| Normal brain aging accompanies alterations in inflammatoryactivities, and one vital this alteration is that increased production ofpro-inflammatory cytokines. It has been demonstrated that activation ofglial cells in the brain responsing to an acute pro-inflammatory cytokineschallenge is enhanced at an advanced age. So far, it remains largelyunknown whether or not there is an age-related change inimmune-associated responses in normal aging brain to an inflammatorychallenge. The change that increased level of pro-inflammatory cytokinesalso contributes to sleep regulation, and this process is impacted bynormal aging. However, little information is available on whether or notsleep loss with aging can elicit same changes in immune-associatedresponses that happen in normal aging brain, as well as this changedepend or not on circulating of pro-inflammatory cytokines level.Objective To investigate the possible roles of pro-inflammatorycytokine in immune-associated responses in normal aging brain, as wellas in sleep regulation during normal aging. For this purpose, theimmune-associated responses in the mouse brain of different ages to anacute pro-inflammatory cytokines challenge was observed, the activationof glial cells, the recruitment of inflammatory cells, and the expressionlevel of pro-inflammatory cytokines in adult rat brain following rapideye-movement (REM) sleep deprivation was also studied.Methods Quantitative real time RT-PCR was employed to determineearly transcriptional changes of genes involved in the response to acuteicv administration of the cytokines tumor necrosis factor (TNF)-αorinterferon (IFN)-λin young (3-4 months) and old (21-23 months) mice.Control experiments were performed in parallel with icv injections ofsaline. After obtaining information on such early response, the expressionof the protein intracellular adhesion molecule (ICAM)-1, which plays akey role in the regulation of blood-brain barrier permeability, wasexamined with Western blotting and immunohistochemistry at 48 h afterTNF-αor saline injections in mice of different ages (3-4 months, 8-9 months and 21-23 months). The recruitment of CD3~+, CD4~+ and CD8~+Tcells in the brain was examined in the same paradigm withimmunohistochemistry. Finally, glial cells and macrophages, majorhistocompatibility complex antigen classⅡ(MHC-Ⅱ) and TNF-αwerestudied in adult rats following 60 h of sleep deprivation.Immunohistochemistry (in bright-field and in fluorescence microscopy,including double immunofluorescence) was used in this part of theinvestigation.Results Real time RT-PCR revealed that the up-regulation elicited byTNF-αon the expression of the ICAM-1 transcript, and by IFN-γon theexpression of MMP-12, SOCS-1 and SOCS-3 transcripts was significantlyincreased in the brain of old mice compared to young ones (P＜0.05). Inaddition, the expression of the TNF receptor TNFR2 was significantlyenhanced in the septum and hippocampus of both cytokine-injected andcontrol (saline-injected) old animals (P＜0.05). At 48 h after TNF-αicvinjections, immunohistochemistry and Western blotting revealed also asignificant up-regulation of the expression of the ICAM-1 protein in thebrain of old mice as compared to younger animal groups (P＜0.05).Double immunofluorescence revealed that ICAM-1 expression in bothastrocytes and microglia.The study of T cell recruitment in the brain pointed out the interestingfinding of enhanced recruitment of lymphocytes in the brain of old micein response to cytokine administration (P＜0.05)In adult rats, after 60 h of sleep deprivation, the activation of bothastrocytes and microglia, the expression of MHC-Ⅱantigen and of TNF-α,the number of activated macrophages were all significantly enhancedcompared with control subjects (P＜0.0001). Double immunostaining ofTNF-αcombined with glial antigens (the astrocyte marker glial fibrillaryacidic protein or the microglial marker OX-42) showed that astrocytesexpressed TNF-αin this paradigm.Conclusionⅰ) The pro-inflammatory effects of pro-inflammatorycytokines are amplified and prolonged in normal aging brain;ⅱ) Animmune hyperresponsiveness and injury susceptibility topro-inflammatory challenge is elicited in normal aging brain,ⅲ) Variation in sleep rhythm exerts impacts on the activation of glial cells and theresponsiveness of inflammatory-associated response during aging, andpro-inflammatory cytokines play an important role in this process,ⅳ)Altogether these results reveal that pro-inflammatory cytokines areclosely correlated with immune-associated responses in normal agingbrain and involved in sleep regulation of aged individuals.
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