Extracting high-quality RNA from formaldehyde-fixed naturally aged neuromusculoskeletal tissues.

Modern approaches to discovering molecular mechanisms and validating treatments for age-related neuromusculoskeletal dysfunction typically rely on high-throughput transcriptome analysis. Previously harvested and fixed tissues offer an incredible reservoir of untapped molecular information. However, obtaining RNA from such formaldehyde-fixed neuromusculoskeletal tissues, especially fibrotic aged tissues, is technically challenging and often results in RNA degradation, chemical modification and yield reduction, prohibiting further analysis. Therefore, we developed a protocol to extract high-quality RNA from formaldehyde-fixed brain, cartilage, muscle and peripheral nerve isolated from naturally aged mice. Isolated RNA produced reliable gene expression data comparable to fresh and flash-frozen tissues and was sensitive enough to detect age-related changes, making our protocol valuable to researchers in the field of aging.

Gait speed is a biomarker of cancer-associated cachexia decline and recovery.

Background: Progressive functional decline is a key element of cancer-associated cachexia. No therapies have successfully translated to the clinic due to an inability to measure and improve physical function in cachectic patients. Major barriers to translating pre-clinical therapies to the clinic include lack of cancer models that accurately mimic functional decline and use of non-specific outcome measures of function, like grip strength. New approaches are needed to investigate cachexia-related function at both the basic and clinical science levels. Methods: Survival extension studies were performed by testing multiple cell lines, dilutions, and vehicle-types in orthotopic implantation of K-ras LSL.G12D/+ ; Trp53 R172H/+ ; Pdx-1-Cre (KPC) derived cells. 128 animals in this new model were then assessed for muscle wasting, inflammation, and functional decline using a battery of biochemical, physiologic, and behavioral techniques. In parallel, we analyzed a 156-subject cohort of cancer patients with a range of cachexia severity, and who required rehabilitation, to determine the relationship between gait speed via six-minute walk test (6MWT), grip strength (hGS), and functional independence measures (FIM). Cachectic patients were identified using the Weight Loss Grading Scale (WLGS), Fearon consensus criteria, and the Prognostic Nutritional Index (PNI). Results: Using a 100-cell dose of DT10022 KPC cells, we extended the survival of the KPC orthotopic model to 8-9 weeks post-implantation compared to higher doses used (p<0.001). In this Low-dose Orthotopic (LO) model, both progressive skeletal and cardiac muscle wasting were detected in parallel to systemic inflammation; skeletal muscle atrophy at the fiber level was detected as early as 3 weeks post-implantation compared to controls (p<0.001). Gait speed in LO animals declined as early 2 week post-implantation whereas grip strength change was a late event and related to end of life. Principle component analysis (PCA) revealed distinct cachectic and non-cachectic animal populations, which we leveraged to show that gait speed decline was specific to cachexia (p<0.01) while grip strength decline was not (p=0.19). These data paralleled our observations in cancer patients with cachexia who required rehabilitation. In cachectic patients (identified by WLGS, Fearon criteria, or PNI, change in 6MWT correlated with motor FIM score changes while hGS did not (r 2 =0.18, p<0.001). This relationship between 6MWT and FIM in cachectic patients was further confirmed through multivariate regression (r 2 =0.30, p<0.001) controlling for age and cancer burden. Conclusion: Outcome measures linked to gait are better associated with cachexia related function and preferred for future pre-clinical and clinical cachexia studies.

Heterogeneous muscle gene expression patterns in patients with massive rotator cuff tears.

Detrimental changes in the composition and function of rotator cuff (RC) muscles are hallmarks of RC disease progression. Previous studies have demonstrated both atrophic and degenerative muscle loss in advanced RC disease. However, the relationship between gene expression and RC muscle pathology remains poorly defined, in large part due to a lack of studies correlating gene expression to tissue composition. Therefore, the purpose of this study was to determine how tissue composition relates to gene expression in muscle biopsies from patients undergoing reverse shoulder arthroplasty (RSA). Gene expression related to myogenesis, atrophy and cell death, adipogenesis and metabolism, inflammation, and fibrosis was measured in 40 RC muscle biopsies, including 31 biopsies from reverse shoulder arthroplasty (RSA) cases that had available histology data and 9 control biopsies from patients with intact RC tendons. After normalization to reference genes, linear regression was used to identify relationships between gene expression and tissue composition. Hierarchical clustering and principal component analysis (PCA) identified unique clusters, and fold-change analysis was used to determine significant differences in expression between clusters. We found that gene expression profiles were largely dependent on muscle presence, with muscle fraction being the only histological parameter that was significantly correlated to gene expression by linear regression. Similarly, samples with histologically-confirmed muscle distinctly segregated from samples without muscle. However, two sub-groups within the muscle-containing RSA biopsies suggest distinct phases of disease, with one group expressing markers of both atrophy and regeneration, and another group not significantly different from either control biopsies or biopsies lacking muscle. In conclusion, this study provides context for the interpretation of gene expression in heterogeneous and degenerating muscle, and provides further evidence for distinct stages of RC disease in humans.

Muscle gene expression patterns in human rotator cuff pathology.

BACKGROUND: Rotator cuff pathology is a common source of shoulder pain with variable etiology and pathoanatomical characteristics. Pathological processes of fatty infiltration, muscle atrophy, and fibrosis have all been invoked as causes for poor outcomes after rotator cuff tear repair. The aims of this study were to measure the expression of key genes associated with adipogenesis, myogenesis, and fibrosis in human rotator cuff muscle after injury and to compare the expression among groups of patients with varied severities of rotator cuff pathology. METHODS: Biopsies of the supraspinatus muscle were obtained arthroscopically from twenty-seven patients in the following operative groups: bursitis (n = 10), tendinopathy (n = 7), full-thickness rotator cuff tear (n = 8), and massive rotator cuff tear (n = 2). Quantitative polymerase chain reaction (qPCR) was performed to characterize gene expression pathways involved in myogenesis, adipogenesis, and fibrosis. RESULTS: Patients with a massive tear demonstrated downregulation of the fibrogenic, adipogenic, and myogenic genes, indicating that the muscle was not in a state of active change and may have difficulty responding to stimuli. Patients with a full-thickness tear showed upregulation of fibrotic and adipogenic genes; at the tissue level, these correspond to the pathologies most detrimental to outcomes of surgical repair. Patients with bursitis or tendinopathy still expressed myogenic genes, indicating that the muscle may be attempting to accommodate the mechanical deficiencies induced by the tendon tear. CONCLUSIONS: Gene expression in human rotator cuff muscles varied according to tendon injury severity. Patients with bursitis and tendinopathy appeared to be expressing pro-myogenic genes, whereas patients with a full-thickness tear were expressing genes associated with fatty atrophy and fibrosis. In contrast, patients with a massive tear appeared to have downregulation of all gene programs except inhibition of myogenesis. CLINICAL RELEVANCE: These data highlight the difficulty in treating massive tears and suggest that the timing of treatment may be important for muscle recovery. Specifically, earlier interventions to address tendon injury may allow muscles to respond more appropriately to mechanical stimuli.

The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration.

INTRODUCTION: Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of the extracellular matrix, and its repair requires both the production of extracellular matrix and the downregulation of proteinase activity. These properties are associated with several growth factors. However, the use of growth factors in clinical practice is limited by their high cost. This cost can be circumvented using synthetic peptides, such as Link N, which can stimulate the synthesis of proteoglycan and collagen by IVD cells in vitro. The purpose of the present study was to evaluate the effect of Link N in vivo in a rabbit model of IVD degeneration. METHODS: New Zealand white rabbits received annular puncture in two lumbar discs. Two weeks after puncture, both punctured discs of each rabbit were injected with either Link N or saline. After 2 weeks, nine rabbits were euthanized and the annulus fibrosus (AF) and nucleus pulposus (NP) of Link N-injected and saline-injected IVDs were removed and used to prepare total RNA. Following reverse transcription, quantitative PCR was performed for aggrecan, COL2A1, COL1A1, ADAMTS-4, ADAMTS-5 and MMP-3. After 12 weeks, 19 rabbits were euthanized and the injected IVDs were removed for biochemical and histological analysis. Proteinase K digests were analyzed for DNA and sulfated glycosaminoglycan content. Disc height was monitored radiographically biweekly. RESULTS: Following needle puncture, disc height decreased by about 25% over 2 weeks, and was partially restored by Link N injection. Puncture of the IVD resulted in a trend towards decreased proteoglycan content in both the NP and AF, and a trend towards partial restoration following Link N injection, although under the time course used this did not achieve statistical significance. Link N did not alter the DNA content of the discs. Link N injection led to a significant increase in aggrecan gene expression and a significant decrease in proteinase gene expression in both the NP and AF, when compared with saline alone. CONCLUSIONS: When administered to the degenerate disc in vivo, Link N stimulated aggrecan gene expression and downregulated metalloproteinase expression, and there was a trend towards increased proteoglycan content of the disc, in both the NP and AF. These are features needed for any agent designed to stimulate disc repair. In principle, therefore, Link N supplementation could be an option for treating disc degeneration.

Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: sustained release of a local antiinflammatory therapeutic.

OBJECTIVE: Interleukin-1 receptor antagonist (IL-1Ra) has been evaluated for the intraarticular treatment of osteoarthritis. Such administration of proteins may have limited utility because of their rapid clearance and short half-life in the joint. The fusion of a drug to elastin-like polypeptides (ELPs) promotes the formation of aggregating particles that form a "drug depot" at physiologic temperatures, a phenomenon intended to prolong the presence of the drug. The purpose of this study was to develop an injectable drug depot composed of IL-1Ra and ELP domains and to evaluate the properties and bioactivity of the recombinant ELP-IL-1Ra fusion protein. METHODS: Fusion proteins between IL-1Ra and 2 distinct sequences and molecular weights of ELP were overexpressed in Escherichia coli. Environmental sensitivity was demonstrated by turbidity and dynamic light scattering as a function of temperature. IL-1Ra domain activity was evaluated by surface plasmon resonance, and in vitro antagonism of IL-1-mediated lymphocyte and thymocyte proliferation, as well as IL-1-induced tumor necrosis factor alpha (TNFalpha) expression and matrix metalloproteinase 3 (MMP-3) and ADAMTS-4 messenger RNA expression in human intervertebral disc fibrochondrocytes. IL-1Ra immunoreactivity was assessed before and after proteolytic degradation of the ELP partner. RESULTS: Both fusion proteins underwent supramolecular aggregation at subphysiologic temperatures and slowly resolubilized at 37 degrees C. Interaction with IL-1 receptor was slower in association but equivalent in dissociation as compared with the commercial antagonist. Anti-IL-1 activity was demonstrated by inhibition of lymphocyte and thymocyte proliferation and by decreased TNFalpha expression and ADAMTS-4 and MMP-3 transcription by fibrochondrocytes. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity as the commercial IL-1Ra. This peptide was more bioactive against lymphocyte proliferation, nearly equivalent to the commercial antagonist. CONCLUSION: The ELP-IL-1Ra fusion protein proved to retain the characteristic ELP inverse phase-transitioning behavior as well as the bioactivity of the IL-1Ra domain. This technology represents a novel drug carrier designed to prolong the presence of bioactive peptides following intraarticular delivery.

Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells.

STUDY DESIGN: In vitro studies of the effects of proinflammatory cytokines on the production of nerve growth factor (NGF) by human intervertebral disc (IVD) cells. OBJECTIVE: To determine the constitutive expression and production of NGF and the effect of cytokines on the expression of NGF by human IVD cells. SUMMARY OF THE BACKGROUND DATA: NGF may play a role in the collateral sprouting of sensory axons, neural survival, and regulation of nociceptive sensory neurons. NGF is known to be up-regulated by proinflammatory cytokines. METHODS: The presence of NGF protein was analyzed by immunohistochemistry using human IVD cells obtained from cadaveric human spines with no known disc disease (MRI Thompson grades 2-4). The effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on NGF production and mRNA expression of NGF by IVD cells were examined. The expression of NGF receptors, trkA and p75, was also assessed immunohistochemically. RESULTS: Cadaveric anulus fibrosus (AF) and nucleus pulposus (NP) cells cultured in vitro in monolayer and in alginate beads positively stained with an anti-NGF antibody. The constitutive production of NGF protein in IVD cells was low (NP) or not detectable (AF). The expression of NGF mRNA was detectable in both cell types. IL-1beta and TNF-alpha up-regulated the NGF mRNA expression and the secretion of NGF protein into the media. TrkA was immunolocalized in AF and NP cells. CONCLUSION: Our results demonstrate, for the first time, that human AF and NP cells constitutively express NGF protein and mRNA, and that the proinflammatory cytokines IL-1beta and TNF-alpha stimulate the production of NGF. The precise role of NGF produced by IVD cells in the generation of discogenic pain or on the metabolism of IVD cells, especially under certain physiologic conditions in which cytokines are up-regulated, needs to be clarified in future experimentation.