[Immune evasion mechanism and its clinical application value in Hodgkin's lymphoma].

Hodgkin's lymphoma (HL) is a unique malignancy in which rare malignant Hodgkin and Reed-Sternberg (HRS) cells are scattered in the inflammatory cell rich microenvironment. This extensive but ineffective inflammatory cell infiltrate indicates that HRS cells have developed mechanisms to evade immune surveillance. The immune escape mechanisms of HL provide prognostic biomarkers and opportunities to develop new drugs. The immune evasion mechanisms in Hodgkin lymphoma include a reduction of human leukocyte antigen (HLA) to affect first signal which is essential for T cell activation; an upregulation of negative co-stimulatory molecules to inhibit T cell activation; resistance to apoptosis or killing by expressing some molecules on HRS cells membrane; an immunosuppressive network formed by HRS cells regulating the microenvironment immune cells. Immune escape mechanisms of HRS cells provide new targets for the development of new drug and the new drug development strategies include drugs on HRS cells and drugs on microenvironment.

Role of spinal cyclooxygenase-2 and prostaglandin E2 in fentanyl-induced hyperalgesia in rats.

BACKGROUND: Accumulated evidence suggests that spinal cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) may be implicated in the development of opioid-induced hyperalgesia. METHODS: Rats received subcutaneous fentanyl injections at different doses (20-80 µg kg-1), four separate times at 15-min intervals. Some rats only received fentanyl (60 µg kg-1 × 4 doses) with or without surgical incision. Fentanyl-induced hyperalgesia was evaluated via a tail-pressure or paw-withdrawal test. The concentrations of spinal COX-2, EP-1 receptor (EP-1R) mRNA, and PGE2 were measured. The effects of the COX-2 inhibitor, parecoxib (intraperitoneal 10 mg kg-1), or the EP-1R antagonist, SC51089 (intraperitoneal 100 µg kg-1), on hyperalgesia and spinal PGE2 were examined. RESULTS: Acute repeated injections of fentanyl dose-dependently induced mechanical hyperalgesia, which reached a peak at the 1st day and persisted for 1-4 days postinjection. This hyperalgesia could be partly or totally prevented by the pretreatment of either parecoxib or SC51089. Consistently, the levels of spinal COX-2 mRNA and PGE2 were also dose-dependently increased, reaching a peak at the first day and persisting for 2 days postinjection. Pretreatment with parecoxib could block the increase in spinal PGE2 and had no effects on spinal COX-2 and EP-1R mRNA. Fentanyl injection enhanced incision-induced mechanical and thermal hyperalgesia. CONCLUSIONS: Acute repeated fentanyl administration dose-dependently produced mechanical hyperalgesia and augmented surgery induced postoperative hyperalgesia. This behavioural change was paralleled with an increase in spinal COX-2 mRNA and PGE2 after fentanyl administration. Inhibition of COX-2 or blockade of EP-1R can partly or totally prevent hyperalgesia.

Interventional effects of squid ink polysaccharides on cyclophosphamide-associated testicular damage in mice.

Cyclophosphamide (CP) is a commonly used antitumour and immunosuppressive drug, but it is inevitable that the chemotherapeutic agent may cause long-term or permanent reproductive damage on young male patients through inducing oxidative stress in the testes. Squid ink polysaccharides (SIP), a newly found marine glycosaminoglycon have been proved to have antioxidant capabilities and chemotherapy-protective activities on model animals in our recent investigations. This study was conducted to assess whether or not SIP could protect male mice against gonadotoxicity during CP exposure. Sexually mature male Kunming mice were allocated to one of four groups. CP was abdominally administered at dose of 15 mg/kg body weight to two groups of mice for ten weeks, once a week, one group of mice received SIP at dose of 80 mg/kg body weight by gavage for ten weeks, once a day. The other two groups comprised a vehicle treated group and an SIP treated group. Toxicity of CP and protective activity of SIP on the testes were assessed by: sperm parameters, organ index, testicular antioxidant ability, activities of marker enzymes, sex hormone content, and histopathological features. Data showed CP-induced, serious negative changes on murine sperm parameters, organ index, testicular antioxidant ability, activities of marker enzymes, sexual hormone contents, and histopathological features which were all significantly impaired by SIP. This study found that SIP were demonstrated to offer protective effects against CP-induced toxicity on testes in mice (Tab. 2, Fig. 3, Ref. 29).

Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants.

BACKGROUND: In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. OBJECTIVE: The objective of this study is to compare BDNF concentrations of subjects with loss-of-function (LOF) and gain-of-function (GOF) MC4R variants with those of controls with common sequence MC4R. METHODS: Circulating BDNF was measured in two cohorts with known MC4R sequence: 148 subjects of Pima Indian heritage ((mean±s.d.): age, 15.7±6.5 years; body mass index z-scores (BMI-Z), 1.63±1.03) and 69 subjects of Hispanic heritage (10.8±3.6 years; BMI-Z, 1.57±1.07). MC4R variants were characterized in vitro by cell surface expression, receptor binding and cyclic AMP response after agonist administration. BDNF single-nucleotide polymorphisms (SNPs) rs12291186, rs6265 and rs7124442 were also genotyped. RESULTS: In the Pima cohort, no significant differences in serum BDNF was observed for 43 LOF subjects versus 65 LOF-matched controls (age, sex and BMI matched; P=0.29) or 20 GOF subjects versus 20 GOF-matched controls (P=0.40). Serum BDNF was significantly associated with genotype for BDNF rs12291186 (P=0.006) and rs6265 (P=0.009), but not rs7124442 (P=0.99); BDNF SNPs did not interact with MC4R status to predict serum BDNF. In the Hispanic cohort, plasma BDNF was not significantly different among 21 LOF subjects, 20 GOF subjects and 28 controls (P=0.79); plasma BDNF was not predicted by BDNF genotype or BDNF-x-MC4R genotype interaction. CONCLUSIONS: Circulating BDNF concentrations were not significantly associated with MC4R functional status, suggesting that peripheral BDNF does not directly reflect hypothalamic BDNF secretion and/or that MC4R signaling is not a significant regulator of the bulk of BDNF expression in humans.

Efficacy of acupuncture in the treatment of limb dyskinesia after stroke: a systematic review and meta-analysis.

OBJECTIVE: This meta-analysis was performed to investigate the effectiveness of acupuncture in post-stroke limb movement disorders. MATERIALS AND METHODS: An electronic search of databases including MEDLINE/PubMed, Web of Science, the Cochrane database, EMBASE, CBM, CNKI, WanFang, and VIP was performed to collect randomized controlled clinical studies on acupuncture administered for post-stroke dyskinesia from inception to April 2023. Data including baseline information, Fugl-Meyer Assessment (FMA) scores, and Barthel Index (BI) were included and analyzed using the meta package in R language. RESULTS: After searching and screening, 17 pieces of literature involving 1,928 participants were included, with 962 participants in the control group and 966 in the study group. Results from the included studies suggested significant benefits provided by acupuncture to improve FMA scores and BI. In specific, incorporation of acupuncture in the treatment of post-stroke limb movement disorders significantly reduced the overall FMA scores of patients by 3.45 (95% CI: 0.22, 6.69) points, the upper extremity FMA scores by 3.63 (95% CI: 0.64, 6.62) points, the lower extremity FMA scores by 3.56 (95% CI: 1.78, 5.35) points, and BI by 7.75 (95% CI: 3.35, 12.16) points. CONCLUSIONS: Acupuncture as an adjunct to the management of post-stroke limb movement disorders contributes significantly to enhancing the motor function and quality of life of patients. However, the evidence of this study is compromised by the limited quantity of the included randomized controlled trials (RCTs) and the mediocre methodological quality. Therefore, high-quality randomized controlled trials are required to validate the benefits of acupuncture on the motor function of patients with post-stroke limb movement disorders.

Pharmacological characterization of three chicken melanocortin-3 receptor mutants.

The melanocortin-3 receptor (MC3R) is a G protein-coupled receptor and potentially important in production traits. Three naturally occurring mutations (M54L, G104S, and L151R) in chicken MC3R (cMC3R) were reported previously to be associated with production traits. Here, we inserted the full-length cMC3R coding sequence into pcDNA3.1(+) and generated the 3 mutations by site-directed mutagenesis. The total and cell surface expression of the receptors was measured by flow cytometry. We analyzed the pharmacological characteristics, including binding and cyclic adenosine monophosphate (cAMP) and mitogen-activated protein kinase (MAPK) signaling, using 6 ligands ([Nle4, D-Phe7]-α-melanocyte stimulating hormone (MSH), α-, ß-, γ-, and D-Trp8-γ-MSHs, and agouti-related peptide). All mutants had similar total and cell surface expression as the wild-type (WT) cMC3R. M54L had similar pharmacological properties as the WT cMC3R. G104S did not exhibit any specific binding but had minimal response to α-, ß-, γ-, and D-Trp8-γ-MSH, although it generated 24% WT response when stimulated by NDP-MSH. Although L151R had normal binding, the responses to agonists were reduced to approximately 25% of that of the WT. In MAPK signaling, all 3 mutants showed significantly increased agonist-stimulated phosphorylation of extracellular signal-regulated protein kinases 1/2, indicating the existence of biased signaling at G104S and L151R. In summary, our studies demonstrated that although all 3 mutations are significantly associated with production traits, only G104S and L151R had severe defects in receptor pharmacology. How M54L might cause production trait differences remains to be investigated.

Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in grass carp (Ctenopharyngodon idella).

Melanocortin-4 receptor (MC4R) plays a pivotal role in the mediation of leptin action on food intake and energy expenditure in mammals. The MC4R has also been identified in several teleosts, and its importance in the regulation of fish energy homeostasis is emerging. We herein reported on the molecular cloning, tissue distribution, and pharmacological characterization of MC4R in grass carp (Ctenopharyngodon idella), an economically and ecologically important fish. We showed that grass carp MC4R (ciMC4R) consisted of a 981 bp open reading frame encoding a protein of 326 amino acids, highly homologous (>95%) to several teleost MC4Rs. Phylogenetic and synteny analysis further indicated ciMC4R was closely related to piscine MC4Rs. Using reverse transcription PCR, we found that mc4r messenger RNA was expressed in the brain as well as various peripheral tissues in grass carp. The pharmacological properties of ciMC4R were investigated using 4 agonists, including α-melanocyte stimulating hormone (α-MSH), ß-MSH, [Nle4, D-Phe7]-MSH (NDP-MSH), and adrenocorticotropic hormone (ACTH). We showed that all 4 ligands could bind to ciMC4R and initiate dose-dependent intracellular cyclic adenosine monophosphate (cAMP) accumulation. Grass carp MC4R had the highest affinity for NDP-MSH. Both NDP-MSH and ACTH (1-24) exhibited higher potencies compared to the other 2 endogenous agonists. The ciMC4R was constitutively active, with significantly increased basal cAMP level compared with that of human MC4R (P < 0.01). The availability of ciMC4R and its pharmacologic characteristics provide a basis for future investigation of its functional roles in regulating diverse physiological processes and novel insights into understanding the mechanism of food habit transition in grass carp.

Geographical pathology of duck livers infected with duck hepatitis B virus from Chiba and Shimane in Japan and Shanghai in China.

In order to evaluate geographical differences in the liver pathology of ducks infected with duck hepatitis B virus (DHBV), ducks in Chiba and Shimane, Japan, and Shanghai, China, were investigated. The numbers (DHBV positive/negative) and the maximum age of the ducks examined were 18/10 at 19 mo, 15/1 at 3 yr 4 mo, and 72/27 at 18 mo, respectively. DHBV infection was induced experimentally in ducks from Chiba and Shimane but was present congenitally in those from Shanghai. Ducks were examined regarding liver function tests, conventional histology, immunohistology, electron microscopy, and molecular hybridization for DHBV DNA in the serum and liver. There was no significant difference between DHBV-positive and -negative ducks in bilirubin and transaminase and alkaline phosphatase activities in the sera. Histologically, while the livers of ducks from Chiba and Shimane did not show necroinflammatory (hepatitis) activity, those from Shanghai frequently did (52.5%). Necroinflammatory activity of the Shanghai ducks was present almost equally in both DHBV-positive and -negative livers. The livers of Shanghai ducks but not the other two areas often (8.3%) had ground-glass inclusions which corresponded ultrastructurally to numerous virus particles in the dilated cisternae of the proliferated endoplasmic reticulum. No advanced liver disease, such as cirrhosis or hepatocellular carcinoma, was observed. There was no significant difference in the amount of DHBV DNA in the sera or in its pattern in the liver tissue among ducks of the three areas. In addition, the livers of Chiba ducks frequently had amyloidosis, while those of Shanghai ducks were contaminated with parasites. In conclusion, DHBV infection did not appear to provoke significant hepatitis activity or advanced liver disease in the examined ducks of all three areas, and the DHBV-positive livers from Shanghai ducks showed a different morphological appearance from those of the other two areas. This variation might reflect the difference in the strain of ducks, subtypes of DHBV, environmental factors, or a combination of these influences.

Mutations in Melanocortin-3 Receptor Gene and Human Obesity.

The prevalence of obesity calls for novel therapeutic targets. The melanocortin-3 receptor (MC3R) has been increasingly recognized as an important regulator of energy homeostasis and MC3R has been intensively analyzed in molecular genetic studies for obesity-related traits. Twenty-seven MC3R mutations and two common polymorphic variants have been identified so far in different cohorts. The mutant MC3Rs demonstrate multiple defects in functional analysis and can be cataloged into different classes according to receptor life cycle based classification system. Although the pathogenic role of MC3R in human obesity remains controversial, recent findings in the noncanonical signaling pathway of MC3R mutants have provided new insights. Potential therapeutic strategies for obesity related to MC3R mutations are highlighted.

Ghrelin Receptor Mutations and Human Obesity.

Growth hormone secretagogue receptor (GHSR) was originally identified as an orphan receptor in porcine and rat anterior pituitary membranes. In 1999, GHSR was deorphanized and shown to be a receptor for ghrelin, a peptide hormone secreted from the stomach. Therefore, GHSR is also called ghrelin receptor. In addition to regulating growth hormone secretion, ghrelin receptor regulates various physiological processes, including food intake and energy expenditure, glucose metabolism, cardiovascular functions, gastric acid secretion and motility, and immune function. Several human genetic studies conducted in populations originated from Europe, Africa, South America, and East Asia identified rare mutations and single nucleotide polymorphisms that might be associated with human obesity and short stature. Functional analyses of mutant GHSRs reveal multiple defects, including cell surface expression, ligand binding, and basal and stimulated signaling. With growing understanding in the functionality of naturally occurring GHSR mutations, potential therapeutic strategies including pharmacological chaperones and novel ligands could be used to correct the GHSR mutants.

Pharmacologic analyses of four chicken melanocortin-4 receptor mutations.

The melanocortin-4 receptor (MC4R) is a critical regulator of mammalian food intake and energy expenditure, with receptor activation resulting in decreased food intake and increased energy expenditure. Recently, studies on role of MC4R in regulation of food intake have been extended to other species, such as chicken. Functional study of mutant MC4Rs is important in proving the causal link between MC4R mutation and production traits. Herein, we cloned chicken MC4R (cMC4R) complementary DNA and generated 4 mutant cMC4Rs (Q18H, G21R, S76L, and L299P) by site-directed mutagenesis and measured their expression by flow cytometry. Pharmacologic characteristics were analyzed with binding and signaling assays using 3 agonists. We showed that G21R had decreased cell surface and total expression (P < 0.05), whereas the other 3 mutants had similar total and cell surface expression levels as wild-type cMC4R. The 4 mutants had either decreased (Q18H, G21R, S76L; P < 0.05) or no (L299P) binding to radiolabeled [Nle(4), D-Phe(7)]-α-melanocyte-stimulating hormone (MSH). In signaling assays, Q18H was constitutively active. Q18H, G21R, and S76L had decreased responses to α-MSH stimulation (P < 0.05). L299P had decreased basal and ligand-stimulated signaling (P < 0.01). Nle(4), D-Phe(7)-MSH was the most potent agonist for cMC4R and therefore would be better suited for further in vivo studies. We conclude that the cloned cMC4R was a functional receptor and provided detailed functional data for these mutations, contributing to a better understanding of cMC4R variants associated with production traits.

Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III.

Whereas numerous mutations of the human lutropin receptor (hLHR) and human TSH receptor (hTSHR) have been shown to cause constitutive activation of these receptors, it has been suggested that either the hFSHR as a whole, or the i3/TM VI region of the hFSHR, is less susceptible to mutation-induced constitutive activation. However, as shown herein, substitution of a highly conserved leucine residue in transmembrane III (TM III) of the hFSHR (Leu 111.18) with arginine causes a 5-fold increase in basal cAMP in transfected cells, consistent with a strong constitutive activation of the hFSHR. Interestingly, this mutant is unresponsive to further hormonal stimulation. Substitutions of hFSHR(L460) with lysine, alanine, or aspartate show that only arginine causes constitutive activation. However, all result in decreased FSH responsiveness, suggesting a role for L460 in FSH-stimulated cAMP production by the hFSHR. Because Leu 111.18 is highly conserved in rhodopsin-like G protein-coupled receptors (GPCRs), we tested the effects of substitution of the comparable leucine in the human beta2-adrenergic receptor (hbeta2-AR). Substitution of L124 in the hbeta2-AR with arginine, lysine, or alanine resulted in constitutive activation as evidenced by increased basal levels of cAMP that could be attenuated by an inverse agonist. In all cases, isoproterenol-stimulated cAMP was unaffected. Taken altogether, our data support a model whereby Leu 111.18 may play a general role in GPCRs by stabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu 111.18 as well as the introduction of a specific residue that serves to stabilize the active state of the receptor.

Activation and up-regulation of spinal cord nitric oxide receptor, soluble guanylate cyclase, after formalin injection into the rat hind paw.

Nitric oxide synthase is expressed abundantly in the spinal cord, and nitric oxide (NO) has been shown to play important roles in the central mechanism of inflammatory hyperalgesia. However, the expression and function of the NO receptor, soluble guanylate cyclase, is not fully understood in this processing at the spinal cord level. In the present study, we report that the soluble guanylate cyclase alpha(1) subunit but not the beta(1) subunit was expressed in rat spinal cord, particularly in the dorsal horn. We showed that intrathecal administration of a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, produced a significant anti-nociception demonstrated by the decrease in the number of flinches and shakes in the formalin-induced inflammatory pain model. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal cord. During formalin-induced long-lasting inflammation, we found that the expression of the alpha(1) subunit of soluble guanylate cyclase was dramatically increased in the lumbar spinal cord on the second and fourth days after formalin injection into the dorsal side of a hind paw. Intraperitoneal pretreatment with an N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine maleate (MK-801), and a neuronal NO synthase inhibitor, 7-nitroindazole, not only significantly blocked formalin-induced secondary thermal hyperalgesia but also suppressed formalin-produced increase in the alpha(1) subunit of soluble guanylate cyclase in the spinal cord. The present results indicate that peripheral inflammation not only initially activates but also later up-regulates soluble guanylate cyclase expression via the NMDA receptor-NO signaling pathway, suggesting that soluble guanylate cyclase might be involved in the central mechanism of formalin-induced inflammatory hyperalgesia in the spinal cord.

Expression and action of cyclic GMP-dependent protein kinase Ialpha in inflammatory hyperalgesia in rat spinal cord.

Several lines of evidence have shown a role for the nitric oxide/cyclic guanosine monophosphate signaling pathway in the development of spinal hyperalgesia. However, the roles of effectors for cyclic guanosine monophosphate are not fully understood in the processing of pain in the spinal cord. The present study showed that cyclic guanosine monophosphate-dependent protein kinase Ialpha but not Ibeta was localized in the neuronal bodies and processes, and was distributed primarily in the superficial laminae of the spinal cord. Intrathecal administration of a selective inhibitor of cyclic guanosine monophosphate-dependent protein kinase Ialpha, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine, produced a significant antinociception demonstrated by the decrease in the number of flinches and shakes in the formalin test. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal dorsal horn. Moreover, cyclic guanosine monophosphate-dependent protein kinase Ialpha protein expression was dramatically increased in the lumbar spinal cord 96 h after injection of formalin into a hindpaw, which occurred mainly in the superficial laminae on the ipsilateral side of a formalin-injected hindpaw. This up-regulation of cyclic guanosine monophosphate-dependent protein kinase Ialpha expression was completely blocked not only by a neuronal nitric oxide synthase inhibitor, 7-nitroindazole, and a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but also by an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate (MK-801). The present results indicate that noxious stimulation not only initially activates but also later up-regulates cyclic guanosine monophosphate-dependent protein kinase Ialpha expression in the superficial laminae via an N-methyl-D-aspartate-nitric oxide-cyclic guanosine monophosphate signaling pathway, suggesting that cyclic guanosine monophosphate-dependent protein kinase Ialpha may play an important role in the central mechanism of formalin-induced inflammatory hyperalgesia in the spinal cord.

Expression of PSD-95/SAP90 is critical for N-methyl-D-aspartate receptor-mediated thermal hyperalgesia in the spinal cord.

PSD-95/SAP90, a molecular scaffold protein, attaches the N-methyl-D-aspartate receptor to cellular signaling pathways through PSD-95/DLG/Z0-1 domain interactions at neuronal synapses.(5,9) This suggests that PSD-95/SAP90 might be involved in many physiological and pathophysiological actions triggered via the N-methyl-D-aspartate receptor in the central nervous system. Here, we present evidence that suppression of the expression of PSD-95/SAP90 in the spinal cord significantly attenuated facilitation of the tail-flick reflex triggered through N-methyl-D-aspartate receptor activation but not baseline tail-flick reflex latency. Moreover, PSD-95/SAP90's messenger RNA and protein were enriched in the spinal cord and selectively distributed in the superficial dorsal horn, where PSD-95/SAP90 overlapped with the N-methyl-D-aspartate receptor. In spinal cord neurons, PSD-95/SAP90 interacted with the N-methyl-D-aspartate receptor subunits 2A/2B. It is indicated that activation of the N-methyl-D-aspartate receptor in spinal hyperalgesia results in association of the N-methyl-D-aspartate receptor with PSD-95/SAP90 and that PSD-95/SAP90 is required for noxious thermal hyperalgesia triggered via the N-methyl-D-aspartate receptor at the spinal cord level. The present findings may provide novel insights into the mechanisms for persistent sensitization of the somatosensory system.

Role of postsynaptic density protein-95 in the maintenance of peripheral nerve injury-induced neuropathic pain in rats.

Our previous work has demonstrated that postsynaptic density protein-95, a molecular scaffolding protein that binds and clusters N-methyl-D-aspartate receptors at neuronal synapses, plays an important role in the development of peripheral nerve injury-induced neuropathic pain. The current study further investigated the possible involvement of postsynaptic density protein-95 in the maintenance of neuropathic pain. Mechanical and thermal hyperalgesia were induced within 3 days and maintained for 15 days or longer after unilateral injury to the fifth lumbar spinal nerve. The rats injected intrathecally with postsynaptic density protein-95 antisense oligodeoxynucleotide every 24 h for 4 days from day 7 to day 10 post-surgery exhibited not only a marked decrease in spinal cord postsynaptic density protein-95 protein expression but also a significant reduction in mechanical and thermal hyperalgesia on day 11 post-surgery. The rats injected with sense oligodeoxynucleotide did not display these changes. However, in the rats without nerve injury, postsynaptic density protein-95 antisense oligodeoxynucleotide given intrathecally every 24 h for 4 days did not affect responses to mechanical and thermal stimulation. In addition, postsynaptic density protein-95 antisense oligodeoxynucleotide did not change locomotor activity of experimental animals. Our results indicate that the deficiency of postsynaptic density protein-95 protein in the spinal cord significantly attenuates nerve injury-induced mechanical and thermal hyperalgesia during both the development and maintenance of chronic neuropathic pain. These results suggest that postsynaptic density protein-95 might be involved in the central mechanisms of chronic neuropathic pain and provide a novel target for development of new pain therapies.

Knockdown of spinal cord postsynaptic density protein-95 prevents the development of morphine tolerance in rats.

The activation of spinal cord N-methyl-D-aspartate (NMDA) receptors and subsequent intracellular cascades play a pivotal role in the development of opioid tolerance. Postsynaptic density protein-95 (PSD-95), a molecular scaffolding protein, assembles a specific set of signaling proteins around NMDA receptors at neuronal synapses. The current study investigated the possible involvement of PSD-95 in the development of opioid tolerance. Opioid tolerance was induced by intrathecal injection of morphine sulfate (20 microg/10 microl) twice a day for 4 consecutive days. Co-administration of morphine twice daily and PSD-95 antisense oligodeoxynucleotide (50 microg/10 microl) once daily for 4 days not only markedly reduced the PSD-95 expression and its binding to NMDA receptors in spinal cord but also significantly prevented the development of morphine tolerance. In contrast, co-administration of morphine twice daily and PSD-95 missense oligodeoxynucleotide (50 microg/10 microl) once daily for 4 days did not produce these effects. The PSD-95 antisense oligodeoxynucleotide at the doses we used did not affect baseline response to noxious thermal stimulation or locomotor function. The present study indicates that the deficiency of spinal cord PSD-95 attenuates the development of opioid tolerance. These results suggest that PSD-95 might be involved in the central mechanisms of opioid tolerance and provide a possible new target for prevention of development of opioid tolerance.

Evidence of neuronal excitatory amino acid carrier 1 expression in rat dorsal root ganglion neurons and their central terminals.

The expression and distribution of the neuronal glutamate transporter, excitatory amino acid carrier-1 (EAAC1), are demonstrated in the dorsal root ganglion neurons and their central terminals. Reverse transcriptase-polymerase chain reaction shows expression of EAAC1 mRNA in the dorsal root ganglion. Immunoblotting analysis further confirms existence of EAAC1 protein in this region. Immunocytochemistry reveals that approximately 46.6% of the dorsal root ganglion neurons are EAAC1-positive. Most EAAC1-positive neurons are small and around 250-750 microm2 in surface area, and some co-label with calcitonin gene-related peptide (CGRP) or isolectin IB4. In the spinal cord, EAAC-1 immunoreactive small dot- or patch-like structures are mainly localized in the superficial dorsal horn, and some are positive for CGRP or labeled by isolectin IB4. Unilateral dorsal rhizotomy experiments further show that EAAC1 immunoreactivity is less intense in superficial dorsal horn on the side ipsilateral to the dorsal rhizotomy than on the contralateral side. The results indicate the presence of EAAC1 in the dorsal root ganglion neurons and their central terminals. Our findings suggest that EAAC1 might play an important role in transmission and modulation of nociceptive information via the regulation of pre-synaptically released glutamate.

Differential roles of neuronal and endothelial nitric oxide synthases during carrageenan-induced inflammatory hyperalgesia.

The present study investigated the role of neuronal nitric oxide synthase (nNOS) in carrageenan-induced inflammatory pain by combining genomic and pharmacological strategies. Intrathecal injection of the nNOS inhibitor 7-nitroindazole dose-dependently inhibited carrageenan-induced thermal hyperalgesia in both early and late phases in wild-type mice. However in nNOS knockout mice, carrageenan-induced thermal hyperalgesia remained intact in the early phase but was reduced in the late phase. Spinal Ca2+ -dependent nitric oxide synthase (NOS) activity in nNOS knockout mice was significantly lower than that in wild-type mice. Following carrageenan injection, although the spinal Ca2+ -dependent NOS activity in both wild-type and knockout mice increased, the enzyme activity in nNOS knockout mice reached a level similar to that in wild-type mice. On the other hand, no significant difference in spinal Ca2+ -independent NOS activity was noted between wild-type and nNOS knockout mice before and after carrageenan injection. Furthermore, intrathecal administration of the endothelial NOS (eNOS) inhibitor L-N5-(1-iminoethyl)-ornithinein nNOS knockout mice inhibited the thermal hyperalgesia in both early and late phases, though this inhibitor had no effect in wild-type mice. Meanwhile, Western blot showed that eNOS expression in the spinal cord of nNOS knockout mice was up-regulated compared with wild-type mice; immunohistochemical staining showed that the spinal eNOS was mainly distributed in superficial laminae of the dorsal horn. Finally, double staining with confocal analysis showed that the enhanced spinal eNOS was expressed in astrocytes, but not in neurons. Our current results indicate that nNOS plays different roles in the two phases of carrageenan-induced inflammatory pain. In this model, enhanced spinal eNOS appears to compensate for the role of nNOS in nNOS knockout mice.