ABSTRACT PURPOSE To compare the concentration of amino acids in subretinal and vitreous fluid of patients with primary rhegmatogenous retinal detachment to that of control vitreous. METHODS

This prospective, observational study measured amino-acid levels in subretinal fluid of patients undergoing scleral buckle placement (_n_=20) and vitreous fluid in patients undergoing pars

plana vitrectomy (_n_=5) for primary retinal detachment. Vitreous fluid from patients undergoing vitrectomy for macular hole (_n_=7) or epiretinal membrane (_n_=3) served as a control.

Subretinal fluid and control vitreous were analysed using high-pressure liquid chromatography. Retinal detachment vitreous was analysed using capillary electrophoresis-laser-induced

(Spearman's correlation coefficient: 0.74, _P_<0.01). Mean arginine levels did not differ significantly between subretinal fluid and control vitreous. Levels of alanine, tyrosine,

valine, isoleucine, leucine, and phenylalanine were significantly lower in subretinal fluid compared to control vitreous (all _P_<0.01). CONCLUSIONS Glutamate levels in subretinal fluid

and vitreous of patients with primary retinal detachment is significantly elevated in comparison to control vitreous. This finding lends further support to the hypothesis that elevated

glutamate levels may result from ischaemia of the outer retina secondary to retinal detachment. SIMILAR CONTENT BEING VIEWED BY OTHERS INCREASED INTRAVITREAL GLUCOSE IN RHEGMATOGENOUS

RETINAL DETACHMENT Article 10 March 2022 STATINS FOR THE PREVENTION OF PROLIFERATIVE VITREORETINOPATHY: CELLULAR RESPONSES IN CULTURED CELLS AND CLINICAL STATIN CONCENTRATIONS IN THE

VITREOUS Article Open access 13 January 2021 POSTOPERATIVE PROLIFERATIVE VITREORETINOPATHY DEVELOPMENT IS LINKED TO VITREAL CXCL5 CONCENTRATIONS Article Open access 14 December 2021

INTRODUCTION Patients who undergo retinal detachment (RD) repair for a macula-on1 or macula-off RD2 often lose vision despite a good anatomic result. The mechanisms underlying this vision

loss are not fully understood. Many abnormal molecular and cellular events occur rapidly after RD and could be responsible for this visual impairment. Photoreceptor outer segment

degeneration appears to be an initial cause of vision loss following RD.3, 4, 5, 6 Studies in detached cat retina show chronic changes in this area that include formation of membrane-bound

sacs and atrophy.3 While reattachment may induce rod and cone outer segments to regenerate, a primate study showed persistent abnormalities such as altered disc stacking in this region of

the retina.7 Other events affecting the photoreceptors include apoptosis and loss of structural integrity, both of which are thought to contribute to the vision impairment following RD.8, 9,

10 Misalignment of photoreceptors, measured by Stiles–Crawford function, may also be a problem after RD repair.11, 12 Müller cells have been shown to react within 15 min of RD,13 with

proliferation and hypertrophy of their processes seen as little as two days post-detachment.14, 15 This abnormal growth leads to their extension into the subretinal space, formation of

intraretinal scars, and irregularities within retinal synaptic connections.14, 16, 17 Visual recovery also may be hampered by an outgrowth of neurites from second- (bipolar and horizontal

cells) and third-order neurons (ganglion cells). This effect, termed sprouting, may contribute to unstable retinal circuitry after RD.18, 19 Elevated levels of glutamate have been shown to

be toxic to retinal neurons, especially retinal ganglion cells.20, 21 Animal studies have shown that alterations within the glutamatergic system of the retina occur early and could be one of

the first biochemical changes associated with RD.22 Excitotoxicity produced by glutamate induces degeneration in the inner retina23 and could be a mediating factor in suboptimal vision

following RD. The separation of the neural retina from the underlying choroidal vasculature creates an ischaemic environment in the outer retina, which can induce pathological metabolic and

cellular alterations.10, 24, 25 Accumulation of glutamate has been observed in animal models of retinal ischaemia,26, 27, 28, 29 and animal studies of RD have shown excess glutamate levels

and lower glutamine synthetase (GS) activity within the retina.30, 31, 32 Moreover, increased levels of glutamate were recently found in the vitreous fluid of human subjects with RD.33 It

was hypothesized that the retinal ischaemia associated with RD leads to the accumulation of glutamate within the retina. To test this hypothesis, the level of glutamate and other amino acids

in subretinal and vitreous fluid of patients with primary RD was measured and compared to that in control vitreous. The control vitreous used in this study was taken from patients

undergoing retinal surgery for either epiretinal membrane or macular hole. We are unaware of any previous study that has examined amino-acid levels in subretinal fibrosis (SRF) of patients

with primary RD. METHODS PATIENTS This is a prospective, observational study of amino-acid levels in SRF of patients undergoing a scleral buckle placement and vitreous fluid in patients

undergoing pars plana vitrectomy for primary RD. The study protocol and consent form was approved by the Human Studies Committee at the Massachusetts Eye and Ear Infirmary. None of the

patients in this study had had previous retinal surgery. Patients undergoing vitrectomy surgery for macular hole or epiretinal membrane served as controls. Demographic information including

age, sex, and affected eye was collected for each study group. Relevant clinical characteristics were recorded for later statistical analysis. Patients were specifically questioned to

estimate the duration between occurrence of the RD and time of sampling. Snellen visual acuity was recorded at the first visit and last follow-up visit and later converted into the logarithm

of the minimal angle of resolution (logMAR) units. LogMAR units was calculated by obtaining the logarithm of the reciprocal of the Snellen visual acuity for vision better than or equal to

20/400. The following conversion was used for patients with vision worse than 20/400: counting fingers=1.6, hand movements=2.0, light perception=2.5, and no light perception=3.0 logMAR

units. SAMPLE COLLECTION Subretinal fluid samples were obtained from 20 patients undergoing scleral buckle placement for RD via modified external needle drainage.34 In this procedure, the

intraocular pressure is temporarily raised to high levels by tightening the scleral buckle. The subretinal space is entered externally, under the bed of the buckle, using a 27G needle on a 3

 cm3 syringe without the plunger. The procedure is directly visualized ophthalmoscopically and the SRF is removed passively. As the drainage proceeds, the needle tip is usually redirected in

the subretinal space to remove remaining SRF and to avoid retinal perforation. Once the SRF drainage is complete, the needle is withdrawn from the eye and the area is inspected. This method

of collecting SRF provides relatively clean samples, free from contamination with blood. In this study, SRF samples were immediately placed on dry ice and frozen at −80°C until amino acid

analysis. Undiluted vitreous specimens (∼1.5 ml) from 5 patients with RD and 10 patients with either an epiretinal membrane (_n_=7) or macular hole (_n_=3) undergoing pars plana vitrectomy

were collected. Vitreous fluid was obtained via a 3 cm3 syringe that was connected to a three-way stopcock system that had been placed in the aspiration line near the vitreous cutter.35

Prior to turning on the infusion, the stopcock lever is directed away from the vitrectomy machine and the vitrectomy cut rate is lowered to 250 cuts per minute. Vitreous was then aspirated

using manual aspiration. After the vitreous sample was obtained, the stopcock lever was returned to its original position and the syringe was removed. For RD-Vit samples, the tube contained

100 _μ_l 1 M acetic acid (pH 2–3) to deactivate protease. The samples were placed on dry ice and immediately stored at −80°C (SRF and Control-Vit) or 4°C (RD-Vit) until amino-acid analysis.

AMINO-ACID ANALYSIS Before being sent for analysis, the samples were given a unique code for identification. Subretinal fluid and vitreous fluid controls were analysed for 10 amino acids

using a Beckman 7300 Amino Acid Analyzer: alanine (Ala), arginine (Arg), aspartate (Asp), glutamate (Glu), glycine (Gly), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), tyrosine

(Tyr), and valine (Val). The Beckman analyzer uses an ion-exchange column to separate amino acids and post-column reaction with ninhydrin/hydrindantin (Beckman Nin-Rx, Beckman Coulter,

Fullerton, CA, USA) for detection at 570 and 440 nm. The samples were vortexed, then spun in a microcentrifuge to pellet the solids. A 100 _μ_l aliquot of the supernatant was taken from each

sample and dried in a Savant SC110A Speed-Vac. The aliquots were dissolved in 100 _μ_l of loading buffer (Beckman Na–S with 2 nmol homoserine per 100 _μ_l as an internal standard), and then

filtered. The recovered volume was measured and diluted back up to 100 _μ_l to load on the analyzer. The results have been corrected to the original 100 _μ_l taken by using the measured

volume as the per cent injected. A collaborating laboratory collected the RD vitreous fluid and performed amino-acid analysis. Calibration curves were constructed using a standard. Analysis

of 2 _μ_l aliquots began with derivatization of vitreous amino acids with 2 _μ_l 20 mM CBQCA (Molecular Probes, Eugene, OR, USA) in the presence of 2 _μ_l 10 mM cyanide. The pH value of the

vitreous samples was between 2 and 3 due to the acetic acid addition. Therefore, the 10 mM cyanide was diluted in 250 mM phosphate buffer (pH 12.0) for the derivatization process. The

reaction mixture of vitreous sample and CBQCA was allowed to react for 2 h at room temperature prior to capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis.36 The running

buffer consisted of 20 mM sodium tetraborate, 20 mM sodium chloride, 45 mM sodium dodecyl sulphate, and 55 mM _β_-cyclodextrin (_β_-CD), and the separation was carried out at 17 kV applied

voltage. The CE-LIF system contained a high-voltage power supply (Spellman, Hauppage, NY, USA), a ZETALIF LIF detector (Picometrics, Paris, France), and an argon ion laser (Coherent, Santa

Clara, CA, USA) at 488 nm. All CE analyses were performed at room temperature with 40 cm × 360 _μ_m OD × 50 _μ_m ID (30 cm effective length) fused-silica capillaries (BioTAQ, Gaithersburg,

MD, USA). In this work, gravimetric sample injection was used with a 30 cm height difference for 5 s. The system operation and data acquisition was performed with a custom LabVIEW (National

Instruments, Austin, TX, USA) program and the data were analysed by using Microsoft Excel and Class Eleganza CE station software V5.5 (Ayer Rajah Industrial Estate, Singapore). STATISTICAL

ANALYSIS Amino-acid concentrations in SRF and RD-Vit were compared with Control-Vit using the Wilcoxon's rank-sum test because the data were not normally distributed. Demographic

information and clinical characteristics were compared using the two-sample _t_-test, Wilcoxon's rank-sum test, or Fisher's exact test depending on the types of variables

(continuous or categorical) and their distributions (normal or non-normal). The association between the concentration of amino acids in the SRF and patient age, sex, affected eye, macular

detachment, RD duration, preop vision, extent of RD, presence of grade C PVR, lens status, and postop vision was assessed either by the Spearman's rank correlation or Wilcoxon's

rank-sum test. A _P_-value of <0.05 was considered statistically significant. We certify that all applicable institutional and governmental regulations concerning the ethical use of human

volunteers were followed during this research. RESULTS Subretinal fluid (_n_=20) and vitreous fluid (_n_=5) samples were obtained from patients with primary RD. Vitreous fluid (_n_=10) from

patients with an epiretinal membrane or macular hole served as controls. Basic demographic information and clinical characteristics of the three groups are summarized in Table 1. Mean age

was 49.5 years (±13.8 years SD) in the SRF group, 66.1 years (±12.1 years SD) in the Control-Vit group, and 63.8 years (±15.5 years SD) in the RD-Vit group. SRF patients were significantly

younger than Control-Vit patients (_P_-value=<0.01). No other significant differences existed in demographic information between the two RD groups and controls. Mean duration of follow-up

time was 20.5 months (±18.7 months SD, range from 1 week to 75 months) for the SRF group and 4.1 months (±1.7 months SD, range from 1.5 to 6 months) for RD-Vit. The mean RD duration was

33.1 days (±68.9 days SD, range from 1 to 270 days) for the SRF group and 52.4 days (±65.4 SD, range from 3 to 150 days) for the RD-Vit group. A significantly better postoperative visual

acuity was observed in the SRF group as compared to the RD-Vit group (_P_-value=<0.05). Comparisons of mean levels of amino acids between Control-Vit, RD-Vit, and SRF are presented in

Table 2. The mean level of glutamate in RD-Vit was 13.4 _μ_M (±11.9 _μ_M SD), which was significantly higher than the Control-Vit glutamate level, 1.7 _μ_M (±0.8 _μ_M SD, _P_-value=0.01).

Glutamate levels in SRF had a mean of 27.0 _μ_M (±19.7 _μ_M SD), and were also significantly elevated relative to Control-Vit (_P_-value <0.01). Aspartate and glycine also had mean

concentrations in SRF that were significantly higher than the control group, Control-Vit (_P_-values: 0.01 and <0.01, respectively). Mean arginine levels did not differ significantly

between SRF and Control-Vit. All other measured amino acids (alanine, tyrosine, valine, isoleucine, leucine, and phenylalanine) were present in SRF at significantly lower concentrations as

compared to Control-Vit (_P_-values all <0.01). Comparing SRF demographic information and clinical characteristics with glutamate levels in SRF did not yield any significant associations

(Table 3). However, the data do reveal a significant, positive association between aspartate levels and glutamate levels in SRF (Spearman's correlation coefficient: 0.74,

_P_-value=<0.01). Glutamate levels in SRF of 11 cases of macula-on detachment did not correlate with postoperative visual acuity (Spearman's correlation coefficient: 0.27,

_P_-value=0.42). Tables 4 and 5 show amino-acid concentrations in subretinal and vitreous fluid for individual patients. DISCUSSION Glutamate was present at significantly higher

concentrations in SRF and vitreous fluid of patients with primary RD as compared to controls. Subretinal aspartate and glycine levels were also found to be significantly elevated.

Furthermore, a significant, positive association was observed between aspartate levels and glutamate levels in SRF. These findings partially support those of a recent clinical investigation

that found elevated glutamate levels in the vitreous of patients with RD.33 However, that study, unlike this one, did not show a significant difference in aspartate or glycine levels between

RD vitreous and controls. These differences might be explained by variations in high-performance liquid chromatography methods. Another study did find elevated aspartate in cat retina after

experimental RD,32 and a rat model of retinal ischaemia has shown elevated levels of glycine in the vitreous.27 Amino-acid levels in the Control-Vit group were comparable to those found by

Honkanen _et al._37 Several plausible theories exist for the accumulation of glutamate in the retina and vitreous following RD. In attached retina, glutamate is released from presynaptic

neurons into the extracellular space, from which most of it is transported into Müller cells via the glutamate/aspartate transporter.38 Glutamate is immediately converted into the non-toxic

amino-acid glutamine by the enzyme GS, and then exported to neighbouring neurons where it is hydrolysed by glutaminase to reform glutamate.39, 40, 41 Disruption of the retinal glutamate

reuptake transporter, glutamate/aspartate transporter, has been shown to lead to increased vitreal glutamate and subsequent ganglion cell death in an animal study.42 Glutamate/aspartate

transporter-deficient mice show a much greater sensitivity to the retinal damage induced by ischaemia.43 Ischaemic disruption of glutamate transport could be caused by elevated extracellular

levels of K+.44 Studies of experimental RD in cat found elevated levels of glutamate coupled with a rapid decrease in GS expression in Müller cells.14, 30 Marc _et al_30 found that

glutamate levels persisted in Müller cells following RD but decreased in the retinal pigment epithelium, a pattern that suggests an alteration in glutamate metabolism rather than a sustained

increase in neuronal glutamate release. GS has also been shown to influence glutamate clearance from the synaptic cleft and amplified expression of GS has provided protection from excess

glutamate.45, 46 Retinal ischaemia secondary to RD may play a role in disrupting the mechanisms of reuptake and degradation responsible for maintaining glutamate homeostasis. Glutamate

toxicity most probably results from osmotic damage and calcium insult. Excess sodium entry into the cell, stimulated by elevated glutamate levels, is coupled with water and chloride entry,

which leads to osmotic cell lysis and neuronal swelling.47 In addition, glutamate has a high affinity for _N_-methyl-D-aspartate (NMDA) receptors, overactivation of which causes a large

influx of calcium into the cell. This increase in calcium leads to protease and lipase activation, endonuclease activation, kinase activation, and cell membrane deterioration, and ultimately

contributes to cell death. Glutamate-mediated NMDA stimulation may also lead to increased nitric oxide production, with a resultant inhibitory effect on energy production in the

mitochondria.27 Because aspartate is also regulated by glutamate/aspartate transporter, the reduced reuptake associated with ischaemia could also account for the increased concentration of

this amino acid after RD.48 Increased neuronal release of aspartate under ischaemic conditions has also been found. Altered glutamate metabolism and transamination reactions associated with

ischaemia could also account for the elevated levels of aspartate, which is a metabolite/precursor for glutamate.49 Both of these explanations can account for the observed positive

association between aspartate and glutamate levels in SRF. Aspartate may also act as a neurotoxin,50 acting through NMDA receptors in a fashion similar to glutamate. Elevated levels of

glycine following RD may result from ischaemic reversal of the glycine transporter, found in Müller cells.51 Since glycine also acts on NMDA receptors and is thought to be required for

channel activation by glutamate, it is possible that this amino acid contributes to some of the toxicity observed after RD. Analysis of demographic information revealed a significantly older

Vit-Control population as compared to the SRF group. This finding is likely due to the fact that the control group was composed of patients with macular hole or epiretinal membrane, ocular

conditions that typically affect older individuals. However, there was no significant difference in age between the RD-Vit and the Control-Vit groups. This may be due to the fact that the

RD-Vit population was disproportionately composed of patients who were pseudophakic at the time of their RD, making the choice of vitrectomy more likely. The data show significantly better

postoperative visual acuity in the SRF group as compared to RD-Vit group. This finding is attributable to the fact that the RD-Vit group possessed a preponderance of those clinical

characteristics that are associated with poor visual outcome: lower mean preoperative visual acuity, increased percentage of patients with macula-off detachment, and increased mean duration

of RD compared to the SRF group. No correlation was observed between SRF glutamate levels and pre or postoperative visual acuity, either overall or in patients with macula-on RD. However,

the small number of patients in this study limits our ability to detect such a correlation, and a larger study will be needed to determine whether there exists a relationship between

glutamate levels and visual impairment following RD. Although the mean duration of RD was shorter for the SRF group as compared to the RD-Vit group, mean glutamate levels were higher in the

SRF group. Glutamate may concentrate more in the smaller subretinal space as compared to the larger vitreous space. Larger studies comprised of groups with paired duration of RD are needed

to further evaluate this finding. There are some limitations to this study that should be kept in mind when interpreting these results. Compared to the study by Diederen _et al_,33 this

study analysed relatively few patients. This small sample size may explain the observed lack of association between glutamate levels and pre and postoperative visual acuity. This study

lacked an ideal SRF control sample for comparison due to the impossibility of collecting SRF in patients without RD. However, the Control-Vit fluid obtained nevertheless offers an

appropriate comparison since SRF is initially composed of liquid vitreous. The two different methods used to measure amino-acid levels will likely yield slightly different means and standard

deviations. The extent of this difference; however, is not likely significant. Comparison of vitreous controls analysed with both methods yielded similar results. Indeed, using two

different techniques, provided their results are similar, reduces the likelihood of a bias in either technique going undetected. Oxygen supplementation may be effective in renormalizing the

glutamate cycle and preventing further excitoxicity seen after RD. In a study by Lewis _et al_,24 a cat model of RD was placed in a hyperoxic environment and showed a stabilization of

glutamate-related neurochemical signatures. NMDA receptor antagonists, such as memantine and dextromethorphan, may also prove to be beneficial in attenuating retinal cell death secondary to

glutamate excitotoxicity.19, 27 Animal studies on the use of NMDA receptor antagonists in RD are needed to investigate these drugs as a possible therapeutic modality. In conclusion, this

study found increased levels of glutamate, glycine, and aspartate in SRF, and of glutamate in vitreous fluid of patients with primary rhegmatogenous RD. This study also found a significant,

positive association between glutamate and aspartate in SRF. Our data do not show an association between any other amino-acid levels and clinical characteristics collected in this study,

including pre and postoperative visual acuity. This study lends further support to the hypothesis that elevated glutamate levels in the vitreous and SRF may result from ischaemia secondary

to RD, expanding upon earlier animal models22, 30, 31, 32 of RD and a recent clinical study of amino-acid levels in vitreous fluid of patients with RD.33 To our knowledge, this is the first

study that has demonstrated elevated levels of glutamate in SRF following RD. Additional studies are needed to delineate the role of glutamate in human RD and to investigate further any

possible association between glutamate levels and visual acuity. REFERENCES * Tani P, Robertson DM, Langworthy A . Rhegmatogenous retinal detachment without macular involvement treated with

scleral buckling. _Am J Ophthalmol_ 1980; 90 (4): 503–508. Article  CAS  PubMed  Google Scholar  * Ross WH . Visual recovery after macula-off retinal detachment. _Eye_ 2002; 16 (4): 440–446.

Article  CAS  PubMed  Google Scholar  * Anderson DH, Stern WH, Fisher SK, Erickson PA, Borgula GA . Retinal detachment in the cat: the pigment epithelial-photoreceptor interface. _Invest

Ophthalmol Vis Sci_ 1983; 24 (7): 906–926. CAS  PubMed  Google Scholar  * Machemer R . Experimental retinal detachment in the owl monkey. IV. The reattached retina. _Am J Ophthalmol_ 1968;

66 (6): 1075–1091. Article  CAS  PubMed  Google Scholar  * Kroll AJ, Machemer R . Experimental retinal detachment in the owl monkey.V. Electron microscopy of the reattached retina. _Am J

Ophthalmol_ 1969; 67 (1): 117–130. Article  CAS  PubMed  Google Scholar  * Kroll AJ, Machemer R . Experimental retinal detachment and reattachment in the rhesus monkey. Electron microscopic

comparison of rods and cones. _Am J Ophthalmol_ 1969; 68 (1): 58–77. Article  CAS  PubMed  Google Scholar  * Guerin CJ, Anderson DH, Fariss RN, Fisher SK . Retinal reattachment of the

primate macula. Photoreceptor recovery after short-term detachment. _Invest Ophthalmol Vis Sci_ 1989; 30 (8): 1708–1725. CAS  PubMed  Google Scholar  * Arroyo JG, Yang L, Bula D, Chen DF .

Photoreceptor apoptosis in human retinal detachment. _Am J Ophthalmol_ 2005; 139 (4): 605–610. Article  PubMed  Google Scholar  * Erickson PA, Fisher SK, Anderson DH, Stern WH, Borgula GA .

Retinal detachment in the cat: the outer nuclear and outer plexiform layers. _Invest Ophthalmol Vis Sci_ 1983; 24 (7): 927–942. CAS  PubMed  Google Scholar  * Yang L, Bula D, Arroyo JG, Chen

DF . Preventing retinal detachment-associated photoreceptor cell loss in Bax-deficient mice. _Invest Ophthalmol Vis Sci_ 2004; 45 (2): 648–654. Article  PubMed  Google Scholar  * Enoch JM,

Van Loo Jr JA, Okun E . Realignment of photoreceptors disturbed in orientation secondary to retinal detachment. _Invest Ophthalmol_ 1973; 12 (11): 849–853. CAS  PubMed  Google Scholar  *

Fitzgerald CR, Birch DG, Enoch JM . Functional analysis of vision in patients after retinal detachment repair. _Arch Ophthalmol_ 1980; 98 (7): 1237–1244. Article  CAS  PubMed  Google Scholar

  * Geller SF, Lewis GP, Fisher SK . FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Muller and RPE cells. _Invest Ophthalmol Vis Sci_ 2001; 42 (6): 1363–1369. CAS 

PubMed  Google Scholar  * Lewis GP, Guerin CJ, Anderson DH, Matsumoto B, Fisher SK . Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment. _Am J

Ophthalmol_ 1994; 118 (3): 368–376. Article  CAS  PubMed  Google Scholar  * Uhlmann S, Bringmann A, Uckermann O, Pannicke T, Weick M, Ulbricht E _et al_. Early glial cell reactivity in

experimental retinal detachment: effect of suramin. _Invest Ophthalmol Vis Sci_ 2003; 44 (9): 4114–4122. Article  PubMed  Google Scholar  * Iandiev I, Uckermann O, Pannicke T _et al_. Glial

cell reactivity in a porcine model of retinal detachment. _Invest Ophthalmol Vis Sci_ 2006; 47 (5): 2161–2171. Article  PubMed  Google Scholar  * Fisher SK, Erickson PA, Lewis GP, Anderson

DH . Intraretinal proliferation induced by retinal detachment. _Invest Ophthalmol Vis Sci_ 1991; 32 (6): 1739–1748. CAS  PubMed  Google Scholar  * Lewis GP, Linberg KA, Fisher SK . Neurite

outgrowth from bipolar and horizontal cells after experimental retinal detachment. _Invest Ophthalmol Vis Sci_ 1998; 39 (2): 424–434. CAS  PubMed  Google Scholar  * Calzada JI, Jones BE,

Netland PA, Johnson DA . Glutamate-induced excitotoxicity in retina: neuroprotection with receptor antagonist, dextromethorphan, but not with calcium channel blockers. _Neurochem Res_ 2002;

27 (1–2): 79–88. Article  CAS  PubMed  Google Scholar  * Olney JW . Glutamate-induced retinal degeneration in neonatal mice: electron microscopy of the acutely evolving lesion. _J

Neuropathol Exp Neurol_ 1969; 28 (3): 455–474. Article  CAS  PubMed  Google Scholar  * Lucas DR, Newhouse JP . The toxic effect of sodium L-glutamate on the inner layers of the retina. _AMA

Arch Ophthalmol_ 1957; 58 (2): 193–201. Article  CAS  PubMed  Google Scholar  * Sherry DM, Townes-Anderson E . Rapid glutamatergic alterations in the neural retina induced by retinal

detachment. _Invest Ophthalmol Vis Sci_ 2000; 41 (9): 2779–2790. CAS  PubMed  Google Scholar  * Sisk DR, Kuwabara T . Histologic changes in the inner retina of albino rats following

intravitreal injection of monosodium L-glutamate. _Graefes Arch Clin Exp Ophthalmol_ 1985; 223 (5): 250–258. Article  CAS  PubMed  Google Scholar  * Lewis G, Mervin K, Valter K, Maslim J,

Kappel PJ, Stone J _et al_. Limiting the proliferation and reactivity of retinal Muller cells during experimental retinal detachment: the value of oxygen supplementation. _Am J Ophthalmol_

1999; 128 (2): 165–172. Article  CAS  PubMed  Google Scholar  * Mervin K, Valter K, Maslim J, Lewis G, Fisher S, Stone J . Limiting photoreceptor death and deconstruction during experimental

retinal detachment: the value of oxygen supplementation. _Am J Ophthalmol_ 1999; 128 (2): 155–164. Article  CAS  PubMed  Google Scholar  * Louzada-Junior P, Dias JJ, Santos WF, Lachat JJ,

Bradford HF, Coutinho-Netto J _et al_. Glutamate release in experimental ischaemia of the retina: an approach using microdialysis. _J Neurochem_ 1992; 59 (1): 358–363. Article  CAS  PubMed 

Google Scholar  * Lagreze WA, Knorle R, Bach M, Feuerstein TJ . Memantine is neuroprotective in a rat model of pressure-induced retinal ischemia. _Invest Ophthalmol Vis Sci_ 1998; 39 (6):

1063–1066. CAS  PubMed  Google Scholar  * Napper GA, Pianta MJ, Kalloniatis M . Localization of amino acid neurotransmitters following _in vitro_ ischemia and anoxia in the rat retina. _Vis

Neurosci_ 2001; 18 (3): 413–427. Article  CAS  PubMed  Google Scholar  * Napper GA, Kalloniatis M . Neurochemical changes following postmortem ischemia in the rat retina. _Vis Neurosci_

1999; 16 (6): 1169–1180. Article  CAS  PubMed  Google Scholar  * Marc RE, Murry RF, Fisher SK, Linberg KA, Lewis GP . Amino acid signatures in the detached cat retina. _Invest Ophthalmol Vis

Sci_ 1998; 39 (9): 1694–1702. CAS  PubMed  Google Scholar  * Sasoh M, Ma N, Yoshida S, Semba R, Uji Y . Immunocytochemical localization of glutamate in normal and detached cat retina.

_Invest Ophthalmol Vis Sci_ 1998; 39 (5): 786–792. CAS  PubMed  Google Scholar  * Sasoh M, Ma N, Ito Y, Esaki K, Uji Y . Changes in localization of amino acids in the detached cat retina.

_Ophthalmic Res_ 2006; 38 (2): 74–82. Article  CAS  PubMed  Google Scholar  * Diederen RM, La Heij EC, Deutz NE, Kijlstra A, Kessels AG, van Eijk HM _et al_. Increased glutamate levels in

the vitreous of patients with retinal detachment. _Exp Eye Res_ 2006; 83 (1): 45–50. Article  CAS  PubMed  Google Scholar  * Jaffe GJ, Brownlow R, Hines J . Modified external needle drainage

procedure for rhegmatogenous retinal detachment. _Retina_ 2003; 23 (1): 80–85. Article  PubMed  Google Scholar  * Arroyo JG, Bula DV, Yang L, Chen DF . Retinal biopsy techniques for the

removal of retinal tissue fragments. _Ophthalmic Surg Lasers Imaging_ 2005; 36 (1): 76–78. PubMed  Google Scholar  * Thongkhao-On K, Kottegoda S, Pulido JS, Shippy SA . Determination of

amino acids in rat vitreous perfusates by capillary electrophoresis. _Electrophoresis_ 2004; 25 (17): 2978–2984. Article  CAS  PubMed  Google Scholar  * Honkanen RA, Baruah S, Zimmerman MB,

Khanna CL, Weaver YK, Narkiewicz J _et al_. Vitreous amino acid concentrations in patients with glaucoma undergoing vitrectomy. _Arch Ophthalmol_ 2003; 121 (2): 183–188. Article  CAS  PubMed

  Google Scholar  * Pow DV, Barnett NL, Penfold P . Are neuronal transporters relevant in retinal glutamate homeostasis? _Neurochem Int_ 2000; 37 (2–3): 191–198. Article  CAS  PubMed  Google

Scholar  * Pow DV, Crook DK . Direct immunocytochemical evidence for the transfer of glutamine from glial cells to neurons: use of specific antibodies directed against the D-stereoisomers

of glutamate and glutamine. _Neuroscience_ 1996; 70 (1): 295–302. Article  CAS  PubMed  Google Scholar  * Van den Berg CJ . _Glutamate and glutamine_, in _Handbook in Neurochemistry_. In: A

Lajtha (ed). Plenum Press: New York, 1970, pp 355–379. Google Scholar  * Kennedy AJ, Voaden MJ, Marshall J . Glutamate metabolism in the frog retina. _Nature_ 1974; 252 (5478): 50–52.

Article  CAS  PubMed  Google Scholar  * Vorwerk CK, Naskar R, Schuettauf F, Quinto K, Zurakowski D, Gochenauer G _et al_. Depression of retinal glutamate transporter function leads to

elevated intravitreal glutamate levels and ganglion cell death. _Invest Ophthalmol Vis Sci_ 2000; 41 (11): 3615–3621. CAS  PubMed  Google Scholar  * Harada T, Harada C, Watanabe M, Inoue Y,

Sakagawa T, Nakayama N _et al_. Functions of the two glutamate transporters GLAST and GLT-1 in the retina. _Proc Natl Acad Sci USA_ 1998; 95 (8): 4663–4666. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Napper GA, Pianta MJ, Kalloniatis M . Reduced glutamate uptake by retinal glial cells under ischemic/hypoxic conditions. _Vis Neurosci_ 1999; 16 (1): 149–158.

Article  CAS  PubMed  Google Scholar  * Shaked I, Ben-Dror I, Vardimon L . Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina. _J Neurochem_ 2002; 83

(3): 574–580. Article  CAS  PubMed  Google Scholar  * Gorovits R, Avidan N, Avisar N, Shaked I, Vardimon L . Glutamine synthetase protects against neuronal degeneration in injured retinal

tissue. _Proc Natl Acad Sci USA_ 1997; 94 (13): 7024–7029. Article  CAS  PubMed  PubMed Central  Google Scholar  * Werth JL, Park TS, Silbergeld DL, Rothman SM . Excitotoxic swelling occurs

in oxygen and glucose deprived human cortical slices. _Brain Res_ 1998; 782 (1–2): 248–254. Article  CAS  PubMed  Google Scholar  * Marcaggi P, Hirji N, Attwell D . Release of L-aspartate by

reversal of glutamate transporters. _Neuropharmacology_ 2005; 49 (6): 843–849. Article  CAS  PubMed  Google Scholar  * Kalloniatis M, Tomisich G . Amino acid neurochemistry of the

vertebrate retina. _Prog Retin Eye Res_ 1999; 18 (6): 811–866. Article  CAS  PubMed  Google Scholar  * Saransaari P, Oja SS . Mechanisms of D-aspartate release under ischemic conditions in

mouse hippocampal slices. _Neurochem Res_ 1999; 24 (8): 1009–1016. Article  CAS  PubMed  Google Scholar  * Phillis JW, O'Regan MH . Characterization of modes of release of amino acids

in the ischemic/reperfused rat cerebral cortex. _Neurochem Int_ 2003; 43 (4–5): 461–467. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS Amino-acid analyses of SRF

and control vitreous fluid were done at the WM Keck Foundation Biotechnology Resource Laboratory of Yale University (New Haven, CT, USA). Retinal detachment vitreous fluid was analysed in

the Department of Chemistry, University of Illinois at Chicago (Chicago, IL, USA). M-JL, JSP, and SAS gratefully acknowledge financial support from NIH EY014908. The authors declare that

they have no proprietary interest in this research. Dr Arroyo is the recipient of an NIH K-23 Physician Training Award. This work was also supported by the Grmstraw-Code WSCZ Charitable

Foundation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Ophthalmology, Harvard Medical School, Boston, MA, USA K M Bertram, D V Bula, J-H Kim, M T Quirk & J G Arroyo *

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA K M Bertram, D V Bula, S Gautam, J-H Kim, M T Quirk & J G Arroyo * Massachusetts Eye and Ear Infirmary,

Harvard Medical School, Boston, MA, USA D V Bula & J G Arroyo * Department of Ophthalmology, Mayo Clinic, Rochester, MN, USA J S Pulido * Department of Chemistry, University of Illinois

at Chicago, Chicago, IL, USA S A Shippy & M-J Lu * General Clinical Research Center and Biometrics, Harvard Medical School, Boston, MA, USA S Gautam * Retina Consultants, Charleston, WV,

USA R M Hatfield Authors * K M Bertram View author publications You can also search for this author inPubMed Google Scholar * D V Bula View author publications You can also search for this

author inPubMed Google Scholar * J S Pulido View author publications You can also search for this author inPubMed Google Scholar * S A Shippy View author publications You can also search for

this author inPubMed Google Scholar * S Gautam View author publications You can also search for this author inPubMed Google Scholar * M-J Lu View author publications You can also search for

this author inPubMed Google Scholar * R M Hatfield View author publications You can also search for this author inPubMed Google Scholar * J-H Kim View author publications You can also

search for this author inPubMed Google Scholar * M T Quirk View author publications You can also search for this author inPubMed Google Scholar * J G Arroyo View author publications You can

also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to J G Arroyo. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Bertram, K., Bula, D., Pulido, J. _et al._ Amino-acid levels in subretinal and vitreous fluid of patients with retinal detachment. _Eye_ 22, 582–589 (2008).

https://doi.org/10.1038/sj.eye.6702993 Download citation * Received: 11 April 2007 * Revised: 04 September 2007 * Accepted: 04 September 2007 * Published: 19 October 2007 * Issue Date: 01

April 2008 * DOI: https://doi.org/10.1038/sj.eye.6702993 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable

link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * amino acids * glutamate * retinal

detachment * subretinal fluid