Research On The Establishment Of Model Of Microglial Activation And Effects Of Piper Kadsura Ohwi

Background:The pathogenesis of Alzheimer's disease (AD) still remains unknown now, but several different theories have been suggested by researchers. The inflammation theory is one of the most possible mechanisms supported by in vitro and in vivo evidences. The activation of microglia can be stimulated by beta-amyloid (Aβ), the main component of neuritic plaques. Up to now, there is still no effective therapy for AD. Piper Kadsura Ohwi is a kind of Chinese herbal drug traditionally used to treat trauma and asthma, etc.. Recent studies revealed that extracts of Piper Kadsura Ohwi may inhibit expression ofβ-amyloid precursor protein (APP) gene and biosynthesis of Aβ, but its effects on the abnormal activation of microglia have not been studied yet.Object:To compare the activation of microglia in vitro induced by oligomeric and fibrillar Aβ25-35, and research the effects of Piper Kadsura Ohwi extracts on the microglial activation.Methods:Cerebrums of neonatal Wistar rats were taken out of the cranial cavity and mixed cells were cultured in vitro. Then the microglia were separated from the mixture and were identified by immunocytochemistry, using the CD11b molecule as a specific marker. Separated microglia were divided randomly into six test groups, intervened by fibrillar Aβ25-35, fibrillar Aβ25-35+ Piper Kadsura Ohwi extracts, fbrilliar Aβ25-35+ dimethyl sulfoxide (DMSO, solvent of Piper Kadsura Ohwi extracts), oligomeric Aβ25-35, oligomeric Aβ25-35+ Piper Kadsura Ohwi extracts and oligomeric Aβ25-35+ DMSO respectively. Also a blank contrast group was set without any intervention.48 hours later, the morphorlogic change of the microglia was observed under the inverted phase contrast microscope. Activation of microglia was tested by immunocytochemistry, using the CD68 molecule as a specific marker of microglial activation. The number of active (known as CD68+) microglia was calculated and the percentages of active microglia in different groups were compared.Results:1) The cells separated from the mixture were verified as microglia, since 98% of the cells express the CD11b molecule, while only 5.4% of cells in the negative contrast group (using PBS as the first antibody) were CD11b+ (P<0.05).2) 48 hours after the different intervents were added into the culture media, microglia in both fibrillar and oligomeric Aβ25-35 groups show the typical morphological change of activation:decreased size of cell body and shortened process (ameboid change), whereas the morphological change of microglia in fibrillar Aβ25-35+ Piper Kadsura Ohwi extracts and oligomeric Aβ25-35+ Piper Kadsura Ohwi extracts group was less apparent.3) CD68 immunocytochemistry shows that each group has a certain proportion of microglia that express CD68, but the positive proportions vary in different groups, P<0.05. Each of the six test groups has a higher positive proportion of CD68 expression than that of the blank contrast (5.1%), P<0.004; positive proportion of the oligomeric Aβ25-35 group (71.2%) is higher than that of the fibrillar Aβ25-35 group (60.8%), P<0.002; positive proportion of fibrillar Aβ25-35+ Piper Kadsura Ohwi extracts group (52.1%) is lower than that of the fibrillar Aβ25-35 group (60.8%), P<0.002; positive proportion of oligomeric Aβ25-35+ Piper Kadsura Ohwi extracts group (67.0%) is not significantly different with that of the oligomeric Aβ25-35 group (71.2%), P=0.005.Conclusions:1) Rat microglia was successfully separated and purified from the cells mixture. CDllb immunocytochemistry results indicated that the purity of microglia is high enough for the subsequent researches.2) It is demonstrated that both fibrillar and oligomeric Aβ25-35 has the ability to activate the silent microglia, but the action of oligomeric Aβ25-35 was more powerful.3) After the possible interference of DMSO, the solvent of Piper Kadsura Ohwi extracts, was precluded, it was revealed that Piper Kadsura Ohwi extracts could inhibit the activation of microglia induced by fibrillar Aβ25-35, but didn't show significant effects on the activation induced by oligomeric Aβ25-35.