Objectives Many rheumatoid arthritis (RA) patients continue to experience persistent pain even after successful management of joint inflammation. Clinical data indicate that RA patients treated with the JAK inhibitor baricitinib consistently achieve pain relief that cannot be entirely attributed to its anti-inflammatory effects. In this study, we investigated the antinociceptive properties of baricitinib using the collagen antibody-induced arthritis (CAIA) model in which mechanical hypersensitivity persists long after resolution of joint inflammation. Methods The effects of baricitinib, etanercept (tumour necrosis factor inhibitor), and LP-922761 (adaptor protein-2 (AP2) associated kinase 1 (AAK1) inhibitor) on pain-like behaviour in CAIA mice were examined. Tissue samples from the late, low-grade inflammatory phase were examined for the effect of the treatments. Additionally, in vitro experiments using dorsal root ganglion (DRG) cells were conducted to assess baricitinib's influence on neuronal excitability and cell morphology. Results Baricitinib reduced CAIA-induced joint inflammation, but its antinociceptive effects were most pronounced during the late phase when etanercept was ineffective. Administering baricitinib both early and late significantly decreased CAIA-induced bone loss, synovial innervation, and baseline STAT3 phosphorylation in ankle joints and DRGs. Unlike etanercept, baricitinib effectively reduced pain-like behaviour and synovial hyperinnervation when administered exclusively in the late phase. Additionally, baricitinib modulated glial cell morphology and neuronal excitability in vitro. Notably, it inhibited AAK1 signalling in DRGs, with AAK1 kinase activity blockade providing an antinociceptive effect in the CAIA model. Conclusions Our data suggests that baricitinib has antinociceptive effects by targeting not only immune cells but also neurons and glia cells via inhibition of 2 signalling pathways linked to chronic pain.

Compartmentalized cell cultures (CCCs) provide the possibility to study mechanisms of neurodegenerative diseases, such as spreading of misfolded proteins in Alzheimer's or Parkinson's disease or functional changes in, e.g., chronic pain, in vitro. However, many CCC devices do not provide the necessary capacity for identifying novel mechanisms, targets, or drugs in a drug discovery context. Here, we present a high-capacity cell culture microtiter microfluidic plate compliant with American National Standard Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) standards that allows to parallelize up to 96 CCCs/experimental units, where each experimental unit comprises three microchannel-connected compartments. The plate design allows the specific treatment of cells in individual compartments through the application of a fluidic barrier. Moreover, the compatibility of the plate with neuronal cultures was confirmed with rodent primary as well as human-induced pluripotent stem cell-derived neurons of the central or peripheral nervous system for up to 14 days in culture. Using immunocytochemistry, we demonstrated that the plate design restricts neuronal soma to individual compartments, while axons, but not dendrites, can grow through the connecting microchannels to neighboring compartments. In addition, we show that neurons are spontaneously active and, as deemed by the appearance of synchronous depolarizations in neighboring compartments, are synaptically coupled. In summary, the design of the microfluidic plate allows for both morphological and functional studies of neurological in vitro cultures with increased capacity to support identification of novel mechanisms, targets, or drugs.

Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overex-pression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low -threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action poten-tials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models. Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues. To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.

Background Alzheimer’s disease (AD) is the most common form of age-related neurodegenerative diseases. Cerebral deposition of Aβ peptides, especially Aβ42, is considered the major neuropathological hallmark of AD and the putative cause of AD-related neurotoxicity. Aβ peptides are produced by sequential proteolytic processing of APP, with β-secretase (BACE) being the initiating enzyme. Therefore, BACE has been considered an attractive therapeutic target in AD research and several BACE inhibitors have been tested in clinical trials, but so far, all have had negative outcomes or even led to worsening of cognitive function. AD can be triggered by Aβ years before the first symptoms appear and one reason for the failures could be that the clinical trials were initiated too late in the disease process. Another possible explanation could be that BACE inhibition alters physiological APP processing in a manner that impairs synaptic function, causing cognitive deterioration. Methods The aim of this study was to investigate if partial BACE inhibition, mimicking the putative protective effect of the Icelandic mutation in the APP gene, could reduce Aβ generation without affecting synaptic transmission. To investigate this, we used an optical electrophysiology platform, in which effects of compounds on synaptic transmission in cultured neurons can be monitored. We employed this method on primary cortical rat neuronal cultures treated with three different BACE inhibitors (BACE inhibitor IV, LY2886721, and lanabecestat) and monitored Aβ secretion into the cell media. Results We found that all three BACE inhibitors tested decreased synaptic transmission at concentrations leading to significantly reduced Aβ secretion. However, low-dose BACE inhibition, resulting in less than a 50% decrease in Aβ secretion, did not affect synaptic transmission for any of the inhibitors tested. Conclusion Our results indicate that Aβ production can be reduced by up to 50%, a level of reduction of relevance to the protective effect of the Icelandic mutation, without causing synaptic dysfunction. We therefore suggest that future clinical trials aimed at prevention of Aβ build-up in the brain should aim for a moderate CNS exposure of BACE inhibitors to avoid side effects on synaptic function.

Diseases such as chronic pain with complex etiologies are unlikely to respond to single, target-specific therapeutics but rather require intervention at multiple points within a perturbed disease system. Such approaches are being enabled by the rise of computational methods to identify key points of intervention and by new screening techniques that focus on a relevant condition or phenotype, rather than a specific target. Here we apply an in silico network pharmacology approach to identify small-molecule compounds with the potential to selectively disrupt the structure of a chronic-pain specific disease network, which we validate using a novel phenotypic screen that recapitulates key aspects of neuronal and pain biology by measuring changes in neuronal excitability in native sensory neurons. The combination of network pharmacology with a phenotypic screen is a powerful approach; we show that hit rates increase from 26% to 42%. This represents a rational approach to the discovery of compounds with a poly-pharmacology based therapeutic value, which will be vital for the discovery of treatments for complex disease.

Censo Biotechnologies and Cellectricon have joined forces to develop a first-in-kind humanized drug discovery platform to target chronic pain. The platform is based on human induced pluripotent stem cell–derived neurons in combination with Cellectricon's Cellaxess ® Elektra screening system. The combination of highly functional screening with a human cell type of relevance for chronic pain has the potential to enable the generation of better drug candidates for this condition and other diseases.

Chronic pain is a disease area with a significant and unmet need for improved treatments. However, due to the complex nature of pain transduction, pain is a notoriously difficult area for drug discovery, where conventional target-based efforts have largely failed to produce new drugs. In this whitepaper, we present a novel phenotypic screening platform based on our proprietary Cellaxess® Elektra technology. This platform provides an exciting opportunity to efficiently identify new classes of compounds that affect neuronal excitability in primary peripheral pain-sensing neurons, while sparing the CNS and cardiovascular system. Combining these three assays means that important decisions about which compounds to advance to further testing and which to eliminate can be made in a seamless operation much earlier along the discovery process, translating to an increase in efficiency, and consequently a decrease in time and costs. Åke Jägervall, Susanne Lardell, Paul Karila, Mattias Karlsson A novel phenotypic screening platform targeting chronic pain drug discovery

Manual patch clamp recordings offer direct insight into ion channel properties through the characterization of ion channel activity. Due to the high quality data it is considered the gold standard for ion channel research. Still, the limited throughput means that maximizing data from each cell, especially sensitive primary cells, is important.We have developed a patch clamp‐based assay for characterization fast‐acting ion channels, in this case exemplified by P2X, in primary dorsal root ganglion (DRG) neurons. This assay is based on the use of primary DRG neurons in culture as a cell model for chronic pain. These neurons retain their sensory functionality and remain responsive to thermal, mechanical and functional stimuli, and when supplemented with nerve growth factor (NGF) they can be used to mimic peripheral sensitization. The assay utilizes a microfluidic perfusion system, Dynaflow Resolve, to facilitate a stable recording situation and fast and programmable solution exchange. At the start of the experiment, compounds and buffer are loaded in the 16 wells of the Dynaflow Resolve chip. Micro‐channels connect each well to a recording chamber where the cells are added. To be able to provide fast solution exchange the DRG neuron is lifted using the patch pipette and positioned in front of the micro‐channel outlets. Then, the cell is scanned through the discrete flow zones formed outside the channels.A comparison between conventional bath perfusion and the automated Dynaflow Resolve system showed that both systems have similar capacity when it comes to obtaining successful high resistance seals. However, for collecting full 5‐8 point dose‐responses from single cells, the success rate for the Dynaflow Resolve superseded the conventional system. This enables a dramatically increased productivity when performing more complex pharmacological characterizations.

In cell‐based high throughput screening applications, the assumption is typically made that all molecules have equal and complete access to the target of interest. In the case of intracellular targets, this is most likely not the case, as the molecules in a diverse screening library will exhibit varying degrees of cell permeability, or even lack thereof. This is likely to result in in false negatives.Electroporation is the use of brief electric pulses to overcome the barrier of the cell membrane. By the application of external electric fields, transient and reversible breakdown of the membrane can be induced. This transient state can be used to load cells with a variety of different molecules. Initially developed for gene transfer, electroporation can be used for delivery of a variety of other molecules ranging from ions to drugs, dyes, tracers and antibodies. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to tumors has been performed.In this work, we have explored the use of a high throughput electroporation platform, Cellaxess Elektra, for unbiased delivery of small molecules into cells in a cell‐based assay. The molecules selected were fluorescent and were representative of those that can be found in a small molecule library. The cell permeability with and without electroporation was characterized by means of fluorescence microscopy, and in the selected collection of ~400 compounds, 20 exhibited low or no cell permeability which could be enhanced by means of electroporation. We believe that this could prove to be a valuable tool in identifying families of new leads previously undiscovered.

AZ465 is a novel selective transient receptor potential cation channel, member A1 (TRPA1) antagonist identified during a focused drug discovery effort. In vitro, AZ465 fully inhibits activation by zinc, O-chlorobenzylidene malononitrile (CS), or cinnamaldehyde of the human TRPA1 channel heterologously expressed in human embryonic kidney cells. Our data using patch-clamp recordings and mouse/human TRPA1 chimeras suggest that AZ465 binds reversibly in the pore region of the human TRPA1 channel. Finally, in an ex vivo model measuring TRPA1 agonist-stimulated release of neuropeptides from human dental pulp biopsies, AZD465 was able to block 50%-60% of CS-induced calcitonin gene-related peptide release, confirming that AZ465 inhibits the native human TRPA1 channel in neuronal tissue.

The projections of enteric neurons showing immunoreactivity for vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and galanin were investigated in the myenteric plexus of the intestine of the Atlantic cod, Gadus morhua. Quantification of immunoreactive material on the proximal and distal side of a myotomy was performed by means of confocal laser scanning microscopy. NOS immunoreactivity was reduced anal to the myotomy, whereas there was an accumulation of immunoreactivity for VIP and for galanin oral to the cut. These results suggest the presence of VIP, NOS and galanin in neurons with oral–to–anal projections along the intestine of the cod. Since descending neurons in the myenteric plexus of many other vertebrates also contain these substances, we conclude that the oral–to–anal projections of neurons containing VIP, NOS and galanin are highly conserved features and important for the descending phase of intestinal peristalsis on an evolutionary basis.

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.

The presence of galanin-like immunoreactivity in nerves to the stomach of the Atlantic cod has been investigated by immunohistochemistry. The distribution of ganglion cells showing galanin-like immunoreactivity was compared with the total distribution in nerves and ganglia. Projection studies were made to determine the origin of the galanin neurons. The effect of galanin was studied in smooth muscle strip preparations of the gut wall and arteries. Galanin-like immunoreactive ganglion cells frequently occurred along the vagal branches to the stomach. Most of them projected cranially. Immunoreactive nerve fibres were present in all layers of the gut and around arterial branches on the surface of the stomach. Ligations of the vagus and splanchnic nerves produced accumulations of immunoreactive material on both sides of the ligature. Galanin produced weak contractile effects unaffected by tetrodotoxin on the gut wall and on gut arteries. It is concluded that a population of the ganglion cells along the vagus nerve in the Atlantic cod contains a galanin-like peptide. Some of these cells may be parts of autonomic parasympathetic pathways innervating the gut of the Atlantic cod, having direct excitatory effects on the smooth muscles of the gut wall and gut arteries.

The innervation of the cod stomach by neurons showing substance P-like immunoreactivity (SPLI), and the effect and mechanism of action of substance P (SP) on the vascularly perfused cod stomach and on isolated muscle strip preparations from the pyloric sphincter have been investigated.Infusion of SP produced a contraction of the stomach wall, which could not be blocked by tetrodotoxin, atropine or methysergide, indicating a direct effect on the stomach smooth muscle. Similarly, the contraction produced by SP on preparations from the pyloric sphincter was unaffected by tetrodotoxin.Nerves showing SPLI were frequent in the myenteric plexus of the whole stomach, and in the submucosa and mucosa of the pyloric part of the stomach. SPLI was also observed in fibres in the intestinal branch of the vagus and occasionally in the splanchnic nerves. Ligation of the nerves showed an accumulation of SPLI above as well as below the ligature, being more prominent proximal to the ligature in the vagus and distal to the ligature in the splanchnic nerve. In the vagus nerve, descending and ascending SPLI-fibres were seen surrounding non-reactive cell bodies. No reduction in intensity of the immunoreaction of the neurons in the stomach wall was observed after ligation or sectioning of the vagosympathetic trunk or the splanchnic nerves, nor were SP-levels measured by radioimmunoassay reduced. After denervation of vagal branches close to the stomach wall an insignificant decrease of immunoreactivity was observed in the myenteric plexus. Capsaicin treatment had no conclusive effect on the distribution of SPLI.It is concluded that the innervation showing SPLI may be of intrinsic as well as extrinsic origin, with pathways in both vagal and splanchnic branches. Only a direct effect of SP on the smooth muscle could be demonstrated.