Lactobacillus Plantarum intake mitigates neuropathic pain behavior via enhancing macrophage M2 polarization in a rat model of peripheral neuropathy.

Pro-inflammatory macrophages (M1-polarized) play a crucial role in neuroinflammation and neuropathic pain following nerve injury. Redirecting macrophage polarization toward anti-inflammatory (M2-polarized) phenotypes offers a promising therapeutic strategy. Recognized for their anti-inflammatory and immunomodulatory properties, probiotics are becoming a focal point of research. This study investigated the effects of Lactobacillus plantarum on macrophage polarization, nerve protection, and neuropathic pain behavior following chronic constriction injury (CCI) of the median nerve. Rats received daily oral doses of L. plantarum for 28 days before and 14 days after CCI. Subsequently, behavioral and electrophysiological assessments were performed. The M1 marker CD86 levels, M2 marker CD206 levels, and concentrations of pro-inflammatory and anti-inflammatory cytokines in the injured median nerve were assessed. L. plantarum administration effectively reduced neuropathic pain behavior and the Firmicutes to Bacteroidetes ratio after CCI. Moreover, L. plantarum treatment increased serum short-chain fatty acids (SCFAs) levels, preserved myelination of the injured median nerve, and suppressed injury-induced discharges. In CCI rats treated with L. plantarum, there was a reduction in CD86 and pro-inflammatory cytokine levels, accompanied by an increase in CD206 and the release of anti-inflammatory cytokines. Furthermore, receptors for anti-inflammatory cytokines were localized on Schwann cells, and their expression was significantly upregulated in the injured nerves of CCI rats receiving L. plantarum. In conclusion, L. plantarum shifts macrophage phenotypes from M1 to M2 by promoting the production of SCFAs and enhancing the release of anti-inflammatory cytokines. Ultimately, this process preserves nerve fiber integrity and impedes the onset of neuropathic pain.

Djulis Hull Enhances the Efficacy of Ferric Citrate Supplementation via Restoring Normal Iron Efflux through the IL-6-Hepcidin-Ferroportin Pathway in High-Fat-Diet-Induced Obese Rats.

Obesity-related functional iron disorder remains a major nutritional challenge. We evaluated the effects of djulis hull (DH) on iron metabolism in 50% high-fat-diet-induced obese rats supplemented with ferric citrate (2 g iron/kg diet) for 12 weeks. DH supplementation (5, 10, 15% dry weight/kg diet) significantly increased serum and hepatic iron but decreased appetite hormones, body weight, hepcidin, and liver inflammation (all p < 0.05). The Spearman correlation showed that appetite hormones were negatively associated with iron but positively correlated with liver hepcidin (all p < 0.05). A Western blot analysis showed that DH significantly downregulated hepatic hepcidin through the IL-6-JAK-STAT3 and enhanced ferroportin (Fpn) via the Keap1-Nrf2 and PHD2-HIF-2α. An in vitro study revealed that major bioactive compounds of DH, hexacosanol, and squalene suppressed LPS-induced IL-6 and hepcidin but enhanced Fpn expression in activated THP-1 cells. In conclusion, DH may exert nutraceutical properties for the treatment of functional iron disorder and restoration of iron efflux may have beneficial effects on weight control.

Curcumin promotes microglial M2 polarization and suppresses chronic constriction: Injury-induced neuropathic pain in a rat model of peripheral neuropathy.

OBJECTIVES: Glia (i.e., astrocyte and microglia) activation in the central nervous system plays a critical role in developing neuropathic pain. Microglia can be activated into proinflammatory (M1) and anti-inflammatory (M2) phenotypes. Switching microglial polarization from M1 to M2 phenotypes represents a novel therapeutic strategy for neuropathic pain. Curcumin has been widely used for its anti-inflammatory and immunomodulatory effects. This study investigated effects of curcumin on astrocyte activation and microglia polarization in the cuneate nucleus (CN) and development of neuropathic pain behavior after chronic constriction injury (CCI) of the median nerve. METHODS: Rats were fed with curcumin once daily at a dose of 40, 80, or 120 mg/kg 30 min before and until 7 d after median nerve CCI. Subsequently, mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filaments and plantar tests, respectively. The levels of astrocyte marker, monoclonal glial fibrillary acidic protein; microglia marker, ionized calcium-binding adapter molecule 1; M1 marker, CD86; and M2 marker, CD206 in the cuneate nucleus were determined. Enzyme-linked immunosorbent assay was applied to measure cytokine concentrations. RESULTS: Curcumin administration dose-dependently reduced mechanical allodynia and thermal hyperalgesia and decreased monoclonal glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 immunoreactivity in the ipsilateral cuneate nucleus after CCI. On ultrastructural observation, curcumin treatment was associated with fewer features of activated astrocytes and microglia. Furthermore, CCI rats given curcumin exhibited a decline in CD86 immunoreactivity and proinflammatory cytokine levels but an increase in CD206 immunoreactivity and release of anti-inflammatory cytokines. CONCLUSIONS: In our findings, curcumin switches microglial phenotypes from M1 to M2 by suppressing astrocytic activation, reducing proinflammatory cytokine release, promoting anti-inflammatory cytokine production, and contributing to relief of neuropathic pain.

The ADAM9/UBN2/AKR1C3 axis promotes resistance to androgen-deprivation in prostate cancer.

Metastatic and castration-resistant disease is a fatal manifestation of prostate cancer (PCa). The mechanism through which resistance to androgen deprivation in PCa is developed remains largely unknown. Our understanding of the tumor microenvironment (TME) and key signaling pathways between tumors and their TME is currently changing in light of the generation of new knowledge with regard to cancer progression. A disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) is a membranous bridge forming cell-cell and cell-matrix connections that regulate tumor aggressiveness and metastasis. However, it is not known whether ADAM9 expressed in the TME contributes to the CRPC phenotype. In this study, we aimed to investigate the expression patterns of ADAM9 in prostate cancer-associated fibroblasts (CAFs). We also intended to elucidate the effects of both stromal cell- and cancer cell-derived ADAM9 on the progression of CRPC and the implicated molecular pathways. By using both clinical specimens and cell lines, we herein showed that unlike the membrane anchored ADAM9 overexpressed by both PCa cells and prostate CAFs, the secreted isoform of ADAM9 (sADAM9) was strongly detected in CAFs, but rarely in tumor cells, and that could be a serum marker for PCa patients. We demonstrated that functionally sADAM9 are characterized as chemoattractant for the directed movement of androgen-independent PCa cells through integrin downstream FAK/AKT pathway, supporting that elevated sADAM9 by prostate CAFs could be responsible for the promotion of CRPC metastasis. Moreover, by stimulating PCa cells with sADAM9, we found that ubinuclein-2 (UBN2) expression was increased. A positive correlation of ADAM9 and UBN2 expression was observed in androgen receptor-expressing PCa cell lines and further confirmed in clinical PCa specimens. Using a genetic modification approach, we identified UBN2 as a downstream target gene of ADAM9 that is critical for the survival of androgen-dependent PCa cells in response to androgen deprivation, through the induction and effect of the aldo-keto reductase family 1 member C3 (AKR1C3). Collectively, our results reveal a novel action of ADAM9 on the transition of androgen-dependent PCa cells into an androgen-independent manner through the UBN2/AKR1C3 axis; the aforementioned action could contribute to the clinically-observed acquired androgen-deprivation therapy resistance.

High-dose ferric citrate supplementation attenuates omega-3 polyunsaturated fatty acid biosynthesis via downregulating delta 5 and 6 desaturases in rats with high-fat diet-induced obesity.

Obesity is associated with an increased risk of an iron deficiency; however, a synergistic relationship between iron and lipid homeostasis was also observed. The aim of this study was to investigate the effects of pharmacological doses of iron supplementation on omega 3 (n-3) and omega 6 (n-6) polyunsaturated fatty acids (PUFAs). Sprague-Dawley (SD) rats were fed a normal diet or a 50% high-fat diet (HFD) without or with pharmacological doses of ferric citrate (0.25, 1, or 2 g ferric iron per kg diet) for 12 weeks, and erythrocyte profiles of n-3 and n-6 PUFAs were quantitated. Ferric citrate supplementation showed dose-related effects on liver inflammation, liver iron accumulation, and increasing circulating levels of iron, erythrocyte degradation biomarkers LVV-hemorphin-7, malondialdehyde (MDA), and insulin. Obese rats supplemented with 2 g ferric iron per kg diet also had decreased levels of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and total n-3 PUFAs compared to rats fed a normal diet or HFD alone. A western blotting analysis revealed that iron-mediated downregulation of n-3 PUFA-converting enzymes (Δ5 and Δ6 desaturases) only occurred at high dosages (≥1 g ferric iron per kg diet). A Spearman correlation analysis showed that total liver iron and serum LVV-hemorphin-7 and MDA were negatively correlated with n-3 PUFAs and their converting enzymes (Δ5 and Δ6 desaturases) (all p < 0.05). In conclusion, obese rats that received high-dose ferric citrate supplementation (>1 g of ferric iron per kg diet) exhibited decreased n-3 PUFA levels via downregulation of expressions of Δ5 and Δ6 desaturase enzymes.

Early neuroimaging and ultrastructural correlates of injury outcome after neonatal hypoxic-ischaemia.

Hypoxic ischaemia encephalopathy is the major cause of brain injury in new-borns. However, to date, useful biomarkers which may be used to early predict neurodevelopmental impairment for proper commencement of hypothermia therapy is still lacking. This study aimed to determine whether the early neuroimaging characteristics and ultrastructural correlates were associated with different injury progressions and brain damage severity outcomes after neonatal hypoxic ischaemia. Longitudinal 7 T MRI was performed within 6 h, 24 h and 7 days after hypoxic ischaemia in rat pups. The brain damage outcome at 7 days post-hypoxic ischaemia assessed using histopathology and MRI were classified as mild, moderate and severe. We found there was a spectrum of different brain damage severity outcomes after the same duration of hypoxic ischaemia. The severity of brain damage determined using MRI correlated well with that assessed by histopathology. Quantitative MRI characteristics denoting water diffusivity in the tissue showed significant differences in the apparent diffusion coefficient deficit volume and deficit ratios within 6 h, at 24 h and 7 days after hypoxic ischaemia among the 3 different outcome groups. The susceptible brain areas to hypoxic ischaemia were revealed by the temporal changes in regional apparent diffusion coefficient values among three outcome groups. Within 6 h post-hypoxic ischaemia, a larger apparent diffusion coefficient deficit volume and deficit ratios and lower apparent diffusion coefficient values were highly associated with adverse brain damage outcome. In the apparent diffusion coefficient deficit areas detected early after hypoxic ischaemia which were highly associated with severe damage outcome, transmission electron microscopy revealed fragmented nuclei; swollen rough endoplasmic reticulum and degenerating mitochondria in the cortex and prominent myelin loss and axon detraction in the white matter. Taken together, different apparent diffusion coefficient patterns obtained early after hypoxic ischaemia are highly associated with different injury progression leading to different brain damage severity outcomes, suggesting the apparent diffusion coefficient characteristics may be applicable to early identify the high-risk neonates for hypothermia therapy.

Melatonin reduces neuropathic pain behavior and glial activation through MT2 melatonin receptor modulation in a rat model of lysophosphatidylcholine-induced demyelination neuropathy.

In this study, we investigated whether melatonin treatment prevents development of neuropathic pain via suppression of glial mitogen-activated protein kinases (MAPKs) activation in the cuneate nucleus (CN) in a lysophosphatidylcholine (LPC)-induced median nerve demyelination neuropathy model. Rats were fed orally with melatonin once a day at a dose of 37.5, 75, or 150 mg/kg 30 min before until 3 days after LPC treatment. Subsequently, behavioral tests were conducted on these animals, and immunohistochemistry and immunoblotting were used for qualitative and quantitative analysis of glia and MAPKs, including ERK, JNK, and p38, activation. Enzyme-linked immunosorbent assays were applied to measure pro-inflammatory cytokine responses. Furthermore, intra-CN microinjection of S26131 (MT1 receptor antagonist), 4P-PDOT (MT2 receptor antagonist), or prazosin (MT3 receptor antagonist) were performed to investigate the association between melatonin receptor subtypes and effects of melatonin on demyelination neuropathy. LPC treatment of the median nerve induced a significant increase in glial fibrillary acidic protein (GFAP; an astrocyte marker) and ED1 (an activated microglia marker) immunoreactivity in the ipsilateral CN and led to development of neuropathic pain behavior. Inspection of GFAP-immunoreactive astrocytes revealed that astrocytic hypertrophy, but not proliferation, contributed to increased GFAP immunoreactivity. Double immunofluorescence showed that both GFAP-immunoreactive astrocytes and ED1-immunoreactive microglia co-expressed p-ERK, p-JNK, and p-p38 immunoreactivity. Melatonin administration dose-dependently reduced neuropathic pain behavior, decreased glial and MAPKs activation, and diminished the release of pro-inflammatory cytokines in the ipsilateral CN after LPC treatment. Furthermore, 4P-PDOT, but not S26131 or prazosin, antagonized the therapeutic effects of melatonin. In conclusion, administration of melatonin, via its cognate MT2 receptor, inhibited activation of glial MAPKs, production of pro-inflammatory cytokines, and development of demyelination-induced neuropathic pain behavior.

Iron and Advanced Glycation End Products: Emerging Role of Iron in Androgen Deficiency in Obesity.

The literature suggests a bidirectional relationship between testosterone (T) and iron, but mechanisms underlying this relationship remain unclear. We investigated effects of iron on advanced glycation end products (AGEs) in obesity-related androgen deficiency. In total, 111 men were recruited, and iron biomarkers and N(ɛ)-(carboxymethyl)lysine (CML) were measured. In an animal study, rats were fed a 50% high-fat diet (HFD) with (0.25, 1, and 2 g ferric iron/kg diet) or without ferric citrate for 12 weeks. Obese rats supplemented with >1 g iron/kg diet had decreased testicular total T compared to HFD alone. Immunohistochemical staining showed that >1 g of ferric iron increased iron and AGE retention in testicular interstitial tissues, which is associated with increased expression of the receptor for AGEs (RAGE), tumor necrosis factor-α, and nitric oxide. Compared with normal weight, overweight/obese men had lower T levels and higher rates of hypogonadism (19% vs. 11.3%) and iron overload (29.8% vs.15.9%). A correlation analysis showed serum total T was positively correlated with transferrin saturation (r = 0.242, p = 0.007) and cathepsin D (r = 0.330, p = 0.001), but negatively correlated with red blood cell aggregation (r = -0.419, p<0.0001) and CML (r = -0.209, p < 0.05). In conclusion, AGEs may partially explain the underlying relationship between dysregulated iron and T deficiency.

Alteration in iron efflux affects male sex hormone testosterone biosynthesis in a diet-induced obese rat model.

This study was motivated by clinical observations that dysmetabolic iron overload syndrome (DIOS) and an androgen deficiency are common features observed in obese adult men; however, the molecular mechanism underlying the effects of DIOS on androgen deficiency remains to be elucidated. We established a DIOS animal model by feeding Sprague-Dawley rats an iron/fat-enriched diet (50% fat plus 0.25, 1, or 2 g ferric iron per kg diet) for 12 weeks to induce iron dysfunction (indicated by decreased tissue iron efflux) in obese rats. Obese rats fed an iron/fat-enriched diet showed decreased levels of testicular total Testosterone (T) and iron exporter ferroportin but increased levels of testicular iron and hepcidin, and these effects were more evident with a >1 g ferric iron per kg diet. A western blot analysis showed that an iron/fat-enriched diet triggered testicular endoplasmic reticular (ER) stress but decreased mitochondrion biogenesis proteins (PGC1α and TFAM) and T-converting proteins (StAR, CYP11A, and 17ß-HSD). TUNEL staining showed that >1 g ferric iron induced apoptosis mainly in germ cells and Leydig's cells. Uncontrolled testicular iron efflux may cause mitochondrial-ER dysfunction and affect T biosynthesis. Future study targeting the testicular hepcidin-ferroportin axis may offer a therapeutic tool to alleviate testicular iron retention and mitochondrial-ER stress in Leydig's cells.

Erythropoietin reduces nerve demyelination, neuropathic pain behavior and microglial MAPKs activation through erythropoietin receptors on Schwann cells in a rat model of peripheral neuropathy.

Neuroprotective effects of erythropoietin (EPO) on peripheral nerve injury remain uncertain. This study investigated the efficacy of EPO in attenuating median nerve chronic constriction injury (CCI)-induced neuropathy. Animals received an intraneural injection of EPO at doses of 1,000, 3,000, or 5,000 units/kg 15 min before median nerve CCI. Afterwards, the behavioral and electrophysiological tests were conducted. Immunohistochemistry and immunoblotting were used for qualitative and quantitative analysis of microglial and mitogen-activated protein kinases (MAPKs), including p38, JNK, and ERK, activation. Enzyme-linked immunosorbent assay and microdialysis were applied to measure pro-inflammatory cytokine and glutamate responses, respectively. EPO pre-treatment dose-dependently ameliorated neuropathic pain behavior, decreased microglial and MAPKs activation, and diminished the release of pro-inflammatory cytokines and glutamate in the ipsilateral cuneate nucleus after CCI. Moreover, EPO pre-treatment preserved myelination of the injured median nerve on morphological investigation and suppressed injury-induced discharges. We also observed that EPO receptor (EPOR) expression was up-regulated in the injured nerve after CCI. Double immunofluorescence showed that EPOR was localized to Schwann cells. Furthermore, siRNA-mediated knockdown of EPOR expression eliminated the therapeutic effects of EPO on attenuating the microglial and MAPKs activation, pro-inflammatory cytokine responses, injury discharges, and neuropathic pain behavior in CCI rats. In conclusion, binding of EPO to its receptors on Schwann cells maintains myelin integrity and blocks ectopic discharges in the injured median nerve, that in the end contribute to attenuation of neuropathic pain via reducing glutamate release from primary afferents and inhibiting activation of microglial MAPKs and production of pro-inflammatory cytokines.

Elevated galanin receptor type 2 primarily contributes to mechanical hypersensitivity after median nerve injury.

In this study, we investigated temporal changes in galanin receptor type 2 (GalR2) expression in NF200-, galanin-, neuropeptide Y (NPY)-, and neuronal nitric oxide synthase (nNOS)-like immunoreactive (LI) dorsal root ganglion (DRG) neurons after median nerve chronic constriction injury (CCI), and the effects of GalR2 on c-Fos expression in the cuneate nucleus (CN). Double immunofluorescence labeling methods were used to appraise changes in GalR2 expression in NF200-LI, galanin-LI, NPY-LI, and nNOS-LI DRG neurons after CCI. The von Frey assay was used to assess the efficiency of intraplantar administration of saline, M871 (a GalR2 antagonist), or AR-M1896 (a GalR2 agonist) on neuropathic signs of rats with CCI. The effects of alterations in c-Fos expression were assessed in all treatments. The percentage of GalR2-LI neurons in lesioned DRGs increased and peaked at 1 week after CCI. We further detected that percentages of GalR2-LI neurons labeled for NF200, galanin, NPY, and nNOS significantly increased following CCI. Furthermore, M871 remarkably attenuated tactile allodynia, but the sensation was slightly aggravated by AR-M1896 after CCI. Consequentially, after electrical stimulation of the CCI-treated median nerve, the number of c-Fos-LI neurons in the cuneate nucleus (CN) was significantly reduced in the M871 group, whereas it increased in the AR-M1896 group. These results suggest that activation of GalR2, probably through NPY or nitric oxide, induces c-Fos expression in the CN and transmits mechanical allodynia sensations to the thalamus.

Ferric Citrate Supplementation Reduces Red-Blood-Cell Aggregation and Improves CD163+ Macrophage-Mediated Hemoglobin Metabolism in a Rat Model of High-Fat-Diet-Induced Obesity.

SCOPE: In adults, >90% of the daily iron requirement is derived from macrophage-mediated heme iron, recycling from senescent red blood cells (RBCs) or free hemoglobin (Hb). Currently, the effects of pharmacological doses of iron supplementation on RBCs and heme iron recycling in obesity are unclear. METHODS AND RESULTS: Sprague Dawley rats are fed a standard diet or a 50% high-fat diet (HFD) with (0.25, 1, and 2 g of ferric iron per kg diet) or without ferric citrate supplementation for 12 weeks. Ferric iron increases hepatic iron accumulation in macrophages and hepatocyte-like cells. Compared with rats that received the standard diet, HFD-fed rats exhibit higher RBC aggregation and serum-free Hb levels but lower LVV-hemorphin-7 levels. These effects are reversed by ferric citrate supplementation. Immunofluorescent staining reveals that ferric iron increases the expression of hepatic CD163+ macrophages and heme oxygenase (HO)-1. A further analysis reveals the dose-related effects of ferric iron on hepatic globin degradation proteins (cathepsin D and glyoxalase 1), cytochrome p450 reductase expression, and HO-1 enzyme activity. CONCLUSIONS: Ferric citrate supplementation reduces RBC aggregation and improves CD163+ macrophage-mediated Hb metabolism in HFD-induced obese rats. These findings suggest that ferric citrate may be explored as an alternative treatment method for RBC dysfunction.

Effects of ferric citrate supplementation on advanced glycation end products in a rat model of streptozotocin/nicotinamide-induced diabetes.

SCOPE: Diabetes is associated with the increased risks of anemia and activation of advanced glycation end products (AGEs) and the receptor for AGEs (RAGE). However, the effects of pharmacological doses of iron supplementation on AGE metabolism are less clear. The aim was to investigate the effect of ferric citrate supplementation on AGE metabolism. METHODS AND RESULTS: Diabetes was induced in overnight starved rats by intraperitoneal injections of 40 mg/kg streptozotocin and 120 mg/kg nicotinamide. Diabetic rats were fed a standard diet or pharmacological doses of ferric citrate (0.5, 1, 2, and 3 g of ferric iron/kg diet) for 10 weeks. Ferric citrate supplementation showed a dose-related effect on the hepatic steatosis score, malondialdehyde, cathepsin D, and glyoxalase I. A Western blot analysis revealed that >1 g of ferric iron suppressed hepatic AGE receptor 1 and high-mobility group-box 1 expressions but increased heme oxygenase-1 and RAGE expressions. Further analysis showed that high doses of ferric iron triggered sterol regulatory element-binding protein 1c, p38-mitogen-activated protein kinase, and nuclear factor-κB protein expressions. CONCLUSION: Overall, the present results suggest a dose-related effect of ferric citrate supplementation on AGE metabolism, and this effect was more evident at high iron doses (>1 g of ferric iron/kg diet).

Neurosteroid Allopregnanolone Suppresses Median Nerve Injury-induced Mechanical Hypersensitivity and Glial Extracellular Signal-regulated Kinase Activation through γ-Aminobutyric Acid Type A Receptor Modulation in the Rat Cuneate Nucleus.

BACKGROUND: Mechanisms underlying neuropathic pain relief by the neurosteroid allopregnanolone remain uncertain. We investigated if allopregnanolone attenuates glial extracellular signal-regulated kinase (ERK) activation in the cuneate nucleus (CN) concomitant with neuropathic pain relief in median nerve chronic constriction injury (CCI) model rats. METHODS: We examined the time course and cellular localization of phosphorylated ERK (p-ERK) in CN after CCI. We subsequently employed microinjection of a mitogen-activated protein kinase kinase (ERK kinase) inhibitor, PD98059, to clarify the role of ERK phosphorylation in neuropathic pain development. Furthermore, we explored the effects of allopregnanolone (by mouth), intra-CN microinjection of γ-aminobutyric acid type A receptor antagonist (bicuculline) or γ-aminobutyric acid type B receptor antagonist (phaclofen) plus allopregnanolone, and allopregnanolone synthesis inhibitor (medroxyprogesterone; subcutaneous) on ERK activation and CCI-induced behavioral hypersensitivity. RESULTS: At 7 days post-CCI, p-ERK levels in ipsilateral CN were significantly increased and reached a peak. PD98059 microinjection into the CN 1 day after CCI dose-dependently attenuated injury-induced behavioral hypersensitivity (withdrawal threshold [mean ± SD], 7.4 ± 1.1, 8.7 ± 1.0, and 10.3 ± 0.8 g for 2.0, 2.5, and 3.0 mM PD98059, respectively, at 7 days post-CCI; n = 6 for each dose). Double immunofluorescence showed that p-ERK was localized to both astrocytes and microglia. Allopregnanolone significantly diminished CN p-ERK levels, glial activation, proinflammatory cytokines, and behavioral hypersensitivity after CCI. Bicuculline, but not phaclofen, blocked all effects of allopregnanolone. Medroxyprogesterone treatment reduced endogenous CN allopregnanolone and exacerbated nerve injury-induced neuropathic pain. CONCLUSIONS: Median nerve injury-induced CN glial ERK activation modulated the development of behavioral hypersensitivity. Allopregnanolone attenuated glial ERK activation and neuropathic pain via γ-aminobutyric acid type A receptors. Reduced endogenous CN allopregnanolone after medroxyprogesterone administration rendered rats more susceptible to CCI-induced neuropathy.

Lysophosphatidylcholine causes neuropathic pain via the increase of neuronal nitric oxide synthase in the dorsal root ganglion and cuneate nucleus.

In this study, we investigated the role of nitric oxide (NO) in lysophosphatidylcholine (LPC) induced peripheral neuropathy by the use of nitric oxide synthase (NOS) inhibitors and NO donor. We found that LPC treatment of the median nerve induced neuropathic pain behaviors (allodynia and hyperalgesia) and nerve demyelination. Immunohistochemistry revealed that the amounts of neuronal NOS-like immunoreative (nNOS-LI) neurons in both the dorsal root ganglion (DRG) and cuneate nucleus (CN) increased and peaked at 1 week after LPC treatment. Following electrical stimulation of the LPC-treated nerve, the number of c-Fos-LI neurons in the ipsilateral CN also increased in a dose-dependent manner following LPC injection and peaked at 1 week. Administration of L-NAME (Nω-Nitro-L-arginine methyl ester) or 7-NI (7-nitroindazole) 1 week after 4% LPC injection attenuated tactile allodynia and thermal hyperalgesia. However, the application of the NO donor S-Nitroso-N-acetylpenicillamine (SNAP) only exacerbated thermal hyperalgesia. After electrical stimulation of the LPC-treated median nerve, the number of c-Fos-LI neurons in the CN diminished in the L-NAME and 7-NI groups, but increased in the SNAP group. Taken together, our findings suggest that advanced NO made by the dramatically increased number of nNOS in the DRG and CN might be involved in the neuropathic sensation and boosted neuronal activity in the CN after LPC treatment.

Changes in GABA and GABA(B) receptor expressions are involved in neuropathy in the rat cuneate nucleus following median nerve transection.

This study examined the relationship between changes in GABA transmission and behavioral abnormalities after median nerve transection. Following unilateral median nerve transection, the percentage of GABA-like immunoreactive neurons in the cuneate nucleus and that of GABA(B) receptor-like immunoreactive neurons in the dorsal root ganglion in the injured side decreased and reached a nadir at 4 weeks after median nerve transection. Four weeks after bilateral median nerve transection and intraperitoneal application with saline, baclofen (2 mg kg⁻¹), or phaclofen (2 mg kg⁻¹) before unilateral electrical stimulation of the injured median nerve, we investigated the level of neuropeptide Y release and c-Fos expression in the stimulated side of the cuneate nucleus. The neuropeptide Y release level and the number of c-Fos-like immunoreactive neurons in the baclofen group were significantly attenuated, whereas those in the phaclofen group had increased compared to the saline group. These findings indicate that median nerve transection reduces GABA transmission, promoting injury-induced neuropeptide Y release and consequently evoking c-Fos expression in cuneate nucleus neurons. Furthermore, this study used the CatWalk method to assess behavioral abnormalities in rats following median nerve transection. These abnormalities were reversed by baclofen treatment. Overall, the results suggest that baclofen treatment block neuropeptide Y release, subsequently lessening c-Fos expression in cuneate neurons and consequently attenuating neuropathic signal transmission to the thalamus.

Decreases of glycine receptor expression induced by median nerve injury in the rat cuneate nucleus contribute to NPY release and c-Fos expression.

AIMS: This study aimed to investigate temporal changes in glycine and its receptor expressions in cuneate neurons after median nerve transection (MNT), and the effects of glycine on neuropeptide Y (NPY) release and c-Fos expression in the cuneate nucleus (CN). MAIN METHODS: Immunohistochemistry methods were used to appraise changes of glycine- and GlyR-like immunoreactive (LI) neurons in the CN after MNT. The alterations in NPY and c-Fos expressions were used to assess the effects of saline, glycine or strychnine treatment. The CatWalk method was used to assess the efficiency of glycine treatment on the neuropathic signs of rats with MNT. KEY FINDINGS: Approximately half of GlyR-LI neurons were fluorogold-labeled cuneothalamic projection neurons in the CN. Following MNT, the number of GlyR-LI neurons significantly decreased in the injured side of CN at 2 and 4 weeks, but the number of glycine-LI neurons remained unchanged. Four weeks after MNT given with electrical stimulation, strychnine significantly decreased the NPY reduction level in the stimulated side CN compared to that of the saline group. However, numbers of c-Fos-LI neurons in the glycine and strychnine groups were both significantly less than that in the saline group. But the paw print width and area in CatWalk analysis showed only a moderate recovery. SIGNIFICANCE: We conjecture that glycine increases glycine-mediated postsynaptic inhibition of cuneate neurons, and also blocks GABAergic neurons containing GlyRs which mediate presynaptic inhibition causing temperate NPY release. Consequently, the compromise results showed a weak reduction in c-Fos expression and a slight amelioration of neuropathic behaviors.

Nitric oxide implicates c-Fos expression in the cuneate nucleus following electrical stimulation of the transected median nerve.

In this study, we investigated whether nitric oxide (NO) modulated injury-induced neuropeptide Y (NPY) releasing and c-Fos expression in the cuneate nucleus (CN) after median nerve transection (MNT). We first examined the temporal changes of neuronal nitric oxide synthase (nNOS) expression in the dorsal root ganglion (DRG) and CN after MNT. Following MNT, the amounts of nNOS-like immunoreactive (nNOS-LI) neurons in the DRG and CN significantly increased as compared with those of the sham-operated rats. Furthermore, 4 weeks after MNT, the increases of nNOS-LI neurons in the DRG and CN were attenuated by pre-emptive lidocaine treatment in a dose-dependent manner. Finally, 4 weeks after MNT, pre-stimulation administration of L-NAME (N (ω)-Nitro-L: -arginine methyl ester) or 7-NI (7-nitroindazole) suppressed the amount of NPY release from the stimulated terminals and thus attenuated c-Fos expression in the CN. Our data implied that NO would modulate neuronal activity in the DRG and CN both after MNT.

Pre-emptive treatment of lidocaine attenuates neuropathic pain and reduces pain-related biochemical markers in the rat cuneate nucleus in median nerve chronic constriction injury model.

This study investigates the effects of lidocaine pre-emptive treatment on neuropathic pain behavior, injury discharges of nerves, neuropeptide Y (NPY) and c-Fos expression in the cuneate nucleus (CN) after median nerve chronic constriction injury (CCI). Behavior tests demonstrated that the pre-emptive lidocaine treatment dose dependently delayed and attenuated the development of mechanical allodynia within a 28-day period. Electrophysiological recording was used to examine the changes in injury discharges of the nerves. An increase in frequency of injury discharges was observed and peaked at postelectrical stimulation stage in the presaline group, which was suppressed by lidocaine pre-emptive treatment in a dose-dependent manner. Lidocaine pretreatment also reduced the number of injury-induced NPY-like immunoreactive (NPY-LI) fibers and c-Fos-LI neurons within the CN in a dose-dependent manner. Furthermore, the mean number of c-Fos-LI neurons in the CN was significantly correlated to the NPY reduction level and the sign of mechanical allodynia following CCI.

Early expression of injury-induced neuropeptide Y in primary sensory neurons and the cuneate nucleus in diabetic rats with median nerve transection.

In this study we examined the temporal changes in neuropeptide Y (NPY) expression in dorsal root ganglion (DRG) neurons and cuneate nucleus (CN) in streptozotocin (STZ)-induced diabetic rats with or without median nerve transection (MNT). Numerous NPY-like immunoreactive (NPY-LI) neurons and fibers were detected in the DRG and CN of the diabetic MNT (DMNT) rats respectively, but not in those with diabetes-alone. Following MNT, the time-course of NPY expression pattern in the diabetic DRG and CN was similar and both peaked at 2 weeks, which was earlier than those in the non-diabetic MNT rats. Consequently, the expression of neurotrophin-3 (NT-3) immunoreactivity in DRG neurons was coincidentally decreased and reached the nadir at 2 weeks in the diabetic MNT rats, which was also earlier than that in the non-diabetic MNT rats. Following electrical stimulation of the injured nerves, the number of NPY-LI fibers became attenuated and the induced c-Fos-LI cells concurrently appeared in the ipsilateral CN. In the diabetic CN, the number of c-Fos-LI cells also peaked at 2 weeks after MNT, which was consistent with the temporal pattern of changes in NPY expression. The results suggest that in diabetes, MNT induced NPY expression via the reduction of NT-3, and electrical stimulation of the injured median nerve evoked the release of NPY and accordingly more c-Fos-LI cells were identified in the CN. Furthermore, this study demonstrated early NPY and c-Fos expression in the diabetic rats after MNT, suggesting that the development of neuropathic signs may be advanced in hyperglycemic rats.