total retinal lysates did not show significant differences among the experimental conditions (data not shown), specific retinal layer quantification showed that the protein, which is strongly expressed by RGC, is increased in the GCL of the treated group, but not when the whole retina is analyzed. These findings suggest that BMMC treatment upregulates Tax1BP1 in the RGC up to at least 14 days after optic nerve crush. 
Tax1BP1 was isolated as a binding partner of Tax1, 42 which is codified by human T-cell leukemia virus type-1 (HTLV-1). Although the function of its binding partners remains elusive, some evidence indicates that Tax1 is a negative regulator of Tax1BP, 43,44 which has antiapoptotic activity, as observed in NIH3T3 cells after tumor necrosis factor (TNF)–induced apoptosis. 34  
Interestingly, TNF-α is secreted by glia and facilitates apoptosis of RGC under different stress conditions. 45 TNF-α has been suggested as a mediator of retinal neuronal death induced by ischemia or glaucoma, 46,47 and also of the secondary degeneration of RGCs following optic nerve crush. 48 In light of these data and our recent findings, we might speculate that the increase of Tax1BP1 in RGC may be related to their augmented survival after nerve crush and cell therapy, as previously reported by our group, 29 considering Tax1BP1 activity against TNF-induced apoptosis. 34  
Whereas Tax1BP1 modulation was observable by immunostaining (Figs. 3G–I), there was no evidence of alterations of Syt IV protein levels among the experimental conditions (Figs. 4A–C), despite the transcript changes (Fig. 2B). This paradoxic finding may be due either to posttranscriptional regulation or to a more complex temporal relationship between the transcription of both genes and protein synthesis. 
Syt IV belongs to a family of membrane-trafficking proteins that contain tandem Ca2 +-binding motifs called C2-domains. 49 Syt IV was originally identified as an immediate early gene that is upregulated following neuronal depolarization. 36,50,51 This particular aspect of Syt IV is interesting because, although we did not identify protein changes, there was an increase in the transcription that was induced by cell therapy after nerve crush. Even though Syt IV may not exert its function without translation to protein, its increased transcription after cell therapy may indicate a possible role of Syt IV after retinal neuronal damage. Indeed, one of the hypotheses of Syt IV upregulation after seizures is that it is part of a protective mechanism to reduce neural activity. 52,53  
It has been proposed that rat Syt IV has evolved as a homeostatic regulator of synaptic acitivity, 54 acting on secretion from large dense core vesicles to downregulate exocytosis. 55 Indeed, Dean and colleagues 56 reported that Syt IV inhibits the release of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, reducing presynaptic vesicle fusion to downregulate synaptic function and affect plasticity. 
Regarding our observations of Syt IV expression by astrocytes in the retina, Zhang and colleagues 38 showed that knockdown of Syt IV impairs glutamate release by cultured astrocytes. Furthermore, Syt IV was proposed to be required for exocytosis of neurotransmitters from astrocytes. 57 Modulation of astrocyte Syt IV after nerve injury and cell therapy may be related to glutamate release and, consequently, to neuronal excitotoxicity. Furthermore, the increase of Syt IV expression at the crush site may be correlated with glial scar formation by astrocytes. 
To our knowledge, this is the first report showing Syt IV expression in the retina and optic nerve, although Syts I/II have been described in photoreceptors and bipolar cells in murine retinas. 58 Syt IV expression and its modulation in glial cells might elucidate key aspects of neuronal degeneration following injury. 
Both Tax1BP1 and Syt IV might be related to cell death modulated by TNF-α: Tax1BP1 because of its antiapoptotic activity 34 and Syt IV because it is required for glutamate release by astrocytes, 38 which may occur in response to extracellular TNF-α increase. 59 Although TNF-α–induced cell death and glutamate excitotoxicity have been widely reviewed, 60,61 the effects of cell therapy on the modulation of these molecules still need to be explained, and the present results suggest that BMMC treatment might regulate these pathways. 
Further studies are needed to elucidate regulation of the other differentially displayed genes. Considering the relationship between protein synthesis and RGC death after axonal damage, 62 it would be interesting to quantify retinal levels of Rps19, which also has extraribosomal functions 63 and is released by apoptotic cells preceding monocyte/macrophage infiltration. 64 In addition, Rps19 interacts with FGF-2, 65 a neurotrophic factor that is upregulated in the retina by cell therapy after optic nerve crush, as we described previously. 29 Thus, interaction between Rps19 and FGF-2 may be related to important pathways for RGC fate in our model. 
Supplementary Materials
Acknowledgments
The authors thank Felipe Marins and Suelen Serio for assistance and Janet Reid for English language review of the manuscript. 
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Footnotes
 Supported in part by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, the Brazilian Ministry of Health, and Financiadora de Estudos e Projetos (EA).
Footnotes
 Disclosure: L.A. Mesentier-Louro, None; J. Coronel, None; C. Zaverucha-do-Valle, None; A. Mencalha, None; B.D. Paredes, None; E. Abdelhay, None; R. Mendez-Otero, None; M.F. Santiago, None
Figure 1. 
 
Labeling and tracking of transplanted BMMC. BMMCs were labeled with CellTrace and analyzed by flow cytometry prior to the injection and after transplant in the vitreous body and retina. (A) Representative dotplot of the labeled BMMCs before injection. Cells were 99.8 ± 0.10% CellTrace+. (B) Representative dotplot of the noninjured and noninjected eye. The detected signal was very low (0.008 ± 0.01%). (C) Representative dotplot of the labeled cells in the injured eye 1 day after injection. The number of CellTrace+ cells was significantly higher than that in the control (6.84 ± 2.3%). (D) Representative dotplot of labeled cells in the injured eye 14 days after transplantation. The presence of CellTrace+ cells was minimal (0.007 ± 0.01%) and comparable to the fellow eye (not shown) and to the noninjured eye (B). The percentage numbers indicated in the plots refer to the rectangular gates.
Figure 1. 
 
Labeling and tracking of transplanted BMMC. BMMCs were labeled with CellTrace and analyzed by flow cytometry prior to the injection and after transplant in the vitreous body and retina. (A) Representative dotplot of the labeled BMMCs before injection. Cells were 99.8 ± 0.10% CellTrace+. (B) Representative dotplot of the noninjured and noninjected eye. The detected signal was very low (0.008 ± 0.01%). (C) Representative dotplot of the labeled cells in the injured eye 1 day after injection. The number of CellTrace+ cells was significantly higher than that in the control (6.84 ± 2.3%). (D) Representative dotplot of labeled cells in the injured eye 14 days after transplantation. The presence of CellTrace+ cells was minimal (0.007 ± 0.01%) and comparable to the fellow eye (not shown) and to the noninjured eye (B). The percentage numbers indicated in the plots refer to the rectangular gates.
Figure 2. 
 
Sequenced genes with mRNA variations among experimental groups. (A) Differentially displayed fragments of retina cDNA in the experimental conditions. The panels show the genes identified after automatic sequencing. (B, C) qRT-PCR analysis of Tax1BP1 (B) and Syt IV (C). mRNA levels were normalized for GAPDH and for the control group. (B) Differences between Tax1BP1 mRNA levels in the retinas of the control (n = 8), untreated (Crush+Saline, n = 4), and treated (Crush+BMMC, n = 4) groups. **P < 0.01, Tukey's Multiple Comparison Test. (C) Differences between Syt IV mRNA levels in the retinas of the control (n = 5), untreated (n = 3), and treated (n = 5) groups. *P < 0.05, Tukey's Multiple Comparison Test.
Figure 2. 
 
Sequenced genes with mRNA variations among experimental groups. (A) Differentially displayed fragments of retina cDNA in the experimental conditions. The panels show the genes identified after automatic sequencing. (B, C) qRT-PCR analysis of Tax1BP1 (B) and Syt IV (C). mRNA levels were normalized for GAPDH and for the control group. (B) Differences between Tax1BP1 mRNA levels in the retinas of the control (n = 8), untreated (Crush+Saline, n = 4), and treated (Crush+BMMC, n = 4) groups. **P < 0.01, Tukey's Multiple Comparison Test. (C) Differences between Syt IV mRNA levels in the retinas of the control (n = 5), untreated (n = 3), and treated (n = 5) groups. *P < 0.05, Tukey's Multiple Comparison Test.
Figure 3. 
 
Tax1BP1 expression in the retina. (AC) Structural illumination microscopy images show that Tax1BP1 (green) is strongly expressed in the GCL, and does not colocalize with GFAP from astrocytes and Müller cell endfeet. (B, C) Structural microscopy Z-stack merged images show Tax1BP1 expression by TUJ1+ cells in the GCL. (DF) Confocal images of Tax1BP1 expression in retinas from control (G), untreated (H), and treated (I) animals. The intensity of labeling at GCL is higher in the control, decreases in the untreated retinas, and increases in the treated retinas. DAPI (AF, blue) or TO-PRO (GI, red) was used for nuclei staining. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm. (J, K) Graphs show the quantification of fluorescence intensity in GCL (J) and total retina (K), expressed by the average mean gray value of Z-stack images. Means of untreated (n = 4) and treated (n = 4) groups were normalized to the control group (n = 4). *P < 0.05, unpaired t-test. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 3. 
 
Tax1BP1 expression in the retina. (AC) Structural illumination microscopy images show that Tax1BP1 (green) is strongly expressed in the GCL, and does not colocalize with GFAP from astrocytes and Müller cell endfeet. (B, C) Structural microscopy Z-stack merged images show Tax1BP1 expression by TUJ1+ cells in the GCL. (DF) Confocal images of Tax1BP1 expression in retinas from control (G), untreated (H), and treated (I) animals. The intensity of labeling at GCL is higher in the control, decreases in the untreated retinas, and increases in the treated retinas. DAPI (AF, blue) or TO-PRO (GI, red) was used for nuclei staining. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm. (J, K) Graphs show the quantification of fluorescence intensity in GCL (J) and total retina (K), expressed by the average mean gray value of Z-stack images. Means of untreated (n = 4) and treated (n = 4) groups were normalized to the control group (n = 4). *P < 0.05, unpaired t-test. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 4. 
 
Syt IV expression in the retina (AF) and optic nerve (GN). Confocal images of the retina and optic nerve. Syt IV is strongly expressed in the GCL, without notable variation among control (A), untreated (B), and treated (C) groups. (DF) Syt IV expression colocalizes with GFAP (green) from astrocytes and/or Müller cells, which are activated by optic nerve injury (DF). Syt IV is expressed along the intraorbital extension of the optic nerve in all groups, but does not colocalize with CTB-488+ axons (green). (G). Syt IV is expressed by GFAP+ astrocytes (green) in the optic nerve (HJ). Arrows in (J) indicate regions of colocalization of Syt IV and GFAP. After injury to the optic nerve, Syt IV is strongly expressed around the crush site (K, M) and is expressed at levels similar to the control at sites distal from the lesion (L, N). Arrows in (M) indicate regions of contact between Syt IV+ astrocytes and CTB-488+ axons (green). TO-PRO was used to stain nuclei (blue) in all panels. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm.
Figure 4. 
 
Syt IV expression in the retina (AF) and optic nerve (GN). Confocal images of the retina and optic nerve. Syt IV is strongly expressed in the GCL, without notable variation among control (A), untreated (B), and treated (C) groups. (DF) Syt IV expression colocalizes with GFAP (green) from astrocytes and/or Müller cells, which are activated by optic nerve injury (DF). Syt IV is expressed along the intraorbital extension of the optic nerve in all groups, but does not colocalize with CTB-488+ axons (green). (G). Syt IV is expressed by GFAP+ astrocytes (green) in the optic nerve (HJ). Arrows in (J) indicate regions of colocalization of Syt IV and GFAP. After injury to the optic nerve, Syt IV is strongly expressed around the crush site (K, M) and is expressed at levels similar to the control at sites distal from the lesion (L, N). Arrows in (M) indicate regions of contact between Syt IV+ astrocytes and CTB-488+ axons (green). TO-PRO was used to stain nuclei (blue) in all panels. Images are representative of at least 3 animals per experimental condition. Scale bar: 20 μm.
Figure 5. 
 
Western blot analysis. Western blot of retinal lysates from the control (n = 8), untreated (n = 4), and treated (n = 4) groups, using specific antibodies against Syt IV. Alpha-tubulin was used as a loading control. Data represent the ratios of densitometric values of the Syt IV band for alpha-tubulin, normalized to the control group. Error bars show the SEM for each experiment. Representative images of the respective bands for Syt IV (top panels) and alpha-tubulin (bottom panels).
Figure 5. 
 
Western blot analysis. Western blot of retinal lysates from the control (n = 8), untreated (n = 4), and treated (n = 4) groups, using specific antibodies against Syt IV. Alpha-tubulin was used as a loading control. Data represent the ratios of densitometric values of the Syt IV band for alpha-tubulin, normalized to the control group. Error bars show the SEM for each experiment. Representative images of the respective bands for Syt IV (top panels) and alpha-tubulin (bottom panels).
Table. 
 
Genes Identified after Automatic Sequencing
Table. 
 
Genes Identified after Automatic Sequencing
GenBank Homology Accession Number Homology (%) e-Value Primers
Tax1 (human T-cell leukemia virus type I) binding protein 1 (Tax1BP1) NM_001004199.1 99% 0.0 A7
Rattus norvegicus synaptotagmin IV (Syt IV) NM_031693.1 98% 2e-24 A7
Rattus norvegicus ribosomal protein S19 (Rps19) NM_001037346.2 97% 2e-113 A6
Rattus norvegicus PDZ domain containing 1 (Pdzk1) NM_031712.1 89% 2e-12 A3
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