Erratum: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain: Erratum.

[This corrects the article DOI: 10.7150/thno.44364.].

The Causal Role of Magnesium Deficiency in the Neuroinflammation, Pain Hypersensitivity and Memory/Emotional Deficits in Ovariectomized and Aged Female Mice.

PURPOSE: Postmenopausal women often suffer from chronic pain, memory decline and mood depression. The mechanisms underlying the neuronal disorders are not fully understood, and effective treatment is still lacking. METHODS: Oral administration of magnesium-L-threonate was tested to treat the neuronal disorders in ovariectomized and aged female mice. The pain hypersensitivity, memory function and depression-like behaviors were measured with a set of behavioral tests. Western blots, immunochemistry and in situ hybridization were used to assess molecular changes. RESULTS: Chronic oral administration of magnesium-L-threonate substantially prevented or reversed the chronic pain and memory/emotional deficits in both ovariectomized and aged female mice. We found that phospho-p65, an active form of nuclear factor-kappaB, tumor necrosis factor-alpha and interleukin-1 beta were significantly upregulated in the neurons of dorsal root ganglion, spinal dorsal horn and hippocampus in ovariectomized and aged mice. The microglia and astrocytes were activated in spinal dorsal horn and hippocampus. Calcitonin gene-related peptide, a marker for peptidergic C-fibers, was upregulated in dorsal horn, which is associated with potentiation of C-fiber-mediated synaptic transmission in the model mice. In parallel with neuroinflammation and synaptic potentiation, free Mg2+ levels in plasma, cerebrospinal fluid and in dorsal root ganglion neurons were significantly reduced. Oral magnesium-L-threonate normalized the neuroinflammation, synaptic potentiation and Mg2+ deficiency, but did not affect the estrogen decline in ovariectomized and aged mice. Furthermore, in cultured dorsal root ganglion neurons, estrogen at physiological concentration elevated intracellular Mg2+, and downregulated phospho-p65, tumor necrosis factor-alpha and interleukin-1 beta exclusively in the presence of extracellular Mg2+. CONCLUSION: Estrogen decline in menopause may cause neuroinflammation by reducing intracellular Mg2+ in neurons, leading to chronic pain, memory/emotional deficits. Supplement Mg2+ by oral magnesium-L-threonate may be a novel approach for treating menopause-related neuronal disorders.

CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain.

Emerging clinical and experimental evidence demonstrates that neuroinflammation plays an important role in cognitive impairment associated with neuropathic pain. However, how peripheral nerve challenge induces remote inflammation in the brain remains largely unknown. Methods: The circulating leukocytes and plasma C-X-C motif chemokine 12 (CXCL12) and brain perivascular macrophages (PVMs) were analyzed by flow cytometry, Western blotting, ELISA, and immunostaining in spared nerve injury (SNI) mice. The memory function was evaluated with a novel object recognition test (NORT) in mice and with Montreal Cognitive Assessment (MoCA) in chronic pain patients. Results: The classical monocytes and CXCL12 in the blood, PVMs in the perivascular space, and gliosis in the brain, particularly in the hippocampus, were persistently increased following SNI in mice. Using the transgenic CCR2RFP/+ and CX3CR1GFP/+ mice, we discovered that at least some of the PVMs were recruited from circulating monocytes. The SNI-induced increase in hippocampal PVMs, gliosis, and memory decline were substantially prevented by either depleting circulating monocytes via intravenous injection of clodronate liposomes or blockade of CXCL12-CXCR4 signaling. On the contrary, intravenous injection of CXCL12 at a pathological concentration in naïve mice mimicked SNI effects. Significantly, we found that circulating monocytes and plasma CXCL12 were elevated in chronic pain patients, and both of them were closely correlated with memory decline. Conclusion: CXCL12-mediated monocyte recruitment into the perivascular space is critical for neuroinflammation and the resultant cognitive impairment in neuropathic pain.

Chronic Oral Administration of Magnesium-L-Threonate Prevents Oxaliplatin-Induced Memory and Emotional Deficits by Normalization of TNF-α/NF-κB Signaling in Rats.

Antineoplastic drugs such as oxaliplatin (OXA) often induce memory and emotional deficits. At present, the mechanisms underlying these side-effects are not fully understood, and no effective treatment is available. Here, we show that the short-term memory deficits and anxiety-like and depression-like behaviors induced by intraperitoneal injections of OXA (4 mg/kg per day for 5 consecutive days) were accompanied by synaptic dysfunction and downregulation of the NR2B subunit of N-methyl-D-aspartate receptors in the hippocampus, which is critically involved in memory and emotion. The OXA-induced behavioral and synaptic changes were prevented by chronic oral administration of magnesium-L-threonate (L-TAMS, 604 mg/kg per day, from 2 days before until the end of experiments). We found that OXA injections significantly reduced the free Mg2+ in serum and cerebrospinal fluid (from ~ 0.8 mmol/L to ~ 0.6 mmol/L). The Mg2+ deficiency (0.6 mmol/L) upregulated tumor necrosis factor (TNF-α) and phospho-p65 (p-p65), an active form of nuclear factor-kappaB (NF-κB), and downregulated the NR2B subunit in cultured hippocampal slices. Oral L-TAMS prevented the OXA-induced upregulation of TNF-α and p-p65, as well as microglial activation in the hippocampus and the medial prefrontal cortex. Finally, similar to oral L-TAMS, intracerebroventricular injection of PDTC, an NF-κB inhibitor, also prevented the OXA-induced memory/emotional deficits and the changes in TNF-α, p-p65, and microglia. Taken together, the activation of TNF-α/NF-κB signaling resulting from reduced brain Mg2+ is responsible for the memory/emotional deficits induced by OXA. Chronic oral L-TAMS may be a novel approach to treating chemotherapy-induced memory/emotional deficits.

Bulleyaconitine A Inhibits Itch and Itch Sensitization Induced by Histamine and Chloroquine.

Itch (pruritus), specifically chronic itch associated with disease conditions, significantly impairs the patient's quality of life. At present, the mechanisms underlying this aversive experience are still unclear, and the effective treatment of itch is largely unmet. Here, we report that intragastrical administration of bulleyaconitine A (BLA), which has been used for treating chronic pain for 30 years in China, inhibited itch-like behaviors induced by intradermal injection of histamine and chloroquine in mice and rats, dose-dependently. We found that a single application of the pruritic agents at the skin region innervated by the sural nerve induced long-term potentiation (LTP) of C-fiber field potentials evoked by the stimulation of the same nerve in the spinal dorsal horn of rats. The spinal LTP was remarkably reversed by the spinal application of either BLA or gastrin-releasing peptide receptor (GRPR) antagonist (PD176252). The effect of PD176252 was completely occluded by BLA, while the effect of BLA was only partially occluded by PD176252. Repetitive injection (daily, for four days) of either histamine or chloroquine in the back of the neck enhanced scratching behaviors progressively, and the itch sensitization persisted for at least one week after the discontinuation of the injections. The behavioral change was accompanied with the potentiation of C-fiber synaptic transmission in the dorsal horn. Both the itch sensitization and synaptic potentiation were substantially attenuated by intragastrical BLA. Together, BLA was effective in inhibiting histamine-dependent and histamine-independent itches, and the mechanisms underlying these effects were involved but not limited to the inhibition of gastrin-releasing peptide (GRP)-GRPR signaling in the spinal dorsal horn.

[A modified protocol of mouse hippocampal primary microglia culture by using manual dissociation, magnetic activated cell sorting and TIC medium].

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-ß, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1ß and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.

Microglia Are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain.

Spinal long-term potentiation (LTP) at C-fiber synapses is hypothesized to underlie chronic pain. However, a causal link between spinal LTP and chronic pain is still lacking. Here, we report that high-frequency stimulation (HFS; 100 Hz, 10 V) of the mouse sciatic nerve reliably induces spinal LTP without causing nerve injury. LTP-inducible stimulation triggers chronic pain lasting for more than 35 days and increases the number of calcitonin gene-related peptide (CGRP) terminals in the spinal dorsal horn. The behavioral and morphological changes can be prevented by blocking NMDA receptors, ablating spinal microglia, or conditionally deleting microglial brain-derived neurotrophic factor (BDNF). HFS-induced spinal LTP, microglial activation, and upregulation of BDNF are inhibited by antibodies against colony-stimulating factor 1 (CSF-1). Together, our results show that microglial CSF1 and BDNF signaling are indispensable for spinal LTP and chronic pain. The microglia-dependent transition of synaptic potentiation to structural alterations in pain pathways may underlie pain chronicity.

Differential regulation of GSK-3ß in spinal dorsal horn and in hippocampus mediated by interleukin-1beta contributes to pain hypersensitivity and memory deficits following peripheral nerve injury.

Accumulating evidence shows that inhibition of glycogen synthase kinase-3beta (GSK-3ß) ameliorates cognitive impairments caused by a diverse array of diseases. Our previous work showed that spared nerve injury (SNI) that induces neuropathic pain causes short-term memory deficits. Here, we reported that GSK-3ß activity was enhanced in hippocampus and reduced in spinal dorsal horn following SNI, and the changes persisted for at least 45 days. Repetitive applications of selective GSK-3ß inhibitors (SB216763, 5 mg/kg, intraperitoneally, three times or AR-A014418, 400 ng/kg, intrathecally, seven times) prevented short-term memory deficits but did not affect neuropathic pain induced by SNI. Surprisingly, we found that the repetitive SB216763 or AR-A014418 induced a persistent pain hypersensitivity in sham animals. Mechanistically, both ß-catenin and brain-derived neurotrophic factor (BDNF) were upregulated in spinal dorsal horn but downregulated in hippocampus following SNI. Injections of SB216763 prevented the BDNF downregulation in hippocampus but enhanced its upregulation in spinal dorsal horn in SNI rats. In sham rats, SB216763 upregulated both ß-catenin and BDNF in spinal dorsal horn but affect neither of them in hippocampus. Finally, intravenous injection of interleukin-1beta that induces pain hypersensitivity and memory deficits mimicked the SNI-induced the differential regulation of GSK-3ß/ß-catenin/BDNF in spinal dorsal horn and in hippocampus. Accordingly, the prolonged opposite changes of GSK-3ß activity in hippocampus and in spinal dorsal horn induced by SNI may contribute to memory deficits and neuropathic pain by differential regulation of BDNF in the two regions. GSK-3ß inhibitors that treat cognitive disorders may result in a long-lasting pain hypersensitivity.

Topical delivery of l-theanine ameliorates TPA-induced acute skin inflammation via downregulating endothelial PECAM-1 and neutrophil infiltration and activation.

l-theanine, the most abundant free amino acid in tea, has been documented to possess many different bioactive properties through oral or intragastrical delivery. However, little is known about the effect of topical delivery of l-theanine on acute inflammation. In the present study, by using 12-O-tetradecanoylphorbol-13-acetate (TPA, 2.5 µg/ear)-induced ear edema model in mice, we first found that single-dose local pretreatment of l-theanine 30 min before TPA time- and dose-dependently suppressed the increases in both skin thickness and weight. Subsequently l-theanine ameliorated TPA-induced erythema, vascular permeability increase, epidermal and dermal hyperplasia, neutrophil infiltration and activation via downregulating the expression of PECAM-1 (a platelet endothelial adhesion molecule-1) in blood vessels and the production of pro-inflammatory cytokines IL-1ß, TNF-α, and mediator cyclooxygenase-2 (COX-2), which is mainly expressed in neutrophils. It highlighted the potential of l-theanine as a locally administrable therapeutic agent for acute cutaneous inflammation.

Interleukin-1ß overproduction is a common cause for neuropathic pain, memory deficit, and depression following peripheral nerve injury in rodents.

BACKGROUND: Chronic pain is often accompanied by short-term memory deficit and depression. Currently, it is believed that short-term memory deficit and depression are consequences of chronic pain. Here, we test the hypothesis that the symptoms might be caused by overproduction of interleukin-1beta (IL-1ß) in the injured nerve independent of neuropathic pain following spared nerve injury in rats and mice. RESULTS: Mechanical allodynia, a behavioral sign of neuropathic pain, was not correlated with short-term memory deficit and depressive behavior in spared nerve injury rats. Spared nerve injury upregulated IL-1ß in the injured sciatic nerve, plasma, and the regions in central nervous system closely associated with pain, memory and emotion, including spinal dorsal horn, hippocampus, prefrontal cortex, nucleus accumbens, and amygdala. Importantly, the spared nerve injury-induced memory deficits, depressive, and pain behaviors were substantially prevented by peri-sciatic administration of IL-1ß neutralizing antibody in rats or deletion of IL-1 receptor type 1 in mice. Furthermore, the behavioral abnormalities induced by spared nerve injury were mimicked in naïve rats by repetitive intravenous injection of re combinant rat IL-1ß (rrIL-1ß) at a pathological concentration as determined from spared nerve injury rats. In addition, microglia were activated by both spared nerve injury and intravenous injection of rrIL-1ß and the effect of spared nerve injury was substantially reversed by peri-sciatic administration of anti-IL-1ß. CONCLUSIONS: Neuropathic pain was not necessary for the development of cognitive and emotional disorders, while the overproduction of IL-1ß in the injured sciatic nerve following peripheral nerve injury may be a common mechanism underlying the generation of neuropathic pain, memory deficit, and depression.