Coupled cell networks are target cells of inflammation, which can spread between different body organs and develop into systemic chronic inflammation

Several organs in the body comprise cells coupled into networks. These cells have in common that they are excitable but do not express action potentials. Furthermore, they are equipped with Ca2+ signaling systems, which can be intercellular and/or extracellular. The transport of small molecules between the cells occurs through gap junctions comprising connexin 43. Examples of cells coupled into networks include astrocytes, keratinocytes, chondrocytes, synovial fibroblasts, osteoblasts, connective tissue cells, cardiac and corneal fibroblasts, myofibroblasts, hepatocytes, and different types of glandular cells. These cells are targets for inflammation, which can be initiated after injury or in disease. If the inflammation reaches the CNS, it develops into neuroinflammation and can be of importance in the development of systemic chronic inflammation, which can manifest as pain and result in changes in the expression and structure of cellular components. Biochemical parameters of importance for cellular functions are described in this review

Nerve growth factor receptors in equine synovial membranes vary with osteoarthritic disease severity

Nerve growth factor (NGF) is a neurotrophin that has been implicated in pain signaling, apoptosis, inflammation and proliferation. The resultant effects depend on interaction with two different receptors; tyrosine kinase A (TrkA) and p75(NTR). NGF increases in synovial fluid from osteoarthritic joints, and monoclonal antibody therapy is trialed to treat osteoarthritis (OA)-related pain. Investigation of the complex and somewhat contradictory signaling pathways of NGF is conducted in neural research, but has not followed through to orthopaedic studies. The objectives of this study were to compare the expression of NGF receptors and the downstream regulator BAX in synovial membranes from joints in various stages of OA. The horse was used as a model. Synovial membranes were harvested from five healthy horses postmortem and from clinical cases with spontaneous OA undergoing arthroscopic surgery for lameness. Four horses with synovitis without gross cartilage changes, four horses with synovitis and cartilage damage, and four horses with synovitis and intracarpal fractures were included. Samples were investigated by immunohistochemistry and results showed that nuclear staining of TrkA, p75(NTR) and BAX increases in OA-associated synovitis. TrkA expression increased in early disease stages whereas increases in p75(NTR) were most prominent in later disease stages with cartilage damage and fibrosis. Clinical significance: Suppression of NGF may result in varied effects depending on different stages of the osteoarthritic disease process

Therapeutic innovation: Inflammatory-reactive astrocytes as targets of inflammation

AbstractThis study aimed to test pharmaceutical compounds targeting astrocytes showing inflammatory dysregulation. The primary rat brain cultures were treated with different batches of serum with or without microglia added to make the cells inflammatory-reactive. Lipopolysaccharide (LPS) and tryptase were used as inflammatory inducers. Expression levels of Toll-like receptor 4 (TLR4), Na+/K+-ATPase, and matrix metalloprotease-13 (MMP-13), as well as actin filament organization, pro-inflammatory cytokines, and intracellular Ca2+ release, were evaluated. LPS combined with tryptase upregulated TLR4 expression, whereas Na+/K+-ATPase expression was downregulated, ATP-evoked Ca2+ transients were increased, actin filaments were reorganized and ring structures instead of stress fibers were observed. Other aims of the study were to prevent astrocytes from becoming inflammatory-reactive and to restore inflammatory dysregulated cellular changes. A combination of the μ-opioid antagonist (−)-naloxone in ultra-low concentrations, the non-addictive μ-opioid agonist (−)-linalool, and the anti-epileptic agent levetiracetam was examined. The results indicated that this drug cocktail prevented the LPS- and tryptase-induced inflammatory dysregulation. The drug cocktail could also restore the LPS- and tryptase-treated cells back to a normal physiological level in terms of the analyzed parameters

Reduced high-intensity training distance in growing horses had no effect on IGF-1 concentrations, but training onset interrupted time-dependent IGF-1 decline

This study investigated plasma insulin like growth factor (IGF)-1 concentrations in 16 young Standardbred horses introduced to systematic high-intensity training at two different levels of intensity. Growth and locomotion asymmetry and correlations between these and plasma IGF-1 concentrations were also examined. From September as 1-year olds to March as 2-year olds (Period 1), all horses were subjected to the same submaximal training program. In March (start of Period 2), the horses were divided into two groups (n=8) and one group was introduced to regular high-intensity training. The other group was introduced to a program where the high-intensity exercise distances were reduced by 30%. These two training programs were maintained for the remaining 21 months of the study (Periods 2, 3, and 4). There was no effect of training group on plasma IGF-1 concentrations. A continuous decline in IGF-1 levels was observed throughout the study (PP>0.05). Front and hind limb asymmetry was elevated in Period 2 compared with Period 1. There were positive correlations between IGF-1 concentrations and changes in body condition score, and a negative correlation between IGF-1 concentration and weight. These results indicate that introduction to high-intensity training induces IGF-1 release in horses, but that a 30% difference in the distances used in high-intensity training does not affect IGF-1 levels. The temporary interruption in decline in IGF-1 release with the onset of high-intensity training may influence growth pattern and locomotion asymmetry, but further studies are needed to assess causality

The expression of nerve growth factor in healthy and inflamed equine chondrocytes analysed by capillary western immunoassay

Nerve Growth Factor (NGF) is a signalling molecule for pain and inflammation. NGF is increased in synovial fluid from osteoarthritic humans and animals, compared to healthy controls. Monoclonal antibody therapy directed against NGF has been approved to treat pain in osteoarthritic dogs but despite many years of trialling, therapy has not been approved for human use. One reason for this is that adverse reactions with rapidly progressing osteoarthritis has occurred in some individuals. More detailed knowledge of NGF expression in joints is needed. In this study, capillary-based Simple Western was used to analyse NGF in cultured equine chondrocytes. Chondrocytes were collected post mortem from three macroscopically healthy intercarpal joints and three intercarpal joints with mild osteoarthritic changes. The chondrocytes were expanded to passage one and seeded in chondrogenic medium to maintain the phenotype. On day four, cells were either stimulated with LPS or kept untreated in medium. All cells were harvested on day five. Wes analysis of lysates did not show mature NGF but two proforms, 40 and 45 kDa, were identified. Results were confirmed with western blot. The same proforms were expressed in chondrocytes from healthy and osteoarthritic joints. Acute inflammation induced by LPS stimulation did not change the forms of expressed NGF. Capillary Simple Western offers a sensitive and sample -sparing alternative to traditional western blot. However, confirmation of peaks is imperative in order to avoid misinterpretation of findings. In addition, in this case the method did not offer the possibility of quantification advertised by the manufacturers

Serotonin-evoked cytosolic Ca2+ release and opioid receptor expression are upregulated in articular cartilage chondrocytes from osteoarthritic joints in horses

Osteoarthritis is a pain-associated progressive disease and pain mediators, such as opioid receptors, expressed in articular cartilage could represent novel therapeutic targets. Acute and chronic stages of OA indicate different metabolic abilities of the chondrocytes depending on inflammatory state.This study aimed to investigate the response of healthy and osteoarthritic chondrocytes and their expression and release of pain mediators in response to acute inflammation.Interleukin-1 beta (IL-beta) and lipopolysaccharide (LPS) were used to induce an acute inflammatory response in cultured equine chondrocytes harvested from healthy joints (HC) and osteoarthritic joints (OAC), the latter representing acute exacerbation of a chronic inflammatory state. Intracellular Ca2+ release was determined after exposure to serotonin (5-hydroxytryptamine (5-HT), glutamate or ATP. Protein expression levels of F- and G-actin, representing actin rearrangement, and opioid receptors were investigated. Glutamate concentrations in culture media were measured. Cartilage was immunohistochemically stained for mu (MOR), kappa (KOR), and delta (DOR) opioid receptors.Upon exposure to acute inflammatory stimuli, OAC showed increased intracellular Ca2+ release after 5-HT stimulation and increased expression of MOR and KOR. When cells were stimulated by inflammatory mediators, glutamate release was increased in both HC and OAC. Immunostaining for MOR was strong in OA cartilage, whereas KOR was less strongly expressed. DOR was not expressed by cultured HC and OAC and immunostaining of OA cartilage equivocal.We show that chondrocytes in different inflammatory stages react differently to the neurotransmitter 5-HT with respect to intracellular Ca2+ release and expression of peripheral pain mediators.Our findings suggest that opioids and neurotransmitters are important in the progression of equine OA. The inflammatory stage of OA (acute versus chronic) should be taken into consideration when therapeutic strategies are being developed

Biglycan neo-epitope (BGN(262)), a novel biomarker for screening early changes in equine osteoarthritic subchondral bone

Objective: Native biglycan (BGN), which can undergo proteolytic cleavage in pathological conditions, is well known to be involved in bone formation and mineralization. This study aimed to delineate the specific cleavage fragment, a neo-epitope for BGN (BGN(262)), in synovial fluid (SF) from young racehorses in training, osteoarthritic (OA) joints with subchondral bone sclerosis (SCBS), and chip fracture joints.Design: A custom-made inhibition ELISA was developed to quantify BGN(262) in SF. Cohort 1: A longitudinal study comprising 10 racehorses undergoing long-term training. Cohort 2: A cross-sectional study comprising joints from horses (N = 69) with different stages of OA and radiographically classified SCBS. Cohort 3: A cross-sectional study comprising horses (N = 9) with chip fractures. Receiver operating characteristic (ROC) curve analysis was performed (healthy joints vs chip joints) to evaluate BGN(262) robustness.Results: Cohort 1: SF BGN(262) levels from racehorses showed a statistical increase during the first 6 months of the training period. Cohort 2: BGN(262) levels were significantly higher in the SF from severe SCBS joints. Cohort 3: SF BGN(262) levels in chip fracture joints showed a significant increase compared to normal joints. The ROC analysis showed an AUC of 0.957 (95% C.I 0.868-1.046), indicating good separation between the groups.Conclusions: The data presented show that BGN(262) levels increase in SF in correlation with the initiation of training, severity of SCBS, and presence of chip fractures. This suggests that BGN262 is a potential predictor and a novel biomarker for early changes in subchondral bone (SCB), aiming to prevent catastrophic injuries in racehorses. (c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/)

Bone Biomarker in Feedlot Cattle from Two Different Production Systems

The rapid growth rate in feedlot cattle is likely to promote joint abnormalities like osteochondrosis dissecans (OCD) and osteoarthritis (OA) with subsequent lameness. We have identified a small leucine-rich repeat proteoglycan (SLRP) biomarker at cleavage site 262GLGHNQIRM (BGN262) arising from the fragmentation of biglycan (BGN) in subchondral bone associated with OA. With a validated custom-made ELISA, BGN262 has been quantified in serum from cattle. The concentration of BGN262 in serum from cattle raised in the conventional and all-natural production systems increased at harvest compared to the starting period. The limitation of the study is the small sample size. However, the promising results encourage a further evaluation of BGN262 and its potential as a biomarker for subchondral bone pathology in cattle

The epidemiology of upper respiratory tract disorders in a population of insured Swedish dogs (2011-2014), and its association to brachycephaly

Upper respiratory tract (URT) disorders are common in dogs but neither general nor breed-related epidemiological data are widely reported. This study ' s aims were to describe the epidemiology of URT disorders in a Swedish population of dogs and to investigate whether brachycephalic breeds were overrepresented among high-risk breeds. A cohort of dogs insured by Agria Djurforsakring in Sweden (2011-2014) was used to calculate overall and breed-specific incidence rate (IR), age at first URT diagnosis and relative risk (RR) for URT disorders. For breeds with high RR for URT disorders, co-morbidities throughout the dog's insurance period and age at death were investigated. The cohort included approximately 450,000 dogs. URT disorders had an overall IR of 50.56 (95% CI; 49.14-52.01) per 10,000 dog years at risk. Among 327 breeds, the English bulldog, Japanese chin, Pomeranian, Norwich terrier and pug had highest RR of URT disorders. Eight of 13 breeds with high RR for URT disorders were brachycephalic. The median age at first URT diagnosis was 6.00 years (interquartile range 2.59-9.78). French bulldogs with URT diagnoses had a significantly shorter life span (median = 3.61 years) than other breeds with URT diagnosis (median = 7.81 years). Dogs with high risk for URT disorders had more co-morbidities than average

Nerve growth factor in the equine joint

Nerve growth factor (NGF) is a neurotrophin with many functions. In humans, it is involved in inflammation, nerve growth, apoptosis and pain signalling. Increased concentrations of NGF in synovial fluid has been shown in humans and dogs with osteoarthritis. Despite osteoarthritis being a common problem in horses, no studies have previously been published on NGF in the equine joint. The aim of this study was to quantify NGF in equine synovial fluid from healthy joints, acutely inflamed septic joints and joints with structural changes associated with osteoarthritis. A secondary aim was to identify the localisation of NGF and its two receptors, TrkA and p75(NTR) in healthy and osteoarthritic articular cartilage. NGF concentrations in synovial fluid from osteoarthritic joints (n = 27), septic joints (n = 9) and healthy joints (n = 16) were determined by ELISA. In addition, articular cartilage from osteoarthritic and healthy joints was examined for NGF, TrkA and P75(NTR) using immunohistochemistry staining. NGF was present in equine synovial fluid and articular cartilage. Compared to synovial fluid from healthy joints, NGF concentration was higher in synovial fluid from joints with structural osteoarthritic changes (P = 0.032) or acute septic inflammation (P = 0.006). In articular cartilage with severe osteoarthritic changes, there was more abundant positive immunohistochemistry staining for NGF and its receptors than in normal articular cartilage. Further studies should focus on identifying precursor forms of NGF, and on receptor expression and downstream signalling of TrkA and P75(NTR) in health and disease. (C) 2020 The Author(s). Published by Elsevier Ltd