Cavadini et al. 10.1073/pnas.0701466104. |
Fig. 6. Photomicrographs of histological sections of liver, lung and kidney with (top row) and without (bottom row) TNF-a treatment: The liver shows an increase in HO-1 positive Kupffer cells. At high power magnification the cells appear also larger. No morphologically apparent liver cell damage is however detected. The lung shows unremarkable numbers of HO-1 positive macrophages in the bronchial mucus layer. No increase in HO-1 positive alveolar macrophages is seen. At high power magnification (hematoxylin eosin staining) increased numbers of megakaryocytes (indicated by arrows) are seen trapped within the alveolar vascular bed. There is, however, no evidence of pulmonary edema or acute pulmonary damage. Sections of the kidney (hematoxylin eosin staining) are unremarkable. The scale bar represents from left to right 400 mm, 50 mm, 200 mm, 50 mm, and 100 mm, respectively.
Fig. 7. At day 3 (ZT 15.5) after minipump insertion, serum was collected and TNF-a was measured by ELISA. Data are given as the mean ± SD of TNF-a serum concentrations measured in six animals per group. Independent samples t test, P = 0.001.
Fig. 8. Decreased concentrations of albumin in serum of TNF-a-treated mice. Serum was collected at day three after minipump implantation at ZT 15.5 and albumin concentrations were measured as described in materials and methods. Data show the mean ±SD of serum albumin concentrations in mice (six mice per group).
Fig. 9. IL1b expression correlates to TNF-a expression, both being increased in TNF-a treated animals (circles with different colors, each individual mouse representing one color) compared to animals infused with PBS (blue open circles) (day 3, at ZT 15.5; 6 mice per group). In contrary, Dbp, Hlf, and Tef are negatively correlated to the relative TNF-a expression.
Fig. 10. TNF-a impairs expression of the clock output genes, Dbp in the SCN. (A and B) Dbp and Bmal1 expression in the SCN at ZT 6 as determined by radioactive in situ hybridization. At the time point of peak expression of Dbp, when Bmal1 expression is lowest, TNF-a leads to a significant reduction by 15% of the Dbp mRNA. (means ± SEM of six consecutive sections per mouse SCN; four mice; paired t test; * P = 0.003).
Fig. 11. (A) Mice treated with TNF-a show increased long rest epochs in the dark phase. Analysis of rest epochs was performed at day 3 of TNF-a and control infused mice; the analysis being performed separately for the light and dark period. The data show the differences in numbers of 1-min episodes with activity = 0 compared to baseline (mean of the three days before minipump insertion). The occurrence of rest was arbitrarily subdivided in episodes for durations with rest = 0 activity counts: up to 1 min rest, between 2 and 5 min, 6 and 60 min, and more than 60 min. (ANOVA for repeated measures, followed by independent-samples t test; * P £0,05). (B) TNF-a does not affect the period length measured in constant darkness. On day 1 after minipump insertion the lights were turned off and the mice were kept in constant darkness. The circadian period of locomotion and running-wheel activity was determined by periodogram analysis over the 5 days in constant darkness. Independent-sample t-tests were performed. TNF-a (1.5 mg/day; light gray bars) or saline as control (dark gray bars).
SI Text
RNA Isolation and Gene Expression Analysis
Whole-cell RNA from cultured cells was extracted using the NucleoSpin-RNA II kit (Macherey-Nagel, Switzerland). RNA from mouse tissues was extracted by homogenization of the organ in TRIzol (Invitrogen) according to the manufacturer's instructions. Subsequently, RNA was reverse-transcribed using random hexamers (Promega, for the in vitro assays; Roche, for the in vivo assays) and AMV reverse transcriptase (Promega, for the in vitro assays) or M-MuLV reverse transcriptase (Roche, for the in vivo assays). The cDNA equivalent to 50 ng of total RNA was PCR-amplified in an ABI PRISM 7700 detection system (PE-Applied Biosystems) using the TaqMan Universal PCR Master Mix (Applied Biosystems) and quantified as follows. Primers and probes for Taqman analysis were either purchased from Applied Biosystems or purchased from Microsynth, Balgach, Switzerland, as described in detail in SI Table 1. The relative levels of each RNA were calculated by the 2-DDCT method (CT standing for the cycle number at which the signal reaches the threshold of detection); 18s rRNA was used as a housekeeping gene. Each CT value used for these calculations is the mean of two duplicates of the same reaction. Relative RNA levels are expressed as x-fold variations compared to ZT = 0 (time course experiments) or as percentages of the average control groups (in the in vitro one-time-point experiments and in the in vivo experiments).
In situ
hybridizationAfter infusion of mice with saline or TNF-a mice were killed at day three at ZT 6. Brains were embedded in paraffin and sectioned at 7-mm-thickness and hybridized with 35S-rUTP labeled riboprobes as described (1). The Bmal1 probe corresponded to nucleotides 654-1290 (accession no. AF015953) and the Dbp probe to nucleotides 2-951 (accession no. NM016974). Quantification was performed by densitometric analysis of autoradiograph films using the Molecular analyst program (Bio-Rad). Data from the SCN were normalized to the lateral hypothalamus next to the SCN. For each treatment three animals were used and six sections per SCN were analyzed. We assessed the relative mRNA abundance values by defining the highest value of each experiment in mock treated animals as 100%.
ELISA
The concentration of albumin and TNF-a in the serum of mice implanted with osmotic minipumps was determined by ELISA (Mouse Albumin ELISA Quantitation Kit, Bethyl Laboratories, TX, and mouse TNF-a ELISA kit, KMC3012, Biosource).
1. Deboer T, Tobler I (2000) J Comp Physiol A 186:969-973.