The primary somatosensory (S1) cortex plays a key role in distinguishing different sensory stimuli. Vibrotactile touch information is conveyed from the periphery to the S1 cortex through three major classes of mechanoreceptors: slowly adapting type 1 (SA1), rapidly adapting (RA), and Pacinian (PC) afferents. It has been a long-standing question whether specific populations in the S1 cortex preserve the peripheral segregation by the afferent submodalities. Here, we investigated whether S1 neurons exhibit specific responses to two distinct vibrotactile stimuli, which excite different types of mechanoreceptors (e.g., SA1 and PC afferents). Using in vivo two-photon microscopy and genetically encoded calcium indicator, GCaMP6s, we recorded calcium activities of S1 L2/3 neurons. At the same time, static (<1 Hz) and dynamic (150 Hz) vibrotactile stimuli, which are known to excite SA1 and PC, respectively, were pseudorandomly applied to the right hind paw in lightly anesthetized mice. We found that most active S1 neurons responded to both static and dynamic stimuli, but more than half of them showed preferred responses to either type of stimulus. Only a small fraction of the active neurons exhibited specific responses to either static or dynamic stimuli. However, the S1 population activity patterns by the two stimuli were markedly distinguished. These results indicate that the vibrotactile inputs driven by excitation of distinct submodalities are converged on the single cells of the S1 cortex, but are well discriminated by population activity patterns composed of neurons that have a weighted preference for each type of stimulus.

Rationale: Neuroinflammation is a primary feature of Alzheimer's disease (AD), for which an increasing number of drugs have been specifically developed. The present study aimed to define the therapeutic impact of a specific subpopulation of T cells that can suppress excessive inflammation in various immune and inflammatory disorders, namely, CD4+CD25+Foxp3+ regulatory T cells (Tregs). Methods: To generate Aβ antigen-specific Tregs (Aβ+ Tregs), Aβ 1-42 peptide was applied in vivo and subsequent in vitro splenocyte culture. After isolating Tregs by magnetic bead based purification method, Aβ+ Tregs were adoptively transferred into 3xTg-AD mice via tail vein injection. Therapeutic efficacy was confirmed with behavior test, Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry staining (IHC). In vitro suppression assay was performed to evaluate the suppressive activity of Aβ+ Tregs using flow cytometry. Thy1.1+ Treg trafficking and distribution was analyzed to explore the infused Tregs migration into specific organs in an antigen-driven manner in AD mice. We further assessed cerebral glucose metabolism using 18F-FDG-PET, an imaging approach for AD biological definition. Subsequently, we evaluated the migration of Aβ+ Tregs toward Aβ activated microglia using live cell imaging, chemotaxis, antibody blocking and migration assay. Results: We showed that Aβ-stimulated Tregs inhibited microglial proinflammatory activity and modulated the microglial phenotype via bystander suppression. Single adoptive transfer of Aβ+ Tregs was enough to induce amelioration of cognitive impairments, Aβ accumulation, hyper-phosphorylation of tau, and neuroinflammation during AD pathology. Moreover, Aβ-specific Tregs effectively inhibited inflammation in primary microglia induced by Aβ exposure. It may indicate bystander suppression in which Aβ-specific Tregs promote immune tolerance by secreting cytokines to modulate immune responses during neurodegeneration. Conclusions: The administration of Aβ antigen-specific regulatory T cells may represent a new cellular therapeutic strategy for AD that acts by modulating the inflammatory status in AD.

Paclitaxel (sold under the brand name Taxol) is a chemotherapeutic drug that is widely used to treat cancer. However, it can also induce peripheral neuropathy, which limits its use. Although several drugs are used to attenuate neuropathy, no optimal treatment is available to date. In this study, the effect of cucurbitacins B and D on paclitaxel-induced neuropathic pain was assessed. Multiple paclitaxel injections (a cumulative dose of 8 mg/kg, i. p.) induced cold and mechanical allodynia from days 10 to 21 in mice, and the i. p. administration of 0.025 mg/kg of cucurbitacins B and D attenuated both allodynia types. However, as cucurbitacin B showed a more toxic effect on non-cancerous (RAW 264.7) cells, further experiments were conducted with cucurbitacin D. The cucurbitacin D dose-dependently (0.025, 0.1, and 0.5 mg/kg) attenuated both allodynia types. In the spinal cord, paclitaxel injection increased the gene expression of noradrenergic (α 1-and α 2-adrenergic) receptors but not serotonergic (5-HT1A and 3) receptors. Cucurbitacin D treatment significantly decreased the spinal α 1- but not α 2-adrenergic receptors, and the amount of spinal noradrenaline was also downregulated. However, the tyrosine hydroxylase expression measured via liquid chromatography in the locus coeruleus did not decrease significantly. Finally, cucurbitacin D treatment did not lower the anticancer effect of chemotherapeutic drugs when co-administered with paclitaxel in CT-26 cell-implanted mice. Altogether, these results suggest that cucurbitacin D could be considered a treatment option against paclitaxel-induced neuropathic pain.

Diabetic neuropathy, a major complication of diabetes mellitus, refers to a collection of clinically diverse disorders affecting the nervous system that may present with pain. Although the number of patients suffering from severe neuropathy is increasing, no optimal treatment method has been developed yet. Acupuncture is well known for its ability to reduce various kinds of pain, and a number of studies have also reported its effect on diabetes mellitus; however, its effect and underlying mechanism against diabetic neuropathy are not yet clearly understood. In this review, ten and five studies performed in humans and animals, respectively, were analyzed. All studies reported that acupuncture significantly relieved diabetic neuropathy. ST36, BL13, BL20, SP6, and SP9 were the most widely used acupoints. Five studies used electro-acupuncture, whereas other studies used manual acupuncture. Furthermore, the effect of acupuncture was shown to be mediated through the various molecules present in the peripheral nerves and spinal cord, such as P65, GPR78, and TRPV1. Five studies reported side effects, such as swelling, numbness, and nausea, but none were reported to be serious. Based on these results, we suggest that acupuncture should be considered as a treatment option for diabetic neuropathy.

Oxaliplatin is a chemotherapeutic agent used for metastatic colon and other advanced cancers. Most common side effect of oxaliplatin is peripheral neuropathy, manifested in mechanical and cold allodynia. Although the analgesic effect of bee venom has been proven to be effective against oxaliplatin-induced peripheral neuropathy, the effect of its major component; melittin has not been studied yet. Thus, in this study, we investigated whether melittin has an analgesic effect on oxaliplatin-induced allodynia. Intraperitoneal single injection of oxaliplatin (6 mg/kg) induced mechanical and cold allodynia, resulting in increased withdrawal behavior in response to von Frey filaments and acetone drop on hind paw. Subcutaneous melittin injection on acupoint ST36 (0.5 mg/kg) alleviated oxaliplatin-induced mechanical and cold allodynia. In electrophysiological study, using spinal in vivo extracellular recording, it was shown that oxaliplatin-induced hyperexcitation of spinal wide dynamic range neurons in response to peripheral stimuli, and melittin administration inhibited this neuronal activity. In behavioral assessment, analgesic effect of melittin was blocked by intrathecal α1- and α2- adrenergic receptor antagonists administration. Based on these results, we suggest that melittin could be used as an analgesic on oxaliplatin-induced peripheral neuropathy, and that its effect is mediated by activating the spinal α1- and α2-adrenergic receptors.

Chronic pain remains an intractable condition in millions of patients worldwide. Spontaneous ongoing pain is a major clinical problem of chronic pain and is extremely challenging to diagnose and treat compared to stimulus-evoked pain. Although extensive efforts have been made in preclinical studies, there still exists a mismatch in pain type between the animal model and humans (i.e., evoked vs. spontaneous), which obstructs the translation of knowledge from preclinical animal models into objective diagnosis and effective new treatments. Here, we developed a deep learning algorithm, designated AI-bRNN (Average training, Individual test-bidirectional Recurrent Neural Network), to detect spontaneous pain information from brain cellular Ca2+ activity recorded by two-photon microscopy imaging in awake, head-fixed mice. AI-bRNN robustly determines the intensity and time points of spontaneous pain even in chronic pain models and evaluates the efficacy of analgesics in real time. Furthermore, AI-bRNN can be applied to various cell types (neurons and glia), brain areas (cerebral cortex and cerebellum) and forms of somatosensory input (itch and pain), proving its versatile performance. These results suggest that our approach offers a clinically relevant, quantitative, real-time preclinical evaluation platform for pain medicine, thereby accelerating the development of new methods for diagnosing and treating human patients with chronic pain.

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward 'shadow imaging' approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.

Cisplatin is a platinum-based chemotherapeutic agent widely used to treat various cancers. However, several side effects have been reported in treated patients. Among these, acute anorexia is one of the most severe secondary effects. In this study, a single oral administration of 100 or 500 mg/kg ginger extract (GE) significantly alleviated the cisplatin-induced decrease in food intake in rats. However, these body weight and water intake decreases were reversed in the 100 mg/kg group rats. To elucidate the underlying mechanism of action, serotonin (5-HT) and 5-HT2C, 3A, and 4 receptors in the nodose ganglion of the vagus nerve were investigated. The results showed that cisplatin-induced increases in serotonin levels in both the blood and nodose ganglion tissues were significantly decreased by100 and 500 mg/kg of GE administration. On 5-HT receptors, 5-HT3A and 4, but not 2C receptors, were affected by cisplatin, and GE 100 and 500 mg/kg succeeded in downregulating the evoked upregulated gene of these receptors. Protein expression of 5-HT3A and 4 receptors were also reduced in the 100 mg/kg group. Furthermore, the injection of 5-HT3A, and 4 receptors antagonists (palonostron, 0.1 mg/kg, i.p.; piboserod, 1 mg/kg, i.p., respectively) in cisplatin treated rats prevented the decrease in food intake. Using high-performance liquid chromatography (HPLC) analysis, [6]-gingerol and [6]-shogaol were identified and quantified as the major components of GE, comprising 4.12% and 2.15% of the GE, respectively. Although [6]-gingerol or [6]-shogaol alone failed to alleviate the evoked anorexia, when treated together, the effect was significant on the cisplatin-induced decrease in food intake. These results show that GE can be considered a treatment option to alleviate cisplatin-induced anorexia.

Although electroacupuncture (EA) relieves various types of pain, individual differences in the sensitivity to EA analgesia have been reported, causing experimental and clinical difficulties. Our functional genomic study using cDNA microarray identified that 5'-AMP-activated protein kinase (AMPK), a well-known factor in the regulation of energy homeostasis, is the most highly expressed gene in the hypothalamus of the rats that were sensitive to EA analgesia ("responder"), as compared to the rats that were insensitive to EA analgesia ("non-responder"). In this study, we investigated the causal relationship between the hypothalamic AMPK and the individual variation in EA analgesia.

Oxaliplatin, a chemotherapy drug, induces acute peripheral neuropathy characterized by cold allodynia, spinal glial activation and increased levels of pro-inflammatory cytokines. Herein, we determined whether Cinnamomi Cortex (C. Cortex), a widely used medicinal herb in East Asia for cold-related diseases, could attenuate oxaliplatin-induced cold allodynia in rats and the mechanisms involved. A single oxaliplatin injection (6 mg/kg, i.p.) induced significant cold allodynia signs based on tail immersion tests using cold water (4 °C). Daily oral administration of water extract of C. Cortex (WECC) (100, 200, and 400 mg/kg) for five consecutive days following an oxaliplatin injection dose-dependently alleviated cold allodynia with only a slight difference in efficacies between the middle dose at 200 mg/kg and the highest dose at 400 mg/kg. WECC at 200 mg/kg significantly suppressed the activation of astrocytes and microglia and decreased the expression levels of IL-1β and TNF in the spinal cord after injection with oxaliplatin. Furthermore, oral administration of coumarin (10 mg/kg), a major phytocompound of C. Cortex, markedly reduced cold allodynia. These results indicate that C. Cortex has a potent anti-allodynic effect in oxaliplatin-injected rats through inhibiting spinal glial cells and pro-inflammatory cytokines. We also suggest that coumarin might play a role in the anti-allodynic effect of C. Cortex.

Mesenchymal stem cells (MSCs) have been identified in multiple types of tissue and exhibit characteristic self-renewal and multi-lineage differentiation abilities. However, the possibility of oncogenic transformation after transplantation is concerning. In this study, we investigated the tumorigenic potential of umbilical cord blood-derived MSCs (hUCB-MSCs) relative to MRC-5 and HeLa cells (negative and positive controls, respectively) both in vitro and in vivo. To evaluate tumorigenicity in vitro, anchorage-independent growth was assessed using the soft agar colony formation assay. hUCB-MSCs and MRC-5 cells formed few colonies, while HeLa cells formed a greater number of larger colonies, indicating that hUCB-MSCs and MRC-5 cells do not have anchorage-independent proliferation potential. To detect tumorigenicity in vivo, hUCB-MSCs were implanted as a single subcutaneous injection into BALB/c-nu mice. No tumor formation was observed in mice transplanted with hUCB-MSCs or MRC-5 cells based on macroand microscopic examinations; however, all mice transplanted with HeLa cells developed tumors that stained positive for a human gene according to immunohistochemical analysis. In conclusion, hUCB-MSCs do not exhibit tumorigenic potential based on in vitro and in vivo assays under our experimental conditions, providing further evidence of their safety for clinical applications.

Oxaliplatin, a chemotherapy drug used to treat colorectal cancer, induces specific sensory neurotoxicity signs that are aggravated by cold and mechanical stimuli. Here we examined the preventive effects of Bee Venom (BV) derived phospholipase A₂ (bvPLA₂) on oxaliplatin-induced neuropathic pain in mice and its immunological mechanism. The cold and mechanical allodynia signs were evaluated by acetone and von Frey hair test on the hind paw, respectively. The most significant allodynia signs were observed at three days after an injection of oxaliplatin (6 mg/kg, i.p.) and then decreased gradually to a normal level on days 7-9. The oxaliplatin injection also induced infiltration of macrophages and upregulated levels of the pro-inflammatory cytokine interleukin (IL)-1β in the lumbar dorsal root ganglia (DRG). Daily treatment with bvPLA₂ (0.2 mg/kg, i.p.) for five consecutive days prior to the oxaliplatin injection markedly inhibited the development of cold and mechanical allodynia, and suppressed infiltration of macrophages and the increase of IL-1β level in the DRG. Such preventive effects of bvPLA₂ were completely blocked by depleting regulatory T cells (Tregs) with CD25 antibody pre-treatments. These results suggest that bvPLA₂ may prevent oxaliplatin-induced neuropathic pain by suppressing immune responses in the DRG by Tregs.

Oxaliplatin, a chemotherapeutic drug for colorectal cancer, induces severe peripheral neuropathy. Bee venom acupuncture (BVA) has been used to attenuate pain, and its effect is known to be mediated by spinal noradrenergic and serotonergic receptors. Morphine is a well-known opioid used to treat different types of pain. Here, we investigated whether treatment with a combination of these two agents has an additive effect on oxaliplatin-induced neuropathic pain in mice. To assess cold and mechanical allodynia, acetone and von Frey filament tests were used, respectively. Significant allodynia signs were observed three days after an oxaliplatin injection (6 mg/kg, i.p.). BVA (0.25, 1, and 2.5 mg/kg, s.c., ST36) or morphine (0.5, 2, and 5 mg/kg, i.p.) alone showed dose-dependent anti-allodynic effects. The combination of BVA and morphine at intermediate doses showed a greater and longer effect than either BVA or morphine alone at the highest dose. Intrathecal pretreatment with the opioidergic (naloxone, 20 μg) or 5-HT3 (MDL-72222, 15 μg) receptor antagonist, but not with α2 adrenergic (idazoxan, 10 μg) receptor antagonist, blocked this additive effect. Therefore, we suggest that the combination effect of BVA and morphine is mediated by spinal opioidergic and 5-HT3 receptors and this combination has a robust and enduring analgesic action against oxaliplatin-induced neuropathic pain.

A single infusion of oxaliplatin, which is widely used to treat metastatic colorectal cancer, induces specific sensory neurotoxicity signs that are triggered or aggravated when exposed to cold or mechanical stimuli. Bee Venom (BV) has been traditionally used in Korea to treat various pain symptoms. Our recent study demonstrated that BV alleviates oxaliplatin-induced cold allodynia in rats, via noradrenergic and serotonergic analgesic pathways. In this study, we have further investigated whether BV derived phospholipase A2 (bvPLA2) attenuates oxaliplatin-induced cold and mechanical allodynia in mice and its mechanism. The behavioral signs of cold and mechanical allodynia were evaluated by acetone and a von Frey hair test on the hind paw, respectively. The significant allodynia signs were observed from one day after an oxaliplatin injection (6 mg/kg, i.p.). Daily administration of bvPLA2 (0.2 mg/kg, i.p.) for five consecutive days markedly attenuated cold and mechanical allodynia, which was more potent than the effect of BV (1 mg/kg, i.p.). The depletion of noradrenaline by an injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4, 50 mg/kg, i.p.) blocked the analgesic effect of bvPLA2, whereas the depletion of serotonin by injecting DL-p-chlorophenylalanine (PCPA, 150 mg/kg, i.p.) for three successive days did not. Furthermore, idazoxan (α2-adrenegic receptor antagonist, 1 mg/kg, i.p.) completely blocked bvPLA2-induced anti-allodynic action, whereas prazosin (α1-adrenegic antagonist, 10 mg/kg, i.p.) did not. These results suggest that bvPLA2 treatment strongly alleviates oxaliplatin-induced acute cold and mechanical allodynia in mice through the activation of the noradrenergic system, via α2-adrenegic receptors, but not via the serotonergic system.

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2β2) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

Cisplatin is used as a potent anticancer drug, but it often causes nephrotoxicity. Bee venom (BV) has been used for the treatment of various inflammatory diseases, and its renoprotective action was shown in NZB/W mice. However, little is known about whether BV has beneficial effects on cisplatin-induced nephrotoxicity and how such effects might be mediated. In the present study, the BV-injected group showed a significant increase in the population of Tregs in spleen. Although there was no significant difference in the numbers of Tregs 3 days after cisplatin injection between the BV- and PBS-injected groups, more migration of Tregs into the kidney was observed 6 hours after cisplatin administration in BV group than in PBS group. In addition, BV-injected mice showed reduced levels of serum creatinine, blood urea nitrogen, renal tissue damage, proinflammatory cytokines, and macrophage infiltration into the kidney 3 days after cisplatin administration. These renoprotective effects were abolished by the depletion of Tregs. The anticancer effect of repeated administrations of cisplatin was not affected by BV injection. These results suggest that BV has protective effects on cisplatin-induced nephrotoxicity in mice, at least in part, through the regulation of Tregs without a big influence on the antitumor effects of cisplatin.

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

Pulmonary fibrosis is a chronic progressive disease with unknown etiology and has poor prognosis. Polyhexamethyleneguanidine phosphate (PHMG-P) causes acute interstitial pneumonia and pulmonary fibrosis in humans when it exposed to the lung. In a previous study, when rats were exposed to PHMG-P through inhalation for 3 weeks, lung inflammation and fibrosis was observed even after 3 weeks of recovery. In this study, we aimed to determine the time course of PHMG-P-induced lung inflammation and fibrosis. We compared pathological action of PHMG-P with that of bleomycin (BLM) and investigated the mechanism underlying PHMG-P-induced lung inflammation and fibrosis. PHMG-P (0.9 mg/kg) or BLM (1.5 mg/kg) was intratracheally administered to mice. At weeks 1, 2, 4 and 10 after instillation, the levels of inflammatory and fibrotic markers and the expression of inflammasome proteins were measured. The inflammatory and fibrotic responses were upregulated until 10 and 4 weeks in the PHMG-P and BLM groups, respectively. Immune cell infiltration and considerable collagen deposition in the peribronchiolar and interstitial areas of the lungs, fibroblast proliferation, and hyperplasia of type II epithelial cells were observed. NALP3 inflammasome activation was detected in the PHMG-P group until 4 weeks, which is suspected to be the main reason for the persistent inflammatory response and exacerbation of fibrotic changes. Most importantly, the pathological changes in the PHMG-P group were similar to those observed in humidifier disinfectant-associated patients. A single exposure of PHMG-P led to persistent pulmonary inflammation and fibrosis for at least 10 weeks.

Whether hematopoietic cell-restricted distribution of antigens affects the degree of thymic negative selection has not been investigated in detail. Here, we show that T cells specific for hematopoietic cell-restricted antigens (HRA) are not completely deleted in the thymus, using the mouse minor histocompatibility antigen H60, the expression of which is restricted to hematopoietic cells. As a result, low avidity T cells escape from thymic deletion. This incomplete thymic deletion occurs to the T cells developing de novo in the thymus of H60-positive recipients in H60-mismatched bone marrow transplantation (BMT). H60-specific thymic deletion escapee CD8+ T cells exhibit effector differentiation potentials in the periphery and contribute to graft-versus-leukemia effects in the recipients of H60-mismatched BMT, regressing H60+ hematological tumors. These results provide information essential for understanding thymic negative selection and developing a strategy to treat hematological tumors.

Only a limited number of neuroimaging studies have explored the effects of ambient air pollution in adults. The prior studies have investigated only cortical volume, and they have reported mixed findings, particularly for gray matter. Furthermore, the association between nitrogen dioxide (NO2) and neuroimaging markers has been little studied in adults.