The AChEI and the inflammation-induced neuropathological changes in mice

Abstract

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD is characterized by two hallmark pathologies: β-amyloid plaque deposition and neurofibrillary tangles (NFT) of hyperphosphorylated tau. For several decades, the amyloid cascade hypothesis has been prevalent. Inflammation was believed to be another factor associated with AD pathogenesis. On the other hand, according to the cholinergic hypothesis of AD, the loss of the cholinergic neurons results in a decrease in acetylcholine level, and in turn induces cognitive and memory deficits. Acetylcholinesterase inhibitors (AChEI) have been the most common prescriptions for AD since 1993. Donepezil is the most commonly used AChEI in the treatment of mild, moderate and severe AD cases and has less adverse effects. According to another hypothesis, the myelin hypothesis, the deposits of Aβ and tau could be by-products of age-related myelin repair processes. Reduced myelin stability may relate to changes in levels of diverse myelin extra components, like lipids and their oxidized products, as well as expression levels of Myelin Basic Protein-MBP. We have successfully established chronic inflammation mouse model in adult, wild-type C57BL/6J mice through subcutaneous, continuous infusion of lipopolysaccharides (LPS) in our lab. We found LPS from the oral bacterium Porphyromonas gingivalis (P. gingivalis) appears to trigger more pronounced neuroinflammation and cognitive impairment than the gut bacterium Escherichia coli (E. coli). Our study aims at a) confirming the contribution of chronic inflammation induced by P.g to the development of AD and the neuropathological changes in mice, b) at exploring the effect of Donepezil on the neuropathological changes in this chronic inflammation mouse model and c) at exploring if there is a difference regarding the presence/expression of MBP among the four groups. Forty-eight male 24-week-old C57BL/6J wild type (C57) mice were divided into 4 treatment groups with 12 mice per group; Group 1: Normal saline injection + normal saline infusion; Group 2: Normal saline injection + P.g.-LPS infusion; Group 3: Donepezil injection + normal saline infusion; Group 4: Donepezil injection + P.g.-LPS infusion. P.g-LPS or normal saline infusion was administered through subcutaneously implanted ALZET® osmotic pumps for 28 days. Donepezil or normal saline injection was administered through the intraperitoneal injection (ip) for 29 days. The Donepezil (1mg/kg/d) or normal saline ip injection was administered one day before the P.g-LPS or normal saline - filled pump implantation. Following 21 days of either P.g.-LPS or normal saline infusion and 22 days of injections, Morris Water Maze (MWM) test was conducted for the assessment of spatial learning and memory of the mice. The experiment lasted until day 26. On day 29 of the experiment, the mice were euthanized by Isoflurane overdose. Serum was isolated after centrifugation of the blood collected by intracardiac puncture. The right hemisphere was snap-frozen for the inflammatory cytokine ELISA assay and the left hemisphere was fixed in 10% formalin overnight, then switched to 70% EtOH for histology and immunohistochemistry (IHC). The sections were immunostained with MBP as a primary antibody. One-way ANOVA showed that there was no statistically significant difference in Day 0 Body Weight after treatment group assignments were made. No adverse effects were observed on the general health of the mice throughout the study. Although there was a statistically significant difference in body weight among groups on Day 7, these body weight differences were transient and did not maintain. The ELISA revealed that there were no significant differences in the IL-1β levels in the brain among all the groups. Regarding MWM, two-way ANOVA revealed no statistically significant differences in the latency to platform between the 4 groups. Nevertheless, a significant difference was noted between the different days. Taken collectively, the results of the spatial learning and reference memory suggest that P. gingivalis-LPS did not induce cognitive dysfunction in these mice. Last but not least, one-way ANOVA revealed no significant differences between the 4 groups in the MBP positive area. In the present study, no statistically significant differences were observed between the 4 groups in the brain IL-1β levels determined by ELISA, spatial learning and reference memory assessed by the MWM, and the MBP protein expression in hippocampus by IHC, respectively. Our previous finding that P.g-LPS induces neuroinflammation and cognitive deficit was not replicated in the current study. We do not know the exact reasons. There is a possibility that the daily injections might affect the neurobehavior performance of mice. This is confirmed by the observation that the mice in the current study had an overall elevated IL-1β in brain, even in the Sal+Sal group. To demonstrate the proposal that the stress from the daily injection might affect the immune responses to chronic LPS, a study with and without daily injection under the same setting will be needed.