Tissue-specific and spatially dependent metabolic signatures perturbed by injury in skeletally mature male and female mice

Abstract

Joint injury is a risk factor for post-traumatic osteoarthritis. However, metabolic and microarchitectural changes within the joint post-injury in both sexes remain unexplored. This study identified tissue-specific and spatially-dependent metabolic signatures in male and female mice using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and LC-MS metabolomics. Male and female C57Bl/6J mice were subjected to non-invasive joint injury. Eight days post-injury, serum, synovial fluid, and whole joints were collected for metabolomics. Analyses compared between injured, contralateral, and naïve mice, revealing local and systemic responses. Data indicate sex influences metabolic profiles across all tissues, particularly amino acid, purine, and pyrimidine metabolism. MALDI-MSI generated 2D ion images of bone, the joint interface, and bone marrow, highlighting increased lipid species in injured limbs, suggesting physiological changes across injured joints at metabolic and spatial levels. Together, these findings reveal significant metabolic changes after injury, with notable sex differences.

Keywords: MALDI imaging; metabolomics; osteoarthritis; sex differences.

Figure 1.. Global metabolomic profiles of whole joints, synovial fluid, and serum are driven by injury status.

(A-C) Partial Least Squares-Discriminant Analysis (PLS-DA) finds some overlap between injured and naïve whole joint and synovial fluid and near-perfect separation of injured and naïve serum. (D-F) Fold change analysis distinguished populations of metabolite features driving separation of metabolomic profiles. (D) Specifically, 250 and 291 metabolite features were highest in injured and naïve whole joints, respectively. (E) 373 and 155 metabolite features were highest in injured and naïve synovial fluid, respectively. (F) 386 and 195 features were highest in injured and naïve serum, respectively. Similarly, PLS-DA reveals overlap between injured, contralateral, and naïve (G) whole joints and (H) synovial fluid with injured samples clustering together between contralateral and naïve samples. To pinpoint pathways driving metabolomic differences between limbs with different injury statuses, median intensity heatmap analyses where injured and contralateral limbs were normalized to naïve limbs were performed. Clusters of co-regulated metabolite features within (I) whole joint and (J) synovial fluid samples were subjected to pathway analyses to identify biological pathways that differ in regulation across limbs in both whole joint and synovial fluid samples. Combined, data provide strong evidence of distinct metabolomic regulation associated with injury status. Columns represent limbs (naïve, injured, contralateral) and rows represent metabolite features. Cooler and warmer colors indicate lower and higher metabolite abundance relative to the mean, respectively. The colors in A-J correspond to: purple = naïve, orange = injured, green = contralateral whole joint; sample types - red = whole joint, blue = synovial fluid, yellow = serum.

Figure 2.. Metabolomic profiles of whole joint, synovial fluid, and serum show sexual dimorphism across injured and naive mice.

(A-C) Partial Least Squares-Discriminant Analysis (PLS-DA) finds (A) complete separation of injured whole joints from males and females and minimal overlap when comparing (B) female and (C) male injured and naïve mice. (D-F) Fold change analysis distinguished populations of whole-joint derived metabolite features driving separation of metabolomic profiles. (G-I) PLS-DA finds minimal overlap when comparing (G) injured SF from males and females, (H) female and (I) male injured and naïve mice. (J-L) Fold change analysis identified populations of synovial fluid metabolite features contributing to the separation of mice that differ by sex and injury. (M-O) PLS-DA finds clear separation with no overlap when comparing (M) injured SF from males and females, (N) female and (O) male injured and naïve mice. (P-R) Fold change analysis identified populations of metabolite features driving separation of serum metabolomic profiles. The colors in A-R correspond to: pink = injured females, peach = naïve females, royal blue = injured males, light blue = naïve males. sample types - red = whole joint, blue = synovial fluid, yellow = serum.

Figure 3.. MALDI-MSI combined ion images from sagittal whole joint sections.

Heatmap analysis of putatively identified molecular species across various tissue structures. Spatial resolution = 100 um. Scale bar = 1 mm. Interval width = 0.35 Da. Colors in panels from left to right: L-carnitine (162.29 m/z, purple), C₃₆H₃₈O₇ (629.61 m/z, orange), (820.41m/z, blue), hydroxyprolyl-isoleucine (245.10 m/z, yellow), and lipid species 18:2/16:0 (758.57 m/z, pink).