Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization

© 2019 The Author(s). Background: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. Methods: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. Results: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. Conclusions: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided

Novel imaging tools for investigating the role of immune signalling in the brain

Abstract not availableJonathan Henry W. Jacobsen, Lindsay M. Parker, Arun V. Everest-Dass, Erik P. Schartner, Georgios Tsiminis, Vasiliki Staikopoulos, Mark R. Hutchinson, Sanam Mustaf

Irinotecan-induced gastrointestinal dysfunction and pain are mediated by common TLR4-dependent mechanisms

Published Online First March 29, 2016Abstract not availableHannah R. Wardill, Rachel J. Gibson, Ysabella Z.A. Van Sebille, Kate R. Secombe, Janet K. Coller, Imogen A. White, Jim Manavis, Mark R. Hutchinson, Vasiliki Staikopoulos, Richard M. Logan, and Joanne M. Bowe

Autofluorescent imprint of chronic constriction nerve injury identified by deep learning

Available online 7 October 2021Our understanding of chronic pain and the underlying molecular mechanisms remains limited due to a lack of tools to identify the complex phenomena responsible for exaggerated pain behaviours. Furthermore, currently there is no objective measure of pain with current assessment relying on patient self-scoring. Here, we applied a fully biologically unsupervised technique of hyperspectral autofluorescence imaging to identify a complex signature associated with chronic constriction nerve injury known to cause allodynia. The analysis was carried out using deep learning/artificial intelligence methods. The central element was a deep learning autoencoder we developed to condense the hyperspectral channel images into a four- colour image, such that spinal cord tissue based on nerve injury status could be differentiated from control tissue. This study provides the first validation of hyperspectral imaging as a tool to differentiate tissues from nerve injured vs non-injured mice. The auto-fluorescent signals associated with nerve injury were not diffuse throughout the tissue but formed specific microscopic size regions. Furthermore, we identified a unique fluorescent signal that could differentiate spinal cord tissue isolated from nerve injured male and female animals. The identification of a specific global autofluorescence fingerprint associated with nerve injury and resultant neuropathic pain opens up the exciting opportunity to develop a diagnostic tool for identifying novel contributors to pain in individuals.Martin E. Gosnell, Vasiliki Staikopoulos, Ayad G. Anwer, Saabah B. Mahbub, Mark R. Hutchinson, Sanam Mustafa, Ewa M. Goldy

In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization

Treating pain by inhibiting ATP activation of P2X3-containing receptors heralds an exciting new approach to pain management, and Afferent's program marks the vanguard in a new class of drugs poised to explore this approach to meet the significant unmet needs in pain management. P2X3 receptor subunits are expressed predominately and selectively in so-called C- and Aδ-fiber primary afferent neurons in most tissues and organ systems, including skin, joints, and hollow organs, suggesting a high degree of specificity to the pain sensing system in the human body. P2X3 antagonists block the activation of these fibers by ATP and stand to offer an alternative approach to the management of pain and discomfort. In addition, P2X3 is expressed pre-synaptically at central terminals of C-fiber afferent neurons, where ATP further sensitizes transmission of painful signals. As a result of the selectivity of the expression of P2X3, there is a lower likelihood of adverse effects in the brain, gastrointestinal, or cardiovascular tissues, effects which remain limiting factors for many existing pain therapeutics. In the periphery, ATP (the factor that triggers P2X3 receptor activation) can be released from various cells as a result of tissue inflammation, injury or stress, as well as visceral organ distension, and stimulate these local nociceptors. The P2X3 receptor rationale has aroused a formidable level of investigation producing many reports that clarify the potential role of ATP as a pain mediator, in chronic sensitized states in particular, and has piqued the interest of pharmaceutical companies. P2X receptor-mediated afferent activation has been implicated in inflammatory, visceral, and neuropathic pain states, as well as in airways hyperreactivity, migraine, itch, and cancer pain. It is well appreciated that oftentimes new mechanisms translate poorly from models into clinical efficacy and effectiveness; however, the breadth of activity seen from P2X3 inhibition in models offers a realistic chance that this novel mechanism to inhibit afferent nerve sensitization may find its place in the sun and bring some merciful relief to the torment of persistent discomfort and pain. The development philosophy at Afferent is to conduct proof of concept patient studies and best identify target patient groups that may benefit from this new intervention

Uterine contractility in the nonpregnant mouse: changes during the estrous cycle and effects of chloride channel blockade

Mechanisms involved in the generation of spontaneous uterine contractions are not fully understood. Kit-expressing interstitial cells of Cajal are pacemakers of contractile rhythm in other visceral organs, and recent studies describe a role for Ca(2+)-activated Cl(-) currents as the initiating conductance in these cells. The existence and role of similar specialized pacemaker cells in the nonpregnant uterus remains undetermined. Spontaneous contractility patterns were characterized throughout the estrous cycle in isolated, nonpregnant mouse uteri using spatiotemporal mapping and tension recordings. During proestrus, estrus, and diestrus, contraction origin predominated in the oviduct end of the uterus, suggesting the existence of a dominant pacemaker site. Propagation speed of contractions during estrus and diestrus were significantly slower than in proestrus and metestrus. Five major patterns of activity were predominantly exhibited in particular stages: quiescent (diestrus), high-frequency phasic (proestrus), low-frequency phasic (estrus), multivariant (metestrus), and complex. Kit-immunopositive cells reminiscent of pacemaking ICCs were not consistently observed within the uterus. Niflumic acid (10 μM), anthracene-9-carboxylic acid (0.1-1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 μM) each reduced the frequency of spontaneous contractions, suggesting involvement of Cl(-) channels in generating spontaneous uterine motor activity. It is unlikely that this conductance is generated by the Ca(2+)-activated Cl(-) channels, anoctamin-1 and CLCA4, as immunohistochemical labeling did not reveal protein expression within muscle or pacemaker cell networks. In summary, these results suggest that spontaneous uterine contractions may be generated by a Kit-negative pacemaker cell type or uterine myocytes, likely involving the activity of a yet-unidentified Cl(-) channel.Kelsi N. Dodds, Vasiliki Staikopoulos, and Elizabeth A.H. Becket

Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury–induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freund's adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased; compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.H.-J. Cho, V. Staikopoulos, J. B. Furness and E. A. Jenning

Amitriptyline pharmacologically preconditions rat hearts against cardiac ischemic-reperfusion injury

Abstract not availableS.M. Lee, M. Hutchinson, V. Staikopoulos, D.A. Sain

AMPK and ACC phosphorylation: Effect of leptin, muscle fibre type and obesity

Crown copyright © 2008 Published by Elsevier Ireland Ltd.Leptin stimulates fatty acid oxidation via the phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl-CoA carboxylase). Obesity is associated with resistance to the effects of leptin. We determined the action of leptin on AMPKα and ACCβ phosphorylation and lipid metabolism in soleus (SOL) and extensor digitorum longus (EDL) muscles from lean and obese Wistar rats after 1 and 100 nM leptin. Both leptin doses stimulated phosphorylation of AMPKα and ACCβ (P ≤ 0.05) only in EDL muscles from lean animals. Malonyl-CoA levels were decreased in EDL muscles from lean animals after 1 and 100 nM leptin and significantly after 100 nM leptin in obese animals (P ≤ 0.05). Long-chain fatty acyl-CoA concentrations were decreased in EDL muscles from both phenotypes after 100 nM leptin. AMPK activation by leptin occurred independently of energy-related metabolites. These data demonstrate that the leptin effect on AMPKα and ACCβ is muscle fibre type dependent and fails in diet-induced obesity.A. Janovská, G. Hatzinikolas, V. Staikopoulos, J. McInerney, M. Mano and G.A. Witterthttp://www.elsevier.com/wps/find/journaldescription.cws_home/506028/description#descriptio