Page 85 - Read Online

Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 7 of 32

vigorously for 15 s, incubated for 4 min at RT, hand-shaken vigorously for 10 s, and then centrifuged in 4
°C at 12,000 × g for 15 min. A portion, 300 µL, of the aqueous layer was extracted and placed into a clean
RNase/DNase/Protease free 1.5 mL tube and 1.5 × aqueous layer (450 µL) of 200 proof EtOH (Sigma-Aldrich;
Cat#E7023) was added to tube, pipetted 4-6 × to mix, moved to collection columns, and centrifuged in
~20 °C at 9,000 × g for 30 s. This was followed by a wash of 700 µL of RWT (provided with Qiagen kit)
and centrifuged (~20 °C at 9,000 × g, 30 s), washed 2 × with 500 µL RPE (provided with Qiagen kit) and
centrifuged (~20 °C at 9,000 × g, 30 s) after each, and washed 2 × with 500 µL 80% EtOH (100% EtOH diluted
with sterile RNase/DNase/Protease free water; Sigma-Aldrich; Cat#W4502), and centrifuged (~20 °C at 9,000 × g,
2 min) after each. Caps were cut from columns and samples were dried by centrifugation (~20 °C at 20,627 ×
g, 12 min), and placed into RNA collection tubes with 14 µL sterile water (provided with Qiagen kit) added
directly to the column filter, and centrifuged (~20 °C at 20,627 × g, 1 min). The concentration and quality of
the total RNA was assessed by NanoDrop (Thermo Scientific, MA, USA).

mRNA Analysis by Quantitative Real-Time PCR
Total RNA samples were diluted to a standardized RNA concentration: 90 ng/μL for SCN, 70 ng/μL for
lumbar dorsal horn, and 100 ng/μL for DRG. Total RNA (0.9-1.2 μg) was used to synthesize cDNA. For
TM
TM
reverse transcription (cDNA), SuperScript IV VILO cDNA Synthesis Kit (Invitrogen) was used per
manufacturer’s instructions. Levels of mRNA transcripts were measured and analyzed, as previously
described [47,55] . The following dilution factors (indicated in parentheses) were applied to cDNA samples for
assessment of transcripts of interest in given tissues: ipsilateral and contralateral LSC (1:2.2), ipsilateral
SCN (1:2.5), and ipsilateral DRG (1:3). The 1:200 dilutions of cDNA were used for assessment of the
normalizer transcripts (18s RNA) for each of the tissue samples. Levels of mRNAs as well as 18s rRNA
(Rn18s) were assayed in triplicate via quantitative real-time PCR (qRT-PCR) with Taqman Gene Expression
Assays (cat# 4351370, ThermoFisher Scientific). In cases of triplicates with standard deviation of more than
0.1, the average value of the two closest replicates were included. All selected gene expression assays were
identified by the manufacturer to be the “best coverage” assays, unless otherwise noted, and designed to
exclude detection of genomic DNA. mRNA levels were analyzed with the formula: C = 2 CT Normalizer /2 CT Target , as
previously described [55,56] .

To test whether BIRT377 treatment influenced the inflammatory milieu in collected tissues, the following
pain-relevant proinflammatory and anti-inflammatory factors were assessed: C-C motif chemokine ligand
2 (CCL2, Ccl2), interleukin-1β (IL-1β, il1b), (TNF, TNFα, Tnf), interleukin-10 (IL-10, Il-10), TGF-β1, Tgfb1.
Monocyte and T cell-specific cytokines and cellular markers were analyzed: integrin alpha M (CD11b,
Itgam, a common monocyte/macrophage marker), cluster of differentiation 3 (CD3; expressed on all T
cells), forkhead box P3 (FOXP3, Foxp3) which is expressed by Treg cells, and Interleukin-17a (IL-17A, Il-17a,
expressed by proinflammatory Th17 cells) [57-60] . To assess whether BIRT377 treatment may lead to allodynia
reversal by modulating glial activation in the LSC, the transcript levels of the microglial specific marker,
transmembrane protein 119 (TMEM119, Tmem119) [47,61] , and the astrocyte activation marker, glial fibrillary
acidic protein (GFAP) were evaluated. All 48 samples (96 for ipsilateral and contralateral LSC) and a “no
template control” sample for each tissue type was processed for the cDNA preparation or real-time PCRs
simultaneously.

Multiplex determination of splenic cytokine and chemokine expression
Frozen spleens were homogenized for 60 s in a buffer with protease inhibitors (MesoScale Discovery)
while kept on ice, and subsequently sonicated (settings: 5 pulses, at 50%, Fisher Scientific). Tissue samples
were then centrifuged at 4200 × g at 4 °C for 10 min to pellet cellular debris. Cellular lysates (supernatant)
TM
were collected in a new set of tubes, and protein concentrations were measured by Quickstart Bradford
Protein Assay Kit (Biorad, CA, USA). Splenic cytokine and chemokine levels were determined using
TM
V-Plex multiplex immunoassays (MesoScale Discovery), as described previously [47,51,62,63] . Briefly,