Measurement of Amino Acid Levels in the Vitreous Humor of Rats after Chronic Intraocular Pressure Elevation or Optic Nerve Transection
Johns Hopkins University, Baltimore, Maryland, United States Journal of Glaucoma
(Impact Factor: 2.11).
10/2002; 11(5):396-405. DOI: 10.1097/00061198-200210000-00005
To investigate whether the levels of free amino acids and protein in the vitreous of rat eyes are altered with chronic intraocular pressure (IOP) elevation or after optic nerve transection.
The concentrations of 20 amino acids in the vitreous humor were measured by high-performance liquid chromatography in both eyes of 41 rats with unilateral IOP elevation induced by translimbal photocoagulation. Eyes were studied 1 day and 1, 2, 4, and 9 weeks after initial IOP elevation. The same amino acids were measured in 41 rats 1 day and 2, 4, and 9 weeks after unilateral transection of the orbital optic nerve. The intravitreal protein level was assayed in additional 22 rats with IOP elevation and 12 rats after nerve transection. Two masked observers evaluated the amount of optic nerve damage with a semiquantitative, light-microscopic technique.
In rats with experimental glaucoma, amino acid concentrations were unchanged 1 day after treatment. At 1 week, 4 of 20 amino acids (aspartate, proline, alanine, and lysine) were higher than in control eyes ( < or = 0.01), but this difference was nonsignificant after Bonferroni correction for multiple simultaneous amino acid comparisons (none achieved < 0.0025). No amino acid was significantly different from control in the nerve transection groups (all > 0.05). Vitreous protein level was significantly higher in glaucomatous eyes than their paired controls at 1 day ( < 0.0001) and 1 week ( < 0.002). One day and 1 week after optic nerve transection, vitreal proteins were significantly elevated compared with control eyes from untreated animals ( < 0.0020 and < 0.0022, respectively), though not compared with their fellow eyes ( = 0.25 and 0.10).
Chronic experimental glaucoma and transection of the optic nerve increase the amount of protein in the rat vitreous above control levels. In the vitreous of rats with experimental glaucoma, a number of free amino acids were transiently elevated to a modest degree, but no significant difference in vitreous glutamate concentration was detected ( > 0.01).
Available from: PubMed Central
- "A recent report using glutamate transporter-knockout mice supports the idea that excessive glutamate concentrations in the retina may induce RGC death . However, only a few reports show glutamate increases in rodent retina due to ocular hypertension [17,18], while other reports show no increase in vitreous glutamate concentration in glaucoma patients [19,20] or in animal models [19,21]. In addition, previous histological analyses of glutamate transporters in ocular hypertensive eyes reported conflicting results; some reported decreases in Gamma-ray Large Area Space Telescope (GLAST) and GLT1 immunoreactivities in RGCs and/or Muller cells [18,22], while others reported increases [15,23]. "
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ABSTRACT: To investigate the effect of hyperbaric pressure on purified retinal ganglion cells (RGCs) and the additive effect of hyperbaric pressure on glutamate-induced RGC death.
An RGC primary culture from 8-day-old Wistar rats was prepared and cultured in a hyperbaric chamber. The RGC survival rate under various pressure conditions and with 5 or 25 µM of glutamate stimulation was determined and compared with that of RGCs under isobaric conditions. First, RGCs were cultured at atmospheric pressure (0 mmHg) and under hyperbaric pressure (+30 and +90 mmHg, with pressure fluctuations varying from 0 to +30 or +60 mmHg). Next, RGCs were cultured at +15, +30, and +90 mmHg with the addition of 5 or 25 µM of glutamate. The effects of N-Methyl-D-aspartic acid (NMDA) and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid (AMPA)/kainate receptor antagonists, MK-801, and 6,7-dinitroquinoxaline-2,3-dione (DNQX), on cell survival were assessed. Additionally, types of cell death and the induction of Bcl-2-associated X protein (BAX) leading to apoptosis were studied under hyperbaric pressure conditions and/or with 5 µM of glutamate.
RGC death was not induced under increasing or fluctuating pressure conditions. RGC death was induced by 25 µM of glutamate and increased as pressure increased. RGC death was not induced by 5 µM of glutamate but was induced by and increased with increasing pressure. MK-801 and DNQX significantly reduced glutamate-induced RGC death, and DNQX was more effective than MK-801. Under hyperbaric pressure conditions, the addition of 5 µM of glutamate resulted in the induction of apoptosis and BAX, which did not occur under hyperbaric pressure conditions or with the addition of glutamate alone.
In a rat RGC culture, hyperbaric pressure alone did not induce RGC death but increased RGC susceptibility to glutamate toxicity, which may be of relevance to ocular diseases with pressure-induced RGC death.
Available from: Richard Dubielzig
- "When using time-efficient approaches, like semiquantitative grading schemes [12,23,24], random sampling [25,26], targeted [14,27], or automated full counts , to assess optic nerve damage, the subjectivity of important steps in the protocol can introduce significant variability in the data. Many of the published studies using these counting methods and grading schemes have not addressed variance between raters and optic nerve slides or attempted to evaluate sources of bias in counting. "
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ABSTRACT: To describe and validate a semi-automated targeted sampling (SATS) method for quantifying optic nerve axons in a feline glaucoma model.
Optic nerve cross sections were obtained from 15 cats, nine with mild to severe glaucoma and six with normal eyes. Optic nerves were dissected, fixed in paraformaldehyde and glutaraldehyde, and processed for light microscopy by resin embedding, sectioning, and staining of axon myelin sheaths with 1% p-phenylenediamine before axon quantification. Commercially available image analysis software was used as a semi-automated axon counting tool (SCT) and was first validated by comparison with a manual axon count (MAC). This counting tool was then used in a SATS method performed by three masked raters and in a semi-automated full count (SAFC) method performed by a single observer. Correlation was assessed between the SCT and MAC using a linear model and analysis of covariance (ANCOVA). Correlation between the SATS and SAFC methods was calculated and the bias, systematic errors, and variance component assessed. The intraclass correlation coefficient (ICC) was determined to establish inter-rater agreement. In addition, the time required to perform the SATS and SAFC methods was evaluated.
Correlation between the axon counts obtained by the SCT and MAC was strong (r = 0.9985). There was evidence of an overcounting of axons by the SCT compared to the MAC with a percentage error rate of 13.0% (95% confidence interval [CI] 11.0%, 15.1%). Both the correlation of SATS count (average per rater) to SAFC (r = 0.9891) and inter-rater agreement (ICC = 0.986) were high. The SATS method presented an overall positive counting error (p<0.001) when compared to the SAFC, consistent with a fixed percentage overestimation of 11.2% (95% CI 8.3%, 14.2%) of the full count. The average time required to quantify axons by the SATS method was 10.9 min, only 27% of that required to conduct the SAFC.
Our data demonstrate that the SATS method provides a practical, rapid, and reliable means of estimating axon counts in the optic nerves of cats with glaucoma.
Available from: Kohichi Tanaka
- "Excitotoxic neuronal cell death is thought to be a final common pathway in various neurological diseases, ranging from acute ischemic stroke to chronic neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis [2-5]. Glutamate excitotoxicity has also been proposed to be an important contributor to the death of retinal ganglion cells (RGCs) in glaucoma and ischemia-related conditions such as vessel occlusion and diabetic retinopathy [6-8], although some investigations have failed to confirm elevated glutamate concentration both in human patients with glaucoma  and in animal models of glaucoma [10,11]. The toxic effects of glutamate on RGCs are predominantly mediated by the overstimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) due to their extreme permeability to calcium ions . "
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ABSTRACT: Loss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A--D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.
GluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.
Our data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.
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