Trigeminal nerve injury can lead to chronic and difficult-to-treat orofacial neuropathic pain. Here, we uncover a key role for the cation channel TRPM3 in the chronic constriction injury of the infraorbital nerve (IoN-CCI) mouse model of trigeminal neuropathic pain. Wild-type (WT) mice develop spontaneous pain and mechanical allodynia for up to 6 weeks following IoN-CCI, whereas Trpm3-/- mice do not develop such symptoms. Using longitudinal RNA sequencing (RNA-seq) analysis, we obtain a detailed time course of transcriptome alterations in trigeminal ganglia during progression of the IoN-CCI model; notably, gene expression regulation is not different between WT and Trpm3-/- mice. Two structurally distinct TRPM3 antagonists, primidone and isosakuranetin, effectively reverse spontaneous pain and mechanical allodynia, whereas mavatrep, a potent TRPV1 antagonist, is without analgesic effect. These data indicate that TRPM3 is essential for ongoing pain and allodynia following trigeminal nerve injury, making it a potential target for treating trigeminally mediated neuropathic pain.

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+-permeable ion channel that plays a pivotal role in peripheral thermosensation and nociception. Moreover, gain-of-function variants in TRPM3 underlie a spectrum of neurodevelopmental and epileptic disorders in humans, indicating an important role of TRPM3 in the central nervous system. Oxidative stress contributes to various neurological disorders of both the central and peripheral nervous system, but it is unknown whether TRPM3 activity is altered by the cellular redox state. Here, we report a direct, bidirectional modification of TRPM3 channel activity by oxidizing and reducing agents. Our data demonstrate a profound effect of the redox state on the channel properties of TRPM3, including a robust shift in the response profile to pharmacology and temperature sensitivity. In addition, we identified two cysteine residues in the extracellular pore loop of TRPM3 that underlie the redox-control of the channel, due to the reversible formation of intra-subunit cysteine bridges. Taken together, these observations raise the hypothesis that TRPM3 could be modulated through an alternative mechanism, potentially affecting pathways involved in pain and neurological function. Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+-permeable ion channel that plays a pivotal role in the peripheral and central nervous systems; however, the effect of cellular redox state on the TRPM3 activity is unknown. Here, the authors reveal that TRPM3 channel activity is bidirectionally modulated by oxidizing and reducing agents, and identify cysteine residues in the extracellular pore loop of TRPM3 that carry the redox-control of the channel.

Chemotherapy-induced peripheral neuropathy is a debilitating pathology affecting a majority of patients who are being treated with specific cytostatic compounds including oxaliplatin. Various in vitro, ex vivo and in vivo preclinical experiments indicate that transient receptor potential ankyrin 1 (TRPA1) plays a crucial role in the symptomatology of chemotherapy-induced peripheral neuropathy. However, it is unclear whether oxaliplatin also modulates the TRPA1 functionality in the skin of rodents or patients. Here, we quantified the vasodilation after topical application of the TRPA1 agonist cinnamaldehyde in a rodent model of chemotherapy-induced peripheral neuropathy (male Sprague Dawley rats, aged 6 weeks) as well as on fingers of patients suffering from chronic chemotherapy-induced peripheral neuropathy after oxaliplatin treatment. Compared to vehicletreated rats, a cumulative dose of oxaliplatin 32 mg/kg enhanced the vasodilation after cinnamaldehyde application on rat abdominal skin. Likewise, also in patients with chronic chemotherapy-induced peripheral neuropathy after oxaliplatin, the response to cinnamaldehyde was significantly higher compared to sex- and agematched healthy controls. Thereby, this study is the first to translate the evidence of increased TRPA1 functionality in vitro or ex vivo in rodents to in vivo conditions in human. The increased TRPA1 functionality in patients with chronic chemotherapy-induced peripheral neuropathy does not only confirm the potential of TRPA1 as target to hit to provide efficacious analgesia, it also paves the way for additional patient stratification on a molecular level and possible treatment response prediction.

Background: Aphasia is a common neurocognitive disorder caused by impaired speech and language, with stroke being the most frequent cause. The neuroanatomical mechanism underlying this condition is not yet fully understood. Case description: This case describes a 74-year-old Caucasian woman admitted with a clinical picture of right total anterior circulation infarct (TACI) and aphasia, scoring 17 on the National Institutes of Health Stroke Scale. Neuroimaging showed a large cortico-subcortical frontotemporoparietal and insular infarct involving the basal ganglia of the right hemisphere and bilateral focal atherosclerotic stenosis on the M1 segment of the middle cerebral artery. There was no left hemispheric lesion or abnormal electric activity on the electroencephalogram. A formal evaluation was compatible with transcortical motor aphasia. The aetiological study revealed atrial fibrillation, and the case was admitted as an ischaemic stroke of undetermined aetiology with two possible causes – intracranial atherosclerotic stenosis or atrial fibrillation. Conclusion: Our patient fulfilled all the formal criteria for crossed aphasia in dextral (CAD): aphasia, a lesion in the right hemisphere coupled with the structural integrity of the left hemisphere, an established preference for right-hand use without a familial history of left-handedness individuals, and an absence of brain damage in childhood. Our patient’s case adds to the evidence that deep structures – alone or in combination with cortical structures – are primarily affected in CAD.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is an adverse effect observed in up to 80% of patients of cancer on treatment with cytostatic drugs including paclitaxel and oxaliplatin. Chemotherapy-induced peripheral neuropathic pain can be so severe that it limits dose and choice of chemotherapy and has significant negative consequences on the quality of life of survivors. Current treatment options for CIPNP are limited and unsatisfactory. TRPM3 is a calcium-permeable ion channel functionally expressed in peripheral sensory neurons involved in the detection of thermal stimuli. Here, we focus on the possible involvement of TRPM3 in acute oxaliplatin-induced mechanical allodynia and cold hypersensitivity. In vitro calcium microfluorimetry and whole-cell patch-clamp experiments showed that TRPM3 is functionally upregulated in both heterologous and homologous expression systems after acute (24 hours) oxaliplatin treatment, whereas the direct application of oxaliplatin was without effect. In vivo behavioral studies using an acute oxaliplatin model for CIPNP showed the development of cold and mechano hypersensitivity in control mice, which was lacking in TRPM3 deficient mice. In addition, the levels of protein ERK, a marker for neuronal activity, were significantly reduced in dorsal root ganglion neurons derived from TRPM3 deficient mice compared with control after oxaliplatin administration. Moreover, intraperitoneal injection of a TRPM3 antagonist, isosakuranetin, effectively reduced the oxaliplatin-induced pain behavior in response to cold and mechanical stimulation in mice with an acute form of oxaliplatin-induced peripheral neuropathy. In summary, TRPM3 represents a potential new target for the treatment of neuropathic pain in patients undergoing chemotherapy.

During pregnancy, the glucose homeostasis of the mother adapts to accommodate the needs of a growing fetus. Decreased insulin sensitivity and increased insulin secretion from the pancreatic beta-cell contribute to this adaptation. Electrophysiological changes in the beta-cell during pregnancy, that could explain the increased insulin secretion, have not yet been identified. The non-selective Transient Receptor Potential Melastatin 4 (TRPM4) cation channel, expressed in beta-cells, may depolarize the beta-cell membrane and promote insulin release. In other organs, TRPM4 ion channel expression fluctuates depending on the relative concentration of estrogen and progesterone, hormones important during pregnancy. These findings suggest a role of TRPM4 in glycemic control, which can be influenced by female reproductive hormones during pregnancy. Using in vivo continuous glucose monitoring throughout the full pregnancy of wildtype (WT) and TRPM4 knockout (KO) mice allows to analyze glycemia during oral glucose tolerance tests (OGTTs) and spontaneous feeding behavior, and to determine glycemic variability. TRPM4 KOmice are hyperglycemic compared to WT mice, mainly before and during the first half of pregnancy. The mean glucose concentration was increased in TRPM4 KO mice in the beginning of pregnancy. In both WT mice and TRPM4 KO mice, the overall glycemia gradually increased during this period, maintaining the difference in glycemia between the two genotypes. Later in pregnancy, the glycemia of both genotypes started gradually decreasing. However, glycemia of TRPM4 KO mice decreased faster, reducing the difference in glycemia between the two genotypes. This indicates a different adaptation to pregnancy. The maximum glucose levels reached during the OGTTs were larger for the TRPM4 KO mice compared to the wildtype mice, which is in line with the general observed hyperglycemia of TRPM4 KOmice. Furthermore, we observed that fasting glucose concentrations, obtained right before the OGTTs, were significantly increased for the TRPM4 KO mice compared to WT mice. Glycemic variability provides information regarding sudden changes and fluctuations in the blood glucose concentration. In general, there were no substantial differences in terms of glycemic variability between both genotypes. To conclude, we could show that TRPM4 plays an active role in glycemic control, before and during pregnancy.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

AIMS:Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear.METHODS AND RESULTS:In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4-/- mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4-/- mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner.CONCLUSION:The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

During the molecular transduction of itch, the stimulation of pruriceptors on sensory fibers leads to the activation or sensitization of ion channels, which results in a consequent depolarization of the neurons. These ion channels mostly belong to the transient receptor potential (TRP) channels, which are involved in nociception and thermosensation. In particular, TRPV1 and TRPA1 were described in the transduction of both thermal nociception as well as histaminergic and non-histaminergic itch. The thermosensitive TRPM3 plays an indispensable role in heat nociception together with TRPV1 and TRPA1. However, the role of TRPM3 in the development of pruritus has not been studied yet. Therefore, in this study we aimed at investigating the potential role of TRPM3 in the transduction of pruritus and pain by investigating itch- and nociception-related behavior of Trpm3+/+ and Trpm3-/- mice, and by studying the activation of somatosensory neurons isolated from trigeminal ganglia upon application of algogenic and pruritogenic substances. Activators of TRPM3 evoked only nocifensive responses, but not itch in Trpm3+/+ animals, and these nocifensive responses were abolished in the Trpm3-/- strain. Histamine and endogenous non-histaminergic pruritogens induced itch in both Trpm3+/+ and Trpm3-/- mice to a similar extent. Genetic deletion or pharmacological blockade diminished TRPM3 mediated Ca2+ responses of sensory neurons, but did not affect responses evoked by pruritogenic substances. Our results demonstrate that, in contrast to other thermosensitive TRP channels, TRPM3 selectively mediates nociception, but not itch sensation, and suggest that TRPM3 is a promising candidate to selectively target pain sensation.

During pregnancy, metabolic adaptations occur to maintain the balance between maternal and foetal growth, including increased insulin secretion and decreased insulin sensitivity. When the body fails to adjust, gestational diabetes mellitus develops. To gain insight in the pregnancy-induced adaptations, we applied continuous glucose monitoring via telemetric transmitters. We show that continuous glucose monitoring in conscious, non-stressed, freely moving mice throughout the full pregnancy is feasible, accurate and safe. We show that healthy mice during a full pregnancy develop adaptations in glucose homeostasis reminiscent of those in pregnant women. Furthermore, continuous glucose monitoring allows the complete analysis of all aspects of glucose excursions associated with spontaneous feeding episodes, and the thorough analysis of glycaemic variability. In conclusion, continuous glucose monitoring allows a detailed description of the glycaemic status during pregnancy, which will help to unravel specific mechanisms for gestational diabetes mellitus.

Pathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. The molecular pathways in the induction of pressure overload LVH are potential targets for therapeutic intervention. Current treatments aim to remove the pressure overload stimulus for LVH, but do not completely reverse adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling remains unresolved. In this study, we show that selective deletion of transient receptor potential melastatin 4 (TRPM4) channels in mouse cardiomyocytes results in an approximately 50% reduction in the LVH induced by transverse aortic constriction. Our results suggest that TRPM4 channel is an important component of the mechanosensory signalling pathway that induces LVH in response to pressure overload and represents a potential novel therapeutic target for the prevention of pathological LVH.

Endometriosis is a prevalent gynecologic disease, defined by dysfunctional endometrium-like lesions outside of the uterine cavity. These lesions are presumably established via retrograde menstruation, i.e., endometrial tissue that flows backwards during menses into the abdomen and deposits on the organs. As ongoing pain is one of the main pain symptoms of patients, an animal model that illuminates this problem is highly anticipated. In the present study, we developed and validated a rat model for ongoing endometriosis-associated pain. First, menstrual endometrial tissue was successfully generated in donor rats, as validated by gross examination, histology and qPCR. Next, endometriosis was induced in recipient animals by intraperitoneal injection of menstrual tissue. This resulted in neuro-angiogenesis as well as established endometriosis lesions, which were similar to their human counterparts, since epithelial and stromal cells were observed. Furthermore, significant differences were noted between control and endometriosis animals concerning bodyweight and posture changes, indicating the presence of ongoing pain in animals with endometriosis. In summary, a rat model for endometriosis was established that reliably mimics the human pathophysiology of endometriosis and in which signs of ongoing pain were detected, thus providing a new research tool for therapy development.

Pregnancy is a challenge for the female body. Increased insulin resistance of the mother allows appropriate nutrient flow to the fetus. This is counterbalanced by increased insulin secretion from pancreatic β‐cells. Gestational diabetes mellitus (GDM) arises when insulin resistance is increased excessively or pancreatic β‐cells fail to adapt.To study basic mechanisms and novel therapeutic strategies for GDM, it is necessary to test glycaemia in pregnant mice. We investigated the potential of continuous glucose monitoring via telemetry as a reliable and stress‐free approach to follow glycaemia during pregnancy. The transmitter is capable of continuous monitoring the temperature, activity, and plasma glucose levels in the arterial blood during full pregnancy. The telemetric implant had no effect on the pregnancy and the litter. Oral glucose tolerance tests were performed before, during and after pregnancy using intermittent blood sampling and telemetry. With both methods, we observed a similar glucose tolerance profile in wildtype mice during pregnancy. However, the use of telemetry results in a large amount of continuous data, which greatly extends the level of detail in which glycaemia during pregnancy can be monitored. We combined camera surveillance and telemetry to observe glycaemia during spontaneous feeding behavior. Once this behavior was known, glucose excursions and their respective parameters could be determined, e.g. amplitude, area under the curve, and time to peak. In addition, several parameters of glycaemic variability, important in diabetes research, were calculated from this dataset. For example, the standard deviation, interquartile range, and continuous net glycaemic action over 30 min seem to be disturbed specifically around delivery. In general, wildtype C57BL/6N mice are able to cope with the extra metabolic demand placed upon the mother by pregnancy. However, changes in glycaemic variability, average glucose levels and activity could be found throughout pregnancy. To conclude, continuous glucose monitoring is a safe method that provides insight in glycaemic control during pregnancy with unprecedented detail.Support or Funding InformationKU Leuven Bijzonder Onderzoeksfonds: TRP Research platform Leuven (TRPLe)Figure 1

The vomeronasal organ (VNO), the sensory organ of the mammalian accessory olfactory system, mediates the activation of sexually dimorphic reproductive behavioral and endocrine responses in males and females. It is unclear how sexually dimorphic and state-dependent responses are generated by vomeronasal sensory neurons (VSNs). Here, we report the expression of the transient receptor potential (TRP) channel Trpm4, a Ca2+-activated monovalent cation channel, as a second TRP channel present in mouse VSNs, in addition to the diacylglycerol-sensitive Trpc2 channel. The expression of Trpm4 in the mouse VNO is sexually dimorphic and, in females, is tightly linked to their reproductive cycle. We show that Trpm4 protein expression is upregulated specifically during proestrus and estrus, when female mice are about to ovulate and become sexually active and receptive. The cyclic regulation of Trpm4 expression in female VSNs depends on ovarian sex hormones and is abolished by surgical removal of the ovaries (OVX). Trpm4 upregulation can be restored in OVX mice by systemic treatment with 17ß-estradiol, requires endogenous activity of aromatase enzyme, and is strongly reduced during late pregnancy. This cyclic regulation of Trpm4 offers a neural mechanism by which female mice could regulate the relative strength of sensory signals in their VSNs, depending on hormonal state. Trpm4 is likely to participate in sex-specific, estrous cycle-dependent and sex hormone-regulated functions of the VNO, and may serve as a previously unknown genetic substrate for dissecting mammalian sexually dimorphic cellular and behavioral responses.

This paper deals with the application of digital technologies to the preservation and exploitation of the heterogeneous documents of the Herbarium Universitatis Mediolanensis of the University of Milan. The collection, dating back to the 19th and 20th Century, is mainly constituted by exsiccata, i.e. specimens (whole plants or plant parts) in dried form mounted on paper sheets; the archive also includes large botanical lithographs originally used in schools as educational tools. The long-term goal of the project is to complete the digitization campaign and make all these documents publicly available via a Web portal; currently, all metadata (23000 files approx.) and about 6000 digital objects are online. In this work, the whole process will be discussed, from digitization to the implementation of the Web portal based on a multimedia relational database.

Urinary tract infections (UTI) affect a large proportion of the population, causing among other symptoms, more frequent and urgent micturition. Previous studies reported that the gram-negative bacterial wall component lipopolysaccharides (LPS) trigger acute epithelial and bladder voiding responses, but the underlying mechanisms remain unknown. The cation channel TRPV4 is implicated in the regulation of the bladder voiding. Since TRPV4 is activated by LPS in airway epithelial cells, we sought to determine whether this channel plays a role in LPS-induced responses in urothelial cells (UCs). We found that human-derived UCs display a fast increase in intracellular Ca2+ concentration upon acute application of Escherichia coli LPS. Such responses were detected also in freshly isolated mouse UCs, and found to be dependent on TRPV4, but not to require the canonical TLR4 signaling pathway of LPS detection. Confocal microscopy experiments revealed that TRPV4 is dispensable for LPS-induced nuclear translocation of NF-κB in mouse UCs. On the other hand, quantitative RT PCR determinations showed an enhanced LPS-induced production of proinflammatory cytokines in TRPV4-deficient UCs. Cystometry experiments in anesthetized wild type mice revealed that acute intravesical instillation of LPS rapidly increases voiding frequency. This effect was not observed in TRPV4-deficient animals, but was largely preserved in Tlr4 KO and Trpa1 KO mice. Our results suggest that activation of TRPV4 by LPS in UCs regulates the proinflammatory response and contributes to LPS-induced increase in voiding frequency. These findings further support the concept that TRP channels are sensors of LPS, mediating fast innate immunity mechanisms against gram-negative bacteria.

Pruritus is one of the most widespread symptom in dermatology. Typically, during the molecular transduction of itch, the activation of metabotropic receptors on the pruriceptive nerve endings stimulated either by exogenous pruritogenic molecules or endogenous signals, leads to the activation or sensitization of an ion channel crucial in the consequent depolarization of the neuronal membrane. Recent research indicated that these ion channels mostly belong to the transient receptor potential (TRP) family of ion channels and show significant overlap with those involved in nociception (TRPV1, TRPA1). TRPM3 was recently identified as a novel thermosensitive channel contributing to nociception. However the role of TRPM3 in the development of pruritus has not been studied yet. Therefore, in the current project we aim at investigating the potential role of TRPM3 in the transduction of pruritus. We investigated the itch inducing effect of various pruritogenic substances on wild type and TRPM3-/- C57/Bl6 mice, in the "cheek model" paradigm, which allows to differentiate the nociceptive and pruriceptive behavioural responses. During this paradigm, pruritogenic substances diluted into 10µl vehicle (PBS+7%TWEEN-80) were injected subcutaneously into the cheek of mouse and reactive behaviour were recorded and analysed quantitatively To investigate the role of TRPM3 in the development of pruritus, we treated n=8-13 wt and TRPM3 mice with the pruritogen histamine, the non-histaminergic serotonin,endothelin-1, chloroquine, the algogen capsaicin and the TRPM3 specific pregnenolone sulphate (PS), compared with vehicle treatment. Our findings argue for the involvement of TRPM3 only in certain pain, but not itch responses: histamine and serotonin evoked similar scratching behaviour on both KO and wt animals; whereas TRPM3 activator PS evoked only pain related responses which were abolished in TRPM3-/- animals.