Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

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We investigated the effect of acetazolamide on retina function by measuring the ERG in mice treated with acetazolamide. We observed that ERG findings in acetazolamide treated mice were analogous to those in human patients with the autosomal dominant rod-cone dystrophy RP17 (Yang et al., 2005). Therefore we caution that long term use of carbonic anhydrase inhibitors may have potential detrimental effects on photoreceptor cells and vision.

2. METHODS

2.1. Animals and Treatment

Mice were housed and handled with the authorization and supervision of the Institutional Animal Care and Use Committee from the University of Utah. Every procedure conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Blk6 mice of 2-3 month of age were given intraperitoneal injections of either normal saline (control group) or acetazolamide at doses of 5, 10, 20, and 30 mg/100 g body weight at a volume of 150 ml at day 0, and day 4 (n = 6 per group). ERG was recorded at day 7.

2.2. ERG Recordings

Under anesthesia with a mixture of ketamine (150 mg/kg i.p.) and xylazine (10 mg/kg i.p.), the mouse head was secured with a stereotaxic head holder and the body temperature monitored through a rectal thermometer and maintained at 38°C using a homeothermic blanket. Pupils were dilated using equal parts of topical phenylephrine (2.5%) and tropicamide (1%). Bupivacaine 0.5% was used as a topical anesthetic to avoid blinking and a drop of 0.9% saline was frequently applied on the cornea to prevent its dehydration and allow electrical contact with the recording electrode (gold wire loop). A platinum subdermal needle (Grass Telefactor, F-E2) inserted under the scalp, between the two eyes, served as the reference electrode. Amplification (at 1-1000 Hz bandpass, without notch filtering), stimulus presentation, and data acquisition were provided by the UTAS-3000 system from LKC Technologies (Gaithersburg, MD).

For dark-adapted ERG recordings, tests consisted of single flash presentations (10 msec duration), repeated 3 to 5 times to verify the response reliability and improve the signal-to- noise ratio, if required. Stimuli were presented at sixteen increasing intensities in one log unit steps varying from -3.6 to 1.4 log cds/m2 in luminance. To minimize the potential bleaching of rods, inter-stimulus intervals were increased as the stimulus luminance was elevated from 10 sec at lowest stimulus intensity up to 2 minutes at highest stimulus intensity.

Following dark-adapted recordings, the animals were light adapted for 15 minutes to assure maximal cone output. Photopic intensity responses (30 cds/m2 background) ranged from -1.6 to 2.9 log cds/m2 (-1.6, -0.6, 0.4, 1.4, 2.4 and 2.9 log cds/m2). Criteria responses were set at 20 mV for a- and b-waves (under scotopic and photopic adaptation). For flicker ERGs, stimuli consisted of white flashes provided by a xenon bulb (luminance of 1.37 log cds/m2), projected on a ganzfeld with a background luminance of 30 cds/m2 presented at 20 Hz. The stimulus was presented during 3 seconds prior to data collection. This guaranteed that the first few responses (not preceded by repeated stimuli and of potentially greater amplitude) were not included in the average. A total of 40 responses were averaged.

The amplitude of the flicker response was defined as the average of the difference between consecutive negative and positive deflections.

2.3. Statistical Analysis

Error values accompanying averages were expressed as standard errors of the mean (SEM). Comparisons between two groups were made using Mann-Whitney U-test. The probability level at which the Null Hypothesis was rejected is represented as the value “p”; statistical significance was set at p < 0.05.

3. RESULTS

Acetazolamide treatment resulted in a depression of both coneand rod-mediated ERG responses, in a dose dependent manner. However, dose dependence differed for cone and rod-related responses; cone related responses were affected at lower dose regimens than rod related responses.

The two lowest dose regimens, studied here, led to depressed cone mediated responses. Photopic b-wave amplitudes at high luminance stimuli under photopic adaptation (Fig. 61.1), as well as cone-mediated 20 Hz flicker amplitudes (Fig. 61.2) were significantly reduced following 5 and 10 mg/100 g acetazolamide treatment. There were tendencies for amplitude reductions at 20 and 30 mg/100 g doses, especially for maximal photopic b-wave amplitudes, but these reductions did not reach statistical significance. Optimal amplitude reductions occurred for the 10 mg/100 g dosage, both for photopic b-waves and 20 Hz flicker. At this dosage, statistically significant reductions in photopic b-wave amplitudes were seen for stimuli of 0.88 log cds/m2 and higher. Photopic b-wave thresholds remained unaffected by acetazolamide, regardless of its dose, and b- amplitudes constantly reached a plateau for stimuli of 1.89 log cds/m2 and of higher luminances.

The results for scotopic ERGs are presented in Fig. 61.3. Statistically significant reductions in scotopic ERG responses (for both maximal a- and b-wave amplitudes) were only obtained with the highest dose regimen, i.e. 30 mg/100 g acetazolamide (Figure 61.3A). In two animals treated at this dosage (scotopic intensity response traces from one of which are presented in Figure 61.3B), the thresholds for a- and b-waves were higher than in PBS injected animals. However, there were no statistically significant changes between acetazolamide (30 mg/100 g) and PBS treated groups. Statistically significant reductions in a- and b-wave amplitudes were seen for stimuli higher than 1.37 log cds/m2 and 0.88 log cds/m2, respectively. There was a significant drop in b-wave amplitudes at the highest luminance tested (326 ± 34 mV at 2.86 log cds/m2) compared with the luminance giving the maximal b-wave amplitude (434 ± 36 at 1.89 log cds/m2). This indication of bleaching was not seen in PBS treated animals.

4. DISCUSSION

This study shows that mice treated with acetazolamide have reduced rod and cone related ERG responsiveness. Treatment with two i.p. injections of 5 or 10 mg/100g (at day 0 and day 4) led to a reduction at day 7 in cone related ERG responses, as reflected by

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amplitudes already 5 hours post-injection of another CAI, methazolamide, at 5 mg/100 g i.p. in rabbits. The findings of decreased ERG responses at time points as early as 5 hours indicate that diminution in ERG responses likely occurs without photoreceptor degeneration and, in the case of depressed rod responsiveness, could be related to acute metabolic changes such as involving an acidification of the retina (Findl et al., 1995). In addition, our findings of optimal depression of cone responses at intermediate doses also argue for a specific change in cell function rather than a direct toxic effect of acetazolamide for the 7 days treatment regimen used here: toxicity should not be expected to diminish as the dosage increases. Still, based on recent findings of an RP form involving a dysfunction of CA4, it is a reasonable assumption that long-term inhibition of this enzyme (and possibly other CAs) might lead to photoreceptor degeneration. Direct injections of acetazolamide in the vitreous of rabbits have indicated that doses of 1 mg or higher not only depressed the b-wave but also resulted in damaged outer segments (Borhani et al., 1994). Assuming that this effect is related to the inhibitory action of acetazolamide on CAs, then a complete inhibition should be expected to have deleterious effects. For instance, topical dorzolamide 3%, which is the optimal dose for treating glaucoma in experimental rabbit, produces a concentration of 35 mM in ocular tissues (Sugrue, 1996), which is higher than the IC50 of 4 mM for CA4 (Ives, 1998), implying a complete inhibition of CA4 enzymatic activity.

CAs are expressed in ocular tissues i.e. in choriocapillaris (CA4) and retina (CA2), their enzymatic action involves catalyzing the reaction of carbon dioxide and water to carbonic acid, which dissociates to bicarbonate and hydrogen ions. Therefore, CAs play a crucial role in regulating tissue pH via their ability to increase the effectiveness of the bicarbonate buffer system. The inhibition of CAs reduces the buffering of the acid reflux from photoreceptors resulting in a lowering of the extracellular pH around these cells. Hydrogen ions have been shown to suppress the dark current of rods in frogs (Liebmann et al., 1984), and acidification was shown to depress photoreceptor response in isolated rat retinae (Winkler, 1972). Hydrogen ions, being elevated in concentration following CAI treatment, might attenuate the amplitude of ERG components by competing with Ca++ for binding sites in rods (Moody, 1984) and/or decreasing the Na+/Ca++ exchange in these cells (Hodgkin and Nunn, 1987), both resulting in elevated intracellular Ca++, which is known to downregulate the light sensitive Na+ conductance in rod outer segments via cGMP gated Na+ channels (Yau, 1994). CAIs might also lead to acidification of the retina by acting on Muller cells, which have been shown to play a crucial role in regulating the extracellular pH (Oakley and Wen, 1989). In brief, the depression of rod photoreceptor response following CA inhibition in mice treated in the present study is likely to involve an acidification of the retina.

Our results indicate that cone function is also affected by acetazolamide treatment; furthermore, cone related ERG responses are depressed at lower doses, 10 mg/100 g, instead of 30 mg/100 g required to diminish scotopic responses. This CAI-induced depression of cone ERG might involve mechanisms other than acidification. Psychophysical tests in human treated with the CAI methazolamide revealed a disturbance of color discrimination which was not correlated with the degree of acidosis (Widengard, 1995). CAIs might act directly on cones themselves and only indirectly on rods (via acidification as explained above). Histochemical analysis of human retinae indicates that cones (with the exception of blue cones) contain CA activity, while rods do not (Nork et al., 1990). In addition, Muller cells, which also contain CAs, are involved in the generation of b-waves via K+-evoked depolarization (Wen and Oakley, 1990), and CA inhibition has been shown to modify the extracellular K+ concentration in isolated salamander Muller cells (Newman, 1996). It is

therefore possible that CAIs might also affect the light induced depolarization of Muller cells, and consequently have an effect on the b-wave amplitude. Finally, acetazolamide decreases the ability of Muller cells for buffering the extracellular fluid compartment (Wen and Oakley, 1990). Changes in fluid balances, such as reported in studies involving mice deficient in the water channel aquaporin-4 (expressed by Muller cells) have been shown to lead to b-wave depression by 10 months of age (Li et al., 2002).

5. CONCLUSIONS

Acetazolamide treatment in mice leads to a depression of both rod and cone related ERG responses as seen in patients with the RP17 rod-cone dystrophy. Therefore, the chronic use of CAIs, particularly pertinent in the treatment of glaucoma, may lead to visual loss, cone function being affected at lower doses than rod function.

6. ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health Grants R01EY14428, R01EY14448 and GCRC M01-RR00064, the Ruth and Milton Steinbach Fund, Ronald McDonald House Charities, the Macular Vision Research Foundation, the Research to Prevent Blindness, Inc., Knights Templar Eye Research Foundation, Grant Ritter Fund, American Health Assistance Foundation, the Karl Kirchgessner Foundation, Val and Edith Green Foundation, and the Simmons Foundation.

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