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, Direct NK cell-mediated lysis of syngenic dorsal root ganglia neurons in vitro, J. Immunol, vol.165, pp.4895-4900
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, Equal amounts of RNA (150-250 ng from whole DRG, 500ng from cell cultures) was reversed-transcribed using M-MLV (200 U/rxn), dNTPs and oligo(dT) 12-18 primers (Invitrogen) in a 20 ml reaction volume according to the manufacturer's instructions. PCR reactions (25 ml) using Go Taq Flexi DNA polymerase (Promega) were then performed from cDNA on a thermal cycler, MJ Mini, BioRad) with the following primer pairs: Raet1a-e universal primers, 2006.
, Life Technologies) and expression was determined relative to a reference gene (Gapdh) in adult DRG for cultures or contralateral DRG for nerve injury experiments using the comparative C t method. Primers were designed using Primer-BLAST software (NIH). Primers were selected based on specificity to the desired target gene upon BLAST search, overlap of the exon-exon boundary, lack of potential hairpin-forming or self-priming regions, single peak in the dissociation curve (single band PCR product) and equivalent amplification efficiency (linear shift in C t value upon serial dilution of cDNA). Products of reverse transcription reactions omitting RNA or M-MLV (-RT) served as negative controls, MicroAmp optical tubes (20ml reaction volume) on a 7500 Real-Time PCR system (Applied Biosystems). The PCR conditions were 50 C (2 min), 95 C (10 min), and
, (forward*) 5 0 -TCCATGACAACTTTGGCATTG-3 0 , (reverse*) 5 0 -CAGTCTTCTGGGTGGCAGTGA-3 0 . Asterisk (*) indicates primers which cross an exon-exon boundary, Gapdh (72bp)
, For total neurite fragmentation gamma high channel images of b-tubulin III immunofluorescence were exported as an unaltered TIFF. In ImageJ images were scale calibrated (1.6 pixels/mm) and brightness threshold set in b/w mode, creating a whole neuron silhouette for selection. Neurite fragments were selected using the Analyze Particles function (size 0.5-25 mm 2 , circularity 0-1) and saved as a drawing. The total area and particular area values (mm 2 ) were obtained using the Measure function. Total DRG cell body area was measured separately and subtracted from the total area to calculate the percent specific neurite fragmentation for each field of view. For analysis of b-tubulin III and STMN2 labeling in sciatic nerve tissue, a composite of the full length nerve was created from individual z section images acquired at x10 magnification and 0.5 zoom along the length of the nerve (LSM700, Zeiss). Images containing crush site, as well as proximal and distal regions (±1-2 mm from the crush site) were exported to ImageJ, scale calibrated (0.8 pixels/mm) and brightness threshold set in b/w mode. The mean pixel density from a 9000 mm 2 selection of the nerve mid-image was recorded from 3-4 images per nerve region, per mouse, per treatment. Co-localized regions of RAE1 double-labeling with b-tubulin III and STMN2 were exported using the co-localization function on the Zen 2012 software after setting for threshold image intensity in both fluorescence channels (1000 units per channel). For in situ hybridization, 3x 2 mm z sections were acquired using x20 apochromat lens at 1x digital zoom. A tile scan of 1024x1024 pixels, 12 bit images was performed with 5% overlap. Image re-stitching was performed offline. Acquisition settings were set to maximize intensity of positive control probe signal while maintaining negligible background on sections, QUANTIFICATION AND STATISTICAL ANALYSIS Laser scanning confocal imaging and analysis For analysis of neurite density in microfluidic co-cultures, single z section low magnification (x10) confocal images at 0.5 zoom (LSM700, Zeiss) were acquired of b-tubulin III immunofluorescence (647nm emission) along the full length of the neurite compartment. Gamma high channel images were exported as an unaltered TIFF using Zeiss imaging software (v8.1, ZEN 2012 SP1, Zeiss)
, Flow cytometry plot of splenic lymphocytes. NKp46+DX5+ NK cells (top-right quadrant) in whole spleen homogenate (left) and after MACS enrichment by negative selection (right). NK cells require cell contact to mediate toxicity against embryonic DRG neurons
, Representative immunostaining images of embryonic DRG neurons (b-tubulin III, magenta) after 4 h co-culture with IL-2 stimulated NK cells
, ELISA detection of granzyme B in media of NK cell-DRG co-cultures. One way ANOVA, F(2,6) = 0.7248, p = 0.5225. n = 3 experimental repeats. Granzyme B was not detected in cultures containing unstimulated NK cells
, Cytotoxicity of IL-2 stimulated NK cells against embryonic DRG neurons after 4 h co-culture in the presence of blocking anti-NKG2D antibody or IgG control antibody
, days in vitro) co-cultured (4 h) with IL-2 stimulated NK cells (2.5x10 5 cells per coverslip) pre-treated with either anti-NKG2D or IgG isotype control antibody. (B) Quantification of DRG neurite fragmentation after NKG2D receptor blockade
, 24 h in culture) and exposed to IL-2 stimulated NK cells in the presence of anti-NKG2D (CX5) blocking antibody or IgG1k isotype control (30 mg/ml) for 4 h. Images representative of b tubulin III labeling of fixed cultures in each condition. (D) Quantification of DRG neurite fragmentation in acute (< 24 h) cultures of L5x injured DRG neuron, L5 DRG neurons were cultured 7 days after L5x injury
, E) Quantification of total DRG cell body area in acute (< 24 h) cultures of sham and L5x injured DRG neuron co-cultured with or without control or IL-2 stimulated NK cells (4 h, One-way ANOVA: Sham, F, p.61
, Flow cytometry of peripheral blood lymphocytes from NKp46-YFP mice labeled with anti-NKp46 and anti-CD3 antibodies
, Immunolabeling with an anti-GFP antibody (green) in spleen tissue sections from wild-type and NKp46-YFP mice. Note enhancement of signal only in labeled NKp46-YFP mouse spleen tissue
, Flow cytometry of peripheral blood lymphocytes from NKp46-DTR or wild-type (WT) mice 24 h after intravenous treatment with DTx (100 ng) or PBS vehicle (100 ml), Cells labeled with fluorescent-conjugated anti-NKp46 and anti-CD3 antibodies
, Sensitivity to mechanical (von Frey filament) stimulation in the injured ipsilateral hind paw before (naive) and after injections (baseline) and at intervals after L5x injury. Ipsilateral: Two-way ANOVA (effect of time) F(6,140) = 62, vol.24
, A) Sciatic nerve tissue sections from adult male NKp46-YFP mice 7 days after sciatic nerve crush injury. Inset shows higher magnification of ipsilateral nerve. Arrows indicate co-localization of NKp46-YFP (anti-GFP, green) and nuclear (DAPI, blue) labeling. (B) Flow cytometry scatterplots of sciatic nerve homogenates obtained from an NKp46-YFP mouse 3 days after sciatic nerve crush injury. (C) Quantification of total number of CD45+/YFP+ double-positive events within lymphocyte FSC/SSC gate from whole sciatic nerve homogenates. Two-way ANOVA
, ELISA quantification of granzyme B content in whole sciatic nerve 7 days after crush injury in wild-type mice
, Student's unpaired t test: t = 8.012, p < 0.0001. (F) NKp46 immunolabeling and nuclear (DAPI) staining in sciatic nerve sections (14 mm) 7 days after forceps crush injury in NKp46-DTR mice treated with DTx or PBS vehicle, NKp46+/DX5+ double-positive lymphocytes in peripheral blood 16 days after sciatic nerve crush
, G) Quantification of NKp46+DAPI+ cells (white arrows) within a 0.4 mm 2 region of the nerve crush site were counted by an investigator blind to the treatment
, ELISA quantification of granzyme B content in whole sciatic nerve 7 days after crush injury in NKp46-DTR mice after treatment with PBS or DTx
, Daily pinprick response score in NKp46-DTR mice treated intravenously with DTx or PBS vehicle every 4 to 5 days. Full sciatic nerve crush on day 0. Two-way ANOVA: Effect of depletion, F(1,352) = 25.20, p < 0.0001). n = 12 mice per group
, Heatmap showing mean sensitivity to pinprick along the lateral hind paw. (K) Peripheral blood sampled 16 days post-injury shows almost a complete loss of NKp46+DX5+ NK cells in DTx-treated NKp46-DTR mice compared to wildtype
, 4030) and CD3+CD4+ (t = 1.971, p = 0.0662) T cell populations were not different between genotypes after DTx treatment, vol.8590, p.0
, wild-type and 10 NKp46-DTR mice received partial crush of the sciatic nerve on day 0 as well as diphtheria toxin (DTx) i.v. starting one day before surgery and continuing every 4 to 5 days. All mice were treated with IL-2/anti-IL-2 antibody complex i
, Daily pinprick response shows a transient reduction in sensitivity in wild-type mice receiving the IL-2/anti-IL-2 antibody complex and DTx treatment but not NKp46-DTR mice. Two-way ANOVA. Effect of genotype: F(1,275) = 12.49, p = 0.0005). Bonferroni post-test
, Heatmap showing mean sensitivity to pinprick along the lateral hind paw