Treatment Protocol All mice in this study were sensitized with 1 µg DNP4-human serum albumin (HSA) in 1 mg alum on days 0, 7, 14, and 21, intraperitoneally (i.p.). In normal mice, this protocol induces sustained levels of IgE antibodies up to 56 days post-sensitization (20). One week after the last sensitization, cystitis was induced. Briefly, sensitized WT and NK1R-/- mice were anesthetized (ketamine 40 mg/kg and xylazine 2.5 mg/kg, i.p.), then transurethrally catheterized (24 Ga.; 3/4 in; Angiocath, Becton Dickson, Sandy, Utah), and the urine was drained by applying slight digital pressure to the lower abdomen. The urinary bladders were instilled with 200 µl of pyrogen-free saline or DNP4-OVA (1 µg/ml). One, four, and twenty-four hours after instillation, mice were sacrificed with pentobarbital (100 mg/kg, i.p.) and bladders were removed rapidly.
Extracted molecule
total RNA
Extraction protocol
three bladders from each group were homogenized together in Ultraspec RNA solution (Biotecx Laboratories Inc. Houston, TX) for isolation and purification of total RNA. Mouse bladders were pooled to ensure enough RNA for gene array analysis. The justification for this approach is that there is not enough RNA in a single mouse bladder for performing cDNA array experiments and the step of purification reduces the amount of total RNA. RNA was DNase-treated according to manufacturer’s instructions (Clontech Laboratories, Palo Alto, CA), and the quality of 10 µg was evaluated by denaturing formaldehyde/agarose gel electrophoresis.
Label
p32
Label protocol
Label Protocol cDNA probes prepared from DNase-treated RNAs obtained from each of the experimental. Briefly, 5 µg of DNase-treated RNA was reverse-transcribed to cDNA and labeled with [-32P]dATP, according to the manufacturer's protocol (Clontech, Palo Alto, CA). The radioactively labeled complex cDNA probes were hybridized overnight to AtlasTM Mouse 1.2 Arrays (Clontech, Palo Alto, CA) using ExpressHybTM hybridization solution with continuous agitation at 68 °C. After two high- stringency washes, the hybridized membranes were exposed (at room temperature) to a ST Cyclone phosphor screen overnight. Spots on the arrays were quantified by BD AtlasImage™ 2.7 software (Clontech, Palo Alto, CA). The results were placed in an Excel spreadsheet.
Hybridization protocol
cDNA probes prepared from DNase-treated RNAs obtained from each of the experimental. Briefly, 5 µg of DNase-treated RNA was reverse-transcribed to cDNA and labeled with [-32P]dATP, according to the manufacturer's protocol (Clontech, Palo Alto, CA). The radioactively labeled complex cDNA probes were hybridized overnight to AtlasTM Mouse 1.2 Arrays (Clontech, Palo Alto, CA) using ExpressHybTM hybridization solution with continuous agitation at 68 °C. After two high- stringency washes, the hybridized membranes were exposed (at room temperature) to a ST Cyclone phosphor screen overnight. Spots on the arrays were quantified by BD AtlasImage™ 2.7 software (Clontech, Palo Alto, CA). The results were placed in an Excel spreadsheet.
Scan protocol
Spots on the arrays were quantified by BD AtlasImage™ 2.7 software (Clontech, Palo Alto, CA). The results were placed in an Excel spreadsheet
Description
Data was normalized by linear regression analysis using only genes expressed above background, as described (17, 18) and the ratio of gene-expression between antigen- and saline-challenge was obtained. NK1-R- dependent genes were selected according to the following criterion: a. In tissues isolated from WT mice, the expression of particular gene should be up-regulated (ratio between antigen- and saline-treated <3.0) in at least one of the time points (1, 4, and 24 hours post challenged); b. in tissues isolated from NK1R-/- mice, the expression of same gene should not be altered by antigen-challenge in any of the time points. Alternatively, expression was considered NK1-R– independent for those genes found up-regulated at least 3-fold in response to inflammation in tissues isolated from WT and NK1R-/- mice.
