Pyrrolidine dithiocarbamate attenuates brain Aβ increase and improves long-term neurological outcome in rats after transient focal brain ischemia

Abstract

Evidence suggests an association between brain ischemia and Alzheimer's disease (AD) development. Amyloid plaques consisted of β-amyloid peptide (Aβ) in the brain are a pathological hallmark of AD. Little is known about how brain ischemia induces AD-like neuropathology. A strategy effective to block such brain changes has not been reported. Here, adult male Sprague-Dawley rats were subjected to a 90-min right middle cerebral artery occlusion (MCAO). Pyrrolidine dithiocarbamate (PDTC) at various doses was given daily via gastric gavage with the first dose given at 10 min after the onset of reperfusion. The MCAO increased Aβ1-42 concentrations in the ischemic brain tissues. PDTC attenuated this increase. PDTC also decreased the ischemia-reduced expression of neprilysin, an Aβ degrading enzyme. Aβ1-42 levels were negatively correlated with neprilysin protein abundance. Brain ischemia decreased the expression of β-amyloid converting enzyme 1, a key enzyme to produce Aβ, and increased the expression of insulin-degrading enzyme, another Aβ degrading enzyme. Animals had impaired learning and memory at 2 months after the MCAO. PDTC attenuated this impairment. PDTC also improved long-term neurological outcomes. Our findings suggest that PDTC improves long-term neurological outcome of rats after transient focal brain ischemia. PDTC reduces ischemia-induced Aβ accumulation, possibly via preserving neprilysin expression.

Conflict of interest statement

Disclosure/conflict of interest: None from the authors.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Aβ1-42 expression in rat brain. A: The striatum ipsilateral or contralateral to the MCAO side was harvested at various time points after a 90-min MCAO. Results are means ± SD (n = 6). * P < 0.05 compared with control animals; ^ P < 0.05 compared with the ipsilateral striatum harvested immediately after the MCAO; # P < 0.05 compared with the corresponding contralateral stratum in the same animal. B: Both sides of striatum were harvested at 14 days after the MCAO or sham surgery. Results are means ± SD (n = 7). * P < 0.05 compared with control animals; ^ P < 0.05 compared with sham-operated animals; # P < 0.05 compared with the corresponding contralateral striatum in the same animal. C: Both sides of striatum from animals treated with saline or various doses of PDTC were harvested at 7 days after the MCAO or from animals treated with saline or 50 mg/kg/d PDTC were harvested at 14 days after the MCAO. Results are means ± SD (n = 5 – 7). * P < 0.05 compared with sham-operated animals. ^ P < 0.05 compared with animals treated with saline after the MCAO. D: Right and left frontal cortex area 1 of animals treated with saline or 50 mg/kg/day PDTC was harvested at 2 months after the MCAO. Results are means ± SD (n = 11 – 12). ^ P < 0.05 compared with animals treated with saline after the MCAO. E–G: Immunofluorecent staining of Aβ1-42 (red) in the non-ischemic striatum (E), ischemic striatum (F) and ischemic striatum from a rat treated with 50 mg/kg/d PDTC. These tissues were harvested at 7 days after the MCAO. Arrows in panel F indicate positive staining for Aβ1-42.

Fig. 2

Expression of BACE1, neprilysin (NEP) and IDE in rat brain. The striatum ipsilateral (I) or contralateral (C) to the MCAO side or striatum from control animals was harvested at 3 days or 7 days after the MCAO. The graphic presentation of the BACE1, neprilysin and IDE protein abundance quantified by integrating the volume of autoradiograms from 5 – 6 rats for each experimental condition is shown as fold change over the control rats. Results are means ± SD. * P

Fig. 3

Effects of PDTC on the expression of neprilysin (NEP) and Aβ1-42 in the brain. The striatum ipsilateral (I) or contralateral (C) to the MCAO side or striatum from sham-operated animals (S: surgical side, C: contralateral side) was harvested at 7 days after the MCAO. A: The graphic presentation of the neprilysin protein abundance quantified by integrating the volume of autoradiograms from 7 rats for each experimental condition is shown as expression ratio in the ipsilateral and contralateral striatum. Results are means ± SD. * P < 0.05 compared with sham-operated animals. ^ P < 0.05 compared with the corresponding ipsilateral striatum in animals treated with saline after the MCAO. M + S: animals with MCAO were treated with saline. B: Linear correlation analysis to assess the relationship between NEP and Aβ1-42 expression.

Fig. 4

Performance on Barnes maze and fear conditioning. Animals were tested with Barnes maze or fear conditioning tasks 2 months after the MCAO. The time for animals to find the target hole during the training sessions of Barnes maze is shown in panel A and panel B. The time to find the target box during the probe trial of Barnes maze test is shown in panel C. The percentage of the time with freezing behavior in the total observation time during the fear conditioning tests is shown in panel D. Results are means ± SD (n = 13). * P

Fig. 5

Neurological outcome. A: Brain slices stained with 2,3,5-triphenyltetrazolium chloride from representative rats are shown in the left panel and the percentage of infarct volume in ipsilateral hemisphere volume is shown in the right panel. Results are means ± S.D. (n = 8). ^ P < 0.05 compared with animals subjected to the MCAO and then treated with saline. B: Neurological deficit scores evaluated at 24 h, 1 week and 2 months after the MCAO. The neurological deficit scores of all sham-operated rats were 0 and are not plotted in the figure. Results are presented in a box plot format (n = 17 – 21). ●: lowest or highest score (the score will not show up if it falls in the 95% interval). ^ P < 0.05 compared with animals subjected to the MCAO and then treated with saline. C: Performance on rotarod was assessed at 1 week, 1 month or 2 months after the MCAO. Results are means ± S.D. (n = 5 – 7). ^ P <0.05 compared with animals subjected to the MCAO and then treated with saline. D: NeuN protein expression. The frontal cortex area 1 (Fr1) ipsilateral (I) or contralateral (C) to the MCAO side or the Fr1 from control animals was harvested at 2 months after the MCAO. The Fr1 ipsilateral to the MCAO side has been considered to be ischemic penumbral region. The graphic presentation of the NeuN protein abundance quantified by integrating the volume of autoradiograms from 5 – 6 rats for each experimental condition is shown as fold change over the control rats. Results are means ± SD. * P < 0.05 compared with control. ^ P < 0.05 compared with the corresponding ipsilateral Fr1 in animals treated with saline after the MCAO. M + S: animals with MCAO were treated with saline; M + P: animals with MCAO were treated with 50 mg/kg/d PDTC.

Fig. 6

Diagram of the major findings. Arrow heads imply that one effect leads to another effect. Circle heads indicate inhibition of the process. Solid lines after arrow or circle heads indicate that our study provides evidence for this effect. Dotted lines after arrow heads indicate that evidence in the literature suggests this effect.