Establishment and identification of a novel HTRA1 mutation mice model.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathyis a rare form of inherited cerebral small vessel disease associated with mutations in the high-temperature requirement serine peptidase A1 gene. As of now, only about 50 cases have been reported. In 2012, our group reported a family with a novel mutant of the high-temperature requirement serine peptidase A1 gene in China for the first time. To further explore the molecular pathogenesis of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a recombination mouse model expressed human high-temperature requirement serine peptidase A1 gene mutant identified by our group was generated using the Donor & Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 system and termed the Mut-high-temperature requirement serine peptidase A1 geneL364P mouse model. Results show that Mut-high-temperature requirement serine peptidase A1 geneL364P mice present similar pathological characteristics to patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, suggesting that the Mut-high-temperature requirement serine peptidase A1 geneL364P mouse model was generated successfully. Moreover, apoptosis was induced in mouse brain vascular smooth muscle cells derived from Mut-high-temperature requirement serine peptidase A1 geneL364P mice. In summary, the cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy mouse model described in this study will be beneficial to demonstrate the pathological mechanism of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy and provide new therapeutic targets for clinical treatment.

The NLRP3/Caspase-1/Interleukin-1ß Axis Is Active in Human Lumbar Cartilaginous Endplate Degeneration.

BACKGROUND: Modic changes are the MRI signal changes of degenerative lumbar vertebral endplate and which lead to or accelerate intervertebral disc degeneration. NLRP3, caspase-1, and interleukin-1ß (IL-1ß) play a pivotal role in the pathogenesis of many inflammatory diseases, such as osteoarthritis. However, the roles of IL-1ß and its activators caspase-1 and NLRP3 are unclear in the degenerative endplate. QUESTIONS/PURPOSES: We asked: (1) What are the degenerative changes of the histologic features and chondrogenic markers' gene expressions between the cartilaginous endplates of patients with Modic changes and trauma (control)? (2) How does the NLRP3/caspase-1/IL-1ß axis in the cartilaginous endplates of patients with Modic changes compare with control (trauma) specimens? METHODS: Surgical specimens of cartilaginous endplates were divided into Modic changes (n = 56) and the trauma control (n = 16) groups. Hematoxylin and eosin and safranin O staining of cartilaginous endplate tissues were done to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed on these tissues to investigate mRNA expression of type II collagen (Col II), SOX-9, matrix metalloproteinase-3, and a disintegrin like and metalloproteinase thrombospondin type I motifs-5. NLRP3, caspase-1, and IL-1ß were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: Hematoxylin and eosin and safranin O staining showed the extracellular matrix degraded in the cartilaginous endplates of patients with Modic changes but not in the control cartilaginous endplates. Chondrogenic Col II (p = 0.024) and SOX9 (p = 0.053) were downregulated in the Modic changes group compared with the control group. In contrast to the control group, the transcriptional levels of NLRP3 (p < 0.001), caspase-1 (p = 0.054), and IL-1ß (p = 0.001) were all upregulated in the Modic changes group. CONCLUSIONS: The expression of NLRP3, caspase-1, and IL-1ß was upregulated in the patients with low back pain and Modic changes on MRI compared with patients with vertebral burst fracture without degenerative changes on MRI. The data suggest the NLRP3/caspase-1/IL-1ß axis may be implicated in lumbar cartilaginous endplate degeneration. CLINICAL RELEVANCE: The NLRP3/caspase-1/IL-1ß axis is active in cartilaginous endplates of patients with Modic changes and inflammatory cascades can exacerbate the cartilaginous endplate degeneration which may act as a trigger for intervertebral disc degeneration and low back pain. If these findings can be confirmed by others, we hope that new and effective therapy could be developed directed against this target.

Expression of cathepsin K and matrix metalloproteinase 1 indicate persistent osteodestructive activity in long-standing ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a common, largely genetically determined, rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis and pathology are still not clear. OBJECTIVE: To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD). METHODS: Bony and soft tissue specimens of 30 patients with AS and 20 with DDD were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin K, matrix metalloproteinase (MMP)1, MMP3 and receptor activator for nuclear factor kappaB (RANK) ligand. RESULTS: Cathepsin K and MMP1-positive cells were only observed in AS specimens. Cathepsin K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP1 was expressed in smaller mononuclear cells attached to bone. Invasion of bone by MMP1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin K-positive. Isolated cells expressing these matrix-degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP3 between AS and DDD. Clear expression of RANK ligand was only detected in one patient with AS. CONCLUSIONS: Cathepsin K is strongly expressed in different regions of the spine in AS. Cathepsin K was mainly expressed by mononuclear cells, fibroblast-like cells and cells attached to bone and at sites of bone remodelling, suggestive of high osteoclastic activity. This supports the role of persistent inflammation in the pathogenesis of AS. How these changes relate to osteoproliferation remains to be determined.

Human disc degeneration is associated with increased MMP 7 expression.

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.

Distinct molecular mechanisms of HTRA1 mutants in manifesting heterozygotes with CARASIL.

OBJECTIVE: To elucidate the molecular mechanism of mutant HTRA1-dependent cerebral small vessel disease in heterozygous individuals. METHODS: We recruited 113 unrelated index patients with clinically diagnosed cerebral small vessel disease. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. RESULTS: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. The mean age at cognitive impairment onset was 51.1 years. Spondylosis deformans was observed in all cases, whereas alopecia was observed in 3 cases; an autopsied case with p.G283E showed arteriopathy in their cerebral small arteries. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (A252T and V297M) did not inhibit wild-type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer-associated HTRA1 activation. CONCLUSIONS: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.

Human group II phospholipase A2 in normal and diseased intervertebral discs.

We measured calcium-dependent phospholipase A2 (PLA2) activity and immunoreactive group II PLA2 levels of 54 normal discs obtained from cadavers and 73 disc samples surgically obtained from patients with spinal disorders, including intervertebral disc herniations, spondylosis, and spondylolisthesis. Both cadaveric and surgical disc specimens contained about two-fold greater PLA2 activity than the ileal mucosa, one of the richest sources of group II PLA2. Discs of middle-aged cases had significantly higher activity than those of younger and elder cases. In cadaveric normal discs, calcium-dependent PLA2 activity was significantly higher in females than in males. Annulus fibrosus and nucleus pulposus contained the same PLA2 levels. In diseased disc, herniated fragments that had extruded or protruded out of the discs possessed lower activity than other parts of discs in the intervertebral space. Immunoreactive group II PLA2 levels of intervertebral discs closely correlated with PLA2 enzymatic activity. We purified a PLA2 from human intervertebral disc to homogeneity to further identify the isozymic nature of discal PLA2. Its NH2-terminal amino acid sequences and molecular weight were identical to those of human group II PLA2. Immunohistochemical analysis using a monoclonal anti-group II PLA2 antibody showed that in both annulus fibrosus and nucleus pulposus chondrocytes contained intense group II PLA2 immunoreactivity in their cytoplasm, and that the matrix contained no substantial immunoreactivity. These results suggest that group II PLA2 in chondrocytes has important physiological roles in discal ordinary metabolism, maintaining discal homeostasis.

Duration of pain and muscular adaptations in patients with dysfunction of the cervical spine.

Biopsies of the sternocleidomastoid and omohyoid muscle were taken from 24 patients who underwent arthrodesis for cervical dysfunction of different etiologies. The two muscles, which are involved differently in movements of the head and cervical spine, were investigated histochemically. Muscle fibers were classified as type I, IIA, IIB, or IIC (transitional fibers) according to the pH lability of myofibrillar ATPase and calculated relative distribution. In both muscles, fiber transformations (as evidenced by an increase in the relative amount of type-IIC fibers) were regularly observed within the first 2 years after the onset of the symptoms. The occurrence of the transformation processes was independent of the patient's age and sex and was the same for the different etiologies. Since the overall fiber composition of the muscles remained essentially unchanged, the fiber transformations must occur alternatingly in both directions (from "slow" to "fast" and the reverse). Muscles of patients with a long case history showed no greater signs of fiber transformation. Therefore, fiber transformations in response to cervical dysfunction occur in the initial stage of the disease and involve different types of muscles. The muscles then return to a "stable" condition, independent of the continuation of the dysfunction and the chronic neck pain.

Collagen synthesis and types I, III, IV, and VI collagens in an animal model of disc degeneration.

STUDY DESIGN: The present study sought to elucidate the changes that occur in collagen chemistry in the early phases of disc degeneration. OBJECTIVE: To monitor the healing process of the injured anulus fibrosus and the secondary degenerative reactions in the nucleus pulposus. SUMMARY OF BACKGROUND DATA: Despite the importance of collagen chemistry under pathologic conditions in the intervertebral disc, knowledge of this aspect is very limited. METHODS: Fourteen pigs were stabbed with a scalpel blade in the anterior part of the anulus fibrosus of a lumbar disc. The animals were killed 2 weeks to 5 months after injury. The activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, the total collagen content, and staining patterns for Types I, III, IV, and VI collagens were analyzed from different parts of the disc. RESULTS: The most active phase of the healing process, assessed from the activities of enzymes involved in collagen biosynthesis, took place during the first month postoperatively. The anular lesion was found to cicatrize through formation of disorganized granulation tissue in which Type I, III, and, IV collagens were deposited. In the nucleus pulposus, activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase and total collagen content increased, and the originally rounded cells became more elongated, resembling fibroblasts. CONCLUSIONS: The results of this study suggest that the altered composition of collagens observed in the degenerate porcine nucleus pulposus results from changes in cell phenotype: Notochondral cells were replaced by fibroblast-like cells. It is likely that trauma to the anulus fibrosus can initiate a progressive degenerative process in the disc tissue.

Serum amylase and lipase elevation is associated with intracranial events.

Serum amylase and lipase elevation has been observed in trauma patients and patients with traumatic intracranial bleeding. However, the causes of this elevation have not been clearly elucidated. A further question remains as to whether other intracranial events are associated with such enzyme elevation as well. We retrospectively reviewed 75 patients consecutively admitted to Cook County Hospital Neurosurgical Intensive Care Unit over a 3-month period for trauma, infection, tumor, or other space-occupying lesions with an unstable condition or neurological deficit. Eleven patients (15%) had elevated amylase and lipase levels. The patients were divided into two groups: Group I (n = 64) had normal and Group II (n = 11) had raised amylase and lipase levels [amylase 402 +/- 444 U/L with normal < or = 125 U/L and lipase 474 +/- 313 U/L with normal < or = 55 U/L]. All Group II patients suffered an intracranial event. Twenty-four Group I (38%) and 10 Group II (91%) patients required craniotomy (P < 0.01). No patients had clinical or radiographic evidence of pancreatitis. In summary, intracranial events are associated with serum amylase and lipase elevation probably through centrally activated pathways. Because of the lack of diagnostic value, routine pancreatic enzyme monitoring should not be performed in this patient population.

Myosin ATPase activity in multifidus muscle from cases of lumbar spinal derangement.

Biopsies of lumbar multifidus muscles were obtained at operation on seventeen patients aged from fifteen to fifty-eight with lumbar spinal derangement, and further material was taken from the cadavers of three subjects aged from nineteen to fifty-one. Sections were prepared to show the presence of ATPase activity, so distinguishing Fast from Slow types of muscle fibre. The normal mosiac pattern arising from the intermingling of fibres from Fast and Slow motor units was seen in sections from cadaveric material and from many of the biopsies. With age and limited lumbar flexibility, the Fast fibres became relatively smaller but with increasing variation in size, suggesting a reduced capacity for phasic activity. The presence of positive root signs was associated with a greater proportion of Slow fibres, and in some patients with the occurrence of atrophied Fast fibres, giving rise to differences in the populations of the two fibres in neighbouring fascicles. The results suggest that multifidus adopts an increasingly postural role with advancing age and with disabling lesions of the lumbar spine.

A novel mutation of the high-temperature requirement A serine peptidase 1 (HTRA1) gene in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL).

OBJECTIVE: Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) gene were studied in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). METHODS: Exons 1-9 of the HTRA1 gene were amplified and bidirectionally sequenced in a Chinese family with CARASIL. Mutation effects were analysed by three-dimensional modelling of the serine protease HTRA1 protein. RESULTS: The proband was found to be homozygous for a novel missense mutation (c.854 C > T) identified in exon 4 of the HTRA1 gene; the parents of the proband were heterozygous for the same missense mutation. This c.854 C > T mutation resulted in a change from proline to leucine (p.P285L) in serine protease HTRA1, and was absent in 260 control chromosomes. Three-dimensional models showed that the change from proline to leucine (p.P285L) could attenuate the hydrogen bond between S284 and S287 residues, which might affect function of serine protease HTRA1. CONCLUSION: Discovery of a novel missense mutation (c.854C>T) associated with CARASIL expands the known CARASIL-related mutations in HTRA1.

Rigid spine syndrome revealing late-onset Pompe disease.

The authors describe a 50-year-old man who was evaluated for a rigid spine syndrome with onset at age 15, and subsequent walking difficulties. Cardiac and pulmonary functions were normal. Deltoid biopsy revealed the presence of small vacuoles and increased glycogen with Periodic Acid Schiff staining in a limited number of fibers. Acid alpha-glucosidase staining was decreased in leucocytes, and genetic analysis identified the presence of two mutations in that gene. This observation suggests that Pompe disease should be considered in the differential diagnosis of rigid spine syndrome, even in patients without respiratory involvement or with a muscle biopsy showing only mild histopathological changes.

Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels.

Intervertebral disc (IVD) degeneration is associated with the increased expression of several matrix metalloproteinases (MMPs), in particular MMP-2. However, little is known about the actual activity of MMP-2 in healthy and degenerated discs, or what mechanisms are involved in its activation. A major activation pathway involves complex formation with MMP-14 and a tissue inhibitor of metalloproteinases-2 (TIMP-2). In a series of 56 human IVDs, obtained at autopsy and graded according to the Thompson score (I-V), we analysed whether MMP-2 activity was increased in different stages of IVD degeneration and to what extent activation was related to the production of MMP-14 and TIMP-2. MMP-2 activation and production were quantified by gelatin zymography. Immunohistochemical staining of MMP-14 and TIMP-2 was quantified with a video overlay-based system. A positive correlation was observed between the amount of active MMP-2 and pro-MMP-2 and degeneration grade (p < 0.001, correlation coefficient (CC) 0.557; and p < 0.001, CC 0.556, respectively). MMP-2 activity correlated positively with MMP-14 and less strongly with TIMP-2 (p = 0.001, CC 0.436; and p = 0.03, CC 0.288, respectively). Moreover, immunopositivity for MMP-14 correlated to degeneration grade (p = 0.002, CC 0.398). IVD degeneration was associated with the activity of MMP-2 and the correlation of its activation with MMP-14 production suggests MMP-14 activates MMP-2 during degeneration. As MMP-14 is capable of activating several other enzymes that are also thought to be involved in IVD degeneration, it may be a key mediator of the degenerative process.

Association between the -1306C/T polymorphism of matrix metalloproteinase-2 gene and lumbar disc disease in Chinese young adults.

Matrix metalloproteinase-2 (MMP-2) has been shown to play a pivotal role in the pathophysiology of lumbar disc disease (LDD). Increased expression and activity of MMP-2 has been documented in degenerative discs. The polymorphism -1306C/T in the promoter region of MMP-2 gene was reported to influence gene transcription and expression. The objective of this study was therefore to investigate the possible association of MMP-2 -1306C/T polymorphism with the occurrence and the clinical characteristics of LDD. MMP-2 genotypes were determined by polymerase chain reaction (PCR) and direct DNA sequencing in a case-control study involving 162 younger patients with LDD and 318 age- and sex-matched healthy adults. The results showed that the frequency of MMP-2 -1306CC genotype was significantly higher in LDD patients when compared with controls. Subjects with the CC genotype had nearly threefold increased risk for LDD (odds ratio 3.08; 95% confidence interval 1.84-5.16) compared with subjects carrying at least one variant T allele. Furthermore, this genotype was found to correlate with more severe grades of disc degeneration observed on magnetic resonance imaging scan. These findings suggest that MMP-2 -1306C/T polymorphism may be a genetic risk factor related to LDD susceptibility in the young adult population.

Senescence in cells of the aging and degenerating intervertebral disc: immunolocalization of senescence-associated beta-galactosidase in human and sand rat discs.

STUDY DESIGN: Human intervertebral disc anulus tissue was obtained in a prospective study of cell senescence. Localization of the senescence biomarker beta-galactosidase (senescence associated beta-galactosidase, SA-beta-gal) was used for quantitative determination of the % senescent cells. Discs were obtained from surgical specimens or control donors. Discs were also studied from the lumbar spine of the sand rat. Experimental studies were approved by the authors' Human Subjects Institutional Review Board and animal use committee. OBJECTIVES: To determine the incidence of cell senescence in human discs with Thompson Grades I through V using immunocytochemistry to quantify the percentage of cells positive for the senescence biomarker SA-beta-gal. SUMMARY OF BACKGROUND DATA: Cell senescence has been recognized as a potential factor playing a role age-related disc degeneration. Senescent cells are viable but have lost the ability to divide. Senescence cells, however, are metabolically active. METHODS: Fifty-seven discs specimens from 54 subjects were examined with immunocytochemistry for anti-SA-beta-gal immunocytochemical localization to identify senescent cells. The fraction of positive cells was determined with quantitative histomorphometry. RESULTS: Quantitative histomorphometry of human discs show an overall incidence of SA-beta-gal-positive cells of 29.9% (+/-24.8, SD), with a range from 0 to 92.01%. Analysis by ANOVA of the % senescent cells grouped by Thompson grade showed significant increases in senescence with increasing disc degeneration (P < 0.0001). Further analysis with Tukey's test showed significant differences between the % senescent cells in Grades I/II versus IV, and versus V. SA-beta-gal-positive cells were also present in discs of the aging sand rat spine. CONCLUSIONS: Quantitative analysis of immunohistochemical localization of SA-beta-gal identified a sizeable population of senescent cells in the aging/degenerating disc. It is important to discover more about the senescent disc cell population because these cells persist and accumulate over time within the disc. Since senescent cells cannot divide, senescence may reduce the disc's ability to generate new cells to replace existing ones lost to necrosis or apoptosis.

Pathogenesis of spinally mediated hyperalgesia in diabetes.

Hyperalgesia to noxious stimuli is accompanied by increased spinal cyclooxygenase (COX)-2 protein in diabetic rats. The present studies were initiated to establish causality between increased spinal COX-2 activity and hyperalgesia during diabetes and to assess the potential involvement of polyol pathway activity in the pathogenesis of spinally mediated hyperalgesia. Rats with 1, 2, or 4 weeks of streptozotocin-induced diabetes exhibited significantly increased levels of spinal COX-2 protein and activity, along with exaggerated paw flinching in response to 0.5% paw formalin injection. Increased flinching of diabetic rats was attenuated by intrathecal pretreatment with a selective COX-2 inhibitor immediately before formalin injection, confirming the involvement of COX-2 activity in diabetic hyperalgesia. Chronic treatment with insulin or ICI222155, an aldose reductase inhibitor (ARI) previously shown to prevent spinal polyol accumulation and formalin-evoked hyperalgesia in diabetic rats, prevented elevated spinal COX-2 protein and activity in diabetic rats. In contrast, the ARI IDD676 had no effect on spinal polyol accumulation, elevated spinal COX-2, or hyperalgesia to paw formalin injection. In the spinal cord, aldose reductase immunoreactivity was present solely in oligodendrocytes, which also contained COX-2 immunoreactivity. Polyol pathway flux in spinal oligodendrocytes provides a pathogenic mechanism linking hyperglycemia to hyperalgesia in diabetic rats.

p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration.

STUDY DESIGN: Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1 beta or TNF-alpha, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. OBJECTIVE: To determine the role of signaling through p38 MAPK in nucleus pulposus cell's response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. SUMMARY OF BACKGROUND DATA: Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1 beta and tumor necrosis factor-alpha are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. MATERIALS AND METHODS: Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-alpha, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. RESULTS: Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-alpha induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-alpha. CONCLUSION: Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.