Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

Phosphatidylethanol in post-mortem brain: correlation with blood alcohol concentration and alcohol use disorder.

Phosphatidylethanol (PEth) is an alcohol derivative that has been employed as a blood-based biomarker for regular alcohol use. This study investigates the utility of phosphatidylethanol (PEth) as a biomarker for assessing alcohol consumption in post-mortem brain tissue. Using samples from the New South Wales Brain Tissue Resource Centre, we analysed PEth(16:0/18:1) levels in the cerebellum and meninges of individuals with varying histories of alcohol use, including those diagnosed with alcohol use disorder (AUD) and controls. Our findings demonstrate a significant correlation between PEth levels and blood alcohol content (BAC) at the time of death, supporting the biomarker's sensitivity to recent alcohol intake. Furthermore, this study explores the potential of PEth levels in differentiating AUD cases from controls, taking into consideration the complexities of diagnosing AUD post-mortem. The study also examined the relationship between PEth levels and liver pathology, identifying a link with the severity of liver damage. These results underscore the value of PEth as a reliable indicator of alcohol consumption and its potential contributions to post-mortem diagnostics and consequently, research into alcohol-related brain damage.

A Survey of Community Perceptions on Brain Donation for Research.

Postmortem brain donation for medical research is a little-known form of organ donation. While most brain research is carried out using animal models, many neurological diseases are uniquely human. Greater availability of human postmortem brain tissue from diseased individuals and controls would therefore improve the development of treatments for neurological and neuropsychiatric diseases. Globally, organ donation for medical research is dwarfed by organ donation for transplantation. In 2021, 36% of Australians were registered organ donors for transplantation, with public "in-principle" support even higher, at 76%. In contrast, there are little data on Australian or international brain donation rates for research. A 30-item online survey was conducted to ascertain knowledge of, and attitudes toward, brain donation in Australia. Of the respondents, 12/237 (5%) were current brain donors and excluded from further analysis. Of the remaining 225, 75% were registered organ donors for transplant. The vast majority (n = 189/225, 84%) of respondents supported or strongly supported the principle of brain donation. However, of those registered for transplantation or whole-body donors, 93/170 (55%) were not aware that brain donation was possible, while 50%, alternatively or also, thought that registering as an organ donor for transplantation rendered them a brain donor by default. Only 9/225 (4%) respondents indicated that they would definitely not donate their brain in the future, while 27 remained unsure. There is prominent public support for brain donation in Australia, with 84% of respondents willing to donate their brain. Yet, the extent of public misconceptions on brain donation for research suggests the need for further education on all types of organ donation, so individuals may make informed decisions.

Increased Neuronal Nitric Oxide Synthase in Alzheimer's Disease Mediates Spontaneous Calcium Signaling and Divergent Glutamatergic Calcium Responses.

Nitrosative stress is a feature of Alzheimer's disease (AD). Aims: We aimed to identify the cause underpinning increased nitric oxide (NO) in neurons and the impact of NO on neuronal function in AD. Results: We analyzed neuronal nitric oxide synthase (nNOS) protein levels in postmortem tissue and induced pluripotent stem cell (iPSC)-derived neurons from Alzheimer's patients and controls by immunohistochemistry and Western blots. Furthermore, we assessed the impact of modulating nNOS function or NO levels on neuronal glutamatergic signaling using calcium imaging. We show that nNOS protein levels are increased in early and severely affected brain regions of AD postmortem tissue, but not late and mildly affected regions, or cognitively normal individuals. The increased nNOS phenotype was also present in iPSC-derived neurons from late-onset Alzheimer's disease (LOAD) patients compared with controls, along with increased levels of nitrite, a stable marker of NO. Innovation: We observed a divergent functional impact of NO that included strengthening the calcium response in control neurons, while dysregulating calcium signaling and altering the amplitude and kinetics of the calcium responses to glutamate in the AD neurons. Pharmacological scavenging of NO or inhibition of nNOS prevented aberrant spontaneous calcium signaling in AD neurons. Conclusion: Together these data identify increases in nNOS protein in AD. Functional data suggest that NO modulation of glutamatergic calcium signaling is neuroprotective under nonpathogenic conditions, with increased nNOS and NO contributing to dysregulated spontaneous calcium signaling in AD neurons.

Biological aging markers in blood and brain tissue indicate age acceleration in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is associated with increased mortality and morbidity risk. A reason for this could be accelerated biological aging, which is strongly influenced by disease processes such as inflammation. As recent studies of AUD show changes in DNA methylation and gene expression in neuroinflammation-related pathways in the brain, biological aging represents a potentially important construct for understanding the adverse effects of substance use disorders. Epigenetic clocks have shown accelerated aging in blood samples from individuals with AUD. However, no systematic evaluation of biological age measures in AUD across different tissues and brain regions has been undertaken. METHODS: As markers of biological aging (BioAge markers), we assessed Levine's and Horvath's epigenetic clocks, DNA methylation telomere length (DNAmTL), telomere length (TL), and mitochondrial DNA copy number (mtDNAcn) in postmortem brain samples from Brodmann Area 9 (BA9), caudate nucleus, and ventral striatum (N = 63-94), and in whole blood samples (N = 179) of individuals with and without AUD. To evaluate the association between AUD status and BioAge markers, we performed linear regression analyses while adjusting for covariates. RESULTS: The majority of BioAge markers were significantly associated with chronological age in all samples. Levine's epigenetic clock and DNAmTL were indicative of accelerated biological aging in AUD in BA9 and whole blood samples, while Horvath's showed the opposite effect in BA9. No significant association of AUD with TL and mtDNAcn was detected. Measured TL and DNAmTL showed only small correlations in blood and none in brain. CONCLUSIONS: The present study is the first to simultaneously investigate epigenetic clocks, telomere length, and mtDNAcn in postmortem brain and whole blood samples in individuals with AUD. We found evidence for accelerated biological aging in AUD in blood and brain, as measured by Levine's epigenetic clock, and DNAmTL. Additional studies of different tissues from the same individuals are needed to draw valid conclusions about the congruence of biological aging in blood and brain.

The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus.

BACKGROUND: The risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65. The hippocampus was chosen as it is highly susceptible to atrophy with ageing and in several neurodegenerative diseases. METHODS: Mass spectrometry-based proteomic and lipidomic analysis of CA1 hippocampus samples from 74 neurologically normal human donors, aged 66-104, was used in combination with multiple regression models and gene set enrichment analysis to identify age-dependent changes in the proteome and lipidome. ANOVA was used to test the effect of major dementia risk alleles in the TMEM106B and APOE genes on the hippocampal proteome and lipidome, adjusting for age, gender, and post-mortem interval. Fibrillar C-terminal TMEM106B fragments were isolated using sarkosyl fractionation and quantified by immunoblotting. RESULTS: Forty proteins were associated with age at false discovery rate-corrected P < 0.05, including proteins that regulate cell adhesion, the cytoskeleton, amino acid and lipid metabolism, and ribosomal subunits. TMEM106B, a regulator of lysosomal and oligodendrocyte function, was regulated with greatest effect size. The increase in TMEM106B levels with ageing was specific to carriers of the rs1990622-A allele in the TMEM106B gene that increases risk for frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and hippocampal sclerosis with ageing. Rs1990622-A was also associated with higher TMEM106B fibril content. Hippocampal lipids were not significantly affected by APOE genotype, however levels of myelin-enriched sulfatides and hexosylceramides were significantly lower, and polyunsaturated phospholipids were higher, in rs1990622-A carriers after controlling for APOE genotype. CONCLUSIONS: Our study demonstrates that TMEM106B protein abundance is increased with brain ageing in humans, establishes that dementia risk allele rs1990622-A predisposes to TMEM106B fibril formation in the hippocampus, and provides the first evidence that rs1990622-A affects brain lipid homeostasis, particularly myelin lipids. Our data suggests that TMEM106B is one of a growing list of major dementia risk genes that affect glial lipid metabolism.

Ranking the risk factors for Alzheimer's disease; findings from the UK Biobank study.

Background: The cause of the most common form of dementia, sporadic Alzheimer's disease (AD), remains unknown. This may reflect insufficiently powered studies to date for this multi-factorial disorder. The UK Biobank dataset presents a unique opportunity to rank known risk factors and determine novel variables. Methods: A custom machine learning approach for high dimensionality data was applied to explore prospectively associations between AD in a sub-cohort of 156,209 UK Biobank participants aged 60-70 including more than 2,090 who were subsequently diagnosed with AD. Results: After the possession of the APOE4 allele, the next highest ranked risk factors were other genetic variants within the TOMM40-APOE-APOC1 locus. When stratified by their apolipoprotein epsilon 4 (APOE4) carrier status, the most prominent risk factors in carriers were AST:ALT ratio, the "number of treatments/ medications" taken as well as "time spent in hospital" while protection was conferred by "Sleeplessness/Insomnia". In non-APOE carriers, lower socioeconomic status and fewer years of education were highly ranked but effect sizes were small relative to APOE4 carriers. Conclusions: Possession of the APOE4 allele was confirmed as the most important risk factor in AD. Other TOMM40-APOE-APOC1 locus variants further moderate the risk of AD in APOE4 carriers. Liver pathology is a novel risk factor in APOE4 carriers while "Sleeplessness/Insomnia" is protective in AD irrespective of APOE4 status. Other factors such as "Number of treatments/ medications" suggest that multimorbidity is an important risk factor for AD. Future treatments aimed at co-morbidities, including liver disease, may concomitantly lower the risk of sporadic AD.

Machine learning analysis of the UK Biobank reveals IGF-1 and inflammatory biomarkers predict Parkinson's disease risk.

INTRODUCTION: Parkinson's disease (PD) is the most common movement disorder, and its prevalence is increasing rapidly worldwide with an ageing population. The UK Biobank is the world's largest and most comprehensive longitudinal study of ageing community volunteers. The cause of the common form of PD is multifactorial, but the degree of causal heterogeneity among patients or the relative importance of one risk factor over another is unclear. This is a major impediment to the discovery of disease-modifying therapies. METHODS: We used an integrated machine learning algorithm (IDEARS) to explore the relative effects of 1,753 measured non-genetic variables in 334,062 eligible UK Biobank participants, including 2,719 who had developed PD since their recruitment into the study. RESULTS: Male gender was the highest-ranked risk factor, followed by elevated serum insulin-like growth factor 1 (IGF-1), lymphocyte count, and neutrophil/lymphocyte ratio. A group of factors aligned with the symptoms of frailty also ranked highly. IGF-1 and neutrophil/lymphocyte ratio were also elevated in both sexes before PD diagnosis and at the point of diagnosis. DISCUSSION: The use of machine learning with the UK Biobank provides the best opportunity to explore the multidimensional nature of PD. Our results suggest that novel risk biomarkers, including elevated IGF-1 and NLR, may play a role in, or are indicative of PD pathomechanisms. In particular, our results are consistent with PD being a central manifestation of a systemic inflammatory disease. These biomarkers may be used clinically to predict future PD risk, improve early diagnosis and provide new therapeutic avenues.

Single-nucleus RNA-sequencing of autosomal dominant Alzheimer disease and risk variant carriers.

Genetic studies of Alzheimer disease (AD) have prioritized variants in genes related to the amyloid cascade, lipid metabolism, and neuroimmune modulation. However, the cell-specific effect of variants in these genes is not fully understood. Here, we perform single-nucleus RNA-sequencing (snRNA-seq) on nearly 300,000 nuclei from the parietal cortex of AD autosomal dominant (APP and PSEN1) and risk-modifying variant (APOE, TREM2 and MS4A) carriers. Within individual cell types, we capture genes commonly dysregulated across variant groups. However, specific transcriptional states are more prevalent within variant carriers. TREM2 oligodendrocytes show a dysregulated autophagy-lysosomal pathway, MS4A microglia have dysregulated complement cascade genes, and APOEε4 inhibitory neurons display signs of ferroptosis. All cell types have enriched states in autosomal dominant carriers. We leverage differential expression and single-nucleus ATAC-seq to map GWAS signals to effector cell types including the NCK2 signal to neurons in addition to the initially proposed microglia. Overall, our results provide insights into the transcriptional diversity resulting from AD genetic architecture and cellular heterogeneity. The data can be explored on the online browser ( http://web.hararilab.org/SNARE/ ).

Spatial analysis for highly multiplexed imaging data to identify tissue microenvironments.

Highly multiplexed in situ imaging cytometry assays have made it possible to study the spatial organization of numerous cell types simultaneously. We have addressed the challenge of quantifying complex multi-cellular relationships by proposing a statistical method which clusters local indicators of spatial association. Our approach successfully identifies distinct tissue architectures in datasets generated from three state-of-the-art high-parameter assays demonstrating its value in summarizing the information-rich data generated from these technologies.

Central markers of neuroinflammation in alcohol use disorder: A meta-analysis of neuroimaging, cerebral spinal fluid, and postmortem studies.

INTRODUCTION AND AIMS: There is emerging evidence that heavy long-term alcohol consumption may alter the neuroimmune profile. We conducted a meta-analysis of the association between alcohol use disorder (AUD) and the extent of neuroinflammation using cerebrospinal (CSF), PET (Positron Emission Tomography), and postmortem studies. DESIGN AND METHODS: A comprehensive search of electronic databases was conducted using the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) for AUD-related terms in combination with neuroinflammatory markers and cytokine- and chemokine-related terms for CSF, PET, and postmortem studies. Participants had to meet established criteria for AUD and/or heavy alcohol consumption with dependence features and be compared with healthy controls. Papers retrieved were assessed for inclusion criteria and a critical appraisal was completed using the Newcastle-Ottawa Scale. A meta-analysis was conducted on postmortem and PET studies. RESULTS: Eleven papers met the inclusion criteria with CSF, PET, and postmortem studies included in the final analysis. Postmortem studies demonstrate significant heterogeneity (𝑄 (14) = 62.02, 𝑝 < 0.001), with the alcohol group showing higher levels of neuroimmune markers than controls (𝑑 = 1.50 [95% CI 0.56, 2.45]). PET studies demonstrated a lower [11 C] PBR28 total volume of distribution (V T ) for translocator protein in the hippocampus (g = -1.95 [95% CI -2.72, -1.18], p < 0.001) of the alcohol group compared to controls. CONCLUSION: There is emerging evidence across multiple diagnostic modalities that alcohol impacts neuroimmune signaling in the human brain.

Vascular Dysfunction Is Central to Alzheimer's Disease Pathogenesis in APOE e4 Carriers.

Alzheimer's disease (AD) is the most common form of dementia and the leading risk factor, after age, is possession of the apolipoprotein E epsilon 4 allele (APOE4). Approximately 50% of AD patients carry one or two copies of APOE4 but the mechanisms by which it confers risk are still unknown. APOE4 carriers are reported to demonstrate changes in brain structure, cognition, and neuropathology, but findings have been inconsistent across studies. In the present study, we used multi-modal data to characterise the effects of APOE4 on the brain, to investigate whether AD pathology manifests differently in APOE4 carriers, and to determine if AD pathomechanisms are different between carriers and non-carriers. Brain structural differences in APOE4 carriers were characterised by applying machine learning to over 2000 brain MRI measurements from 33,384 non-demented UK biobank study participants. APOE4 carriers showed brain changes consistent with vascular dysfunction, such as reduced white matter integrity in posterior brain regions. The relationship between APOE4 and AD pathology was explored among the 1260 individuals from the Religious Orders Study and Memory and Aging Project (ROSMAP). APOE4 status had a greater effect on amyloid than tau load, particularly amyloid in the posterior cortical regions. APOE status was also highly correlated with cerebral amyloid angiopathy (CAA). Bulk tissue brain transcriptomic data from ROSMAP and a similar dataset from the Mount Sinai Brain Bank showed that differentially expressed genes between the dementia and non-dementia groups were enriched for vascular-related processes (e.g., "angiogenesis") in APOE4 carriers only. Immune-related transcripts were more strongly correlated with AD pathology in APOE4 carriers with some transcripts such as TREM2 and positively correlated with pathology severity in APOE4 carriers, but negatively in non-carriers. Overall, cumulative evidence from the largest neuroimaging, pathology, and transcriptomic studies available suggests that vascular dysfunction is key to the development of AD in APOE4 carriers. However, further studies are required to tease out non-APOE4-specific mechanisms.

The Key Factors Predicting Dementia in Individuals With Alzheimer's Disease-Type Pathology.

Dementia affects millions of individuals worldwide, yet there are no effective treatments. Alzheimer's disease, the most common form of dementia, is characterized by amyloid and tau pathology with amyloid accumulation thought to precipitate tau pathology, neurodegeneration, and dementia. The Religious Orders Study and Memory and Aging Project (ROSMAP) cohort is a unique resource with quantitative pathology from multiple brain regions, RNA sequencing, and longitudinal cognitive data. Our previous work applying machine learning to the RNA sequencing data identified lactoferrin (LTF) as the gene most predictive of amyloid accumulation with a potential amyloidogenic mechanism identified in vitro and with cell-culture models. In the present study, we examined which pathologies and genes were related to cognitive status (dementia, mild impairment, and no cognitive impairment) and rate of cognitive decline. Tau load in the anterior cingulate and ADAMTS2, encoding a metallopeptidase, were the respective regional pathology and gene most associated with cognitive decline, while PRTN3, encoding a serine protease, was the key protective feature. ADAMTS2, but not PRTN3, was related to amyloid and tau load in the previous study while LTF was not related to cognitive decline here. These findings confirm a general relationship between tau pathology and dementia, show the specific importance of tau pathology in the anterior cingulate cortex and identify ADAMTS2 as a potential target for slowing cognitive decline.

Overlap between Central and Peripheral Transcriptomes in Parkinson's Disease but Not Alzheimer's Disease.

Most neurodegenerative disorders take decades to develop, and their early detection is challenged by confounding non-pathological ageing processes. Therefore, the discovery of genes and molecular pathways in both peripheral and brain tissues that are highly predictive of disease evolution is necessary. To find genes that influence Alzheimer's disease (AD) and Parkinson's disease (PD) pathogenesis, human RNA-Seq transcriptomic data from Brodmann Area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC), whole blood (WB), and peripheral blood mononuclear cells (PBMC) were analysed using a combination of differential gene expression and a random forest-based machine learning algorithm. The results suggest that there is little overlap between PD and AD, and the AD brain signature is unique mainly compared to blood-based samples. Moreover, the AD-BA9 was characterised by changes in 'nervous system development' with Myocyte-specific enhancer factor 2C (Mef2C), encoding a transcription factor that induces microglia activation, a prominent feature. The peripheral AD transcriptome was associated with alterations in 'viral process', and FYN, which has been previously shown to link amyloid-beta and tau, was the prominent feature. However, in the absence of any overlap with the central transcriptome, it is unclear whether peripheral FYN levels reflect AD severity or progression. In PD, central and peripheral signatures are characterised by anomalies in 'exocytosis' and specific genes related to the SNARE complex, including Vesicle-associated membrane protein 2 (VAMP2), Syntaxin 1A (STX1A), and p21-activated kinase 1 (PAK1). This is consistent with our current understanding of the physiological role of alpha-synuclein and how alpha-synuclein oligomers compromise vesicle docking and neurotransmission. Overall, the results describe distinct disease-specific pathomechanisms, both within the brain and peripherally, for the two most common neurodegenerative disorders.

Lipidome changes in alcohol-related brain damage.

Alcohol-related brain injury is characterized by cognitive deficits and brain atrophy with the prefrontal cortex particularly susceptible. White matter in the human brain is lipid rich and a major target of damage from chronic alcohol abuse; yet, there is sparse information on how these lipids are affected. Here, we used untargeted lipidomics as a discovery tool to describe these changes in the prefrontal, middle temporal, and visual cortices of human subjects with alcohol use disorder and controls. Significant changes to the lipidome, predominantly in the prefrontal and visual cortices, and differences between the white and grey matter of each brain region were identified. These effects include broad decreases to phospholipids and ceramide, decreased polyunsaturated fatty acids, decreased sphingadiene backbones, and selective decreases in cholesteryl ester fatty acid chains. Our findings show that chronic alcohol abuse results in selective changes to the neurolipidome, which likely reflects both the directs effects on the brain and concurrent effects on the liver.

The acute phase protein lactoferrin is a key feature of Alzheimer's disease and predictor of Aß burden through induction of APP amyloidogenic processing.

Amyloidogenic processing of the amyloid precursor protein (APP) forms the amyloid-ß peptide (Aß) component of pathognomonic extracellular plaques of AD. Additional early cortical changes in AD include neuroinflammation and elevated iron levels. Activation of the innate immune system in the brain is a neuroprotective response to infection; however, persistent neuroinflammation is linked to AD neuropathology by uncertain mechanisms. Non-parametric machine learning analysis on transcriptomic data from a large neuropathologically characterised patient cohort revealed the acute phase protein lactoferrin (Lf) as the key predictor of amyloid pathology. In vitro studies showed that an interaction between APP and the iron-bound form of Lf secreted from activated microglia diverted neuronal APP endocytosis from the canonical clathrin-dependent pathway to one requiring ADP ribosylation factor 6 trafficking. By rerouting APP recycling to the Rab11-positive compartment for amyloidogenic processing, Lf dramatically increased neuronal Aß production. Lf emerges as a novel pharmacological target for AD that not only modulates APP processing but provides a link between Aß production, neuroinflammation and iron dysregulation.

Periodontitis induces endothelial dysfunction in mice.

The treatment of periodontitis has numerous positive effects on established chronic health conditions, including cardiovascular disease and diabetes. However, ethical considerations do limit the establishment of human trials to investigate whether periodontitis promotes the early stages of chronic conditions. Therefore, the aim of this study was to investigate whether periodontitis induces endothelial dysfunction in hyperlipidemic apolipoprotein E gene-deficient (ApoE-/-) mice. Forty-five 8-week-old ApoE-/- mice were challenged by oral lavage with Porphyromonas gingivalis and Streptococcus gordonii for 4 weeks. A subgroup of animals (n = 15-17/group) was placed in a metabolic chamber immediately before euthanasia at 4 weeks to measure VO2/CO2 concentrations and voluntary locomotion. In infected and control animals alveolar bone levels were measured by x-ray imaging and endothelial function was determined by measuring endothelial-dependent vasorelaxation of aortic rings. The mRNA expression levels of serum amyloid A and tumor necrosis factor were determined in liver tissues by qRT PCR and protein concentrations in serum by ELISA. Caecal contents were analysed by sequencing to determine changes to the gut microbiota to investigate linkages between microbiome and systemic changes. The results showed that oral lavage of P. gingivalis and S. gordonii for 4 weeks, initiated periodontitis in ApoE-/- mice, similar to the human situation. The oral inflammation was accompanied by a significant increase in mRNA expression of pro-inflammatory mediators serum amyloid A1 and tumor necrosis factor in the liver. Mice with periodontitis also exhibited impaired endothelial-dependent vasorelaxation responses to acetylcholine. This systemic response was connected to increased energy expenditure, locomotion and respiratory quotient. No differences were detected in caecal microbiota between the infected and control animals. Overall, this is the first report that provide evidence that periodontitis induces endothelial dysfunction in mice. Other systemic responses observed in response to the local reaction need further investigation. The study suggests that early prevention of periodontitis may help limit the early stages of endothelial dysfunction that is linked to atherogenesis in humans.