Journal: The Journal of Neuroscience

Article Title: Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

doi: 10.1523/JNEUROSCI.2856-15.2016

Figure Lengend Snippet: P2Y1-targeted siRNA injections into the median and ulnar nerves selectively knock down ischemia-induced protein overexpression in the DRGs. A, Ischemia (BAO or PenCON+BAO) induces an increase in the expression levels of P2Y1 compared with Naive mice and this is prevented by injections of P2Y1-targeted siRNAs (PenY1+BAO) into the ulnar and median nerves 2 d before the ischemic injury. B, Representative Western blot for all conditions assessed showing inhibition of P2Y1 upregulation during BAO or PenCON+BAO by P2Y1-targeting siRNA injections. One-way ANOVA/Tukey's post hoc, *p < 0.01 versus Naive and PenY1+BAO.

Article Snippet: Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al. (2004 ) and Jankowski et al. (2006 , 2010 , 2012a , b ).

Techniques: Over Expression, Expressing, Western Blot, Inhibition

Journal: The Journal of Neuroscience

Article Title: Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

doi: 10.1523/JNEUROSCI.2856-15.2016

Figure Lengend Snippet: Ischemic insult increases spontaneous and evoked pain-related behaviors, reduces grip strength, and increases mean arterial blood pressure after exercise. A, One day after BAO or PenCON injection with BAO, paw-guarding scores are significantly increased compared with Naive mice; however, selective knockdown of P2Y1 (PenY1+BAO) partially prevents the observed injury-induced changes in guarding. B, Mechanical withdrawal thresholds are also found to be reduced after ischemic injury, but P2Y1 knockdown also partially prevents the development of mechanical hypersensitivity. C, Grip strength was significantly reduced compared with baseline in the BAO and PenCON+BAO groups, but not in the Naive or P2Y1-knockdown mice. D, The BAO and PenCON+BAO groups also showed a significant increase in systemic MBP specifically when low-intensity exercise was combined with the ischemic injury, but this was also prevented by afferent-specific inhibition of P2Y1. Two-way repeated-measures ANOVA with Bonferroni's post hoc, *p < 0.05 vs 1 d BAO, **p < 0.01 vs baseline and 1 d Naive.

Article Snippet: Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al. (2004 ) and Jankowski et al. (2006 , 2010 , 2012a , b ).

Techniques: Injection, Inhibition

Journal: The Journal of Neuroscience

Article Title: Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

doi: 10.1523/JNEUROSCI.2856-15.2016

Figure Lengend Snippet: Inhibition of P2Y1 during BAO prevents the injury-induced alterations in chemosensitive muscle afferent prevalence. A, A decrease in the percentage of cells responsive to low metabolite concentrations is observed after ischemic injury (BAO or PenCON+BAO) compared with Naive mice, but this is not observed in the selective P2Y1-knockdown group. B, There are no significant alterations in the numbers of group III/IV muscle afferents that respond to high concentrations of metabolites in any condition. C, The observed increase in afferents that respond to both high and low concentrations of metabolites in the BAO and PenCON+BAO groups is also prevented by selective knockdown of P2Y1. χ-Squared analysis, *p < 0.05, **p < 0.01, ***p < 0.001 versus Naive and PenY1+BAO.

Article Snippet: Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al. (2004 ) and Jankowski et al. (2006 , 2010 , 2012a , b ).

Techniques: Inhibition

Journal: The Journal of Neuroscience

Article Title: Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

doi: 10.1523/JNEUROSCI.2856-15.2016

Figure Lengend Snippet: Knockdown of P2Y1 during BAO blocks the increased firing in response heat stimuli in muscle afferents. A, Increased mean peak instantaneous frequencies in response to heat stimulation of the muscles is observed in mice with BAO and mice with PenCON+BAO compared with Naives, but this is not observed in the PenY1+BAO group. B, Examples of responses to heat stimulation of the muscles during ex vivo recording in Naive, BAO, and PenY1+BAO mice. Kruskall–Wallis one-way ANOVA on ranks with Dunn's post hoc test, *p < 0.05 vs Naive and PenY1.

Article Snippet: Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al. (2004 ) and Jankowski et al. (2006 , 2010 , 2012a , b ).

Techniques: Ex Vivo

Journal: The Journal of Neuroscience

Article Title: Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

doi: 10.1523/JNEUROSCI.2856-15.2016

Figure Lengend Snippet: Immunostaining of different intracellularly filled and functionally characterized muscle afferents from ex vivo recording in Naive, BAO Control, and PenY1+BAO conditions. A–D, Example of a characterized muscle afferent from a Naive mouse that responded only to low metabolites. This cell was negative for ASIC3 and P2X3. E–H, Another cell from a Naive mouse that responded to mechanical and heat stimuli in addition to high metabolites was positive for ASIC3 but not P2X3. I–L, A cell from a mouse with BAO that responded not only to mechanical stimulation but also to low and high mixtures of metabolites was positive for both ASIC3 and P2X3. M–T, A cell from a P2Y1-knockdown animal after ischemic injury that responded to mechanical stimulation and also to the high mixture of metabolites was found to be positive for ASIC3 and P2X3 (M–P), while another cell from this condition that only responded to the low-metabolite mixture was also immunopositive for ASIC3 but not P2X3 (Q–T). U, V, Positive (U) and negative (V) immunohistochemistry controls show specific binding of ASIC3 antibody.

Article Snippet: Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al. (2004 ) and Jankowski et al. (2006 , 2010 , 2012a , b ).

Techniques: Immunostaining, Ex Vivo, Immunohistochemistry, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

doi: 10.1523/JNEUROSCI.2856-15.2016

Figure Lengend Snippet: Selective knockdown of P2Y1 in muscle afferents does not alter the de novo expression of P2X3 or ASIC3 in DRGs after BAO but does reduce the membrane expression of ASIC3. A–C, The average number of cells per section that were positive for P2X3 (A) and ASIC3 (B) and that coexpressed ASIC3 and P2X3 (C) was significantly increased during ischemia (BAO and PenCON+BAO) compared with Naive, but this was not reversed by the P2Y1 knockdown (PenY1+BAO). Kruskal–Wallis with Dunn's post hoc; *p < 0.02 vs Naive. D, Examples of immunostaining in DRGs from Naive, BAO, and PenY1+BAO conditions. E, The membrane protein fraction isolated from DRGs of ischemic mice (BAO and PenCON+BAO) shows increased expression of ASIC3 versus Naive mice; inhibition of P2Y1 partially blocked this increase in membrane-bound ASIC3 in DRGs during BAO (one-way ANOVA with Holm–Sidak post hoc; **p < 0.02 vs Naive and PenY1+BAO; *p < 0.03 vs Naive and p = 0.069 vs PenY1+BAO). F, Examples of DRG membrane fraction Western blots for ASIC3 in Naive, BAO, PenCON+BAO, or PenY1+BAO mice.

Article Snippet: Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al. (2004 ) and Jankowski et al. (2006 , 2010 , 2012a , b ).

Techniques: Expressing, Immunostaining, Isolation, Inhibition, Western Blot