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    Alomone Labs chelerytrine
    PKC activity modulation in axonal loss. ( A ) shows the percentage of singly-, doubly-, and triply- (or more) innervated NMJs in the control mice (PBS) and after four applications of one of the following substances: the PKC paninhibitors <t>Chelerytrine</t> (CHE) and CaC, and the PKC panstimulators (BRY, at 1 and 10 nM) and PMA. CHE and CaC action results in the persistence of many polyinnervated synapses. Accordingly, both PKC stimulators PMA and BRY increase the number of monoinnervated junctions and clearly decrease the percentage of doubly-innervated junctions in the case of BRY. ( B ) shows the percentage of singly-, doubly-, and triply (or more) innervated NMJs after exposure to the cPKCβI and nPKCε isoform selective inhibitors βIV 5–3 and εV 1–2 , and the cPKCβI and nPKCε selective activators dPPA and FR 236,924 respectively. The selective inhibitors similarly increase the doubly- and triply-innervated synapses with the corresponding reduction in the monoinnervated junctions. The activators considerably accelerate nerve terminal elimination. Data were presented as percentages of NMJ ± SD (for each treatment and PBS control: n pups = 6–9; n = 11–18 LALs; n NMJ: PBS: 2538; Bry 1 nM: 1232 Bry 10 nM:1384; PMA:1247; CaC: 1573; Che:1499; βIV 5–3 : 1275; dPPA: 1121; εV 1–2 :1663 and FR 236924: 1367). Fisher’s test: * p
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    1) Product Images from "Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination"

    Article Title: Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination

    Journal: Cells

    doi: 10.3390/cells8111304

    PKC activity modulation in axonal loss. ( A ) shows the percentage of singly-, doubly-, and triply- (or more) innervated NMJs in the control mice (PBS) and after four applications of one of the following substances: the PKC paninhibitors Chelerytrine (CHE) and CaC, and the PKC panstimulators (BRY, at 1 and 10 nM) and PMA. CHE and CaC action results in the persistence of many polyinnervated synapses. Accordingly, both PKC stimulators PMA and BRY increase the number of monoinnervated junctions and clearly decrease the percentage of doubly-innervated junctions in the case of BRY. ( B ) shows the percentage of singly-, doubly-, and triply (or more) innervated NMJs after exposure to the cPKCβI and nPKCε isoform selective inhibitors βIV 5–3 and εV 1–2 , and the cPKCβI and nPKCε selective activators dPPA and FR 236,924 respectively. The selective inhibitors similarly increase the doubly- and triply-innervated synapses with the corresponding reduction in the monoinnervated junctions. The activators considerably accelerate nerve terminal elimination. Data were presented as percentages of NMJ ± SD (for each treatment and PBS control: n pups = 6–9; n = 11–18 LALs; n NMJ: PBS: 2538; Bry 1 nM: 1232 Bry 10 nM:1384; PMA:1247; CaC: 1573; Che:1499; βIV 5–3 : 1275; dPPA: 1121; εV 1–2 :1663 and FR 236924: 1367). Fisher’s test: * p
    Figure Legend Snippet: PKC activity modulation in axonal loss. ( A ) shows the percentage of singly-, doubly-, and triply- (or more) innervated NMJs in the control mice (PBS) and after four applications of one of the following substances: the PKC paninhibitors Chelerytrine (CHE) and CaC, and the PKC panstimulators (BRY, at 1 and 10 nM) and PMA. CHE and CaC action results in the persistence of many polyinnervated synapses. Accordingly, both PKC stimulators PMA and BRY increase the number of monoinnervated junctions and clearly decrease the percentage of doubly-innervated junctions in the case of BRY. ( B ) shows the percentage of singly-, doubly-, and triply (or more) innervated NMJs after exposure to the cPKCβI and nPKCε isoform selective inhibitors βIV 5–3 and εV 1–2 , and the cPKCβI and nPKCε selective activators dPPA and FR 236,924 respectively. The selective inhibitors similarly increase the doubly- and triply-innervated synapses with the corresponding reduction in the monoinnervated junctions. The activators considerably accelerate nerve terminal elimination. Data were presented as percentages of NMJ ± SD (for each treatment and PBS control: n pups = 6–9; n = 11–18 LALs; n NMJ: PBS: 2538; Bry 1 nM: 1232 Bry 10 nM:1384; PMA:1247; CaC: 1573; Che:1499; βIV 5–3 : 1275; dPPA: 1121; εV 1–2 :1663 and FR 236924: 1367). Fisher’s test: * p

    Techniques Used: Activity Assay, Mouse Assay

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    Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. 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( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected <t>with</t> <t>RTX</t> <t>(s.c.,</t> 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). 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    Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend Snippet: Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

    Article Snippet: RTX (ref. ; Alomone Labs, R-400) was injected (subcutaneously; s.c.) in three dosages (30, 70 and 100 μg kg−1 ) into the right flank of Rag1 −/ − and C57BL/6J mice of around three weeks of age.

    Techniques: Injection, Mouse Assay, Activation Assay, FACS, Whisker Assay

    Secondary neuroinflammation in the spinal dorsal horn 7 days after ITS or ITC. (A) Representative immunofluorescent photomicrographs showing the expression of Iba-1, GFAP, SDF1, and CXCR4 at the spinal dorsal horn contralateral to ITS or ITC. (B) Quantification of the mean immunofluorescent intensity of Iba-1, GFAP, SDF1, and CXCR4-labeled cellular profiles. *** p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Secondary damage and neuroinflammation in the spinal dorsal horn mediate post-thalamic hemorrhagic stroke pain hypersensitivity: SDF1-CXCR4 signaling mediation

    doi: 10.3389/fnmol.2022.911476

    Figure Lengend Snippet: Secondary neuroinflammation in the spinal dorsal horn 7 days after ITS or ITC. (A) Representative immunofluorescent photomicrographs showing the expression of Iba-1, GFAP, SDF1, and CXCR4 at the spinal dorsal horn contralateral to ITS or ITC. (B) Quantification of the mean immunofluorescent intensity of Iba-1, GFAP, SDF1, and CXCR4-labeled cellular profiles. *** p

    Article Snippet: After blocking with 2% BSA in 0.1 M PBS, the sections were incubated overnight at 4°C with the primary antibodies: mouse anti-NeuN (1:200, Abcam), rabbit anti-Bax (1:200, Abcam), rabbit anti-Bcl-2 (1:200, Abcam), goat anti-Iba1(1:200, Abcam), mouse anti-GFAP(1:200, Millipore), mouse anti-SDF1 (1:200, Santa), and rabbit anti-CXCR4 (1:400, Alomone) (for details refer to ).

    Techniques: Expressing, Labeling

    Suppressing effects of i.t. antineuroinflammation treatment on expression levels of Iba-1, GFAP, SDF1, and CXCR4 in the spinal dorsal horn following ITS or ITC. Western blot assays (A) and quantitative analysis (B–C) of Iba1, GFAP, SDF-1, and CXCR4 expressions in the dorsal cord following i.t. administration of vehicle and drugs 10 days after ITS or ITC (3 days after i.t. catheterization). ITC-Veh, rats with ITC receiving i.t. administration of vehicle; ITC-MC, rats with ITC receiving i.t. administration of MC; ITC-FC, rats with ITC receiving i.t. administration of FC; ITC-AMD, rats with ITC receiving i.t. administration of AMD3100. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Secondary damage and neuroinflammation in the spinal dorsal horn mediate post-thalamic hemorrhagic stroke pain hypersensitivity: SDF1-CXCR4 signaling mediation

    doi: 10.3389/fnmol.2022.911476

    Figure Lengend Snippet: Suppressing effects of i.t. antineuroinflammation treatment on expression levels of Iba-1, GFAP, SDF1, and CXCR4 in the spinal dorsal horn following ITS or ITC. Western blot assays (A) and quantitative analysis (B–C) of Iba1, GFAP, SDF-1, and CXCR4 expressions in the dorsal cord following i.t. administration of vehicle and drugs 10 days after ITS or ITC (3 days after i.t. catheterization). ITC-Veh, rats with ITC receiving i.t. administration of vehicle; ITC-MC, rats with ITC receiving i.t. administration of MC; ITC-FC, rats with ITC receiving i.t. administration of FC; ITC-AMD, rats with ITC receiving i.t. administration of AMD3100. * p

    Article Snippet: After blocking with 2% BSA in 0.1 M PBS, the sections were incubated overnight at 4°C with the primary antibodies: mouse anti-NeuN (1:200, Abcam), rabbit anti-Bax (1:200, Abcam), rabbit anti-Bcl-2 (1:200, Abcam), goat anti-Iba1(1:200, Abcam), mouse anti-GFAP(1:200, Millipore), mouse anti-SDF1 (1:200, Santa), and rabbit anti-CXCR4 (1:400, Alomone) (for details refer to ).

    Techniques: Expressing, Western Blot

    Expression of NALCN increases in DRG and spinal dorsal horn after CFA injection in rats. (A) Representative immunoblotting image and statistical analysis indicated that the level of NALCN protein in DRG was upregulated at 8 h after CFA injection ( n = 5, by two-way ANOVA, Holm–Sidak test). (B) The level of NALCN protein in spinal cord was upregulated at day 3 after CFA injection ( n = 5, by two-way ANOVA, Holm–Sidak test). (C) Double immunofluorescence staining performed with an NALCN antibody in DRG at 8 h after CFA injection. NALCN (red) and neuronal marker NeuN (green) immunoreactivity showed NALCN mostly colocalized with NeuN. (D) Double immunofluorescence staining of spinal cord at day 3 after CFA injection showed NALCN immunoreactivity mostly colocalized with NeuN. (E) Double immunostaining shows colocalization of NALCN (red) with IB4 (green), NF200 (green), SP (green), and TRPV1 (green) in DRG neurons. (F) Percent of neuronal markers of the total population of DRG neurons. Five animals were used to obtain counts (unpaired t -test). IB4, isolectin B4; NF200, neurofilament 200 kDa; SP, substance P; TRPV1, transient receptor potential vanilloid 1. Data are presented as mean ± SD. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Elevated Expression and Activity of Sodium Leak Channel Contributes to Neuronal Sensitization of Inflammatory Pain in Rats

    doi: 10.3389/fnmol.2021.723395

    Figure Lengend Snippet: Expression of NALCN increases in DRG and spinal dorsal horn after CFA injection in rats. (A) Representative immunoblotting image and statistical analysis indicated that the level of NALCN protein in DRG was upregulated at 8 h after CFA injection ( n = 5, by two-way ANOVA, Holm–Sidak test). (B) The level of NALCN protein in spinal cord was upregulated at day 3 after CFA injection ( n = 5, by two-way ANOVA, Holm–Sidak test). (C) Double immunofluorescence staining performed with an NALCN antibody in DRG at 8 h after CFA injection. NALCN (red) and neuronal marker NeuN (green) immunoreactivity showed NALCN mostly colocalized with NeuN. (D) Double immunofluorescence staining of spinal cord at day 3 after CFA injection showed NALCN immunoreactivity mostly colocalized with NeuN. (E) Double immunostaining shows colocalization of NALCN (red) with IB4 (green), NF200 (green), SP (green), and TRPV1 (green) in DRG neurons. (F) Percent of neuronal markers of the total population of DRG neurons. Five animals were used to obtain counts (unpaired t -test). IB4, isolectin B4; NF200, neurofilament 200 kDa; SP, substance P; TRPV1, transient receptor potential vanilloid 1. Data are presented as mean ± SD. * p

    Article Snippet: Sections were double labeled by incubating them overnight at 4°C with primary antibodies against NALCN (1:400, Alomone Labs, Jerusalem, Israel), TRPV1 (1:800, Abcam, Cambridge, United Kingdom), SP (1:200, Abcam, Cambridge, United Kingdom), neurofilament 200 (NF200, 1:800, Millipore, Burlington, MA, United States), isolectin B4 (IB4)-FITC-conjugated 488 (1:400, Sigma, St. Louis, MO, United States), NeuN (1:400, Sigma, St. Louis, MO, United States), and vesicular glutamate transporter 2 (VGLUT2, 1:200, Novus, Centennial, CO, United States).

    Techniques: Expressing, Injection, Double Immunofluorescence Staining, Marker, Double Immunostaining