Journal: Cell Reports Medicine

Article Title: α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

doi: 10.1016/j.xcrm.2024.101525

Figure Lengend Snippet: L3–5 DRG neurons innervate eWAT (A) Schematic of the AAV-based tracing strategy to visualize L3–5 DRG projections to eWAT. (B) Representative fluorescence image of the unsectioned mouse L4 DRG transduced by AAV-tdTomato. Scale, 100 μm. (C) Mouse eWAT before and after clearing. Scale, 2 mm. (D) Representative fluorescence images of the unsectioned mouse eWAT 21 days after AAV injection into the sciatic nerve and 7 days after SCI. Scale, 500 μm (inset: 50 μm). (E) Representative fluorescence images of L4 DRG. Arrowheads indicate Fluoro-Gold retrogradely labeled CGRP-positive DRG neurons projecting to eWAT. Scale, 50 μm.

Article Snippet: CGRP content was calculated using the Mouse CGRP ELISA kit (NBP3-00522, Novus Biologicals) following the manufacturer’s instructions.

Techniques: Fluorescence, Injection, Labeling

Journal: Cell Reports Medicine

Article Title: α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

doi: 10.1016/j.xcrm.2024.101525

Figure Lengend Snippet: SCI leads to increased α2δ1 and RAMP1 expression in L3–5 DRG neurons and eWAT, respectively (A) Scatterplot shows α2δ1 expression in Fluoro-Gold retrogradely labeled L3–5 DRG neurons at 7DPO (sham n = 4 and SCI n = 5 biological replicates, 419–486 neurons/experimental condition). Inset: α2δ1 expression in small-diameter (<20 μm) DRG neurons. Mean and SEM (mixed model with a random subject intercept to account for within-subject correlation ∗∗∗ p < 0.001). (B) Distribution of α2δ1 expression in retrogradely labeled CGRP-positive L3–5 DRG neurons at 7DPO. These neurons are a subset of (A) (two-sample Kolmogorov-Smirnov test ∗∗∗ p < 0.001; sham n = 4 and SCI n = 5 biological replicates; 161–233 neurons/experimental condition). (C) Representative fluorescence images of L3–5 DRG at 7DPO. Arrowheads indicate Fluoro-Gold retrogradely labeled CGRP-positive DRG neurons whose centrally projecting axon was severed after SCI. Scale, 50 μm. (D) Distribution of Cav2.1 expression in retrogradely labeled CGRP-positive L3–5 DRG neurons at 7DPO (two-sample Kolmogorov-Smirnov test ∗∗∗ p < 0.001; sham n = 4 and SCI n = 5 biological replicates; 109–236 neurons/experimental condition). (E) CGRP concentration in eWAT at 7DPO. Mean and SEM (unpaired two-tailed Student’s t test ∗ p < 0.05, sham n = 4 and SCI n = 4 biological replicates). (F) Representative fluorescence images of eWAT at 7DPO. Scale, 100 μm. (G) Quantification of (F). Mean and SEM (unpaired two-tailed Student’s t test ∗∗ p < 0.01, sham n = 4 and SCI n = 3 biological replicates). (H) Representative fluorescence images and 3D reconstruction of nociceptive nerve terminals in the unsectioned eWAT at 7DPO. Scale, 500 μm. (I) Quantification of (H). Mean and SEM (unpaired two-tailed Student’s t test, ns [not significant], sham n = 3 and SCI n = 3 biological replicates). (J) ex vivo eWAT lipolysis under basal (vehicle) and CGRP-stimulated (1 μM) conditions. Scatterplot shows fold change in glycerol release (paired two-tailed Student’s t test ∗∗ p < 0.01, n = 4 biological replicates). (K) Representative fluorescence and DIC images of cultured adult eWAT adipocytes loaded with the calcium indicator Fluo-4 AM. Scale, 100 μm. (L) Calcium response in primary eWAT adipocyte after vehicle (dH 2 O) and CGRP (43 μM) administration (vehicle n = 81 and CGRP n = 100 eWAT adipocyte traces from 5 independent experiments/experimental condition). (M) Quantification of (L). Mean and SEM (unpaired two-tailed Student’s t test ∗∗∗ p < 0.001, vehicle n = 81 and CGRP n = 100 eWAT adipocyte traces from 5 independent experiments/experimental condition).

Article Snippet: CGRP content was calculated using the Mouse CGRP ELISA kit (NBP3-00522, Novus Biologicals) following the manufacturer’s instructions.

Techniques: Expressing, Labeling, Fluorescence, Concentration Assay, Two Tailed Test, Ex Vivo, Cell Culture

Journal: Cell Reports Medicine

Article Title: α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

doi: 10.1016/j.xcrm.2024.101525

Figure Lengend Snippet: Cacna2d1 conditional deletion in DRG neurons normalizes eWAT lipolysis after SCI (A) Experimental scheme and timeline of AAV injection to transduce L3–5 DRG. (B) Real-time qPCR shows Cacna2d1 normalized expression in adult L3–5 DRG from Cacna2d1 fl/fl mice injected with either AAV-GFP or AAV-Cre. Mean and SEM (unpaired two-tailed Student’s t test ∗ p < 0.05, AAV-Ctr n = 4 and AAV-Cre n = 4 biological replicates). (C) Representative hematoxylin and eosin images of eWAT at 7 days post injury (DPI). Scale, 100 μm. (D) Quantification of adipocyte area in (C). Mean and SEM (two-way ANOVA ∗∗ p < 0.01 and ∗∗∗ p < 0.001, ns [not significant], AAV-GFP/SCI n = 3 and AAV-Cre/SCI n = 3 biological replicates; 2,063–2,322 adipocytes/experimental condition). (E) Ex vivo eWAT lipolysis at 7DPI. Mean and SEM (Kruskal-Wallis test followed by Dunn’s multiple comparisons test ∗∗ p < 0.01, ns [not significant], AAV-GFP n = 6, AAV-GFP/SCI n = 5, and AAV-Cre/SCI n = 6 biological replicates). The AAV-GFP samples are the same as in Figure S4 E. (F) Immunoblots show pHSL, HSL, and ATGL expression in eWAT at 7DPI. GAPDH is used as a loading control. (G) Quantification of (F). Mean and SEM (unpaired two-tailed Student’s t test ∗∗ p < 0.01, AAV-GFP/SCI n = 4 and AAV-Cre/SCI n = 4 biological replicates). (H) CGRP concentration in eWAT at 7DPI. Mean and SEM (unpaired two-tailed Student’s t test ∗∗ p < 0.01, AAV-GFP/SCI n = 4 and AAV-Cre/SCI n = 4 biological replicates). (I) Representative hematoxylin and eosin images of eWAT at 7DPI. Scale, 100 μm. (J) Quantification of adipocyte area in (I). Mean and SEM (two-way ANOVA ∗∗∗ p < 0.001, ns [not significant], Na v 1.8-Cre n = 5 and Na v 1.8-Cre/ Cacna2d1 fl/fl n = 5 biological replicates; 2,732–3,170 adipocytes/experimental condition). (K) Ex vivo eWAT lipolysis at 7DPI. Mean and SEM (unpaired two-tailed Student’s t test ∗ p < 0.05, Na v 1.8-Cre n = 4 and Na v 1.8-Cre/ Cacna2d1 fl/fl n = 5 biological replicates). (L) Immunoblots show pHSL, HSL, and ATGL expression in eWAT at 7DPI. GAPDH is used as a loading control. (M) Quantification of (L). Mean and SEM (unpaired two-tailed Student’s t test ∗ p < 0.05, Na v 1.8-Cre n = 4 and Na v 1.8-Cre/ Cacna2d1 fl/fl n = 5 biological replicates). (N) CGRP concentration in eWAT at 7DPI. Mean and SEM (unpaired two-tailed Student’s t test ∗∗ p < 0.01, Na v 1.8-Cre n = 8 and Na v 1.8-Cre/ Cacna2d1 fl/fl n = 10 biological replicates).

Article Snippet: CGRP content was calculated using the Mouse CGRP ELISA kit (NBP3-00522, Novus Biologicals) following the manufacturer’s instructions.

Techniques: Injection, Transduction, Expressing, Two Tailed Test, Ex Vivo, Western Blot, Control, Concentration Assay

Journal: Cell Reports Medicine

Article Title: α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

doi: 10.1016/j.xcrm.2024.101525

Figure Lengend Snippet: α2δ1 pharmacological blockade via GBP administration normalizes eWAT lipolysis and reduces ectopic lipid accumulation in the liver after SCI (A) Experimental scheme. (B) Mouse body weight. Mean and SEM (mixed model with a random subject intercept to account for within-subject correlation ∗ p < 0.05 and ∗∗∗ p < 0.001, ns [not significant], sham n = 10, vehicle/SCI n = 11, and GBP/SCI n = 11 biological replicates). (C) Representative images of hematoxylin and eosin-stained eWAT sections 28DPO. Scale, 100 μm. (D) Quantification of adipocyte area in (C). Mean and SEM (two-way ANOVA ∗ p < 0.05 and ∗∗∗ p < 0.001, ns [not significant], vehicle/SCI n = 5 and GBP/SCI n = 4 biological replicates; 654–1,051 adipocytes/experimental condition). (E) Ex vivo eWAT lipolysis at 30DPI (unpaired two-tailed Student’s t test ∗∗∗ p < 0.001, vehicle/SCI n = 6 and GBP/SCI n = 6 biological replicates). (F) Glycerol concentration in the serum at 30DPI. Mean and SEM (unpaired two-tailed Student’s t test ∗ p < 0.05, vehicle/SCI n = 5 and GBP/SCI n = 4 biological replicates). (G) Immunoblots show pHSL, HSL, and ATGL expression in eWAT at 30DPI. GAPDH is used as a loading control. (H) Quantification of (G). Mean and SEM (unpaired two-tailed Student’s t test ∗ p < 0.05, ns [not significant], vehicle/SCI n = 3 and GBP/SCI n = 3 biological replicates). (I) CGRP concentration in the serum at 7DPI. Mean and SEM (unpaired 2-tailed Student’s t test ∗ p < 0.05, vehicle/SCI n = 6 and GBP/SCI n = 5 biological replicates). (J) Representative images of liver sections stained with oil red O to visualize lipid accumulation. Scale, 50 μm. (K) Quantification of (J). Mean and SEM (two-way ANOVA ∗ p < 0.05 and ∗∗∗ p < 0.001, ns [not significant], vehicle/SCI n = 4 and GBP/SCI n = 5 biological replicates). (L) Metabolic cage assessment (CLAMS) of oxygen consumption (VO 2 ) and respiratory exchange rate (RER) at 1 month after SCI. Mean and SEM (mixed model with a random subject intercept to account for within-subject correlation, p values for group/period interaction are provided, sham n = 4, SCI n = 4, vehicle/SCI (middle) n = 4, GBP/SCI n = 5, vehicle/SCI (right) n = 4, and dGBP/SCI n = 5).

Article Snippet: CGRP content was calculated using the Mouse CGRP ELISA kit (NBP3-00522, Novus Biologicals) following the manufacturer’s instructions.

Techniques: Staining, Ex Vivo, Two Tailed Test, Concentration Assay, Western Blot, Expressing, Control

Journal: Cell Reports Medicine

Article Title: α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

doi: 10.1016/j.xcrm.2024.101525

Figure Lengend Snippet:

Article Snippet: CGRP content was calculated using the Mouse CGRP ELISA kit (NBP3-00522, Novus Biologicals) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Recombinant, Plasmid Preparation, Saline, Protease Inhibitor, Staining, Enzyme-linked Immunosorbent Assay, H&E Stain, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Software, Imaging, Microscopy, Real-time Polymerase Chain Reaction, Blocking Assay, Membrane