Abstract
INTRODUCTION
Alzheimer’s disease (AD) is the most common acquired cerebral degeneration and the main cause of dementia in Western countries (50% – 66%). It interferes in personal, social, and work activities. Its pathophysiology is based on molecular and cellular neuronal abnormalities that include senile plaques and neurofibrillary tangles with a chronic inflammatory background [1]. One of the main factors involved in the development and progression of AD is a cerebral inflammatory process. Some inflammatory biochemical markers seem to be elevated in patients with AD when compared with healthy subjects of the same age. The plasmatic levels of interleukin 6 (IL-6) and the cerebral levels of IL-6, interleukin 1 Beta (IL-1β) and tumor necrosis factor alpha (TNF-α), were increased in these patients [2].
Dental infections, especially periodontitis, are associated with dementia and are risk factors for the onset or the aggravation of AD [3 –6]. Chronic periodontitis is an infectious-inflammatory disease caused by bacteria that destroy the connective and bone tissue of the periodontium [7]. This chronic infection generates chronic inflammation with the release of local and systemic inflammatory and immune mediators such as cytokines, which are elevated in the serum of patients [8]. Besides, oral infections at the mucosa, such as candidiasis, are more frequent in AD. They are also able to increase serum IL-6 and TNF-α levels [9]. Thus, the objective of this study was to investigate the prevalence of oral infections and serum levels of IL-6 and TNF-α in patients with AD, mild cognitive impairment (MCI), and non-demented elderly.
MATERIALS AND METHODS
Subjects
Twenty-five patients with AD and 19 patients with MCI diagnosed according to the criteria of the National Institute for Communicative Disorders and Stroke (Alzheimer’s Disease and Related Disorders Association) (NINCDS-ADRDA) [10] were evaluated. The diagnosis was performed by an experienced neurologist of the Group of Behavior and Cognitive Neurology of the Hospital das Clinicas (Medical School of the University of Sao Paulo). The patients were compared with 21 healthy subjects. They were non-demented elderly that had been followed at the Group of Multidisciplinary Assistance of Elderly of the Geriatric Clinic at the same hospital. All patients, controls, relatives and/or guardians were informed about the purposes of the study and all signed the informed consent. The local Ethics Committee approved the protocol.
Patients with AD, MCI, and controls were matched according to educational status, age, gender and the prevalence of hypertension and diabetes (p > 0.05; SPSS 17.0; SPSS Inc., Illinois, USA). A single trained neurologist who was blinded to the dentist evaluation performed the neurological evaluation of all subjects.
Exclusion criteria were other neurodegenerative or neurological conditions (e.g., Parkinson’s disease, multiple sclerosis, other causes of dementia, neural infections such as Hansen’s disease, stroke) and cerebral, facial, or cervical tumors.
Protocol of evaluation
A single trained dentist performed the complete oral exam and clinical questionnaire to identify absences of teeth and aspects of the oral mucosa, tongue, and orofacial pain [11]. The decayed, missing, and filled tooth index was determined as well as the O’Leary plaque index (PI). The periodontal probing was performed to determine the gingival bleeding index (BI), probing pocket depth (PPD), the clinical attachment level (CAL), and cement-enamel junction distance (CEJ) [12, 13].
The serum level of cytokines (IL-6, IL-1β, and TNF-α) were investigated by venous blood samples of the subjects, acquired concomitant to clinical exam. The blood samples were obtained in dry tubes of 9 mL (Vacuette®) and immediately centrifuged, and the serum was immediately stored in a –80°C freezer. After the storage of all samples, a multiplex panel (MILLIPLEX map High Sensitivity Human Cytokine Panel®) was used to perform the cytokine analysis.
Statistical analysis
Data were tabulated and analyzed according to means, standard deviations, frequencies, and normality tests for the distribution of quantitative variables (Shapiro-Wilk test and Q-Q plot). Variables with normal distribution were analyzed with one-way ANOVA, MANOVA and Pearson’s correlation coefficient. Non-parametric chi square test was also performed. The level of significance was 5%.
RESULTS
The demographic characteristics and the results of cognitive and functional evaluation are illustrated in Table 1. Patients with AD had higher impairment of cognition and function than the other groups (p < 0.001).
AD patients showed higher prevalence of candidiasis as well as coated tongue (Table 2). There were no differences in periodontal indexes. Patients with AD and MCI had more absent teeth (p = 0.032) and a longer duration of edentulism (p = 0.032) than healthy elderly (Table 3). The main cause for tooth loss in AD patients was periodontal disease (P < 0.001); in opposition, decay was the main cause of tooth loss of the MCI group and the control group (Table 3).
The cytokine levels (IL-1β, TNF-α, and IL-6) can be observed in Fig. 1. Patients with AD had significantly higher IL-6 levels than controls (p = 0.029), and patients with periodontal disease had significantly higher TNF-α levels than patients with healthy periodontal tissues (p = 0.005). The multivariate analysis showed an association between IL-6 and TNF-α in patients with AD or MCI and periodontal disease (p = 0.023).
The concentration of IL-6 was negatively correlated with the MMSE score (p = 0.018; Pearson) and positively correlated with the Pfeffer’s Functional Assessment Questionnaire (FAQ) (p < 0.001; Pearson), which means that the high serum IL-6 level was observed in subjects with low performance in cognitive tests. The concentration of TNF-α was positively correlated with the periodontal indexes PPD, CAL, BI (p < 0.001; Pearson), and PI (p = 0.008; Pearson), which means that patients with worse periodontal condition had higher serum TNF-α level. Finally, IL-6 and TNF-α were positively correlated in all studied groups (p < 0.001; Pearson).
DISCUSSION
This study shows that the elevated concentration of IL-6 in patients with AD was associated with high serum TNF-α level of subjects with periodontal disease. This finding suggests that immune-inflammatory mechanisms of periodontitis may underlie its role in the onset, progression or aggravation of AD. IL-6 is a biomarker for cognitive impairment [14], and TNF-α might be a biomarker of the association of periodontitis with AD by these results.
The etiology of AD is not yet completely elucidated, and the involvement of chronic inflammation in its pathophysiology is still controversial. The cerebral inflammatory process could be a consequence of the deposits of senile plaques and neurofibrillary tangles, and its immune-inflammatory mediators might accelerate the neurodegenerative process [15, 16]. This hypothesis is supported by the evidence of IL-6 and C reactive protein as biomarkers of cognitive impairment [14 , 18].
Common immune-inflammatory backgrounds of neurodegeneration in AD and bone destruction in periodontitis supports their overlapping. Oral infections are frequent in the adult population and their systemic impact is important, supporting prevention and early treatment to avoid complications, such as AD, at old age [3 , 19–21]. Systemic inflammation of periodontitis is implicated in the progression and aggravation of several chronic illnesses [5], and it includes dementia.
The present results are in agreement with previous observations showing the association of early tooth loss due to periodontal disease with dementia [22]. The main cause of tooth loss was periodontitis, which corroborates to the association of cytokine expression and AD. The impact of tooth loss on nutrition, especially when occurring under the age of 35 is another risk factor of AD [23]. Oral rehabilitation with prostheses is compulsory to improve nutritional status that have an implication in general health and in prevention of cognitive deficit. Thus, oral diseases may play a role in AD by immune-inflammatory and indirect nutritional consequences [24]. Besides systemic inflammatory mechanisms, oral bacteria can reach the cerebral tissue and induce chronic local infection and inflammation in the brain [25].
In the present population studied, there were no statistical differences in the prevalence of dental diseases and periodontal parameters, except infections of the oral mucosa, a finding similar to earlier observations, which was the subject of a previous publication [11]. In that previous publication, the severity of periodontitis was not different but its prevalence was higher in patients with AD. Although the current evaluation showed similar prevalence of periodontitis between studied groups, the previous history of causal factors for dental extractions showed periodontitis as the reason for teeth loss only in AD patients. However, candidiasis can also increase the evaluated cytokines in blood serum [9]. We suggest monitoring blood serum levels of IL-6 and TNF-α and evaluating the clinical signs of periodontitis and other oral infections in AD is essential.
TNF-α, IL-1β, and IL-6 are capable of stimulating the synthesis of Aβ-amyloid 1-42 peptide (Aβ42) and the phosphorylation of tau protein (P-tau), and Aβ42 and P-Tau can stimulate the production of TNFα, IL-1β, and IL-6 by glial cells [26]. Thus, periodontitis and oral mucosa infections, as potential causes of TNF-α increase in the blood serum, is a candidate for a modifying factor for the development of AD [19, 21].
The world’s population got older in the last century, and as dementia is becoming more prevalent, it is becoming a public health problem. Co-morbidities are frequent and may play a role in the aggravation of symptoms. Non-modifiable risk factors include age, presence of APOE ɛ4, and family history [27]. Oral infections can be assessed, treated, and prevented [11 , 29], and their blood serum biomarkers (IL-6 and TNF-α) may help in the analysis of their risks in dementia.
In conclusion, this case-control transversal study shows evidence of the association between cytokines expression in the blood serum with oral infections and AD. The next step is to investigate the role of periodontitis and oral mucosa infections in the incidence of dementia in longitudinal studies, to elucidate the mechanisms underlying the overlap between TNF-α and IL-6 in neurodegeneration. Examining clinical implications is necessary to prevention, and the treatment of oral infections in adults and the elderly will improve the quality of aging because these infections potentially have a role as risk factors for AD.
