[Efficacy Analysis of Bendamustine-Based Combination Regimen in Treatment of Patients with Relapsed/Refractory Non-Hodgkin Lymphoma].

OBJECTIVE: To investigate the efficacy and safety of bendamustine combined with gemcitabine, vinorelbine，glucocorticoids (BeGEV)±X regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma. METHODS: A total of 18 relapsed/ refractory non-Hodgkin lymphoma patients at the age of 18 years or older hospitalized in the First People's Hospital of Changzhou from March 2020 to March 2021 were selected. They received two or more cycles of BeGEV±X regimen. X could be anti-CD20 monoclonal antibody, PD-1-blocking antibodies, lenalidomide, BTK inhibitor, Bcl-2 inhibitor and so on according to patients' disease feature. The clinical efficacy and adverse effects were observed. RESULTS: In total, 18 patients completed two or more cycles of BeGEV±X regimen, including 14 with diffuse large B-cell lymphoma, one with low-grade follicular lymphoma, one with follicular lymphoma grade 3b, one with angioimmunoblastic T-cell lymphoma and one with peripheral T-cell lymphoma, not otherwise specified. 11 patients were male. The median age of the patients was 64 years old. 17 patients had modified Ann Arbor stage Ⅲ/Ⅳ disease. 13 patients had high- intermediate risk or high risk IPI score, while 15 patients had high-intermediate high risk or high risk NCCN-IPI score. 14 cases had extranodal sites of disease. And 6 cases had bulky disease. 12 patients experienced refractory disease, while 8 patients had received 3 line or more prior treatment. After two or three cycles of chemotherapy, the complete response rate was 6/18, the partial response rate was 3/18, and the objective response rate was 9/18. From the beginning of salvage chemotherapy to the end of follow-up, the median progression-free survival time was 130 days, and the median overall survival was 152 days. The most common grade 3 to 4 adverse events were hematologic toxicities, infection and febrile neutropenia. CONCLUSION: BeGEV±X is an effective salvage regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma, while adverse events such as hematologic toxicities and infection should be closely monitored.

Access management strategy of large medical equipment based on the government function reform and market development effect / 中国卫生政策研究

Based on the relationship between government and market,this paper analyzed the development process of large medical equipment configuration management strategy,and forecasted the trend of management.It is found that the development process of large equipment management in China can be divided into four stages:sprouting stage,management window period,growth stage,and transition period.The management model of the four stages,respectively,are integrated management,market-oriented management,quantitative control-based comprehensive supervision,and legal supervision.As per the analysis of this work,it was found that the sprouting of the regulation of industry standards,training professionals,management efficiency are low;market-oriented led to rapid growth in the number of equipment,with a low configuration efficiency;growth period of the number of regulatory norms of the market and equipment is reasonable to use;and the legal supervision in transition is taking shape.Currently,the lack of rational allocation of large-scale equipment in the hospitals is the main factor leading to market failure.In the legal framework,the government intervention with configuration permits is the major large-scale equipment configuration management model in the future.

Access management strategy of large medical equipment based on the government function reform and market development effect / 中国卫生政策研究

Based on the relationship between government and market,this paper analyzed the development process of large medical equipment configuration management strategy,and forecasted the trend of management.It is found that the development process of large equipment management in China can be divided into four stages:sprouting stage,management window period,growth stage,and transition period.The management model of the four stages,respectively,are integrated management,market-oriented management,quantitative control-based comprehensive supervision,and legal supervision.As per the analysis of this work,it was found that the sprouting of the regulation of industry standards,training professionals,management efficiency are low;market-oriented led to rapid growth in the number of equipment,with a low configuration efficiency;growth period of the number of regulatory norms of the market and equipment is reasonable to use;and the legal supervision in transition is taking shape.Currently,the lack of rational allocation of large-scale equipment in the hospitals is the main factor leading to market failure.In the legal framework,the government intervention with configuration permits is the major large-scale equipment configuration management model in the future.

Allogeneic hemopietic stem cell transplants for the treatment of B cell acute lymphocytic leukemia.

OBJECTIVE: Explore the feasibility of allo- hemopietic stem cell transplants in treating patients with B cell acute lymphocytic leukemia. METHODS: Between september 2006 and February 2011, fifteen patients with B cell acute lymphocytic leukemia (ALL) were treated by allo-hemopietic stem cell transplants (HSCT). Stem cell sources were peripheral blood. Six patients were conditioned by busulfan (BU) and cyclophosphamide (CY) and nine patients were conditioned with TBI and cyclophosphamide (CY). Graft versus host disease (GVHD) prophylaxis regimen consisted of cyclosporine A (CSA), methotrex ate (MTX) and mycophenolatemofetil (MMF). RESULTS: Patients received a median of 7.98×108·kg⁻¹ (5.36-12.30×108·kg⁻¹) mononuclear cells (MNC). The median time of ANC>0.5×109/L was day 12 (10-15), and PLT>20.0×109/L was day 13 (11-16). Extensive acute GVHD occurred in 6 (40.0%) patients, and extensive chronic GVHD was recorded in 6 (40.0%) patients. Nine patients were alive after 2.5-65 months follow-up. CONCLUSION: Allogeneic stem cell transplant could be effective in treating patients with B cell acute lymphocytic leukemia.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors.

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.

Spinal phosphinositide 3-kinase-Akt-mammalian target of rapamycin signaling cascades in inflammation-induced hyperalgesia.

Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.

Release of prostaglandin E(2) and nitric oxide from spinal microglia is dependent on activation of p38 mitogen-activated protein kinase.

BACKGROUND: The spinal release of prostaglandins (PGs), nitric oxide (NO), and cytokines has been implicated in spinal nociceptive processing. Microglia represent a possible cell of origin for these proexcitatory mediators. Spinal microglia possess Toll-like receptor 4 (TLR4) and neurokinin 1 (NK1) receptors, and both receptors play a significant role in peripheral nerve injury- and inflammation-induced spinal sensitization. Accordingly, we examined the properties of the cascades activated by the respective targets, which led to the release of PGE(2) and an increase in nitrite (NO(2)(-)) (a marker of NO) from cultured rat spinal microglia. METHODS: Spinal microglia isolated from Sprague-Dawley neonatal rats were cultured with lipopolysaccharide (LPS) or substance P (SP) alone, with LPS in combination with SP, and with LPS in the presence of each inhibitor of cyclooxygenase (COX), NO synthase 2 (NOS2) or p38 mitogen-activated protein kinase (p38), or minocycline for 24 hours and 48 hours. Concentrations of PGE(2) and NO(2)(-) in culture supernatants were measured using an enzyme immunoassay and a colorimetric assay, respectively. RESULTS: Application of LPS (a TLR4 ligand, 0.1 to 10 ng/mL) to cultured microglia produced a dose- and time-dependent increase in PGE(2) and NO(2)(-) production, whereas no effects were observed after incubation with SP (an NK1 agonist, up to 10(-5) M) alone or in combination with LPS. Antagonist studies with SC-560 (COX-1 inhibitor) and SC-236 (COX-2 inhibitor) showed that LPS-induced PGE(2) release was generated from both COX-1 and COX-2. LPS-induced NO release was suppressed by 1400W, an inhibitor of NOS2. Minocycline, an agent blocking microglial activation, and SB203580, an inhibitor of p38, both attenuated the LPS-induced PGE(2) and NO release. The 1400W, at the doses that suppressed NO release, also blocked increased PGE(2) release. CONCLUSIONS: Our findings suggest that (a) activation of spinal microglia via TLR4 but not NK1 receptors produces PGE(2) and NO release from these cells; (b) the evoked PGE(2) release is generated by both COX-1 and COX-2, and (c) the COX-PGE(2) pathway is regulated by p38 and NOS2. Taken together with our previous in vivo work, the current findings emphasize that p38 in spinal microglia is a key player in regulating production of pronociceptive molecules, such as PGE(2) and NO.

Inflammatory hyperalgesia induces essential bioactive lipid production in the spinal cord.

Lipid molecules play an important role in regulating the sensitivity of sensory neurons and enhancing pain perception, and growing evidence indicates that the effect occurs both at the site of injury and in the spinal cord. Using high-throughput mass spectrometry methodology, we sought to determine the contribution of spinal bioactive lipid species to inflammation-induced hyperalgesia in rats. Quantitative analysis of CSF and spinal cord tissue for eicosanoids, ethanolamides and fatty acids revealed the presence of 102 distinct lipid species. After induction of peripheral inflammation by intra-plantar injection of carrageenan to the ipsilateral hind paw, lipid changes in cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) signaling pathways peaked at 4 h in the CSF. In contrast, changes occurred in a temporally disparate manner in the spinal cord with LOX-derived hepoxilins followed by COX-derived prostaglandin E(2), and subsequently the ethanolamine anandamide. Systemic treatment with the mu opioid agonist morphine, the COX inhibitor ketorolac, or the LOX inhibitor nordihydroguaiaretic acid significantly reduced tactile allodynia, while their effects on the lipid metabolites were different. Morphine did not alter the lipid profile in the presence or absence of carrageenan inflammation. Ketorolac caused a global reduction in eicosanoid metabolism in naïve animals that remained suppressed following injection of carrageenan. Nordihydroguaiaretic acid-treated animals also displayed reduced basal levels of COX and 12-LOX metabolites, but only 12-LOX metabolites remained decreased after carrageenan treatment. These findings suggest that both COX and 12-LOX play an important role in the induction of carrageenan-mediated hyperalgesia through these pathways.

Role of spinal p38alpha and beta MAPK in inflammatory hyperalgesia and spinal COX-2 expression.

Pharmacological studies indicate that spinal p38 mitogen-activated protein kinase plays a role in the development of hyperalgesia. We investigated whether either the spinal isoform p38alpha or p38beta is involved in peripheral inflammation evoked pain state and increased expression of spinal COX-2. Using intrathecal antisense oligonucleotides, we show that hyperalgesia is prevented by downregulation of p38beta but not p38alpha, whereas increases in spinal COX-2 protein expression at 8 hours are mediated by both p38alpha and beta isoforms. These data suggest that early activation of spinal p38beta isoform may affect acute facilitatory processing, and both p38beta and alpha isoforms mediate temporally delayed upregulation of spinal COX-2.

Acetaminophen prevents hyperalgesia in central pain cascade.

Acetaminophen is an analgesic and antipyretic drug believed to exert its effect through interruption of nociceptive processing. In order to determine whether this effect is due to peripheral or central activity, we studied the efficacy of systemic (oral) and intrathecal (IT) application of acetaminophen in preventing the development of hyperalgesia induced through the direct activation of pro-algogenic spinal receptors. Spinal administration of substance P (SP, 30 nmol, IT) in rats produced a decreased thermal threshold, indicating centrally mediated hyperalgesia. Pretreatment of rats with oral acetaminophen (300 mg/kg), but not vehicle, significantly attenuated IT SP-induced hyperalgesia. Acetaminophen given IT also produced a dose-dependent (10-200 microg) antinociceptive effect. In addition, oral acetaminophen suppressed spinal PGE(2) release evoked by IT SP in an in vivo IT dialysis model. The ability of IT as well as oral acetaminophen to reverse this spinally initiated hyperalgesia emphasizes the likely central action and bioavailability of the systemically delivered drug. Jointly, these data argue for an important central antihyperalgesic action of acetaminophen.

Inhibition of spinal constitutive NOS-2 by 1400W attenuates tissue injury and inflammation-induced hyperalgesia and spinal p38 activation.

Nitric oxide (NO) and its synthesizing enzymes, including NO synthase-2 (NOS-2, also called inducible NOS, iNOS), have been implicated in spinal nociception. 1400W is a highly selective NOS-2 inhibitor, as compared with either NOS-1 (neuronal NOS, nNOS) or NOS-3 (endothelial NOS). Here we examined the anti-nociceptive effects of intrathecal (IT) administration of 1400W in two experimental models of hyperalgesia (formalin and carrageenan models), in addition to the effect of 1400W on stimulation-induced activation of spinal p38 mitogen-activated protein kinase (p38). IT treatment of rats with 1400W produced a dose-dependent inhibition of paw formalin-induced phase II flinches, and attenuated carrageenan-induced thermal hyperalgesia. In contrast, IT injection of a selective inhibitor of NOS-1, nNOS inhibitor-I, had no effect in either model. Furthermore, 1400W at a dose that suppressed formalin-induced flinching behavior also blocked formalin-evoked p38 phosphorylation (activation) in the spinal cord, while nNOS inhibitor-I displayed no activity. The prompt effects of IT 1400W suggest involvement of constitutively expressed NOS-2 in spinal nociception. The NOS-2 protein in rat spinal cords was undetectable by Western blotting. However, when the protein was immunoprecipitated prior to Western blotting, NOS-2-immunoreactive bands were detected in the tissues, including naïve spinal cords. The presence of constitutive spinal NOS-2 was further confirmed by reverse transcriptase-polymerase chain reaction. Taken together, the present studies suggest that constitutively expressed spinal NOS-2 mediates tissue injury and inflammation-induced hyperalgesia, and that activation of p38 is one of the downstream factors in NO-mediated signaling in the initial processing of spinal nociception.

Descending serotonergic facilitation of spinal ERK activation and pain behavior.

Serotonin (5-HT) derived from bulbo-spinal projection is released by nociceptive input into the spinal dorsal horn. Here we report that formalin injection in the paw produced pain behavior (flinching) and phosphorylation of spinal ERK1/2 (P-ERK1/2, indicating activation) in rats. Depletion of spinal 5-HT by intrathecal (IT) 5,7-DHT, a serotonergic neurotoxin, profoundly reduced formalin evoked flinching and the increase in P-ERK1/2. Ondansetron (a 5-HT3 receptor antagonist) at IT doses that inhibited flinching also attenuated spinal ERK activation. These findings reveal that primary afferent-evoked activation of spinal ERK requires the input from an excitatory 5-HT descending pathway.

Sensory neuronal phenotype in galanin receptor 2 knockout mice: focus on dorsal root ganglion neurone development and pain behaviour.

Galanin is a 29-amino-acid peptide expressed in dorsal root ganglion (DRG) neurones and spinal dorsal horn neurones. It affects pain threshold and has developmental and trophic effects. Galanin acts at three G-protein-coupled receptors, galanin receptors (GalR1-3), each expressed in the DRGs as suggested by in situ hybridization and/or reverse transcriptase-polymerase chain reaction. The GalR2 knockout (-/-) mice permit studies on the contributions of this receptor subtype to the role of galanin at the spinal level. At 1 week after sciatic nerve transection (axotomy), there were 16-20% fewer neurones in intact and contralateral DRGs of -/- mice as compared with wild-type (WT) mice. In addition, a significant neurone loss (26% reduction) was found in the ipsilateral DRGs of WT mice, whereas no further neurone loss was seen in -/- mice. Expression of several peptides has been examined after axotomy, including galanin, neuropeptide Y and two of its receptors as well as substance P, and no significant differences were found between -/- and WT mice in either ipsi- or contralateral DRGs, respectively. After thermal injury and spinal nerve ligation, onset and duration of hyperalgesia in the injured paw were similar in GalR2-/- and WT animals. Recovery from spinal nerve ligation-caused allodynia had the same kinetics in -/- and WT animals. These data are in line with earlier observations from the peripheral and central nervous system, suggesting that galanin actions mediated by GalR2 subtype are of importance in neurodevelopment and neuroprotection.

Systemic and intrathecal effects of a novel series of phospholipase A2 inhibitors on hyperalgesia and spinal prostaglandin E2 release.

Phospholipase A(2) (PLA(2)) forms are expressed in spinal cord, and inhibiting spinal PLA(2) induces a potent antihyperalgesia. Here, we examined the antihyperalgesic effects after systemic and i.t. delivery of four compounds constructed with a common motif consisting of a 2-oxoamide with a hydrocarbon tail and a four-carbon tether. These molecules were characterized for their ability to block group IVA calcium-dependent PLA(2) (cPLA(2)) and group VIA calcium-independent PLA(2) (iPLA(2)) in inhibition assays using human recombinant enzyme. The rank ordering of potency in blocking group IVA cPLA(2) was AX048 (ethyl 4-[(2-oxohexadecanoyl)amino]butanoate), AX006 (4-[(2-oxohexadecanoyl)amino]butanoic acid), and AX057 (tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate) > AX010 (methyl 4-[(2-oxohexadecanoyl)amino]butanoate) and for inhibiting group VIA iPLA(2) was AX048, AX057 > AX006, and AX010. No agent altered recombinant cyclooxygenase activity. In vivo, i.t. (30 mug) and systemic (0.2-3 mg/kg i.p.) AX048 blocked carrageenan hyperalgesia and after systemic delivery in a model of spinally mediated hyperalgesia induced by i.t. substance P (SP). The other agents were without activity. In rats prepared with lumbar i.t. loop dialysis catheters, SP evoked spinal prostaglandin E(2) (PGE(2)) release. AX048 alone inhibited PGE(2) release. Intrathecal SR141617, a cannabinoid CB1 inhibitor at doses that blocked the effects of i.t. anandamide had no effect upon i.t. AX048. These results suggest that AX048 is the first systemically bioavailable compound with a significant affinity for group IVA cPLA(2), which produces a potent antihyperalgesia. The other agents, although demonstrating enzymatic activity in cell-free assays, appear unable to gain access to the intracellular PLA(2) toward which their action is targeted.

Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.

Activation of p38 mitogen-activated protein kinase (p38) in spinal microglia is implicated in spinal nociceptive processing. Minocycline, a tetracycline derivative, displays selective inhibition of microglial activation, a function that is distinct from its antibiotic activity. In the present study we examined antinociceptive effects of intrathecal (IT) administration of minocycline in experimental models of inflammation-evoked hyperalgesia in addition to the effect of minocycline on stimulation-induced activation of p38 in spinal microglia. Intrathecal minocycline produced a dose-dependent reduction of formalin-evoked second-phase flinching behaviour in rats, and prevented thermal hyperalgesia induced by carrageenan injection into the paw. In contrast, systemic delivery (intraperitoneally) of minocycline inhibited the first but not the second phase of formalin-induced flinching, and it had no effect on carrageenan-induced hyperalgesia. Centrally mediated hyperalgesia induced by IT delivery of N-methyl-d-aspartate was completely blocked by IT minocycline. An increase in phosphorylation (activation) of p38 (P-p38) was observed in the dorsal spinal cord after carrageenan paw injection, assessed by both Western blotting and immunohistochemistry. The increased P-p38 immunoreactivity was seen primarily in microglia but also in a small population of neurons. Minocycline, at the IT dose that blocked carrageenan-induced hyperalgesia, also attenuated the increased P-p38 in microglia. In addition, minocycline suppressed lipopolysaccharide-evoked P-p38 in cultured spinal microglial cells. Taken together, these findings show that minocycline given IT produces a potent and consistent antinociception in models of tissue injury and inflammation-evoked pain, and they provide strong support for the idea that this effect is mediated by direct inhibition of spinal microglia and subsequent activation of p38 in these cells.

Prostaglandin E2 release evoked by intrathecal dynorphin is dependent on spinal p38 mitogen activated protein kinase.

Spinal dynorphin has been hypothesized to play a pivotal role in spinal sensitization. Although the mechanism of this action is not clear, several lines of evidence suggest that spinal dynorphin-induced hyperalgesia is mediated through an increase in spinal cyclooxygenase products via an enhanced N-methyl-D-aspartate (NMDA) receptor function. Spinal NMDA-evoked prostaglandin release and nociception has been linked to the activation of p38 mitogen activated protein kinase (p38). In the present work, we show that intrathecal delivery of an N-truncated fragment of dynorphin A, dynorphin A 2-17 (dyn2-17), which has no activity at opioid receptors, induced a 8-10-fold increase in phosphorylation of p38 in the spinal cord. The increase in phosphorylated p38 was detected in laminae I-IV of the dorsal horn. Moreover, confocal microscopy showed that the activation of p38 occurred in microglia, but not in neurons or astrocytes. In awake rats, prepared with chronically placed intrathecal loop dialysis catheters, the concentration of prostaglandin E2 in lumbar cerebrospinal fluid was increased 5-fold by intrathecal administration of dyn2-17. Injection of SD-282, a selective p38 inhibitor, but not PD98059, an ERK1/2 inhibitor, attenuated the prostaglanin E2 release. These data, taken together, support the hypothesis that dynorphin, independent of effects mediated by opioid receptors, has properties that can induce spinal sensitization and indicates that dyn2-17 effects may be mediated through activation of the p38 pathway. These studies provide an important downstream linkage where by dynorphin may act through a non-neuronal link to induce a facilitation of spinal nociceptive processing.

Resting and evoked spinal substance P release during chronic intrathecal morphine infusion: parallels with tolerance and dependence.

Spinal opiate analgesia is associated with presynaptic inhibition of release of excitatory neurotransmitters/neuromodulators, e.g., substance P (SP), from primary afferent terminals. Chronic intrathecal (i.t.) administration of opiates such as morphine results in an initial analgesia followed by tolerance and a state of dependence. In this study, we examined the resting and evoked neurokinin 1 receptor (NK1r) internalization, indicative of endogenous SP release, in dorsal horn neurons of the lumbar spinal cord by immunocytochemistry during chronic i.t. infusion of morphine in rats. Noxious mechanical stimulation (compression) applied to unilateral hind paw evoked a significant increase in NK1r internalization in lamina I neurons in the ipsilateral dorsal horn. Intrathecal morphine infusion (40 nmol/microl/h) for 1 day possessed similar analgesic efficacy as acute morphine and blocked compression-induced spinal NK1r internalization. After 5 days of morphine infusion, thermal escape latencies were the same as in preinfusion animals or saline-infused controls, and compression-evoked NK1r internalization was no longer suppressed. Systemic administration of naloxone to rats on day 6 of morphine infusion resulted in prominent withdrawal behaviors and a concomitant increase in NK1r internalization in dorsal horn. The naloxone-induced internalization was blocked by NK1r antagonist L-703,606 [cis-2-(diphenylmethyl)-N-[(2-iodophenyl)methyl]-1 azabicyclo[2.2.2]octan-3-amine] or pretreatment with capsaicin, confirming that the internalization is due to the endogenous SP release from the primary afferents. We conclude that inability to suppress release of excitatory neurotransmitters/neuromodulators from primary afferents by morphine after chronic exposure is an important component in spinal morphine tolerance, and excessive release from these afferents contributes to the spinal morphine withdrawal syndrome.

Inhibition by spinal mu- and delta-opioid agonists of afferent-evoked substance P release.

Opioid mu- and delta-receptors are present on the central terminals of primary afferents, where they are thought to inhibit neurotransmitter release. This mechanism may mediate analgesia produced by spinal opiates; however, when they used neurokinin 1 receptor (NK1R) internalization as an indicator of substance P release, Trafton et al. (1999) noted that this evoked internalization was altered only modestly by morphine delivered intrathecally at spinal cord segment S1-S2. We reexamined this issue by studying the effect of opiates on NK1R internalization in spinal cord slices and in vivo. In slices, NK1R internalization evoked by dorsal root stimulation at C-fiber intensity was abolished by the mu agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) (1 microM) and decreased by the delta agonist [D-Phe2,5]-enkephalin (DPDPE) (1 microM). In vivo, hindpaw compression induced NK1R internalization in ipsilateral laminas I-II. This evoked internalization was significantly reduced by morphine (60 nmol), DAMGO (1 nmol), and DPDPE (100 nmol), but not by the kappa agonist trans-(1S,2S)-3,4-dichloro-N-mathyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride (200 nmol), delivered at spinal cord segment L2 using intrathecal catheters. These doses of the mu and delta agonists were equi-analgesic as measured by a thermal escape test. Lower doses neither produced analgesia nor inhibited NK1R internalization. In contrast, morphine delivered by percutaneous injections at S1-S2 had only a modest effect on thermal escape, even at higher doses. Morphine decreased NK1R internalization after systemic delivery, but at a dose greater than that necessary to produce equivalent analgesia. All effects were reversed by naloxone. These results indicate that lumbar opiates inhibit noxious stimuli-induced neurotransmitter release from primary afferents at doses that are confirmed behaviorally as analgesic.