Real-time monitoring of cortical brain activity in response to acute pain using wide-area Ca2+ imaging.

Previous human and rodent studies indicated that nociceptive stimuli activate many brain regions that is involved in the somatosensory and emotional sensation. Although these studies have identified several important brain regions involved in pain perception, it has been a challenge to observe neural activity directly and simultaneously in these multiple brain regions during pain perception. Using a transgenic mouse expressing G-CaMP7 in majority of astrocytes and a subpopulation of excitatory neurons, we recorded the brain activity in the mouse cerebral cortex during acute pain stimulation. Both of hind paw pinch and intraplantar administration of formalin caused strong transient increase of the fluorescence in several cortical regions, including primary somatosensory, motor and retrosplenial cortex. This increase of the fluorescence intensity was attenuated by the pretreatment with morphine. The present study provides important insight into the cortico-cortical network during pain perception.

Investigating the Influence of Morphine and Cocaine on the Mesolimbic Pathway Using a Novel Microimaging Platform.

Dopamine (DA)'s relationship with addiction is complex, and the related pathways in the mesocorticolimbic system are used to deliver DA, regulating both behavioral and perceptual actions. Specifically, the mesolimbic pathway connecting the ventral tegmental area (VTA) and the nucleus accumbens (NAc) is crucial in regulating memory, emotion, motivation, and behavior due to its responsibility to modulate dopamine. To better investigate the relationship between DA and addiction, more advanced mapping methods are necessary to monitor its production and propagation accurately and efficiently. In this study, we incorporate dLight1.2 adeno-associated virus (AAV) into our latest CMOS (complementary metal-oxide semiconductor) imaging platform to investigate the effects of two pharmacological substances, morphine and cocaine, in the NAc using adult mice. By implanting our self-fabricated CMOS imaging device into the deep brain, fluorescence imaging of the NAc using the dLight1.2 AAV allows for the visualization of DA molecules delivered from the VTA in real time. Our results suggest that changes in extracellular DA can be observed with this adapted system, showing potential for new applications and methods for approaching addiction studies. Additionally, we can identify the unique characteristic trend of DA release for both morphine and cocaine, further validating the underlying biochemical mechanisms used to modulate dopaminergic activation.

Multi-Region Microdialysis Imaging Platform Revealed Dorsal Raphe Nucleus Calcium Signaling and Serotonin Dynamics during Nociceptive Pain.

In this research, we combined our ultralight micro-imaging device for calcium imaging with microdialysis to simultaneously visualize neural activity in the dorsal raphe nucleus (DRN) and measure serotonin release in the central nucleus of the amygdala (CeA) and the anterior cingulate cortex (ACC). Using this platform, we observed brain activity following nociception induced by formalin injection in the mouse's hind paw. Our device showed that DRN fluorescence intensity increased after formalin injection, and the increase was highly correlated with the elevation in serotonin release in both the CeA and ACC. The increase in calcium fluorescence intensity occurred during the acute and inflammatory phases, which suggests the biphasic response of nociceptive pain. Furthermore, we found that the increase in fluorescence intensity was positively correlated with mouse licking behavior. Lastly, we compared the laterality of pain stimulation and found that DRN fluorescence activity was higher for contralateral stimulation. Microdialysis showed that CeA serotonin concentration increased only after contralateral stimulation, while ACC serotonin release responded bilaterally. In conclusion, our study not only revealed the inter-regional serotonergic connection among the DRN, the CeA, and the ACC, but also demonstrated that our device is feasible for multi-site implantation in conjunction with a microdialysis system, allowing the simultaneous multi-modal observation of different regions in the brain.

Astrocyte-neuron lactate shuttle sensitizes nociceptive transmission in the spinal cord.

Astrocytes play a key role in the maintenance of synaptic transmission by producing L-lactate via the astrocyte-neuron lactate shuttle (ANLS). Astrocyte activation in the spinal cord is involved in the expression of neuropathic pain. We investigated the role of the ANLS in the spinal cord on hyperalgesia in neuropathic pain in mice. Specific activation of dorsal horn astrocytes induced mechanical hyperalgesia, which was attenuated by α-cyano-4-hydroxycinnamate (4-CIN), an inhibitor of monocarboxylate transporters that deliver L-lactate from astrocytes to neurons. Intrathecal L-lactate administration lowered the mechanical nociceptive threshold, which was attenuated by pretreatment with 4-CIN and isosafrole (a lactate dehydrogenase inhibitor), but not gliotoxin. Intrathecal L-lactate administration significantly upregulated c-Fos and cofilin phosphorylation, which was reversed by 4-CIN. The lowered mechanical nociceptive threshold was significantly attenuated by intrathecal fluorocitrate (an astrocyte-specific Krebs cycle inhibitor), 4-CIN, and isosafrole treatment. Thus, these results suggested that, in neuropathic pain, mechanical hyperalgesia was maintained by excessive L-lactate supplied by activated astrocytes via an aberrant ANLS.

Involvement of l-lactate in hippocampal dysfunction of type I diabetes.

Hippocampal neurons play a crucial role in memory formation. Accumulating evidence raises the possibility that hippocampal sharp-wave ripples (SW-Rs) are involved in memory consolidation. Here, we examined in an animal model of diabetes and found the amplitude of SW-Rs in diabetic mice were smaller than control group and were rescued by acute application of l-lactate, a major neural energy source. The cognitive impairment in diabetic mice was alleviated by intracerebroventricular l-lactate treatment. Our results suggested that l-lactate is important for hippocampal dysfunction in diabetes.

Ethanol-induced enhancement of inhibitory synaptic transmission in the rat spinal substantia gelatinosa.

Recent studies have shown that ethanol produces a widespread modulation of neuronal activity in the central nervous system. It is not fully understood, however, how ethanol changes nociceptive transmission. We investigated acute effects of ethanol on synaptic transmission in the substantia gelatinosa (lamina II of the spinal dorsal horn) and mechanical responses in the spinal dorsal horn. In substantia gelatinosa neurons, bath application of ethanol at low concentration (10 mM) did not change the frequency and amplitude of spontaneous inhibitory postsynaptic currents. At medium to high concentrations (20-100 mM), however, ethanol elicited a barrage of large amplitude spontaneous inhibitory postsynaptic currents. In the presence of tetrodotoxin, such enhancement of spontaneous inhibitory postsynaptic currents was not detected. In addition, ethanol (20-100 mM) increased the frequency of spontaneous discharge of vesicular GABA transporter-Venus-labeled neurons and suppressed the mechanical nociceptive response in wide-dynamic range neurons in the spinal dorsal horn. The present results suggest that ethanol may reduce nociceptive information transfer in the spinal dorsal horn by enhancement of inhibitory GABAergic and glycinergic synaptic transmission.

Role of microglia in mechanical allodynia in the anterior cingulate cortex.

Plastic changes that increase nociceptive transmission are observed in several brain regions under conditions of chronic pain. Synaptic plasticity in the anterior cingulate cortex (ACC) is particularly associated with neuropathic pain. Glial cells are considered candidates for the modulation of neural plastic changes in the central nervous system. In this study, we aimed to investigate the role of ACC glial cells in the development of neuropathic pain. First, we examined the expression of glial cells in the ACC of nerve-ligated mice. The expression of astrocytes and microglia was increased in the ACC of nerve-ligated mice, which was reversed by intracerebroventricular (i.c.v) treatment with the microglia inhibitor minocycline. Then, we examined the effect of minocycline on mechanical allodynia in nerve-ligated mice. I.c.v. and intra-ACC treatment with minocycline partially inhibited mechanical allodynia in the nerve-ligated mice. The expression of phosphorylated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit at Ser831, but not at Ser845, was increased in the ACC of the nerve-ligated mice compared to sham-operated mice, which was attenuated by minocycline administration. These results suggest that the activation of microglia in the ACC is involved in the development of hyperalgesia in mice with neuropathic pain.

Oxaliplatin treatment changes the function of sensory nerves in rats.

Oxaliplatin (L-OHP) is a platinum-based chemotherapy drug, used in standard treatment of colorectal cancer. L-OHP frequently causes acute peripheral neuropathies. These adverse effects limit cancer therapy with L-OHP. The present study was designed to reveal the changes in sensory nerve function in L-OHP-injected rats. Mechanical static allodynia, dynamic allodynia, and cold allodynia were evaluated using the von Frey test, brush test, and acetone test, respectively. Sensory nerve fiber responsiveness was measured using a Neurometer. The fifth lumbar ventral root was sectioned to record multi-unit efferent discharges. Single intraperitoneal administration of L-OHP induced mechanical static allodynia, dynamic allodynia, and cold allodynia in Wistar/ST rats. The thresholds for paw withdrawal induced by 2000 Hz (Aß-fiber) and 5 Hz (C-fiber), but not 250 Hz (Aδ-fiber) sine-wave electrical stimulation were reduced in L-OHP-treated rats. Multi-unit efferent discharges were increased by mechanical stimulation using a von Frey filament applied to the plantar surface of the hindpaw. The discharges during and after stimulation were increased in the L-OHP-treated rats. Cold stimulation, but not brush stimulation, increased the discharges in L-OHP-treated rats. These results suggest that sensitization of Aß- and C-fibers, but not Aδ-fibers, contributes to the development of L-OHP-induced mechanical and cold allodynia.

Involvement of renal sympathetic nerve activation on the progression of ischemic acute kidney injury in the mouse.

Renal ischemia produces renal sympathoexcitation that is responsible for the development of ischemic acute kidney injury. The present study examined changes in the sympathetic nerve function in mice. Ischemic acute kidney injury was induced by occlusion of both renal pedicles. Renal ischemia/reperfusion increased blood urea nitrogen and plasma creatinine and expression of tyrosine hydroxylase, a rate-limiting enzyme for the biosynthesis of noradrenaline, in the kidney. Renal immunoreactivity of tyrosine hydroxylase was observed along with vessel and tubular structure both in the sham-operated and the ischemic acute kidney injury mice. The prominent morphological change was that tyrosine hydroxylase immunoreactivity was observed in the glomeruli of the ischemic acute kidney injury mice, whereas there are almost no tyrosine hydroxylase immunoreactivity signals in the glomeruli of the sham-operated mice. This tyrosine hydroxylase immunoreactivity in the glomeruli is colocalized with synapsin I immunoreactivity in the ischemic acute kidney injury mice. Intraperitoneal pretreatment with DSP-4 (50 mg/kg) attenuated these changes induced by renal ischemia/reperfusion. These results suggest that morphological and functional changes of glomerulus adrenergic nerve terminal are involved in the pathophysiology of ischemia/reperfusion-induced ischemic acute kidney injury.

Gabapentin prevents oxaliplatin-induced mechanical hyperalgesia in mice.

Oxaliplatin, a platinum-based chemotherapy drug, frequently causes acute and chronic peripheral neuropathies including mechanical hyperalgesia. These adverse effects hinder anticancer therapy with the drug. In this study, we examined several drugs that might prevent oxaliplatin-induced peripheral neuropathy. Single intraperitoneal (i.p.) injection of oxaliplatin (10 mg/kg) induced cold allodynia (acetone test) and mechanical hyperalgesia (von Frey test). Gabapentin, but not simvastatin and atorvastatin, prevented oxaliplatin-induced mechanical hyperalgesia without affecting cold allodynia. Moreover, oxaliplatin caused phosphorylation of cofilin protein in the spinal cord, which has been shown to be involved in the neuropathic hyperalgesia. This increased phosphorylation of cofilin was also attenuated by gabapentin treatment. These results suggest that gabapentin is useful for relieving oxaliplatin-induced mechanical hyperalgesia and that the pathogenic mechanisms of cold allodynia and mechanical hyperalgesia differ.

Exercise Suppresses Head and Neck Squamous Cell Carcinoma Growth via Oncostatin M.

Major advances have been made in cancer treatment, but the prognosis for elderly cancer patients with sarcopenia and frailty remains poor. Myokines, which are thought to exert preventive effects against sarcopenia, have been reported to be associated with the prognosis of various cancers, but their effect on head and neck squamous cell carcinoma (HNSCC) is unknown. The aim of this study was to clarify the influence of exercise on the control of HNSCC and to examine the underlying mechanism involved. Mice were injected with HSC-3-M3 cells, a human cell line of highly metastatic and poorly differentiated tongue cancer, at the beginning of the study. Just prior to transplantation, blood was collected from the mice, and the levels of myokines were measured by ELISA. Oncostatin M (OSM), a selected myokine, was added to HSC-3-M3 cells, after which the cell proliferation ability, cell cycle, and protein expression were analyzed in vitro. Tumor cell viability was lower (control: 100%, exercise: 75%), tumors were smaller (control: 26.2 mm3, exercise: 6.4 mm3), and survival was longer in the exercise group than in the control group in vivo. OSM inhibited HSC-3-M3 cell proliferation in a concentration-dependent manner in vitro. The addition of OSM increased the proportion of cells in the G0/G1 phase, decreased the proportion of cells in the G2/M phase, and increased the expression of the CDK inhibitors p21 and p27. These results indicate that exercise may directly inhibit the proliferation of HNSCC cell lines via OSM.

Contribution of TRPV1 receptor-expressing fibers to spinal ventral root after-discharges and mechanical hyperalgesia in a spared nerve injury (SNI) rat model.

Neuropathic pain induces allodynia and hyperalgesia. In the spared nerve injury (SNI) model, marked mechanical hyperalgesia is manifested as prolongation of the duration of paw withdrawal after pin stimulation. We have previously reported that spinal ventral root discharges (after-discharges) after cessation of noxious mechanical stimulation applied to the corresponding hindpaw were prolonged in anesthetized spinalized rats. Since these after-discharges occurred through transient receptor potential (TRP) V1-positive fibers, these fibers could contribute to mechanical hyperalgesia. Therefore, we examined whether selective deletion of TRPV1-positive fibers by resiniferatoxin, an ultrapotent TRPV1 agonist, would affect the behavioral changes and ventral root discharges in SNI rats. Mechanical allodynia in the von Frey test, mechanical hyperalgesia after pin stimulation, and enhancement of ventral root discharges, but not thermal hyperalgesia in the plantar test, appeared in Wistar rats with SNI. Mechanical hyperalgesia was abolished by treatment with resiniferatoxin, whereas mechanical allodynia was not affected. Moreover, resiniferatoxin eliminated after-discharges completely. These results show that TRPV1-positive fibers do not participate in the mechanical allodynia caused by sensitization of Aß-fibers, but contribute to the enhancement of after-discharges and mechanical hyperalgesia following SNI. It is suggested that the mechanisms responsible for generating mechanical allodynia differ from those for prolongation of mechanical hyperalgesia.

Combined treatment with basalt stone and placenta extract to improve the chronic pain and scar after breast cancer surgery: a case report.

BACKGROUND: The changes in body image caused by breast deformities and postoperative pain have a detrimental influence on the physical and mental health of patients with breast cancer. The postoperative quality of life (QOL) of these patients reduces significantly owing to the changes in the breast, an organ unique to women, that occur following breast cancer surgery. CASE PRESENTATION: This case report presents the case of a Asian woman in her early 40 s with postoperative hypertrophic scarring and contraction of the scar following mastectomy; the patient presented with decreased range of motion of the upper arm, hyperpigmentation from radiation burns, changes in breast shape, and chronic pain. The patient received a combination therapy comprising Basalt Stone Treatment and the application of horse placenta extract. As a result of a total of eight sessions conducted once every two weeks, the patient's pain and scar improved. No adverse events were observed after the therapy. CONCLUSION: Combination therapy with Basalt Stone Treatment and horse placenta extract improved the chronic pain and scar after breast cancer surgery.

Reinstating olfactory bulb-derived limbic gamma oscillations alleviates depression-like behavioral deficits in rodents.

Although the etiology of major depressive disorder remains poorly understood, reduced gamma oscillations is an emerging biomarker. Olfactory bulbectomy, an established model of depression that reduces limbic gamma oscillations, suffers from non-specific effects of structural damage. Here, we show that transient functional suppression of olfactory bulb neurons or their piriform cortex efferents decreased gamma oscillation power in limbic areas and induced depression-like behaviors in rodents. Enhancing transmission of gamma oscillations from olfactory bulb to limbic structures by closed-loop electrical neuromodulation alleviated these behaviors. By contrast, silencing gamma transmission by anti-phase closed-loop stimulation strengthened depression-like behaviors in naive animals. These induced behaviors were neutralized by ketamine treatment that restored limbic gamma power. Taken together, our results reveal a causal link between limbic gamma oscillations and depression-like behaviors in rodents. Interfering with these endogenous rhythms can affect behaviors in rodent models of depression, suggesting that restoring gamma oscillations may alleviate depressive symptoms.

Effect of spinally administered simvastatin on the formalin-induced nociceptive response in mice.

Clinical and experimental observations indicated that 3-hydroxy-3-methylglutaryl CoA reductase inhibitor statins have pleiotropic effects. The present study determined the antinociceptive property of centrally administered simvastatin on the formalin-induced nociception in the mouse. Intrathecal administration of simvastatin at doses of 0.5 - 50 nmol dose-dependently attenuated the second, but not the first, phase of the formalin-induced nociception, which was partially reversed by mevalonate (5 µmol). Intracerebroventricular injection of simvastatin (50 nmol) did not affect the formalin-induced nociception. These results suggest that simvastatin-induced antinociception is mediated by attenuation of the sensitization of spinal nociceptive transmission.

Peripheral neuropathy in the pre-diabetic state of the type 2 diabetes mouse model (TSOD mice) involves TRPV1 expression in dorsal root ganglions.

Peripheral neuropathy, which is a complication of diabetes mellitus (DM), is thought to occur in the pre-DM state, being known as impaired glucose tolerance (IGT) neuropathy, although its pathogenesis is unknown. Since it is reversible, an effective treatment at the pre-DM stage could stop the progression of peripheral neuropathy and improve patients' quality of life and reduce medical costs. We investigated the hypersensitivity to mechanical and thermal stimuli during the pre-DM state in Tsumura Suzuki Obese Diabetes (TSOD) mice, a type 2 DM mouse model. The expression pattern of the Transient Receptor Potential Vanilloid 1 (TRPV1)-positive cells in the dorsal root ganglia (DRG) was examined in TSOD mice, which showed a pre-DM state at 5-12 weeks of age and decreased mechanical and thermal nociceptive thresholds. Additionally, the size of TRPV1-positive cells in TSOD mice increased compared with that in non-diabetic controls (Tsumura Suzuki Non-Obesity; TSNO). Furthermore, the expression of TRPV1 on myelinated nerve fibers (neurofilament heavy-positive cells) had significantly increased. Thus, TSOD mice in the pre-DM state at 5-12 weeks of age could be a useful animal model of IGT neuropathy. We also hypothesized that the development of IGT neuropathy may involve a switch in TRPV1 expression from small, unmyelinated neurons to large, myelinated neurons in the DRG.

The dipeptide Phe-Phe amide attenuates signs of hyperalgesia, allodynia and nociception in diabetic mice using a mechanism involving the sigma receptor system.

BACKGROUND: Previous studies have demonstrated that intrathecal administration of the substance P amino-terminal metabolite substance P1-7 (SP1-7) and its C-terminal amidated congener induced antihyperalgesic effects in diabetic mice. In this study, we studied a small synthetic dipeptide related to SP1-7 and endomorphin-2, i.e. Phe-Phe amide, using the tail-flick test and von Frey filament test in diabetic and non-diabetic mice. RESULTS: Intrathecal treatment with the dipeptide increased the tail-flick latency in both diabetic and non-diabetic mice. This effect of Phe-Phe amide was significantly greater in diabetic mice than non-diabetic mice. The Phe-Phe amide-induced antinociceptive effect in both diabetic and non-diabetic mice was reversed by the σ1 receptor agonist (+)-pentazocine. Moreover, Phe-Phe amide attenuated mechanical allodynia in diabetic mice, which was reversible by (+)-pentazocine. The expression of spinal σ1 receptor mRNA and protein did not differ between diabetic mice and non-diabetic mice. On the other hand, the expression of phosphorylated extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 proteins was enhanced in diabetic mice. (+)-Pentazocine caused phosphorylation of ERK1 and ERK2 proteins in non-diabetic mice, but not in diabetic mice. CONCLUSIONS: These results suggest that the spinal σ1 receptor system might contribute to diabetic mechanical allodynia and thermal hyperalgesia, which could be potently attenuated by Phe-Phe amide.

Application of Kampo medicines for the palliation of cancer cachexia.

Cancer cachexia results in the discontinuation of aggressive cancer therapy, and halting its progression has a significant effect on the survival rate and quality of life of patients with cancer. Currently, there are few therapies to control or slow down the progression of cancer cachexia. Although traditional Japanese Kampo medicine is widely used to support aggressive cancer therapy, the relevant scientific evidence is limited. Additionally, Kampo medicines are based on historical experience. In recent years, there have been widespread attempts to prove the efficacy of Kampo medicines through basic research, and an increasing number of studies have clarified the mechanism of action of Kampo medicines at the molecular level. It has been proposed that the improvement of cancer cachexia by Kampo medicines might involve enhancement of feeding via the central nervous system, improvement of protein maintenance in the skeletal muscle, and suppression of inflammatory cytokine production. In particular, among Kampo medicines, tonifying formulae, called "hozai" in Japanese, have been shown to be effective in alleviating cancer cachexia. In this review, we summarize the recent progress of basic and clinical research in Kampo medicines on cancer cachexia, and introduce Kampo medicines that are expected to be attractive supportive cancer medication.

The Medial Septum as a Potential Target for Treating Brain Disorders Associated With Oscillopathies.

The medial septum (MS), as part of the basal forebrain, supports many physiological functions, from sensorimotor integration to cognition. With often reciprocal connections with a broad set of peers at all major divisions of the brain, the MS orchestrates oscillatory neuronal activities throughout the brain. These oscillations are critical in generating sensory and emotional salience, locomotion, maintaining mood, supporting innate anxiety, and governing learning and memory. Accumulating evidence points out that the physiological oscillations under septal influence are frequently disrupted or altered in pathological conditions. Therefore, the MS may be a potential target for treating neurological and psychiatric disorders with abnormal oscillations (oscillopathies) to restore healthy patterns or erase undesired ones. Recent studies have revealed that the patterned stimulation of the MS alleviates symptoms of epilepsy. We discuss here that stimulus timing is a critical determinant of treatment efficacy on multiple time scales. On-demand stimulation may dramatically reduce side effects by not interfering with normal physiological functions. A precise pattern-matched stimulation through adaptive timing governed by the ongoing oscillations is essential to effectively terminate pathological oscillations. The time-targeted strategy for the MS stimulation may provide an effective way of treating multiple disorders including Alzheimer's disease, anxiety/fear, schizophrenia, and depression, as well as pain.

GI-SleepNet: A Highly Versatile Image-Based Sleep Classification Using a Deep Learning Algorithm.

Sleep-stage classification is essential for sleep research. Various automatic judgment programs, including deep learning algorithms using artificial intelligence (AI), have been developed, but have limitations with regard to data format compatibility, human interpretability, cost, and technical requirements. We developed a novel program called GI-SleepNet, generative adversarial network (GAN)-assisted image-based sleep staging for mice that is accurate, versatile, compact, and easy to use. In this program, electroencephalogram and electromyography data are first visualized as images, and then classified into three stages (wake, NREM, and REM) by a supervised image learning algorithm. To increase its accuracy, we adopted GAN and artificially generated fake REM sleep data to equalize the number of stages. This resulted in improved accuracy, and as little as one mouse's data yielded significant accuracy. Due to its image-based nature, the program is easy to apply to data of different formats, different species of animals, and even outside sleep research. Image data can be easily understood; thus, confirmation by experts is easily obtained, even when there are prediction anomalies. As deep learning in image processing is one of the leading fields in AI, numerous algorithms are also available.