Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: hM4Di and hM3Dq expression in TRPV1 + DRG neurons. A , Schematic representation of the transgenic TRPV1-hM4Di mouse design. B , HA-tagged hM4Di expression was confirmed by HA immunostaining in different subsets of neurons from T10-L1 and L6-S1 levels. Scale bars equal 50 μm. C , Dot plot showing the percentage of peptidergic (TRPV1 and CGRP) and non-peptidergic (IB4) neurons that co-express HA-tagged hM4Di in DRG from TRPV1-hM4Di mice (n = 3). D , Schematic representation of the transgenic TRPV1-hM3Dq mouse design. E , mCherry-tagged hM3Dq visualization in different subsets of neurons from T10-L1 and L6-S1 levels. Scale bars equal 50 μm. F , Dot plot showing the percentage of peptidergic (TRPV1 and CGRP) and non-peptidergic (GFRα2) neurons co-expressing hM3Dq-mCherry in DRG from TRPV1-hM3Dq mice (n = 3).

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Expressing, Transgenic Assay, Immunostaining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Effect of CNO on TRPV1 + DRG neuron excitability. A , Representative traces showing the effect of CNO (10 μM) on capsaicin-induced (100 nM) depolarization and AP firing in a DRG neurons from TRPV1-hM4Di or TRPV1-hM3Dq mice. Note the hyperpolarization and inhibition of AP firing in TRPV1-hM4Di, but the depolarization and increased AP firing in TRPV1-hM3Dq neurons. B , CNO exposure hyperpolarizes or depolarizes the resting membrane potential (RMP) of TRPV1-hM4Di and TRPV1-hM3Dq neurons, respectively (TRPV1-hM4Di: −56.6 ± 2.47 mV vs −64.5 ± 2.28 mV, n = 9; TRPV1-hM3Dq: −56.01 ± 0.7 mV vs −47.7 ± 0.99 mV, n = 14). Statistical analysis was performed using the paired t test (∗∗ P < .01). C , CNO decreases or increases the action potential threshold of capsaicin-responsive TRPV1-hM4Di and TRPV1-hM3Dq neurons, respectively (TRPV1-hM4Di: −32.6 ± 1.7 mV vs −24.7 ± 0.95 mV, n = 12; TRPV1-hM3Dq: −30.4 ± 1.7 mV vs −39.7 ± 2.0 mV, n = 11). Statistical analysis was performed using the paired t test (∗∗ P < .01). D , Representative traces of AP firing evoked by 200 pA of injected current (1 second pulse) in the absence and presence of CNO (10 μM) in TRPV1-hM4Di ( top ) and TRPV1-hM3Dq ( bottom ) neurons. E , The AP frequency evoked by increasing the amount of injected current was greater in TRPV1-hM3Dq but lower in TRPV1-hM4Di neurons (TRPV1-hM4Di: 12.7 ± 2.04 Hz vs 6.0 ± 1.32 Hz, n = 6; TRPV1-hM3Dq: 11.6 ± 2.5 Hz vs 23.8 ± 4.1 Hz, n = 7, elicited by 300 pA of current). Statistical analysis was performed using 2-way analysis of variance followed by the Bonferroni post hoc test (∗∗ P < .05 vs TRPV1-hM4Di; $$ P < .01 vs TRPV1-hM3Dq). Three independent experiments were performed.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Inhibition, Membrane, Injection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Inhibition of TRPV1 + neurons prevents VHS, neuronal and microglial activation in the spinal cord of colitis mice. A , Schematic representation of the experimental design. Colitis was induced by adding 2.5% DSS in the drinking water (water as control) of TRPV1-hM4Di mice while receiving an i.p. injection of CNO twice daily (1 mg/kg, DSS + CNO) or vehicle (DSS + Veh) for 7 days. B , Representative traces of CRD test showing the effect of chronic CNO on DSS-induced VHS in TRPV1-hM4Di mice. C , VMR to colorectal distension in DSS colitis mice after treatment with CNO or Veh (Water ( n = 4), DSS + Veh ( n = 7), or DSS + CNO ( n = 6)). Statistical analysis was performed using 2-way analysis of variance followed by Tukey post hoc test (∗ P < .05 vs water; $$ P < .01 vs DSS + CNO). D , Representative confocal images of c-Fos positive neurons immunostained in the thoracolumbar (T10–L1) and lumbosacral (L6–S1) spinal cord sections. Scale bar = 50 μm. E , Dot plot showing the average number of c-Fos positive neurons in the spinal dorsal horn of water ( n = 3), DSS + Veh ( n = 4–5), and DSS + CNO ( n = 3–5) treated mice. Statistical analysis was performed using 1-way analysis of variance followed by the Tukey post hoc test (∗∗ P < .01; ∗∗∗ P < .001). F , Representative confocal images of microglia immunoreactive to Iba-1 quantified in the T10–L1 and L6–S1 spinal cord sections. Scale bar = 50 μm. G , Dot plot showing the intensity of Iba-1 immunostaining in the spinal dorsal horn of water ( n = 3), DSS + Veh ( n = 5), and DSS + CNO ( n = 3–4) treated mice. Statistical analysis was performed using 1-way analysis of variance followed by the Tukey post hoc test (∗ P < .05). Three independent experiments were performed.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Inhibition, Activation Assay, Control, Injection, Immunostaining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Chemogenetic silencing of TRPV1 + neurons is not sufficient to reduce DSS-induced colitis. Body weight monitoring ( A ), representative images of colon ( B ), and macroscopic damage ( C ) of TRPV1-hM4Di water ( n = 4), DSS + Veh ( n = 8), and DSS + CNO ( n = 9) mice. TNF-α ( D ), IL-1β ( E ), and IL-6 ( F ) levels were determined by luminex technology in the colon of TRPV1-hM4Di water ( n = 3–5), DSS + Veh ( n = 7), and DSS + CNO ( n = 6–7) treated mice. Statistical analysis was performed using 2-way analysis of variance followed by the Tukey post hoc test ( A ), the Kruskal-Wallis test followed by the Dunn post hoc test (∗ P < .05; ∗∗ P < .01) ( B , D , E ), or 1-way analysis of variance followed by the Tukey post hoc test ( F ). Three independent experiments were performed.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Luminex

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: List of the Cytokines Assessed in Colonic Samples of TRPV1-hM4Di and hM4Di Littermate Control Mice

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Activation of TRPV1 + visceral afferents induces microglial activation and VHS. A , Schematic representation of the experimental design. TRPV1-hM3Dq mice received CNO (0.1 mg/kg) or vehicle, once daily, by gavage for 7 days. B , VMR to colorectal distension measured after treatment with Veh ( n = 10) or CNO ( n = 12). Statistical analysis was performed using 2-way analysis of variance followed by the Bonferroni post hoc test (∗ P < .05). C , Representative confocal images of c-Fos positive neurons immunostained in the T10–L1 and L6–S1 spinal cord sections. D , Dot plot showing the average number of c-Fos positive neurons in the spinal dorsal horn of Veh ( n = 4–5) and CNO ( n = 6–7) treated mice. Statistical analysis was performed using t test (∗ P < .05). E , Representative confocal images of microglia immunoreactive to Iba-1 in the T10–L1 and L6–S1 spinal cord sections. F , Dot plot showing the intensity of Iba-1 immunostaining in the spinal dorsal horn of Veh ( n = 5–7) and CNO ( n = 4–6) treated mice. Statistical analysis was performed using the Mann-Whitney or t test (∗∗ P < .01; ∗∗∗∗ P < .0001). Three independent experiments were performed.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Activation Assay, Immunostaining, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Chemogenetic activation induces neurogenic inflammation in naive mice. Body weight monitoring ( A ), representative images of colon ( B ), and macroscopic damage ( C ) of TRPV1-hM3Dq Veh ( n = 11) and CNO ( n = 12) treated mice. TNF-α ( D ), Il-1β ( E ), and IL-6 ( F ) levels were determined by luminex technology in the colon of TRPV1-hM3Dq Veh ( n = 5–6) and CNO ( n = 6–7) treated mice. Statistical analysis was performed using 2-way analysis of variance followed by the Bonferroni post hoc test (∗∗ P < .01), Mann-Whitney test, or t test (∗ P < .05; ∗∗ P < .01). Three independent experiments were performed.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Activation Assay, Luminex, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: List of the Cytokines Assessed in Colonic Samples of TRPV1-hM3Dq and hM3Dq Littermate Control Mice

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Activation of TRPV1 + neurons induces ATP release and P2RY12 upregulation in the spinal cord. A , ATP levels in supernatant of control (Veh, n = 5) or CNO-treated (10 μM, n = 5) TRPV1-hM3Dq neurons. Data are normalized to control treated neurons. Statistical analysis was performed using the t test (∗∗ P < .01). B , Purinergic receptor P2RY12 protein level was determined by Western blot in the T10–L1 and L6–S1 spinal cord sections of Veh ( n = 3–6) and CNO-treated ( n = 3–7) TRPV1-hM3Dq mice. C , Bar graph representing P2RY12 quantification in T10–L1 and L6–S1 spinal cord sections. Data are normalized to Veh mice. Statistical analysis was performed using the Mann-Whitney or t test (∗ P < 0.05; ∗∗∗ P < .001). D , Purinergic receptor P2RY12 protein level was determined by Western blot in the T10–L1 and L6–S1 spinal cord sections of control ( n = 8) and DSS ( n = 8–9) wild type mice. E , Bar graph representing P2RY12 quantification in T10–L1 and L6–S1 spinal cord sections. Data are normalized to control mice. Statistical analysis was performed using the t test (∗ P < .05; ∗∗∗ P < .001). Two independent experiments were performed.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Activation Assay, Control, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor

doi: 10.1016/j.jcmgh.2021.12.012

Figure Lengend Snippet: Primer Sequences

Article Snippet: Subsequently, sections were incubated overnight at 4°C with primary antibody mix in 1% BSA in 0.02 % Triton X-100 (rabbit anti-TRPV1, 1:250, Alomone, Cat. No. ACC-030; rabbit anti-CGRP, 1:1000, Sigma-Aldrich, Cat. No.PC205L; goat anti-GFRα2, 1:500, R&D Systems, Cat. No. AF429; anti-IB4-coupled Alexa 594, 1:1000, Invitrogen, Cat. No. I21412; anti-HA, 1:500, Biolegend, Cat. No. 901502).

Techniques: Sequencing, Mutagenesis

Journal: Scientific Reports

Article Title: Central expression of synaptophysin and synaptoporin in nociceptive afferent subtypes in the dorsal horn

doi: 10.1038/s41598-019-40967-y

Figure Lengend Snippet: Co-localization of synaptophysin and synaptoporin with IB4-binding C fibers in the thoracic dorsal horn. IB4 binding C fibers distributed in lamina II of T7 and T13 dorsal horns and expressed synaptophysin and/or synaptoporin as shown in a representative montage of confocal images from T7. ( A ) Areas of IB4 binding projections within the investigated region of interest were analyzed at both thoracic levels.( B ) Colocalization of IB4 binding axon terminals with synaptophysin or with synaptoporin was assessed by double labeling quantification ( C ) to find significantly different expression levels of synaptoporin and synaptophysin in this C fiber subpopulation (n = 4, p < 0.05, Kruskal-Wallis test with multiple comparison test, Cohen’s d > 1.3).

Article Snippet: The primary antibodies included goat anti-IB4 (1:400, Vector, Burlingame, CA), goat anti-CTB (1:2000, List Biological Laboratories, Campbell, CA), mouse anti-synaptophysin (1:1000, Synaptic Systems), rabbit anti-synaptoporin (1:300, Synaptic Systems), and Guinea pig anti-CGRP (1:2000, Peninsula Laboratories International, San Carlos, CA).

Techniques: Binding Assay, Labeling, Expressing

Journal: Scientific Reports

Article Title: Central expression of synaptophysin and synaptoporin in nociceptive afferent subtypes in the dorsal horn

doi: 10.1038/s41598-019-40967-y

Figure Lengend Snippet: Expression of synaptophysin and synaptoporin in dorsal cutaneous C fibers in the T7 and T13 spinal cord. Cutaneous C fibers were labeled with IB4 injections into DCNs. ( A ) DCN-specific C fibers projected laterally at the inner layer of laminae II overlaid by the synaptoporin distribution area as shown in representative montages of confocal images at T7. ( B ) Overlapping areas of DCN-specific IB4+ C fibers with synaptoporin and synaptophysin were quantified at every 200 μm across the sections centered at the dorsal horn entry zone (DREZ) at T7 and T13. ( C ) Bar graphs show averaged percent overlapping areas across those serial sections from 3 rats ( D ).

Article Snippet: The primary antibodies included goat anti-IB4 (1:400, Vector, Burlingame, CA), goat anti-CTB (1:2000, List Biological Laboratories, Campbell, CA), mouse anti-synaptophysin (1:1000, Synaptic Systems), rabbit anti-synaptoporin (1:300, Synaptic Systems), and Guinea pig anti-CGRP (1:2000, Peninsula Laboratories International, San Carlos, CA).

Techniques: Expressing, Labeling

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3β inhibition-independent pathways

doi: 10.1177/0271678X16647738

Figure Lengend Snippet: Lithium stimulates peri-infarct cell proliferation, neurogenesis, angiogenesis, and axonal plasticity. Mice were exposed to 45 min of intraluminal MCA occlusion, intraperitoneally treated with normal saline (control) or lithium (1 mg/kg bolus, followed by 2 mg/kg/day) starting at 6 h after reperfusion over up to 7 days and sacrificed after 56 days. (a) Assessment of postischemic cell proliferation by bromodeoxyuridine (BrdU) incorporation in the ischemic striatum, (b–d) differentiation analysis of BrdU+ cells in the ischemic striatum based on co-expression of neuronal markers Dcx (in b) and NeuN (in c) (indicative of new-born neurons, i.e. neurogenesis) or endothelial marker CD31 (in d) (indicative of new-born endothelium, i.e. angiogenesis), as well as (e) determination of axonal density in the peri-infarct cortex after contralesional injection of the anterograde tract tracer biotinylated dextran amine (BDA) (n = 12–13 per group). Data for axonal densities are given as percentage of proportional areas as indicated in the materials and methods section. *Significantly different from controls with p:0.029 (a), p:0.005 (b), p:0.013 (c), p:0.045 (d), and p:0.008 (e). Scale bars: 50 µm.

Article Snippet: For analysis of activated microglia, sections were labeled with biotinylated anti-Ib4 antibody (1:25, Vector, USA) that was detected by Alexa488-conjugated streptavidine (1:50, Invitrogen).

Techniques: BrdU Incorporation Assay, Expressing, Marker, Injection

Journal: Experimental neurology

Article Title: NEUROTROPHIN SELECTIVITY IN ORGANIZING TOPOGRAPHIC REGENERATION OF NOCICEPTIVE AFFERENTS

doi: 10.1016/j.expneurol.2015.06.007

Figure Lengend Snippet: Expression of neurotrophin in the DRG failed to promote regeneration of sensory afferents through the DREZ. Lentivirus encoding GFP, NGF or Artemin shows very good expression 20 days after direct injection into the DRG. Within the DRG numerous neurons, Schwann cells and axons could be observed labeled by GFP (A) and a generalize artemin staining surrounding neurons was identify using the Flag epitope antibody (B). Western blot analysis was also used to confirm expression of either artemin or NGF within DRGs (C). Although, we consistently observed good expression of GFP (D & G), NGF (E & H) or artemin (F & I), this expression within the DRG failed to support regeneration of either CGRP or IB4 sensory afferents into the spinal cord. Scale bars A) 100μm; B 50μm; D – I) 300μm

Article Snippet: Visualization was achieved by incubating sections in biotinylated secondary antibody (except for IB4 as it is already biotinylated) followed by vectastain Elite ABC reagents (Vector laboratories, Burlingame, CA), and developed using the peroxidase substrate, 3,3-diaminobenzidine to generate brown color.

Techniques: Expressing, Injection, Labeling, Staining, FLAG-tag, Western Blot

Journal: Experimental neurology

Article Title: NEUROTROPHIN SELECTIVITY IN ORGANIZING TOPOGRAPHIC REGENERATION OF NOCICEPTIVE AFFERENTS

doi: 10.1016/j.expneurol.2015.06.007

Figure Lengend Snippet: Artemin and GDNF enhanced regeneration of IB4+ axons while NGF had no effect on IB4 axons. Non-injured Sham controls show a normal distribution of IB4 axons within the inner region of laminae II (A). Dorsal root crush and injections of lenti-GFP resulted in a complete abolishment of IB4+ axons and absence of regeneration (B). NGF expression in the dorsal horn did not have any effect on IB4+ axon regeneration and looked similar to GFP lesion controls (C). Artemin produced modest regeneration of IB4+ axons just past the entry zone (arrows; D). GDNF induced robust regeneration of IB4+ axons (arrows) into the appropriate laminae (right side E). Quantification of IB4+ axon occupying area within laminae I – III (F). Compared to control and NGF, artemin and GDNF produced a statistically significant regeneration of IB4+ axons (*p<.05, Tukey’s post hoc test) and the effect of GDNF was significantly higher compared to all treatment groups after dorsal root injury (δ p<.05). Scale bar = 300 μm.

Article Snippet: Visualization was achieved by incubating sections in biotinylated secondary antibody (except for IB4 as it is already biotinylated) followed by vectastain Elite ABC reagents (Vector laboratories, Burlingame, CA), and developed using the peroxidase substrate, 3,3-diaminobenzidine to generate brown color.

Techniques: Expressing, Produced

Journal: Experimental neurology

Article Title: NEUROTROPHIN SELECTIVITY IN ORGANIZING TOPOGRAPHIC REGENERATION OF NOCICEPTIVE AFFERENTS

doi: 10.1016/j.expneurol.2015.06.007

Figure Lengend Snippet: Co-expression of artemin with NGF, GFRα3 or GDNF augmented the regeneration of IB4+ axons. NGF (A, a), GFRα3 (B, b) or GDNF (C, c) when co-expressed with artemin produced robust regeneration of IB4+ axons while, co-expression of GFRα1 with GDNF (D, d) did not increase IB4+ axon regeneration compared to GDNF alone. Magnified images of the boxed region from respective ipsilateral dorsal horn (a, b, c, & d). Scale bar = 300 μm A, B, C, & D. Scale bar = 100 μm for a, b, c, & d. E, Quantification of IB4+ axon occupying area in artemin+NGF, artemin+GFRα3, artemin+GDNF and GDNF+GFRα1 animals compared to artemin and GDNF from figure 3. Co-expression of GFRα3 or GDNF along with artemin produced a statistically significant increase in regeneration of IB4+ axons compared to artemin alone and artemin+NGF (one way ANOVA, Tukey’s post hoc test, *p<.05).

Article Snippet: Visualization was achieved by incubating sections in biotinylated secondary antibody (except for IB4 as it is already biotinylated) followed by vectastain Elite ABC reagents (Vector laboratories, Burlingame, CA), and developed using the peroxidase substrate, 3,3-diaminobenzidine to generate brown color.

Techniques: Expressing, Produced