TrkB Signaling in Retinal Glia Stimulates Neuroprotection after Optic Nerve Injury
2015; Elsevier BV; Volume: 185; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2015.08.005
ISSN1525-2191
AutoresChikako Harada, Yuriko Azuchi, Takahiko Noro, Xiaoli Guo, Atsuko Kimura, Kazuhiko Namekata, Takayuki Harada,
Tópico(s)Retinopathy of Prematurity Studies
ResumoBrain-derived neurotrophic factor (BDNF) regulates neural cell survival mainly by activating TrkB receptors. Several lines of evidence support a key role for BDNF-TrkB signaling in survival of adult retinal ganglion cells in animal models of optic nerve injury (ONI), but the neuroprotective effect of exogenous BDNF is transient. Glial cells have recently attracted considerable attention as mediators of neural cell survival, and TrkB expression in retinal glia suggests its role in neuroprotection. To elucidate this point directly, we examined the effect of ONI on TrkBflox/flox:glial fibrillary acidic protein (GFAP)–Cre+ (TrkBGFAP) knockout (KO) mice, in which TrkB is deleted in retinal glial cells. ONI markedly increased mRNA expression levels of basic fibroblast growth factor (bFGF) in wild-type (WT) mice but not in TrkBGFAP KO mice. Immunohistochemical analysis at 7 days after ONI (d7) revealed bFGF up-regulation mainly occurred in Müller glia. ONI-induced retinal ganglion cell loss in WT mice was consistently mild compared with TrkBGFAP KO mice at d7. On the other hand, ONI severely decreased TrkB expression in both WT and TrkBGFAP KO mice after d7, and the severity of retinal degeneration was comparable with TrkBGFAP KO mice at d14. Our data provide direct evidence that glial TrkB signaling plays an important role in the early stage of neural protection after traumatic injury. Brain-derived neurotrophic factor (BDNF) regulates neural cell survival mainly by activating TrkB receptors. Several lines of evidence support a key role for BDNF-TrkB signaling in survival of adult retinal ganglion cells in animal models of optic nerve injury (ONI), but the neuroprotective effect of exogenous BDNF is transient. Glial cells have recently attracted considerable attention as mediators of neural cell survival, and TrkB expression in retinal glia suggests its role in neuroprotection. To elucidate this point directly, we examined the effect of ONI on TrkBflox/flox:glial fibrillary acidic protein (GFAP)–Cre+ (TrkBGFAP) knockout (KO) mice, in which TrkB is deleted in retinal glial cells. ONI markedly increased mRNA expression levels of basic fibroblast growth factor (bFGF) in wild-type (WT) mice but not in TrkBGFAP KO mice. Immunohistochemical analysis at 7 days after ONI (d7) revealed bFGF up-regulation mainly occurred in Müller glia. ONI-induced retinal ganglion cell loss in WT mice was consistently mild compared with TrkBGFAP KO mice at d7. On the other hand, ONI severely decreased TrkB expression in both WT and TrkBGFAP KO mice after d7, and the severity of retinal degeneration was comparable with TrkBGFAP KO mice at d14. Our data provide direct evidence that glial TrkB signaling plays an important role in the early stage of neural protection after traumatic injury. Glaucoma is a neurodegenerative disease and one of the leading causes of vision loss in the world.1Quigley H.A. Broman A.T. The number of people with glaucoma worldwide in 2010 and 2020.Br J Ophthalmol. 2006; 90: 262-267Crossref PubMed Scopus (5280) Google Scholar Glaucoma is characterized by progressive degeneration of retinal ganglion cells (RGCs) and their axons. The optic nerve injury (ONI) model mimics some aspects of glaucoma, including RGC death induced by excitotoxicity and oxidative stress; therefore, it is a useful animal model for glaucoma.2Katome T. Namekata K. Guo X. Semba K. Kittaka D. Kawamura K. Kimura A. Harada C. Ichijo H. Mitamura Y. Harada T. Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury.Cell Death Differ. 2013; 20: 270-280Crossref PubMed Scopus (69) Google Scholar, 3Semba K. Namekata K. Kimura A. Harada C. Katome T. Yoshida H. Mitamura Y. Harada T. Dock3 overexpression and p38 MAPK inhibition synergistically stimulate neuroprotection and axon regeneration after optic nerve injury.Neurosci Lett. 2014; 581: 89-93Crossref PubMed Scopus (13) Google Scholar Previous studies have found that trophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), and basic fibroblast growth factor (bFGF), protect RGCs and promote axon regeneration in an ONI model.4Mansour-Robaey S. Clarke D.B. Wang Y.-C. Bray G.M. Aguayo A.J. Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells.Proc Natl Acad Sci U S A. 1994; 91: 1632-1636Crossref PubMed Scopus (698) Google Scholar, 5Koeberle P.D. Ball A.K. Effects of GDNF on retinal ganglion cell survival following axotomy.Vision Res. 1998; 38: 1505-1515Crossref PubMed Scopus (150) Google Scholar, 6Parrilla-Reverter G. Agudo M. Sobrado-Calvo P. Salinas-Navarro M. 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Functions of the two glutamate transporters GLAST and GLT-1 in the retina.Proc Natl Acad Sci U S A. 1998; 95: 4663-4666Crossref PubMed Scopus (217) Google Scholar We reported that loss of GLAST in mice leads to RGC and optic nerve degeneration that is similar to retinal degeneration observed in normal tension glaucoma.12Harada T. Harada C. Nakamura K. Quah H.M. Okumura A. Namekata K. Saeki T. Aihara M. Yoshida H. Mitani A. Tanaka K. The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma.J Clin Invest. 2007; 117: 1763-1770Crossref PubMed Scopus (251) Google Scholar The response of Müller glia to retinal damage may be mediated by growth factors produced by retinal cells, such as BDNF and bFGF.13Harada T. Harada C. Nakayama N. Okuyama S. Yoshida K. Kohsaka S. Matsuda H. Wada K. 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Cellular signaling and factors involved in Müller cell gliosis: neuroprotective and detrimental effects.Prog Retin Eye Res. 2009; 28: 423-451Crossref PubMed Scopus (488) Google Scholar BDNF is known to regulate neural cell survival and axonal outgrowth mainly by activating TrkB receptors, which stimulates various signaling cascades, such as the mitogen-activated protein kinase pathway, the phosphatidylinositol 3-kinase pathway, and Fyn-mediated actin polymerization in RGCs.17Namekata K. Harada C. Taya C. Guo X. Kimura H. Parada L.F. Harada T. Dock3 induces axonal outgrowth by stimulating membrane recruitment of the WAVE complex.Proc Natl Acad Sci U S A. 2010; 107: 7586-7591Crossref PubMed Scopus (67) Google Scholar, 18Blum R. Konnerth A. Neurotrophin-mediated rapid signaling in the central nervous system: mechanisms and functions.Physiology (Bethesda). 2005; 20: 70-78Crossref PubMed Scopus (191) Google Scholar, 19Nikoletopoulou V. Lickert H. Frade J.M. Rencurel C. Giallonardo P. 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Kielczewski J. Valenta D. Baumrind L. Pease M.E. Klein R.L. Hauswirth W.W. Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model.Invest Ophthalmol Vis Sci. 2003; 44: 4357-4365Crossref PubMed Scopus (319) Google Scholar However, the effect of exogenous BDNF in delaying RGC death is transient, and so, when used alone, its therapeutic value seems limited. On the basis of the evidence revealing the importance of glial cells in neuroprotection, TrkB signaling in retinal glia may play a role in neuroprotection, but the effect of TrkB signaling in retinal glia in vivo remains unclear. In this study, we used a conditional knockout (KO) mouse strain in which TrkB was deleted from retinal glia. These mice enabled us to examine the effects of glial TrkB signaling on ONI-induced RGC death. We monitored changes in retinal morphologic features for 2 weeks after ONI using spectral-domain optical coherence tomography (SD-OCT), which permits noninvasive, longitudinal, and quantitative assessment of retinal structures in live animals.2Katome T. Namekata K. Guo X. Semba K. Kittaka D. Kawamura K. Kimura A. Harada C. Ichijo H. Mitamura Y. Harada T. Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury.Cell Death Differ. 2013; 20: 270-280Crossref PubMed Scopus (69) Google Scholar, 3Semba K. Namekata K. Kimura A. Harada C. Katome T. Yoshida H. Mitamura Y. Harada T. Dock3 overexpression and p38 MAPK inhibition synergistically stimulate neuroprotection and axon regeneration after optic nerve injury.Neurosci Lett. 2014; 581: 89-93Crossref PubMed Scopus (13) Google Scholar, 23Semba K. Namekata K. Guo X. Harada C. Harada T. Mitamura Y. Renin-angiotensin system regulates neurodegeneration in a mouse model of normal tension glaucoma.Cell Death Dis. 2014; 5: e1333Crossref PubMed Scopus (40) Google Scholar, 24Semba K. Namekata K. Kimura A. Harada C. Mitamura Y. Harada T. Brimonidine prevents neurodegeneration in a mouse model of normal tension glaucoma.Cell Death Dis. 2014; 5: e1341Crossref PubMed Scopus (42) Google Scholar, 25Noro T. Namekata K. Kimura A. Guo X. Azuchi Y. Harada C. Nakano T. Tsuneoka H. Harada T. Spermidine promotes retinal ganglion cell survival and optic nerve regeneration in adult mice following optic nerve injury.Cell Death Dis. 2015; 6: e1720Crossref PubMed Scopus (65) Google Scholar, 26Noro T. Namekata K. Azuchi Y. Kimura A. Guo X. Harada C. Nakano T. Tsuneoka H. Harada T. Spermidine ameliorates neurodegeneration in a mouse model of normal tension glaucoma.Invest Ophthalmol Vis Sci. 2015; 56: 5012-5019Crossref PubMed Scopus (41) Google Scholar We found that TrkB signaling in retinal glia plays an important role in the early phase of neuroprotection after ONI. Experiments were performed using C57BL/6J mice (CLEA Japan, Tokyo, Japan), TrkBflox/flox:glial fibrillary acidic protein (GFAP)–Cre+ (TrkBGFAP KO) mice,27Harada C. Guo X. Namekata K. Kimura A. Nakamura K. Tanaka K. Parada L.F. Harada T. Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration.Nat Commun. 2011; 2: 189Crossref PubMed Scopus (80) Google Scholar and GFAP-Cre:ROSA-tdTomato mice,28Madisen L. Zwingman T.A. Sunkin S.M. Oh S.W. Zariwala H.A. Gu H. Ng L.L. Palmiter R.D. Hawrylycz M.J. Jones A.R. Lein E.S. Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (3877) Google Scholar in accordance with the Tokyo Metropolitan Institute of Medical Science Guidelines for the Care and Use of Animals. Light intensity inside the cages ranged from 100 to 200 lux, and a 12-hour light/12-hour dark cycle was maintained. Mice were deeply anesthetized with isoflurane (Intervet, Tokyo, Japan), placed on a stereotaxic frame, and injected with 2 μL of Fluoro-Gold (FG; 2% in phosphate-buffered saline; Fluorochrome LLC, Denver, CO) into the superior colliculus.20Kimura A. Namekata K. Guo X. Noro T. Harada C. Harada T. Valproic acid prevents NMDA-induced retinal ganglion cell death via stimulation of neuronal TrkB receptor signaling.Am J Pathol. 2015; 185: 756-764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Harada C. Nakamura K. Namekata K. Okumura A. Mitamura Y. Iizuka Y. Kashiwagi K. Yoshida K. Ohno S. Matsuzawa A. Tanaka K. Ichijo H. Harada T. Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo.Am J Pathol. 2006; 168: 261-269Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Ten days after FG application, mice were anesthetized with sodium pentobarbital (87.5 mg/kg) before ONI. The optic nerve was exposed intraorbitally and crushed at approximately 0.5 to 1.0 mm from the posterior pole of the eyeball with fine surgical forceps for 5 seconds.2Katome T. Namekata K. Guo X. Semba K. Kittaka D. Kawamura K. Kimura A. Harada C. Ichijo H. Mitamura Y. Harada T. Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury.Cell Death Differ. 2013; 20: 270-280Crossref PubMed Scopus (69) Google Scholar, 3Semba K. Namekata K. Kimura A. Harada C. Katome T. Yoshida H. Mitamura Y. Harada T. Dock3 overexpression and p38 MAPK inhibition synergistically stimulate neuroprotection and axon regeneration after optic nerve injury.Neurosci Lett. 2014; 581: 89-93Crossref PubMed Scopus (13) Google Scholar, 25Noro T. Namekata K. Kimura A. Guo X. Azuchi Y. Harada C. Nakano T. Tsuneoka H. Harada T. Spermidine promotes retinal ganglion cell survival and optic nerve regeneration in adult mice following optic nerve injury.Cell Death Dis. 2015; 6: e1720Crossref PubMed Scopus (65) Google Scholar On day 7 (d7) and day 14 (d14) after ONI, eyes were enucleated, and retinas were isolated for whole mount preparation. Retinas were fixed in 4% paraformaldehyde in 0.1M phosphate-buffered saline solution for 20 minutes, mounted on a glass slide with a mounting medium (Vectashield; Vector Laboratories Inc., Burlingame, CA), and the RGC density was examined with a fluorescent microscope. Three standard areas (0.04 mm2) of each retina at a point 0.1 mm from the optic disk were randomly chosen. FG-labeled cells were counted, and the mean number of RGCs per square millimeter was calculated. Mice were anesthetized by i.p. injection of sodium pentobarbital (87.5 mg/kg), and SD-OCT examinations using an RS-3000 system (Nidek, Gamagori, Japan) were performed to monitor retinal degeneration in live mice.2Katome T. Namekata K. Guo X. Semba K. Kittaka D. Kawamura K. Kimura A. Harada C. Ichijo H. Mitamura Y. Harada T. Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury.Cell Death Differ. 2013; 20: 270-280Crossref PubMed Scopus (69) Google Scholar, 3Semba K. Namekata K. Kimura A. Harada C. Katome T. Yoshida H. Mitamura Y. Harada T. Dock3 overexpression and p38 MAPK inhibition synergistically stimulate neuroprotection and axon regeneration after optic nerve injury.Neurosci Lett. 2014; 581: 89-93Crossref PubMed Scopus (13) Google Scholar, 23Semba K. Namekata K. Guo X. Harada C. Harada T. Mitamura Y. Renin-angiotensin system regulates neurodegeneration in a mouse model of normal tension glaucoma.Cell Death Dis. 2014; 5: e1333Crossref PubMed Scopus (40) Google Scholar, 24Semba K. Namekata K. Kimura A. Harada C. Mitamura Y. Harada T. Brimonidine prevents neurodegeneration in a mouse model of normal tension glaucoma.Cell Death Dis. 2014; 5: e1341Crossref PubMed Scopus (42) Google Scholar, 25Noro T. Namekata K. Kimura A. Guo X. Azuchi Y. Harada C. Nakano T. Tsuneoka H. Harada T. Spermidine promotes retinal ganglion cell survival and optic nerve regeneration in adult mice following optic nerve injury.Cell Death Dis. 2015; 6: e1720Crossref PubMed Scopus (65) Google Scholar, 26Noro T. Namekata K. Azuchi Y. Kimura A. Guo X. Harada C. Nakano T. Tsuneoka H. Harada T. Spermidine ameliorates neurodegeneration in a mouse model of normal tension glaucoma.Invest Ophthalmol Vis Sci. 2015; 56: 5012-5019Crossref PubMed Scopus (41) Google Scholar For fundus imaging, polymethyl methacrylate contact lenses optimal for mice (UNICON, Osaka, Japan) were placed on the corneas. Use of the contact lenses prevents anesthesia-induced cataract progression. A 60-D adaptor lens was placed on the objective lens of the Multiline OCT to focus on the mouse retina. All the images were location matched, scanning vertically through the center of the optic nerve head at 3-disk-diameter lengths above the optic nerve head. The mean thickness of the ganglion cell complex (GCC), between the internal limiting membrane and the interface of the inner plexiform layer and the inner nuclear layer, was measured. In this study, the maximum number of B-scans set by the manufacturer (50 for line scans) was used for averaging. Paraffin-embedded retinal sections of 7-μm thickness were cut through the optic nerve and stained with hematoxylin and eosin. The RGC number and the extent of retinal degeneration were quantified in two ways.29Harada C. Nakamura K. Namekata K. Okumura A. Mitamura Y. Iizuka Y. Kashiwagi K. Yoshida K. Ohno S. Matsuzawa A. Tanaka K. Ichijo H. Harada T. Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo.Am J Pathol. 2006; 168: 261-269Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 30Harada C. Namekata K. Guo X. Yoshida H. Mitamura Y. Matsumoto Y. Tanaka K. Ichijo H. Harada T. ASK1 deficiency attenuates neural cell death in GLAST-deficient mice, a model of normal tension glaucoma.Cell Death Differ. 2010; 17: 1751-1759Crossref PubMed Scopus (83) Google Scholar First, the number of neurons in the ganglion cell layer (GCL) was counted from one ora serrata through the optic nerve to the other ora serrata. Second, in the same sections, the thickness of the inner retinal layer, between the internal limiting membrane and the interface of the outer plexiform layer and the outer nuclear layer, was analyzed. Retinas were examined by immunostaining as reported previously.23Semba K. Namekata K. Guo X. Harada C. Harada T. Mitamura Y. Renin-angiotensin system regulates neurodegeneration in a mouse model of normal tension glaucoma.Cell Death Dis. 2014; 5: e1333Crossref PubMed Scopus (40) Google Scholar, 27Harada C. Guo X. Namekata K. Kimura A. Nakamura K. Tanaka K. Parada L.F. Harada T. Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration.Nat Commun. 2011; 2: 189Crossref PubMed Scopus (80) Google Scholar Immunohistochemistry (IHC) was performed using the following primary antibodies: glutamine synthetase (GS) (1:500; MAB302; Chemicon, Temecula, CA), NeuN (1:1000; MAB377, Chemicon), TrkB (1:200; sc-8316, Santa Cruz, Santa Cruz, CA), bFGF (1:200; sc-79, Santa Cruz), and GFAP (ready-to-use; ab929, Abcam, Cambridge, MA). The sections were examined with a microscope (BX51; Olympus, Tokyo, Japan) equipped with Plan Fluor objectives connected to a DP70 camera (Olympus). For quantification of the TrkB and bFGF expression levels, intensity of the immunolabeling at two random areas (0.05 mm2) at the GCL and inner nuclear layer 500 μm away from the optic nerve head were analyzed using ImageJ version 1.46r (NIH, Bethesda, MD). Sections from six different eyes were analyzed. Immunoblotting was performed for whole retina protein extracts from six different eyes as reported previously.31Namekata K. Harada C. Guo X. Kimura A. Kittaka D. Watanabe H. Harada T. Dock3 stimulates axonal outgrowth via GSK-3beta-mediated microtubule assembly.J Neurosci. 2012; 32: 264-274Crossref PubMed Scopus (71) Google Scholar Membranes were incubated with an antibody against TrkB (1:200; Santa Cruz), GLAST (1:1000; Af660, Frontier Institute, Hokkaido, Japan), NeuN (1:1000; Chemicon), or actin (1:1000; 612656, BD Bioscience, San Diego, CA). For quantification of the TrkB expression levels, band intensities were analyzed using ImageJ version 1.46r and normalized to the expression levels of actin. Real-time quantitative PCR for whole retina RNA extracts from six different eyes was performed using an ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA) with a Power SYBR Green PCR Master Mix (Applied Biosystems) as previously reported.20Kimura A. Namekata K. Guo X. Noro T. Harada C. Harada T. Valproic acid prevents NMDA-induced retinal ganglion cell death via stimulation of neuronal TrkB receptor signaling.Am J Pathol. 2015; 185: 756-764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Complementary DNA reverse-transcribed from total RNA was amplified using primers specific for nerve growth factor (sense: 5′-CGACTCCAAACACTGGAACTCA-3′; antisense: 5′-GCCTGCTTCTCATCTGTTGTCA-3′), BDNF (sense: 5′-ATGCCGCAAACATGTCTATGAG-3′; neurotrophin-4/5 (NT-4/5) (sense: 5′-AGTATGCGACGCAGTGAGTG-3′; antisense: 5′-TTGCATTCTGAGAGCCAGTG-3′), antisense: 5′-TGACCCACTCGCTAATACTGTCA-3′), ciliary neurotrophic factor (sense: 5′-CACTGATCGCTGGAGTGAGA-3′; antisense: 5′-AGGCAGAAACTTGGAGCGTA-3′), GDNF (sense: 5′-GACTTGGGTTTGGGCTATGA-3′; antisense: 5′-AACATGCCTGGCCTACTTTG-3′), bFGF (sense: 5′-CACCAGGCCACTTCAAGGA-3′; antisense: 5′-GATGGATGCGCAGGAAGAA-3′), TNF-α (sense: 5′-CGTCAGCCGATTTGCTATCT-3′; antisense: 5′-CGGACTCCGCAAAGTCTAAG-3′), and glyceraldehyde-3-phosphate dehydrogenase (sense: 5′-TGCACCACCAACTGCTTAG-3′; antisense: 5′-GGATGCAGGGATGATGTTC-3′). Data were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA. For statistical comparison of two samples, we used a two-tailed t-test. Data are presented as means ± SEM. P < 0.05 was regarded as statistically significant. Previous studies have found that the GFAP-Cre transgenic strain, in which Cre expression is regulated by the human GFAP promoter, expresses Cre recombinase in glial cells.27Harada C. Guo X. Namekata K. Kimura A. Nakamura K. Tanaka K. Parada L.F. Harada T. Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration.Nat Commun. 2011; 2: 189Crossref PubMed Scopus (80) Google Scholar, 32Zhuo L. Theis M. Alvarez-Maya I. Brenner M. Willecke K. Messing A. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo.Genesis. 2001; 31: 85-94Crossref PubMed Scopus (492) Google Scholar, 33Malatesta P. Hack M.A. Hartfuss E. Kettenmann H. Klinkert W. Kirchhoff F. Götz M. Neuronal or glial progeny: regional differences in radial glia fate.Neuron. 2003; 37: 751-764Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar To examine expression of the GFAP-Cre transgene in the adult retina, we crossed GFAP-Cre mice with ROSA-tdTomato reporter mice.28Madisen L. Zwingman T.A. Sunkin S.M. Oh S.W. Zariwala H.A. Gu H. Ng L.L. Palmiter R.D. Hawrylycz M.J. Jones A.R. Lein E.S. Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (3877) Google Scholar In these mice, tdTomato expressing cells were double-labeled with GS (a marker of Müller glial cells) (Figure 1A) but not with NeuN (a marker of neurons including RGCs) (Figure 1B). These results indicate that expression of Cre recombinase is restricted to retinal glia in GFAP-Cre mice.27Harada C. Guo X. Namekata K. Kimura A. Nakamura K. Tanaka K. Parada L.F. Harada T. Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration.Nat Commun. 2011; 2: 189Crossref PubMed Scopus (80) Google Scholar To selectively eliminate TrkB from retinal glia, we next crossed TrkBflox/flox mice34Luikart B.W. Nef S. Virmani T. Lush M.E. Liu Y. Kavalali E.T. Parada L.F. TrkB has a cell-autonomous role in the establishment of hippocampal Schaffer collateral synapses.J Neurosci. 2005; 25: 3774-3786Crossref PubMed Scopus (130) Google Scholar with GFAP-Cre mice. Resultant double transgenic TrkBflox/flox;GFAP-Cre+ mice were termed TrkBGFAP KO mice. Retinal structure and expression of NeuN and GS were normal compared with wild-type (WT) mice (Figure 2, A and B). IHC analysis revealed that TrkB protein expression in Müller glia was eliminated in TrkBGFAP KO mice (Figure 2C). We also examined the expression levels of TrkB in the whole retina by immunoblot analyses. Expression patterns of full length and truncated form (T1) of TrkB were similar, but their expression levels were significantly lower in TrkBGFAP KO mice (Figure 3, A and B). In contrast, expression levels of GLAST, which is expressed in Müller glia,11Harada T. Harada C. Watanabe M. Inoue Y. Sakagawa T. Nakayama N. Sasaki S. Okuyama S. Watase K. Wada K. Tanaka K. Functions of the two glutamate transporters GLAST and GLT-1 in the retina.Proc Natl Acad Sci U S A. 1998; 95: 4663-4666Crossref PubMed Scopus (217) Google Scholar, 12Harada T. Harada C. Nakamura K. Quah H.M. Okumura A. Namekata K. Saeki T. Aihara M. Yoshida H. Mitani A. Tanaka K. The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma.J Clin Invest. 2007; 117: 1763-1770Crossref PubMed Scopus (251) Google Scholar and NeuN were similar in both WT and TrkBGFAP KO mice (Figure 3C). These findings indicate that GFAP-Cre virtually eliminated TrkB expression from retinal glia in vivo.Figure 3Western blot analysis of retinas from wild-type (WT) and TrkBflox/flox:glial fibrillary acidic protein (GFAP)–Cre+ (TrkBGFAP) knockout (KO) mice. A: Immunoblot analyses of TrkB. T1 reveals a truncated form of TrkB. Arrow indicates nonspecific bands. B: Quantitative analyses of both full-length and truncated forms of TrkB expression levels in A. Data are normalized to band intensities of TrkB in normal WT mice (100%). C: Immunoblot analyses of glutamate transporter glutamate/aspartate transporter (GLAST), NeuN, and actin. Each lane was loaded with 30 μg of proteins. Data are presented as means ± SEM of six samples for each experiment. ∗P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By using TrkBGFAP KO mice, we investigated the effects of TrkB signaling in retinal glia after ONI. For this purpose, we specifically labeled RGCs by retrograde-labeling 10 days before ONI and quantified the RGC count at d7 and d14 (Figure 4A). On d7, RGC loss was detected in WT mice, and the RGC number was significantly lower in TrkBGFAP KO mice (Figure 4, B and C). RGC degeneration was progressive in WT mice between d7 and d14, and by d14 the RGC number was similar in both strains (Figure 4, B and C). We then examined histopathologic features of the retina at d7 and d14. By d7, the cell count in the GCL was reduced in WT mice, and the cell count was significantly lower in TrkBGFAP KO mice (Figure 5, A and B). In addition, the thickness of the inner retinal layer was significantly decreased in TrkBGFAP KO mice compared with WT mice at d7 (Figure 5, A and C). Consistent with the time course of RGC loss, the cell count in the GCL and inner retinal layer thickness were similar in both strains at d14 (Figure 5, A–C). We also visualized retinal layers using OCT, a noninvasive imaging technique that can be used to acquire cross-sectional tomographic images of the retina in vivo. This technique is very useful in monitoring the changes in retinal structures for a period after injury in live animals.2Katome T. Namekata K. Guo X. Semba K. Kittaka D. Kawamura K. Kimura A. Harada C. Ichijo H. Mitamura Y. Harada T. Inh
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