Increased glutamate levels in the vitreous of patients with retinal detachment

Experimental Eye Research 83 (2006) 45e50 www.elsevier.com/locate/yexer Increased glutamate levels in the vitreous of patients with retinal detachment* Roselie M.H. Diederen a,*, Ellen C. La Heij a, Nicolaas E.P. Deutz b, Aize Kijlstra a, Alfons G.H. Kessels c, Hans M.H. van Eijk b, Albert T.A. Liem a, Suzanne Dieudonné a, Fred Hendrikse a a Department of Ophthalmology, University Hospital Maastricht, 6202 AZ, Maastricht, The Netherlands b Department of Surgery, University of Maastricht, The Netherlands c Department of Clinical Epidemiology and Medical Technology Assessment, University Hospital Maastricht, The Netherlands Received 13 February 2005; accepted in revised form 22 October 2005 Available online 10 March 2006 Abstract Experimental models have implicated glutamate in the irreversible damage to retinal cells following retinal detachment. In this retrospective study we investigated a possible role for glutamate and other amino acid neurotransmitters during clinical rhegmatogenous retinal detachment (RRD). Undiluted vitreous samples were obtained from 176 patients undergoing pars plana vitrectomy. The study group consisted of 114 patients (114 eyes) with a rhegmatogenous retinal detachment. Controls included 52 eyes with an idiopathic macular hole or idiopathic epiretinal membrane and 10 eyes with a traction retinal detachment due to proliferative diabetic retinopathy. Vitreous concentrations of glutamate, gammaaminobutyric acid (GABA), taurine, glycine, and aspartate were determined by high-pressure liquid chromatography (HPLC). Multivariate analysis was used to examine a possible association between amino acid neurotransmitter levels and several clinical variables including visual acuity. The mean vitreous concentration of glutamate in eyes with a rhegmatogenous retinal detachment (16.6
5.6 mM) was significantly higher as compared to the controls (13.1
5.2 mM) (P ¼ 0.001). Taurine levels were also increased in RRD, whereas no significant difference could be observed in glycine, aspartate and GABA levels when comparing RRD with controls. A correlation was found between increased vitreous glutamate and a lower pre-operative visual acuity. No association was, however, observed between post-operative visual acuity and the level of any of the five amino acid neurotransmitters. RRD was associated with a significantly increased vitreous glutamate concentration. Using visual acuity as a functional parameter in this study, we could not demonstrate a correlation between vitreous glutamate, or any of the other tested amino acid neurotransmitters and visual outcome. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: retinal detachment; glutamate; excitotoxicity; amino acid; vitreous 1. Introduction Glutamate is an excitatory neurotransmitter in the retina, which after its release from neurons is cleared from the * This study was supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid. The authors have no proprietary interest related to this article. * Corresponding author. Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital Maastricht, P.O. Box 5800, P. Debyelaan 25, 6202 AZ, Maastricht, The Netherlands. Tel.: þ31 43 3875646; fax: þ31 43 3875343. E-mail address: r.diederen@np.unimaas.nl (R.M.H. Diederen). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.10.031 extracellular environment via uptake by Müller cells. Müller cells subsequently transform glutamate into glutamine by the enzyme glutamine synthetase (GS). Ischemia of the retina leads to changes in the localization of the retinal amino acid neurotransmitters glutamate and GABA as well as to accumulation of glutamate and GABA in Müller cells (Napper et al., 2001; Napper and Kalloniatis, 1999). Glutamate toxicity is considered to be caused by an excessive activation of the NMDA glutamate receptor, leading to an increased calcium influx, finally resulting in cell death (Lipton, 2004). In cat eyes with experimental retinal detachment, a marked decrease in the expression of glutamine synthetase (GS) 46 R.M.H. Diederen et al. / Experimental Eye Research 83 (2006) 45e50 activity has been found, suggesting that the clearance capacity of glutamate by Müller cells may be lost after retinal detachment (Lewis et al., 1989). Also, there is evidence for large shifts in intracellular glutamate concentrations in the retina following experimental retinal detachment (Sherry and Townes-Anderson, 2000). Furthermore, in experimental intervention studies, it has been shown that glutamate receptor antagonists were able to decrease retinal damage after experimentally induced retinal ischemia (Vorwerk et al., 1996; Mosinger et al., 1990; Yoon and Marmor, 1989). Experiments detecting changes in neurotransmitter levels after retinal detachment have mostly been performed in animal models. The role of neurotransmitters in human retinal detachment is not yet known and determining their role was therefore the purpose of our study. Since it is not possible to collect retinal samples from patients we decided to study the level of glutamate in vitreous. To address this issue, vitreous fluid was obtained from retinal detachment patients undergoing a vitrectomy. Using high-performance liquid chromatography (HPLC), we determined vitreous fluid levels of all five amino acid neurotransmitters that are known in the central nervous system (glutamate, GABA, aspartate, glycine, and taurine). Glutamate, GABA, and glycine also act as neurotransmitters in the mammalian retina, but there is scant evidence for a retinal transmitter function for aspartate and taurine (Marc et al., 1995). Previous research has shown higher glutamate levels in vitreous fluid of patients with proliferative diabetic retinopathy without detachment (Ambati et al., 1997). Therefore, we included eyes with proliferative diabetic retinopathy as a positive control. We found increased levels of glutamate and taurine in eyes with rhegmatogenous retinal detachment. To examine a role for these amino acids in retinal cell damage, visual acuity was used as a functional parameter. We could, however, not demonstrate a correlation between vitreous glutamate, nor any of the other tested amino acid neurotransmitters and visual outcome. were used as controls. We also collected vitreous fluid from eyes with a traction retinal detachment due to proliferative diabetic retinopathy (PDR). The study was performed with the agreement of the institutional ethics committee; all patients gave their informed consent prior to inclusion in the study and after the nature of the study was explained. The study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The following pre-operative clinical characteristics of the study patients were collected for statistical analysis: age, sex, eye affected, the number of detached quadrants of the retina, whether or not the central area of the macula (foveal region) was involved in the detachment, pre-operative corrected visual acuity, intraocular pressure (IOP), whether the patient used anti-glaucoma medication and the number of anti-glaucoma medications, whether the patient had diabetes mellitus, the number of prior eye operations, including cataract surgery, scleral buckling, prior endolaser or cryocoagulation, and follow-up time. By carefully questioning the patient, the approximate length of time between the occurrence of the retinal detachment and time of sampling was established. The following variables related to the vitrectomy were collected: whether intraocular endotamponade was necessary with oil or gas and whether or not a re-detachment occurred. At final follow-up we recorded visual acuity and the anatomic result. 2.2. Sample collection Undiluted vitrectomy samples (approximately 0.5 ml) were obtained by a conventional three-port closed vitrectomy technique by manual suction at the beginning of the vitrectomy, before opening the infusion line of Balanced Salt Solution (BSS, Alcon Laboratories, Texas, USA), as described earlier (La Heij et al., 2002). The samples were transferred to Eppendorf tubes and stored at 80  C until the time of amino acid analysis. 2.3. Amino acid analysis 2. Methods 2.1. Patients In a prospective study, vitreous fluid samples were collected from patients with a rhegmatogenous retinal detachment (RRD). All patients were operated in our department between May 1999 and January 2003 with a vitrectomy. Eyes with uveitis, trauma or vitreous hemorrhage were excluded. Only patients with a minimum follow-up of three months were included in the analysis. We operate eyes with a rhegmatogenous retinal detachment with up to proliferative vitreoretinopathy (PVR) grade C1 with a conventional scleral buckling technique. Eyes with PVR grade C2 and higher are operated on with a primary vitrectomy, as described earlier (La Heij et al., 2001). Vitreous samples from patients with idiopathic macular hole or idiopathic epiretinal membranes Amino acid analysis of vitreous fluid was performed using high-performance liquid chromatography (HPLC), as described earlier (Van Eijk et al., 1993). Vitreous analysis was performed in a masked fashion, using only the sample number without the technician knowing the clinical history of the patient. The following five amino acid neurotransmitters were analyzed: aspartate, gamma-aminobutyrate (GABA), glutamate, glycine, and taurine. Amino acid analysis was performed following precolumn derivatization with o-phthaldehyde (OPA). Samples (50 ml) were first centrifuged at 50,000  g. Next, 4 ml supernatant was pipetted into a glass 300 ml insert, containing 194 ml water and 2 ml norvaline solution (500 mmol/l; used as an internal standard). From this mixture, 5 ml was mixed automatically with 5 ml of OPA reagent, incubated for 2 min and injected onto a 150  4.6 mm (i.d.) column filled with Allsphere 3 mm (Alltech, Breda, Netherlands) using a WISP 715 47 R.M.H. Diederen et al. / Experimental Eye Research 83 (2006) 45e50 sample processor (Waters, Etten-Leur, Netherlands). Amino acid derivatives were eluted using 25 mmol/l citric acid buffer pH 6.8 containing 3% tetrahydrofuran as starting solvent, followed by gradient elution using a linear addition to 100% of solvent B (same buffer, now containing 40% acetonitril and 5% tetrahydrofuran) within 30 min. Fluorescence was monitored with a Jasco Model 820FP fluorescence detector (B&L Systems, Zoetermeer, Netherlands). Besides an external standard, norvaline was used as an internal standard. Measurements were made at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. Data were collected online and processed using Turbochrom Software (PerkineElmer, Oosterhout, Netherlands). 2.4. Statistical analysis For statistical analysis, we consulted a professional statistician at our University Hospital (AGH Kessels, co-author). Levels of all amino acid neurotransmitters were compared between groups using Student’s t-test. Because the GABA concentration was skewed, the statistics for GABA were performed after a log transformation. Comparisons for sex, glaucoma and prior eye surgery between patients with RRD and controls were performed using the Chi-squared test. The Pearson correlation test was used to test the association between the vitreous concentration of the five amino acid neurotransmitters and age, intraocular pressure, the number of quadrants the retina was detached, whether the patient had diabetes mellitus, the number of prior eye operations, including cataract surgery, scleral buckling, prior endolaser or cryocoagulation, the approximate length of time between occurrence of retinal detachment and time of sampling, whether a re-detachment occurred, and the anatomic result. The BonferronieHochberg correction for multiple comparisons was applied in all tests (Hochberg, 1988). For statistical analysis, Snellen visual acuities were converted to a logarithmic scale (LogMAR, i.e. the logarithm of the minimal angle of resolution), as described earlier (Ferris et al., 1982). The association between the levels of the various amino acid neurotransmitters, and pre-operative visual acuity was investigated with a multiple linear regression analysis using the status of the macula as co-variable. The association between the levels of the various amino acid neurotransmitters, and final post-operative visual acuity was investigated with a multiple linear regression analysis using the preoperative visual acuity as co-variable. Secondly, these associations were investigated with a multivariate linear regression model adjusting for nine variables: pre-operative visual acuity, diabetes, glaucoma, prior scleral buckling, prior endolaser, prior cryocoagulation, duration of detachment, status of the macula and number of quadrants of retinal detachment. 3. Results 23 months (SD
13 months). The mean duration of retinal detachment was 44 days (ranged from 1 to 365 days; Table 1). The control group consisted of 52 patients with an epiretinal membrane or macular hole. The mean age in this group was 68 years (SD
8 years). In addition, vitreous samples were obtained from a third patient group of 10 patients with traction retinal detachment due to proliferative diabetic retinopathy (PDR). The mean age in this third group was 52 years (SD
14 years). No significant differences were found between the age of patients and controls. Baseline clinical characteristics of all three groups are summarized in Table 1. The mean level of glutamate in vitreous of eyes with RRD was 16.6
5.6 mM, which was significantly higher than in the control group (13.1
5.2 mM; P ¼ 0.001; Table 2). The mean vitreous concentration of taurine in eyes with RRD was 26.0
7.8 mM and was also significantly higher than in the control group (22.6
6.6 mM; P ¼ 0.04). The mean GABA concentration in the vitreous fluid samples of RRD patients (1.9
0.0 mM) was not significantly different compared to controls (2.0
0.5 mM; P ¼ 0.82; Table 2). The mean concentrations of the other two neurotransmitters, glycine and Table 1 Basic clinical and ocular characteristics Age (years)a Maleb Femaleb Right eyeb Left eyeb IOP (mmHg)a Diabetesb Glaucomab Aphakicb Pseudophakicb Prior scleral bucklingb Prior endolaserb Prior cryocoagulationb Duration of detachment (days)a Macular detachmentb Number of quadrants of retinal Detachmenta Re-detachmentb Pre-operative visual acuitya,d Post-operative visual acuitya,d Duration of follow-up (months)a a Controls (n ¼ 52) PDRc (n ¼ 10) 58.2
15.1 69 (61%) 45 (39%) 61 (54%) 51 (46%) 14.5
5.7 3 (3%) 7 (6%) 6 (5%) 31 (27%) 62 (54%) 66.2
8.1 26 (50%) 26 (50%) 29 (56%) 23 (44%) 16.9
5.9 3 (6%) 4 (8%) 1 (2%) 12 (23%) 3 (6%) 51.3
14.0 5 (50%) 5 (50%) 5 (50%) 5 (50%) 12.7
5.1 10 (100%) 0 0 0 0 32 (28%) 41 (36%) 11 (21%) 3 (6%) 7 (70%) 0 16.6
14.1 18.0
9.6 43.6
72.1 70 (61%) 2.6
1.1 37 (33%) 1.7
0.9 1.4
1.0 22.9
13.0 Numbers are noted in mean
SD (standard deviation). Number (%). c RRD, rhegmatogenous retinal detachment; PDR, proliferative diabetic retinopathy. d Snellen visual acuities were converted to a logarithmic scale (LogMAR, i.e. the logarithm of the minimal angle of resolution). b Vitreous fluid samples were collected from 114 eyes (114 patients) with rhegmatogenous retinal detachment. The mean age was 58 years (SD
15 years). Mean follow-up time was RRDc (n ¼ 114) 48 R.M.H. Diederen et al. / Experimental Eye Research 83 (2006) 45e50 Table 2 Vitreous amino acid concentration (mM) Aspartate Glutamate Glycine Taurine GABA a b Controls (n ¼ 52) Eyes with RRDb (n ¼ 114) P value P valuea Eyes with PDRb (n ¼ 10) P value P valuea 6.6
2.5 13.1
5.2 49.4
36.3 22.6
6.6 2.0
0.5 6.7
2.8 16.6
5.6 42.5
32.0 26.0
7.8 1.9
0.5 0.795 0.001 0.223 0.008 0.819 >0.99 0.001 >0.99 0.040 >0.99 8.9
3.4 19.5
6.5 67.5
23.1 28.1
11.0 2.1
0.4 0.053 0.001 0.135 0.154 0.900 0.265 0.001 >0.99 >0.99 >0.99 After BonferronieHochberg correction for multiple comparisons. RRD, rhegmatogenous retinal detachment; PDR, proliferative diabetic retinopathy. aspartate, also did not differ significantly between patients with RRD and controls. The mean level of glutamate in the vitreous fluid of eyes with traction retinal detachment due to PDR was 19.5
6.5 mM, which was significantly higher than in the control group (13.1
5.2 mM; P ¼ 0.001; Table 2). No significant difference for glutamate was found between eyes with RRD and PDR. The mean concentrations of the other neurotransmitters, GABA, glycine, taurine and aspartate, did not differ significantly between the PDR group and controls (Table 2). In the control group, we found no statistically significant difference in the vitreous concentrations of all five amino acid neurotransmitters between patients with a macular hole and patients with an epiretinal membrane. Of all clinical variables, such as prior scleral buckling, prior endolaser, prior cryocoagulation, duration of detachment, status of the macula and number of quadrants and retinal detachment, analyzed in the present study, none did significantly correlate with the concentration of any of the amino acid neurotransmitters, except for glutamate. There we found a significant correlation between a higher vitreous glutamate concentration and a lower pre-operative visual acuity (P ¼ 0.039) in patients with RRD, using the status of the macula as co-variable. No significant correlation was found between any of the five amino acid neurotransmitter levels, taken at the time of retinal detachment and post-operative visual acuity recorded at final follow-up in patients with RRD after using multivariate analysis. Finally, of the clinical variables, only pre-operative visual acuity was associated with final visual outcome. 4. Discussion In this study we report a significantly elevated concentration of glutamate in the vitreous of eyes with rhegmatogenous retinal detachment compared to controls. Similar to RRD, glutamate levels in eyes with a traction retinal detachment due to proliferative diabetic retinopathy were also significantly higher than controls. This latter observation confirms an earlier study, in which also raised glutamate levels were detected in the vitreous of eyes with proliferative diabetic retinopathy as compared to controls (Ambati et al., 1997). Glutamate is the primary excitatory amino acid neurotransmitter within the retina, and excessive levels of glutamate can cause excitotoxicity. This may, at least in part, be due to excessive activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors and hence excessive Ca2þ influx through the receptor’s associated ion channel (Lipton, 2004). After its release from neurons glutamate is cleared from the extracellular environment via uptake by Müller cells. In these Müller cells, high-affinity glutamate transporters, like GLAST are believed to be essential for terminating synaptic transmission and for keeping the extracellular glutamate concentration below neurotoxic levels (Harada et al., 1998; Barnstable, 1993). Excessive glutamate levels can cause neurotoxicity via overproduction of nitric oxide (NO) which leads to toxic free radical formation. This produces cell death by causing DNA damage and decreased energy production by inhibition of mitochondrial function (Dawson and Dawson, 1996). The increased level of glutamate in the vitreous of eyes with clinical retinal detachment as shown in the current study is consistent with earlier experimental findings in cat eyes (Marc et al., 1998). In this cat study, the glutamate content of Müller cells was found to be increased and even remained elevated for many weeks after experimental retinal detachment. Other animal studies have provided evidence that extracellular glutamate can cause retinal ganglion cell death (Luo et al., 2001). In a clinical setting, retinal ganglion cell damage may be demonstrated by decreased visual acuity, or preferably by visual field loss. Because the current study was performed retrospectively and visual field testing is not a routine procedure in patients with RRD, we used visual acuity as a functional parameter. We also analyzed various other clinical variables to detect a possible association with neurotransmitter level. Although we found a correlation between higher vitreous glutamate levels and a lower pre-operative visual acuity, we were not able to demonstrate an association between any of the tested amino acid neurotransmitters and post-operative visual acuity. Similar to glutamate, we found a significantly higher concentration of taurine in eyes with an RRD compared to control eyes. These findings partly reflect results of the cat study by Marc et al. (1998) in which the authors found a supernormal restoration of taurine levels in Müller cells after an initial decline, in eyes with a retinal detachment. Ischemia has been shown to induce a glutamate-mediated release of GABA from amacrine cells in the rabbit retina (Osborne and Herrera, 1994). In the current study, rhegmatogenous retinal detachment was not found to be associated with a significant difference in vitreous GABA concentrations R.M.H. Diederen et al. / Experimental Eye Research 83 (2006) 45e50 compared to controls. This finding suggests that the glutamatemediated release of GABA from amacrine cells is not altered after rhegmatogenous retinal detachment. Glutamate acts as a precursor for GABA. After the glutamate transporter has taken up glutamate in the glial cells the enzyme glutamic acid decarboxylase (GAD) catalyzes the decarboxylation of glutamic acid to form GABA. The glutamate transporter needs ATP and therefore oxygen or glucose availability. A possible explanation as to why vitreous GABA levels are not to increase in patients with retinal detachment could be the lack of ATP in the Müller cells, due to the hypoxia in the detached retina. This may lead to an extracellular glutamate accumulation and less conversion of glutamate to GABA. Eyes with a traction retinal detachment due to PDR also did not have a significantly higher level of GABA in their vitreous compared to controls. This latter finding, however, is in contrast with a previous study in which the authors reported a significantly elevated level of GABA in the vitreous of patients compared to controls (Ambati et al., 1997). In this study 22 eyes were included with PDR, but these eyes did not have traction retinal detachment which was the main difference with the current study (Ambati et al., 1997). In contrast with the current study, the study by Asensio Sanchez et al. (2002), reported no significant differences in the glutamate or taurine levels between patient groups with RRD or macular hole. These authors, however, included only a small number of patients with a macular hole (n ¼ 5), as compared to the current study, in which 25 eyes with macular hole were investigated. Furthermore, the vitreous amino acid levels of our patient and control group were two to three times higher than vitreous amino acid levels reported earlier by Honkanen et al. (2003) and Asensio Sanchez et al. (2002), but were comparable to the levels found by Dreyer et al. (1996). It should be noted that, in the study reported by Dreyer et al. patients with cataract were used as controls, whereas we used patients with idiopathic macular holes or idiopathic epiretinal membranes as controls. Variation in the vitreous glutamate concentration between this study and the current study may be explained by differences in sample collection or in HPLC technique. In the study of Honkanen et al. samples were centrifuged before they were stored, while the samples in the current study and in the study by Dreyer et al. were centrifuged after thawing just before HPLC analysis. Cells that may be present in the vitreous fluid samples may release intracellular glutamate during freezing and thawing prior to centrifugation. Our patient group included 11 patients with glaucoma, seven patients with RRD and four eyes with a macular hole. Since it was shown that glaucoma might be associated with higher levels of vitreous glutamate (Dreyer et al., 1996), we also investigated whether these patients had a significantly higher level of glutamate in their vitreous fluid. We could not detect any differences, which were consistent with previous findings (Honkanen et al., 2003). Moreover, excluding these eyes did not significantly alter the difference in glutamate concentration between patient eyes and control eyes (16.5
5.6 vs 13.2
5.3 mM, patients with RRD vs controls). 49 In conclusion, in this clinical study we found a significantly elevated concentration of glutamate and taurine in the vitreous of eyes with retinal detachment, which confirms earlier experimental retinal detachment studies in the cat (Marc et al., 1998). Although we found a correlation between higher vitreous glutamate levels and a lower pre-operative visual acuity, we were not able to demonstrate an association between any of the tested amino acid neurotransmitters and post-operative visual acuity. References Ambati, J., Chalam, K.V., Chawla, D.K., D’Angio, C.T., Guillet, E.G., Rose, J.S., et al., 1997. Elevated g-aminobutyric acid, glutamate and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch. Ophthalmol. 115, 1161e1166. Asensio Sanchez, V.M., Corral Azor, A., Aguirre Aragon, B., De Paz Garcia, M., 2002. Amino acid concentrations in the vitreous body in control subjects. 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