Tottori J. Clin. Res. 1(1), 152-167, 2008

Cytokine expression profiles in the brain of non-demented control patients withincreasing Alzheimer’s disease pathology, in comparison with normal control andAlzheimer’s disease patients
Yoshihiro Konishi1, 2)*Haruhisa Satoh1) Keiko Harano3) Teruo Harano4) Ikuo Tooyama1)

1) Molecular Neuroscience Research Center, Shiga University of Medical Science2) Department of Clinical Research, Tottori National Medical Center3) Laboratory of Chemistry, Department of Clinical Nutrition,Kawasaki University of Medical Welfare
4)

Department of Biochemistry, Kawasaki Medical School*Correspondence: ykonishi@tottori-iryo.hosp.go.jp

Abstract
Evidence of the critical importance of inflammatory mediators in Alzheimer’s disease (AD) pathology has
been provided by basic, clinical and epidemiological studies. These studies have indicated that nonsteroidal
anti-inflammatory drugs (NSAIDs) delay the onset and slow the progression of AD. Herein we examined what
inflammatory mediators are involved in AD pathology and how early they begin to be linked to the developmentof AD pathology before clinical manifestation of overt dementia. To address this issue, we analyzed cytokine
expression profiles of the brain, with focus on nondemented control patients with increasing AD pathology,
referred to as high pathology control (HPC) cases. If some cytokines are more primarily involved in HPC cases,
which provide an intermediate subset between AD and normal control cases, the target of inflammatory mediators
implicated more in the progress of AD could be pinpointed for the development of AD therapies. Withsemi-quantitative analysis of cytokine mRNA, we found that interleukin-1 (IL-1) β and IL-1 receptor antagonist
showed somewhat of an increment in the HPC; IL-6, IL-10 and tumor necrosis factor (TNF) α, expressed undernormal conditions, had a tendency to decrease in the HPC, leading to the assumption that temporal expression ofthe IL-1 family and the decline in IL-6, IL-10 and TNFα expression play key roles in the manifestation of overt
dementia with AD progression. Tottori J. Clin. Res. 1(1), 152-168, 2008
Key words : cytokines, Alzheimer’s disease, neuroinflammation, dementia
Introduction

widely suggested that conventional nonsteroidal

The pathogenesis of AD has typically not beenanti-inflammatory drugs (NSAIDs) may delay theexplained by alterations in immune function. Several

onset and slow the progression of AD. The hallmarks

studies have shown the importance of T cell andof AD, amyloid β peptide (Aβ), neurofibrillary tangles

macrophage

provided(NFT), and neuronal degeneration, are the most likely

criticalsources for inflammation in the AD brain2). Actually,

importance of inflammatory mediators in AD1). Direct

β-plated, fibrillar Aβ and tau-containing NFT haveand tangential evidence of a neurodegenerative role

been shown to directly activate the classicalfor the inflammatory process in the AD brain has been

complement pathway fully in vitro, and to do so evenprovided from basic research. Clinical research has

in the absence of antibodies3,4). A variety of molecules,

secretory

epidemiologicalproducts,

evidence

detailingand
the152

defined as key mediators in peripheral immune

control7-9). Low pathology control (LPC) cases, also

reactions, have been found to occur in highreferred to as ND patients, have limited AD pathology2)

concentration in the AD brain .
Thus,neuroinflammationand no prior medical history of dementia.
clearlyoccurs

inpathologically vulnerable regions of the AD brain, andMaterials and methods

itPatient samples

generally seems

tobeacceptedthat ADneuroinflammation significantly contributes to AD

From among the 100 routine brain autopsies per2, 5)

pathogenesis. However, many issues regarding thisyear of patients who had prospectively enrolled in thecontribution require clarification. Do inflammatory

Sun Health Research Institute Tissue Donation

mechanisms actually cause damage in the AD brain?

Program and given premortem consent, four to six

Are they more primary AD pathologic processes thattypical cases were selected from each of the early andoccur before neuronal damage becomes significant? Islate stages of AD, HPC and LPC patients, based onneuroinflammation always a secondary response to thepriorhallmarks of AD, occurring in the later symptomatic

neuropsychological test scores, including data of thestages of AD6)? In fact, HPC patients without a history

Folstein Mini Mental Status Examination (MMSE)

of dementia but who nonetheless exhibit sufficientand the Clinical Dementia Rating Scale (CDR), andneocortex Aβ deposits and entorhinal cortex NFT at

postmortem neuropathological

autopsy to otherwise qualify for the diagnosis of AD

Consortium to establish a registry for AD (CERAD)

show elevations of inflammatory markers, althoughpathological criteria10) and the Braak staging11). In

only modest and partial, that are significantly greater

addition, our study was approved by the ethicalthan in nondemented elderly (ND) patients but

committees of both the Shiga University of Medical

7-9)

dramatically less than in AD patients
particularly

importantbecause

ifmedicalrecordswithrecords

antemortemwiththe. This matter isScience and the National Hospital Organization Tottori

inflammatory

Medical Center.
Tissue preparation

mediators begin to change in the latent phase of AD,
anti-inflammatory therapy may interrupt or slow theBrain tissue sections, which had originally beenpathological progression of latent AD and thereby

prepared for immunohistochemical procedures, were6)

delay or even prevent the onset of dementia . In

used for the present study. Briefly, brain tissue wasaddition, investigations of what inflammatory or

removed within four hours of death, sectioned

immune mechanisms, or mediators are earlier or morecoronally at 1 cm intervals, fixed for 48 hours insignificantly involved in the developmental process of

ice-cold 4% paraformaldehyde in a 0.1 M phosphate

AD pathology could be expected to lead to thebuffer (pH 7.4), cut at 40 µm on a freezing microtome,

development of therapeutic strategies to pinpoint thecyroprotected for 36-48 hours in a 0.1 M phosphateinflammatory

buffer (pH 7.4) containing 2% dimethylsulfoxidemechanisms

involvedinAD

pathological progression.
In(DMSO) and 10% glycerol, and stored in a 20 mM

the present study,

we examined whatphosphate buffer (pH 7.4) containing 30% ethylene

inflammatory mediators are involved in AD pathology

glycol and 30% glycerol at -20oC until use, asand how early they begin to be linked to thedescribed elsewhere in detail8).

development

Extraction of total RNA

of AD

pathology before

clinicalmanifestation of overt dementia. To address this issue,

Three sections from the temporal cortex of each

we analyzed cytokine expression profiles of the brain,case were incubated for one hour at 45oC with 200 µl

with focus on HPC cases. HPC cases provide anof a homogenate buffer consisting of 3.5 mg/ml ofintermediate subset between AD and a low pathology

proteinase K (Wako Pure Chemicals), 2% sodium153