Female rats have a different healing phenotype than males after anterior cruciate ligament rupture with no intervention

Abstract

Little is known on the sex-specific healing responses after an anterior cruciate ligament (ACL) rupture. To address this, we compared male and female Sprague-Dawley rats following non-surgical ACL rupture. Hematology, inflammation, joint swelling, range of motion, and pain-sensitivity were analyzed at various times over 31-days. Healing was assessed by histopathology and gene expression changes in the ACL remnant and adjacent joint tissues. In the first few days, males and females showed similar functional responses after rupture, despite contrasting hematology and systemic inflammatory profiles. Sex-specific differences were found in inflammatory, immune and angiogenic potential in the synovial fluid. Histopathology and increased collagen and fibronectin gene expression revealed significant tissue remodeling in both sexes. In the ACL remnant, however, Acta2 gene expression (α-SMA production) was 4-fold higher in males, with no change in females, indicating increased fibroblast-to-myofibroblast transition with higher contractile elements (stiffness) in males. Females had 80% lower Pparg expression, which further suggests reduced cellular differentiation potential in females than males. Sex differences were also apparent in the infrapatellar fat pad and articular cartilage. We conclude females and males showed different patterns of healing post-ACL rupture over 31-days, which may impact timing of reconstruction surgery, and possibly clinical outcome.

Adenosine, lidocaine, and magnesium therapy augments joint tissue healing following experimental anterior cruciate ligament rupture and reconstruction

“…Non-invasive ACL rupture of the right hind limb was performed on anaesthetized animals, as described previously. 15 Three days after ACL rupture, animals underwent remnant-sparing ACLR surgery on the right knee using a tail tendon autograft, with postoperative assessment over 28 days ( Figure 1 ). 14 , 15 All surgeries were performed by a single surgeon (PCM) on eight to ten animals per surgery day, with equal numbers of animals per treatment group, blinding of the surgical team and data analyst (JLM) to treatment assignment, and randomization of the order of animals undergoing surgery.…”

“…For pain assessment, the paw withdrawal threshold (PWT) was evaluated in the operated and non-operated knee (Saline, n = 10 per sex; ALM, n = 11 per sex) at day 5, 8, 14, and 28 postoperatively, using an up-down protocol for von Frey filaments, as previously described. 15 Data show the PWT for the operated and non-operated limb after ACLR surgery.…”

“…At day 28 postoperatively, complete blood cell counts were performed (VetScan HM5 hematology analyzer; Abaxis, USA), and inflammatory cytokines and chemokines were measured in plasma and synovial wash (interleukin (IL)-1β, IL-6, interferon-inducible protein (IP)-10, lipopolysaccharide-inducible CXC chemokine (LIX), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein-1 α (MIP-1α), MIP-2, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), tumour necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF)) using custom Milliplex Rat Cytokine/Chemokine Magnetic Bead Panels (Abacus ALS, Australia), as described previously. 15 Baseline ranges for haematology parameters (n = 10 per sex), and plasma and synovial wash inflammatory mediators (n = 8 per sex), were determined in healthy male and female animals.…”

“… 20 Synovial lavage was performed on operated and non-operated knees. 15 Quadriceps atrophy was assessed by comparing wet weights of quadriceps muscles from operated and non-operated hind limbs. Samples of the ACL graft and articular cartilage of the medial femoral condyle (MFC) were collected for gene expression studies (Saline, n = 6 per sex; ALM, n = 7 per sex).…”

“…Immunohistochemical localization of the macrophage marker, CD68 (ED1, 1:125 dilution), and α-SMA (1A4, 1:1,000 dilution) was performed using methods described previously. 14 , 15 , 20 …”