Tnfaip8 and Tipe2 Gene Deletion Ameliorates Immediate Proteoglycan Loss and Inflammatory Responses in the Injured Mouse Intervertebral Disc.

OBJECTIVE: TNFAIP8 and TIPE2 belong to TNFa-induced protein 8 (TNFAIP8/TIPE) family. They control apoptosis and direct leukocyte migration. Nucleus pulposus (NP) cell loss is a hallmark of intervertebral disc (IVD) degeneration in response to injury, and inflammation may cause pain. Here, we examined the effects of TNFAIP8/TIPE2 deficiency on the IVDs in mice with these genes deleted. DESIGN: Tail IVDs in Tnfaip8 or Tipe2 single and double knockout mice (Tnfaip8-/-, Tipe2-/-, and Tnfaip8/Tipe2 dko), and wild type (WT) controls were injured. The spine motion segments were stained with Safranin O to reveal proteoglycans. Macrophages were identified by immunostaining, and selected inflammatory marker and collagen gene expression was examined by Real Time PCR. RESULTS: The injured tail IVDs of Tnfaip-/-, Tipe2-/-, and Tnfaip8/Tipe2 dko mice all displayed higher levels of proteoglycans than WT controls. Fewer macrophages were found in the injured IVDs of Tipe2-/- and Tnfaip8/Tipe2 dko mice than WT. Il6, Adam8 and Col1 gene expression was downregulated in the injured IVDs of Tnfip8/Tipe2 dko mice. CONCLUSIONS: TNFAIP8 and TIPE2 loss of function ameliorated proteoglycan loss and inflammation in the injured IVDs. They may serve as molecular targets to preserve disc structure and reduce inflammation.

Tnfa , Il6 , Cxcl1 , and Adam8 Genes Are the Early Markers After Mouse Tail Intervertebral Disc Injury.

OBJECTIVES: The early molecular events after intervertebral disc injury remain unclear. In this study, we aimed to compare inflammatory markers from 1 day to 4 wks after injury to have a comprehensive understanding of the intervertebral disc response to injury. DESIGN: Mouse tail intervertebral disc injury was induced by a needle puncture. Inflammatory marker gene expression and morphological changes were recorded at 1 day, 1 wk, and 4 wks after injury. RESULTS: Tnfa , Il6 , and Cxcl1 gene expression peaked at day 1 post-needle puncture of the mouse intervertebral disc, Adam8 gene expression peaked at 1-wk time point, while Tipe2 gene expression was upregulated at week 4 postinjury. F4/80 positive cells, likely to be macrophages, are present as early as day 1 in the injured intervertebral discs and consistently present at week 4 postinjury. Loss of Safranin O staining and increased histological scores of the injured intervertebral discs are consistent with progressive degeneration after injury. CONCLUSIONS: Inflammatory cytokines including Tnfa precede Tipe2 , suggesting that Tipe2 is likely induced by Tnfa . Upregulation of Adam8 and Cxcl1 gene expression persisted at week 4, suggesting that they play a role in the transition to chronic phase of intervertebral disc degeneration.

Expression of Human Interleukin 8 in Mice Alters Their Natural Behaviors.

Objective: To examine the effects of human interleukin (IL) 8 expression on mouse behavior. Methods: A mouse line expressing human IL8 in the intervertebral discs (IVD) and cartilaginous tissues (hIL8+ ) was generated. Mouse spontaneous behaviors, including locomotion, climbing, rearing, grooming, eating, drinking, and immobility were recorded with a fully automatic, non-invasive platform. Results: Distance traveled by the hIL8+ mice declined with age compared with control littermates, and male hIL8+ mice traveled a shorter distance than male controls and females of either genotype (p <0.05). The hIL8+ mice also spent less time in locomotion than control mice (p <0.01), and male hIL8+ mice spent the least amount of time and had lowest count in locomotion compared with the other 3 groups at 12 weeks of age or greater (p <0.05). The hIL8+ mice spent less time climbing than controls, and male mice spent less time climbing than female mice of the same genotype (p <0.01). The hIL8+ mice spent more time eating and less time drinking than controls, and all mice spent less time eating and more time drinking with increasing age. Finally, hIL8+ mice spent more time immobile than controls, and male hIL8+ mice spent more time immobile than any other group (p <0.05). Conclusion: The hIL8+ mice, especially hIL8+ males, showed reduced ambulation and climbing. Mice showed age-related decrease in eating and increase in drinking and grooming time that was also influenced by expression of hIL8. These changes in natural behaviors in control mice are consistent with functional decline with age. Effects of hIL8 superimposed on the natural aging process could involve systemic (e.g., on the brain) and local (e.g., in the spine and joint tissues) mechanisms. Future exploration of these mechanisms might be productive.

Elevated inflammatory gene expression in intervertebral disc tissues in mice with ADAM8 inactivated.

We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.

The Inner Annulus Fibrosus Encroaches on the Nucleus Pulposus in the Injured Mouse Tail Intervertebral Disc.

OBJECTIVE: The aim was to identify the source of cells within the center of the abnormal fibrocartilage tissue of the degenerative intervertebral disc after injury. DESIGN: Cross-breeding of mice with an inducible type II promoter collagen construct (Col2CreER) to Rosa26-TdTomato mice has been shown to result in Cre-recombinase activity and Tomato expression in inner annulus fibrosus cells after tamoxifen injection. To investigate the role of the inner annulus fibrosus in the intervertebral disc injury response, tail intervertebral discs of Col2CreER/tdTomato mice were punctured with a needle and examined 1-4 wks after injury. N-cadherin was examined by immunostaining. RESULTS: After the injury, the fibrocartilage in the degenerative intervertebral disc consisted of residual diseased nucleus pulposus cells and encroaching inner annulus fibrosus cells. The residual nucleus pulposus cells had lost their epithelial cell-like morphology and instead became oval shaped, with reduced adhesion to neighboring nucleus pulposus cells. This change in cellular morphology coincided with a loss of N-cadherin, which contributes to maintenance of healthy nucleus pulposus cell morphology. As expected, injured tail intervertebral discs showed reduced compressive properties as determined by biomechanical assessments. CONCLUSIONS: The cellular composition of the degenerative intervertebral disc has been defined here, which is an important step in developing future treatments.

TNFAIP8 family gene expressions in the mouse tail intervertebral disc injury model.

INTRODUCTION: The TNF-α-induced protein-8 (TNFAIP8, also known as TIPE) family of molecules comprises four members: TNFAIP8 and TIPEs1-3. Since the first description of these proteins, their roles in fine-tuning inflammation and in directing leukocyte migration have been described in several organ systems. However, their relationship with intervertebral disc (IVD) is unknown. MATERIALS AND METHODS: Here, we describe the expression of TNFAIP8 family genes in the nucleus pulposus (NP) and annulus fibrosus (AF) of the normal adult murine IVD. We further describe the expression of these genes in the injured male and female murine IVD. RESULTS: Tnfaip8 gene expression was decreased, and Tipe1 gene expression was essentially unchanged, in response to injury. Tipe2 and Tipe3 gene expression was markedly elevated in response to IVD injury, along with those encoding known inflammatory markers (ie, Tnfa, Il6, Cxcl1, and Adam8). Additionally, sex-related differences were also observed for some of these genes in intact and injured mouse IVDs. Future studies include examining tissue distribution of TNFAIP8 family proteins and identifying cells that produce them. In addition, examining mice that are deficient in TNFAIP8 molecules, in relation to gene expression, tissue morphology and mouse behavior, may further delineate the roles of these molecules in IVD inflammation and degeneration.

Functional Deficits in Mice Expressing Human Interleukin 8.

We showed previously that inflammatory mediators, including IL8, in intervertebral disc tissues from patients with discogenic back pain may play a key role in back pain. To investigate the molecular mechanism of IL8 signaling in back pain, we generated a mouse model that conditionally expresses human (h) IL8. We hypothesized that hIL8 levels affect mouse activity and function. Briefly, hIL8 cDNA was inserted into the pCALL2 plasmid, linearized, and injected into mouse embryos. Resulting pCALL2-hIL8 mice were then bred with GDF5-Cre mice to express the transgene in cartilage and intervertebral disc (IVD) tissues. Functional capacities including nest-making and other natural behaviors were measured. Both male and female mice expressing hIL8 showed lower nesting scores than did littermates that did not express hIL8 (n = 14 to 16 per group). At 28 wk of age, mice expressing hIL8 (n = 35) spent more time immobile and eating during each night than littermate controls (n = 33). Furthermore, hIL8-expressing mice traveled shorter distances and at a lower average speed than littermate controls. Thus, in an initial effort to investigate the relationship between this chemokine and mouse behavior, we have documented changes in normal activities in mice conditionally expressing hIL8.

Influence of Genetic Background and Sex on Gene Expression in the Mouse (Mus musculus) Tail in a Model of Intervertebral Disc Injury.

To facilitate rational experimental design and fulfill the NIH requirement of including sex as a biologic variable, we examined the influences of genetic background and sex on responses to intervertebral disc (IVD) injury in the mouse tail. The goal of this study was to compare gene expression and histologic changes in response to a tail IVD injury (needle puncture) in male and female mice on the DBA and C57BL/6 (B6) backgrounds. We hypothesized that extracellular matrix gene expression in response to IVD injury differs between mice of different genetic backgrounds and sex. Consistent changes were detected in gene expression and histologic features after IVD injury in mice on both genetic backgrounds and sexes. In particular, expression of col1a1 and adam8 was higher in the injured IVD of DBA mice than B6 mice. Conversely, col2a1 expression was higher in B6 mice than DBA mice. Sex-associated differences were significant only in B6 mice, in which col2a1 expression was greater in male mice than in female. Histologic differences in response to injury were not apparent between DBA and B6 mice or between males and females. In conclusion, mouse tail IVD showed sex- and strain-related changes in gene expression and histology after needle puncture. The magnitude of change in gene expression differed with regard to genetic background and, to a lesser degree, sex.

Functional muscle hypertrophy by increased insulin-like growth factor 1 does not require dysferlin.

INTRODUCTION: Dysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice. METHODS: Muscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice. RESULTS: Muscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles. DISCUSSION: We conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies.

Extracellular Matrix and Adhesion Molecule Gene Expression in the Normal and Injured Murine Intervertebral Disc.

OBJECTIVE: The aim of the study was to determine the transcription profile of the mouse nucleus pulposus and annulus fibrosus with an unbiased method. Furthermore, pathophysiological relevance of selected genes was demonstrated in the mouse tail intervertebral disc injury model. DESIGN: Paired normal mouse nucleus pulposus and annulus fibrosus tissue from C57BL/6j mice was examined by a polymerase chain reaction array. Key gene expression in the normal and injured intervertebral discs was confirmed by real-time polymerase chain reaction. RESULTS: Among the 84 genes studied, 63 were expressed higher in annulus fibrosus than in nucleus pulposus; only four genes were expressed higher in nucleus pulposus than in annulus fibrosus (n = 4, P ≤ 0.05). Real-time polymerase chain reaction confirmed that cadherin (cdh) 2 gene expression was higher in nucleus pulposus than in annulus fibrosus, and type I collagen (col1) gene expression was higher in the annulus fibrosus than in nucleus pulposus (n = 8, P < 0.01). One week after tail intervertebral disc injury, cdh2 gene expression decreased, while col1 expression increased (n = 8, P < 0.01). CONCLUSIONS: This is the first study to examine the relative expression of 84 genes in normal mouse nucleus pulposus and annulus fibrosus. Key genes in the normal and injured mouse intervertebral discs were confirmed with real-time polymerase chain reaction. This information should be useful for studying the mouse model of intervertebral disc degeneration and guide future cell therapy approaches.

Spatial distribution of type II collagen gene expression in the mouse intervertebral disc.

Genetic tools such as the Cre-Lox reporter system are powerful aids for tissue-specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre-recombinase expression in mice: (a) Col2-Cre, which allows constitutive Cre-recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2-CreER, which contains a shorter promoter/enhancer region than Col2-Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre-recombinase expression. The goal of the study is to characterize Cre-recombinase distribution pattern in Col2-Cre and Col2-CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real-time PCR. We crossed Col2-Cre mice or Col2-CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2-CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre-recombinase. TdTomato in the constitutively active Col2-Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2-CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2-Cre/tdTomato and Col2-Cre-ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.

Intervertebral Disc Degeneration in a Percutaneous Mouse Tail Injury Model.

OBJECTIVES: Intervertebral disc (IVD) degenerates progressively with age and after injuries. In this study, we aimed to characterize early molecular events underlying disc degeneration using a mouse tail IVD injury model. DESIGN: We have established a transcutaneous minimally invasive approach to induce mouse tail IVD injury under fluoroscopic guidance. Morphological and molecular changes in the injured IVDs are compared with the baseline features of adjacent intact levels. RESULTS: After needle puncture, tail IVDs exhibited time-dependent histological changes. The aggrecan neoepitope VDIPEN was evident from 2 days to 4 wks after injury. A disintegrin and metalloproteinase domain-containing protein 8 (adam8) is a surface protease known to cleave fibronectin in the IVD. Gene expression of adam8 was elevated at all time points after injury, whereas the increase of C-X-C motif chemokine ligand (cxcl)-1 gene expression was statistically significant at 2 days and 2 wks after injury. Type 1 collagen gene expression decreased initially at day 2 but increased at 2 wks after injury, whereas no significant change in type 2 collagen gene expression was observed. The extracellular matrix gene expression pattern is consistent with fibrocartilage formation after injury. CONCLUSIONS: Mouse tail IVDs degenerate after needle puncture, as demonstrated by histological changes and aggrecan degradation. The minimally invasive tail IVD injury model should prove useful to investigators studying mechanisms of IVD degeneration and repair.

Recessive and dominant mutations in COL12A1 cause a novel EDS/myopathy overlap syndrome in humans and mice.

Collagen VI-related myopathies are disorders of connective tissue presenting with an overlap phenotype combining clinical involvement from the muscle and from the connective tissue. Not all patients displaying related overlap phenotypes between muscle and connective tissue have mutations in collagen VI. Here, we report a homozygous recessive loss of function mutation and a de novo dominant mutation in collagen XII (COL12A1) as underlying a novel overlap syndrome involving muscle and connective tissue. Two siblings homozygous for a loss of function mutation showed widespread joint hyperlaxity combined with weakness precluding independent ambulation, while the patient with the de novo missense mutation was more mildly affected, showing improvement including the acquisition of walking. A mouse model with inactivation of the Col12a1 gene showed decreased grip strength, a delay in fiber-type transition and a deficiency in passive force generation while the muscle seems more resistant to eccentric contraction induced force drop, indicating a role for a matrix-based passive force-transducing elastic element in the generation of the weakness. This new muscle connective tissue overlap syndrome expands on the emerging importance of the muscle extracellular matrix in the pathogenesis of muscle disease.

Isometric and eccentric force generation assessment of skeletal muscles isolated from murine models of muscular dystrophies.

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Leupeptin-based inhibitors do not improve the mdx phenotype.

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Sexual dimorphism of murine masticatory muscle function.

OBJECTIVE: To determine if gender distinctions of force generating capacity existed in murine masticatory muscles. DESIGN: In order to investigate the effect of sex on force generating capacity in this muscle group, an isolated muscle preparation was developed utilising the murine anterior deep masseter. Age-matched male and female mice were utilized to assess function, muscle fibre type and size in this muscle. RESULTS: Maximum isometric force production was not different between age-matched male and female mice. However, the rate of force generation and relaxation was slower in female masseter muscles. Assessment of fibre type distribution by immunohistochemistry revealed a three-fold decrease in the proportion of myosin heavy chain 2b positive fibres in female masseters, which correlated with the differences in contraction kinetics. CONCLUSIONS: These results provide evidence that masticatory muscle strength in mice is not affected by sex, but there are significant distinctions in kinetics associated with force production between males and females.