aav 8 cmv egfp  (Addgene inc)

Journal: Anesthesia and Analgesia

Article Title: Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice

doi: 10.1213/ANE.0000000000005768

Figure Lengend Snippet: Overexpression of neuronal GRK2 in spinal cord–alleviated cisplatin-induced peripheral neuropathy. A, Western blot analysis of GRK2 after 5 injections of cisplatin. Results are normalized to β-actin and shown as ratios to saline-treated mice. Values are mean ± SEM. * P < .05, P = .0259. B, Experimental strategy for neuronal GRK2 overexpression. Neuron-specific promoter hSyn targeted Cre-ERT2 recombination activated by tamoxifen. C, Tamoxifen-activated Cre-ERT2 upregulated the expression of GRK2 in the spinal cord. Results are normalized to β-actin. Values are mean ± SEM. * P < .05, P = .0334. D, Representative images show NeuN labeling (red) and eGFP-tagged (green) AAV-GRK2 in the spinal cord. Scale bar, 100 μm. E, Upregulation of neuronal GRK2 prevented cisplatin-induced reduction of PWT. Values are mean ± SEM. * P < .05, ** P < .01. Saline versus cisplatin P = .0011; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0198; cisplatin versus cis + AAV-GRK2 P = .0024. F, Upregulation of neuronal GRK2 shortened cisplatin-induced prolonged responsive time. Values are mean ± SEM. * P < .05, ** P < .01. Saline versus cisplatin P = .0016; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0178; cisplatin versus cis + AAV-GRK2 P = .0014. G, Immunofluorescence staining of PGP 9.5 positive nerve fibers (white arrow) in the hind paw (scale bar =100 mm). H, Upregulation of neuronal GRK2 inhibited cisplatin-induced IENFs loss. Values are mean ± SEM. ** P < .01, *** P < .001. Saline versus cisplatin P = .0027; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0002; cisplatin versus cis + AAV-GRK2 P = .0004. AAV indicates adeno-associated virus; Cre-ERT2, cyclization recombinase estrogen receptor tamoxifen 2; eGFP, enhanced green fluorescent protein; GRK2, G protein–coupled receptor kinase 2; hSyn, human synapsin; IENFs, intraepidermal nerve fibers; NeuN, neuronal nuclei; PCR, polymerase chain reaction; PGP, protein gene product; PWT, paw withdrawal threshold; SEM, standard error of the mean; WB, Western blot.

Article Snippet: For overexpression of the neuronal GRK2 in spinal cord, mice were injected with a 2:1 volume mixture of an adeno-associated virus (AAV)2/8 carrying CMV-DIO-GRK2-2A-EGFP and an AAV2/8 carrying human synapsin-cyclization recombinase estrogen receptor tamoxifen 2 (hSyn-CreERT2) (OBiO Technology Co Ltd) by a CRE-dependent manner, and CRE activity was induced by single tamoxifen (1 mg in 100 μL, i.p.) injections for 5 days after 3 weeks; tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil.

Techniques: Over Expression, Western Blot, Expressing, Labeling, Immunofluorescence, Staining, Polymerase Chain Reaction

Journal: Anesthesia and Analgesia

Article Title: Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice

doi: 10.1213/ANE.0000000000005768

Figure Lengend Snippet: Upregulation of neuronal GRK2 altered the proinflammatory activity of spinal microglia and the expression of TREM2/DAP12. Western blot analysis of TREM2 (A) and DAP12 (B) on week 3 after the first cisplatin injection. Results are normalized to β-actin and shown as ratios to saline-treated mice. Values are mean ± SEM. * P < .05, ** P < .01. A, Saline versus cisplatin P = .0149; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0187; cisplatin versus cis + AAV-GRK2 P = .0012. B, saline versus cisplatin P = .0482; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0168; cisplatin versus cis + AAV-GRK2 P = .0107. Real-time PCR analysis of pain-related molecules at week 3 after the first cisplatin injection. These molecules were included: IL-1β (C), IL-6 (D), iNOS (E), CD16 (F), IL-4 (G), IL-10 (H), and CD206 (I). Results are normalized to GAPDH. Values are mean ± SEM. * P < .05, ** P < .01. C, Saline versus cisplatin P = .0321; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0333; cisplatin versus cis + AAV-GRK2 P = .0054. D, Saline versus cisplatin P = .0078; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0440; cisplatin versus cis + AAV-GRK2 P = .0092. E, Saline versus cisplatin P = .0293; cis + AAV-eGFP versus cis + AAV-GRK2 P = .0218; cisplatin versus cis + AAV-GRK2 P = .0485. F, Saline versus cisplatin P = .0395; cisplatin versus cis + AAV-GRK2 P = .0240. I, Cis + AAV-eGFP versus cis + AAV-GRK2 P = .0392. AAV indicates adeno-associated virus; CD, cluster of differentiation; DAP12, DNAX-activating protein of 12 kDa; eGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRK2, G protein–coupled receptor kinase 2; IL, interleukin; iNOS, inducible nitric oxide synthase; PCR, polymerase chain reaction; SEM, standard error of the mean; TREM2, triggering receptor expressed on myeloid cells 2.

Article Snippet: For overexpression of the neuronal GRK2 in spinal cord, mice were injected with a 2:1 volume mixture of an adeno-associated virus (AAV)2/8 carrying CMV-DIO-GRK2-2A-EGFP and an AAV2/8 carrying human synapsin-cyclization recombinase estrogen receptor tamoxifen 2 (hSyn-CreERT2) (OBiO Technology Co Ltd) by a CRE-dependent manner, and CRE activity was induced by single tamoxifen (1 mg in 100 μL, i.p.) injections for 5 days after 3 weeks; tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil.

Techniques: Activity Assay, Expressing, Western Blot, Injection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Anesthesia and Analgesia

Article Title: Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice

doi: 10.1213/ANE.0000000000005768

Figure Lengend Snippet: Multiple EA treatment for prevention of cisplatin-induced peripheral neuropathy and regulation of GRK2 expression. A, EA prevented the decreased in paw withdrawal threshold induced by cisplatin in von Frey test. Values are mean ± SEM. * P < .05, ** P < .01. Cis versus cis + EA P = .0021; cis + shEA versus cis + EA P = .0264. B, EA reduced the prolonged response time in adhesive removal test. Values are mean ± SEM. * P < .05, ** P < .01. Cis versus cis + EA P = .0387; cis + shEA versus cis + EA P = .0040. C, Immunofluorescence staining of PGP 9.5 positive nerve fibers (white arrow) in the hind paw (scale bar = 100 μm). D, EA inhibited cisplatin-induced IENFs loss. Values are mean ± SEM. *** P < .001. Cis versus cis + EA P = .0002; cis + shEA versus cis + EA P = .0005. E, EA regulated the expression of GRK2 in spinal dorsal horn. Western blot analysis of GRK2 levels after EA. EA treatment reversed the decline of spinal GRK2 induced by cisplatin. Results are normalized to β-actin and shown as ratios to cisplatin-treated mice. Values are mean ± SEM. * P < .05. Cis versus cis + EA P = .0278. EA indicates electroacupuncture; GRK2, G protein–coupled receptor kinase 2; IENFs, intraepidermal nerve fibers; PGP, protein gene product; SEM, standard error of the mean; shEA, sham electroacupuncture.

Article Snippet: For overexpression of the neuronal GRK2 in spinal cord, mice were injected with a 2:1 volume mixture of an adeno-associated virus (AAV)2/8 carrying CMV-DIO-GRK2-2A-EGFP and an AAV2/8 carrying human synapsin-cyclization recombinase estrogen receptor tamoxifen 2 (hSyn-CreERT2) (OBiO Technology Co Ltd) by a CRE-dependent manner, and CRE activity was induced by single tamoxifen (1 mg in 100 μL, i.p.) injections for 5 days after 3 weeks; tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot

Journal: Anesthesia and Analgesia

Article Title: Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice

doi: 10.1213/ANE.0000000000005768

Figure Lengend Snippet: Neuronal GRK2 mediated EA alleviating cisplatin-induced CIPN. A, Western blot analysis of down regulated GRK2 protein expression in the spinal dorsal horn of mice 3 wk after the virus administration. Results are normalized to β-actin and shown as ratios to syn-eGFP-treated mice. Values are mean ± SEM. * P < .05. syn-eGFP versus syn-GRK2 shRNA P = .0441. B, Representative images showed NeuN labeling (red) and eGFP-tagged (green) AAV-GRK2 shRNA in the spinal cord. Scale bar = 100 μm. C, Downregulation of neuronal GRK2 in the spinal cord inhibited the analgesic effect of EA in cisplatin-induced mechanical allodynia. Values are mean ± SEM. ** P < .01. Cis + EA + syn-eGFP versus cis + EA + syn-GRK2 shRNA P = .0010. D, Downregulation of neuronal GRK2 inhibited the alleviative effect of EA in cisplatin-induced sensory deficits. Values are mean ± SEM. *** P < .001. Cis + EA+ syn-eGFP versus cis + EA+ syn-GRK2 shRNA P = .0006. E, Immunofluorescence staining of PGP 9.5 positive nerve fibers (white arrow) in the hind paw (scale bar =100 mm). F, Downregulation of neuronal GRK2 inhibited the preventive effect of EA on cisplatin-induced IENFs loss. Values are mean ± SEM. ** P < .01. Cis + EA+ syn-eGFP versus cis + EA + syn-GRK2 shRNA P = .0043. AAV indicates adeno-associated virus; CIPN, chemotherapy-induced peripheral neuropathy; EA, electroacupuncture; eGFP, enhanced green fluorescent protein; GRK2, G protein–coupled receptor kinase 2; IENFs, intraepidermal nerve fibers; NeuN, neuronal nuclei; PGP, protein gene product; SEM, standard error of the mean; shRNA, short hairpin RNA.

Article Snippet: For overexpression of the neuronal GRK2 in spinal cord, mice were injected with a 2:1 volume mixture of an adeno-associated virus (AAV)2/8 carrying CMV-DIO-GRK2-2A-EGFP and an AAV2/8 carrying human synapsin-cyclization recombinase estrogen receptor tamoxifen 2 (hSyn-CreERT2) (OBiO Technology Co Ltd) by a CRE-dependent manner, and CRE activity was induced by single tamoxifen (1 mg in 100 μL, i.p.) injections for 5 days after 3 weeks; tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil.

Techniques: Western Blot, Expressing, shRNA, Labeling, Immunofluorescence, Staining

Journal: Anesthesia and Analgesia

Article Title: Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice

doi: 10.1213/ANE.0000000000005768

Figure Lengend Snippet: Neuronal GRK2 mediated the regulation of EA on microglia activation. Real-time PCR analysis of pain-related molecules at week 3 after the first cisplatin injection. These molecules were included IL-1β (A), IL-6 (B), TNF-α (C), iNOS (D), CD16 (E), IL-10 (F), IL-4 (G), and TGF-β (H). Results are normalized to GAPDH. Values are mean ± SEM. * P < .05, ** P < .01, **** P < .0001. A, P < .0001. B, P = .0055. C, P = .0093. D, P = .0064. E, P = .0391. F, P = .7324. G, P = .0825. H, P = .1118. I, P = .5767. J, P = .0082. K, P = .0273. Downregulation of neuronal GRK2 inhibited the downregulation of EA on TREM2 (J) and DAP12 (K) mRNA. Downregulation of neuronal GRK2 inhibited the downregulation of EA on TREM2 (L) and DAP12 (M) protein expression. Results are normalized to GAPDH (TREM2) or β-actin (DAP12) and shown as ratios to syn-eGFP treated mice. Values are mean ± SEM. * P < .05. L, P = .0180. M, P = .0372. CD indicates cluster of differentiation; EA, electroacupuncture; eGFP,enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRK2, G protein–coupled receptor kinase; IL, interleukin; iNOS, inducible nitric oxide synthase; ns, nonsignificant; PCR, polymerase chain reaction; SEM, standard error of the mean; shRNA, short hairpin RNA; TGF, tumor growth factor; TNF, tumor necrosis factor; TREM2, triggering receptor expressed on myeloid cells 2.

Article Snippet: For overexpression of the neuronal GRK2 in spinal cord, mice were injected with a 2:1 volume mixture of an adeno-associated virus (AAV)2/8 carrying CMV-DIO-GRK2-2A-EGFP and an AAV2/8 carrying human synapsin-cyclization recombinase estrogen receptor tamoxifen 2 (hSyn-CreERT2) (OBiO Technology Co Ltd) by a CRE-dependent manner, and CRE activity was induced by single tamoxifen (1 mg in 100 μL, i.p.) injections for 5 days after 3 weeks; tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Injection, Expressing, Polymerase Chain Reaction, shRNA