The incidence and prevalence of glaucoma increases exponentially with age.1 However, the most well-defined risk factor for glaucoma, elevated intraocular pressure (IOP), shows only a modest increase with age.2 This suggests that aging influences IOP-independent pathways to increase the risk of neuronal injury. Studies in the area of stroke provide evidence that older brains are less capable of recovery from ischemic insult than younger ones.3,4 DiNapoli et al5 have shown that older rats exhibit a deterioration in the blood-brain barrier, which they propose could account for the greater neuronal damage and slower recovery of older brains after stroke.
Consistent with these studies, Kawai et al6 have shown that there is an age-related decline in retinal ganglion cell numbers (2-month-old vs. 24-month-old Fischer rats). They demonstrated that the remaining retinal ganglion cells in 2-year-old rats were more sensitive to ischemia reperfusion injury induced by IOP elevation than those in the 2-month-old rats. Katano et al7 considered the effect of a short period of acute IOP elevation on retinal function in young and old Wistar rats and found that 18-month-old rats failed to fully recover from an IOP insult of 80 mm Hg for 120 min, whereas 4-month-old rats showed complete recovery. These studies suggest that older eyes are more susceptible to severe IOP elevation. What is not known is whether older eyes show increased sensitivity and impaired recovery to more moderate levels of IOP elevation and whether older eyes are able to recover from repeated episodes of IOP elevation.
This study will investigate the susceptibility of 3- and 18-month-old rats to repeated IOP elevation (60 mm Hg for 1 h). This combination of this IOP magnitude and duration has been shown to produce the largest separation between attenuation of inner and outer retinal responses.8 Data from Zhi et al9 suggest that inner retinal blood flow is compromised at 60 mm Hg, but choroidal flow is not, which may account for the observations of Bui and Fortune.8 Retinal function in young rats can completely recover from one episode of IOP elevation as high as 70 mm Hg,10,11 but it is unclear whether multiple episodes of a similar magnitude will produce a cumulative retinal dysfunction. Given that studies have demonstrated that older neurons are more susceptible to stress, we will investigate whether repeated episodes of IOP challenge produces greater functional deficits in older eyes.
All experimental procedures abide by the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and approval was obtained from our facility’s animal ethics committee. Two cohorts of Sprague-Dawley rats (3-month old, n = 16, and 18-month old, n = 16) were housed in a 12:12-h light:dark cycle, with a maximum illuminance of 50 lux. Food and water were available ad libitum.
Before experimentation, animals were anesthetized by intramuscular injection of ketamine:xylazine (60:5 mg/kg; Troy Laboratories Pty Ltd., New South Wales, Australia) followed by topical application of proxymetacaine (0.5%; Alcon Laboratories, New South Wales, Australia) for corneal anesthesia. Mydriasis was achieved by application of tropicamide (0.5%; Alcon Laboratories) and phenylephrine hydrochloride (2.5%; Chauvin Pharmaceuticals Ltd., United Kingdom).
All animals underwent overnight dark adaptation to allow for electroretinogram (ERG) measurement (see later), immediately after which animals underwent anterior chamber cannulation. This was repeated for the next 3 weeks for a total of four insults, with 7 days of recovery between each IOP challenge. At the fifth week, only dark-adapted ERG was measured, followed by animal termination. Thus, each functional assessment allowed for 7 days of recovery after the previous IOP challenge. Repeated cannulation appeared to have little effect on pupil size in 3- or 18-month-old rats.
Each animal had one eye randomly assigned to either IOP elevation (60 mm Hg) or sham (15 mm Hg) for 1 h. Throughout the treatment, body temperature was maintained by a circulating water pad. Core temperature and pulse were monitored continuously, and blood pressure was measured every 15 min (tail cuff sphygmomanometer, ML125; ADInstruments Pty Ltd., New South Wales, Australia).
Repeated IOP Elevations
Intraocular pressure elevation was achieved by cannulating the anterior chamber using beveled glass pipettes (75-μm tip; Harvard Apparatus, Kent, United Kingdom). We have previously calibrated our glass pipettes and shown that resistance across the tip is likely to have negligible effect at 60 mm Hg.12 Each glass pipette was attached via polyethylene tubing (1.27 × 0.97 mm; Microtube Extrusions, New South Wales, Australia) to a Hanks balanced salt solution reservoir (Sigma-Aldrich, Castle Hill, New South Wales, Australia). A pressure transducer was placed in a series to continuously monitor IOP (Transpac; Abbot Critical Care Systems, Sligo, Ireland). The height of the reservoir was precalibrated to give a pressure of either 15 mm Hg (sham baseline) or 60 mm Hg (IOP challenge). The beveled glass pipette was then inserted with the aid of micromanipulators (KITE-R; World Precision Instruments, Sarasota, FL) into the rodent’s anterior chamber, perpendicular to the corneal center, avoiding contact with the iris and lens.
Retinal function was assessed with the full-field ERG, after dark adaptation (>12 h). Electrode configuration and flash characteristics have been described previously.10 Briefly, purpose-built chlorided silver electrodes were used for ERG recording, where the active (0.8-mm diameter) was positioned on the corneal apex and the ring-shaped reference was placed around the sclera near the equator. The ground electrode (F-E2-30; Grass Telefactor, West Warwick, RI) was inserted subcutaneously into the tail. Electrical contact and corneal hydration were maintained with 10-mg/mL carmellose sodium (Celluvisc; Allergan, Irvine, CA). The ERG stimulus was generated by light-emitting diodes (5 W, 5500 K; Luxeon, Calgary, Alberta, Canada) in a Ganzfeld sphere (Photometric Solutions International Pty Ltd., Huntingdale, Victoria, Australia). Electroretinograms were collected over a range of luminous energies (−6.53 to 2.07 log scotopic cd.s.m−2). Noise was reduced by averaging 20 signals at the dimmest light levels, with progressively fewer waveforms averaged with increasing luminous energy. Interstimulus interval was lengthened from 2 to 120 s to allow for total recovery between successive flashes.
The leading edge of scotopic α wave is modeled using a delayed Gaussian equation.13,14
In Equation 1, the P3 (μV) response for a given luminous energy (i, log cd.s.m−2) is expressed as a function of time (t, s), saturated amplitude (RmP3), and sensitivity (S, m2.cd−1.s−3) after a brief delay (td, s). All parameters were optimized by minimizing the sum-of-square merit function with the Solver module in Excel (Microsoft, Redmond, WA) across an ensemble of waveforms returned from the top three luminous energies (1.78, 1.95, and 2.07 log cd.s.m−2), which elicits saturated a-wave amplitude.15
Bipolar Cell Response
To isolate the bipolar cell component (P2) of the ERG, the modeled P3 (Equation 1) is subtracted from the raw ERG.16 The amplitude of the isolated putative P2 as a function of luminous energy is modeled with a saturating hyperbolic function (Equation 2).17
The P2 amplitude (V, μV) is defined as a function of luminous energy (i, log cd.s.m−2); Vmax (μV), a saturated amplitude; and the semisaturation constant (k, log cd.s.m−2), the inverse of which gives sensitivity (K = 1/k). Using the Solver module of an Excel spreadsheet (Microsoft), Vmax and k were optimized to minimize the sum-of-square merit function.
Retinal Ganglion Cell Response
The amplitude of the ganglion cell–dominated scotopic threshold response (STR)8 is measured at a fixed time of 130 ms (positive STR [pSTR]) after stimulus onset. Because of the small amplitude of STR components, data were averaged across three luminous energies (−5.14, −5.00, and −4.78 log cd.s.m−2).
Data Analysis and Statistics
Data for each eye (i.e., sham treated, 15 mm Hg, or IOP treated, 60 mm Hg) is expressed as a percentage relative to its own baseline recorded immediately before the first cannulation. A repeated-measures two-way analysis of variance (ANOVA) established that there was no interaction between time (weeks, nested factor) and treatment (15/60 mm Hg, between factor); thus, data are collapsed across the 4 weeks. Data across ages could then be compared with a repeated-measures two-way ANOVA (between factor age, within factor 15/60 mm Hg). A separate two-way ANOVA was used to compare treatment effect across ERG parameters within an age group (between factor ERG parameters, within factor 15/60 mm Hg).
Fig. 1A and C shows that four repeated treatments (either 15 or 60 mm Hg) produce minimal functional change in 3-month-old rats. After four episodes of cannulation, the 18-month-old rats show a reduction in the b wave (Fig. 1B) and, in particular, the positive component of STR (Fig. 1D) for both sham and IOP-elevated eyes.
Fig. 2 illustrates the effect of treatment (sham and IOP elevation) by expressing ERG parameters for 3- and 18-month-old rats as a percentage change relative to each eye’s own baseline. For the 3-month-old rats, repeated-measures ANOVA reveals that there was no treatment effect (sham vs. IOP elevation) for photoreceptoral amplitude (RmP3, Fig. 2A; F1,19 = 0.02, P = 0.90), bipolar cell P2 amplitude (Vmax, Fig. 2C; F1,19 = 0.12, P = 0.73), or ganglion cell amplitude (pSTR, Fig. 2E; F1,18 = 0.01, P = 0.92). Furthermore, there was no effect across the number of cannulations (RmP3 F1,19 = 0.85, P = 0.47; Vmax F1,19 = 1.88, P = 0.14; pSTR F1,18 = 2.70, P = 0.05), indicating that repeated anterior chamber insults (cannulation and/or IOP elevation) do not affect the ERG output in 3-month-old rats. In contrast, Fig. 2G shows that, in 3-month-old rats, phototransduction sensitivity (S) was significantly greater in IOP-elevated eyes than in sham-treated eyes (F1,18 = 4.58, P < 0.05). This increase in sensitivity was not constant across the number of cannulations (F1,18 = 16.41, P < 0.0001). Analysis of raw phototransduction sensitivity in 3-month-old rats shows that there was a significant increase from baseline in both sham-treated (F4 = 2.9, P = 0.03) and IOP-treated (F4 = 5.2, P = 0.002) eyes.
Bipolar sensitivity (K, Fig. 2I) both for 3-month-old sham and IOP-elevated eyes was increased, but there was no difference between the two groups (F1,15 = 0.81, P = 0.38). A significant effect across the number of cannulations was also observed (F1,15 = 4.83, P < 0.01), with bipolar sensitivity increasing significantly from baseline after cannulations 2 to 4.
In the older rats, Fig. 2B shows that there was a decrease in RmP3 for both sham and IOP-elevated eyes across all cannulations when compared with baseline. This relative decrease was the same for sham and IOP-elevated groups (F1,23 = 0.31, P = 0.58). However, there was a significant effect across the number of cannulations (F1,23 = 7.04, P < 0.001). This finding is similar for the bipolar cell Vmax, which declines with the number of cannulations (Fig. 2D; IOP effect F1,23 = 1.12, P = 0.30; cannulation effect F1,23 = 20.13, P < 0.0001). In Fig. 2F, there was also no difference between IOP-elevated and sham eyes (F1,21 = 0.02, P = 0.90); however, the pSTR did decline significantly after the first cannulation (F1,21 = 6.99, P < 0.001).
Unlike the improvement in sensitivity seen in 3-month-old rats, photoreceptor sensitivity in older rats (Fig. 2H) decreases after the fourth repeated cannulation (F1,21 = 3.23, P < 0.05) in both sham-treated and IOP-treated cohorts (F1,21 = 0.15, P = 0.71). Bipolar cell sensitivity (K, Fig. 2J) in older rats also shows a significant decline with repeated cannulation (F1,21 = 12.89, P < 0.0001) for both sham and IOP-elevated eyes (F1,21 = 0.01, P = 0.94).
Fig. 3 shows the overall effect of repeated injury by averaging across the four cannulations. Two-way ANOVA between age group and treatments revealed a significant age effect for all ERG parameters (RmP3 F1,1 = 6.02, P < 0.05; Vmax F1,1 = 3.05, P < 0.0001; pSTR F1,1 = 12.03, P < 0.01; S F1,1 = 8.80, P < 0.01; K F1,1 = 47.62, P < 0.0001). There was no IOP elevation effect in either 3- or 18-month-old rats (RmP3 F1,1 = 0.09, P = 0.76; Vmax F1,1 = 0.50, P = 0.48; pSTR F1,1 = 0.27, P = 0.61; S F1,1 = 0.47, P = 0.49; K F1,1 = 0.20, P = 0.65).
To investigate whether an ERG component amplitude was more affected than others, a two-way ANOVA comparing relative changes in RmP3, Vmax, and pSTR across sham-treated and IOP-treated eyes was performed for both ages. In the 3-month-old rats, there was no significant difference between ERG components (F1,2 = 1.31, P = 0.28). However, the decrease in amplitude across RmP3, Vmax, and pSTR was not uniform in the older rats (F1,2 = 15.16, P < 0.0001), as a Bonferroni post hoc test reveals that the ganglion cell–dominated pSTR was significantly more attenuated than the photoreceptoral RmP3.
In 3-month-old rats, bipolar cell sensitivity showed a larger increase with repeated cannulation than did photoreceptoral sensitivity (F1,1 = 12.00, P < 0.01, comparison across Fig. 3D and E). In contrast, older rats showed a greater reduction in bipolar cell sensitivity when compared with the photoreceptoral sensitivity (F1,1 = 106.00, P < 0.0001, comparison across Fig. 3D and E).
This study shows that repeated injury has little effect on the retinal function of 3-month-old albino rats but produces dysfunction in 18-month-old rats. The observation that there was no dysfunction in younger animals is consistent with previous findings that younger rats can completely recover from a single episode of IOP challenge similar in magnitude to that used here (70 mm Hg for 1 h).10,11,18 Our study extends previous reports to show that younger rats are able to resist repeated moderate IOP insults of short duration separated by 1 week.
Our data provide a possible explanation for the functional resilience of the inner retina in 3-month-old rats. In particular, ganglion cell dysfunction arising from repeated injury could be masked if the upstream input to the ganglion cells were to be increased. One way for greater input to be achieved is by means of an improvement in photoreceptor and/or bipolar cell sensitivity to light, as is observed in our data (Fig. 2). This means that, at dim light levels, the same number of photons captured would produce a larger ganglion cell response (leftward shift of the luminance energy response function). The mechanism underlying this observed improvement in sensitivity is unclear. Studies in the brain show that new neurons can proliferate at the site of ischemic injury.19 However, an increase in the number of photoreceptors should produce larger photoreceptoral amplitude without a change in sensitivity, which was not the case in our data. Therefore, the upregulation of sensitivity in 3-month-old rats reflects other mechanisms, possibly alterations in the efficiency of phototransduction proteins.20
Our data also show that, in 3-month-old rats, bipolar cell sensitivity (mean [SEM] 52.8% [16.3%], F1,1 = 12.00, P < 0.01) increased even more than phototransduction sensitivity (mean [SEM] 14.0% [4.0%]). This is consistent with the observation of Aleman et al21 that rod-bipolar cell synapses can increase after retinal injury. A consequence of a greater number of bipolar cell dendrites with the same number of photoreceptors (same phototransduction amplitude) would be increased convergence, which could manifest as an improved sensitivity to light in the rod pathway. We propose that upregulation of sensitivity within the phototransduction cascade and between the photoreceptor and bipolar cell synapses in 3-month-old rats reflects a compensatory mechanism to maintain normal retinal output in response to repeated injury. The data from 18-month-old rats suggest that such compensatory mechanisms are insufficient. Our previous study22 showed that aging itself in 18-month-old compared with 3-month-old rats causes increased sensitivity. This is consistent with a previous report that, in older mice, there is evidence of proliferation of rod-bipolar and horizontal cell dendrites toward rod photoreceptor spherules.23 It is possible that 18-month-old rats have already reached their maximal capacity to increase sensitivity and, hence, in response to repeated stress, cannot further compensate. This manifests as loss in ganglion cell output with repeated insult in the older cohort.
Note that, in the older eyes, both repeated sham cannulation and IOP elevation produced similar functional deficits. Based on this observation, we believe that there is a common mechanism of injury in both sham and IOP-elevated eyes. Although the exact mechanism of injury in the older eyes from cannulation is unclear, based on previous reports, we speculate that inflammation may be involved. Hoyng et al24 have reported that cannulation of the rabbit anterior chamber resulted in classic signs of inflammation, which were observable on the anterior ocular surface as well as in the aqueous humor. More recently, Chinnery et al25 showed that corneal trauma followed by topical application of a lipopolysaccharide in mice produced retinal inflammation. It is also well established that basal inflammation increases with age26–28 as demonstrated by accumulations of macrophages29 and microglia25 in the subretinal space of older mice. Thus, it is possible that inflammatory processes contribute to the nonspecific functional losses arising from repeated insults to older eyes.
In 3-month-old animals, four repeated cannulations with or without IOP elevation did not decrease the amplitude of retinal responses. There was a significant improvement in the sensitivity of outer retinal components, which we believe represents a compensatory mechanism to account for the lack of inner retinal dysfunction in 3-month-old rats. In older eyes, repeated trauma (both sham treatment and IOP elevation) produced a cumulative loss of retinal function with the number of cannulations, with the exact mechanism for injury unknown. However, we propose that, as the older eyes have already upregulated retinal sensitivity to counteract age-related cell loss, there is no longer a buffer to protect against additional injury.
Bang V. Bui
Department of Optometry and Vision Science
University of Melbourne
Parkville 3010 Victoria
This work was supported by National Health and Medical Research Council project grant 537077.
This manuscript has been presented as a poster at The Association for Research in Vision and Ophthalmology at Fort Lauderdale, Florida, in May of 2012; Vingrys AJ, Charng J, Nguyen CT, Jobling AI, Bui BV. The effect of repeated intracameral injection on young and old rat eyes. Invest Ophthalmol Vis Sci. ARVO E-Abstract 2012.
Received June 19, 2012; accepted November 26, 2012.
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