Mihai Radu

Neurovascular unit in chronic pain.

Chronic pain is a debilitating condition with major socioeconomic impact, whose neurobiological basis is still not clear. An involvement of the neurovascular unit (NVU) has been recently proposed. In particular, the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB), two NVU key players, may be affected during the development of chronic pain; in particular, transient permeabilization of the barrier is suggested by several inflammatory- and nerve-injury-based pain models, and we argue that the clarification of molecular BBB/BSCB permeabilization events will shed new light in understanding chronic pain mechanisms. Possible biases in experiments supporting this theory and its translational potentials are discussed. Moving beyond an exclusive focus on the role of the endothelium, we propose that our understanding of the mechanisms subserving chronic pain will benefit from the extension of research efforts to the NVU as a whole. In this view, the available evidence on the interaction between analgesic drugs and the NVU is here reviewed. Chronic pain comorbidities, such as neuroinflammatory and neurodegenerative diseases, are also discussed in view of NVU changes, together with innovative pharmacological solutions targeting NVU components in chronic pain treatment.

Nonsteroidal anti-inflammatory drugs in clinical and experimental epilepsy

Current antiepileptic drugs have limited efficacy and provide little or no benefits in 30% of the patients. Given that a role for brain inflammation in epilepsy has been repeatedly reported in recent years, the potential of anti-inflammatory drugs should be explored in depth, as they may provide new therapeutical approaches in preventing or reducing epileptogenesis. Here, we review preclinical (both in vivo and in vitro) and clinical epilepsy studies in which nonsteroidal antiinflammatory drugs (NSAIDs), i.e. cyclooxygenase-2 (COX-2) selective inhibitors (COXIBs) and nonselective NSAIDs, were used for seizure control. The effects of NSAIDs are reviewed in animal models of both chemical (pilocarpine, kainic acid, pentylenetetrazol, or carbachol administration) and electrical (tetanic hippocampal stimulation, electroshock) seizure induction. In the pilocarpine model, NSAIDs are neuroprotective, reduce mossy fiber sprouting or diminish P-glycoprotein upregulation, but only rarely protect against seizures. While neuroprotective effects have also been observed in the kainic acid model, NSAIDs tend in general to worsen seizure activity. Effects of COXIB administration in the pentylenetetrazol-induced seizures model are variable, alternating from protection against seizures to null effects or even increased incidence of convulsions. Moreover, NSAIDs tested in the tetanic hippocampal stimulation model diminished the seizure-associated P-glycoprotein upregulation, but were not very effective in seizure control. NSAIDs efficacy in experimental in vivo epilepsy studies may be influenced by multiple factors, including the timing of administration (before or after status epilepticus induction), the animal model of epilepsy or some of the signaling pathways involved in cyclooxygenase induction (e.g. prostaglandins and their receptors). On the other hand, the few clinical studies on the use of NSAIDs in neurological pathologies accompanied/characterized by seizures indicate that nonselective NSAIDs (e.g. aspirin) in prolonged, low-dose treatments may offer protection against seizures and stroke-like events. No clinical trials in epileptic patients using COXIBs have been conducted so far, as several international drug-control authorities have withdrawn these drugs from the market; future studies should focus on improved COXIB formulations. We argue that, while the available evidence is still inconclusive, the potential therapeutic benefits of controlling and diminishing brain inflammation in the treatment of epilepsy should be actively explored.

Mechanisms of Ceftazidime and Ciprofloxacin Transport through Porins in Multidrug-Resistance Developed by Extended-Spectrum Beta-Lactamase E.coli Strains

Resistance towards antibiotics stands out today as a major issue in the clinical act of treatment of bacterial-generated infections. This process was characterized in proteoliposomes reconstituted from an E.coli strain isolated from invasive infections (blood culture) occurred in patients with a cardio-vascular device admitted for surgery. Fluorescence spectroscopy and patch-clamp technique have been used. Two types of antibiotics have been targeted: ceftazidime and ciprofloxacin. Antibiotics addition in proteoliposomes suspension undergoes a quenching in tryptophan residues from outer membrane porins structure, probably due to the formation of a transient non-fluorescent porin-antibiotic complex. Patch-clamp recordings revealed strong ion current blockages for both antibiotics, reflecting antibiotic–channel interactions but with varying strength of interaction. The present study puts forward the mechanism of multidrug-resistance in extended-spectrum beta-lactamase E.coli strains, as being caused by alterations of the antibiotics transport across the porins of the outer bacterial membrane.

Calcium Signaling in Interstitial Cells: Focus on Telocytes

In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca2+ oscillations, the inositol triphosphate (IP3)/Ca2+ signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies.

All muscarinic acetylcholine receptors (M 1 -M 5 ) are expressed in murine brain microvascular endothelium

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

Acid-sensing ion channels as potential pharmacological targets in peripheral and central nervous system diseases

Acid-sensing ion channels (ASICs) are widely expressed in the body and represent good sensors for detecting protons. The pH drop in the nervous system is equivalent to ischemia and acidosis, and ASICs are very good detectors in discriminating slight changes in acidity. ASICs are important pharmacological targets being involved in a variety of pathophysiological processes affecting both the peripheral nervous system (e.g., peripheral pain, diabetic neuropathy) and the central nervous system (e.g., stroke, epilepsy, migraine, anxiety, fear, depression, neurodegenerative diseases, etc.). This review discusses the role played by ASICs in different pathologies and the pharmacological agents acting on ASICs that might represent promising drugs. As the majority of above-mentioned pathologies involve not only neuronal dysfunctions but also microvascular alterations, in the next future, ASICs may be also considered as potential pharmacological targets at the vasculature level. Perspectives and limitations in the use of ASICs antagonists and modulators as pharmaceutical agents are also discussed.

Are they in or out? The elusive interaction between Qtracker(®)800 vascular labels and brain endothelial cells

AIM Qtracker(®)800 Vascular labels (Qtracker(®)800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown. MATERIALS & METHODS Qtracker(®)800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker(®)800 exposure on mouse and human endothelial cells by calcium imaging. RESULTS Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker(®)800. CONCLUSION Qtracker(®)800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging.

Endothelial cells are key-players in pilocarpine-induced epileptogenesis

In recent years, the concept of the neurovascular unit (NVU) has emerged as a new paradigm for investigating both physiology and pathology in the CNS. This concept proposes that a purely neurocentric focus is not sufficient, and emphasizes that all cell types in the brain including neuronal, glial and vascular components (endothelial cells, blood cells, including immunity cells) must be examined in an integrated context. Cell–cell signaling and coupling between these different compartments thus form the basis for normal function (Lok et al. 2007). We tested the hypothesis that disordered signaling and perturbed coupling of these different components can be the basis for epileptogenesis in the pilocarpine model of epilepsy. We thus determined that pilocarpine can act on endothelial cells via receptors, comparing the response of the same stimulation in neurons as well.

Quantum dots as new guests in the body: structural and functional data

Many promising applications of quantum dots (QDs) in nanomedicine and in vivo imaging for further diagnostic are being developed. Despite the immense potential for the medical applications of QDs, little is known about the bioavailability and health consequences of QDs in animals and humans. Although some investigators reported that QDs do not appear to cause toxicity, others demonstrated a variety of cytotoxic effects. In this study, QDs800 (InVitrogen) have been used. Previous data from our group evaluated the bio-distribution by optical imaging, transmission electron microscopy, inductively coupled plasma mass spectroscopy analysis in mice, and the effects on novel object recognition memory, EEG activity, and some histopatological analysis on mice in different organs (liver, spleen, lungs, testis, brain). Here, we studied the systemic inflammation caused by QDs in different organs, and then focussed our attention to the brain. It is known that brain inflammation leads to microglia and astrocyte activation, which in turn are sensitive to the changes in the CNS microenvironment and rapidly activated in all conditions that affect normal neuronal functions. We demonstrated that the presence of QDs could impair synaptic response and neuronal excitability; secondly, we are currently investigating whether the electrical changes are induced by QDs by themselves or by the inflammation induced by their presence.