Animal Models Of Post-traumatic Osteoarthritis Research Articles

Comparative transcriptomic analysis of articular cartilage of post-traumatic osteoarthritis models.

Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here, skeletally mature C57Bl6 mice were subjected to either rapid-onset non-surgical mechanical rupture of the anterior cruciate ligament (ACL) or to surgical destabilisation of the medial meniscus (DMM). Transcriptome profiling of micro-dissected cartilage at day7 or day42 following ACL or DMM procedure, respectively, showed that the two models were comparable and highly correlative. Gene ontology (GO) enrichment analysis identified similarly enriched pathways that were overrepresented by anabolic terms. To address the transcriptome changes more completely in the ACL model, we also performed small RNA sequencing, describing the first microRNA profile of this model. miR-199-5p was amongst the most abundant, yet differentially expressed, microRNAs, and its inhibition in primary human chondrocytes led to a transcriptome response that was comparable to that observed in both human 'OA damaged vs intact cartilage' and murine DMM cartilage datasets. We also experimentally verified CELSR1, GIT1, ECE1 and SOS2 as novel miR-199-5p targets. Together, these data support the use of the ACL rupture model as a non-invasive companion to the DMM model.

A pilot study to determine the optimal dose of scAAVIL-1ra in a large animal model of post-traumatic osteoarthritis

Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related increase in IL-1ra levels with a decrease in PGE2 levels was observed, with the peak IL-1ra concentration being observed 7 days post-treatment in all groups. Perivascular infiltration with mononuclear cells was observed within the synovial membrane of the joint treated with the highest viral dose of 5 × 1012 vg, but this was absent in the lower-dosed joints. The second-highest dose of scAAVeqIL-1ra 5 × 1011 vg demonstrated elevated IL-1ra levels without any cellular response in the synovium. Taken together, the data suggest that the 10-fold lower dose of 5 × 1011vg scAAVIL-1ra would be a safe therapeutic dose in an equine model of PTOA.

IMPACT OF EXERCISE ON POSTTRAUAMTIC OSTEOARTHRITIS AFTER KNEE JOINT INJURY: A SYSTEMATIC REVIEW OF ANIMALS WITH ANTERIOR CRUCIATE LIGAMENT OR MEDIAL MENISCUS

Purpose: A previous meta-anlysis of studies involving healthy animals demonstrated that a moderate daily dose of exercise may be chondroprotective. However, it remains unknown if exercise following joint injury may either accelerate posttraumatic changes or have a chondroprotective effect in animal models of posttraumatic osteoarthritis (PTOA). The purpose of the current systematic review and meta-analysis was to evaluate the role of exercise on knee cartilage changes in animals with either anterior cruciate ligament or medial meniscus transection.

In vivo multimodal imaging of hyaluronan-mediated inflammatory response in articular cartilage

One driving factor in the progression to posttraumatic osteoarthritis (PTOA) is the perpetuation of the inflammatory response to injury into chronic inflammation. Molecular imaging offers many opportunities to complement the sensitivity of current imaging modalities with molecular specificity. The goal of this study was to develop and characterize agents to image hyaluronan (HA)-mediated inflammatory signaling. We developed optical (Cy5.5-P15-1) and magnetic resonance contrast agents (Gd-DOTA-P15-1) based in a hyaluronan-binding peptide (P15-1) that has shown anti-inflammatory effects on human chondrocytes, and validated them in vitro and in vivo in two animal models of PTOA. In vitro studies with a near infrared (NIR) Cy5.5-P15-1 imaging agent showed a fast and stable localization of Cy5.5-P15-1 on chondrocytes, but not in synovial cells. In vivo NIR showed significantly higher retention of imaging agent in PTOA knees between 12 and 72h (n=8, Cohen's d>2 after 24h). NIR fluorescence accumulation correlated with histologic severity in cartilage and meniscus (ρ between 0.37 and 0.57, P<0.001). By using in vivo magnetic resonance imaging with a Gd-DOTA-P15-1 contrast agent in 12 rats, we detected a significant decrease of T1 on injured knees in all cartilage plates at 48h (-15%, 95%-confidence interval (CI)=[-18%,-11%]) while no change was observed in the controls (-2%, 95%-CI=[-5%,+1%]). This study provides the first in vivo evidence that hyaluronan-related inflammatory response in cartilage after injury is a common finding. Beyond P15-1, we have demonstrated that molecular imaging can provide a versatile technology to investigate and phenotype PTOA pathogenesis, as well as study therapeutic interventions.

Experimental animal models of post-traumatic osteoarthritis of the knee.

Due to the complex and dynamic nature of osteoarthritis (OA) and post-traumatic osteoarthritis (PTOA), animal models have been used to investigate the progression and pathogenesis of the disease. Researchers have used different experimental models to study OA and PTOA. With an emphasis on the knee joint, this review will compare and contrast the existing body of knowledge from anterior cruciate ligament transection models, meniscectomy models, combination models, as well as impact models in large animals to see how tissues respond to these different approaches to induce experimental OA and PTOA. The tissues discussed will include articular cartilage and the meniscus, with a focus on morphological, mechanical and histological assessments. The goal of this review is to demonstrate the progressive nature of OA by indicating the strong correlation between progressive tissue degeneration, change of mechanical properties, and loss of biochemical integrity and to highlight key differences between the most commonly used experimental animal models.

High resolution digital imaging of India ink stained (HiReD-IIIs) rabbit knee articular cartilage reveals site-specific damage patterns at different stages of progression

Purpose: Human articular cartilage (hAC) exhibits degeneration that is site-specific and exhibits six characteristic patterns of early surface damage. These hAC patterns were distinguished by staining the cartilage surface with India Ink and photographing en face with film providing ∼(4 um)2 pixel resolution. However, the mechanisms for the initiation and progression of such damage are unclear. Modern 2-D digital imaging allows quantitative imaging over large areas, and 3D histology (3DH) allows structural visualization at ∼(0.5-1 μm)3 voxel resolution. Animal models of post-traumatic osteoarthritis (PTOA) may help elucidate the underlying mechanical and/or biological mechanisms of such damage. In the rabbit ACL transection (rACLT) model of PTOA, cartilage damage progresses steadily over 9 weeks. The aims of the present study were to analyze site-specific AC damage at four weeks after rACLT by (1) High Resolution Digital Imaging of India Ink Staining (designated here as HiReD-IIIS, “hired eyes”), (2) elucidate the 3D pattern of surface damage by 3DH, and (3) assess the extent of inhomogeneity and anisotropy of cartilage surface damage at weight-bearing sites. Methods: Rabbit Samples. With IACUC approval, ten adult female New Zealand White rabbits (11-19 mo, weight = 3.8-5.2 kg) were subjected to unilateral (right knee) ACLT to induce PTOA. At day 28, rabbits were sacrificed, the ACLT and contralateral non-operated left (CTRL) knees were harvested, and the FCs were exposed. High Resolution Digital Imaging. AC surfaces were aligned, dabbed with a 1:5 dilution of India Ink in phosphate buffered saline (PBS) + proteinase inhibitors, and digitally imaged at high resolution (4 μm)2/pixel. Custom Image Processing. Gray-scale images were normalized to 0 (darkest) and 1 (brightest) using standards, and cropped to isolate the weight bearing regions: posteromedial [PostMed] and posterolateral [PostLat] on each condyle. Surface damage area was calculated in two locations of the size ∼15mm2 on a) medial and b) lateral aspects of each weight bearing region (PostMed-M, PostMed-L, PostLat-M and PostLat-L) using 2-D morphometric analysis (adaptive thresholding by mean, despeckling, and morphological operations [closing]), normalized to the overall selected condyle area. The overall surface damage orientation, defined relative to horizontal axis ranging from [-90o, 90o], was approximated by vector analysis. Statistics. Damage area data are shown as mean ± SE. At each weight bearing region, ACLT and CTRL were compared for normalized damage area by two-way ANOVA, and for damage area and orientation association by Pearson correlation. Significance was taken as p<0.05. Results: HiReD-IIIS revealed site-specific articular cartilage (AC) deterioration patterns (Figure 1) including [1-TCL] transverse curvilinear lines (perpendicular to articulation axis), [2-PCL] parallel curvilinear lines (parallel to articulation axis), [3-RST] reticular sawtooth, and [4-R] ravine (tortuous/curvilinear contours), in the (L)ateral and (M)edial femoral condyle (FC) load-bearing regions, confirmed by 3DH. ACLT induced detectable damage, increasing normalized damage area (Figure 2A) by 4-30% in PostLat-M site, with 80% of the FCs’ lesion area concentrated between 5-25%, as compared to CTRL (p<0.0001). Surface damage patterns in all locations had an increasing trend in area with PostLat-M and PostMed-L reaching a maximum of 35% and 23%, respectively. Damage area was negatively correlated with the overall lesion orientation in the PostLat-L region (r = -0.66). Surface damage, R, was the primary pattern at PostLat-M, appearing at different states of progression. On PostLat-L, normalized damage areas of <5% and 5-18% corresponded with TCL and RST patterns, respectively (Figure 2B). On PostMed-L, lesion areas of <10% matched with PCL and TCL, and ≥10% with RST patterns. Conclusions: The integrated high-resolution image acquisition and morphometric lesion analysis revealed (1) a trajectory for evolution of TCL into RST in the PostLat-L and of TCL and PCL into RST on the PostMed-L regions, and (2) progressive occurrence of site-specific lesions that are more prominent on the PostMed-L and PostLat-M, which could be indicative of altered contact between joint constituents, leading to abnormal loading modes and directions in a destabilized joint.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis

To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

How to establish an expected animal model of post-traumatic osteoarthritis?

Abstract Background Animal models of osteoarthritis (OA), including post-traumatic osteoarthritis and spontaneous osteoarthritis, have been established in many ways. In recent years, there have been many reports in various foreign academic journals, but animal models of post-traumatic osteoarthritis (distinct from spontaneous osteoarthritis) have rarely been established or summarized in these reports. Animal models of post-traumatic osteoarthritis show different characteristics depending on the animal species and modeling methods used, which is why we have written this article. Objective To summarize the research progress and research status of animal models of post-traumatic osteoarthritis. Methods A retrospective review of the animal model of post-traumatic osteoarthritis (OA) was conducted on the basis of reports retrieved from the PubMed database with the keywords for searching “animal model, post-traumatic osteoarthritis (PTOA)” from October 2006 to October 2016 and confided English language. A total of 80 academic articles on the study of animal models of traumatic osteoarthritis were retrieved, and 34 of them were included in this literature review after reading the free full-text of them. Results Different PTOA models based on different modeling methods and different animal species had their own characteristics. Different modeling methods should be selected according to different modeling animals. Conclusions Considering the project funds, experimental objectives and technical conditions, appropriate experimental animal and modeling method should be selected based on synthetic considerations to obtain an appropriate PTOA model and ideal experimental results.

A Biomechanical Study of Two Distinct Methods of Anterior Cruciate Ligament Rupture, and a Novel Surgical Reconstruction Technique, in a Small Animal Model of Posttraumatic Osteoarthritis.

Small animal models are critical for studies of sports-related knee injury and disease such as posttraumatic osteoarthritis (PTOA) following anterior cruciate ligament (ACL) rupture. In such models, ACL damage can be achieved by surgical transection or, using a more recent innovation, by noninvasive biomechanical means. Whether these approaches differentially alter normal mechanics is unknown. Furthermore, while surgical reconstruction of ruptured ACL can greatly improve joint stability, its effect on PTOA development is also unclear. Our primary purpose was to characterize rodent knee joint mechanics in two models of ACL rupture using a novel quantitative laxity mechanical test. Our secondary aim was to characterize a new reconstruction technique using autograft tail tendon, and to assess its effect on joint mechanics. Our hypothesis was that surgical ACL transection would have a greater effect on joint mechanics. A total of 24 rat knee specimens underwent surgical or biomechanical ACL rupture and were stabilized using a new reconstruction technique using autograft tail tendon. Joint mechanics were assessed three times; preinjury, postinjury, and again after reconstruction, using quantitative joint laxity testing. Primary test readouts were maximum anteroposterior (AP) laxity, loading curve slope, and energy absorption. Student's t-tests were performed to identify intragroup differences. All surgical transections were completed successfully; maximum load in the biomechanical model was 67 ± 7.7 N, with a coefficient of variation of 11.43%. Surgical transection caused increased AP laxity, while biomechanical injury nonsignificantly increased this parameter. In both cases, these changes recovered to baseline by reconstruction. Loading curve slope was reduced in both models and was also returned to baseline by repair. Energy absorption followed the same pattern except it remained significantly different from baseline postreconstruction in the surgical group. This study supports our hypothesis knee joint mechanics is differentially affected by injury mechanism in a small animal model. We also report a novel reconstruction technique in this model, using autograft tail tendon.

Automated Bone Segmentation and Surface Evaluation of a Small Animal Model of Post-Traumatic Osteoarthritis.

MicroCT imaging allows for noninvasive microstructural evaluation of mineralized bone tissue, and is essential in studies of small animal models of bone and joint diseases. Automatic segmentation and evaluation of articular surfaces is challenging. Here, we present a novel method to create knee joint surface models, for the evaluation of PTOA-related joint changes in the rat using an atlas-based diffeomorphic registration to automatically isolate bone from surrounding tissues. As validation, two independent raters manually segment datasets and the resulting segmentations were compared to our novel automatic segmentation process. Data were evaluated using label map volumes, overlap metrics, Euclidean distance mapping, and a time trial. Intraclass correlation coefficients were calculated to compare methods, and were greater than 0.90. Total overlap, union overlap, and mean overlap were calculated to compare the automatic and manual methods and ranged from 0.85 to 0.99. A Euclidean distance comparison was also performed and showed no measurable difference between manual and automatic segmentations. Furthermore, our new method was 18 times faster than manual segmentation. Overall, this study describes a reliable, accurate, and automatic segmentation method for mineralized knee structures from microCT images, and will allow for efficient assessment of bony changes in small animal models of PTOA.

How to establish an expected animal model of post-traumatic osteoarthritis?

How to establish an expected animal model of post-traumatic osteoarthritis?

Utility of circulating serum miRNAs as biomarkers of early cartilage degeneration in animal models of post-traumatic osteoarthritis and inflammatory arthritis

The purpose of this study was to determine if serum microRNA (miRNA) signatures were biomarkers of early cartilage degeneration in preclinical mouse models of post-traumatic osteoarthritis (OA) and inflammatory arthritis. Cartilage degeneration was induced in 10-12 week old male C57BL6 mice by destabilization of the medial meniscus (DMM) or intra-articular injection of methylated-bovine-serum-albumin (AIA), with sham-operated or saline-injected control animals (n=6/treatment/time). Total serum RNA and knee joints were isolated at 1, 4 and 16 weeks post-induction. Cartilage degeneration was scored histologically. Serum miRNA expression profiling was performed using Agilent microarrays and validated by qPCR. DMM-operated and AIA mice had characteristic cartilage degeneration (proteoglycan loss, chondrocyte hypertrophy, structural damage), that increased significantly with time compared with controls, and with distinct temporal differences between arthritis models. However, expression profiling revealed no statistically significant dysregulation of serum miRNAs between AIA vs saline-injected or DMM vs sham-operated control mice at the critical early disease stages. The inability to detect DMM or AIA serum miRNA signatures compared with controls was not due to the insensitivity of the expression profiling approach since significant changes were observed in miRNA expression between the arthritis models and between time points. While distinct patterns of progressive cartilage degradation were induced in the arthritis models, we were unable to identify any serum miRNAs that were significantly dysregulated in early stages of disease compared with controls. This suggests circulating serum miRNAs may not be useful as cartilage biomarkers in distinguishing the early or progressive stages of arthritis cartilage degeneration.

Comparison of micro-CT post-processing methods for evaluating the trabecular bone volume fraction in a rat ACL-transection model

Trabecular bone volume fraction assessments are likely sensitive to the analysis method and selection of the region of interest. Currently, there are several methods for selecting the region of interest to analyze trabecular bone in animal models of post-traumatic osteoarthritis. The objective of this study was to compare three published methods for determining the trabecular bone volume fraction of the medial tibial epiphyses in ACL transected and contralateral ACL intact knees. Micro-computed tomography images of both knees were obtained five weeks post-operatively and evaluated using three methods: (1) the Whole Compartment Method that captured the entire medial compartment, (2) the centrally located Single Core Method, and (3) the Triplet Core Method that averaged focal locations in the anterior, central, and posterior regions. The Whole Compartment Method detected significant bone loss in the ACL transected knee compared to the ACL intact knee (p<0.001), with a loss of 15.2±3.9%. The Single Core and the Triplet Core Methods detected losses of 7.5±10.5% (p=0.061) and 14.1±13.7%(p=0.01), respectively. Details regarding segmentation methods are important for facilitating comparisons between studies, and for selecting methods to document trabecular bone changes and treatment outcomes. Based on these findings, the Whole Compartment Method is recommended, as it was least variable and more sensitive for detecting differences in the bone volume fraction in the medial compartment.

Articular Cartilage Injury and Potential Remedies.

Osteoarthritis affects millions of people worldwide, is associated with joint stiffness and pain, and often causes significant disability and loss of productivity. Osteoarthritis is believed to occur as a result of ordinary "wear and tear" on joints during the course of normal activities of daily living. Posttraumatic osteoarthritis is a particular subset of osteoarthritis that occurs after a joint injury. Developing clinically relevant animal models will allow investigators to delineate the causes of posttraumatic osteoarthritis and develop means to slow or prevent its development after joint injury. Chondroprotectant compounds, which attack the degenerative pathways at a variety of steps, are being developed in an effort to prevent posttraumatic osteoarthritis and offer great promise. Often times, cartilage degradation after joint injury occurs despite our best efforts. When this happens, there are several evolving techniques that offer at least short-term relief from the effects of posttraumatic osteoarthritis. Occasionally, these traumatic lesions are so large that dramatic steps must be taken in an attempt to restore articular congruity and joint stability. Fresh osteochondral allografts have been used in these settings and offer the possibility of joint preservation. For patients presenting with neglected displaced intra-articular fractures that have healed, intra-articular osteotomy techniques are being developed in an effort to restore joint congruity and function. This article reviews the results of a newly developed animal model of posttraumatic osteoarthritis, several promising chondroprotectant compounds, and also cartilage techniques that are used when degenerative cartilage lesions develop after joint injury.

Development and reliability of a multi-modality scoring system for evaluation of disease progression in pre-clinical models of osteoarthritis: celecoxib may possess disease-modifying properties

We sought to develop a comprehensive scoring system for evaluation of pre-clinical models of osteoarthritis (OA) progression, and use this to evaluate two different classes of drugs for management of OA. Post-traumatic OA (PTOA) was surgically induced in skeletally mature rats. Rats were randomly divided in three groups receiving either glucosamine (high dose of 192 mg/kg) or celecoxib (clinical dose) or no treatment. Disease progression was monitored utilizing micro-magnetic resonance imaging (MRI), micro-computed tomography (CT) and histology. Pertinent features such as osteophytes, subchondral sclerosis, joint effusion, bone marrow lesion (BML), cysts, loose bodies and cartilage abnormalities were included in designing a sensitive multi-modality based scoring system, termed the rat arthritis knee scoring system (RAKSS). Overall, an inter-observer correlation coefficient (ICC) of greater than 0.750 was achieved for each scored feature. None of the treatments prevented cartilage loss, synovitis, joint effusion, or sclerosis. However, celecoxib significantly reduced osteophyte development compared to placebo. Although signs of inflammation such as synovitis and joint effusion were readily identified at 4 weeks post-operation, we did not detect any BML. We report the development of a sensitive and reliable multi-modality scoring system, the RAKSS, for evaluation of OA severity in pre-clinical animal models. Using this scoring system, we found that celecoxib prevented enlargement of osteophytes in this animal model of PTOA, and thus it may be useful in preventing OA progression. However, it did not show any chondroprotective effect using the recommended dose. In contrast, high dose glucosamine had no measurable effects.

In vivo fluorescence reflectance imaging of protease activity in a mouse model of post-traumatic osteoarthritis

Joint injuries initiate a surge of inflammatory cytokines and proteases that contribute to cartilage and subchondral bone degeneration. Detecting these early processes in animal models of post-traumatic osteoarthritis (PTOA) typically involves ex vivo analysis of blood serum or synovial fluid biomarkers, or histological analysis of the joint. In this study, we used in vivo fluorescence reflectance imaging (FRI) to quantify protease, matrix metalloproteinase (MMP), and Cathepsin K activity in mice following anterior cruciate ligament (ACL) rupture. We hypothesized that these processes would be elevated at early time points following joint injury, but would return to control levels at later time points. Mice were injured via tibial compression overload, and FRI was performed at time points from 1 to 56 days after injury using commercially available activatable fluorescent tracers to quantify protease, MMP, and cathepsin K activity in injured vs uninjured knees. PTOA was assessed at 56 days post-injury using micro-computed tomography and whole-joint histology. Protease activity, MMP activity, and cathepsin K activity were all significantly increased in injured knees relative to uninjured knees at all time points, peaking at 1-7 days post-injury, then decreasing at later time points while still remaining elevated relative to controls. This study establishes FRI as a reliable method for in vivo quantification of early biological processes in a translatable mouse model of PTOA, and provides crucial information about the time course of inflammation and biological activity following joint injury. These data may inform future studies aimed at targeting these early processes to inhibit PTOA development.

Non-terminal animal model of post-traumatic osteoarthritis induced by acute joint injury

Develop a non-terminal animal model of acute joint injury that demonstrates clinical and morphological evidence of early post-traumatic osteoarthritis (PTOA). An osteochondral (OC) fragment was created arthroscopically in one metacarpophalangeal (MCP) joint of 11 horses and the contralateral joint was sham operated. Eleven additional horses served as unoperated controls. Every 2 weeks, force plate analysis, flexion response, joint circumference, and synovial effusion scores were recorded. At weeks 0 and 16, radiographs (all horses) and arthroscopic videos (OC injured and sham joints) were graded. At week 16, synovium and cartilage biopsies were taken arthroscopically from OC injured and sham joints for histologic evaluation and the OC fragment was removed. OC fragments were successfully created and horses were free of clinical lameness after fragment removal. Forelimb gait asymmetry was observed at week 2 (P=0.0012), while joint circumference (P<0.0001) and effusion scores (P<0.0001) were increased in injured limbs compared to baseline from weeks 2 to 16. Positive flexion response of injured limbs was noted at multiple time points. Capsular enthesophytes were seen radiographically in injured limbs. Articular cartilage damage was demonstrated arthroscopically as mild wear-lines and histologically as superficial zone chondrocyte death accompanied by mild proliferation. Synovial hyperemia and fibrosis were present at the site of OC injury. Acute OC injury to the MCP joint resulted in clinical, imaging, and histologic changes in cartilage and synovium characteristic of early PTOA. This model will be useful for defining biomarkers of early osteoarthritis and for monitoring response to therapy and surgery.