December 2010
Volume 51, Issue 12
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Glaucoma  |   December 2010
Altered Calcium Signaling in an Experimental Model of Glaucoma
Author Affiliations & Notes
  • Minna Niittykoski
    From the Department of Ophthalmology, Institute of Clinical Medicine, and
  • Giedrius Kalesnykas
    From the Department of Ophthalmology, Institute of Clinical Medicine, and
  • Kim P. Larsson
    Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland;
  • Kai Kaarniranta
    From the Department of Ophthalmology, Institute of Clinical Medicine, and
    Department of Ophthalmology, Kuopio University Hospital, Kuopio, Finland;
  • Karl E. O. Åkerman
    Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland;
    Physiology, Institute of Biomedicine, University of Helsinki, Helsinki, Finland;
  • Hannu Uusitalo
    From the Department of Ophthalmology, Institute of Clinical Medicine, and
    Department of Ophthalmology, Medical School, University of Tampere, Tampere, Finland; and
    Department of Ophthalmology, Tampere University Hospital, Tampere, Finland.
  • Corresponding author: Minna Niittykoski, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P. O. Box 1627, FIN-70211 Kuopio, Finland; mijoni@gmail.com
Investigative Ophthalmology & Visual Science December 2010, Vol.51, 6387-6393. doi:10.1167/iovs.09-3816
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      Minna Niittykoski, Giedrius Kalesnykas, Kim P. Larsson, Kai Kaarniranta, Karl E. O. Åkerman, Hannu Uusitalo; Altered Calcium Signaling in an Experimental Model of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2010;51(12):6387-6393. doi: 10.1167/iovs.09-3816.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate calcium signaling in a rat experimental model of glaucoma.

Methods.: A method for labeling ganglion cell layer (GCL) neurons with the calcium indicator Fura-2 in flat-mounted retinas of adult rats was established. Pharmacologically evoked responses in laser-induced glaucomatous and control retinas were imaged 2 weeks after the initial laser treatment. The optic nerves of the same eyes were evaluated for neurodegenerative changes.

Results.: After laser treatment, intraocular pressure (IOP) was elevated 1.5- to 4.9-fold (24.70 ± 15.57 mm Hg) compared with control eyes (8.71 ± 1.53 mm Hg), and the area of neurodegenerative axons in optic nerve sections of laser-treated eyes was increased by 1.2- to 13.3-fold. The basal intracellular Ca2+ level, as revealed by the Fura-2 ratio, was elevated in GCL neurons of laser-treated eyes compared with controls. This might suggest a mild degree of damage at the level of the soma in the GCL neurons of eyes with elevated IOP. Although glaucomatous GCL neurons remained functional as assessed pharmacologically, analysis of imaging data revealed that responses evoked by a brief application of ATP were slightly reduced rather than increased in the cells of laser-treated eyes compared with controls. No significant relationships were found between IOP/optic nerve damage and functional characteristics (basal intracellular Ca2+ level or response to carbachol/elevated K+/ATP) within cells of laser-treated eyes.

Conclusions.: Ca2+ imaging is a useful tool to map altered physiological characteristics of individual GCL neurons in the glaucomatous eye.

Retinal ganglion cells (RGCs) are the final output neurons that relay visual information from the retina to the brain and are affected by still poorly understood pathologic mechanisms in glaucoma, a group of neurodegenerative diseases of the eye that is one of the leading causes of blindness. 1,2 Both apoptosis and excitotoxicity have been considered as mechanisms through which RGC death occurs in glaucoma. 1,3 The typical findings in glaucoma patients include damage to the optic nerve axons, as indicated by nerve fiber layer defects and cupping of the optic disc, and functionally by reduced visual fields. 1 Several risk factors, such as increased intraocular pressure (IOP) level, are now widely recognized, but the functional status of individual RGCs during glaucoma remains poorly understood. 
Calcium regulates and maintains many cellular functions such as cell proliferation and development, neurotransmitter release, metabolism, and apoptotic and necrotic forms of cell death. 4,5 One approach that can be used to examine calcium signaling at the cellular level utilizes optical recording in combination with Ca2+-sensitive fluorescence indicators. 6 Several published studies have imaged mixed retinal cell and RGC cultures and individual RGCs or retinal layers in situ. 7 18 In contrast, only one report on experimental glaucoma has used calcium imaging ex vivo. 19 In that study, induced IOP elevation did not impair retinal glutamate uptake. 19  
We hypothesized that responsiveness to various pharmacologic stimuli would be affected in glaucomatous RGCs and, furthermore, that the functional changes would depend on IOP exposure and the extent of the structural damage. To test this hypothesis, we investigated calcium signaling at the single cell level in a rat experimental model of glaucoma. We studied retinas from rats with a unilateral IOP increase while the other eye served as a control. The validity of glaucomatous damage was confirmed in optic nerve sections from the same rats. 
Methods
Glaucoma Model
Adult male Wistar rats (n = 22; weight, 458 ± 13 g; National Laboratory Animal Centre, University of Kuopio, Kuopio, Finland) were housed in a controlled environment (temperature +21°C ± 1°C, 12-hour light/12-hour dark cycle). Food and water were available ad libitum. These animal studies were approved by the Committee for the Welfare of Laboratory Animals of the University of Kuopio and adhered to the European Communities Council Directive (86/609/EEC) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Glaucoma was induced in anesthetized (75 mg/kg ketamine [Ketalar; Pfizer Oy Animal Health, Espoo, Finland]; 0.5 mg/kg medetomidine [Domitor, Orion Oyj, Espoo, Finland]; administered intraperitoneally) rats by laser photocoagulation of episcleral and limbal veins, as previously described. 20,21 Laser treatment was performed twice, with a 1-week interval. The amount of energy in an argon laser (Oculight; Iris Medical, Mountain View, CA) was set to 1 W for 0.2 seconds, and a total of 190 to 240 spots were delivered (50–100 μm spot size). The contralateral eye of each rat remained untreated, thus serving as a normotensive control. After this procedure, oxybuprocaine (Oftan Obucain, 4 mg/mL; Santen Oy, Tampere, Finland) was administered into the eyes and rats were aroused with atipamezole (1 mg/kg, administered subcutaneously; Antisedan; Orion Oyj, Espoo, Finland). 
During the follow-up period of 2 weeks (beginning from the first laser treatment), IOP was measured on the day after the laser treatments and then every third day using a tonometer (TonoLab TV02; Tiolat Oy, Helsinki, Finland). The last IOP measurement was performed before kill. Oxybuprocaine (Oftan Obucain, 4 mg/mL; Santen Oy) was applied into the eyes of fully anesthetized rats, and five readings were taken from both eyes in each time point. 
Quantitative Fluorescence Imaging
Anesthetized rats were decapitated, and their eyes were quickly transferred into chilled, oxygenated HEPES-buffered (20 mM, Fluka; BioChemika, Buchs, Switzerland) Ames' medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany; pH 7.4) supplemented with 0.5 mM probenecid (Sigma-Aldrich). Anterior segments were removed, and four slits were cut to the remaining eye cup. Then the retinas were separated from the sclera and placed onto a carrier (black HABP filter; Millipore, Billerica, MA) with the GCL uppermost. Each carrier was immediately transferred into a preincubation chamber (+30°C), and retinas were allowed to recover in the dark for 1 hour before loading was initiated. The loading method was modified from a previous study. 7 Injection and incubation bath solutions contained 0.01% pluronic F-127 (Molecular Probes, Eugene, OR) and 0.1% bovine serum albumin (Sigma-Aldrich) in HEPES-buffered Ames' medium supplemented with probenecid. A solution containing the calcium indicator dye Fura-2 acetoxymethyl (AM) ester (500 μM; Invitrogen, Molecular Probes) was injected (16 μL unilaterally) through the retinal layers into several spots near the centers of retinas with a Hamilton syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland). The retinas were then incubated in 10 μM Fura-2 AM containing solution for 3 hours. We have previously shown that 72% of RGCs in the glaucomatous eyes are functionally viable for loading after elevated IOP for 2 weeks. 21 After loading, the retinas were rinsed four times with oxygenated medium and placed for 20 to 30 minutes into the preincubation chamber before imaging was initiated. 
Pieces from retina (approximately one-quarter of the retina) were cut with an ophthalmic scalpel blade and placed into the recording chamber (0.2-mL vol) at +30°C with the GCL downward. This procedure was selected because it minimized cutting of the cellular processes before loading. Retinas were constantly perfused with oxygenated HEPES-buffered (20 mM) Ames' medium supplemented with 0.5 mM probenecid at a flow rate 1.5 mL/min. The microscope (TMS; Nikon, Tokyo, Japan) was focused on labeled GCL neurons. No imaging was performed in the close vicinity of the edges (peripheral rim and radius of quarter) because the tissue preparation might have caused some damage to the cells near the border. Furthermore, no imaging was taken from the centers of the retinas (i.e., the site in which injections were made). Circular regions of interest were outlined around somas of cells (small cells were not imaged). Before imaging was started, the background fluorescence was obtained according to instructions provided by the manufacturer (Intracellular Imaging Inc., Cincinnati, OH). Experiments were performed using UV light (340 and 380 nm) with a filter exchanger under the control of a fluorescence imaging system (InCyt Im2; Intracellular Imaging Inc.) and a 430-nm dichroic mirror (Nikon). Emission was measured through a 510-nm barrier filter (Nikon) with a charge-coupled device camera (Cohu, San Diego, CA). The ratio (F340/F380) was used as an indicator of the free intracellular calcium concentration. 6 Sampling frequency was set to 0.75 Hz. After baseline stabilization, cells were stimulated with 1 mM carbachol (2 minutes; Aldrich), 50 mM KCl (1.5 minutes; J. T. Baker, Deventer, Holland), and 400 μM ATP (1.5 minutes; adenosine 5′-triphosphate, magnesium salt; Sigma-Aldrich), with a washout period after each drug exposure (5 minutes, 10 minutes, and 5 minutes, respectively). Retinal pieces were imaged from both eyes of each rat, and a total of two to four imaging sessions per rat were performed. The sequence of imaged retinal pieces was alternated. Every other day, the first imaged piece was from a control retina and the next was from a glaucomatous retina. 
After imaging, the retinas were photographed with the same system. In some cases, the retinas were placed onto glass slides with the GCL facing upward and coverslipped. They were then viewed through an Olympus microscope (BX40; Olympus Europa GmbH, Hamburg, Germany) with 20× and 40× objectives and an Olympus filter set (U-MWU2; excitation filter 330–385 nm; emission filter 420 nm; dichromatic filter 400 nm; Olympus Europa GmbH). The system was equipped with an Olympus camera (DP50; Olympus Europa GmbH) and personal computer software (Viewfinder Lite; Pixera Corporation, Egham, UK). 
Imaging data were analyzed using graphing (Origin, version 7.5; Microcal Software Inc., Northampton, MA) and spreadsheet (Excel 2002; Microsoft Corporation, Redmond, WA) programs. Only the rats in which IOP remained elevated for 2 weeks and the cells that responded to elevated K+ were included in this study. Poorly labeled cells or cells with unstable basal calcium levels, cells with decreasing or increasing ratio levels throughout the experiment or showing irreversible responses despite the washout periods, and cells in which all three pharmacologic treatments were not successful were rejected from the analysis. Peak responses were measured, and results were expressed as a change in the ratio compared with basal (prestimulation) ratio, which was calculated by averaging 30 values (during the last 40 seconds before pharmacologic stimulation). Cells were also classified according to the types of responses. Classification was performed by comparing the extent of the increase by one treatment in relation to the responses evoked by the other two pharmacologic stimulations in the same cell (elevated K+ > carbachol > ATP, elevated K+ > ATP > carbachol, carbachol > elevated K+ >ATP). 
Histology
Optic nerves were isolated after kill and were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 hours (+4°C). They were then stored in tissue collection solution (30% ethylene glycol and 25% glycerol in 0.05 M phosphate buffer, pH 7.4) until they were washed overnight with 0.1 M phosphate buffer (pH 7.4), embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA), and cut with a cryostat (CM 3050S; Leica Microsystems Nussloch GmbH, Nussloch, Germany). Optic nerve sections (5-μm thick) were stained with slightly modified Gallyas silver staining to detect neurodegenerative axons of nonfunctional RGCs. 22 Optic nerve sections were viewed with a light microscope (BX40; Olympus) through 20× and 40× objectives. One section containing a cross-section of optic nerve was selected from each eye, and photographs from nonoverlapping fields in each quarter of the optic nerve section were captured with the 20× objective. Because of the capturing of nonoverlapping fields, the cross-shaped area containing the most central region was not analyzed, whereas analyzed areas covered peripheral edges. Photographs were transformed to grayscale (Photoshop CS2, version 9.0; Adobe Systems, San Jose, CA), and the areas of degenerative axons as well as the total area of optic nerves were measured (Scion Image for Windows software, release Alpha 4.0.3.2; Scion Corporation, Frederick, MD). Those areas were used to calculate the percentage of degenerated axons out of the total optic nerve area. 
Statistical Analysis
Data were statistically analyzed (SPSS for Windows software, version 14.0; SPSS Inc., Chicago, IL), and IOPs and imaging data (basal level, a change in the ratio) were normalized using log10 transformation before the analysis. Statistical evaluation of these data was conducted using the linear mixed-model procedure. Other imaging data, which were based on a classification (i.e., type of response), were averaged for each rat, and subsequently statistical comparisons were performed using the Mann-Whitney U test (exact method, two-tailed values). Areas of neurodegenerative axons in optic nerves were analyzed using the Mann-Whitney exact test (two-tailed). In the correlation analysis, data from each rat were averaged. Spearman correlation coefficients (two-tailed) were calculated to determine associations among IOPs, areas of neurodegenerative axons in optic nerves, and imaging data. Results are presented as mean ± SD unless otherwise stated. The level of statistical significance was set at P < 0.05. 
Results
Basal IOPs before the first laser treatment were 9.53 ± 1.48 mm Hg. After laser treatment, IOPs were significantly increased in the laser-treated eyes compared with the control eyes (linear mixed-model analysis, F 1, 148 = 180.588; P < 0.001; Fig. 1). The average IOPs at 2 weeks were 8.71 ± 1.53 mm Hg (range, 7.55–10.80 mm Hg) in control eyes and 24.70 ± 15.57 mm Hg (range, 13.20–40.10 mm Hg) in the laser-treated eyes. The IOP increase (mean of 2 weeks) in the laser-treated eyes was 2.9-fold (range, 1.5- to 4.9-fold) compared with control eyes. 
Figure 1.
 
IOPs (mean ± SD) in laser-treated (■) and control eyes (□) during 2-week period. Laser treatments were performed on days 0 and 7. These data are from 14 rats.
Figure 1.
 
IOPs (mean ± SD) in laser-treated (■) and control eyes (□) during 2-week period. Laser treatments were performed on days 0 and 7. These data are from 14 rats.
The area of neurodegenerative axons was determined in optic nerves from the eyes that had elevated IOP for 2 weeks to confirm the validity of the rat glaucoma model. Analysis of Gallyas silver-stained sections (Fig. 2) revealed that the area of neurodegenerative axons ipsilateral to laser treatment (1.57% ± 1.51%) was increased by an average of 3.9-fold (range, 1.2- to 13.3-fold) compared with controls (0.41% ± 0.21%; Mann-Whitney exact test; P = 0.001). The area of neurodegenerative axons in optic nerves increased as IOP increased in laser-treated eyes (IOP increase, fold above control, Spearman correlation, P < 0.05; cumulative IOP during 2 weeks, Spearman correlation, P < 0.01; Fig. 3). With respect to the different parameters related to IOP, the peak IOPs (43.83 ± 14.39 mm Hg; range, 22.00–72.20 mm Hg) were the poorest predictors of the area of neurodegenerative axons in optic nerves of laser-treated eyes (Spearman correlation, P > 0.05; data not shown). 
Figure 2.
 
Bright-field photographs of Gallyas silver-stained sections of optic nerves from laser-treated (A) and control eyes (B). The IOP increase (mean of 2 weeks) was 3.9-fold, and the area of neurodegenerative axons in optic nerves was 5.3% in the laser-treated eye. Small black dots (arrows) represent degenerated axons. Scale bar, 20 μm.
Figure 2.
 
Bright-field photographs of Gallyas silver-stained sections of optic nerves from laser-treated (A) and control eyes (B). The IOP increase (mean of 2 weeks) was 3.9-fold, and the area of neurodegenerative axons in optic nerves was 5.3% in the laser-treated eye. Small black dots (arrows) represent degenerated axons. Scale bar, 20 μm.
Figure 3.
 
Relationships between different parameters of IOP and area of neurodegenerative axons in optic nerves in individual rats. (A) IOP increase (fold above control, mean of 2 weeks, Spearman correlation coefficient 0.622, P < 0.05) and (B) cumulative IOP over the period 2 week (Spearman correlation coefficient 0.666, P < 0.01). These data are from 14 laser-treated eyes.
Figure 3.
 
Relationships between different parameters of IOP and area of neurodegenerative axons in optic nerves in individual rats. (A) IOP increase (fold above control, mean of 2 weeks, Spearman correlation coefficient 0.622, P < 0.05) and (B) cumulative IOP over the period 2 week (Spearman correlation coefficient 0.666, P < 0.01). These data are from 14 laser-treated eyes.
Representative examples of labeled GCL neurons are shown in Figure 4. Our labeling method was found to be suitable for examining Ca2+ responses in this rat model of glaucoma. A total of 451 cells of 765 imaged cells were accepted for the statistical evaluation (195 cells of 334 cells in controls and 256 cells of 431 cells in cells exposed to elevated IOP). Statistical analysis was based on 82.4% of imaged control retinas and 73.9% of imaged retinas from eyes exposed to elevated IOP. The basal level before pharmacologic applications differed between the control cells (F 340/F 380; 0.74 ± 0.33) and cells of laser-treated eyes that had been exposed to elevated IOP for 2 weeks (F 340/F380; 0.81 ± 0.38; linear mixed-model analysis, F 1, 436.712 = 21.270, P < 0.001; Table 1). Application of carbachol, a cholinergic receptor agonist, induced moderate intracellular Ca2+ responses, as revealed by Fura-2 ratios (Fig. 4). In the overall analysis, Δ Fura-2 ratios in control cells and cells exposed to elevated IOP did not differ (linear mixed-model analysis, F 1, 441.897 = 3.694, P > 0.05; Table 2). When cells were challenged with elevated K+, peak responses were larger and more obvious than those evoked by carbachol or ATP (Fig. 4). In fact, most cells responded in such a way that the changes evoked by elevated K+ were larger than those obtained with carbachol and ATP (elevated K+ > carbachol > ATP, ∼64% in controls and ∼76% in cells exposed to elevated IOP; elevated K+ > ATP > carbachol, ∼34% in controls and ∼16% in cells exposed to elevated IOP; Mann-Whitney exact test; P > 0.05). A minority of cells (∼1% in controls and ∼8% in exposed cells; Mann-Whitney exact test; P > 0.05) responded such that the changes to elevated K+ were the second largest, and none of imaged cells responded with smaller K+-induced changes compared to both of the other two stimuli. The changes evoked by elevated K+ were not significantly larger in control cells than in cells that had been exposed to elevated IOP (linear mixed-model analysis, F 1, 439.517 = 1.930, P > 0.05; Table 2), whereas Δ Fura-2 ratios evoked by ATP in control cells differed from those seen in the cells exposed to elevated IOP (linear mixed-model analysis, F 1, 441.531 = 38.030, P < 0.001; Table 2). 
Figure 4.
 
Photographs of Fura-2–labeled GCL neurons as well as examples of carbachol, elevated K+, and ATP-induced responses in a rat model of glaucoma. Images were captured from coverslipped retinas that were obtained from laser-treated (3.2-fold IOP increase) and control eyes. Scale bar, 10 μm. To enhance the clarity of the figures, the contrast and brightness were adjusted in the digital photographs. Examples of effects of pharmacologic manipulations on Fura-2 ratios (F 340/F 380) in cells of laser-treated eye exposed to elevated IOP for 2 weeks (black lines) and control cells of the contralateral eyes (gray lines) are also illustrated. IOP increases (mean of 2 weeks) were 3.9-fold (carbachol and ATP) and 1.6-fold (elevated K+) in the laser-treated eyes.
Figure 4.
 
Photographs of Fura-2–labeled GCL neurons as well as examples of carbachol, elevated K+, and ATP-induced responses in a rat model of glaucoma. Images were captured from coverslipped retinas that were obtained from laser-treated (3.2-fold IOP increase) and control eyes. Scale bar, 10 μm. To enhance the clarity of the figures, the contrast and brightness were adjusted in the digital photographs. Examples of effects of pharmacologic manipulations on Fura-2 ratios (F 340/F 380) in cells of laser-treated eye exposed to elevated IOP for 2 weeks (black lines) and control cells of the contralateral eyes (gray lines) are also illustrated. IOP increases (mean of 2 weeks) were 3.9-fold (carbachol and ATP) and 1.6-fold (elevated K+) in the laser-treated eyes.
Table 1.
 
Basal Level in Cells Obtained from a Rat Model of Glaucoma
Table 1.
 
Basal Level in Cells Obtained from a Rat Model of Glaucoma
Fura-2 Ratio (F340/F380, Mean ± SD) 95% Confidence Intervals (F340/F380) Percentiles (F340/F380)
Lower Upper 25th 50th 75th
Exposed to elevated IOP (n = 256) 0.81 ± 0.38* 0.52 0.82 0.52 0.66 0.93
Control (n = 195) 0.74 ± 0.33 0.50 0.78 0.55 0.63 0.78
Table 2.
 
Summary of Pharmacologically Induced Responses in Cells Obtained from a Rat Model of Glaucoma
Table 2.
 
Summary of Pharmacologically Induced Responses in Cells Obtained from a Rat Model of Glaucoma
Δ Fura-2 Ratios (Change ± SD) 95% Confidence Intervals (Change) Percentiles (Change)
Lower Upper 25th 50th 75th
Carbachol
    Exposed to elevated IOP (n = 256) 0.123 ± 0.139 0.040 0.096 0.046 0.092 0.140
    Control (n = 195) 0.122 ± 0.116 0.046 0.111 0.032 0.089 0.175
Elevated K+
    Exposed to elevated IOP (n = 256) 0.388 ± 0.384 0.122 0.276 0.169 0.240 0.408
    Control (n = 195) 0.398 ± 0.363 0.130 0.295 0.140 0.247 0.616
ATP
    Exposed to elevated IOP (n = 256) 0.062 ± 0.054* 0.023 0.047 0.022 0.048 0.077
    Control (n = 195) 0.065 ± 0.041 0.033 0.066 0.032 0.056 0.093
To investigate the nature of the glaucoma-induced changes in more detail, relationships between functional characteristics, different parameters of IOP, and optic nerve damage were evaluated in cells of laser-treated eyes. Neither basal level before pharmacologic applications nor changes evoked by carbachol, elevated K+, or ATP were correlated with the IOP increase, cumulative IOP, peak IOP, or area of neurodegenerative fibers in optic nerve sections (Table 3). 
Table 3.
 
Correlation Coefficients in Cells Obtained from Laser-Treated Eyes Exposed to Elevated IOP
Table 3.
 
Correlation Coefficients in Cells Obtained from Laser-Treated Eyes Exposed to Elevated IOP
Correlation Basal Level Carbachol Elevated K+ ATP
IOP increase −0.115 −0.309 −0.297 0.091
Cumulative IOP −0.139 −0.394 −0.200 0.067
Peak IOP −0.333 −0.600 −0.515 0.139
Area of neurodegenerative axons 0.455 0.248 0.079 0.636
Discussion
This study attempted to elucidate some of the functional alterations occurring in glaucomatous retina. Thus, single cells were examined by fluorescence imaging ex vivo in retinal tissue obtained from rats with experimentally induced glaucoma. We demonstrate here novel findings on basal intracellular calcium levels (i.e., Fura-2 ratio) and cellular responses that were evoked by a brief application of pharmacologic stimuli in surviving, functional cells of rats having at least a 1.5-fold IOP increase and at least a 1.2-fold increase in the area of neurodegenerative axons in optic nerves compared with controls. The functional characteristics as indicated by basal intracellular calcium levels and responses to pharmacologic stimuli in the cells of rats with glaucomatous eyes were not, however, associated with IOP level or with the area of neurodegenerative axons in optic nerve sections. 
In this study, tissue was obtained from rats that were followed up for 2 weeks after the initial laser operation. Our recent study 21 using the same model demonstrated that the laser-induced IOP increase caused a severe loss of RGCs. In the present study, exposure to elevated IOP resulted in a significant increase in the area of neurodegenerative axons in optic nerve sections of laser-treated eyes compared with control. In agreement with the present findings, IOP elevation and axonal injury, 20,23 26 as well as the correlations between the extent of optic nerve damage and different parameters of IOP exposure, have been reported in experimental glaucoma models. 23,25,27  
Next we showed that GCL neurons of adult rats could be labeled with calcium-indicatory dye. Bundles of labeled RGC axons were also observed in our study as in the reports of Hartwick et al. 13,19 in which adult rats were also used. In the present study, labeling did not occur without the injection step, and it is believed to involve retrograde transport of the dye into RGC somas by way of cut RGC axons passing through the injection site. Furthermore, to increase the specificity of these experiments, imaging was not performed near the injection site in which bright background fluorescence and unclear cellular boundaries were present. Because the imaged cells were located in the peripheral area of the retina rather than the central area of the retina and small cells were not imaged, it is likely that the imaged cells included RGCs, but displaced amacrine cells cannot be ruled out because Fura-2 AM was used (for diameters of soma and dendrites/processes, see Refs. 28 30). 
In the imaging experiments, we found that the basal intracellular calcium level was increased in cells of the exposed eyes, suggestive of a mild degree of damage at the level of the soma. This finding might reflect a somewhat reduced capacity to maintain low intracellular calcium levels in these cells exposed to elevated pressure. This basal elevation was, however, not reflected as a change in responsiveness to the stimuli, with one possible exception: the response to ATP. Over the long run, elevation of the basal intracellular calcium level may enhance cellular vulnerability to damage. As far as we are aware, the basal calcium level in glaucoma models has not been reported previously, but increased calcium levels were observed in RGC cultures after treatments inducing apoptosis and under hypoxic conditions. 14,31  
We have, for the first time, studied the effect of carbachol on calcium levels in GCL neurons of the rat retina. 32 34 Responses induced by carbachol and high K+, however, were unaltered by increased IOP and thus did not seem to be affected in the glaucoma model used in this study. Responsiveness to high K+ and ATP has been reported in previous retinal imaging studies. 9,35 39 For example, analogous to our data, Taschenberger et al. 37 demonstrated that KCl (35 mM) could elicit larger responses than ATP (300 μM) in cultured postnatal rat RGCs. The responsiveness to high K+ has been linked to neurons, whereas glial cells are unresponsive to K+ but more often responsive to ATP than neurons. 40 Surprisingly, the responsiveness to ATP was slightly reduced rather than increased in the GCL neurons that remained alive after IOP elevation. One possible explanation for the effect of ATP and the lack of significant difference in responses evoked by carbachol and high K+ is that the population of cells studied was not the same in the control retinas and the retinas that had been exposed to high IOP (i.e., the experimental design did not allow within-cell comparison). Without a more detailed investigation, it is difficult to judge the relevance of increased baseline Ca2+ and whether the decreased response to ATP is related to abnormal calcium dynamics in GCL neurons in eyes with elevated IOP. However, purinergic receptors might have a minor role in the pathophysiology of glaucoma. A role for endogenous extracellular ATP and P2X receptors in the acute pressure-induced RGC damage was recently demonstrated, 41 but the effect of sustained pressure elevation on P2 receptors has not been examined in detail. The reduction in the response to ATP might thus reflect some loss or selective damage of ATP-responsive cells. The P2 receptors have been associated not only with excitotoxic and apoptotic degeneration and glial activation but also with regeneration/cell survival and neuroprotective mechanisms. 42  
Finally, in contrast to our expectations, we found no evidence that physiological characteristics were correlated with IOP level or neurodegenerative axons of optic nerves. This would indicate that although elevated IOP caused the degeneration of axons, there is little impact on the level of the soma. It should be noted that there are a few previous studies that have examined the consequences of IOP pressure or structural damage on the functional properties. For example, a reduction in the amplitudes of pattern electroretinogram responses and scotopic threshold responses, which reflect impairment of the function of the inner retinal (mostly ganglion) cells in vivo, were detected in glaucoma models and after optic nerve transaction. 25,43 46 Data in humans indicate that the pattern electroretinogram can even be used to predict the development of glaucomatous visual field changes in patients with elevated IOP. 47  
In conclusion, the present results demonstrate that optical recording in combination with pharmacologic stimulation is an appropriate tool for mapping the physiological characteristics of individual GCL neurons. Fluorescence imaging revealed that the basal intracellular calcium level was increased whereas pharmacologic responsiveness to ATP was slightly reduced in surviving cells that had been exposed to elevated IOP. Further studies on functional changes, including Ca2+ influx and efflux mechanisms as well as Ca2+ buffering and sequestration in glaucoma and the relationship of functional characteristics to IOP exposure and structural damage, are warranted. Understanding pathophysiological signaling mechanisms may reveal novel therapeutic approaches for protecting surviving cells within glaucomatous tissue. 
Footnotes
 Supported by the Finnish Funding Agency for Technology and Innovations Grant 2396/31/03, the Finnish Cultural Foundation of Northern Savo, Else May Björn Eye Research Fund, the Finnish Eye Research Foundation, the Academy of Finland, and the Sigrid Jusélius Foundation.
Footnotes
 Disclosure: M. Niittykoski, None; G. Kalesnykas, None; K.P. Larsson, None; K. Kaarniranta, None; K.E.O. Åkerman, None; H. Uusitalo, None
The authors thank Anne-Mari Haapaniemi and Tiina Sistonen for technical assistance, Marja-Leena Hannila for statistical assistance, and Ewen MacDonald for revising the language of the manuscript. 
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Figure 1.
 
IOPs (mean ± SD) in laser-treated (■) and control eyes (□) during 2-week period. Laser treatments were performed on days 0 and 7. These data are from 14 rats.
Figure 1.
 
IOPs (mean ± SD) in laser-treated (■) and control eyes (□) during 2-week period. Laser treatments were performed on days 0 and 7. These data are from 14 rats.
Figure 2.
 
Bright-field photographs of Gallyas silver-stained sections of optic nerves from laser-treated (A) and control eyes (B). The IOP increase (mean of 2 weeks) was 3.9-fold, and the area of neurodegenerative axons in optic nerves was 5.3% in the laser-treated eye. Small black dots (arrows) represent degenerated axons. Scale bar, 20 μm.
Figure 2.
 
Bright-field photographs of Gallyas silver-stained sections of optic nerves from laser-treated (A) and control eyes (B). The IOP increase (mean of 2 weeks) was 3.9-fold, and the area of neurodegenerative axons in optic nerves was 5.3% in the laser-treated eye. Small black dots (arrows) represent degenerated axons. Scale bar, 20 μm.
Figure 3.
 
Relationships between different parameters of IOP and area of neurodegenerative axons in optic nerves in individual rats. (A) IOP increase (fold above control, mean of 2 weeks, Spearman correlation coefficient 0.622, P < 0.05) and (B) cumulative IOP over the period 2 week (Spearman correlation coefficient 0.666, P < 0.01). These data are from 14 laser-treated eyes.
Figure 3.
 
Relationships between different parameters of IOP and area of neurodegenerative axons in optic nerves in individual rats. (A) IOP increase (fold above control, mean of 2 weeks, Spearman correlation coefficient 0.622, P < 0.05) and (B) cumulative IOP over the period 2 week (Spearman correlation coefficient 0.666, P < 0.01). These data are from 14 laser-treated eyes.
Figure 4.
 
Photographs of Fura-2–labeled GCL neurons as well as examples of carbachol, elevated K+, and ATP-induced responses in a rat model of glaucoma. Images were captured from coverslipped retinas that were obtained from laser-treated (3.2-fold IOP increase) and control eyes. Scale bar, 10 μm. To enhance the clarity of the figures, the contrast and brightness were adjusted in the digital photographs. Examples of effects of pharmacologic manipulations on Fura-2 ratios (F 340/F 380) in cells of laser-treated eye exposed to elevated IOP for 2 weeks (black lines) and control cells of the contralateral eyes (gray lines) are also illustrated. IOP increases (mean of 2 weeks) were 3.9-fold (carbachol and ATP) and 1.6-fold (elevated K+) in the laser-treated eyes.
Figure 4.
 
Photographs of Fura-2–labeled GCL neurons as well as examples of carbachol, elevated K+, and ATP-induced responses in a rat model of glaucoma. Images were captured from coverslipped retinas that were obtained from laser-treated (3.2-fold IOP increase) and control eyes. Scale bar, 10 μm. To enhance the clarity of the figures, the contrast and brightness were adjusted in the digital photographs. Examples of effects of pharmacologic manipulations on Fura-2 ratios (F 340/F 380) in cells of laser-treated eye exposed to elevated IOP for 2 weeks (black lines) and control cells of the contralateral eyes (gray lines) are also illustrated. IOP increases (mean of 2 weeks) were 3.9-fold (carbachol and ATP) and 1.6-fold (elevated K+) in the laser-treated eyes.
Table 1.
 
Basal Level in Cells Obtained from a Rat Model of Glaucoma
Table 1.
 
Basal Level in Cells Obtained from a Rat Model of Glaucoma
Fura-2 Ratio (F340/F380, Mean ± SD) 95% Confidence Intervals (F340/F380) Percentiles (F340/F380)
Lower Upper 25th 50th 75th
Exposed to elevated IOP (n = 256) 0.81 ± 0.38* 0.52 0.82 0.52 0.66 0.93
Control (n = 195) 0.74 ± 0.33 0.50 0.78 0.55 0.63 0.78
Table 2.
 
Summary of Pharmacologically Induced Responses in Cells Obtained from a Rat Model of Glaucoma
Table 2.
 
Summary of Pharmacologically Induced Responses in Cells Obtained from a Rat Model of Glaucoma
Δ Fura-2 Ratios (Change ± SD) 95% Confidence Intervals (Change) Percentiles (Change)
Lower Upper 25th 50th 75th
Carbachol
    Exposed to elevated IOP (n = 256) 0.123 ± 0.139 0.040 0.096 0.046 0.092 0.140
    Control (n = 195) 0.122 ± 0.116 0.046 0.111 0.032 0.089 0.175
Elevated K+
    Exposed to elevated IOP (n = 256) 0.388 ± 0.384 0.122 0.276 0.169 0.240 0.408
    Control (n = 195) 0.398 ± 0.363 0.130 0.295 0.140 0.247 0.616
ATP
    Exposed to elevated IOP (n = 256) 0.062 ± 0.054* 0.023 0.047 0.022 0.048 0.077
    Control (n = 195) 0.065 ± 0.041 0.033 0.066 0.032 0.056 0.093
Table 3.
 
Correlation Coefficients in Cells Obtained from Laser-Treated Eyes Exposed to Elevated IOP
Table 3.
 
Correlation Coefficients in Cells Obtained from Laser-Treated Eyes Exposed to Elevated IOP
Correlation Basal Level Carbachol Elevated K+ ATP
IOP increase −0.115 −0.309 −0.297 0.091
Cumulative IOP −0.139 −0.394 −0.200 0.067
Peak IOP −0.333 −0.600 −0.515 0.139
Area of neurodegenerative axons 0.455 0.248 0.079 0.636
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