Neuropathic pain caused by miswiring and abnormal end organ targeting

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.The research leading to these results has received funding from the following sources: an ERC Advanced Investigator grant to R.K. (Pain Plasticity 294293); grants from the Deutsche Forschungsgemeinschaft to R.K. (SFB1158, projects B01, B06), to T.K. (SFB1158, project B08), to S.G.L. (SFB1158, project A01) and to V.G. (SFB1158, project A03); a grant to B.O. (project number 371923335); and grant CIDEGENT/2020/052 from Generalitat Valenciana to F.J.T. R.K. is a member of the Molecular Medicine Partnership Unit of the European Molecular Biology Laboratory and Medical Faculty Heidelberg. V.G. and T.A.N. were partially supported by a post-doctoral fellowship and physician scientist fellowship, respectively, from the Medical Faculty Heidelberg. D.M. was partially supported by a post-doctoral fellowship from Excellence Cluster CellNetworks. We acknowledge support from the Interdisciplinary Neurobehavioral Core (INBC) for the behavioural experiments, the data storage service SDS@hd and bwMLS&WISO HPC supported by the state of Baden-Württemberg and the German Research Foundation (DFG) through grants INST 35/1314-1 FUGG and INST 35/1134-1 FUGG, respectively.Peer reviewe

Sensorimotor Activity Partially Ameliorates Pain and Reduces Nociceptive Fiber Density in the Chronically Injured Spinal Cord

A large proportion of patients suffering from spinal cord injury (SCI) develop chronic central neuropathic pain. Previously, we and others have shown that sensorimotor training early after SCI can prevent the development of mechanical allodynia. To determine whether training initiated in the subchronic/chronic phase remains effective, correlates of below-level neuropathic pain were analyzed in the hindpaws 5-10 weeks after a moderate T11 contusion SCI (50 kDyn) in adult female C57BL/6 mice. In a comparison of SCI and sham mice 5 weeks post-injury, about 80% of injured animals developed mechanical hypersensitivity to light mechanical stimuli, whereas testing of noxious stimuli revealed hypo-responsiveness. Thermal sensitivity testing showed a decreased response latency after injury. Without intervention, mechanical and thermal hyper-responsiveness were evident until the end of the experiment (10 weeks). In contrast, treadmill training (2 × 15 min/day; 5 × /week) initiated 6 weeks post-injury resulted in partial amelioration of pain behavior and this effect remained stable. Analysis of calcitonin gene-related peptide (CGRP)-labeled fibers in lamina III-IV of the lumbar dorsal horn revealed an increase in labeling density after SCI. This was not due to changes in the number or size distribution of CGRP-labeled lumbar dorsal root ganglion neurons. Treadmill training reduced the CGRP-labeling density in the spinal cord of injured mice, whereas the density of non-peptidergic isolectin-B4 (IB4)+ fibers showed no changes in lamina IIi and a slight reduction of sparse IB4 labeling in laminae III-IV. Thus, sensorimotor activity initiated in the subchronic/chronic phase of SCI remains effective in ameliorating pain behavior and influencing structural changes of the nociceptive system

CD8+ T Cells in OA Knee Joints Are Differentiated into Subsets Depending on OA Stage and Compartment

Osteoarthritis (OA) is no longer considered a purely degenerative disease. OA is defined as a disease of the entire joint, in which inflammation occurs in various joint tissues. The overall aim of this study was to analyze the presence and polarization of CD8+ T cell subsets in OA knee joints, in relation to the OA stage and compartment (synovial fluid (SF), synovial membrane (SM,) peripheral blood (PB)). A quantitative flow analysis of CD8+ T cell subsets to compare the SF, SM, PB, was performed in patients with different stages of OA (early, unicondylar and bicondylar OA). Samples of the SF, SM and PB were harvested from a total of 55 patients at the time of surgery. Early OA was confirmed by independent surgeons intraoperatively. Uni- and bicondylar OA was confirmed and graded by two plane radiographs. Samples were analyzed by flow cytometry for surface markers, and cytokines by intracellular staining (ICS). CD8+ T cells were shown to be differentiated into pro-inflammatory IFN-γ producing Tc1 and IL-17A producing Tc17, as well as anti-inflammatory IL-4 producing Tc2. All CD8+ T cell subsets (Tc1, Tc17, and Tc2) were detected in both the SM and SF. The percentage of CD8+ T cell subsets of the total CD8+ T cell population was dependent on the OA stage and compartment. Compared with the peripheral blood (PB), the proportion of CD8+IFN-γ+ Tc1 and CD8+IL-17A+ Tc17 was significantly increased in OA SF. This was confirmed in our data for both early OA and end-stage OA. In the SM samples of end-stage OA patients, the proportion of CD8+IL-17A+ Tc17 was significantly increased compared to the PB. Comparing SF and SM samples of end-stage OA patients, the proportion of CD8+IFN-γ+ Tc1 was significantly increased in SF, whereas there were no differences concerning CD8+IL-4+ Tc2 and CD8+IL-17A+ Tc17. End-stage OA samples showed a significant increase of CD8+IL-4+ Tc2 in the SM for both unicondylar and bicondylar OA compared to early OA. CD8+ T cells infiltrating the SM and SF in OA knees are differentiated into IFN-γ-, IL-17A-, and IL-4-producing CD8+ T cell subsets (Tc1, Tc17, Tc2). This differentiation depends on the OA stage and OA compartment. Further investigation of CD8+ T cell subsets and their interaction with other inflammatory cells such as CD4+ T cells and macrophages may help to identify novel therapeutic anti-inflammatory strategies for containing OA progression

Rheological Behavior of Different Calf Sera before, during and after Biomechanical Testing

Due to different rheological behavior of human synovial fluid and the test mediums for in vitro examinations, wear tests cannot replicate the in vivo situation completely. The standards for wear testing indicate calf serum as in vitro test medium. However, these standards do not contain precise information on the main constituent components and the rheological properties. In this study, bovine calf serum and newborn calf serum with a protein concentration of 20 g/L, both approved for wear testing defined by the International Organization for Standardization (ISO), were characterized according to their rheological properties to detect differences before and during tribological simulation. The rheological behavior was determined at five defined intervals of a tribological test. The two test fluids differ in their rheological properties before and during the test and can therefore lead to deviating results in tribological testing. Furthermore, both test media changes considerably over test duration. At a test duration of 0.5 Mio cycles, both fluids have changed so that there is no longer any difference between them in terms of rheological properties. These changes could be attributed to denaturation and degradation of proteins. Thus, the choice of medium impacts tribological test results which should be considered for the interpretation of these studies

Intrinsically disordered intracellular domains control key features of the mechanically-gated ion channel PIEZO2

A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function.This study was supported by the DFG grants LE3210/3-1 and SFB1158/1 to S.G.L. We thank Ms. Anke Niemann for technical assistance. We also thank Dr. Ulrike Engel for help with setting up the TRIF experiments at the Nikon Imaging Facility at Heidelberg University.Open Access funding enabled and organized by Projekt DEAL.Peer reviewe

Rheological Behavior of Different Calf Sera before, during and after Biomechanical Testing

Due to different rheological behavior of human synovial fluid and the test mediums for in vitro examinations, wear tests cannot replicate the in vivo situation completely. The standards for wear testing indicate calf serum as in vitro test medium. However, these standards do not contain precise information on the main constituent components and the rheological properties. In this study, bovine calf serum and newborn calf serum with a protein concentration of 20 g/L, both approved for wear testing defined by the International Organization for Standardization (ISO), were characterized according to their rheological properties to detect differences before and during tribological simulation. The rheological behavior was determined at five defined intervals of a tribological test. The two test fluids differ in their rheological properties before and during the test and can therefore lead to deviating results in tribological testing. Furthermore, both test media changes considerably over test duration. At a test duration of 0.5 Mio cycles, both fluids have changed so that there is no longer any difference between them in terms of rheological properties. These changes could be attributed to denaturation and degradation of proteins. Thus, the choice of medium impacts tribological test results which should be considered for the interpretation of these studies

In Vitro Wear Behavior of Knee Implants at Different Load Levels: The Impact of the Test Fluid

Calf serum is defined as a test fluid for in vitro knee wear simulation studies in the ISO standard. However, protein degradation typically occurs during in vitro wear simulation. The current study should indicate whether increased loads change the rheological properties of the test fluid and may, therefore, lead to favorable tribological behavior and reduced wear. Three different load levels were simulated in a displacement-controlled knee wear simulation study. The gravimetric wear rates were determined, pressure measurements were performed, and the dynamic viscosity of the test fluids were analyzed after the simulation of 0.5 × 106 cycles. The lowest load level led to the lowest wear rate, and the lowest contact pressure and contact area, compared to the medium and high-load level. Although, the high-load level led to the highest contact pressure and contact area, the wear rates were comparable to the medium-load level. The rheological measurements revealed the highest dynamic viscosity for the high-load level and no differences could be found between the medium and low loading condition. To perform realistic wear simulation studies, the reproduction of the in vivo interrelationships between the shear forces and wear are necessary

Early-onset treadmill training reduces mechanical allodynia and modulates calcitonin gene-related peptide fiber density in lamina III/IV in a mouse model of spinal cord contusion injury

Abstract: Below-level central neuropathic pain (CNP) affects a large proportion of spinal cord injured individuals. To better define the dynamic changes of the spinal cord neural network contributing to the development of CNP after spinal cord injury (SCI), we characterized the morphological and behavioral correlates of CNP in female C57BL/6 mice after a moderate T11 contusion SCI (50 kdyn) and the influence of moderate physical activity. Compared with sham-operated animals, injured mice developed mechanical allodynia 2 weeks post injury when tested with small-diameter von Frey hair filaments (0.16 g and 0.4 g filament), but presented hyporesponsiveness to noxious mechanical stimuli (1.4 g filament). The mechano-sensory alterations lasted up to 35 days post injury, the longest time point examined. The response latency to heat stimuli already decreased significantly 10 days post injury reaching a plateau 2 weeks later. In contrast, injured mice developed remarkable hyposensitivity to cold stimuli. Animals that underwent moderate treadmill training (2 × 15 minutes; 5 d/wk) showed a significant reduction in the response rate to light mechanical stimuli as early as 6 days after training. Calcitonin gene-related peptide (CGRP) labeling in lamina III-IV of the dorsal horn revealed significant increases in CGRP-labeling density in injured animals compared with sham control animals. Importantly, treadmill training reduced CGRP-labeling density by about 50% (P < 0.01), partially reducing the injury-induced increases. Analysis of IB4-labeled nonpeptidergic sensory fibers revealed no differences between experimental groups. Abnormalities in temperature sensation were not influenced by physical activity. Thus, treadmill training partially resolves signs of below-level CNP after SCI and modulates the density of CGRP-labeled fibers

Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis

Background!#!Investigating the pathophysiological mechanisms of early osteoarthritis (OA) is of utmost interest since this stage holds the strongest promise for therapeutic interventions. The aims of this study were to analyze if synovial inflammation is already present in early OA and to characterize the involved cell populations, by investigating synovial fluid (SF) and synovial membrane (SM) of early OA patients for the presence and polarization status of CD4 T cells.!##!Methods!#!A quantitative analysis of CD4!##!Results!#!SF and SM showed a distinct infiltration of CD4!##!Conclusions!#!Early OA joints show already significant inflammation through CD