Efficacy assessment of a combined anticholinergic and oxime treatment against topical sarin-induced miosis and visual impairment in rats

Abstract

Background and purpose: Eye exposure to the organophosphorus (OP) irreversible cholinesterase inhibitor sarin results in long-term miosis and impaired visual function. We have previously shown that tropicamide is better at ameliorating this insult than topical atropine or cyclopentolate. However, to minimize side effects associated with repeated tropicamide applications and high treatment doses, we evaluated the effects of oximes (ChE re-activators) alone and combined with tropicamide at ameliorating OP-induced ocular impairments.

Experimental approach: Rats were topically exposed to sarin, followed by topical treatment with various oximes alone or in combination with tropicamide. Pupil width and light reflex were measured by an infrared-based digital photograph system, while visual performance was assessed by employing the cueing version of the Morris water maze (MWM).

Key results: Oxime treatment following sarin ocular exposure induced a slow persistent pupil widening with efficacy in the order of HLö-7 > HI-6 > obidoxime = TMB-4 = MMB-4. In the light reflex test, the ability of the iris to contract following oxime treatment was mostly impaired at 1 h and was back to normal at 4 h following sarin exposure. All oxime treatments ameliorated the sarin-induced visual impairment as tested in the visual task (MWM). The combined topical treatment of tropicamide with an oxime induced a rapid improvement in pupil widening, light reflex and visual performance, and enabled a reduction in tropicamide dose.

Conclusions and implications: The use of tropicamide combined with an oxime should be considered as the topical treatment of choice against the toxic effects of ocular OP exposure.

Keywords: HI-6; HLö-7; Long-Evans rats; MMB-4; TMB-4; anticholinergic treatments; cued MWM; miosis; obidoxime; organophosphates; oxim; pupillary light reflex; sarin; tropicamide; visual impairment.

Figure 1

Pupil width alteration following topical sarin exposure and oxime treatment. Rats were exposed topically to PG (A), 0.2 (B) or 1 μg (C) sarin followed by two drops of topical oxime (2.5%) treatment (except for one treatment group, in which one drop of HI-6 was used, indicated as HI-6X1). Pupil width is presented as % relative to baseline of the same animal before exposure. Each point is the mean ± SEM of 12 rats. All differences, between groups, at different time points post-exposure are statistically significant (by anova), at a level of *P < 0.01.

Figure 2

Effect of oxime treatments on the pupillary light reflex following sarin exposure. Treated eyes, as described in Figure 1 (two drops of topical 2.5% oxime), were illuminated with 350 lux for 2–3 s, 1 and 4 h following exposure to PG (A), 0.2 μg (B) or 1 μg (C) sarin. Each point is presented as % change relative to the pre-reflex pupil width and represents the mean ± SEM of 12 animals. Normal light reflex range is indicated by the dotted horizontal lines. Differences between treatments following exposure of 0.2 or 1 μg sarin at both time points post-exposure are statistically significant (by anova), at a level of *P < 0.01 (vs. saline at the same time point).

Figure 3

Visual performance test following topical sarin exposure and oxime treatment. Both eyes were topically exposed to 1 μg sarin or to 0.66% PG (control). Fifteen minutes following exposure, pupil diameter was measured to confirm pupil constriction (not shown). Eyes were then treated with saline or 2.5% oximes as indicated. Twenty minutes following treatment, visual performance was evaluated under controlled room light conditions (∼170 lux) using the cued MWM paradigm. The parameters examined and compared between groups were escape latency (A), path length (B) and swimming speed (C). The first block shows the average of the initial four trials of 12 rats, and the second block represents the following four trials average for the same animals. Differences in escape latency and path length between non-treated sarin-exposed group and each of the other groups are statistically significant (by manova) at a level of *P < 0.001 (vs. sarin; saline in the same block).

Figure 4

Evaluation of the combined tropicamide and oxime treatment on pupil width following topical sarin exposure. Rats were exposed topically to PG (A) or 1 μg sarin (B) and 20 min later were given a combined topical treatment of tropicamide (Trop) with two drops of 2.5% TMB-4, 2.5% obidoxime, 2.5% MMB-4 or 2.5% HI-6. Pupil width was determined at the indicated time points and presented as % relative to baseline of the same animal before exposure. Each point is the mean ± SEM of 12 rats. Differences between groups, at various time points post-exposure, are statistically significant (by anova) at a level of *P < 0.05.

Figure 5

Effect of the combined tropicamide and oxime treatments on the pupillary light reflex following sarin exposure. Eyes were illuminated with 350 lux for 2–3 s, 1 and 4 h following the combined treatment (tropicamide with two drops of 2.5% oxime as indicated) and after exposure to PG (A) or 1 μg (B) sarin. Each point (mean ± SEM) is presented as % change relative to the pre-reflex pupil width and represents the average performance of 12 animals. Physiological light reflex range is indicated by the dotted horizontal lines. Differences in pupillary light reflex between groups are statistically significant (by anova) at a level of *P < 0.001 (vs. sarin; saline in the same block) as indicated.

Figure 6

Visual performance evaluation using the combined treatment following sarin exposure. Both rat eyes were treated topically with the indicated drugs, tropicamide (Trop) with two drops of various oximes (2.5%), 20 min following PG (A, C, E) or 1 μg sarin (B, D, F) exposure. Ocular measurements and the visual test were conducted 15 min later. Visual performance was evaluated under controlled light conditions (170 lux) using the ‘cued MWM’ paradigm. The parameters examined and compared between groups were escape latency (A), path length (B) and swimming speed (C); n = 12 for each group. The first block shows the average of the initial four trials of 12 rats, and the second block represents the following four trials average for the same animals. Significant (by manova) differences between groups are indicated by asterisks at a level of *P < 0.001. (B, D) *P < 0.001, versus sarin; saline in the same block; (E) *P < 0.001, versus Trop + HI-6 in the same block.