document: glaucoma , a neurodegenerative disease , is characterized by gradual loss of retinal ganglion cells ( rgcs ) and optic nerve atrophy.1 multiple factors are related to glaucoma , such as high intraocular pressure ( iop)2 and low cerebral spinal fluid pressure.3 mller cells are the major type of glial cells in the mammalian retina that can support and nourish retinal neurons , maintain extracellular ion homeostasis , glutamate recycling , and interaction in synaptic transmission.4,5 therefore , mller cells are involved in retinal function . 
   investigations indicate that mller cells not only play an important physiological role , but they are also involved in multiple pathological retinal diseases such as glaucoma.68 it has been reported that reactive mller cells can aggravate retinal damage by releasing cytokines such as tumor necrosis factor- ( tnf-).9 growing evidence supports that increased glial production of tnf- contributes to the neurodegeneration in glaucoma.10 tnf- is a secreted inflammatory cytokine that is responsible for apoptosis , necrosis , and inflammation.11,12 tnf- is increased in the retina following ischemia or damage and other neurodegenerative disorders.1317 -aminoadipic acid ( aaa ) , a six - carbon homolog of glutamate , is a well - known compound that induces specific glial toxicity through blocking the glutamate uptake.1820 the use of aaa to interfere with glial influence on neuronal tissues is reported.21,22 however , the effects of aaa inhibition on mller cell gliosis in glaucoma are unknown . the current study aimed to determine if aaa treatment inhibits mller cell gliosis and protects against retinal abnormalities induced in a rat model of acute ocular hypertension ( aoh ) mimicking glaucoma 
   adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . 
   the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . 
   all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , 
   the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . 
   sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . 
   time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . 
   animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . 
   aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 tissues were fixed with 4% paraformaldehyde . 
   eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , 
   sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . 
   sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . 
   slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . 
   the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) 
   optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . 
   briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . 
   the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . 
   retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in 
   all , 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . 
   the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . 
   after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . 
   the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . 
   the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . 
   analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student  newman 
   wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . 
   statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . 
   adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . 
   the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . 
   all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , hnenberg , switzerland ) . 
   the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . 
   sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . 
   time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . 
   animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . 
   aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 
   eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , tokyo , japan ) . 
   sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . 
   sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . 
   slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . 
   the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) . 
   optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . 
   the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . 
   the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . 
   retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in all , 
   40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . 
   the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . 
   after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , 
   waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . 
   the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . 
   the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . 
   analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student  newman 
   wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . 
   statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . 
   values were reported as mean  sd . a value of p<0.05 was considered as statistically significant . 
   to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . 
   the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . 
   aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa 
   attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . 
   in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . 
   aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) 
   , the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . in the rat model of aoh , 
   aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and 
   gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . 
   after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . 
   aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . 
   activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . 
   importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . 
   to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . 
   the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . 
   aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa 
   attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . 
   in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . 
   aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) , 
   the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . 
   in the rat model of aoh , aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and astrocytes mainly locate along gcl . 
   gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . 
   after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . 
   aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . 
   as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . 
   activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . 
   importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . 
   glaucoma is a leading cause of irreversible vision loss characterized by progressive death of rgcs , and elevated iop is a major risk factor.28 a rodent model of aoh is well established for acute angle closure glaucoma , and it has been widely used to investigate the pathogenesis of death of rgcs.29 in the current rat model of aoh , we found induced cell apoptosis and decreased number of rgcs with iop induction versus sham operation control . 
   importantly , treatment of aaa versus pbs control significantly attenuated rgc apoptosis and rescued the reduced number of rgcs , demonstrating protective effects on aoh retinas ( figure 5 ) . 
   activated mller cells contribute to the progression of glaucoma.30 mller cells perform a multitude of important regulatory and supportive roles , including secretion of trophic factors , removal of metabolic waste , and neurotransmitter recycling.31,32 we found that mller cells were activated with induced gfap immunoreactivity in the rat model of aoh versus control retinas . 
   gfap , an intermediate filament protein , is considered as a marker of reactive mller cell gliosis,27 which is not or less expressed in mller cells in normal retinas and expressed highly at ischemic,33 light - induced retinal degeneration,34 and retinal detachment.35 activation of mller cells so far was demonstrated to have both protective and detrimental effects.3638 especially early after injury , mller cell gliosis is believed to be neuroprotective and promotes the repair of neurons in response to injury,36 with gfap upregulation to provide additional structural integrity to the retina at the site of injury.39 however , activated mller cells also express tnf-,40 monocyte chemoattractant protein,41 and nitric oxide42 to induce death of rgcs . 
   tnf- induction contributes to inflammation , apoptosis , and necrosis , leading to cell death . 
   we observed that aaa treatment attenuated gfap immunoreactivity in mller cell processes across ipl and reduced retinal tnf- levels in the rat model of aoh . 
   these findings suggested that aaa promoted survival of rgcs in the rat model of aoh , likely through the inhibition of aoh - induced mller cell gliosis and in turn downregulation of tnf- protein production . 
   we found that aaa could effectively protect retina against loss of rgcs and apoptosis in aoh retinas through attenuating mller cell gliosis and downregulating tnf- production . 
   these observations suggested that aaa might be a potential therapeutic target in the treatment of neurodegeneration in glaucoma . 
   ( a ) reprehensive results of hoechst staining in rat retina after 1 d , 3 d , and 5 d of aoh ; ( b ) quantitative analysis of the retinal thickness . 
   notes : the results showed that the thickness of ipl and inl layers decreased significantly following the reperfusion time after aoh treatment ( scale bar = 50 m ) . * p<0.05 compared with the control group ( n=4 per group ) . 
   abbreviations : aoh , acute ocular hypertension ; ipl , inner plexiform layer ; inl , inner nuclear layer ; ctrl , control ; d , day ; gcl , ganglion cell layer ; onl , outer nuclear layer . 
   note : strong expression of gfap was shown in the gs - positive mller cell after 5 d of aoh compared with the control group ( scale bar = 50 m ) . 
   abbreviations : gs , glutamine synthetase ; gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ctrl , control ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; d , days . increased gfap immunoreactivity after aoh induction in rat retinas . 
   notes : ( a ) immunofluorescent staining showed that aoh increased gfap immunoreactivity in the mller cell processes across ipl . 
   abbreviations : gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ipl , inner plexiform layer ; d , day ; pbs , phosphate - buffered saline ; aaa , -aminoadipic acid .
summary: objectiveocular hypertension is an important risk factor for glaucoma . 
   the purpose of this study was to investigate the gliotoxic effects of -aminoadipic acid ( aaa ) in a rat model of aoh and its underlying mechanisms.materials and methodsin the rat model of acute ocular hypertension ( aoh ) , intraocular pressure was increased to 110 mmhg for 60 minutes . 
   animals were divided into four groups : sham operation ( ctrl ) , aoh , aoh + phosphate - buffered saline ( pbs ) , and aoh + aaa . 
   cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay , and retinal ganglion cells ( rgcs ) immunostained with thy-1 were counted . 
   mller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein . 
   tumor necrosis factor- ( tnf- ) was examined using western blot.resultsin the rat model of aoh , cell apoptosis was induced in the ganglion cell layer and the number of rgcs was decreased . 
   mller cell gliosis in the retinas of rats was induced , and retinal protein levels of tnf- were increased . 
   intravitreal treatment of aaa versus pbs control attenuated these retinal abnormalities to show protective effects in the rat model of aoh.conclusionin the retinas of the rat model of aoh , aaa treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of rgcs , likely through the attenuation of mller cell gliosis and suppression of tnf- induction . 
   our observations suggest that aaa might be a potential therapeutic target in glaucoma .