Journal: Brain

Article Title: Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

doi: 10.1093/brain/awab408

Figure Lengend Snippet: NMDARs at female SDH synapses are not potentiated or upregulated by the ex vivo BDNF or in vivo CFA models of pathological pain. ( A ) Baseline lamina I mEPSCs do not differ between male and female adult SD rats. Left : Saline - treated male vs female lamina I neurons, created using BioRender.com. Middle : Average mEPSCs at +60 mV in lamina I neurons from male (green) and female (purple) adult rats. Right : Peak amplitude, decay constant and charge transfer of the NMDAR component of mEPSCs at +60 mV do not differ between male and female SD rats. n = 10 for males and n = 9 for females. Compared using independent samples t -test. ( B ) Male lamina I NMDAR mEPSCs are potentiated following CFA hindpaw injection and ex vivo BDNF treatment. Left : Experimental paradigm showing male in vivo CFA versus ex vivo BDNF models, created using BioRender.com. Middle : NMDAR mEPSCs from male rat lamina I neurons; control in black, CFA in green, BDNF in blue. Right : Charge transfer of NMDAR mEPSCs for groups shown to left . n = 10 for control, n = 8 for CFA and n = 6 for BDNF. Compared using Welch’s test followed by Games–Howell comparison. ( C ) Female lamina I NMDAR mEPSCs are not potentiated following CFA hindpaw injection or ex vivo BDNF treatment. Left : Experimental paradigm showing female in vivo CFA versus ex vivo BDNF models, created using BioRender.com. Middle : NMDAR mEPSCs from female rat lamina I neurons; control in black, CFA in purple, BDNF in blue. Right : Charge transfer of NMDAR mEPSCs shown to left. n = 10 for control, n = 8 for CFA and n = 8 for BDNF. Compared using the Kruskal–Wallis one-way ANOVA. ( D ) Ex vivo BDNF treatment model elicits no effect in female rat SDH synaptosomes. Plots ( left ) and representative western blots ( right ) from female rat SDH synaptosomes of tissue treated with either control saline (lilac, n = 8) or 50 ng/ml recombinant BDNF for 70 min (purple, n = 8; compared using paired-samples t -test). * P < 0.05.

Article Snippet: After tissue preparation, rat or human spinal tissue was placed in oxygenated, room temperature saline containing 50–100 ng/ml recombinant BDNF (Alomone Labs) or control saline for 70 min. A subset of female rat experiments was carried out using an incubation time of 2–4.5 h. Treatment of spinal tissue with BDNF and the kinase inhibitor PP2 (Calbiochem) was carried out using the same approach.

Techniques: Ex Vivo, In Vivo, Injection, Western Blot, Recombinant

Journal: Brain

Article Title: Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

doi: 10.1093/brain/awab408

Figure Lengend Snippet: In contrast to males, ex vivo BDNF treatment does not activate markers of disinhibition or facilitated excitation at SDH synapses of female human spinal tissue. ( A ) The ex vivo BDNF model elicits KCC2 internalization in adult male human SDH neurons. Left : Experimental paradigm showing treatment of human SDH tissue in either saline or BDNF, created using BioRender.com. Top right : Average KCC2 intensity values from SDH neurons of male human donor tissue ( n = 12) incubated in saline (light green) versus BDNF (dark green). Bottom right : Comparisons of membrane and intracellular regions by the extra sum-of-squares F -test method; the membrane component is fitted to a Gaussian distribution, while the intracellular component is fitted to an exponential decay. ( B ) The ex vivo BDNF model has no effect on KCC2 internalization in adult female human SDH neurons. Left : Experimental paradigm showing treatment of human SDH tissue in either saline or BDNF, created using BioRender.com. Top right : Average KCC2 intensity values from SDH neurons of saline (lilac) versus BDNF-treated (purple) spinal segments of 10 female human donors. Bottom right: Comparisons of membrane and intracellular regions by the extra sum-of-squares F -test method; the membrane component is fitted to a Gaussian distribution, while the intracellular component is fitted to an exponential decay. ( C and D ) Representative confocal images of male ( C ) and female ( D ) human superficial dorsal horn incubated in saline or BDNF. KCC2 (red), CGRP (green) and NeuN (blue). A zoomed region ( top right ) shows a neuron expressing KCC2 together with the delineation of the membrane and the distance to the membrane of each pixel analysed in a colour-coded distance map. KCC2 intensity (i.u.) versus distance to the membrane profile ( bottom ). Scale bar = 50 µm; inset = 10 µm. ( E ) The ex vivo BDNF treatment model elicits no effect in human female SDH synaptosomes. Plots ( left ) and representative western blots ( right ) from SDH synaptosomes of human female spinal cord treated with either control saline (lilac, n = 6) or 100 ng/ml recombinant BDNF for 70 min (purple, n = 6). Comparisons were made using paired samples t -tests in all cases except for aSTEP 61 , where the Wilcoxon signed-rank test was used. * P < 0.05.

Article Snippet: After tissue preparation, rat or human spinal tissue was placed in oxygenated, room temperature saline containing 50–100 ng/ml recombinant BDNF (Alomone Labs) or control saline for 70 min. A subset of female rat experiments was carried out using an incubation time of 2–4.5 h. Treatment of spinal tissue with BDNF and the kinase inhibitor PP2 (Calbiochem) was carried out using the same approach.

Techniques: Ex Vivo, Incubation, Expressing, Western Blot, Recombinant

Journal: Brain

Article Title: Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

doi: 10.1093/brain/awab408

Figure Lengend Snippet: Ovariectomy triggers BDNF-mediated NMDAR potentiation by the KCC2/STEP 61 /SFK pathway in SDH neurons of female rats. ( A ) Ovariectomy (OVX) has no effect on baseline lamina I NMDAR mEPSCs of female SD rats. Left : Saline-treated naïve female versusOVX female lamina I neurons, created using BioRender.com. Middle : Average mEPSCs at +60 mV in lamina I neurons of naïve (purple) and ovariectomized (lilac) female rats. Right : Peak amplitude (compared using Mann–Whitney test), decay constant (compared using independent samples t -test) and charge transfer (compared using independent samples t -test) of the NMDAR component of mEPSCs do not differ between naïve ( n = 10) and OVX ( n = 8) female rats. ( B ) In OVX rats, NMDAR mEPSCs in lamina I neurons are potentiated following ex vivo BDNF treatment. This potentiation is blocked using co-treatment with the SFK inhibitor, PP2. Left : Recordings from lamina I neurons were compared for saline- versus BDNF-treated slices from OVX rats, created using BioRender.com. Middle: Average mEPSCs at +60 mV from OVX female rat lamina I neurons; control in black, BDNF in purple, BDNF+PP2 in blue. Right : Charge transfer of NMDAR mEPSCs shown on left . n = 8 for control, n = 9 for BDNF and BDNF + PP2. Comparisons were made using one-way ANOVA, followed by Tukey HSD when P < 0.05. ( C ) Ex vivo BDNF treatment in OVX rat SDH synaptosomes results in upregulation of pY 420 Fyn and downregulation of KCC2 and STEP 61 . Plots ( left ) and representative western blots ( right ) from OVX female rat SDH synaptosomes of tissue treated with either control saline (lilac, n = 8) or 50 ng/ml recombinant BDNF for 70 min (purple, n = 8, compared using paired samples t -tests). * P < 0.05.

Article Snippet: After tissue preparation, rat or human spinal tissue was placed in oxygenated, room temperature saline containing 50–100 ng/ml recombinant BDNF (Alomone Labs) or control saline for 70 min. A subset of female rat experiments was carried out using an incubation time of 2–4.5 h. Treatment of spinal tissue with BDNF and the kinase inhibitor PP2 (Calbiochem) was carried out using the same approach.

Techniques: MANN-WHITNEY, Ex Vivo, Western Blot, Recombinant

Journal: The Journal of Neuroscience

Article Title: Matrix Metalloproteinase-9 Contributes to Kindled Seizure Development in Pentylenetetrazole-Treated Mice by Converting Pro-BDNF to Mature BDNF in the Hippocampus

doi: 10.1523/JNEUROSCI.3118-11.2011

Figure Lengend Snippet: Effects of TrkB-Fc and pro-BDNF on PTZ-induced kindling in wild-type and MMP-9(−/−) mice. A, B, Mice received microinjections of TrkB-Fc or IgG1-Fc into the right ventricle 30 min before each PTZ treatment (25 mg/kg). Values are means ± SE (n = 10 for A; n = 8 for B). *p < 0.05 versus IgG1-Fc-injected wild-type mice. C, D, Mice received microinjections of pro-BDNF or PBS bilaterally into the hippocampal DG 30 min before each PTZ treatment (25 mg/kg). Values are means ± SE (n = 8–10 for A; n = 7 for B). *p < 0.05 versus PBS-injected wild-type mice.

Article Snippet: TrkB-Fc (R&D Systems) or IgG 1 -Fc (R&D Systems) was microinjected at a dose of 50 ng/μl and a rate of 1 μl/min (final volume, 2 μl) according to previous reports ( Yajima et al., 2005 ; Zhou et al., 2010 ) with minor modifications. pro-BDNF (50 ng/μl; #B-240, Alomone Labs) or the vehicle PBS was bilaterally microinjected at a rate of 1 μl/min (final volume, 1 μl) according to the previous studies ( Shirayama et al., 2002 ; Yajima et al., 2005 ) with minor modifications.

Techniques: Injection

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p < 0.05, **p < 0.01 versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( B ) H&E staining (a and d) and immunohistochemsitry (b,c and e–g) of proBDNF in the foot skin at 3 h post-formalin injection. proBDNF is expressed in the epidermis, basal layer and subcutaneous layers in the foot skin (b,c); Higher magnification (box in b) showing proBDNF is also mildly expressed in the nerve fibers in the control plantar (c); Responding to peripheral inflammation by 5% formalin intra-plantar injection, intensive proBDNF immunoreactivity is observed and mainly localized in the inflammatory cells (f, black arrows) and nerve fiber-like structures (g). Scale bars: 50 μm, 3 replicates, n = 3 per group. ( C ) a, Representative Western blot of proBDNF and mBDNF; b–d, Semi-quantitative analyses of mBDNF, proBDNF and their ratio in the inflamed tissue after Complete Freund Adjuvant (CFA, 10 μL) intra-plantar injection into Kunming mice (*p < 0.05, **p < 0.01versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( D ) Histological staining (a) and proBDNF immunohistochemistry (b,c) in the plantar at 1 day post-CFA injection; c, higher magnification of box in b showing that proBDNF is highly expressed in the inflammatory cells. Scale bar, 100 μm, 3 replicates, n = 3 per group. Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Western Blot, Injection, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P < 0.05, **p < 0.01 versus baseline, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 10–12 per group). ( B ) Ectopic overexpression of proBDNF by intra-plantar injection of Ad-proBDNF or Ad-EGFP reduces PWT dramatically in Kunming mice. (a) Representative proBDNF Western blot (upper panel) and its semi-quantitative analysis (lower panel, *P < 0.05, ***p < 0.001 versus control, ### p < 0.001 versus indicated groups, one-way ANOVA followed by Tukey’s Multiple Comparison post hoc test, n = 4 per group); (b) Representative fluorescent images after delivery of Ad-EGFP . Scale bar, 50 μm, 3 replicates, n = 3 per group; (c) PWT at 7 days post-injection of Ad-proBDNF or Ad-EGFP control (***p < 0.001 versus Ad-EGFP , student’s t test, n = 7 per group). ( C ) Co-injection of proBDNF (0.1 μg), but not mBDNF (0.1 μg) restored the biphasic nociceptive response after low-concentration of formalin (0.5%) intra-plantar injection (*p < 0.05versus vehicle, two-way ANOVA followed by Bonferroni’s Multiple Comparison post hoc test, n = 10–12 per group). ( D ) Exogenous proBDNF (1 μg) intra-plantar injection induces ERK activation in the ipsilateral spinal cord dorsal horn at 3 h post-injection, Scale bar, 50 μm, n = 3 per group, 3 replicates. ( E ) Spinal p-ERK expression at 3 h after proBDNF (1 μg) intra-plantar injection. (a) Representative Western blot and (b,c) their semi-quantitative analyses of p-ERK, (*p < 0.05, **p < 0.01 versus proBDNF-R, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Injection, Over Expression, Western Blot, Concentration Assay, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P < 0.001 versus control, # p < 0.05 versus indicated group, one-way ANOVA followed by Tukey’s Multiple Comparison post hoc test). Neurospheres treated with proBDNF (100 ng/mL) showed dormancy without any neuronal migration and neurospheres had no morphological changes. Neurospheres treated with 2B11 (100 ng/ml) showed strong migration capability comparing with other groups. Neurospheres treated with 2B11 and proBDNF (100 ng/ml) showed similar ability of migration with sheep anti-proBDNF antibody treatment group.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Migration