The protective role of tacrine and donepezil in the retina of acetylcholinesterase knockout mice

Abstract

Aim: To determine the effect of different concentrations of the acetylcholinesterase (AChE) inhibitors tacrine and donepezil on retinal protection in AChE(+/-) mice (AChE knockout mice) of various ages.

Methods: Cultured ARPE-19 cells were treated with hydrogen peroxide (H2O2) at concentrations of 0, 250, 500, 1000 and 2000 µmol/L and protein levels were measured using Western blot. Intraperitoneal injections of tacrine and donepezil (0.1 mg/mL, 0.2 mg/mL and 0.4 mg/mL) were respectively given to AChE(+/-) mice aged 2mo and 4mo and wild-type S129 mice for 7d; phosphate buffered saline (PBS) was administered to the control group. The mice were sacrificed after 30d by in vitro cardiac perfusion and retinal samples were taken. AChE-deficient mice were identified by polymerase chain reaction (PCR) analysis using specific genotyping protocols obtained from the Jackson Laboratory website. H&E staining, immunofluorescence and Western blot were performed to observe AChE protein expression changes in the retinal pigment epithelial (RPE) cell layer.

Results: Different concentrations of H2O2 induced AChE expression during RPE cell apoptosis. AChE(+/-) mice retina were thinner than those in wild-type mice (P<0.05); the retinal structure was still intact at 2mo but became thinner with increasing age (P<0.05); furthermore, AChE(+/-) mice developed more slowly than wild-type mice (P<0.05). Increased concentrations of tacrine and donepezil did not significantly improve the protection of the retina function and morphology (P>0.05).

Conclusion: In vivo, tacrine and donepezil can inhibit the expression of AChE; the decrease of AChE expression in the retina is beneficial for the development of the retina.

Keywords: AChE+/− animal models; acetylcholinesterase; acetylcholinesterase inhibitors; apoptosis; retinal pigment epithelium.

Figure 1. After different concentrations of H₂O₂ were incubated for 2h, the AChE protein was detected by Western blot

At higher concentrations of H₂O₂, expression of cleaved-PARP (A, B) and AChE increased (C) Western blot grey values were quantitatively analyzed (ImageJ software); ^aP<0.05 was considered as statistically significant.

Figure 2. Apoptosis induced RPE cells treated with H₂O₂ as observed by morphology and TUNEL analysis

In the control group, HoChest 33258 staining was positive; AChE immunofluorescence and TUNEL staining were negative. In the H₂O₂ (1000 µmol/L) group, AChE immunofluorescence and TUNEL were all positive (400×).

Figure 3. Identification of AChE knockout mice

A: PCR-based genotyping of WT and AChE deficient mice; B: Western blot analysis of AChE protein expression in normal retinal tissues of WT and AChE deficient mice.

Figure 4. Retina sections from the tacrine and donepezil groups of mice or the PBS control, stained with H&E

Retina cells of the WT group were in normal. The PBS group showed widespread disorder of the retinal structural. The tacrine and donepezil groups showed improvement on the PBS group but the number of stained cells was still reduced (40×).