rabbit scad  (Proteintech)

Journal: CNS Neuroscience & Therapeutics

Article Title: Downregulation of Mfn2 Contributes to Chronic Postsurgical Pain via Inducing the Pyroptosis of GABAergic Neurons in the Spinal Cord

doi: 10.1111/cns.70508

Figure Lengend Snippet: Experimental designs and animal groups. Experiment 1: The changes of the NLRP3/Caspase‐1/GSDMD pathway, GABAergic neurons, and mitochondrial dynamics‐related proteins in the spinal cord of rats with SMIR. Experiment 2: The effects of GSDMD inhibitor (DSF) on mechanical allodynia and pyroptosis of GABAergic neurons in rats with SMIR. Experiment 3: The effects of Mfn2 activator (MASM7) on mechanical allodynia, mitochondrial function, and the pyroptosis of GABAergic neurons in rats with SMIR. DSF, disulfiram ; i.t., intrathecal injection; IF, immunofluorescence; MMP, mitochondrial membrane potential; mtDNA, mitochondrial DNA; PWT, paw withdrawal threshold; qPCR, quantitative polymerase chain reaction; ROS, reactive oxygen species; SMIR, skin/muscle incision and retraction; WB, western blot.

Article Snippet: The final mitochondrial pellet was resuspended in an appropriate volume of storage buffer, and protein concentration was determined using the BCA protein assay kit (Boster, Wuhan, China).

Techniques: Injection, Immunofluorescence, Membrane, Real-time Polymerase Chain Reaction, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Downregulation of Mfn2 Contributes to Chronic Postsurgical Pain via Inducing the Pyroptosis of GABAergic Neurons in the Spinal Cord

doi: 10.1111/cns.70508

Figure Lengend Snippet: SMIR rats exhibited mitochondrial dysfunction and reduced expression of Mfn2 in the spinal cord. (A, B) The representative images of MitoSox in the spinal of rats in sham and SMIR rats. Scale bar: 100 μm and 50 μm. Quantitative analysis show that the mitochondrial ROS generation was increase in the SMIR rats compared with the Sham group ( n = 5). Student's t ‐test, ** p < 0.01 compared with the Sham group. (C) JC‐1 staining and quantitative analysis show that MMP was decreased in the SMIR rats compared with the Sham group ( n = 5). Student's t ‐test, ** p < 0.01 compared with the Sham group. (D) mtDNA level in the cytosol of the SMIR group was higher than that in the Sham group, while there was no significant change in nuclei DNA level ( n = 6). Student's t ‐test, **** p < 0.0001 compared with the Sham group. (E) The mitochondrial ultrastructure in spinal neurons exhibited mitochondrial swelling, destruction and disappearance of mitochondrial cristae compared with the Sham group ( n = 3). The labeled red boxes highlight the mitochondrial ultrastructure. The red arrow represents the mitochondria. Scale bar: 1 μm and 500 nm. (F) Representative Western blot images of the mitochondrial dynamics‐related proteins in the sham or SMIR rats. (G–I) Quantitative analysis showed that the protein levels of Mfn2 was significantly decreased and p‐Drp1 was significantly increased on day 1 and sustained on day 21. While no significant difference was observed in Mfn1 between the sham and SMIR groups ( n = 6). One‐way ANOVA, followed by Bonferroni's post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with the Sham group. (J) Double immunofluorescence of Mfn2 with NeuN, Iba1, GFAP in the spinal cord of the sham and SMIR rats. Scale bar: 100 μm and 50 μm for detail. (K–M) Statistical analysis indicated that the immunoreactivity of Mfn2 + NeuN + in the SMIR group were significantly decreased compared with the Sham group, while there was no different in the immunoreactivity of Mfn2 + Iba1 + and GSDMD + GFAP + between Sham and SMIR groups ( n = 4). Student's t ‐test, **** p < 0.0001 compared with the Sham group.

Article Snippet: The final mitochondrial pellet was resuspended in an appropriate volume of storage buffer, and protein concentration was determined using the BCA protein assay kit (Boster, Wuhan, China).

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

Journal: CNS Neuroscience & Therapeutics

Article Title: Downregulation of Mfn2 Contributes to Chronic Postsurgical Pain via Inducing the Pyroptosis of GABAergic Neurons in the Spinal Cord

doi: 10.1111/cns.70508

Figure Lengend Snippet: Mfn2 activator alleviated SMIR‐induced mechanical allodynia through reducing mitochondrial dysfunction. (A) Repeated injections of MASM7 (10 μg, i.t.) reversed the established mechanical allodynia in SMIR rats ( n = 6). Generalized Estimating Equations (GEE) model, followed by Bonferroni's post hoc test, **** p < 0.0001 compared with the Sham + Vehicle group, #### p < 0.0001 compared with the SMIR + Vehicle group. (B) Western blot indicated treating with MASM7 increased the expression levels of Mfn2 compared with the SMIR + Vehicle group ( n = 6). One‐way ANOVA, followed by Bonferroni's post hoc test, ** p < 0.01 compared with the Sham + Vehicle group, ## p < 0.01 compared with the SMIR + Vehicle group, (C, D) The representative images of MitoSox of rats in vehicle or MASM7‐treated SMIR rats. Scale bar: 100 μm and 50 μm. Quantitative analysis showed that treated with MASM7 significantly decreased the generation of mitochondrial ROS induced by SMIR compared with the Vehicle group ( n = 5). One‐way ANOVA, followed by Bonferroni's post hoc test, **** p < 0.0001 compared with the Sham + Vehicle group, ## p < 0.01 compared with the SMIR + Vehicle group. Scale bar: 100 μm and 50 μm for detail. (E) JC‐1 staining and quantitative analysis show that MMP was revered in the SMIR + MASM7 group compared with the SMIR + Vehicle group ( n = 5). One‐way ANOVA, followed by Bonferroni's post hoc test, ** p < 0.01 compared with the Sham + Vehicle group, # p < 0.05 compared with the SMIR + Vehicle group. (F) The abnormal release of cytosolic mitochondrial DNA in the SMIR + MASM7 group was significantly decreased compared with the SMIR + Vehicle group ( n =5). One‐way ANOVA, followed by Bonferroni's post hoc test, **** p < 0.0001 compared with the Sham + Vehicle group, #### p < 0.0001 compared with the SMIR + Vehicle group. (G) Representative electron morphological changes of the mitochondrial showed the mitochondrial morphological injury in spinal neurons caused by SMIR was alleviated by MASM7 treatment ( n = 3). The labeled red boxes highlight the mitochondrial ultrastructure. The red arrow represents the mitochondria. Scale bar: 1 μm and 500 nm.

Article Snippet: The final mitochondrial pellet was resuspended in an appropriate volume of storage buffer, and protein concentration was determined using the BCA protein assay kit (Boster, Wuhan, China).

Techniques: Western Blot, Expressing, Staining, Labeling