Introduction 1 2 3 4 5 Taken together, it is likely that IE is related to both the influence of NO and glial cells changes. Nevertheless, there have been no reports on a relationship between NOS-2 and glial cells within the brain after infection with the influenza virus. Thus, the aim of these studies is to better understand the clinical state of IE by focusing on NO and astrocytes in the central nervous system (CNS) after influenza virus infection. By using the A/NWS/33 influenza virus infected BALB/c model mouse, we were able to demonstrate that influenza virus infection leads to an up regulation of NOS-2 and astrocytes, mostly around capillary blood vessels of the hippocampus and olfactory bulb, starting at an early stage of the disease. Although IE has been reported for more than a decade, a therapy for IE has not yet been defined. We expect that our results will provide evidence which will aid in the development of a novel IE therapy. Experimental procedures Virus Influenza A/NWS virus, which is a mouse brain adapted type of human influenza A/NWS/33 virus was used. (A kind gift from Dr. K. Hayashi, Toyama University, Japan.) Experimental animals In total, 82 male BALB/c mice (5-week-old) were purchased from Japan Charles River Co. and given free access to food and water (Oriental Yeast, Co., Tokyo) and acclimatized for at least a week before the experiment. Anesthetics 7 5 Body weight Body weight changes were monitored daily. Euthanasia All mice were decapitated whilst under ether inhalational anesthesia. Each brain was divided into eight regions (frontal cortex, occipital cortex, cerebellum, medullar oblongata, hippocampus, corpus striatum, thalamus/hypothalamus and olfactory bulb). Then each section was immediately frozen with liquid nitrogen. 2 − 3 − 2 3 x 3 2 3 RNA extraction RNA was extracted from each part of the brain by using a QIA amp RNA extraction kit (QIAGEN K.K., Tokyo), according to the protocol suggested by the manufacturer. cDNA synthesis cDNA was synthesized from purified RNA by using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland). Random hexamer primer was used and the protocol was as follows; after annealing for 10 min at 25°C, and incubating 30 min at 55°C, the reaction was heated to 85°C for 5 min and chilled on ice. Real time reverse transcriptase-polymerase chain reaction (RT-PCR) Messenger RNA levels of NOS-1, NOS-2 and NOS-3 were quantified by using a Light Cycler Fast Start DNA Master HybProbe (Roche). This was done according to the following protocol; Pre-incubation was 95°C for 30 s. This was followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 15 s, amplification at 72°C for 8 s for 45 cycles. Cycles were followed by 30 s 40°C cooling program. The primers and probes were designed by the Light Cycler primer and probe set (Roche Diagnostics Applied Science, Tokyo). NOS-2, NOS-3 and NOS-1 primer sequences were designed on GenBank database NM_0100927, NM_008713, and NM_008712 respectively. All data were normalized by dividing with the corresponding GAPDH mRNA from the same sample. Immunohistochemistry 2 2 2 2 All sections were counterstained with Mayer’s hematoxylin (SIGMA, MI). Statistical analysis Fisher’s exact test was used when comparing body weights. All results obtained from the real time RT-PCR were subjected to one- or two-way analysis of variance (ANOVA) and differences among the means were analyzed by two-way ANOVA followed by a Newman–Keuls range test at the 0.05 significance level. Results are expressed as mean ± standard error. Results Body weight 1 P Fig. 1    Viral titers 4 Real-time RT-PCR for NOS-2, NOS-3 and NOS-1 2 2 2 Fig. 2 P a a b b  Changes in brain NO levels between non-infected and infected mice 3 2 3 3 2 2 3 P P Fig. 3 P  Immunohistochemistry 4 4 4 4 4 Fig. 4 Anti-NOS-2 immunohistochemistry and GFAP immunohistochemistry in the hippocampus at 1 (Fig. 4a) and 6 (Fig. 4b) days p.i  Discussion 6 1 5 7 The reason why we determined the amounts of influenza virus in this experiment is as followed. During the past experiment, we had experienced that the mice die within 3 days p.i. when inoculating any more than the virus we used this time and when we use fewer viruses, there were hardly any difference in the non-infected and infected behavioral change. 2 4 2 8 4 9 10 2 3 11 5 12 16 17 18 4 19 17 20 21 4 22