Peripheral-central network analysis of cancer cachexia status accompanied by the polarization of hypothalamic microglia with low expression of inhibitory immune checkpoint receptors.

While the excessive inflammation in cancer cachexia is well-known to be induced by the overproduction of inflammatory mediators in the periphery, microflora disruption and brain dysfunction are also considered to contribute to the induction of cancer cachexia. Hypothalamic microglia play a crucial role in brain inflammation and central-peripheral immune circuits via the production of inflammatory mediators. In the present study, we evaluated possible changes in excessive secretion of gut microbiota-derived endotoxin and the expression timeline of several inflammation-regulatory mediators and their inhibiting modulators in hypothalamic microglia of a mouse model of cancer cachexia following transplantation of pancreatic cancer cells. We demonstrated that the plasma level of lipopolysaccharide (LPS) was significantly increased with an increase in anaerobic bacteria, especially Firmicutes, in the gut at the late stage of tumor-bearing mice that exhibited dramatic appetite loss, sarcopenia and severe peripheral immune suppression. At the early stage, in which tumor-bearing mice had not yet displayed "cachexia symptoms", the mRNA expression of pro-inflammatory cytokines, but not of the neurodegenerative and severe inflammatory modulator lipocalin-2 (LCN2), was significantly increased, whereas at the late "cachexia stage", the level of LCN2 mRNA was significantly increased along with significant decreases in levels of inhibitory immune checkpoint receptors programmed death receptor-1 (PD-1) and CD112R in hypothalamic microglia. In addition, a high density of activated neurons in the paraventricular nucleus (PVN) of the hypothalamus region and a significant increase in corticosterone secretion were found in cachexia model mice. Related to the cachexia state, released corticosterone was clearly increased in normal mice with specific activation of PVN neurons. A marked decrease in the natural killer cell population was also observed in the spleen of mice with robust activation of PVN neurons as well as mice with cancer cachexia. On the other hand, in vivo administration of LPS in normal mice induced hypothalamic microglia with low expression of inhibitory immune checkpoint receptors. These findings suggest that the induction of cancer cachexia may parallel exacerbation of the hypothalamic inflammatory status with polarization to microglia expressed with low levels of inhibitory immune checkpoint receptors following LPS release from the gut microflora.

Chronic pain-induced astrocyte activation in the cingulate cortex with no change in neural or glial differentiation from neural stem cells in mice.

Pain pathways terminate in discrete brain areas that monitor the sensory and affective qualities of the initiating stimulus and show remarkable plasticity. Here, we found that chronic pain by sciatic nerve ligation caused a dramatic increase in glial fibrillary acidic protein (GFAP)-like immunoreactivity (IR), which is located in the dendritic astrocytes, with its expanding distribution in the cingulate cortex (CG) of mice. The branched GFAP-like IR in the CG of nerve-ligated mice was overlapped with S100beta-like IR, which is highly limited to the cell body of astrocytes, whereas there was no difference of S100beta-like IR between sham-operated and nerve-ligated mice. The number of BrdU-positive cells on the CG was not changed by sciatic nerve ligation. Furthermore, subventricular zone (SVZ)-derived neural stem cells marked by pEGFP-C1 did not migrate toward the CG after sciatic nerve ligation. In the behavioral assay, the thermal hyperalgesia observed on the ipsirateral side in nerve-ligated mice was significantly suppressed by a single pre-microinjection of a glial-modulating agent propentofylline into the CG 24 h before nerve ligation. These results suggest that chronic painful stimuli induces astrocyte activation in the CG, whereas they do not affect the cell proliferation/differentiation from neural stem cells in the CG and the migration of neural stem cells from the SVZ area. The astrocyte activation in the CG may, at least in part, contribute to the development of a chronic pain-like state following sciatic nerve ligation in mice.

Chronic pain-induced emotional dysfunction is associated with astrogliosis due to cortical delta-opioid receptor dysfunction.

It has been widely recognized that chronic pain could cause physiological changes at supraspinal levels. The delta-opioidergic system is involved in antinociception, emotionality, immune response and neuron-glia communication. In this study, we show that mice with chronic pain exhibit anxiety-like behavior and an increase of astrocytes in the cingulate cortex due to the dysfunction of cortical delta-opioid receptor systems. Using neural stem cells cultured from the mouse embryonic forebrain, astrocyte differentiation was clearly observed following long-term exposure to the selective delta-opioid receptor antagonist, naltrindole. We also found that micro-injection of either activated astrocyte or astrocyte-conditioned medium into the cingulate cortex of mice aggravated the expression of anxiety-like behavior. Our results indicate that the chronic pain process promotes astrogliosis in the cingulate cortex through the dysfunction of cortical delta-opioid receptors. This phenomenon may lead to emotional disorders including aggravated anxiety under chronic pain-like state.

Chronic pain induces anxiety with concomitant changes in opioidergic function in the amygdala.

Clinically, it has been reported that chronic pain induces depression, anxiety, and reduced quality of life. The endogenous opioid system has been implicated in nociception, anxiety, and stress. The present study was undertaken to investigate whether chronic pain could induce anxiogenic effects and changes in the opioidergic function in the amygdala in mice. We found that either injection of complete Freund's adjuvant (CFA) or neuropathic pain induced by sciatic nerve ligation produced a significant anxiogenic effect at 4 weeks after the injection or surgery. Under these conditions, the selective mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO)- and the selective delta-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-stimulated [35S]GTPgammaS binding in membranes of the amygdala was significantly suppressed by CFA injection or nerve ligation. CFA injection was associated with a significant increase in the kappa-opioid receptor agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (ICI199,441)-stimulated [35S]GTPgammaS binding in membranes of the amygdala. The intracerebroventricular administration and microinjection of a selective mu-opioid receptor antagonist, a selective delta-opioid receptor antagonist, and the endogenous kappa-opioid receptor ligand dynorphin A caused a significant anxiogenic effect in mice. We also found that thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery. In the light-dark test, the time spent in the lit compartment was not changed at 8 weeks after surgery. Collectively, the present data constitute the first evidence that chronic pain has an anxiogenic effect in mice. This phenomenon may be associated with changes in opioidergic function in the amygdala.