rabbit polyclonal antibody against rat tnf α  (Novus Biologicals)

Journal: JOR Spine

Article Title: Intradiscal Cutibacterium acnes Sustains Modic Type 1‐Like Lesions Over Time in a Rat Lumbar Endplate Injury Model

doi: 10.1002/jsp2.70182

Figure Lengend Snippet: Schematic overview of the study design and experimental timeline. The surgery involved (1) an anterior abdominal approach, followed by (2) EP injury, and (3) intradiscal injection of either TNF‐α or C. acnes , such that all outcome measures reflect the combined effects of both the EP injury and the respective injectate. Pain‐like behavior was evaluated biweekly using the von Frey assay to assess hind paw mechanical allodynia. Post‐euthanasia assessments included ex vivo spinal MRI using T1w and T2w sequences as well as histological analysis of spine and spinal cord. The timeline depicts the three experimental cohorts and the corresponding analyses time points. C. acnes, Cutibacterium acnes ; EP, endplate; MRI, magnetic resonance imaging; T1w, T1‐weighted.

Article Snippet: Sections were then incubated for 1 h at room temperature with one of the following primary antibodies: (i) rabbit polyclonal antibody against rat TNF‐α (1:500 dilution, #NBP1‐19532, Novus Biologics, MN, USA), (ii) rabbit recombinant multiclonal antibody against rat NE (1:500 dilution, #ab314916, Abcam, Waltham, MA, USA), or (iii) rabbit recombinant polyclonal antibody against rat CD19 (1:150 dilution, #27949‐1‐AP, ThermoFisher, Waltham, MA, USA).

Techniques: Injection, Ex Vivo, Magnetic Resonance Imaging

Journal: JOR Spine

Article Title: Intradiscal Cutibacterium acnes Sustains Modic Type 1‐Like Lesions Over Time in a Rat Lumbar Endplate Injury Model

doi: 10.1002/jsp2.70182

Figure Lengend Snippet: Degree of disc degeneration was increased in both EP injury + C. acnes and EP injury + TNF‐α discs compared to Sham without difference between injury groups. (A) Representative T1w and T2w MRI of discs per group and timepoint. (B) Both EP injury + C. acnes and EP injury + TNF‐α discs were significantly more degenerated than Sham at 8‐ and 14 weeks post‐injury, with no differences between the two injury groups at any time point. Disc degeneration increased over time in the EP injury + C. acnes group. Bars represent median with IQR. Significance bars represent results of Tukey's post hoc analysis of pairwise comparisons after detecting main effects with a two‐way ANOVA. Timepoints week 1, 8, and 14: Sham: N = 8 discs, EP injury + TNF‐α: N = 12–14 discs, EP injury + C. acnes : N = 10–12 discs. (C) Representative histological images of whole discs per group and timepoint, SafO/F/H staining. (D) Quantification of histological disc degeneration score. Discs from both EP injury groups were significantly more degenerated than Sham at all‐time points (weeks 1, 8, and 14). No significant differences were observed between EP injury + TNF‐α versus EP injury + C. acnes groups at any time point. Bars represent median with IQR. Significance bars represent results of Tukey's post hoc analysis of pairwise comparisons after detecting main effects with a two‐way ANOVA. Timepoints week 1, 8, and 14: Sham: N = 6–7 discs, EP injury + TNF‐α: N = 10–12 discs, EP injury + C. acnes : N = 9–12 discs. ANOVA, analysis of variance; C. acnes , Cutibacterium acnes ; EP, endplate; IQR, interquartile range; SafO/F/H, Safranin‐O/fastgreen/hematoxylin; T1w, T1‐weighted.

Article Snippet: Sections were then incubated for 1 h at room temperature with one of the following primary antibodies: (i) rabbit polyclonal antibody against rat TNF‐α (1:500 dilution, #NBP1‐19532, Novus Biologics, MN, USA), (ii) rabbit recombinant multiclonal antibody against rat NE (1:500 dilution, #ab314916, Abcam, Waltham, MA, USA), or (iii) rabbit recombinant polyclonal antibody against rat CD19 (1:150 dilution, #27949‐1‐AP, ThermoFisher, Waltham, MA, USA).

Techniques: Staining

Journal: JOR Spine

Article Title: Intradiscal Cutibacterium acnes Sustains Modic Type 1‐Like Lesions Over Time in a Rat Lumbar Endplate Injury Model

doi: 10.1002/jsp2.70182

Figure Lengend Snippet: Intradiscal injectate following EP injury determined MC subtype prevalence over time. (A) Representative T1w and T2w MR images showing MC1‐ (top), MC2‐ (middle), and MC3‐like (bottom) lesions in EP injury + TNF‐α (left) or EP injury + C. acnes (right) discs. All 3 different MC subtypes developed in both EP injury + TNF‐α and EP injury + C. acnes groups. MC1‐like lesions: T1w: Hypo‐, or isointense; T2w: Hyperintense. MC2‐like lesions: T1w and T2w: Hyperintense. MC3‐like lesions: T1w and T2w: Hypointense. Red arrows surround bone marrow lesions. Images are from time points 8‐ and 14‐weeks post‐injury. (B) Representative histological images of features associated with MC1 (cellular infiltrates), MC2 (fatty replacement of normal bone marrow), and MC3 (sclerotic bone) in EP injury + TNF‐α and EP injury + C. acnes groups. In both injury groups, all 3 histological features of MC subtypes were found. #: Normal bone marrow region. 1: Cellular infiltrates; 2: Fatty replacement of normal bone marrow; 3: Increased bone structure. SafO/F/H staining. (C) Quantification of MC1‐like lesion prevalence (% of total number of EPs: L6 cranial, L5 caudal, L5 cranial, L4 caudal) revealed that EP injury + intradiscal C. acnes injection resulted in a significantly higher prevalence of MC1‐like lesions across all 3 time points compared to both EP injury + TNF‐α and Sham. The EP injury + TNF‐α group tended to have significantly more MC1‐like lesions compared to Sham. (D) MC2‐like lesions increased in the EP injury + TNF‐α group over time and were significantly higher than EP injury + C. acnes and Sham at 14‐weeks post‐injury. (E) MC3‐like lesions increased over time in the EP injury + C. acnes group. Bars represent median with IQR. Significance bars represent pairwise comparisons from Tukey's post hoc analysis following two‐way ANOVA. Timepoints week 1, 8, and 14: Sham: N = 4 rats, EP injury + TNF‐α: N = 5–7 rats, EP injury + C. acnes : N = 5–6 rats. * p < 0.05, ** p < 0.01, *** p < 0.001. ANOVA, analysis of variance; C. acnes , Cutibacterium acnes ; EP, endplate; IQR, interquartile range; MC, modic change; SafO/F/H, Safranin‐O/fastgreen/hematoxylin; T1w, T1‐weighted.

Article Snippet: Sections were then incubated for 1 h at room temperature with one of the following primary antibodies: (i) rabbit polyclonal antibody against rat TNF‐α (1:500 dilution, #NBP1‐19532, Novus Biologics, MN, USA), (ii) rabbit recombinant multiclonal antibody against rat NE (1:500 dilution, #ab314916, Abcam, Waltham, MA, USA), or (iii) rabbit recombinant polyclonal antibody against rat CD19 (1:150 dilution, #27949‐1‐AP, ThermoFisher, Waltham, MA, USA).

Techniques: Staining, Injection

Journal: JOR Spine

Article Title: Intradiscal Cutibacterium acnes Sustains Modic Type 1‐Like Lesions Over Time in a Rat Lumbar Endplate Injury Model

doi: 10.1002/jsp2.70182

Figure Lengend Snippet: Intradiscal C. acnes versus TNF‐α injection following EP injury determined adjacent bone marrow lesion immune cell response. (A) Representative images of intradiscal inflammatory burden measured as TNF‐α‐ir in Sham (left), TNF‐α (middle) and C. acnes (right)‐injected discs. Upper right images represent magnified areas of the overview section. Arrows indicate TNF‐α positive cells. (B) Both EP injury + TNF‐α and EP injury + C. acnes groups led to increased discal TNF‐α‐ir at all 3 time points without a difference between injury groups. Bars represent median with IQR. Significance bars represent pairwise comparisons from Tukey's post hoc analysis. Timepoints week 1, 8, and 14: Sham: N = 6–8 discs, EP injury + TNF‐α: N = 8–10 discs, EP injury + C. acnes : 10–12 discs. (C) Representative images of NE‐ir (top) and CD19‐ir (bottom). Red arrows indicate NE‐positive cells, orange arrows indicate CD19‐positive cells. (D) Bone marrow lesions adjacent to EP injury + C. acnes ‐ versus EP injury + TNF‐α‐ discs showed increased NE‐ir and CD19‐ir. Bars represent median with IQR. Significance bars represent results from Mann–Whitney U ‐test. All time points combined: EP injury + TNF‐α: N = 12 bone marrow lesions, EP injury + C. acnes : N = 16 bone marrow lesions. * p < 0.05, ** p < 0.01, *** p < 0.001. C. acnes , Cutibacterium acnes ; IQR, interquartile range; NE, neutrophil elastase.

Article Snippet: Sections were then incubated for 1 h at room temperature with one of the following primary antibodies: (i) rabbit polyclonal antibody against rat TNF‐α (1:500 dilution, #NBP1‐19532, Novus Biologics, MN, USA), (ii) rabbit recombinant multiclonal antibody against rat NE (1:500 dilution, #ab314916, Abcam, Waltham, MA, USA), or (iii) rabbit recombinant polyclonal antibody against rat CD19 (1:150 dilution, #27949‐1‐AP, ThermoFisher, Waltham, MA, USA).

Techniques: Injection, MANN-WHITNEY

Journal: JOR Spine

Article Title: Intradiscal Cutibacterium acnes Sustains Modic Type 1‐Like Lesions Over Time in a Rat Lumbar Endplate Injury Model

doi: 10.1002/jsp2.70182

Figure Lengend Snippet: Pain‐like behavior and spinal cord sensitization was increased in both EP injury groups and SubP was significantly higher in the EP injury + C. acnes group. (A) Normalized hind paw withdrawal thresholds (% baseline) measured using von Frey testing over 13 weeks following EP injury with TNF‐α (blue squares) or C. acnes (pink triangles) injection, or sham surgery (black circles). Both EP injury groups demonstrated a significant and sustained reduction in mechanical thresholds compared to Sham, indicating long‐lasting mechanical hypersensitivity. No significant difference was observed between the EP injury + TNF‐α and EP injury + C. acnes groups. Data are shown as mean ± SD. Mixed‐effect analysis with Tukey post hoc analysis. * p < 0.05 for EP injury + TNF‐α versus Sham; # p < 0.05 for EP injury + C. acnes versus Sham. Time points 1–13 weeks: Sham: N = 4–12 rats, EP injury + TNF‐α: 6–19 rats, EP injury + C. acnes : 6–18 rats. (B) Random forest model showed that MC1‐like presence and area contributed strongest to pain‐like behavior. Graph shows %IncMSE. p values are FDR‐adjusted permutation‐based values. Black bars: p < 0.05. (C) Representative images of SubP expression spinal cord dorsal horn. (D) Quantification of SubP‐ir in the spinal cord dorsal horn area at 1‐, 8‐, and 14‐weeks. SubP levels were significantly increased in EP injury + TNF‐α and EP injury + C. acnes groups compared to Sham at 8‐ and 14‐weeks. SubP was significantly higher in EP injury + C. acnes versus EP injury + TNF‐α groups at 14‐WKs post‐injury. Significance bars represent results from Tukey's post hoc analysis. Time points weeks 1, 8, 14: Sham: N = 4, EP injury + TNF‐α: N = 6–7, EP injury + C. acnes : N = 5–6 spinal cords. (E) Representative images of GFAP expression in spinal cord dorsal horn. (F) Quantification of GFAP‐ir in the spinal cord dorsal horn area at 1‐, 8‐, and 14‐weeks. GFAP expression increased significantly in both EP injury + TNF‐α and EP injury + C . acnes groups compared to Sham, with a progressive increase from week 1 to week 14. There was no difference between injury groups. Significance bars represent results from Tukey post hoc analysis. Time points weeks 1, 8, 14: Sham: N = 4, EP injury + TNF‐α: N = 6–7, EP injury + C. acnes : N = 5–6. Bars represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. C. acnes , Cutibacterium acnes ; EP, endplate; GFAP, glial fibrillary acidic protein; IncMSE, percent increase in mean standard error; IQR, interquartile range; MC, modic change; SubP, substance P.

Article Snippet: Sections were then incubated for 1 h at room temperature with one of the following primary antibodies: (i) rabbit polyclonal antibody against rat TNF‐α (1:500 dilution, #NBP1‐19532, Novus Biologics, MN, USA), (ii) rabbit recombinant multiclonal antibody against rat NE (1:500 dilution, #ab314916, Abcam, Waltham, MA, USA), or (iii) rabbit recombinant polyclonal antibody against rat CD19 (1:150 dilution, #27949‐1‐AP, ThermoFisher, Waltham, MA, USA).

Techniques: Injection, Expressing