Unger et al. data

<p>Three-dimensional imaging of swine stifle</p> <p>Support was provided by the National Institute of Neurological Disorders and Stroke (NINDS) under R01NS100725 (to A.S.B.) and by the Richard M. Schulze Family Foundation (to T.P.M. and A.S.B.).</p> <p> </p

Graded destruction of articular cartilage.

<p>Articular cartilage was seen on multiplanar MR images as a band hyperintense (white) signal between the intraarticular space and subchondral bone. The thickness of this band was compared throughout its length and the quality and degree of its thinning was used to grade each cartilage lesion. Grade 1 damage overlying the tibial plateau (A and B) and patella (C), as indicated by nearly normal appearance with superficial indentation (arrowheads), here from one 4 mg knee at week 8. Grade 2 damage overlying the femoral condyle (D), tibial plateau (E), and trochlea (F), as indicated by cartilage thinning of less than 50% (arrowheads), here from two 4 mg knees at weeks 8 and 9. Grade 3 damage overlying the tibial plateau (G and H) and patella (I), as indicated by loss of cartilage of at least 50% and up to subchondral bone but not deeper (arrowheads), here from two 4 mg knees at weeks 6 and 8. Grade 4 damage overlying the femoral condyle (J and K) and trochlea (L), as indicated by complete loss of cartilage with associated underlying damage to subchondral bone (arrowheads), here from one 4 mg knee at week 12. Representative Grade 0 images from one non-injected knee contralateral to a 4 mg knee at week 12 (M, N, O) demonstrate full thickness cartilage throughout all articular surfaces. Images are representative of n = 587 cartilage lesions identified. Coronal views: top row. Sagittal views: middle row. Axial views: bottom row.</p

Progression of structural joint damage quantified by serial MRI.

<p>(A) Idealized knee depicts the anatomical relationship between paired articular surfaces and bones comprising the knee joint in humans and swine. A total of n = 4,560 MRI measurements are summarized as anatomical heat maps. The coloring of articular surfaces, subchondral bone, and the intraarticular space relates lesion severity and location shown in the remaining panels. Lesion severity is indicated by different colors. Detailed methods for computing numerical grades and converting grades into color is described in the methods section. Minimal or no damage was seen at the outset of the study, reflecting healthy knees (lighter colors) for cartilage damage (B), bone marrow edema (C), subchondral erosion (D), and joint effusion (E). Lesion severity increased in each category with time and dose. Ligaments and menisci were also evaluated (n = 1,440 MRI measurements) but neither observer recorded damage to either structure in any knee at any time point.</p

Bone marrow edema.

<p>Edema is seen in subchondral bone as a hyperintense focus extending from the cartilage-bone interface into underlying bone. The deepest, leading edge of edematous signal is serpiginous and irregular. Grade 1 bone marrow edema is evidenced by relatively small hyperintense foci (arrowheads), extending here into subjacent subchondral bone of the femoral condyle (A and B) and the trochlea (C), here from two 4 mg knees at week 7. Grade 2 bone marrow edema (arrowheads) is relatively moderate in size, shown at the posterior aspect of the femoral condyle (D and E) and within the patella (F), here from one 4 mg knee at week 10. Grade 3 bone marrow edema was not found in any knee throughout the study. Representative Grade 0 images from one non-injected knee contralateral to a 4 mg knee at week 12 (G, H, I) demonstrate absence of subchondral hyperintensity. Images are representative of bone marrow edema identified in n = 412 images. Coronal views: top row. Sagittal views: middle row. Axial views: bottom row.</p

Inter-rater reliability of MRI grading.

<p>Inter-rater reliability of MRI grading.</p

Intraarticular joint effusion.

<p>Joint effusions were observed as hyperintense fluid collections (arrowheads) within the joint space of MIA-injected knees. Control knees never demonstrated an effusion. Grade 1 effusions were small and observed as focal fluid collections present along the margins of the femoral condyles (A and B, upper arrowhead and C) and the tibial condyle (B, lower arrowhead), here from one 4 mg knee at week 12. Grade 1 lesions did not appear to distend the intraarticular capsule. Grade 2 effusions were of moderate size and formed multiple, discrete collections in a single joint, for instance, along the tibia plateau (D), femoral condyle, and femoral shaft (E), here from one 4 mg knee at week 8. Grade 2 effusions distended the intraarticular capsule in several locations, including at the suprapatellar space (E, upper arrowhead and F). Grade 3 lesions formed circumferential collections around intraarticular bone (G and H) and displaced extraarticular soft tissue (I), here from one 4 mg knee at week 12. Examples of an intact intraarticular tendon (B, arrow) and intact meniscus (D, arrow) are shown for reference. Representative Grade 0 images from one non-injected knee contralateral to a 4 mg knee at week 12 (J, K, L) demonstrate absence of intraarticular hyperintense fluid. Images are representative of n = 97 joint effusions identified. Coronal views: top row. Sagittal views: middle row. Axial views: bottom row.</p

Gross assessment of MIA-, sham-, and non-injected knees.

<p>(A) Representative ultrasound image demonstrates application of the clinical intraarticular injection technique in swine. The needle tip (arrowhead) was guided in real time to the intraarticular space defined by the interface between hyperechoic cortical bone (femoral condyle, fc) and anechoic synovial fluid (arrow). (B) Representative image of a control knee (n = 29) showing intact articular cartilage of the patellofemoral (top) and tibiofemoral (bottom) articulations. The trajectory of the needle in panel A is illustrated (dotted arrow). (C) A non-injected knee illustrates traumatic, focal disruption of articular cartilage (n = 1) at the femoral condyle. The cartilage fragment shown outside the knee was found free-floating within the joint and tracing of its crisp borders showed it to match the focal area of exposed subchondral bone. This post-traumatic lesion was not factored into grading for this specimen. (D–G) Representative images of MIA-injected knees (n = 25) where articular cartilage destruction was unique compared to controls. (D) 40 mg MIA, n = 2. (E) 12 mg MIA, n = 2. (F) 4 mg MIA, n = 12. (G) 1.2 mg MIA, n = 9.</p

Subchondral bone erosion.

<p>(A) Coronal view and (B) sagittal view of an erosion in the femoral condyle (arrowheads). The lesion is a discrete, scalloped indentation in the subchondral bone associated with the absence of overlying cartilage. (C) Axial view of another erosion located in the patellar subchondral bone (arrowhead), here from one 4 mg knee at week 12. The overlying articular cartilage is completely missing. Images are representative of n = 292 erosions identified.</p

Plasticity of DNA methylation in a nerve injury model of pain

<div><p>The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (<i>P</i> < 10⁻⁴) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (<i>P</i> < 10⁻¹⁵) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings—obtained in mixed tissue—show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.</p></div