Structural Consequences of a Partial Anterior Cruciate Ligament Injury on Remaining Joint Integrity: Evidence for Ligament and Bone Changes Over Time in an Ovine Model.

BACKGROUND: Severe injury to the knee joint often results in accelerated posttraumatic osteoarthritis (PTOA). In an ovine knee injury model, altered kinematics and degradation of the cartilage have been observed at 20 and 40 weeks after partial anterior cruciate ligament (ACL) transection (p-ACL Tx) surgery. However, changes to the integrity of the remaining intact intra-articular ligaments (posterolateral [PL] band and posterior cruciate ligament [PCL]) as well as the subchondral bone after anteromedial (AM) band Tx remain to be characterized. PURPOSE: (1) To investigate histological alterations to the remaining intact intra-articular ligaments, the synovium, and the infrapatellar fat pad (IPFP) and (2) to quantify subchondral bone changes at the contact surfaces of the proximal tibia at 20 and 40 weeks after AM band Tx. STUDY DESIGN: Descriptive laboratory study. METHODS: Mature female Suffolk cross sheep were allocated into 3 groups: nonoperative controls (n = 6), 20 weeks after partial ACL transection (p-ACL Tx; n = 5), and 40 weeks after p-ACL Tx (n = 6). Ligament, synovium, and IPFP sections were stained and graded. Tibial subchondral bone microarchitecture was assessed using high-resolution peripheral quantitative computed tomography. RESULTS: p-ACL Tx of the AM band led to significant change in histological scores of the PL band and the PCL at 20 weeks after p-ACL Tx (P = .031 and P = .033, respectively) and 40 weeks after p-ACL Tx (P = .011 and P = .029) as compared with nonoperative controls. Alterations in inflammatory cells and collagen fiber orientation contributed to the greatest extent of the combined histological score in the PL band and PCL. p-ACL Tx did not lead to chronic activation of the synovium or IPFP. Trabecular bone mineral density was strongly inversely correlated with combined gross morphological damage in the top and middle layers of the subchondral bone in the lateral tibial plateau for animals at 40 weeks after p-ACL Tx. CONCLUSION: p-ACL Tx influences the integrity (biology and structure) of remaining intact intra-articular ligaments and bone microarchitecture in a partial knee injury ovine model. CLINICAL RELEVANCE: p-ACL Tx leads to alterations in structural integrity of the remaining intact ligaments and degenerative changes in the trabecular bone mineral density, which may be detrimental to the injured athlete's knee joint in the long term.

Correlation of damage score in PTOA with changes in stress on cartilage in an ovine model.

Objective: There is a high risk of developing osteoarthritis (OA) following traumatic injury to the knee. Severe ligament injuries can disrupt the integrity of the multicomponent knee at both biological and biomechanical levels. We hypothesize changes in cartilages stresses could lead to tissue damage and development of OA. Design: The in-vivo gait kinematics of the stifle (knee) joint of four adult female ovine subjects were recorded prior to and at ten-and-twenty weeks following partial ACL-MCL transection. The subjects were sacrificed and the experimental joint from each subject was mounted on a parallel robotic system programmed with the kinematic findings. Ten custom-built Fibre Bragg Grating optic sensors were arranged to measure contact stresses on the surface of the tibial plateau articular cartilage. These sensors provide the first accurate stress measurements in a joint during gait replication using the previously recorded in-vivo kinematics. The relationship between the results obtained and observed focal damage was assessed. Results: The locations on the tibial plateaus that experienced the greatest change in contact stresses corresponded with the locations of focal damage development. No direct link was detected between individual animal differences in kinematics and variations in stress magnitudes or the development of focal cartilage damage. Conclusions: The findings highlight the importance of mechanical stress determinants in the integrated set point for the knee (with individual variation), and how injury-related stress changes correlate with development of PTOA.

Posttraumatic Osteoarthritis Development and Progression in an Ovine Model of Partial Anterior Cruciate Ligament Transection and Effect of Repeated Intra-articular Methylprednisolone Acetate Injections on Early Disease.

BACKGROUND: Partial anterior cruciate ligament (p-ACL) ruptures are a common injury of athletes. However, few preclinical models have investigated the natural history and treatment of p-ACL injuries. PURPOSE: To (1) demonstrate whether a controlled p-ACL injury model (anteromedial band transection) develops progressive gross morphological and histological posttraumatic osteoarthritis (PTOA)-like changes at 20 and 40 weeks after the injury and (2) investigate the efficacy of repeated (0, 5, 10, and 15 weeks) intra-articular injections of methylprednisolone acetate (MPA; 80 mg/mL) in the mitigation of potential PTOA-like changes after p-ACL transection. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty-one 3- to 5-year-old female Suffolk-cross sheep were allocated to 4 groups: (1) nonoperative controls (n = 5), (2) 20 weeks after p-ACL transection (n = 5), (3) 40 weeks after p-ACL transection (n = 6), and (4) 20 weeks after p-ACL transection + MPA (n = 5). Gross morphological grading and histological analyses were conducted. mRNA expression levels for inflammatory, degradative, and structural molecules were assessed. RESULTS: p-ACL transection led to significantly more combined gross damage ( P = .008) and significant aggregate histological damage ( P = .009) at 40 weeks after p-ACL transection than the nonoperative controls, and damage was progressive over time. Macroscopically, MPA appeared to slightly mitigate gross damage at 20 weeks after p-ACL transection in some animals. However, microscopic analysis revealed that repeated MPA injections after p-ACL transection led to significant loss in proteoglycan content compared with the nonoperative controls and 20 weeks after p-ACL transection ( P = .008 and P = .008, respectively). CONCLUSION: p-ACL transection led to significant gross and histological damage by 40 weeks, which was progressive over time. Multiple repeated MPA injections were not appropriate to mitigate injury-related damage in a p-ACL transection ovine model as significant proteoglycan loss was observed in MPA-treated knees. CLINICAL RELEVANCE: A p-ACL injury leads to slow and progressive PTOA-like joint damage, and multiple repeated injections of glucocorticoids may be detrimental to the knee joint in the long term.

Location and gene-specific effects of methylprednisolone acetate on mitigating IL1ß-induced inflammation in mature ovine explant knee tissue.

OBJECTIVE AND DESIGN: To determine the ability of methylprednisolone acetate (MPA) to influence interleukin 1ß (IL1ß)-induced gene expression in ovine knee joint tissues. MATERIAL OR SUBJECTS: Ovine articular cartilage, synovium, and infrapatellar fat pad (IPFP) explants. TREATMENT: Explants were treated with 10-3 M or 10-4 M MPA. METHODS: Explant treatment groups: (1) control (DMEM); (2) inflammation (IL1ß); (3) IL1ß + 10-3 M MPA; or (4) IL1ß + 10-4 M MPA. Cell viability was assessed pre- and post-treatment. Expression of mRNA levels for inflammatory, degradative, anabolic, innate immunity, and adipose-related molecules was quantified via qPCR, and analyzed via the comparative C T method. RESULTS: Except for IL8 in a subset of cartilage locations, matrix metalloproteinases (MMPs) were the only genes consistently affected by MPA. MPA mitigated IL1ß-induced MMP3 expression levels in all regions of the articular cartilage, and in the synovium and IPFP, while MMP1 mRNA expression levels were significantly decreased with MPA after IL1ß in the tibial plateau and synovium, but paradoxical increases in the IPFP. MMP13 mRNA expression levels exhibited significant decreases with MPA after IL1ß in the femoral condyles, tibial plateau, synovium, and IPFP. CONCLUSIONS: MPA treatment suppressed IL1ß-induced mRNA levels for MMPs in articular cartilage, synovium, and IPFP and was found to be tissue-, location-, and gene-specific.

Use of pre-clinical surgically induced models to understand biomechanical and biological consequences of PTOA development.

Post-traumatic osteoarthritis (PTOA) development is often observed following traumatic knee injuries involving key stabilising structures such as the cruciate ligaments or the menisci. Both biomechanical and biological alterations that follow knee injuries have been implicated in PTOA development, although it has not been possible to differentiate clearly between the two causal factors. This review critically examines the outcomes from pre-clinical lapine and ovine injury models arising in the authors' laboratories and differing in severity of PTOA development and progression. Specifically, we focus on how varying severity of knee injuries influence the subsequent alterations in kinematics, kinetics, and biological outcomes. The immediate impact of injury on the lubrication capacity of the joint is examined in the context of its influence on biomechanical alterations, thus linking the biological changes to abnormal kinematics, leading to a focus on the potential areas for interventions to inhibit or prevent development of the disease. We believe that PTOA results from altered cartilage surface interactions where biological and biomechanical factors intersect, and mitigating acute joint inflammation may be critical to prolonging PTOA development. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:454-465, 2017.

The infrapatellar fat pad is affected by injury induced inflammation in the rabbit knee: use of dexamethasone to mitigate damage.

OBJECTIVE AND DESIGN: The health of the infrapatellar fat pad (IFP) has been linked to pain, joint inflammation, and the onset of post-traumatic osteoarthritis. Thus, early inflammation effects on the IFP could have long term sequelae on joint integrity. This study was designed to characterize the natural history of the IFP in a model of surgically induced knee injury and inflammation, and to test the efficacy of one intra-articular (IA) administration of dexamethasone (DEX) immediately following surgery. METHODS: An IA bone drill hole injury to the rabbit knee was conducted and immediately treated with DEX (n = 12). Early and late post-surgical time-points were investigated (48 h and 9 weeks) and the outcome measures were analysis of IFP histology, mRNA levels for relevant molecules, and protein levels for a subset of cytokines. Data were analyzed against a surgical control (injury without treatment; n = 12), a surgical sham (capsular incision only; n = 12), and normal control (n = 6). TREATMENT: Single IA injection of DEX (0.5 mg/kg), administered at the completion of surgery. RESULTS: IFPs from injured joints exhibited significantly increased cellularity and early fibrosis at 48 h post surgery. While the histological inflammation from a capsular incision alone resolved, knee injured animals progressed to a significantly more fibrotic IFP by 9 weeks. DEX significantly lowered histological scores at 48 h, but not at the 9 weeks. DEX did not influence mRNA levels for IL-1ß, 6, and 8, however, protein analysis indicated that IL-8 levels were lower in DEX treated joints. DEX resulted in significantly elevated expression of mRNA for MCP-1, leptin, and VEGF. CONCLUSION: One IA administration of a glucocorticoid appears to mitigate the initial inflammation within the joint, but is not sufficient to protect the joint to 9 weeks post-surgery.

Single intra-articular dexamethasone injection immediately post-surgery in a rabbit model mitigates early inflammatory responses and post-traumatic osteoarthritis-like alterations.

Despite surgical reconstruction of the anterior cruciate ligament, a significant number of patients will still develop post-traumatic osteoarthritis (PTOA). Our objective was to determine if mitigating aspects of the acute phase of inflammation following a defined knee surgery with a single administration of a glucocorticoid could prevent the development of PTOA-like changes within an established rabbit model of surgically induced PTOA. An early and late post-surgical time-point was investigated in this study (48 h and 9 weeks post-surgery) in which the following groups were repeated (each n=6, for a total of 24 rabbits per time-point, and 48 rabbits used in the study): control (age/sex matched), sham (arthrotomy), drill injury (arthrotomy+two drill holes to a non-cartilaginous area of the femoral notch), and drill injury+single intra-articular (IA) injection of dexamethasone (DEX). At 48 h post-surgery, DEX treatment significantly lowered the mRNA levels for a subset of pro-inflammatory mediators, and significantly lowered the histological grade. Nine weeks post surgery, DEX treatment significantly lowered the histological scores (presented as effect size) for synovium (3.8), lateral femoral condyle (3.9), and lateral tibial cartilage (5.1) samples. Thus, DEX likely acts to prevent injury induced inflammation that could contribute to subsequent joint damage.

Alterations in Hoffa's fat pad induced by an inflammatory response following idealized anterior cruciate ligament surgery.

OBJECTIVE: To determine whether inflammation following anterior cruciate ligament (ACL) reconstruction leads to long-term pathological changes in the infrapatellar fat pad (IPFP or Hoffa's fat pad) which could compromise the integrity of the knee joint. MATERIALS AND METHODS: Sixteen mature sheep underwent anatomic idealized ACL reconstruction surgery (ACL-R) and were sacrificed at 2 weeks (n = 9) and 20 weeks (n = 7) post-ACL-R. Five additional animals served as unoperated controls. A histological grading protocol was developed to quantify the changes in the IPFP post-injury. mRNA expression levels for key markers of inflammation, angiogenesis and tissue regeneration were assessed by qPCR. RESULTS: The IPFP exhibited altered cellularity and fibrosis at 2 and 20 weeks post-ACL-R. Immunohistochemistry detected macrophage-like cells in the IPFP which were increased at 20 weeks. Specific pro-inflammatory cytokines and IPFP specific adipokines exhibited changes indicating early inflammation mediated alterations. Elevations in CD105 mRNA levels at 2 weeks corroborated the increases in neovascularization observed in the IPFP following injury. CONCLUSIONS: Sustained long-term pathological changes stemming from inflammation are present in IPFP tissue after ACL-R surgery and may compromise the long-term integrity of the knee joint.

Normal sheep synovium has similar appearances and constitutive expression of inflammatory cytokines within and between knee joints: a baseline histological and molecular analysis.

Abstract Clinical evidence suggests that synovium can add to adjacent articular cartilage damage, potentially contributing to the development of osteoarthritis (OA). Inflammation of the synovium (synovitis) is dependent on the type of injury sustained, the time after injury and concomitant changes in other joint tissues. To define the role of synovitis in OA development, there is a need for baseline measures that can reliably distinguish synovial inflammation from normal synovium both within and between joints. This study tested the hypothesis that normal synovium from distinct anatomical locations in young and adult sheep is homogeneous with respect to consistently low molecular expression of the inflammatory mediators - tumour necrosis factor alpha (TNF-α) and interleukins (IL) such as IL-1ß, IL-1Ra, IL-6 and IL-8. Additionally, maturation will not influence the expression of these select inflammatory biomarkers. Samples of synovium from four anatomic locations (medial and lateral margins, suprapatellar pouch (patella region), posterior to the posterior cruciate ligament, from each joint of 5 adult and 4 immature animals were graded histologically or analyzed for mRNA expression of inflammatory cytokines. Histologically, no evidence of synovitis was noted although some variance in sub-intimal fibrosis was observed between sample locations in mature sheep. Molecular expression of all inflammatory mediators was low and homogeneously expressed at constitutive levels in all sample locations. These findings confirm the hypothesis that the normal sheep synovium is a homogeneous tissue throughout the joint and establishes the baseline expression levels for several pro-inflammatory mediators in both immature and mature sheep.

Estrogen receptor beta and truncated variants enhance the expression of transfected MMP-1 promoter constructs in response to specific mechanical loading.

BACKGROUND: Joint diseases such as osteoarthritis (OA) predominantly afflict post-menopausal women, suggesting a pertinent role for female hormones. Estrogen receptor beta (ER-ß) has been detected in connective tissues of the knee joint suggesting that these tissues are responsive to the hormone estrogen. Matrix metalloproteinase-1 (MMP-1) activity contributes to cartilage degradation, a key factor leading to OA development in synovial joints. Two polymorphic forms of MMP-1 exist due to a deletion/insertion of the guanine residue in the promoter, and the 2G allelic variant of MMP-1 exhibits more activity than the 1G allele. Previous studies have demonstrated that the polymorphic forms of the human MMP-1 are influenced by the modulating effects of estrogen receptor isoforms. In addition to hormonal influences, physiological factors such as altered mechanical loading are also contributory features of OA. In the present study, the combined influence of biomechanical and hormonal variables on the activity of MMP-1 isoforms was evaluated. We hypothesized that the combined effects of ER-ß and sheer stress will differentially activate the two allelic forms of MMP-1 in a hormone-independent manner. METHODS: HIG-82 synoviocytes were transiently transfected with 1G or 2G alleles (±) ER-ß and subjected to either shear or equibiaxial stress. Next, 1G/2G promoter activity was measured to determine the combined influence of physiological stimuli. Truncated ER-ß constructs were used to determine the importance of different domains of ER-ß on 1G/2G activation. RESULTS: The 2G allele exhibited a constitutively higher activity than the 1G allele, which was further increased when the transfected cells were subject to shear stress, but not equibiaxial stress. Moreover, the combination of ER-ß and shear stress further increased the activity levels of the 1G/2G allelic variants. Additionally, select AF-2 truncated ER-ß variants led to increased activity levels for the 2G allele, indicating the AF-1 domain was likely involved in the response to mechanical stimulation. CONCLUSIONS: These results suggest that the 1G/2G alleles of MMP-1 are influenced by specific mechanical stimuli like shear stress, as well as the ER-ß receptor. These findings contribute to the potential allelic involvement in connective tissue diseases such as OA in females compared to males.

Characterization of proteoglycan 4 and hyaluronan composition and lubrication function of ovine synovial fluid following knee surgery.

The objective of this study was to determine changes in (1) proteoglycan 4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, and (3) cartilage boundary lubricating ability of synovial fluid (SF) from surgical sham (SHAM), anterior cruciate ligament (ACL)/medial collateral ligament (MCL) transection, and lateral meniscectomy (MEN) in a post-knee surgery ovine model. Ovine SF (oSF) was collected at euthanization 20 weeks after surgery, with the contralateral joint serving as the non-operative control. PRG4 and HA concentration in oSF was measured by sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubricating ability of oSF was measured by a cartilage-cartilage friction test. PRG4 and HA concentration in SHAM, ACL/MCL, and MEN oSF were similar in comparison to the contralateral control (CTRL) oSF. The HA MW distribution in the operated oSF for all ranges were similar to the respective CTRL oSF. The kinetic coefficients of friction in operated and CTRL oSF were similar in all groups, and were significantly lower than saline. These results indicate oSF lubricant composition and function at 20 weeks post-knee surgery were similar to contralateral CTRL, and suggest earlier time points post surgery warrant further investigation.

Carrageenan-induced transient inflammation in a rabbit knee model: molecular changes consistent with an early osteoarthritis phenotype.

OBJECTIVE: Inflammation following a knee injury is one of the factors associated with initiation of cartilage degeneration leading to osteoarthritis (OA). The hypothesis tested was that inflammation results in elevated expression of proteinases implicated in OA. METHODS: Mature female rabbits received a single carrageenan injection to the right hind knee and the left knee served as the control. Five animals were killed at time points of 1, 2 and 4 weeks. The synovium and cartilage from both knees were collected and analysed for specific mRNA levels. RESULTS: Interleukin (IL)-1ß and IL-6 mRNA levels peaked at 2 weeks and returned to normal levels in tissues by 4 weeks post-carrageenan treatment. Matrix metalloproteinase (MMP)-13, MMP-1, MMP-3 and cathepsin K followed the trend set by the inflammatory cytokines. Both synovium and cartilage tissues exhibited similar patterns of molecular expression, with cartilage from the tibial plateau responding more strongly than the femoral condyles. CONCLUSIONS: The acute inflammatory milieu controls the transient expression of many degradative proteinases in the knee. However, a single acute exposure to inflammation in the rabbit knee is insufficient to create a chronic inflammatory environment and other complementary factors, such as persistent mechanical instability and/or injury, may contribute to the establishment of OA.

Post-natal molecular adaptations in anteromedial and posterolateral bundles of the ovine anterior cruciate ligament: one structure with two parts or two distinct ligaments?

The human anterior cruciate ligament (ACL) is a composite structure of two anatomically distinct bundles: an anteromedial (AM) and posterolateral (PL) bundles. Tendons are often used as autografts for surgical reconstruction of ACL following severe injury. However, despite successful surgical reconstruction, some people experience re-rupture and later development of osteoarthritis. Understanding the structure and molecular makeup of normal ACL is essential for its optimal replacement. Reportedly the two bundles display different tensions throughout joint motion and may be fundamentally different. This study assessed the similarities and differences in ultrastructure and molecular composition of the AM and PL bundles to test the hypothesis that the two bundles of the ACL develop unique characteristics with maturation. ACLs from nine mature and six immature sheep were compared. The bundles were examined for mRNA and protein levels of collagen types I, III, V, and VI, and two proteoglycans. The fibril diameter composition of the two bundles was examined with transmission electron microscopy. Maturation does alter the molecular and structural composition of the two bundles of ACL. Although the PL band appears to mature slower than the AM band, no significant differences were detected between the bundles in the mature animals. We thus reject our hypothesis that the two ACL bundles are distinct. The two anatomically distinct bundles of the sheep ACL can be considered as two parts of one structure at maturity and material that would result in a structure of similar functionality can be used to replace each ACL bundle in the sheep.

Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

Early joint tissue changes are highly correlated with a set of inflammatory and degradative synovial biomarkers after ACL autograft and its sham surgery in an ovine model.

While impossible in humans, the mechanisms of early cartilage, bone and meniscal damage can be quantified after anterior cruciate ligament (ACL) injury in animal models. We utilized an ovine model to determine if the mRNA expression of inflammatory and degradative molecules (IL-1ß, IL-6, MMP-1, 2, 3, and 13) in the synovium correlated to changes in joint tissues 2 weeks post-ACL surgery, to test the hypothesis that synovial inflammation is a marker of these changes and possibly their originator. Nine "idealized" ACL autografts were performed and compared with three sham and six normal animals. Using validated protocols, early osteophyte formation, articular cartilage, and meniscal damage were quantified. Synovium was harvested and mRNA expression quantified using qPCR. Multiple linear regression analysis (MLRA) was utilized to correlate synovial mRNA expression in treated and contra-lateral limbs, from all treatment groups with corresponding joint scores. Synovial mRNA expression was significantly elevated in all experimental and sham joints. The MLRA model was a significant predictive tool (p = 0.001, R(2) = 0.70) of gross tissue scores with significant contributions from IL-1ß, IL-6, and MMP-3. Findings suggest that this set of synovial biomarkers is predictive (p < 0.009) of early gross changes of joint tissues after arthrotomy and likely directly involved in the relevant mechanisms, particularly early osteophyte formation, in vivo.

Molecular events surrounding collagen fibril assembly in the early healing rabbit medial collateral ligament--failure to recapitulate normal ligament development.

??Although injuries to the medial collateral ligament (MCL) can heal functionally without surgical intervention, the collagen fibers in the healing tissue remain compromised. The molecular basis for this poor healing potential was investigated by examining extracellular matrix-modifying molecules such as bone morphogenetic protein 1 (BMP-1), procollagen C proteinase enhancer (PCOLCE), lysyl oxidase (LOX), and transforming growth factor beta 1 (TGF-ß1) involved in collagen fibrillogenesis during normal early postnatal ligament maturation and at comparable intervals after MCL injury. Samples of midsections of rabbit MCLs were collected from 3-, 6-, 14-, and 52-week-old normal animals and at 3, 6, and 14 weeks postinjury. Harvested midsubstance tissues were analyzed for collagen fibril diameter by transmission electron microscopy (TEM), and mRNA levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR). Results showed different patterns of expression between normal MCL maturation and during scar maturation. BMP-1 and PCOLCE mRNA levels were upregulated in the 3?14-week period during maturation of normal ligaments but decreased at skeletal maturity. The scar tissue exhibited a 3.5-fold increase in PCOLCE mRNA levels during the early healing phase, but these decreased with time. After injury, BMP-1 mRNA levels in scars were low and did not change during healing. Both LOX and TGF-ß1 mRNA levels were low during normal MCL development compared with levels at maturity and exhibited elevated mRNA levels during early healing that decreased with time postinjury. These results suggest that gene expression in scars during MCL healing does not recapitulate expression in normal ligament fibroblasts during maturation.

Influence of bilateral medial collateral ligament injury on mRNA expression in distal corneal tissues of control and ovariohysterectomized rabbits.

PURPOSE: Corneal tissues are reported to be impacted by physiological changes (eg, menopause), systemic autoimmune diseases, and osteoarthritic-like conditions. In this study, changes in specific mRNA levels in the cornea after a ligament injury in normal and rabbits subjected to surgical menopause were examined. METHODS: Skeletally mature female rabbits were either sham-operated (control) or were subjected to surgical menopause (OVX). Eight weeks post-OVX, subsets of control and OVX animals were subjected to bilateral injuries to their medial collateral ligaments (MCL) of the knee, and 6 and 14 weeks postinjury, corneal tissues were harvested. Using reverse transcriptase-polymerase chain reaction, mRNA levels for several relevant molecules, including matrix molecules, growth factors, cytokines, proteinases, and hormone receptors, were assessed. RESULTS: mRNA levels for estrogen receptor, decorin, collagens, several growth factors, and inflammatory cytokines decreased in central corneal tissue 6 weeks after distal MCL injury in control animals. The central corneal tissues of animals subjected to OVX alone also exhibited decreases in mRNA levels for a similar set of molecules. When OVX animals were further subjected to MCL injury, the mRNA levels for many of these molecules did not vary from those in the uninjured OVX group. Interestingly, mRNA levels for most molecules were still altered 14 weeks post-MCL injury in the control and OVX animals, a time when the MCL has healed. CONCLUSIONS: Corneal tissues respond to changes resulting from OVX and/or injury. OVX combined with a ligament injury does not appear to have an additive impact on corneal mRNA levels for most of the molecules assessed.

Distinct roles for AF-1 and -2 of ER-alpha in regulation of MMP-13 promoter activity.

Previous studies have indicated that ER-alpha can influence the activity of the MMP-13 promoter. ER-alpha activity is mediated by two separate transcriptional activation domains (AF-1 and AF-2). The present study focused on analyzing the roles of these domains on the activation of the MMP-13 promoter. Transfection of synoviocytes with an ER-alpha construct lacking the C-terminus AF-2 domain led to significant elevation in MMP-13 promoter activity compared to wild type ER-alpha. Progressive deletions in the N-terminal AF-1 domain led to significant losses in MMP-13 promoter activity. MMP-13 promoter mutagenesis indicated that an AP-1 regulatory site was essential for ER-alpha mutant activity. Thus, both AF-1 and AF-2 domains of ER-alpha are required for regulation of MMP-13 promoter activity. As ER variants and ER related proteins have been implicated in bone and joint disorders, these findings provide understanding of the possible role of ER variants in the development of such conditions.

Influence of timing (pre-puberty or skeletal maturity) of ovariohysterectomy on mRNA levels in corneal tissues of female rabbits.

PURPOSE: Corneal thickness and curvature are reported to be influenced by hormonal changes associated with menstrual cycle, pregnancy, or menopause. However, the molecular mechanisms leading to these alterations are not clearly understood. The present study focuses on gene expression patterns (mRNA levels) in corneal tissues following surgically induced menopause in an animal model. The impact of lower hormone levels on mRNA levels in corneal tissues after pre-puberty ovariohysterectomy (OVX) was compared to that in skeletally mature adult animals. METHODS: Skeletally mature adult female rabbits were either left unoperated (control) or were subjected to OVX at 54 weeks of age using an approved protocol. The central (approximately 6 mm) and the peripheral corneal tissues were harvested from normal and OVX rabbits eight weeks after surgery. In a second study, young sexually immature rabbits at eight weeks of age were subjected to OVX and corneal tissues were collected when the animals were 22 and 32 weeks of age. In both experiments, RNA was isolated from corneal tissues and RT-PCR was used to assess mRNA levels for several relevant molecules. RESULTS: When mature animals were examined eight weeks after OVX, mRNA levels for molecules such as the estrogen receptor, decorin, collagen I, collagen V, and several growth factors were found to be significantly decreased in central corneal tissues. Interestingly, no corresponding changes in mRNA levels were observed for these same molecules in peripheral corneal tissues. When young, pre-pubertal animals were subjected to OVX, mRNA levels were found to be mainly unchanged for the OVX animals at 22 weeks of age i.e., after 14 weeks of low hormone conditions. However, significant decreases in mRNA levels for a similar subset of molecules were observed when the animals were at least 32 weeks of age, i.e., after 24 weeks of a low hormone environment. Examination of peripheral corneal tissues did not show significant changes in mRNA levels due to OVX at either 22 or 32 weeks of age except for collagens I and V at 32 weeks of age. CONCLUSIONS: These results indicate significant alterations in mRNA levels in the central corneal tissues of rabbits following OVX. Interestingly, peripheral corneal tissues show very little alteration in mRNA levels for the same molecules. Furthermore, OVX had a more rapid impact on mRNA levels in mature animals than in skeletally immature animals. Thus, loss of hormone producing tissues during growth and maturation apparently delayed the impact of hormone removal compared to loss after maturity had been attained and growth stimuli are likely absent. Therefore, specific areas of the cornea are more responsive to hormone levels than others. The impact of the loss of hormones is influenced by the maturation state of the rabbit, but mRNA levels for a similar subset of genes are affected by OVX in both age groups.

Polymorphisms in the promoter regions for human MMP-1 and MMP-13 lead to differential responses to the alpha and beta isoforms of estrogen receptor and their ligand in vitro.

Estrogen receptors (ER) are present in connective tissues and therefore it is possible that the loss of estrogen after menopause influences the integrity of these tissues, contributing to development of degenerative conditions such as osteoporosis and osteoarthritis in a subset of women. Aberrant expression of matrix metalloproteinases (e.g. MMP-1 and MMP-13) has been implicated in the progression of these diseases. The present study investigated potential molecular mechanisms involved in the regulation of expression of MMP-1 and MMP-13 promoter variants by ER-alpha and ER-beta (+/-estrogen) in a transient transfection system. The results demonstrate that the activity of human MMP-1 and MMP-13 polymorphic variants is elevated in the presence of ER-alpha and ER-beta, and the single nucleotide polymorphisms present in the promoters of MMP-1 and MMP-13 variants leads to differential activities in response to the ER isoforms. Furthermore, the influence of 17-beta estradiol also varies depending upon whether the alpha or the beta isoform of ER is the modulator of these polymorphic variants. These findings support the conclusion that ER isoforms may be contributing to disease development and/or progression in genetically distinct subsets of women following menopause, and provide mechanistic insights into how such contributions are manifested.