BACKGROUND: The results from cross sectional and longitudinal studies show that 
periodontitis is closely associated with cognitive impairment (CI) and 
Alzhemer's Disease (AD). Further, studies using animal model of periodontitis 
and human post-mortem brain tissues from subjects with AD strongly suggest that 
a gram-negative periodontal pathogen, Porphyromonas gingivalis (Pg) and/or its 
product gingipain is/are translocated to the brain. However, neuropathology 
resulting from Pg oral application is not known. In this work, we tested the 
hypothesis that repeated exposure of wild type C57BL/6 mice to orally 
administered Pg results in neuroinflammation, neurodegeneration, microgliosis, 
astrogliosis and formation of intra- and extracellular amyloid plaque and 
neurofibrillary tangles (NFTs) which are pathognomonic signs of AD.
METHODS: Experimental chronic periodontitis was induced in ten wild type 8-week 
old C57BL/6 WT mice by repeated oral application (MWF/week) of Pg/gingipain for 
22 weeks (experimental group). Another 10 wild type 8-week old C57BL/6 mice 
received vehicle alone (control group) MWF per week for 22 weeks. Brain tissues 
were collected and the presence of Pg/gingipain was determined by 
immunofluorescence (IF) microscopy, confocal microscopy, and quantitative PCR 
(qPCR). The hippocampi were examined for the signs of neuropathology related to 
AD: TNFα, IL1β, and IL6 expression (neuroinflammation), NeuN and Fluoro Jade C 
staining (neurodegeneration) and amyloid beta1-42 (Aβ42) production and 
phosphorylation of tau protein at Ser396 were assessed by IF and confocal 
microscopy. Further, gene expression of amyloid precursor protein (APP), 
beta-site APP cleaving enzyme 1 (BACE1), a disintegrin and metalloproteinase 
domain-containing protein10 (ADAM10) for α-secretase and presenilin1 (PSEN1) for 
ɣ-secretase, and NeuN (rbFox3) were determined by RT-qPCR. Microgliosis and 
astrogliosis were also determined by IF microscopy.
RESULTS: Pg/gingipain was detected in the hippocampi of mice in the experimental 
group by immunohistochemistry, confocal microscopy, and qPCR confirming the 
translocation of orally applied Pg to the brain. Pg/gingipain was localized 
intra-nuclearly and peri-nuclearly in microglia (Iba1+), astrocytes (GFAP+), 
neurons (NeuN+) and was evident extracellularly. Significantly greater levels of 
expression of IL6, TNFα and IL1β were evident in experimental as compared to 
control group (p<0.01, p<0.00001, p<0.00001 respectively). In addition, 
microgliosis and astrogliosis were evident in the experimental but not in 
control group (p <0.01, p<0.0001 respectively). Neurodegeneration was evident in 
the experimental group based on a fewer number of intact neuronal cells assessed 
by NeuN positivity and rbFOX3 gene expression, and there was a greater number of 
degenerating neurons in the hippocampi of experimental mice assessed by Fluoro 
Jade C positivity. APP and BACE1 gene expression were increased in experimental 
group compared with control group (p<0.05, p<0.001 respectively). PSEN1 gene 
expression was higher in experimental than control group but the difference was 
not statistically significant (p = 0.07). ADAM10 gene expression was 
significantly decreased in experimental group compared with control group 
(p<0.01). Extracellular Aβ42 was detected in the parenchyma in the experimental 
but not in the control group (p< 0.00001). Finally, phospho-Tau (Ser396) protein 
was detected and NFTs were evident in experimental but not in the control group 
(p<0.00001).
CONCLUSIONS: This study is the first to show neurodegeneration and the formation 
of extracellular Aβ42 in young adult WT mice after repeated oral application of 
Pg. The neuropathological features observed in this study strongly suggest that 
low grade chronic periodontal pathogen infection can result in the development 
of neuropathology that is consistent with that of AD.