Regulation of Toll-Like Receptors in the Choroid Plexus in the Immature Brain After Systemic Inflammatory Stimuli

C57BL/6J mice were bought from Charles River (Germany). Mice were housed in a 12-h light–dark cycle and bred at Laboratory for Experimental Biomedicine (University of Gothenburg, Gothenburg, Sweden) and were provided with a standard laboratory chow diet (B&K, Solna, Sweden) and drinking water ad libitum. All animal experiments were approved by the Animal Ethics Committee of Gothenburg (No. 374-09).

Postnatal day (PND) 8 mice were injected intraperitoneally (i.p.; 10 μl/g body weight) with PAM (tlrl-pms; 5 mg/kg), a TLR-1/2 ligand; Poly I:C (Poly(I:C)-LMW, InvivoGen; 10 mg/kg), a TLR-3 ligand; LPS (List Biological Laboratories, Inc., ultrapure #423; 0.3 mg/kg), a TLR-4 ligand; or vehicle (LPS free saline, S8776, Sigma-Aldrich, Sweden) and were then deeply anesthetized and sacrificed 14 h postinjection for tissue sampling. TLR agonist doses and time for sacrifice were based on our previous studies showing biological effects at these concentrations and at this particular time point. In particular, we have found that a single injection, 14 h before hypoxia–ischemia, of these ligands at the doses used in the present study, sensitizes the neonatal brain to hypoxic–ischemic damage ([22] and Stridh et al., unpublished).

Brains were rapidly dissected out and placed in phosphate-buffered saline (PBS). Choroid plexuses from the lateral ventricles were dissected out under a microscope, snap frozen, and stored at −80 °C until analysis. To achieve enough RNA for expression analysis, choroid plexus from two animals were pooled for each sample.

Choroid plexus tissues were homogenized and total RNA was extracted using the RNeasy Micro Kit (Qiagen, Sweden) according to the manufacturer’s instructions. RNA was measured in a spectrophotometer at 260 nm absorbance. QuantiTect Reverse Transcription Kit (Qiagen, Sweden) was used to synthesize first strand cDNA according to the manufacturer’s instructions. Each PCR (20 μl) contained 2 μl cDNA, 10 μl Quanti Fast SYBR Green PCR Master Mix (Qiagen, Sweden), 2 μl PCR primer, and 6 μl H₂O. The following primers were used: TLR-1 (QT00157430), TLR-2 (QT00129752), TLR-3 (QT00122983), TLR-4 (QT00259042), claudin-1 (QT00159278), occludin (QT00111055), zonula occludens-1 (ZO-1; QT00493899), interleukin-1β (IL-1β; QT01048355), TNF-α (QT00104006), monocyte chemotactic protein-1 (MCP-1; QT00167832), GAPDH (QT01658692), and YWHAZ (QT00105350), all from Qiagen, Sweden.

The amplification protocol comprised an initial 5-min denaturation at 95 °C, followed by 40 cycles of denaturation for 10 s at 95 °C and annealing/extension for 30 s at 60 °C on a LightCycler 480 (Roche, Sweden). Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimating amplification efficiency, a standard curve was generated using increasing concentrations of cDNA. The amplification transcripts were quantified with the relative standard curve and normalized against the reference genes GAPDH and YWHAZ.

A separate group of pups were injected with TLR ligands along with saline-injected controls, as described above, and animals were killed 14 h later at PND 9 (n = 2 for each group; from two mixed litters). Pups were killed by an i.p. injection of pentobarbital and immediately transcardially perfused with cold heparinized saline for about 30 s followed by cold Histofix (Histolab, Gothenburg, Sweden) for 5 min using a syringe pump using a flow rate to minimize the risk of damaging cerebral vessels. Brains were dissected out and left in Histofix overnight in the fridge before being dehydrated and embedded in paraffin. Coronal 10-μm sections were cut from the hemisphere and two sections containing the lateral choroid plexus were used from each animal for immunohistochemistry. Paraffin on sections was removed by xylene and sections were rehydrated in decreasing ethanol concentrations. Sections were washed in PBS containing 0.1 % Tween 20, which was also used for all subsequent washes. Antigen presentation was enhanced by proteinase treatment (10 μg/ml; proteinase K, Roche Diagnostics, Germany) for 9 min. Blocking was performed by incubation in 3 % H₂O₂ in PBS for 10 min and serum-free protein block (Dako, Sweden) for 1 h. Rabbit anti-occludin (1:100; Invitrogen, Cat#71-1500) and anti ZO-1 (1:100, Invitrogen Cat#61-7300) were used as primary antibodies and incubated overnight (4 °C), and sections were subsequently incubated for 2 h with biotinylated secondary antibodies against the appropriate species (1:250; Vector Laboratories, USA) at room temperature. All sections were then incubated with avidin–peroxidase complex (ABC Kit PK-6100; Vector Laboratories, USA) for 1 h and developed with the DAB Kit (SK-4100, Vector Laboratories, USA). Control sections, with omission of primary antibodies, generated near to blank sections. Before coverslipping, sections were counterstained with hematoxylin (2 min).