Surgical neuromodulation through implantable devices allows for stimulation delivery to subcortical regions, crucial for symptom control in many debilitating neurological conditions. Novel closed-loop algorithms deliver therapy tailor-made to endogenous physiological activity, however rely on precise sensing of signals such as subcortical oscillations. The frequency of such intrinsic activity can vary depending on subcortical target nucleus, while factors such as regional anatomy may also contribute to variability in sensing signals. While artefact parameters have been explored in more ‘standard’ and commonly used targets , characterisation in novel candidate nuclei is still under investigation. One such important area is the brainstem, which contains nuclei crucial for arousal and autonomic regulation. The brainstem provides additional implantation targets for treatment indications in disorders of consciousness and sleep, yet poses distinct anatomical challenges compared to central subcortical targets. Here we investigate the region-specific artefacts encountered during activity and rest while streaming data from brainstem implants with a cranially-mounted device in two patients. Such artefacts result from this complex anatomical environment and its interactions with physiological parameters such as head movement and cardiac functions. The implications of the micromotion-induced artefacts, and potential mitigation, are then considered for future closed-loop stimulation methods. IInvasive central nervous system neuromodulation may be achieved through implantable devices, interfacing with sub-cortical structures through contact arrays of various configurations. In their most common clinically applied iteration, these are situated on linear leads, which are surgically inserted in the target area for stimulation . DBS is a treatment method in a variety of pharmacoresistant neurological disorders, with subcortical targets ranging from clinical use in basal ganglia to the investigational exploration of the brainstem.Novel neuromodulation methods utilise sensing capabilities in closed-loop strategies, where endogenous oscillations are targeted , 2]. Cl. Cl2]. CThe brainstem is encased in a system of spaces filled with cerebrospinal fluid (CSF), namely the quadrigeminal cistern, the cisterna pontis and interpeduncularis as well as the fourth ventricle towards its caudal end. Its extension, the medulla oblongata, is also dorsally surrounded by CSF through the cisterna cerebellomedullaris, while ventrally encased in the caudal portion of the cisterna interpeduncularis . This suWhile studies of brainstem kinematics are mainly indicated in cases of pathology, such as Chiari malformations, cervical canal stenosis or instability at the craniocervical junction –12], br, br12], Another source of displacement arising from the brainstem’s vicinity to CSF-filled spaces is that pulsatile fluid flows may cause additional motion of neural tissue. As dictated by the Monroe-Kelly doctrine, intracranial volume − comprised of CSF, parenchymal and intravessel (blood) volumes − is constant, therefore any change in the volume of one of its components requires compensation . During Brainstem motion associated to cardiac systole may be relatively small in scale (0.1−0.5 mm) but occur periodically with each cardiac cycle . SimilarBased on the above results and anatomical considerations, implants situated in the brainstem and following a trajectory parallel to its rostrocaudal axis will also have a degree of mobility following natural target micromotions. We set out to examine the impact of positional differences and cardiac activity on the quality of streamed local field potentials (LFPs) from two patients with brainstem DBS leads. Since the novel iteration of this device would be capable of closed-loop stimulation , we wereIIAThe Pedunculopontine Nucleus (PPN) of the upper brain-stem was bilaterally implanted, as part of a clinical trial examining autonomic and motor function modulation in multiple systems atrophy, a lethal neurodegenerative condition (MINDS NCT05197816). We used the Harvard Ascending Arousal Network Atlas  to visuaBWe used DBS leads with four 1.5 mm annular contacts (with 1.5mm spacing)  as part of a cranially mounted system with LFP-streaming capability (at a sampling frequency of 1 kHz). The four contacts were sampled as series of dipoles, and located on a helically extruded lead body for optimal force distribution. These characteristics optimise lead viability by minimising shear stress in the implantation environment.CWe streamed LFPs with simultaneous electroencephalogram (EEG), electromyogram (EMG) and electrocardiogram . For motion artefact capture, we instructed patients to nod rhythmically, recording head motion artefacts on our extracranial electrode system, followed by a period of stable head position. Streaming was initiated prior to nodding and consisted of ‘sweeps’ of an average of 49.52 s (ranging 18.3−54.54 s).For signal analyses, LFP files from two patients were loaded into Matlab , and pre-processed with a set of 3rd order Butterworth filters, a high-pass set at 0.1 Hz, and a low-pass at 180 Hz. For EEG, a band-stop for the rejection of 50 Hz line noise and its harmonics was also used. Dropped packages during the streaming process were accounted for and interpolated based on adjacent values. We did not initially de-trend the LFP signal, since we were interested in the presence of drifts as artefact. ECG was additionally processed with an adaptive thresholding operation and decision rules to identify QRS complexes and inter-beat intervals (IBI) , 26]..26].Both intracranial and extracranial signals were visually inspected to assess for common grossly visible artefacts and verification of arrhythmia versus noise presence in the processed ECG trace. Signals were time-locked based on common presence of QRS complexes and gross motion artefact, with results verified using known (marked) time lags between recording and streaming systems. Furthermore, frequencies of motion  as well as cardiac pulsation (as defined by IBI during periods without arrhythmia) were calculated and subsequently defined as frequencies of interest (FOIs).LFPs were further band-passed to artefact FOI, with power spectra visualised after a short-time Fourier transform , then averaged across the FOI spectrum and compared across different lead contact dipoles. For constant cardiac artefacts, signal power within the artefact FOI was compared to power within the beta (15−30 Hz) and gamma (35−50 Hz) bands, as based on prior reports of human recordings these correspond to physiological PPN activity , 27]. P. P27]. PWith regards to motion, we noted that rhythmical head movements generated associated LFP artefacts , which wBased on IBI after any ectopic heartbeat exclusion (and accounting for heart rate variability within patient) the artefact FOI was calculated, with upper limits that did not exceed 1.2 Hz and lower limit also affected by filtering properties . We noteMore specifically, when caudal dipoles consisting of the adjacent lead contacts of two patients were compared between hemispheres, there was no significant difference between mean power in the IBI FOI . When adjacent dipoles were compared to mid-lead contact dipole, which was less encased by a greater ratio of CSF to brain matter, the latter was more contaminated . Finally, when adjacent dipoles were compared to the largest potential dipole within the lead (top to bottom contacts), IBI FOIs differed significantly  with the largest streaming dipole being the most contaminated.We subsequently explored the effects of cardiac artefacts on signal-to-noise ratio, especially during periods of optimal streamed signal quality without motion artefact. Mean power-in-band across all pooled dipole STFT time bins for the head stationary part of each sweep revealed that cardiac FOI is significantly larger compared to intrinsic frequencies . Both patients exhibited the same phenomenon, without differences between cases . Both within patient and across dipoles and time, power within the cardiac artefact FOI band dominated the signal . The artefact FOI was distinct from intrinsic activity frequency bands, which were still identifiable − albeit with lower mean power-in-band .IIIWhile the most frequent targets for invasive subcortical neuromodulation are located in the basal ganglia, brainstem nuclei are highly important in arousal regulation and therefore present an additional attractive target for intervention in disorders of sleep and consciousness. Previously, central thalamic stimulation showed promise in ameliorating behavioural impairments after severe traumatic brain injury , while bBipolar re-referencing while acquiring and analysing signals from basal ganglia nuclei is an efficient strategy for artefact reduction , 32]; h; h32]; hSpecifically, we suggest that streaming and sampling from dipoles consisting of contacts which are unequally affected by CSF-distributed micromotion artefact should be discouraged except in cases of specific sub-regional interest, since the distribution of artefact FOI will not be eliminated from the signal. We showed that this phenomenon is present in brainstem sub-regions while at head rest due to artefact associated with the cardiac cycle (which therefore cannot be attenuated). However, in relatively stable dipoles consisting of adjacent contacts (thus maximising elimination of volume conduction) in regions further away from the pontine and quadrigeminal cisterns, the two hemispheres are not only comparable across patients but also minimally affected by low frequency artefacts.Endogenous frequencies were still present in streamed LFP signals, albeit with a lower power-in-band, as endogenous PPN activity occurs mainly in the beta and gamma range, ensuring minimal overlap with cardiac IBI FOI. However, there are brainstem regions where tonic firing rates have been reported within that FOI, such as locus coeruleus (LC) baseline firing  . To our One of the main future challenges is the development of responsive stimulation, delivering state-specific therapy in accordance with endogenous brain activity. Such closed-loop systems of complex physiological states could be additionally optimised with the use of integrated extracranial activity sensing (such as motion detection through accelerometry). Such an additional sensor integration could lead to further artefact reduction in the case of brainstem targets, through gross motion artefact cancellation during streaming. In addition, while a closed-loop stimulation mode is active, an accelerometer could ensure safety and efficacy through a ‘fall-back’ mode activated by head motion. This could ensure state-specific stimulation  while avoiding sensing contaminants, especially in targets where the intrinsic activity frequency bands may be compromised during head motion or pose a risk of amplifier saturation.Designing bioelectronics systems with advanced modulatory capabilities relies on identifying mitigations posed by physiological activity , and − aIVOur study is primarily limited by the small number of subjects and the specific patient pathology. Diurnal cycles of the CSF system  suggest VThe introduction of closed-loop control policies presents significant potential for the refinement of neuromodulation; however, it relies on the accurate sensing of μV−scale physiological signals . While t


Lynx pardinus) (n = 22), we compared the growth of captive versus wild born and/or reared individuals. Captive-born individuals gained weight as a function of calorie intake, unlike wild-born individuals. When compared with females reared in the wild, captive-reared females achieved a larger body size, without evidence of obesity. Captivity-associated changes to metabolic programming may compromise survival in the wild if an increased body size incurs a greater energy requirement. Large body size may also confer a competitive advantage over smaller, wild-born individuals, disrupting the social organisation of existing wild populations, and inferring long-term implications for the phenotypic composition of wild populations.Reintroduction of captive-bred individuals into the wild is an important conservation activity. However, environmental conditions can influence developmental programming, potentially causing metabolic disorders in adults. These effects are investigated here for the first time in an endangered species. Using body weight and feed intake data for Iberian lynx ( Controlled experiments with animals, as well as human epidemiological studies, have demonstrated a developmental programming effect, whereby early  nutrition can influence metabolic processes in later life, such as alterations to growth, glucose homeostasis, insulin sensitivity, energy balance, lipid metabolism and obesity, as well as impairment of cardiac and endocrine function5. This metabolic programming may represent either a deleterious functional impairment that arises from compromised development6, or an adaptive response of the foetus to the maternal environment that enables offspring to cope with the environment to which they are exposed after birth7. Under the ‘coping’ hypothesis, challenges to development, such as food deprivation, may be counteracted by short-term metabolic changes7. These often carry longer-term fitness costs that present as impaired physiological performance in the adult and may even persist into subsequent generations9. Equally, the ‘developmental origins of health and disease’ concept, which considers a range of potential mechanisms for metabolic programming, predicts that disease prevention interventions implemented in adulthood (e.g. lifestyle changes in exercise and diet) may be less effective for metabolically programmed individuals than would be expected in the absence of such programming7. Hence, factors that elevate risk of poor health, including increased appetite, certain food preferences or reduced propensity to exercise, may have more serious consequences in developmentally programmed individuals7.The environmental and physiological conditions experienced by organisms during sensitive periods of foetal and early post-natal development can exert profound effects on individuals, including irreparable disruption to normal development or the stimulation of alternative adult phenotypes, including those with increased susceptibility to certain diseasesAcinonyx jubatus) populations, but rarely detected in wild populations10. Similarly, iron storage disease (ISD) and obesity are captivity-specific conditions causing morbidity and mortality in a variety of species; frugivorous and browsing avian and mammalian species are affected by ISD12, and species ranging from lemurs (Varecia spp.)13 to elephants (Loxodonta africana and Elephas maximus)14 are affected by diseases associated with obesity.In the context of endangered species conservation, a number of health and disease concerns are known to uniquely affect captive populations. For example, gastrointestinal disease is prevalent in captive cheetah 24, evaluation of the programming effect of rearing or birth environment is less well understood.The key changes reported in captive bred animals, compared to wild conspecifics, are alterations to behaviour25. An outcome of this high food availability is often an alteration to the growth and development of animals in captivity; for example, captive lions (Panthera leo) typically grow faster and possess larger skulls than wild lions26. If metabolic programming disruption occurs under captive conditions, where food resources differ from the wild, the potentially negative implications of this process necessitate careful consideration in reintroduction programmes using captive-born or reared animals. If the same mechanism is present in captive-born or reared animals that are subsequently released into the wild, a potential outcome is compromised post-release health, survival, and reproductive success via altered metabolic rate and associated increased body size, obesity and hyperphagia27.The potential for metabolic programming effects are of particular concern for non-domestic species since they typically experience greater food availability in captivity compared to wild conspecificsLynx pardinus). Captive breeding and reintroduction programmes are key components of the conservation action plan for this species and have played an important role in improving its population status28 to the point that it was recently downgraded from critically endangered to endangered on the International Union for Conservation of Nature (IUCN) Red List29. Nonetheless, the species remains the most endangered felid in the world and is restricted to habitats in southern Spain and Portugal, where only two populations (Doñana and Sierra Morena) remained prior to reintroduction efforts. These two populations had been isolated since the 1950s30, with the smallest of these (Doñana) subsequently shown to be affected by inbreeding depression32. Current conservation efforts are focused on the preservation of the remnant populations, together with a reintroduction programme to recover the species’ historical distribution33.To date, no studies have investigated the existence of metabolic programming in captive endangered species. Here we address this knowledge gap by utilising a historically collected dataset for the Iberian lynx  was 34%39, although earlier research indicated mortality within the first 18 months of release was even higher (60%37). Importantly, captive-bred animals have a higher post-release mortality rate (52%) than wild-born animals (29%)36.A captive breeding programme for Iberian lynx was initiated in 2003, as part of the conservation strategy for the species to ensure a healthy captive stock and eventually provide individuals for release. The captive stock was initially founded with wild-caught individuals that started breeding in 2005Our study explores the possibility that metabolic programming may be occurring within the captive population of Iberian lynx. If so, this would represent a potentially important, but overlooked, variable contributing to conservation outcomes. We tested the ability of pre- and post-natal environment to predict energy intake or body weight and whether an association existed between them. Specifically, we predicted that captive-born and captive-reared individuals would express greater body size than wild caught and wild-reared individuals as a function of energy intake.There was a statistically important interaction between sex and rearing environment on lynx body weight Table . OverallThis sex difference was more pronounced if animals were reared in the wild; females were heavier when reared in captivity. Under the captive feeding regime , there was a statistically important interaction between birth environment and calorie intake on body weight Table . CaptiveFemale body weight did not predict the number of cubs produced by female lynx after controlling for the number of litters produced Table .Table 2PLynx body weight as a function of height and length was not affected by age or birth and rearing environment Table . Sex was40. However, no previous studies have explored such disorders in the context of captive breeding of an endangered species for the purpose of reintroduction.Developmental programming leading to metabolic disorders is well recognised in human medicine and has been demonstrated in laboratory studies of model animalsOur results suggest a metabolic programming effect of pre- and post-natal environment as reflected in birth and rearing conditions in the Iberian lynx. Three key findings were evident from this study: (a) captive-born lynx displayed a higher rate of energy intake than wild-born individuals under a captive feeding regime tailored to ensure good body condition; (b) wild-born individuals maintained a stable body weight against energy intake whereas captive-born individuals gained weight; and (c) captive-reared females achieved a larger body size than wild-reared females. None of these differences in body weight and energy intake arose from changes in body proportions and we found no evidence that female reproductive success in captivity was affected by body weight.25 in comparison with the natural environment; the higher energy expenditure required in nature for foraging, predator avoidance, reproduction, and other natural behaviours is effectively eliminated in captivity19. It follows that captive animals tend to be larger than wild conspecifics, attributed in some cases to the provision of larger rations in captivity25 and often associated with obesity-related health concerns. Our captive wild-born lynx achieved similar energy intakes to free-ranging conspecifics, i.e. a dietary intake of 1218 kcal day−1  for non-reproductive Iberian lynx41. Although a diet reflecting that of wild individuals is often considered an appropriate benchmark for captive feeding, this feeding strategy may oversupply energy to captive-born animals. Unlike their wild-born captive conspecifics, captive-born lynx in our study had a higher daily energy intake  and diets manipulated accordingly. As such, it is feasible that under a scenario of less efficient dietary modification in response to observed body condition changes, captive-born lynx may have been at risk of obesity. This outcome is particularly important in light of the variable daily energy intake of lynx . This variability reflects the rapid and frequent response of animal managers to changes in lynx body condition. In this regard, wild-born lynx appeared to stabilise body condition faster than captive-born animals and, therefore, required less frequent adjustments to meal size  could not be tested here but warrant further investigation.Diets that cause maternal obesity can lead to offspring resistance to leptin-signalling as a result of exposure to high concentrations of leptin from maternal milk, resulting in hyperphagia and consequent higher body weight51. Therefore, maladaptive captive-born or -reared phenotypes have the potential to compromise the fitness not only of the reintroduced individuals, but also of subsequent generations despite those being born and reared in the wild. Longitudinal studies will hence be necessary to best appreciate the extent and implications of this apparent developmental programming.Disruptions to metabolic programming also have the potential to alter phenotypes through epigenetic effects across multiple generations36. Because lynx have smaller home ranges in areas with higher densities of rabbits53, if reintroduced lynx are larger and have higher energy requirements, this will likely translate into a requirement for a larger territory, or more prey-dense habitat, in order to meet their elevated nutritional demands. Larger territories would reduce lynx density in these areas, increasing the total area required to maintain a viable population. Larger territories also increase the probability of encounters with traffic and, thus, increase the possibility of mortalities from vehicle collisions, which is already the major cause of death for released lynx33. Mortality rates of juveniles are particularly high during the dispersal phase52, and food availability is linked to the successful settlement of juveniles54, hence individuals with increased dietary requirements are under additional pressure. This is particularly concerning given that low prey availability is already cited as a key cause of population declines, to the extent that supplementary feeding has been implemented in some areas to support conservation efforts54. The potentially greater supplementary food provisioning required to meet increased nutritional requirements of captive-born released lynx has implications for conservation resource investment. Moreover, the larger body size of captive-born reintroduced individuals confers a competitive advantage in accessing higher quality habitats such as those with higher prey density and/or feeding stations55 potentially leading to the exclusion of smaller, wild-born animals.The implications of our findings for species conservation require consideration and incorporation into future breeding and reintroduction programme planning. It has already been shown that post-release mortality is higher in captive-bred animals than wild-born animalsA further conservation concern arises in relation to the potential impact of metabolic programming on reproductive output, or survival to reproductive age. Although we found no evidence for a change in reproductive success as a function of body size in captive Iberian lynx Table , no anim2, may limit lifetime reproductive potential via increased morbidity or mortality, or even intraspecific competitive factors. Iberian lynx are a sexually dimorphic species, with body size the main morphological difference between the sexes56. Territoriality in female mammals has been explained through intra-sexual competition for food resources57 to cover the higher energy requirements of females during gestation, lactation, and cub rearing. The Iberian lynx is a solitary felid, with strong intra-sexual territoriality58. The larger body size of captive-reared females could provide them a competitive advantage against smaller, wild females which may be excluded from higher quality territories and subsequent reproduction, thereby incurring indirect consequences for social organisation within extant wild population and/or stimulating genetic bias towards captive-reared phenotypes.The potential health implications associated with a larger body size, or metabolic disorders demonstrated in laboratory studies of other speciesMustela nivalis), in which females are also smaller than males and males do not participate in the rearing of the offspring, support the hypothesis that female body size is limited by the elevated energy requirements of reproduction and cub rearing59. Reproductive success may also decline in response to limited food availability as for the Canadian lynx (Lynx canadensis)60, and larger females may be more sensitive to minor changes in food availability than smaller females. Furthermore, female Eurasian lynx (Lynx lynx) with cubs reduce their territory size during the first weeks after birth61, such that protection of the litter may occur at the expense of adequate prey acquisition.Alternatively, reproduction of released captive-reared females may be compromised if females have higher nutritional needs for maintenance. Data on the energy requirements for the weasel  that were included in the breeding stock for the centre between 23/03/2010 and 16/04/2017. The animals were categorised according to birth environment as wild-born (n = 12) and captive-born (n = 10). Rearing environment was similarly designated as either wild or captive; the cut-off point for classification of the rearing period was at 54 days because the lynx weaning process occurs from 54 to 72 days old63. Wild-reared lynx (n = 7) were those that entered captivity older than 3 months of age, and captive-reared lynx (n = 15) were either born in captivity , or entered captivity before the start of the weaning process .All data used in this study were obtained from El Acebuche Breeding Centre . The captive population initially comprised 26 wild-caught Iberian lynx that were brought into captivity as founders between 2002 and 2008Oryctolagus cuniculus) meat mixed with milk. The European rabbit is the lynx’s main prey41 and its proportion in the mixture was gradually increased until the cubs were eating only whole prey at an age of approximately 100 days64. Starting between 9 and 12 months of age, lynx were fed 6 days week−1 and fasted on the seventh day. The lynx’s rations were reviewed weekly to ensure good body condition; readjustments were made when lynx were observed to be over-weight or under-weight, according to the body condition standards65. This assessment included a visual  assessment of the animal’s body fat and aligned with published body condition scoring systems for felids67. This feeding adjustment protocol was applied comparably among individuals regardless of birth or rearing origin, and the frequency or extent of adjustments to ration were decided on the basis of animal condition and response to food provisioning.Hand-rearing of cubs occurred occasionally when a cub’s survival was considered critical to the breeding programme. Hand-rearing was used for captive-born cubs when mothers showed inefficient maternal care, abandoned their cub, or died. Wild-born cubs have also been hand-reared, and consequently introduced into the captive breeding programme due to the mother’s death or improbability of survival in the wild. In these cases cubs were bottle-fed with artificial milk until the age of 30 days when they were offered small pieces of farmed European rabbit  and whole partridge (Alectoris rufa). The total metabolisable energy content of the diet  was calculated using Atwater factors for each food type and the quantity of each consumed per month. The Atwater factors used for beef, chicken breast, and rabbit muscle meat were unmodified (i.e. 4 × crude protein (CP), 9 × crude fat (CF), and 4 × nitrogen-free extract (NFE)), as recommended for raw foods69, whilst modified Atwater factors  were used for feed items with predicted lower digestibility 70. The nutrient composition of each dietary component was determined from the published literature . Total energy contribution was subsequently calculated based on the proportional contribution of each food type to the consumed meal. The mean daily energy intake of each individual was calculated for the 30 days prior to each weighing data point.As part of the management system of El Acebuche Breeding Centre, individual husbandry records are maintained for each lynx, including daily feed intake recorded to the nearest gram, calculated as amount offered less uneaten remains. In accordance with wild lynx dietary intake65, as per methods used in other species78.Body weight (kg ± 0.1) data were obtained during periodic routine husbandry checks. Animals were weighed prior to feeding on an opportunistic or routine basis . Individual body height and length measurements (cm) were obtained during veterinary examination under general anaesthesia. Body height was measured as the distance from the metacarpal pad to the shoulder, and body length as the distance from the tip of the nose to the base of the tail following the body shapeData were only used from lynx that were fed individually and observed daily, to ensure certainty of the quantity of food ingested. Similarly, data during periods of gestation, lactation and cub growth until 2 years of age were excluded because monitoring individually ingested quantities of food is imprecise during these periods due to husbandry protocols preventing close contact with the animals. The dataset comprised 1160 records, with details of individual birth environment , rearing environment , body weight, and estimated daily energy intake. Age data were calculated from birth date which was accurate to the day  or month . Wild-born lynx entered captivity either as cubs, or as adults which had been monitored since birth and, therefore, year of birth was known for all of them. Cubs obtained during the birthing season had day of birth estimated on the basis of developmental stage . To investigate potential impact on reproductive success, data for the total cumulative number of litters and cubs surviving until 2018 produced by each female were used as a proxy for reproductive success. An additional subset of data (55 records) comprised body height, length and weight, measured at irregular intervals, for every individual.81.To make inferences about model parameters a Bayesian approach was used. Bayesian inference is robust in dealing with unbalanced data, dependency due to repeated measures, and a non-normal response variable. This approach also avoids reliance on hypothesis testing and P-values, which are increasingly recognised as unreliable statistical tools for any but the simplest models82 with models fitted in a Bayesian framework using integrated nested Laplace approximation (R-INLA)83. To accommodate temporal dependency in the data, body weight was modelled using a random walk (RW1) trend model fitted for age following a gamma distribution, which assumed body weight was strictly positive and continuous. All measured variables were included in an initial model with an optimal fixed structure identified with a backward selection procedure based on Watanabe-Akaike Information Criterion (WAIC)84. To assess final model sensitivity to priors, we re-ran models with PC and half-Cauchy prior distributions on hyperparameters, which yielded qualitatively identical outcomes in all cases (results not shown).Data were modelled using R version 3.5.2Wtijk is the body weight on day i for lynx j at age (age) k, which assumes body weight follows a gamma distribution with mean μ and precision ϕ. Sexijk is a categorical covariate corresponding with sex, male and female. The variables Birthijk and Rearijk are also categorical covariates, each with two levels, corresponding with birth , and rearing provenance . The variable kcalijk, is a continuous covariate corresponding with daily estimated kilocalories consumed by an individual lynx during the 30 days prior to weight measurement. The random intercept lynxj was included to introduce a correlation structure between weight measurements for the same individual, with variance σlynx distributed normally and equal to 0.The best-fitting model for lynx body weight, identified by backward selection, was:Cubsi is the number of offspring produced by female lynx i assuming a negative binomial distribution with mean μ and dispersion k. The variables Wti and Litteri are continuous covariates corresponding with lynx body weight (kg) and total number of litters, respectively.Data were also fitted to a model for the total number of cubs produced by each female, which took the form:Lynx body weight was modelled using height and length data to examine whether body proportions, along with sex, birth and rearing provenance influenced body size.ageij was lynx age on day i for lynx j, assuming body weight (Wtij) follows a gamma distribution with mean μ and precision ϕ. The variables heightij and lengthij were continuous covariates corresponding with body height and length respectively. Individual lynx (lynxj) were included as a random term in the model.The model took the form:


Roughly 6% of healthy myocardium is composed of pure extracellular matrix (ECM) such as collagen fibers and, to a lesser extent, elastic fibers. This interstitial extracellular mixture—mainly synthesized by fibroblast and myofibroblasts—creates a three-dimensional cardiac skeleton which allows the cardiomyocytes to perform their contractile functions  resident quiescent cardiac fibroblasts inhabit myocardial tissue; (ii) during myocardial injury (MI), fibroblasts differentiate into myofibroblasts, which actively proliferate and secrete ECM until the injury is resolved; (iii) finally the site of injury is populated by not proliferative and terminally differentiated matrifibrocytes secreting ECM in order to maintain the integrity of the scar , activated myofibroblasts , and matrifibrocytes   treatment, which reduced cardiac fibrosis and ameliorated cardiomyocytes mitochondrial function through Apelin/Sirt3 pathway. Zhao et al. described how muscarinic acetylcholine receptor 3 (M3R) signaling after choline activation represents another important axes that controls cardiac fibrosis through TGF-β1/Smad2-3/p38 MAPK pathway.Thomas and Grisanti reviewed the extensive crosstalk between inflammation and cardiac fibrosis contributing to the progression of heart failure (HF). Following myocardial injury and cardiomyocytes death, local inflammatory cells  infiltrate the site of injury and secrete pro-inflammatory mediators , which play an important regulatory role in the transition from quiescent resident fibroblasts into active and proliferative myofibroblasts, initiating the production of ECM components. Narrowing the focus to one specific mediator in the crosstalk between the inflammatory response and development of fibrosis, Okyere and Tilley described the role of leukocytes in the regulation of cardiac remodeling. Both innate and adaptive leukocytes critically influence pathological fibrotic remodeling.Rogers et al. described a cell-free therapeutic application based on EVs secreted from stem/progenitor cells that can directly stimulate the trans-differentiation of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages play a crucial role. The interplay among macrophages, fibroblasts, and endothelial cells represents a major driving force of myocardial fibrosis. Marek-Iannucci et al. established a minimally invasive and cost-effective model of cooling pericardial perfusion in swine, which highlights the potential therapeutic effects of hypothermia post-ischemia/reperfusion injury in reducing cardiac fibrosis, inflammation and immune cell recruitment.Farini et al. showed that cardiac expression of the inflammatory mediator Pentraxin 3 (PTX3) influences inflammatory/fibrotic pathways in an animal model of Duchenne Muscular Dystrophy and may be an interesting therapeutic target.Degradation of large areas of replacement fibrosis could be catastrophic unless accompanied by robust cardiac regeneration, due to the negligible endogenous regenerative potential of the adult heart. Upon cardiac injury, the compensatory but maladaptive fibrotic response mediated by cardiac fibroblast can vary significantly among different injury types  fibrosis with increased arrhythmogenesis following isoproterenol (ISO) injections for 5 weeks in rats. The anti-fibrotic and anti-inflammatory agent pirfenidone administered 1 week after completion of ISO decreased LA fibrosis and arrhythmia leading to an improvement in cardiac function.De Simone et al. showed that cardiac myofibroblast are not excitable cells, electronically coupled to cardiomyocytes and their function is not limited solely to ECM production and protecting the architecture of the heart, but is also important for regulating the cardiac electrical conduction. After excessive collagen deposition, only scarce cellular structures populate the sclerotic area, which likely is not capable of creating electrically efficient fibroblast/cardiomyocyte couplings and causing arrhythmogenesis. Overexpressing β1AR antibodies, which act as βAR agonists, impacted electrophysiological properties in terms of atrial effective refractory period (AERP), AF inducibility, and electrical conduction in rabbits.The implication of early delivery of therapies post-MI will most certainly differ from the treatment for non-ischemic diastolic dysfunction with a stiff left ventricle and even more so from the treatment of end-stage heart failure with more pronounced fibrosis. While the fibrotic response following myocardial injury may initially be compensatory, could eventually be maladaptive if the large cardiac functional reserve is compromised. Therapeutic intervention in this setting should aim to prevent the fine sclerotic tissue deposition between the cardiomyocytes. This could be implemented with personalized anti-fibrotic therapy with specific timing of the intervention and tailoring it to the type of injury rather than completely blocking the fibrosis pathway.GP substantially contributed to the conception and design of the editorial, literature search, drafting the article, and revising the article critically for important intellectual content. CL contributed in literature search, drafting the article, and revising the article critically for important intellectual content. MW contributed in literature search, drafting the article, and revising the article critically for important intellectual content. DC contributed in literature search and drafting the article and revising the article critically for important intellectual content. All the authors approved the final version of the article to be published.MW was employed by the company Center for Biomarker Research in Medicine, CBmed GmbH, Austria. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this paper, an approach to determine the thermal impedance of a multi-chip silicon carbide (SiC) power module is proposed, by fusing optical measurement and multi-physics simulations. The tested power module consists of four parallel SiC metal-oxide semiconductor field-effect transistors (MOSFETs) and four parallel SiC Schottky barrier diodes. This study mainly relies on junction temperature measurements performed using fiber optic temperature sensors instead of temperature-sensitive electrical parameters (TESPs). However, the fiber optics provide a relatively slow response compared to other available TSEP measurement methods and cannot detect fast responses. Therefore, the region corresponding to undetected signals is estimated via multi-physics simulations of the power module. This method provides a compensated cooling curve. We analyze the thermal resistance using network identification by deconvolution (NID). The estimated thermal resistance is compared to that obtained via a conventional method, and the difference is 3.8%. The proposed fusion method is accurate and reliable and does not require additional circuits or calibrations. Power modules with high power densities are increasingly in demand for various applications, including electrified vehicles, transportations, and renewable energy systems ,2,3. TheIt is expected that such parallel SiC power modules would encounter various thermal reliability issues originating from their high power density. Common thermal reliability issues include thermal stress, thermal fatigue, and wire bond lift-off caused by thermal expansion coefficient mismatches and large temperature fluctuations ,13,14,15The thermal impedance is composed of thermal resistance and thermal capacitance. The thermal impedance can be derived from the junction temperature cooling curve. Thus, for an accurate thermal impedance characterization, device junction temperature monitoring is essential. Junction temperatures are primarily monitored by either measuring temperature-sensitive electrical parameters (TSEPs) or employing additional temperature sensors ,22,23,24Vgs) is also an effective parameter because its reduction is linear with an increase or decrease in the junction temperature ) is from the device junction to the base plate. The red line is the structure function extracted based on the temperature curve measured with thermal grease, while the green line is that without thermal grease. The blue circle where the separation starts corresponds to Rth j-case the junction to the case. A comparison of the proposed and conventional methods shows an error of 3.8%.The cumulative structure function is calculated using a commercial software provided from the T3ster Master from Mentor Graphics. The compensated temperature cooling curve in In this paper, we proposed a thermal impedance characterization method based on optical measurements assisted by a multi-physics simulation for a multi-chip SiC MOSFET power module. Fiber optics was used to accurately measure the junction temperature cooling curve. The region that was not detected owing to the relatively slow response of the fiber optic sensor to the TSEP was estimated via the multi-physics simulation. After compensation by the simulation result, the cooling curve was extracted to obtain the thermal resistance and thermal capacitance via the NID method. The results were validated using the conventional measurement method, and the error was within 3.8%. In addition to the high accuracy, the proposed method has the following advantages: (1) simple measurement without employing complex calibrations, (2) measurement availability without removing the silicone gel of the power module, and (3) not requiring additional circuits or additional device pin structures. Thus, this method could be an attractive solution for measuring the thermal impedance in applications entailing multi-chip SiC power modules with anti-parallel diodes.


Osteoarthritis (OA) is one of the most debilitating diseases and is associated with a high personal and socioeconomic burden. So far, there is no therapy available that effectively arrests structural deterioration of cartilage and bone or is able to successfully reverse any of the existing structural defects. Efforts to identify more tailored treatment options led to the development of strategies that enabled the classification of patient subgroups from the pool of heterogeneous phenotypes that display distinct common characteristics. To this end, the classification differentiates the structural endotypes into cartilage and bone subtypes, which are predominantly driven by structure-related degenerative events. In addition, further classifications have highlighted individuals with an increased inflammatory contribution (inflammatory phenotype) and pain-driven phenotypes as well as senescence and metabolic syndrome phenotypes. Most probably, it will not be possible to classify individuals by a single definite subtype, but it might help to identify groups of patients with a predominant pathology that would more likely benefit from a specific drug or cell-based therapy. Current clinical trials addressed mainly regeneration/repair of cartilage and bone defects or targeted pro-inflammatory mediators by intra-articular injections of drugs and antibodies. Pain was treated mostly by antagonizing nerve growth factor (NGF) activity and its receptor tropomyosin-related kinase A (TrkA). Therapies targeting metabolic disorders such as diabetes mellitus and senescence/aging-related pathologies are not specifically addressing OA. However, none of these therapies has been proven to modify disease progression significantly or successfully prevent final joint replacement in the advanced disease stage. Within this review, we discuss the recent advances in phenotype-specific treatment options and evaluate their applicability for use in personalized OA therapy. For many years, strong and cost-intensive efforts have been undertaken to develop therapies to improve care, quality of life, and pain relief for OA patients. Therapeutic approaches predominantly addressed symptoms and tried to modify/improve structural features of affected joint tissues. Despite this, no therapies have been able to halt or delay OA progression satisfactorily or provided effective and long-lasting symptomatic relief. Currently, joint replacement with an artificial prosthesis is the most effective measure to improve pain sensation and quality of life in patients. The development of novel therapeutic approaches targeting the osteoarthritic degradative and inflammatory processes in cartilage, synovium, or bone requires a deep understanding of the disease status of these joint tissues at the time of the intervention. It is crucial to apply therapies at the early stages of the disease prior to major structural and functional alterations in the osteochondral unit; otherwise, these interventions will not be successful3. Structural and clinical features of OA are characterized by a high interpatient variability. This heterogeneity is considered to be a major factor associated with the complexity of OA and the ongoing difficulties to identify “one size fits all” therapies5.Osteoarthritis (OA) is a multi-factorial, mostly slowly progressing, and primarily non-inflammatory degenerative disorder of the synovial joints that is often age related and/or trauma induced. Degradative processes eventually lead to the irreversible destruction of the articular cartilage and other tissues of the joints. Although OA is the most common musculoskeletal condition worldwide that causes significant health, economic, and social problems, research efforts so far have not been able to define its exact etiology. Age-related wear of articular cartilage and subchondral bone, limb overuse, overloading and mal-alignment, genetic disorders, and metabolic syndromes  are important players in the onset and progression of OA5. Several attempts have been made to group and identify OA phenotypes according to pathobiological mechanisms. Felson defines criteria for characterizing OA phenotypes via an epidemiological approach4. He discriminates between generalized arthritis and joint-specific OA, secondary and primary OA, and incident and progressive OA. Another intriguing perspective is presented by Berenbaumet al., who provide a novel definition of OA as a “mismatch disease” commonly referred to by evolutionary biologists6. This is defined as a condition that is more common today than in the past because the human body is not well adapted to certain features of modern environments. These features are high levels of physical inactivity, chronic low-grade inflammation, high body mass index, and obesogenic diets (processed foods which are high in sugar and saturated fat and low in fiber content). Other distinctions include the following phenotypes: chronic pain with central sensitization, inflammation, metabolic syndrome, bone and cartilage metabolism, and mechanical and minimal joint disease7. Devezaet al. divide according to mechanistic subgroups including senescence, inflammation, pain, and metabolic endotypes5.It is accepted that defining OA subgroups based on risk factors is too simple, so it is of high clinical interest to identify specific OA phenotypes  and endotypes , which are the basis of accurate prognosis and development of personalized therapies8. Brandtet al. pointed out in a commentary that they feel all OA is secondary due to the accumulation of intra-articular (i.a.) stress and all OA is primarily driven by mechanical stress on the joint9. They postulate a basic mechanical etiopathogenesis for common OA and would rather categorize it on the basis of the underlying mechanical abnormality like post-traumatic, failure to absorb repetitive impulsive loading, and congenital or developmental anatomic incongruities.However, one thing has to be kept in mind when trying to define subgroups of OA. Although OA can be initiated by multiple factors at multiple sites, mechanical overloading is still the key feature of its pathogenesis. Joint tissues are sensitive to physical stimuli, and OA may result from excessively aberrant or physiologically normal mechanical stresses on initially healthy or pathologically impaired articular cartilage, bone, and ligaments, respectivelyThis review will select key OA pheno-endotypes according to relevant literature and current clinical trials/therapies that have been the most promising targets for recent clinical or pre-clinical studies.11. Cartilage matrix degeneration products are well investigated for drug target discovery. Several key anabolic and catabolic pathway enzymes are dysregulated in OA cartilage, providing the opportunity to identify and validate new drug targets12. To this end, novel combinations addressing existing known targets may be identified. This also includes combinations of therapeutics that are anti-catabolic and those that target anabolic signaling pathways. If investigated, this might lead to identifying novel efficacious add-ons from combining known drugs and targets. For better classification, there should be discrimination between disease-modifying OA drugs (DMOADs), which target pathways of cartilage catabolism and anabolism, and regenerative strategies based on stem cells and their components.OA is characterized by the degradation of articular cartilage and bone matrix components. Among the earliest are type II collagen and the proteoglycan aggrecan, leading to the loss of cartilage structure and function13. Sprifermin is currently being studied in a phase II multicenter randomized dose-finding clinical study . The i.a. administration of 100 μg of sprifermin to participants with symptomatic radiographic knee OA every 6 or 12 months vs. placebo resulted in an improvement in total femorotibial joint cartilage thickness after a follow up period of 2 years. This improvement was statistically significant, but clinical importance was not clear. Application of a lower dose, 30 μg of sprifermin, every 6 or 12 months vs. placebo did not result in a significant difference and it was uncertain whether the response was long lasting14  investigated the administration of BMP-7 in patients with knee OA. Two trials were completed without results being posted (NCT01133613 and NCT01111045), and one was completed with results published (NCT00456157)in pain .46, might be another way to delay cartilage destruction. A human clinical trial (NCT00041756) involving knee OA patients receiving the MMP inhibitor PG-116800 (PG-530742), which has low affinity for both MMP-1 and MMP-7, was unfortunately terminated because of musculoskeletal toxicity12  and BMP-7 have promising pro-anabolic effects on cartilage tissue, whereas the inhibition of catabolic factors such as proteases has not shown beneficial effects in cartilage so far owing to adverse effects .www.clinicaltrials.gov, suggesting regenerative medicine may be a promising therapy for future OA management. At first, case reports described the transplantation of mesenchymal stem cells (MSCs) through an invasive approach (surgery). Later, the introduction of autologous MSCs within the joint by i.a. injection, which represents a less-invasive strategy, was reported to be feasible and safe. Indeed, after i.a. injection into SCID mice, MSCs engrafted directly in the injured site, which has been suggested to avoid systemic distribution and toxicity as well as to promote longer survival of the cells48. Currently, mostly autologous MSCs and adipose-derived stem cells (ASCs) are applied for knee OA therapy, but very few approaches are targeting hip OA. Almost all of the trials are phase Ia/b safety and dose-finding studies with a low number of participants. About 45 of the listed studies are completed, but only six of them report results. A total of 55 of the trials that are recruiting or not yet recruiting indicate strongly increasing interest in stem cell therapy not only for traumatic cartilage injury but also for late-stage OA.A total of 144 clinical trials investigating the therapeutic impact of stem cells on OA and on cartilage trauma have been reported to date atOur literature search shows that stem cells, specifically autologous stem cells derived from bone marrow (BMSCs) and adipose tissue (ASCs), are preferred over other cell types for regenerative strategies. However, there is doubt among surgeons and researchers about whether or not stem cells are really the optimal tool for regenerative therapy.49. When exposed to inflammatory signals, MSCs develop and show a rich secretory profile. After stimulation of these cells with tumor necrosis factor (TNF)-α, a proteomic approach identified 118 proteins, which were differentially expressed by human ASCs50. However, paracrine effects are not limited to soluble factors, as stem cells and other cell types produce extracellular vesicles (EVs), which are small phospholipid-bilayer-enclosed particles carrying many cytoplasmic components52. EVs play a role in a number of different cellular activities, such as communication between cells via horizontal transfer of mRNA and proteins, and they are distinguished according to size. EVs are attractive options for therapeutic use because of their unique physical and biological characteristics, which include high biocompatibility and intrinsic targeting activity53. Exosomes, the most-studied group of EV, can be as small as 30–150 nm in diameter, so it may be possible for them to passively diffuse through tissues54. Overall, the consensus is that stem cell secretomes and EVs applied for the treatment of cartilage pathology and knee OA had pleiotropic and overall positive effects55.In vitro, anti-catabolic, immunomodulatory, and regenerative properties were assigned to the secretome and EVs. Pre-clinicalin vivo studies resulted in positive effects on the joint and confirmed the effectiveness of EV injections as a minimally invasive therapy56. Exosome injections partially improved the gait abnormality patterns in an OA mouse model57, and MSC secretome injections provided early (day seven) pain reduction in treated mice58. All together, these data support the translational potential of this regenerative approach. The promisingin vitro andin vivo results support the potential of this new treatment strategy, opening up new perspectives for cell component-based therapies. EVs are proposed as next-generation biomarkers to predict the pathophysiological state of the joint55, assigning an important role for EVs in future therapies for the treatment of joint disorders. Remarkably, they constitute a simpler, and most of all safer, alternative to actual cell-based therapeutic strategies, as they are cell derived but not living cells and thus cannot proliferate or form tumors. As known for cells, EVs can also be combined with scaffolds, either bound on their surface or embedded within the biomaterial matrices. Specific activation signals such as ultrasound may enable the controlled release of specific subpopulations of EVs, i.e. exosomes.After administration, stem cells tend to disappear quickly from the target tissue; however, their chondroprotective and immunomodulatory effects are long lasting. Presumably, the therapeutic effects are mainly mediated in a paracrine manner, as they appear to be independent of the engrafted cells60. Therefore, any changes affecting bone cell metabolism, structural integrity, and architecture might render the bone more susceptible to aberrant loading or even induce abnormal reactions to normal physiological load. OA-related changes in subchondral bone structure were long regarded as an adaptation of bone to the biomechanical changes observed in articular cartilage. Recently, several pre-clinical and clinical studies demonstrated that alterations in bone structure might even precede and instead mediate cartilage pathology62 and that OA progression is associated with temporal changes in bone structure60. In early OA, accelerated bone turnover leads to bone plate thinning and increased porosity, whereas the trabecular compartment shows increased trabecular spacing and decreased bone volume fraction. Progression of OA is accompanied by subchondral bone plate thickening, increased trabecular thickness, and increased bone volume fraction60. Bone marrow lesions (BMLs), a hallmark of OA, appear early on MRI and are associated with increased pain and cartilage degeneration63.Besides nutrient supply and metabolism, physiological and non-physiological shock absorption and support of overlying cartilage are the main functions of subchondral bone18. There are indications that a specific patient subgroup might respond to BP use: intravenous zoledronic acid successfully reduced BML size and visual analogue scale (VAS) pain score after 6 months in a randomized controlled trial, though a second multicenter trial could not confirm the results17. Furthermore, a meta-analysis by Vaysbrotet al. identified similar effects in a trial using oral risedronate treatment in a patient subgroup with BMLs18. An active phase III trial (NCT02746068) using AXS-02 (disodium zoledronate tetrahydrate) in knee OA with associated BMLs provided promising results in the reduction of pain supposedly by suppression of local acid and pro-inflammatory cytokine production64. The ongoing phenotype debate in OA raised the question of whether the effectiveness of BPs has been confounded owing to the heterogeneity of the patient group enrolled in clinical trials so far64. Indeed, BPs might be especially beneficial in patients with BML or high bone turnover in an early state of OA. Interestingly, pharmacologic agents like BPs that directly affect osteoclast activity effectively reduced pain. Recent research identified osteoclasts as the inducers of OA bone pain by induction of sensory innervation in a murine OA model65. Determination of more of these connections and more precise categorization of patient subgroups (like bone pain due to BML development or increased osteoclast activity/bone turnover) might lead to the repurposing of already existing drugs for new targets.Bisphosphonates (BPs) effectively slow down bone turnover by inhibiting osteoclast activity in osteoporosis, but their usability in OA remains uncertain66 and slowed bone and cartilage degeneration in a phase IIa multicenter trial of primary knee OA20. With 26 weeks’ duration, the study was relatively short, and MIV-711 did not reduce pain during this time .New therapeutic approaches include neutralization of cathepsin K, the major osteolytic protease released by osteoclasts. The “small molecule” cathepsin K inhibitor MIV-711 effectively attenuated joint pathology in a rabbit OA modelis time . Denosum3  might be an option to target and strengthen bone in OA owing to its ability to increase calcium and phosphate uptake from the gut and its direct effect on bone cell metabolism70. Numerous trials for vitamin D3 supplementation in OA patients can be found at clinicaltrials.gov, but there are contradictory reports about a relationship between vitamin D levels and a higher risk for OA incidence and progression71. A meta-analysis of randomized controlled trials revealed a reduction in WOMAC pain and improved joint function in OA patients after vitamin D3 intake, but only at a concentration of 2,000 IU72. Cartilage degradation was not affected. Generally, vitamin D3 intake might be beneficial for a large proportion of the population, as its deficiency is a worldwide problem and elderly people, who are also at an increased risk of OA, are often affected73.Additional dietary supplementation of vitamin DRestoration of bone metabolism and structure might be a worthwhile goal because of the huge importance of this structure as a mechanic buffer for proper load perception and distribution. A detailed knowledge of timely changes in OA-related bone metabolism might enable a more precise use of bone anabolic and anti-catabolic therapies to restore or prevent bone degradation. Maintenance of bone structure and shock-absorbing abilities might prevent cartilage alterations and therefore put a hold on subsequent degradative events culminating in joint failure.74. Synovitis is a common feature of inflammatory OA, and technical progress in imaging technologies like ultrasound and MRI revealed synovitis in a large number of patients at different disease stages76. A plethora of triggers including aberrant mechanical forces, metabolic syndrome, increased age, and fragments of cartilage extracellular matrix (ECM) or crystals might induce the release of these mediators from various responsive joint tissue cell types. A large number of recent review articles address cells and components of the innate immune system as the main drivers of OA inflammatory processes78. Non-steroidal anti-inflammatory drugs and glucocorticoids are commonly used to treat OA but are not optimal owing to moderate effectiveness and serious side-effects in long-term use80.It is now commonly accepted that OA has an inflammatory component that might be more dominant in specific patient subgroups and joint tissues. The release of various pro-inflammatory mediators like prostaglandins, cytokines, and chemokines has been demonstrated in numerous pre-clinical OA animal models and in patients81 and was associated with hip OA over knee OA in a study analyzing mononuclear cell contribution to synovial inflammation82. The anti-GM-CSF antibody otilimab (GSK3196165) was enrolled in a study for erosive hand OA and yielded promising results (NCT02683785)83. Mavrilimumab, a GM-CSF receptor inhibitory antibody effective in RA84, might therefore also have potential as an OA drug. The predominantly low-grade and non-systemic inflammation observed in OA might explain the limited success of single cytokine blockade. Concentration on specific OA subsets like erosive hand OA associated with more pronounced inflammation potentially presents a responsive patient group for anti-cytokine biologics. Identification of a single potent driver of OA inflammation seems to be difficult, and broader approaches targeting general pro-inflammatory signaling pathway components might be more favorable (Most biologics used to treat OA-related inflammation were developed for rheumatoid arthritis (RA), a disease associated with more pronounced inflammation. So far, biologics targeting the inflammatory cytokines interleukin (IL)-1 and -6 as well as TNF-α have not been useful in the prevention of pain or structural progression of OA . Granulovorable .85. Several studies using HC in hand OA have failed to show effects so far31, and results from a study in knee OA are not obtainable yet (NCT01645176). TLR downstream targets include molecules like MyD88, TRAF3/6, p38 MAPK, Janus kinases, and transcription factors like NF-κB86. Several attempts have been made to interfere with signaling molecules to inhibit inflammation in OA. The I-κB kinase inhibitor SAR113945 was tested in four trials of knee OA , but, though the compound showed a good safety and tolerability profile, a larger proof-of-concept study failed to show superior efficacy32. A small subgroup of patients with effusion at baseline elicited a reduced WOMAC pain score after 56 days. Local delivery of a potent p38 MAPK inhibitor (PH-797804) reduced joint destruction and inflammation in a murine destabilization OA model87. The efficacy of PH-797804 compared to naproxen was evaluated in a clinical trial involving knee OA patients, but the results have not yet been disclosed (NCT01102660). FX-005, another therapeutic p38 MAPK inhibitor with sustained-release kinetics, was evaluated in a phase I/II knee OA trial where it promoted pain relief superior to placebo after 4 weeks (NCT01291914) , a chloroquine derivative used to treat malaria and inflammatory autoimmune disorders like RA, supposedly exerts its effects via Toll-like receptor (TLR) 7/9291914) . Direct 90. An increasing number of drugs targeting different factors of the complement cascade are available and were tested in the clinic for various diseases91, but, to the best of our knowledge, none of them has been tested in OA patients so far. Similarly, there is increasing awareness that adaptive immune mechanisms might be involved in OA pathophysiology93, but these insights have not been translated into therapeutic approaches so far. Identification of more immune system entities contributing to OA development and progression will provide even greater numbers of targets for potential OA therapeutics but requires meticulous analysis of timely and spatial involvement in disease-related processes.The complement system might also be a potential source of therapeutic targets for OA therapy94. Virus-related delivery systems might provide superior performance over classical delivery systems but, clearly, safety aspects  have to be taken seriously and analyzed carefully in relation to effectiveness before considering any kind of genetic therapy95. In general, data from an equine study using AAV2.5-delivered IL-1Ra showed promising results regarding pharmacodynamics and safety profile96. As mentioned above, first-in-human clinical trials will evaluate the safety profile of gene-related therapies and will give a general hint regarding the applicability of gene therapies for OA.Novel genetic approaches are currently under evaluation for safety in clinical phase I studies. They include i.a. injection of recombinant adeno-associated virus type 2/5 (rAAV2.5) vector encoding IL-1 receptor antagonist (IL-1Ra) into one knee joint of patients with moderate OA of the knee (NCT02790723) and FX201, a helper-dependent non-integrating adenovirus, containing the human IL-1Ra gene under the control of an inflammation-sensitive promoter (NCT04119687). Moreover, new gene therapeutic targets include interferon (INF)-β  in subjects with RA or OA and active arthritis of the hand (NCT02727764) as well as XT-150, a plasmid DNA carrying a variant of the human IL-10 transgene (NCT03477487). Although results of these studies are not available yet, gene therapy offers great therapeutic potential as it is aimed at prolonged, site-specific, and controlled release of treatments in the target joints. Confining anti-cytokine or any anti-inflammatory treatment to specific joints could potentially prevent the side-effects observed with a more classic biologic treatment plana priori reason why patients see a physician. Huge effort has been put into OA-related pain research to identify underlying mechanisms, but, because of its complexity, no general guidelines could be identified for its effective treatment98. The sensation of pain in OA does not show uniform appearance among patients and during progression. The source of OA pain includes nociceptive pain komma inflammatory pain and neuropathic pain as well as processes of peripheral and central sensitization. Structural features like BMLs, synovitis, and joint effusion show a strong association with pain intensity100.Apart from structural deterioration in OA joints, pain is a dominant and probably the most debilitating hallmark of OA pathology and theClassical treatments include the use of acetaminophen (paracetamol), NSAIDs, and opioids, which induce a plethora of unwanted side effects. Here komma we will discuss the latest developments in therapies directly targeting neuronal structures to alleviate OA pain.105. After the first promising clinical tests using anti-NGF antibodies, the FDA stopped ongoing trials owing to reports of serious adverse side effects with fast progression of OA and increased demand for knee replacement surgery. After the identification of risk factors (NSAID use) and dose modifications, the hold was lifted in 2015 and clinical trials continued . NGF might be released upon mechanical stimulation or inflammatory mediators from different cell types including osteoclasts, osteocytes, chondrocytes, synovial fibroblasts, and macrophages in pre-clinical OA studies and human OANTR. TrkA inhibition effectively reduced pain behavior in several pre-clinical OA models110. Various molecules have been developed to target the TrkA receptor: pan Trk inhibitor GZ389988 (NCT02424942 and NCT02845271), AR786 , ASP7962 , ONO-4474 (pan Trk inhibitor), and VM902A . Only GZ389988 exhibited modest pain reduction compared to control after 4 weeks but no prolonged efficacy after 12 weeks34 , is currently being investigated in a phase I clinical trial (NCT03227796), but results are not available yet.New strategies to inhibit NGF-induced pain concentrate on the antagonization of its receptors tropomyosin-related kinase A (TrkA) and p75eeks34 . NGF rec115. TRP vanilloid 1 (TRPV1) channels expressed by sensory neurons might be targeted using receptor agonists like capsaicin or resiniferatoxin. In various animal models, these molecules resulted in long-term desensitization or nerve degradation, thus reducing pain and neuropeptide release, but conflicting observations have been reported114. Currently, several active clinical trials are analyzing the use of topical and i.a. trans-capsaicin (CNTX-4975) in knee OA. Stevenset al. described a dose-dependent effect of i.a. CNTX-4975 that reduced pain compared to placebo over 24 weeks in patients with moderate-to-severe knee OA , a selective competitive TRPV1 receptor antagonist, was evaluated in phase I studies and successfully reduced pain and improved functional score in knee OA patients after 7 days, though future studies require dose adjustment owing to adverse events involving thermal perception37 -induced OA121. Furthermore, Vertex’s sodium-channel blocker VX-150 showed efficacy in acute pain reduction and was granted a breakthrough therapeutic potential by the FDA, though results of a completed phase IIa clinical study have not been published yet (NCT02660424)122.Furthermore, ion channels like transient receptor potential (Trp) and Nav1.7/Nav1.8 voltage-gated sodium channels gained attention as potential drug targetsknee OA 35. The tion37 . Animal 38  agonists with an improved safety profile targeting the µ, δ, and κ subtypes are in development. Selectively targeting the peripheral κ-OR might avoid side effects observed when drugs target the µ-OR. Cara Therapeutics developed the selective κ-OR agonist CR845 and evaluated its efficacy in knee and hip OA (NCT02524197 and NCT02944448), reporting dose-dependent efficacy in the reduction of pain in hip OA over knee OA38 .123. Cebranopadol (GRT6005), a dual agonist for the NOP/µ-OR, proved to be safe and efficient in chronic back pain124 and was recently tested in patients with painful knee OA (NCT01357837 and NCT01709214). Other nerve-associated targets include the bradykinin B2 receptor  and the CB2 cannabinoid receptor . Direct blockade of the sensory neuropeptide α-calcitonin gene-related peptide using LY2951742  failed to reduce pain in patients with mild-to-moderate OA39 e OA39 . CR4056,type40 .Despite huge efforts invested in the development of new OA analgesics and although several candidates look promising and more and more potential drug targets are identified, pain reduction in OA is still relatively unsuccessful. The complex and diverse underlying mechanisms of OA pain, the timely and spatial alterations of pain types and sensitization, and the interaction of nerves and OA-related structural changes, immune reactions, and altered metabolic conditions still require more intense interdisciplinary research to achieve effective pain management.128.Metabolism can be altered in OA, and there are multiple metabolic components underlying metabolic dysregulation. The metabolic syndrome, characterized by excessive and long-term positive energy balance, is defined by several cardio-metabolic factors that commonly are found together with obesity, which are central adiposity, dyslipidemia, impaired fasting glucose levels, and hypertension. People with metabolic syndrome are prone to developing a variety of disorders, especially cardiovascular diseases, type 2 diabetes mellitus (T2DM), and some forms of tumor. The increase in prevalence of metabolic syndrome in industrialized nations, and an association with obesity, together with the fact that it was a rare disease in pre-industrial populations, leads to the hypothesis that the metabolic syndrome might be a major risk factor for OA nowadays129, T2DM and knee OA often coexist and are known for common risk factors such as obesity and aging. The mechanical impact of excess body weight on joints may explain lower limb OA. However, it is unclear whether T2DM is linked to OA independently of excess weight and whether T2DM is involved in OA pathology. A coexistence between the occurrence of T2DM and OA was shown, but a causal link is not yet established130. T2DM clearly has an unhealthy effect on OA via two pathways: (1) chronic hyperglycemia, which is connected to oxidative stress, abnormal production of pro-inflammatory cytokines and advanced glycation end products (AGEs) in joint tissues, and (2) insulin resistance, which may have local effects but may also maintain a systemic low-grade inflammatory state131.Besides hypertension, which seems to provide an elevated risk of knee OA119. OA chondrocytes where AMP kinase (AMPK) has been removed exhibit increased catabolic responses to pro-inflammatory cytokines and biochemical injury. These effects are attenuated by molecules that activate AMPK, indicating that decreased AMPK activity is associated with cartilage degradation121. Possibly, AMPK-activating drugs such as methotrexate, metformin, and sodium salicylate might be good candidates to combat OA progression. A Taiwanese study examined whether or not the use of a COX-2 inhibitor with metformin in OA patients with T2DM was related to fewer joint replacement surgeries than the use of a COX-2 inhibitor only122. At the end of a 10-year follow up period, fewer joint replacement surgeries seemed to be needed in the case group compared to the control group. This outcome may be attributed to the fact that a combination therapy decreases pro-inflammatory factors associated with OA progression much more than one without metformin therapy  and mitochondrial dysfunction. Hypertrophic and degradative markers were upregulated in chondrocytes, resulting in increased breakdown of the cartilage matrix. The authors reported that the severity of these diet-induced OA symptoms was reduced by the application of atorvastatin and a mitochondrial-targeting antioxidant, thus implicating that hypercholesterolemia promotes OA progression by mitochondrial dysfunction in chondrocytes, which was in part a result of increasing ROS production and apoptosis.Some pre-clinical and clinical data are available regarding the effects of statin usage on OA progression. Farnahgi42. Another trial investigated the association between statin therapy initiation and incidence of hand OA, but no association was observed in this study43. A pooled analysis based on time-to-event analysis of four population-based large cohorts demonstrated that statin use is not associated with reduced risk of consultation or surgery for OA of the hip or knee44  promoter141. Introducing OA in this mouse strain after anterior cruciate ligament transection (ACLT) allowed the selective following and removal of senescent cells. I.a. injection of a senolytic molecule, UBX0101, which selectively kills senescent cells, attenuated the development of post-traumatic OA, reduced pain and the production of SASP factors, and improved the development, phenotype, and function of human OA chondrocytes in 3D pellet culture. The half-life of UBX0101 was low, preventing systemic exposure, but was still effective in blocking cartilage degradation by eliminating senescent cells rather than blocking their SASP secretion.Cellular senescence leads to the senescence-associated secretory phenotype (SASP), which is characterized by cell-cycle arrest, enhanced production of pro-inflammatory cytokines and other factorsTargeting cell cycle inhibitors appears to be an intriguing new strategy to halt OA progression by addressing a risk factor, aging, that is closely associated to OA.142. The prevention of mitochondrial peroxiredoxin (PRX) 3 hyperoxidation-induced expression of mitochondrial catalase abrogated p38-mediated cell death and restored homeostatic signaling to maintain the viability of aging chondrocytes143. Another promising target could be superoxide dismutase 2 (Sod2), as deletion of Sod2 enhanced the severity of OA in mice144. Another interesting target for counteracting oxidative stress-induced tissue damage might be nuclear receptor erythroid 2 related factor (Nrf2). Nrf2 is a key transcription factor that regulates the expression of phase II antioxidant enzymes. These enzymes protect against oxidative stress and tissue damage. Cai and colleagues explored the role of Nrf2 in OA pathogenesis and the effects of Nrf2 acetylation for histone deacetylase inhibitor (HDACi) protection. For this, they took advantage of two murine OA models: i.a. injection of MIA and destabilization of the medial meniscus (DMM)145. In order to analyze the efficacy of HDACi on protection from cartilage damage, a pan-HDACi, trichostatin A (TSA), was applied. TSA promoted the induction of Nrf2 downstream proteins in mouse joint tissues and reduced the expression of OA-associated proteins like several MMPs and pro-inflammatory cytokines. TSA markedly ameliorated the cartilage damage in both OA models but offered no significant protection in Nrf2-knockout mice, suggesting that the protective effect of HDACi on OA progression was Nrf2 dependent.An important therapeutic target might be redox-signaling pathways and associated mitochondrial dysfunction in OA. It is accepted that increasing levels of ROS contribute to age-related diseases by promoting cellular dysfunction and abolishing physiological cell signaling pathwaysAddressing redox-signaling pathways and mitochondrial dysfunction will enable exciting novel strategies to combat cellular senescence in general and thereby eliminate a major risk factor for OA: age.et al. treated these mice for 3 months with SFX-01®, a synthetic stable variant of sulforaphane, a naturally occurring antioxidant146. SFX-01® treatment both modifies bone architecture in the STR/Ort mice and likely reduces OA pain and improves gait without improving articular cartilage lesion severity and occurrence of osteophytes in the joints of these mice. These findings strengthen the possibility that bone-targeting therapies with antioxidants may have some merit and exert osteotrophic effects possibly not only in OA.Antioxidants may also have bone-protective effects in OA pathology. Using a spontaneous OA model, the STR/Ort mice, JavaheriFor a few years, a novel concept considered OA as a multi-faceted disease involving the whole joint and not only cartilage or synovium. This offers new options to identify and develop novel therapeutics and to re-profile candidate drugs. Recent advances in OA pathology have enlightened key roles of several new pathways, which can be targeted. However, as OA is a highly heterogeneous disease, a single therapeutic targeting a single joint tissue may not be effective and no “one size fits all” drug/therapy will ever be developed. Improved patient stratification in combination with advanced DMOADs and cell-based therapies might lead to the development of personalized OA therapeutics.Attention to temporal changes in disease progression like the transition from high bone turnover in early OA to decreased bone turnover in the late stages or timely changes in the pain type requires precise knowledge of the underlying mechanistic alterations. Choosing appropriate medication for selective disease time-points might help tailor individual treatment regimens for each patient in the future. Additionally, OA might present itself with overlapping endotypes like, for instance, an inflammatory pain endotype that could benefit from a combination of pharmaceuticals addressing both pain and inflammation.Many clinical studies have been conducted in OA that address mainly structural targets like cartilage and bone in combination with reduction of inflammation and pain. In general, success was marginal, and only very few drugs, i.e. sprifermin or some BPs, resulted in improvement of joint structure and function. In addition, targeting TrkA or TRPV1 led to pain relief; however, no pharmacological treatment was able to halt or reverse OA progression long term.Data from the application of MSCs/ASCs generated some guarded optimism; however, so far, only cartilage lesions were addressed with cell-based therapies, whereas subchondral bone, tendons, and other joint tissues were not included. This is a crucial shortcoming, as OA is recognized as a whole-joint disease.One central point to be considered for all future therapeutic approaches, either regenerative or pharmacological, is the mechanical status of the joint. With this in mind, it is strongly suggested that the altered joint mechanics that cause OA are addressed in a first-line therapy. If altered OA joint mechanics are not normalized and original biomechanical pathways are not restored, it will be most likely that pharmacological or biological treatments of articular cartilage or inflammatory processes will not be efficacious. Furthermore, modulation of derailed cellular mechanoreceptive pathways might provide new opportunities to halt structural tissue deterioration. OA is not a single disease with a common pathophysiological pathway, as many pathways and risk factors lead to mechanical failure of the joint , so idenConsidering these central points, personalized OA therapy is the ultimate goal, and recent advances in phenotype classification and targeted drug development might provide a pool of suitable therapeutic options in the future.