Astrocyte activation in hindlimb somatosensory cortex contributes to electroacupuncture analgesia in acid-induced pain.

Background: Several studies have confirmed the direct relationship between extracellular acidification and the occurrence of pain. As an effective pain management approach, the mechanism of electroacupuncture (EA) treatment of acidification-induced pain is not fully understood. The purpose of this study was to assess the analgesic effect of EA in this type of pain and to explore the underlying mechanism(s). Methods: We used plantar injection of the acidified phosphate-buffered saline (PBS; pH 6.0) to trigger thermal hyperalgesia in male Sprague-Dawley (SD) rats aged 6-8 weeks. The value of thermal withdrawal latency (TWL) was quantified after applying EA stimulation to the ST36 acupoint and/or chemogenetic control of astrocytes in the hindlimb somatosensory cortex. Results: Both EA and chemogenetic astrocyte activation suppressed the acid-induced thermal hyperalgesia in the rat paw, whereas inhibition of astrocyte activation did not influence the hyperalgesia. At the same time, EA-induced analgesia was blocked by chemogenetic inhibition of astrocytes. Conclusion: The present results suggest that EA-activated astrocytes in the hindlimb somatosensory cortex exert an analgesic effect on acid-induced pain, although these astrocytes might only moderately regulate acid-induced pain in the absence of EA. Our results imply a novel mode of action of astrocytes involved in EA analgesia.

Beneficial effect of electroacupuncture on the distribution of foreign substances in the brain of rats developing depression-like behavior.

We used low and high molecular weight fluorescence tracers to investigate the entry of foreign solutes into the brain parenchyma and their exit from it by the glymphatic system, during experimentally induced depressive-like behavior in rats. The tail suspension test (TST), as an acute stressor, is known to induce such a type of behavior, considered to model the human major depressive disorder (MDD). Electroacupuncture (EAP) relieves both depressive-like behavior in rodents and the symptoms of MDD in humans. Here we report that 180 min after the intracisternal injection of the low molecular weight tracer Fluorescein-5-Isothiocianate Conjugated Dextran (FITC-d3), a 15-min duration TST tended to increase the control fluorescence in the brain of rats. Both EAP and sham EAP decreased the fluorescence of FITC-d3 in comparison with the TST, but not the control value. In addition, EAP and sham EAP counteracted the effects of TST. The high molecular weight tracer Ovalbumin Alexa Fluor 555 Conjugate (OA-45) failed to enter the brain parenchyma and accumulated at more superficial sites; however, EAP or sham EAP modified the distribution of fluorescence under TST application in a similar manner as that observed during the use of FITC-d3. It is concluded that EAP is possibly a valid treatment to slow down the entry of foreign solutes into the brain; in view of the comparable effects of EAP on FITC-d3 and OA-45 distribution, EAP seems to act before FITC-d3 passes the astroglial aquaporin-4 water channels, which are a critical constituent of the glymphatic system.

P2X7 receptor-mediated depression-like reactions arising in the mouse medial prefrontal cortex.

Major depressive disorder is a frequent and debilitating psychiatric disease. We have shown in some of the acute animal models of major depressive disorder (tail suspension test and forced swim test) that depression-like behavior can be aggravated in mice by the microinjection into the medial prefrontal cortex of the P2X7R agonistic adenosine 5'-triphosphate or its structural analog dibenzoyl-ATP, and these effects can be reversed by the P2X7R antagonistic JNJ-47965567. When measuring tail suspension test, the prolongation of immobility time by the P2YR agonist adenosine 5'-[ß-thio]diphosphate and the reduction of the adenosine 5'-(γ-thio)triphosphate effect by P2Y1R (MRS 2179) or P2Y12R (PSB 0739) antagonists, but not by JNJ-47965567, all suggest the involvement of P2YRs. In order to elucidate the localization of the modulatory P2X7Rs in the brain, we recorded current responses to dibenzoyl-ATP in layer V astrocytes and pyramidal neurons of medial prefrontal cortex brain slices by the whole-cell patch-clamp procedure; the current amplitudes were not altered in preparations taken from tail suspension test or foot shock-treated mice. The release of adenosine 5'-triphosphate was decreased by foot shock, although not by tail suspension test both in the hippocampus and PFC. In conclusion, we suggest, that in the medial prefrontal cortex, acute stressful stimuli cause supersensitivity of P2X7Rs facilitating the learned helplessness reaction.

ATP indirectly stimulates hippocampal CA1 and CA3 pyramidal neurons via the activation of neighboring P2X7 receptor-bearing astrocytes and NG2 glial cells, respectively.

There is ongoing dispute on the question whether CNS neurons possess ATP-sensitive P2X7 receptors (Rs) or whether only non-neuronal cells bear this receptor-type and indirectly signal to the neighboring neurons. We genetically deleted P2X7Rs specifically in astrocytes, oligodendrocytes and microglia, and then recorded current responses in neurons to the prototypic agonist of this receptor, dibenzoyl-ATP (Bz-ATP). These experiments were made in brain slice preparations taken from the indicated variants of the P2X7R KO animals. In hippocampal CA3, but not CA1 pyramidal neurons, the deletion of oligodendrocytic (NG2 glial) P2X7Rs abolished the Bz-ATP-induced current responses. In contrast to the Bz-ATP-induced currents in CA3 pyramidal neurons, current amplitudes evoked by the ionotropic glutamate/GABAAR agonists AMPA/muscimol were not inhibited at all. Whereas in the CA3 area NG2 glia appeared to mediate the P2X7R-mediated stimulation of pyramidal neurons, in the CA1 area, astrocytic P2X7Rs had a somewhat similar effect. This was shown by recording the frequencies and amplitudes of spontaneous excitatory currents (sPSCs) in brain slice preparations. Bz-ATP increased the sPSC frequency in CA1, but not CA3 pyramidal neurons without altering the amplitude, indicating a P2X7R-mediated increase of the neuronal input. Micro-injection of the selective astrocytic toxin L-α-aminoadipate into both hippocampi, or the in vitro application of the GABAAR antagonistic gabazine, completely blocked the frequency increases of sPSCs. Hence, CA1 and CA3 pyramidal neurons of the mouse did not possess P2X7Rs, but were indirectly modulated by astrocytic and oligodendrocytic P2X7Rs, respectively.

A Possible Causal Involvement of Neuroinflammatory, Purinergic P2X7 Receptors in Psychiatric Disorders.

P2X7 receptors (Rs) are prominent members of the P2XR family, which after binding ATP, open non-selective cationic channels, thereby allowing the transmembrane passage of Na+, Ca2+, and K+. Long-lasting and repetitive stimulation of the receptor by its agonist leads to the formation of large membrane pores permeable for organic cations of up to 900 Da molecular size. These pores are believed to play a role in apoptosis and inflammation. P2X7Rs are located primarily at peripheral macrophages and microglial cells, the resident macrophages of the CNS. The coactivation of toll-like receptors 4 (TLR4) by lipopolysaccharide, a constituent of the cell membrane of gram-negative bacteria, and the P2X7R by ATP leads to the generation and release of the proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α. Together with the microglial release of chemokines, reactive oxygen and nitrogen species, proteases, and excitotoxic glutamate, these cytokines result in neurodegeneration. P2X7Rs were found not only to amplify various neurodegenerative illnesses, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis, but also to participate in a range of psychiatric diseases, such as major depression, bipolar disorder, schizophrenia, and autism spectrum disorder. Based on the prevention/reversal of neuroinflammation, pharmacological antagonists of P2X7Rs and their genetic deletion in animal experiments counteract these deleterious psychiatric conditions. Hence, brain penetrant P2X7R antagonists are potential therapeutics for psychiatric diseases, although the available evidence still needs to be extended and validated by further clinical data.

High, in Contrast to Low Levels of Acute Stress Induce Depressive-like Behavior by Involving Astrocytic, in Addition to Microglial P2X7 Receptors in the Rodent Hippocampus.

Extracellular adenosine 5'-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans.

Increasing Efficiency of Repetitive Electroacupuncture on Purine- and Acid-Induced Pain During a Three-Week Treatment Schedule.

Acupuncture (AP) is an important constituent of the therapeutic repertoire of traditional Chinese medicine and has been widely used to alleviate chronic painful conditions all over the world. We studied in rats the efficiency of electroacupuncture (EAP) applied to the Zusanli acupoint (ST36) as an analgesic treatment over a 3-week period of time on purine (α,ß-methylene ATP, dibenzoyl-ATP)- and acid (pH 6.0 medium)-induced pain in the rat paw. The two ATP derivatives stimulated P2X3 and P2X7 receptors, respectively, while the slightly acidic medium stimulated the "acid-sensitive ion channel 3" (ASIC3). It was found that the P2X7 receptor and ASIC-mediated pain was counteracted by EAP with greater efficiency at the end than at the beginning of the treatment schedule, while the P2X3 receptor-mediated pain was not. Our findings have important clinical and theoretical consequences, among others, because they are difficult to reconcile with the assumption that AP is primarily due to the release of peripheral and central opioid peptides causing the well-known tolerance to their effects. In consequence, AP is a convenient therapeutic instrument to treat subacute and chronic pain.

Inherent P2X7 Receptors Regulate Macrophage Functions during Inflammatory Diseases.

Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophage-localized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1ß (pro-IL-1ß)-degrading caspase-1 to lead to IL-1ß release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn's disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.

Surgeon case volume and 5 years survival rate for colorectal cancer.

AIM: Many factors that influence patient outcome in colorectal surgery are not editable, and these are related either the tumor, the patient and the treatment. The surgeon- and hospital-related factors are independent predictors of outcome for colorectal cancer surgery and these are supervision, teaching/training, specialization in colorectal surgery, high caseload, high hospital caseload. MATERIALS OF STUDY: We evaluated the impact of the experience of 4 surgeons on the 5 years survival rate of patients with colon and rectal cancer and we valued if the surgeons' experience plays an equal role in both. RESULTS: Four experienced surgeons operated 384 patients with colorectal cancer. Surgeon with the major experience and colorectal-dedicated presented a slightly better total 5 years survival rate, comparing to other surgeons, although he had a considerably better 5 years survival rate in rectal operations. CONCLUSIONS: We concluded that surgeon- and hospital-related factors directly influence the surgeon learning curve and are therefore rightly considered predictors of outcome in colorectal cancer surgery. A low surgeon or hospital caseload may be compensated by intensified supervision or by improved training and teaching. KEY WORDS: Colon cancer, Colectomy, Surgeon volume.

Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNS.

Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood-brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer's Disease, Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5'-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation.

The P2X7 receptor: a new therapeutic target in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a neurodegenerative illness with genetic risk as an etiological factor in a subset of cases. In AD with autosomal dominant inheritance, the extracellular ß-amyloid (Aß) aggregates and intracellular neurofibrillary tangles which consist of hyperphosphorylated tau, appear to be involved in the neuronal damage; however, other forms of AD may have a polygenetic causality. Microglial cells orchestrate pathophysiological events responsible for neuronal damage in AD. They surround Aß aggregates and the stimulation of microglial P2X7 receptors (P2X7Rs) by high local concentrations of ATP which originates from damaged CNS cells, results in degeneration of nearby neurons. Areas covered: We discuss the pathogenesis of Alzheimer's disease, the role of P2X7 receptors and their potential as therapeutic targets. We also address the fundamental hurdles in the development of new therapeutic strategies for Alzheimer's disease. Expert opinion: There are many difficulties associated with the development of efficient pharmacological strategies for AD; the lack of good animal and cellular models for this illness is a key obstacle. None of the pharmacological strategies developed so far have led to an improvement of the treatment of AD. Hence, the consideration of blood-brain barrier-permeable P2X7R antagonists as possible therapeutic agents in AD is a must.

P2X receptors and acupuncture analgesia.

Purinergic signaling has recently been suggested to constitute the cellular mechanism underlying acupuncture-induced analgesia (AA). By extending the original hypothesis on endogenous opioids being released during AA, Geoffrey Burnstock and Maiken Nedergaard supplied evidence for the involvement of purinoceptors (P2 and P1/A1 receptors) in the beneficial effects of AA. In view of certain pain states (e.g. neuropathic pain) which respond only poorly to therapy with standard analgesics, as well as with respect to the numerous unwanted effects of opioids and non-steroidal anti-inflammatory drugs, it is of great significance to search for alternative therapeutic options. Because clinical studies on AA yielded sometimes heterogeneous results, it is of eminent importance to relay on experiments carried out on laboratory animals, by evaluating the data with stringent statistical methods including comparison with a sufficient number of control groups. In this review, we summarize the state of the art situation with respect to the participation of P2 receptors in AA and try to forecast how the field is likely to move forward in the future.

Author Correction: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel.

The originally published version of this article contained an error in the name of the author Flóra Gölöncsér, which was incorrectly given as Flóra Göröncsér. This has now been corrected in both the PDF and HTML versions of the article.

The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel.

Two subclasses of acid-sensing ion channels (ASIC3) and of ATP-sensitive P2X receptors (P2X3Rs) show a partially overlapping expression in sensory neurons. Here we report that both recombinant and native receptors interact with each other in multiple ways. Current measurements with the patch-clamp technique prove that ASIC3 stimulation strongly inhibits the P2X3R current partly by a Ca2+-dependent mechanism. The proton-binding site is critical for this effect and the two receptor channels appear to switch their ionic permeabilities during activation. Co-immunoprecipation proves the close association of the two protein structures. BN-PAGE and SDS-PAGE analysis is also best reconciled with the view that ASIC3 and P2X3Rs form a multiprotein structure. Finally, in vivo measurements in rats reveal the summation of pH and purinergically induced pain. In conclusion, the receptor subunits do not appear to form a heteromeric channel, but tightly associate with each other to form a protein complex, mediating unidirectional inhibition.

Neuronal P2X7 Receptors Revisited: Do They Really Exist?

P2X7 receptors (Rs) constitute a subclass of ATP-sensitive ionotropic receptors (P2X1-P2X7). P2X7Rs have many distinguishing features, mostly based on their long intracellular C terminus regulating trafficking to the cell membrane, protein-protein interactions, and post-translational modification. Their C-terminal tail is especially important in enabling the transition from the nonselective ion channel mode to a membrane pore allowing the passage of large molecules. There is an ongoing dispute on the existence of neuronal P2X7Rs with consequences for our knowledge on their involvement in neuroinflammation, aggravating stroke, temporal lobe epilepsy, neuropathic pain, and various neurodegenerative diseases. Whereas early results appeared to support the operation of P2X7Rs at neurons, more recently glial P2X7Rs are increasingly considered as indirect causes of neuronal effects. Specific tools for P2X7Rs are of limited value because of the poor selectivity of agonists, and the inherent failure of antibodies to differentiate between the large number of active and inactive splice variants, or gain-of-function and loss-of-function small nucleotide polymorphisms of the receptor. Unfortunately, the available P2RX7 knock-out mice generated by pharmaceutical companies possess certain splice variants, which evade inactivation. In view of the recently discovered bidirectional dialogue between astrocytes and neurons (and even microglia and neurons), we offer an alternative explanation for previous data, which assumedly support the existence of P2X7Rs at neurons. We think that the unbiased reader will follow our argumentation on astrocytic or microglial P2X7Rs being the primary targets of pathologically high extracellular ATP concentrations, although a neuronal localization of these receptors cannot be fully excluded either.