Sex differences in pain-related behaviors and clinical progression of disease in mouse models of visceral pain

Previous studies have reported sex differences in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) patients, including differences in visceral pain perception. Despite this, sex differences in behavioral manifestations of visceral pain and underlying pathology of the gastrointestinal tract have been largely understudied in preclinical research. In this study, we evaluated potential sex differences in spontaneous visceral nociceptive responses, referred abdominal hypersensitivity, disease progression and bowel pathology in mouse models of acute and persistent colon inflammation. Our experiments show that females exhibit more visceral nociceptive responses and referred abdominal hypersensitivity than males in the context of acute but not persistent colon inflammation. We further demonstrate that, following acute and persistent colon inflammation, visceral pain-related behavioral responses in females and males are distinct, with increases in licking of the abdomen only observed in females and increases in abdominal contractions only seen in males. During persistent colon inflammation, males exhibit worse disease progression than females, which is manifested as worse physical appearance and higher weight loss. However, no measurable sex differences were observed in persistent inflammation-induced bowel pathology, stool consistency or fecal blood. Overall, our findings demonstrate that visceral pain-related behaviors and disease progression in the context of acute and persistent colon inflammation are sex-dependent, highlighting the importance of considering sex as a biological variable in future mechanistic studies of visceral pain as well as in the development of diagnostics and therapeutic options for chronic gastrointestinal diseases.

Chrysin-Loaded Microemulsion: Formulation Design, Evaluation and Antihyperalgesic Activity in Mice

Chrysin is a bioactive flavonoid found in pollens, passion flowers, honey, royal jelly, and propolis, which is commonly used as an ingredient in natural food supplements and is primarily responsible for their pharmacological properties. A transparent chrysin-loaded microemulsion (CS-ME) prepared through a ternary phase diagram was evaluated for use as an antihyperalgesic formulation. It was formulated with 40% Labrasol® (surfactant), 5% isopropyl myristate (oil phase) and 55% water (aqueous phase) and classified as an oil-in-water (O/W) microsized system (74.4 ± 15.8 nm). Its negative Zeta potential (−16.1 ± 1.9 mV) was confirmed by polarized light microscopy and dynamic light scattering analysis. In vitro studies in Franz-type static diffusion cells showed that chrysin release from CS-ME followed zero-order kinetics. Oral administration of CS-ME in mice resulted in a statistically significantly reduction (p < 0.05) in carrageenan-induced mechanical hyperalgesia compared to the control group. Treatment with CS-ME also showed anti-inflammatory activity by significantly decreasing the TNF-α level (p < 0.01) and increasing that of IL-10 (p < 0.05) compared to the control group. These results suggest that the proposed microsystem is a promising vector for the release of chrysin, being able to improve its capacity to modulate inflammatory and nociceptive responses.

Morphine Perinatal Exposure Induces Long-Lasting Negative Emotional States in Adult Offspring Rodents

Psychoactive substances during pregnancy and lactation is a key problem in contemporary society, causing social, economic, and health disturbance. In 2010, about 30 million people used opioid analgesics for non-therapeutic purposes, and the prevalence of opioids use during pregnancy ranged from 1% to 21%, representing a public health problem. This study aimed to evaluate the long-lasting neurobehavioral and nociceptive consequences in adult offspring rats and mice exposed to morphine during intrauterine/lactation periods. Pregnant rats and mice were exposed subcutaneously to morphine (10 mg/kg/day) during 42 consecutive days (from the first day of pregnancy until the last day of lactation). Offspring were weighed on post-natal days (PND) 1, 5, 10, 15, 20, 30, and 60, and behavioral tasks (experiment 1) or nociceptive responses (experiment 2) were assessed at 75 days of age (adult life). Morphine-exposed female rats displayed increased spontaneous locomotor activity. More importantly, both males and female rats perinatally exposed to morphine displayed anxiety- and depressive-like behaviors. Morphine-exposed mice presented alterations in the nociceptive responses on the writhing test. This study showed that sex difference plays a role in pain threshold and that deleterious effects of morphine during pre/perinatal periods are nonrepairable in adulthood, which highlights the long-lasting clinical consequences related to anxiety, depression, and nociceptive disorders in adulthood followed by intrauterine and lactation morphine exposure.

Steroid hormone signaling activates thermal nociception during Drosophila peripheral nervous system development

Sensory neurons enable animals to detect environmental changes and avoid harm. An intriguing open question concerns how the various attributes of sensory neurons arise in development. Drosophila melanogaster larvae undergo a behavioral transition by robustly activating a thermal nociceptive escape behavior during the second half of larval development (3rd instar). The Class 4 dendritic arborization (C4da) neurons are multimodal sensors which tile the body wall of Drosophila larvae and detect nociceptive temperature, light, and mechanical force. In contrast to the increase in nociceptive behavior in the 3rd instar, we find that ultraviolet light-induced Ca2+ activity in C4da neurons decreases during same period of larval development. Loss of ecdysone receptor has previously been shown to reduce nociception in 3rd instar larvae. We find that ligand dependent activation of ecdysone signaling is sufficient to promote nociceptive responses in 2nd instar larvae and suppress expression of subdued (encoding a TMEM16 channel). Reduction of subdued expression in 2nd instar C4da neurons not only increases thermal nociception but also decreases the response to ultraviolet light. Thus, steroid hormone signaling suppresses subdued expression to facilitate the sensory switch of C4da neurons. This regulation of a developmental sensory switch through steroid hormone regulation of channel expression raises the possibility that ion channel homeostasis is a key target for tuning the development of sensory modalities.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models

Background Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. Methods In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. Results Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. Conclusion Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

ASSESMENT OF ANTI-NOCICEPTIVE ACTIONS OF ADIPOSE-DERIVED MESEMCHYMAL STEM CELLS IN EXPERIMENTAL PERIPHERAL NEUROPATHIC PAIN

Objective. To estimate an anti-nociceptive and regenerative potential of adipose-derived mesenchymal stem cells in experimental post-traumatic neuropathy in rats. Methods. Neuropathic pain was induced by axotomy technique in rat left hind paw (Wistar rats (n=113)). The respective group of subjects received ADMSCs dose of 1×10⁶ cells/kg and 2×10⁶ cells/kg into the site of sciatic nerve injury at 2 regimens: single (7^th day post-surgery) and twice (7^th and 14^th day post-surgery). Nociceptive responses, as well as histological changes of sciatic nerve and perineural tissue were assessed in dynamics. Results. Sciatic nerve axotomy led to a significant increase of mechanical nociceptive sensitivity of ipsilateral hind paw by 7^th day, as well as to fibrotic changes of peri- and epineural areas of damaged nerve fibers and to denervation of surrounding muscle tissue and fascia. Local administration of ADMSCs effectively abolished mechanical hyperalgesia by 14^th day after first injection at all regimens tested. Among tested regimens, the most pronounced anti-nociceptive and regenerative effects were induced by single injection of ADMSCs (1×10⁶ cells/kg). As the dose and frequency of ADMSCs administration elevated, their reparative and anti-inflammatory properties reduced. Conclusion. Obtained results testify anti-nociceptive potential of ADMSCs and feasibility of its further investigation on the experimental models of neuropathy. What this paper adds For the first time the impact of different regimen of allogenic adipose-derived mesenchymal stem cells (ADMSCs) transplantation on nociceptive sensitivity and microstructure changes of sciatic nerve in rats with peripheral neuropathy has been studied. Allogenic transplantation of mesenchymal stem cells at a dose of 1×10⁶ cells/kg has been found out to exhibit the most powerful anti-nociceptive and regenerative effects with a single local injection confirmed by algometry and histological study.

Remote targeted electrical stimulation

The ability to generate electric fields in deep tissues remotely, without surgically implanting electrodes, could transform diagnoses and treatments of nervous system disorders. Here, we show that focal electrostimulation effects can be elicited remotely by combining two noninvasive forms of energies---magnetic and focused ultrasonic fields. The approach, based in the Lorentz equation and referred to as Lstim, electrically stimulates specified tissue targets with the precision of deep brain or spinal cord stimulation, but does not require electrode implantation. Lstim potentiated the responses of human nerves, enhancing the neuromodulatory effects of ultrasound by 74% on average. The effects showed a double dissociation---a significant and substantial increase in nociceptive responses, yet a significant reduction in tactile responses. In line with the Lorentz equation, Lstim was only observed when nerves were oriented perpendicularly to the magnetic and ultrasonic fields. A sham condition showed no effects. Both the ultrasonic and the induced electric fields were well below the respective safety indices, and no detrimental effects were detected. Lstim uniquely integrates noninvasiveness, sharp focus, and the efficacy of electrical stimulation. The approach has the potential to provide a noninvasive tool to dissect brain function in humans and to diagnose the neural circuits involved in nervous system disorders. Moreover, this effect should be taken into account when ultrasound is applied inside MRI.

Delineating neural responses and functional connectivity changes during vestibular and nociceptive stimulation reveal the uniqueness of cortical vestibular processing

Vestibular information is ubiquitous and often processed jointly with visual, somatosensory and proprioceptive information. Among the cortical brain regions associated with human vestibular processing, area OP2 in the parietal operculum has been proposed as vestibular core region. However, delineating responses uniquely to vestibular stimulation in this region using neuroimaging is challenging for several reasons: First, the parietal operculum is a cytoarchitectonically heterogeneous region responding to multisensory stimulation. Second, artificial vestibular stimulation evokes confounding somatosensory and nociceptive responses blurring responses contributing to vestibular perception. Furthermore, immediate effects of vestibular stimulation on the organization of functional networks have not been investigated in detail yet. Using high resolution neuroimaging in a task-based and functional connectivity approach, we compared two equally salient stimuli—unilateral galvanic vestibular (GVS) and galvanic nociceptive stimulation (GNS)—to disentangle the processing of both modalities in the parietal operculum and characterize their effects on functional network architecture. GNS and GVS gave joint responses in area OP1, 3, 4, and the anterior and middle insula, but not in area OP2. GVS gave stronger responses in the parietal operculum just adjacent to OP3 and OP4, whereas GNS evoked stronger responses in area OP1, 3 and 4. Our results underline the importance of considering this common pathway when interpreting vestibular neuroimaging experiments and underpin the role of area OP2 in central vestibular processing. Global network changes were found during GNS, but not during GVS. This lack of network reconfiguration despite the saliency of GVS may reflect the continuous processing of vestibular information in the awake human.

PRDM12 Is Transcriptionally Active and Required for Nociceptor Function Throughout Life

PR domain-containing member 12 (PRDM12) is a key developmental transcription factor in sensory neuronal specification and survival. Patients with rare deleterious variants in PRDM12 are born with congenital insensitivity to pain (CIP) due to the complete absence of a subtype of peripheral neurons that detect pain. In this paper, we report two additional CIP cases with a novel homozygous PRDM12 variant. To elucidate the function of PRDM12 during mammalian development and adulthood, we generated temporal and spatial conditional mouse models. We find that PRDM12 is expressed throughout the adult nervous system. We observed that loss of PRDM12 during mid-sensory neurogenesis but not in the adult leads to reduced survival. Comparing cellular biophysical nociceptive properties in developmental and adult-onset PRDM12 deletion mouse models, we find that PRDM12 is necessary for proper nociceptive responses throughout life. However, we find that PRDM12 regulates distinct age-dependent transcriptional programs. Together, our results implicate PRDM12 as a viable therapeutic target for specific pain therapies even in adults.