Heat-killed Mycobacterium Butyricum Research Articles

Fentanyl dose-sparing in polyarthritic rats requires full agonism at 5-HT1A receptors: Comparison between NLX-112, (±)8-OH-DPAT, and buspirone.

NLX-112 (a.k.a. F13640, befiradol) is a highly selective and fully efficacious agonist at 5-hydroxytryptamine (5-HT1A) receptors. It has been shown to be robustly and potently active in nociceptive, neuropathic and traumatic pain models in rats and mice. In particular, NLX-112 decreases oral fentanyl self-administration (FSA) in polyarthritic rats, ie, it has opioid dose-sparing effects. To examine if the dose-sparing effects of NLX-112 in polyarthritic rats are shared by other 5-HT1A ligands: the prototypical 5-HT1A receptor agonist 8-HYDROXY-2-(DI-n-PROPYLAMINO)TETRALIN ((±)8-OH-DPAT), and the 5-HT1A receptor partial agonist and weak dopamine D2 receptor blocker, -buspirone. Polyarthritis was induced by inoculating rats with heat-killed Mycobacterium butyricum. They then had access to either a fentanyl (0.008 mg/mL) or a sweetened solution in their home cage. NLX-112, (±)8-OH-DPAT, or buspirone was administered via an osmotic minipump (5 µL/h) during a 2-week infusion period from day 14 to day 28 post-inoculation with Mycobacterium butyricum. Control infusions consisted of sterile 0.9 percent NaCl. NLX-112 (0.63 mg/day) significantly decreased FSA by 47 percent and increased total fluid consumption (TFC) by 7 percent (vehicle-loaded minipumps as controls). Both (±)8-OH-DPAT and buspirone (0.63 and 2.5 mg/day, respectively) failed to reduce FSA; (±)8-OH-DPAT did not modify TFC, while buspirone significantly decreased it by 17 percent. These results suggest that oral FSA dose-sparing effect, in this rat polyarthritis pain model, requires high efficacy activation of 5-HT1A receptors, such as that afforded by NLX-112. By contrast, the agonist efficacy of (±)8-OH-DPAT and buspirone seems insufficient for FSA dose-sparing.

Could omega-3 fatty acids preserve endothelial function in a rat model of rheumatoid arthritis?

Could omega-3 fatty acids preserve endothelial function in a rat model of rheumatoid arthritis? Background: Endothelial dysfunction is claimed to be the cause of increased risk for cardiovascular diseases in rheumatoid arthritis (RA) patients. Omega-3 fatty acids is a promising drug in this field; however, its exact effects on endothelial functions are not fully understood. Aim of the work: This study aimed to evaluate the effect of omega-3 fatty acids on endothelial reactivity and some markers of endothelial dysfunction [Vascular cell adhesion molecule-1 (VCAM-1), tumour necrosis factor alpha (TNF-α), Malondialdehyde (MDA) and NOS activity] in a rat model of rheumatoid arthritis. Material & methods: Thirty male rats were divided into 3 groups (control, untreated RA, and RA group with omega-3 supplementation). RA induction was done by intradermal injection of heat-killed Mycobacterium butyricum and was confirmed by clinical signs of arthritis on day 11. In the treated group, omega-3 was given daily via gastric gavage starting from day 11 until the end of the study. The study duration was 30 days for all groups starting from the day of induction. At the end of the study, rats were sacrificed, blood samples were collected for VACM-1, TNF-α, MDA and nitrite measurement and thoracic aortae were taken to test vascular reactivity. Results: All markers increased while vascular reactivity decreased significantly after RA induction. Omega-3 treatment significantly decreased all biochemical markers and restored normal vascular reactivity in the treated group.

Presynaptic glutamatergic transmission and feedback system of oxytocinergic neurons in the hypothalamus of a rat model of adjuvant arthritis.

The neurohypophysial hormone oxytocin (OXT) is synthesized in the hypothalamic paraventricular and supraoptic nuclei. Recently, some studies have considered OXT to be important in sensory modulation and that the OXT protein is upregulated by acute and chronic nociception. However, the mechanism by which OXT is upregulated in neurons is unknown. In this study, we examined the resting membrane potentials and excitatory postsynaptic currents in OXT-ergic neurons in the paraventricular nucleus in adjuvant arthritis rat model, a model of chronic inflammation, using whole-cell patch-clamping. Transgenic rats expressing OXT and monomeric red fluorescent protein 1 (mRFP1) fusion protein to visualize the OXT-ergic neurons were used, and the OXT-mRFP1 transgenic rat model of adjuvant arthritis was developed by injection of heat-killed Mycobacterium butyricum. Furthermore, the feedback system of synthesized OXT was also examined using the OXT receptor antagonist L-368,899. We found that the resting membrane potentials and frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-monomeric red fluorescent protein 1 neurons in the paraventricular nucleus were significantly increased in adjuvant arthritis rats. Furthermore, L-368,899 dose-dependently increased the frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-ergic neurons. Following bath application of the GABAA receptor antagonist picrotoxin and the cannabinoid receptor 1 antagonist AM 251, L-368,899 still increased the frequency of miniature excitatory postsynaptic currents. However, following bath application of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride, L-368,899 did not alter the miniature excitatory postsynaptic current frequency. Thus, it is suggested that OXT-ergic neuron activity is upregulated via an increase in glutamate release, and that the upregulated OXT neurons have a feedback system with released endogenous OXT. It is possible that nitric oxide, but not GABA, may contribute to the feedback system of OXT neurons in chronic inflammation.

Aminobisphosphonate (YM175) inhibits bone destruction in rat adjuvant arthritis

This study was designed to examine the effects of an aminobisphosphonate (YM175, which is also called incadronate) on bone destruction in rat adjuvant arthritis (AA). Thirty-five female Lewis rats were given an intradermal injection of heat-killed Mycobacterium butyricum and randomly allocated to five groups (seven rats/group). In the three YM175-treated (0.01, 0.1 and 1 mg/kg per day) groups, YM175 was injected subcutaneously every day from day 0 to day 42. The effects of YM175 in AA rats were evaluated according to an arthritis score, hind paw volume, and radiological and histological examinations. The results showed that YM175 suppressed the radiological and histopathological changes, as well as the joint swelling, in rat AA in a dose-dependent manner. The number of tartrate- resistant acid phosphatase (TRAP)-positive cells (osteoclasts and preosteoclasts or osteoclast precursors) in bone marrow spaces and granulation tissue in the YM175-treated groups was also reduced in a dose-dependent manner. This study provides the first evidence that YM175, among aminobisphosphonates, not only inhibits bone destruction in rat AA, probably by reducing osteoclast numbers, but that it also suppresses joint inflammation. These results suggest that YM175 may be a useful drug for the prophylactic treatment of both bone destruction and joint inflammation in patients with rheumatoid arthritis.

Proteasome inhibitor MG132 modulates inflammatory pain by central mechanisms in adjuvant arthritis

In rheumatoid arthritis (RA), pain and inflammation are initial symptoms followed by various degrees of bone and cartilage destruction. Previously, we have shown that reversible proteasome inhibitor MG132 attenuates pain and joint inflammation in a rat model of adjuvant-arthritis. Our present study aims to study the effects of MG132 on molecular changes in the dorsal root ganglia (DRG) and in the spinal cord (SC) using the same animal model. Arthritis was induced by heat-killed Mycobacterium butyricum in rats. The expression of substance P (SP) was analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in DRG and in the SC. The nuclear factor-κB (NF-κB) DNA-binding activity in the SC was analyzed by electromobility shift assay. Arthritic rats treated daily with MG132 demonstrated a marked reduction of SP gene expression in the DRG and number of SP-positive cells was reduced. In the spinal cord of arthritic rats elevated SP messenger RNA levels were normalized and NF-κB-DNA-binding activity was down-regulated in arthritic rats treated with MG132. Our results indicate that proteasome inhibitor MG132 attenuates pain in adjuvant arthritis by targeting the sensory neuropeptide substance P in the peripheral and central nervous systems. These effects may be mediated through the inhibition of NF-κB activation.

The natural stilbenoid pinosylvin and activated neutrophils: effects on oxidative burst, protein kinase C, apoptosis and efficiency in adjuvant arthritis.

To investigate the effects of the naturally occurring stilbenoid pinosylvin on neutrophil activity in vitro and in experimental arthritis, and to examine whether protein kinase C (PKC) activation served as an assumed target of pinosylvin action. Fresh human blood neutrophils were isolated. The oxidative burst of neutrophils was evaluated on the basis of enhanced chemiluminescence. Neutrophil viability was evaluated with flow cytometry, and PKC phosphorylation was assessed by Western blotting analysis. Adjuvant arthritis was induced in Lewis rats with heat-killed Mycobacterium butyricum, and the animals were administered with pinosylvin (30 mg/kg, po) daily for 21 d after arthritis induction. In isolated human neutrophils, pinosylvin (10 and 100 μmol/L) significantly decreased the formation of oxidants, both extra- and intracellularly, and effectively inhibited PKC activation stimulated by phorbol myristate acetate (0.05 μmol/L). The inhibition was not due to neutrophil damage or increased apoptosis. In arthritic rats, the number of neutrophils in blood was dramatically increased, and whole blood chemiluminescence (spontaneous and PMA-stimulated) was markedly enhanced. Pinosylvin administration decreased the number of neutrophils (from 69 671 ± 5588/μL to 51 293 ± 3947/μL, P=0.0198) and significantly reduced the amount of reactive oxygen species in blood. Pinosylvin is an effective inhibitor of neutrophil activity, and is potentially useful as a complementary medicine in states associated with persistent inflammation.

Cocoa intake attenuates oxidative stress associated with rat adjuvant arthritis

Cocoa contains flavonoids with antioxidant properties. The aim of this study was to ascertain the effect of cocoa intake on oxidative stress associated with a model of chronic inflammation such as adjuvant arthritis. Female Wistar rats were fed with a 5% or 10% cocoa-enriched diet or were given p.o. a quercetin suspension every other day for 10days. Arthritis was induced by a heat-killed Mycobacterium butyricum suspension. Reactive oxygen species (ROS) produced by macrophages, and splenic superoxide dismutase (total, cytoplasmic and mitochondrial) and catalase activities were determined. Clinically, joint swelling in arthritic rats was not reduced by antioxidants; however, the 5% cocoa diet and quercetin administration reduced ROS production. Moreover, the 5% cocoa diet normalized the activities of superoxide dismutase and catalase. In conclusion, a cocoa diet reduces the oxidative stress associated with a chronic inflammatory pathology, although it was not enough to attenuate joint swelling.

Effect of cocoa-enriched diets on lymphocytes involved in adjuvant arthritis in rats

Cocoa and its flavonoids have potential anti-inflammatory properties in vitro and in acute inflammation models in vivo. The aim of the present study was to ascertain the effects of two cocoa-enriched diets on adjuvant arthritis (AA) in rats, considering not only clinical and biochemical inflammatory indices, but also antibody response and lymphocyte composition. Female Wistar rats were fed with a 5 or 10 % cocoa-enriched diet beginning 2 weeks before arthritis induction and until the end of the study. AA was induced by an intradermal injection of heat-killed Mycobacterium butyricum suspension. The hind-paw swelling (plethysmometry), serum anti-mycobacterial antibody concentration (ELISA), blood and inguinal lymph node lymphocyte subset percentage (flow cytometry), and IL-2, interferon γ and PGE₂ released from splenocytes (ELISA) were assessed. Although the cocoa diets had no significant effect on hind-paw swelling, a tendency to reduce it was observed at the end of the study. Cocoa-enriched diets were able to decrease the serum anti-mycobacterial antibody concentration and the splenocyte PGE2 production, as well as the proportion of T-helper (Th) lymphocytes in blood and regional lymph nodes, which probably includes cells responsible for the arthritic process. The cocoa diets prevented a decrease in the proportion of regulatory T-cells in blood and a disequilibrium between inguinal lymph node natural killer (NK) CD8⁺ and NK CD8⁻ subsets. In conclusion, the cocoa-enriched diets during AA were not able to significantly decrease joint inflammation but modified Th-cell proportions and prevented specific antibody synthesis.

Attenuation of pain and inflammation in adjuvant‐induced arthritis by the proteasome inhibitor MG132

In rheumatoid arthritis (RA), pain and joint destruction are initiated and propagated by the production of proinflammatory mediators. Synthesis of these mediators is regulated by the transcription factor NF-kappaB, which is controlled by the ubiquitin proteasome system (UPS). The present study explored the effects of the proteasome inhibitor MG132 on inflammation, pain, joint destruction, and expression of sensory neuropeptides as markers of neuronal response in a rat model of arthritis. Arthritis was induced in rats by injection of heat-killed Mycobacterium butyricum. Arthritis severity was scored, and nociception was evaluated by mechanical pressure applied to the hind paw. Joint destruction was assessed by radiologic and histologic analyses. NF-kappaB DNA-binding activity was analyzed by electromobility shift assay, and changes in the expression of the p50 NF-kappaB subunit and the proinflammatory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry. Arthritic rats treated with MG132 demonstrated a marked reduction in inflammation, pain, and joint destruction. The elevated DNA-binding activity of the NF-kappaB/p50 homodimer and p50, as well as the neuronal expression of SP and CGRP, observed in the ankle joints of arthritic rats were normalized after treatment with MG132. In arthritic rats, inhibition of proteasome reduced the severity of arthritis and reversed the pain behavior associated with joint inflammation. These effects may be mediated through the inhibition of NF-kappaB activation and may possibly involve the peripheral nervous system. New generations of nontoxic proteasome inhibitors may represent a novel pharmacotherapy for RA.

Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition

Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1–100 µM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane.

Response of arginine vasopressin-enhanced green fluorescent protein fusion gene in the hypothalamus of adjuvant-induced arthritic rats

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.

Additions of Killed Whole Cell Bacteria Preparations to Freund Complete Adjuvant Alter Laying Hen Antibody Response to Soluble Protein Antigen

Passive transfer of antibodies from hen to egg has value to both the producer of commercial polyclonal egg antibody and the producer of hatching eggs. Water-in-oil emulsions are commonly amended with immune stimulants such as Mycobacteria (e.g., Freund complete adjuvant; FCA) to increase antibody production to soluble protein antigens (SPA). Recent discoveries of the mechanisms by which microbial products act as adjuvants led us to hypothesize that additions of killed whole cell bacteria (bacterins) to FCA could improve antibody responses to SPA. All injections used in each experiment were water-in-oil emulsions (50:50) containing 3 mg/mL of phospholipase A(2) (PLA(2)) immunogen. Additionally, all primary control and treatment injections contained heat-killed Mycobacterium butyricum immunogens from FCA. In addition to PLA(2) and FCA, primary treatment injections contained various microbial bacterin immunogens. Hence, the experimental treatment of all experiments was addition of a commercial source of microbial bacterin to FCA for the primary injection only. Booster injections were the same as the primary control injections except Freund incomplete adjuvant replaced FCA. Anti-body titers to PLA(2) in yolk were determined by ELISA. Bacterins tested as additives to FCA were Escherichia coli, Staphylococcus aureus, Streptococcus suis, and Corynebacterium pseudotuberculosis. Escherichia coli bacterin added to FCA decreased egg yolk antibody titer to SPA by 23% in hens of different ages and strains (P < 0.0001). In a second experiment, a 51% decrease in antibody production associated with E. coli bacterin was sustained for several weeks after the primary immunization (P = 0.003). Staphylococcus aureus or Streptococcus suis combined with FCA increased egg yolk antibody 62 and 51%, respectively (P < 0.05), and Corynebacterium pseudotuberculosis had no effect. In conclusion, the addition of bacterin to FCA can influence hen antibody response to SPA as measured in egg yolks. It is hypothesized that the difference in antibody production may be related to the composition of various pathogen associated molecular patterns in the primary injection.

Freund's complete adjuvant induces arthritis in mice lacking a functional interferon‐γ receptor by triggering tumor necrosis factor α–driven osteoclastogenesis

To investigate the hypothesis that Freund's complete adjuvant (CFA) plays an essential role in the induction of collagen-induced arthritis in mice, by testing whether CFA by itself is able to induce arthritis in interferon-gamma receptor-knockout (IFNgammaR-KO) mice. IFNgammaR-KO and wild-type mice were sensitized with a single intradermal injection of CFA containing heat-killed Mycobacterium butyricum. Flow cytometric analysis and in vitro osteoclastogenesis assays were performed on blood, spleen, and bone marrow cells. Tumor necrosis factor (TNF) levels were measured in the serum, and levels of RANKL, osteoprotegerin (OPG), and TNFalpha in the synovium were determined by quantitative reverse transcriptase-polymerase chain reaction. Effects of treatment with the TNFalpha antagonist etanercept were assessed. Symptoms of arthritis appeared in IFNgammaR-KO mice but not in wild-type mice, and reached an incidence of 55%. The onset coincided with an expansion of CD11b+ splenocytes that spontaneously produced TNFalpha and with increased osteoclastogenesis in spleen and blood cells. Expansion of CD11b+ splenocytes and osteoclast precursor cells was more pronounced in arthritic than in nonarthritic mice. There was a >100-fold increase in the RANKL:OPG ratio in the synovia of CFA-sensitized mice compared with those of naive animals. Treatment with etanercept prevented the development of arthritis and mitigated the increased expansion of myeloid cells as well as the increase in osteoclast precursor numbers in the spleen and blood. These results indicate that sensitization of mice with CFA creates a condition in which dysregulation of a single cytokine leads to arthritis by triggering TNFalpha-driven osteoclastogenesis.

Incadronate disodium inhibits joint destruction and periarticular bone loss only in the early phase of rat adjuvant-induced arthritis

Destruction of articular cartilage and subchondral bone loss in the affected joints of rat adjuvant arthritis have never been quantified histologically. This study aimed to evaluate the effect of incadronate disodium on joint destruction and periarticular bone loss, using histomorphometric measurements. Seven-week-old female Lewis rats were injected with 0.1 mg of heat-killed Mycobacterium butyricum into the tail base. Immediately after sensitization, vehicle, or incadronate at 10 or 100 microg/kg per day, was administered subcutaneously, three times per week. Hind-paw volume was measured weekly and the animals were killed at 2, 4, 6, and 10 weeks after sensitization. After taking X-rays, decalcified sagittal sections of the ankle joint were prepared and stained with toluidine blue and tartarate-resistant acid phosphatase. Articular cartilage destruction and subchondral bone loss were evaluated histomorphometrically. At 2 weeks after sensitization, no radiographic or histologic changes were observed. However, at 4 weeks, severe articular cartilage destruction and subchondral bone loss were found in the arthritic control group, while these changes were inhibited dose-dependently by incadronate treatment. At 6 and 10 weeks, both the destructive changes and the bone loss had further progressed, and they were not inhibited by incadronate treatment. Incadronate dose-dependently inhibited articular cartilage destruction and subchondral bone loss at 4 weeks after sensitization in this adjuvant arthritis model. However, the suppressive effects of incadronate did not continue until 6 and 10 weeks.

Induction of galanin-like peptide gene expression in the arcuate nucleus of the rat after acute but not chronic inflammatory stress

Galanin-like peptide (GALP) has been recently isolated from the porcine hypothalamus. The GALP mRNA is restricted to neurons in the hypothalamic arcuate nucleus (Arc) and pituicytes in the posterior pituitary gland (PP), but physiological functions of the GALP remains unclear in both areas. We examined the effects of acute and chronic inflammatory stresses on the GALP mRNA levels in the rat Arc using in situ hybridization histochemistry. Intraperitoneal (i.p.) injection of bacterial endotoxin lipopolysaccharide (LPS) caused a marked increase of the GALP mRNA levels in the Arc. The effects of i.p. injection of LPS on the GALP mRNA levels in the Arc were significantly attenuated by pretreatment with i.p. injection of indomethacin cyclooxygenase inhibitor. Adjuvant arthritis caused by subcutaneous (s.c.) injection of heat-killed Mycobacterium butyricum as chronic inflammatory stress did not affect the GALP mRNA levels in the Arc, though the GALP mRNA levels in the pituicytes of the PP were markedly increased by two peaks at 12 h and 15 days after s.c. injection of heat-killed M. butyricum. Enzymeimmunoassay showed that the plasma concentration of GALP was not affected by these inflammatory stresses. These results suggest that acute inflammatory stress might be a potent stimulant to increase the GALP mRNA levels in the Arc of the rat via synthesis of prostaglandins.

Loss of muscarinic M 4 receptors in spinal cord of arthritic rats: implications for a role of M 4 receptors in pain response

Changes in the levels of muscarinic M 4 receptors in spinal cord of acute and chronic arthritic rats (animal models of pain) were studied by receptor autoradiography using muscarinic M 4 receptor subtype selective ligand. Arthritis was induced in female Lewis rats by single intradermal injection of heat-killed Mycobacterium butyricum and sacrificed 12 days (acute group) and 30 days (chronic and control groups) after induction of arthritis. Our results demonstrate significant reduction in the level of M 4 receptors in the spinal cord (Rexed laminae I–X) of acute and chronic arthritic rats compared to controls. These findings suggest that the muscarinic M 4 receptor subtype may be involved in cholinergic mechanisms of analgesia.

Inhibition of the nucleoside transporter inhibits disease progression in the rat adjuvant‐induced arthritis model

AbstractWe investigated the in vivo effects of the nucleoside transporter inhibitor nitrobenzylmercaptopurine (NBMPR) on rat adjuvant‐induced arthritis. This agent inhibits uptake of adenosine into cells, presumably leading to increased levels of extracellular adenosine. Arthritis was induced in 7‐week‐old male Lewis rats by injection of incomplete Freund's adjuvant containing heat‐killed Mycobacterium butyricum into the base of the tail on day 0. The rats were then treated from day 0 with daily intraperitoneal injections of NBMPR (50 mg/kg/d in 1 ml of saline) or saline alone (untreated). The onset and progression of arthritis was evaluated daily using the arthritis severity index (0–4) for swelling and redness in four joints (bilateral front‐ and hind‐paws), with a score of 0 representing no rheumatoid changes and a score of 4 representing the most severe changes. Body weights were also measured to monitor the systemic antiarthritic effects of NBMPR as well as its systemic toxicity. On the last day (day 35), the rats were killed, and radiographic and histopathological assessments of their hind‐paws were conducted. The onset of arthritis was detected on the same day in both NBMPR‐treated and untreated arthritic rats, but subsequent disease progression was suppressed significantly in NBMPR‐treated rats. Radiographic (e.g., bone erosion and destruction) and histopathological (e.g., cell infiltration and cartilage erosion) findings confirmed the improved condition of the NBMPR‐treated rats. There was no significant difference in body weight among the rats. Thus, increasing extracellular adenosine with NBMPR appears to suppress the progression of arthritis. Drug Dev. Res. 58:479–485, 2003. © 2003 Wiley‐Liss, Inc.

Long-Lasting Regulation of Galanin, Opioid, and Other Peptides in Dorsal Root Ganglia and Spinal Cord during Experimental Polyarthritis

Mechanisms involved in transition from acute to chronic pain are still not well understood and our means to therapeutically influence this transition are limited. Moreover, very little is known about long-lasting consequences of prolonged exposure to painful stimuli with regard to phenotypic changes and pain experience. In this study we have analyzed long term behavioral and neurochemical effects of intradermal tail injection of heat-killed mycobacterium butyricum suspended in complete Freund's adjuvant. Calcitonin gene-related peptide (CGRP) and galanin mRNA levels were investigated in dorsal root ganglia of polyarthritic rats during the acute (21-) and the remission stage (79 days postinjection), and opioid peptide mRNAs and receptors were studied in the spinal cord. Most of the increases in peptide mRNA levels observed during the acute stage of arthritis were still present in the remission stages. Thus, CGRP and galanin mRNAs in DRGs, and opioid peptide mRNAs and opioid receptors in the spinal cord were still strongly up-regulated, when animals do not exhibit spontaneous pain behavior and inflammation. Hot-plate test in the presence of naloxone, performed in the remission stage, indicated that opiates participate in pain threshold regulation after prolonged painful condition. Finally, X-ray examination revealed a complete destruction of joint structure, thus suggesting a parallel lesion of peripheral nerve endings. These results suggest that in the remission stage of chronic joint inflammation several types of mechanisms are activated aiming at counteracting both inflammatory and neuropathic pain. Thus, opioid systems in the dorsal horn as well as galanin in DRG neurons are upregulated, both alternating pain.

A new spin on an old model: in vivo evaluation of disease progression by magnetic resonance imaging with respect to standard inflammatory parameters and histopathology in the adjuvant arthritic rat.

To noninvasively examine the pathogenesis of rat adjuvant-induced arthritis (AIA) by magnetic resonance imaging (MRI), and to correlate MRI indices of disease progression with classic inflammatory parameters and histologic evaluation. AIA was established in male Lewis rats following subcutaneous injection in the right hindpaw with 0.5 mg of heat-killed Mycobacterium butyricum suspended in light mineral oil. In vivo MRI evaluations of soft tissue and bony changes in AIA rats with matched histopathology were correlated with changes in left hindpaw volumes, circulating leukocytes, acute-phase reactants, and urinary collagen crosslinks throughout the disease process. MRI of arthritic tibiotarsal joints of the uninjected left hindpaws from AIA rats demonstrated 2 distinct phases of disease activity. The first phase, apparent between days 10 and 18, was characterized by periarticular inflammation with marked synovitis, synovial fibroplasia, and distension of the joint capsule into the surrounding tissue. The secondary phase, occurring between days 18 and 30, was marked by continued soft tissue inflammation, periostitis with osteolysis, and periosteal new bone formation progressing to a state of near complete ankylosis by day 30. These 2 phases of disease activity observed by MRI paralleled biochemical, cellular, and histologic markers of disease progression. MRI can be used to noninvasively detect, monitor, and quantify the chronic synovitis and progressive destruction of soft tissue and bone in live AIA rats, thereby improving the ability to evaluate disease progression in this preclinical animal model of rheumatoid arthritis.

Sirolimus (rapamycin, Rapamune) and combination therapy with cyclosporin A in the rat developing adjuvant arthritis model: correlation with blood levels and the effects of different oral formulations.

To determine whole blood levels of sirolimus, a macrolide antibiotic in the rat developing adjuvant arthritis (AA) model after dosing orally with two different vehicles, and whether combinational doses of sirolimus and cyclosporin A (CsA) produced additive or synergistic inhibitory effects in this model. Male Lewis rats (150-180g). Arthritis was induced by the injection (0.5 mg/ rat) of heat-killed Mycobacterium butyricum suspended in light mineral oil. Drugs were administered orally either in fine suspension (0.5% Tween 80) or in emulsion (phosal 50 PG in 1% Tween 80) at doses of 0.1 to 5 mg/kg in a 7 day, MWF or daily regimen. Paw volumes (ml) were measured by automated mercury plethysmograph and sirolimus concentrations in whole blood were quantitated by liquid chromatography/ mass spectroscopy. At 72h (7 days after adjuvant) after receiving the third oral dose (4.5 mg/kg p.o.), the phosal vehicle resulted in higher sirolimus blood levels (2.5 ng/ml) than in Tween 80 (1.6 ng/ml). After the rats received the last oral dose on day 14, (7 total doses of sirolimus at 4.5 mg/kg) the sirolimus blood levels (2h after the last dose) were about 2 times higher for the phosal dosed rats (9.8 ng/ml) compared to Tween 80 dosed rats (4.6ng/ml). Even 24h after the last dose, sirolimus blood levels were still elevated in the phosal dosed rats (0.8 ng/ml) relative to 0.5% Tween 80 dosed rats (0.5 ng/ml). At day 16 in the rat developing model, sirolimus, when given in phosal vehicle, produced an ED50 of 0.28 mg/ kg (i.e. inhibition of uninjected paw edema) that was about 5.5 times lower than using 0.5% Tween 80 as the suspending agent (ED50 = 1.6mg/kg). When combining sirolimus and CsA using precalculated doses for producing an additive effect in this adjuvant model, an additive inhibitory effect on uninjected paw edema was observed at equal combinational doses of 0.5 and 2 mg/kg, respectively. The phosal vehicle used in administering sirolimus increases the absorption and whole blood levels in the rat and the elevated blood levels correlated positively with the therapeutic effect in the rat developing AA model. In addition, combination therapy using sirolimus and CsA produced an additive effect in rat developing AA.