Freund's Complete Adjuvant Administration Research Articles

Peripheral inflammation effects on retinal phagocytic system

AbstractPurposeTo study how a unilateral arthritic inflammatory process, affecting the peripheral nervous system, can activate the mononuclear phagocytic system at retinal level.MethodsAn experimental model of peripheral inflammatory process produced by an intraplantar injection of Freund's complete adjuvant (FCA) in the left hind limb was induced to adult C57BL/6J lysozyme M‐eGFP‐knock‐in (LysM‐eGFP) mice that express LysM‐eGFP selectively in myelomonocytic cells. FCA, sham and naïve mice were processed at different survival times (ranging 1–10 days post‐lesion). Retinas were prepared as whole‐mounts and histologically analyzed for microglia markers and LysM‐eGFP expression.ResultsAfter FCA administration morphological signs compatibles with microglial activation were observed in the eGFP+ cells in the innermost layer of the retina, persisting along the time‐course of study. The three sub‐population of eGFP+ cells identified, amoeboid, ramified and peripheral, showed significant and relevant changes in the distribution pattern respect to that observed in untreated retinas from sham or naïve mice. The quantitative analysis showed statistically significant changes in the expression of eGFP+ and Iba‐1+ for all cell sub‐population in the course of time. However, the number of eGFP+ and Iba‐1 immunonegative amoeboid cells was constant and similar to those of sham or control retinas.ConclusionsInduction of a peripheral inflammatory process by administration of FCA elicits a response from the peripheral bone marrow‐derived monocyte‐macrophage population at retinal level, where these cells are recruited and transformed into microglia.

Juglanin attenuated adjuvant-induced arthritis via inactivating NF-κB/IκBα and a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeat pathways in experimental rats

Background: Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease affecting multiple joints and worsening the quality of life. Juglanin, a natural aldose reductase inhibitor, has shown to exhibit anti-inflammatory, anti-nociceptive, and anti-oxidant properties. Objective: The present study was undertaken to evaluate their possible mechanism of action against adjuvant-induced arthritis (AIA), i.e., Freund's complete adjuvant (FCA)-induced arthritis, in experimental rats. Materials and Methods: FCA (0.1 mL) was administered into the subplantar region of female Wistar rats paw to induced AIA. The rats were treated with vehicle (distilled water, 10 mL/kg) or leflunomide (10 mg/kg), or juglanin (10, 20, and 40 mg/kg, respectively) orally for the next 16 days. Various biochemical, molecular, and histological parameters were evaluated to determine the potential of juglanin. Results: Subplantar administration of FCA resulted in a statistically significant (P

Effect of umbelliferone on adjuvant-induced arthritis in rats by MAPK/NF-κB pathway.

PurposeUmbelliferone (Umb), a member of coumarin family, is found in many plants and is a promising molecule with potential anti-inflammatory, anti-oxidative, and anti-tumor activities. However, the effect of Umb on arthritis remains unclear.MethodsA rat model with Freund’s complete adjuvant (FCA)-induced arthritis was developed and used to test the efficacy of Umb on arthritis rats. Rats were given an intragastric injection of Umb (20 and 40 mg/kg) once daily from days 21 to 28 after the administration of FCA. Hind paw volume was assessed using a volume meter. The pro-inflammatory cytokine levels and prostaglandin E2 (PEG2) level in serum and synovial fluid were detected by ELISA. HE staining was used to determine representative histological changes in joint tissues, and Western blot analysis was employed to study the effects of Umb on MAPK/NF-κB signaling pathway.ResultsOur results showed that Umb suppressed the release of IL-6, IL-1β, tumor necrosis factor-alpha, and PEG2. In addition, Umb could also dramatically ameliorate the pathological changes observed in rat joints. Based on the results of Western blot, we also observed that Umb could strikingly suppress the expression of MAPK/NF-κB pathway molecules.ConclusionThese results proved that treatment with Umb is very effective for arthritis and inhibiting the MAPK/NF-κB signaling pathway may be a potential therapeutic target for treatment of arthritis.

Влияние криоконсервирования на функциональный статус стволовых кроветворных и мезенхимальных клеток костного мозга животных с аутоиммунной патологией

The application of cryopreserved autologous bone marrow (BM) is the reconstructive therapy tool for autoimmune diseases (AIDs). The BM use efficiency is determined by preservation rate of its hematopoietic and mesenchymal stem cells (HSCs and MSCs, respectively). This fact determines the need to design the optimal cryopreservation regimens for the BM of AIDs donors. Here we assessed a functional status of bone marrow HSCs and MSCs of mice with adjuvant arthritis (AA), initiated by Freund's complete adjuvant administration. The bone marrow of CBA/H mice to day 14 of AA (chronic stage) and that of healthy animals were cryopreserved under dimethyl sulfoxide protection. The content of HSCs of various differentiation extent was determined by spleen colony formation in vivo to days 8 (CFU-S-8) and 14 (CFU-S-14). Functional activity of MSCs in BM was studied in vitro by the content of fibroblast colony forming units (CFU-F). There was established the fact of altering the functional potential of hematopoietic progenitor cells of BM of various differentiation levels (CFU-S-8, CFU-S-14) and their stromal microenvironment (CFU-F) during clinical manifestation of AA. After cryopreservation of AA animals' BM cells with 7% DMSO a functional potential of CFU-S and CFU-F was established to be similar to the healthy animals' BM, cryopreserved with 10% DMSO. The cryopreserved with the proposed regimen autologous BM may be used for a targeted recovery of lymphohemopoietic system in AA animals. Probl Cryobiol Cryomed 2016; 26(1):63-72.

Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

Hypoferremia observed during systemic inflammatory disorders is regulated by hepcidin. Hepcidin up-regulation is particularly important during acute inflammation, as it restricts the availability of iron, which is necessary for pathogenic microorganism growth before adaptive immunity occurs. The aim of this study was to evaluate the clinical findings and hepatic hepcidin mRNA expression in horses using a Freund's complete adjuvant (FCA) model of inflammation. The expression of hepcidin mRNA in the liver was determined in healthy horses following two intramuscular injections of FCA at 0 h and 12 h. Plasma iron and fibrinogen concentrations were measured at multiple time points between 0 h and 240 h post-FCA injection (PI). Hepcidin mRNA expression was determined by RT-qPCR using liver biopsy samples performed at 0 h (control), 6 h and 18 h PI. The mean plasma fibrinogen level was significantly different from the control values only between 120 and 216 h PI. The mean plasma iron level was significantly lower than the control between 16 and 72 h PI, reaching the lowest levels at 30 h PI (33 % of the initial value), and returned to the reference value from 96 h PI to the end of the experiment. Hepcidin mRNA expression increased at 6 h PI and remained high at 18 h PI. The iron plasma concentration was an earlier indicator of inflammatory processes in horses when compared with fibrinogen and might be useful for the early detection of inflammation in the horse. FCA administration caused the rapid onset of hypoferremia, and this effect was likely the result of up-regulated hepatic hepcidin gene expression. This study emphasizes the importance of hepcidin and iron metabolism during inflammation in horses.

The antinociceptive effects of estradiol on adjuvant-induced hyperalgesia in rats involve activation of adrenergic and serotonergic systems

Estradiol is a female hormone required for maintaining pregnancy and developing follicles in the ovary. Estradiol has been shown to perform a variety of physiological activities, including pain reduction. In this study, we tested the hypothesis that estradiol exerts antinociceptive effects in a rat model of inflammatory hyperalgesia. We established a subacute hyperalgesia model using plantar injection of Freund's complete adjuvant (FCA) in Sprague-Dawley rats. We administered estradiol every 24 h, beginning 12 h after FCA administration, and used the plantar test to determine its effect on hyperalgesia. To determine the mechanism of action of estradiol, we evaluated the role of the opioid antinociceptive system using naloxone and the role of the descending pain inhibitory system using the α-2-receptor antagonist yohimbine and the serotonin receptor antagonist methysergide. Administration of FCA induced hyperalgesia, which was significantly reduced by estradiol treatment compared to controls. Moreover, this effect was not antagonized by naloxone, but was attenuated by α-2-receptor and serotonin-receptor antagonists. Estradiol is known to perform a variety of physiological functions. Our findings suggest that one such function is antinociception via an interaction with α-2 receptors and serotonin receptors.

New Anthranilic Acid Derivative, EAntS-GS, Attenuates Freund’s Complete Adjuvant-Induced Acute Pain in Rats

Acute and chronic pain commonly accompany various clinical conditions such as contusion, fracture, osteoarthritis, peripheral neuropathy, and postherpetic neuralgia. Recent studies have found that antioxidative drugs can have analgesic effects. The present study tested the hypothesis that a new anthranilic acid derivative, EAntS-GS, exerts antinociceptive effects on inflammatory pain in a rat model. We induced subacute pain with a plantar injection of Freund's complete adjuvant (FCA) in Sprague-Dawley rats. EAntS-GS (1 mg/kg subcutaneous injection or 1% application) was administered every 12 h beginning 24 h after FCA administration, and the plantar test was used to determine its effect on pain. Levels of myeloperoxidase, inducible nitric oxide synthase (iNOS), and protease activated receptor 2 (PAR2) were measured to elucidate the mechanism of action of EAntS-GS. EAntS-GS significantly reduced FCA-induced pain and myeloperoxidase, iNOS, and PAR2 levels. Our findings suggest that the new anthranilic acid derivative, EAntS-GS, exerts antinociceptive effects, and that the mechanism involves iNOS and PAR2. We conclude that EAntS-GS should be considered a new therapeutic tool to treat acute and chronic pain.

Comparison of antiallodynic effect of intrathecal morphine, brimonidine and rilmenidine between neuritis and ligation injury induced neuropathic pain.

Mechanical allodynia is generally resulted from nerve damage by direct injury or inflammation. Thus, this study was designed to compare the antiallodynic effect of morphine, brimonidine and rilmenidine in two models of neuropathic pain, that is, induced by nerve ligation and neuritis. Rats were prepared with tight ligation of the L5/L6 spinal nerves (SNL group) or with Freund's complete adjuvant (FCA) administration evoked sciatic inflammatory neuritis (SIN group). Antiallodynic effects by intrathecal morphine, brimonidine and rilmenidine were measured by applying von Frey filaments to the lesioned hind paw. Thresholds for withdrawal response were assessed and converted to % MPE to obtain an effective dose 50% (ED 50) and a dose response curve. Either SNL group or SIN group showed marked mechanical allodynia in the lesioned hind paw. Antiallodynic effects of morphine were different between two groups. That is ED 50 was 0.16 microgram (SIN) and 8.12 microgram (SNL), and dose response curve of the SIN group shifted left from that of the SNL group. The difference between SIN and SNL groups was statistically significant (P < 0.05). With the brimonidine or rilmenidine administration, ED 50 s were 0.12 microgram (SNL) and 0.37 microgram (SIN) and 2.16 microgram (SIN) and 11.46 microgram (SNL), respectively. And the shift to left of dose response curve from the SNL group is more prominent with rilmenidine administration. These results suggest morphine and rilmenidine showed a better effect on reducing the mechanical allodynia induced by FCA administration.

Selective Downregulation of Hepatic Cytochrome P450 Expression and Activity in a Rat Model of Inflammatory Pain

This study was designed to examine the effect of Freund's complete adjuvant (FCA)-induced inflammation on liver P450 expression and activities in the first 7 days that followed a single FCA injection in the rat hindpaw. Rats were humanely sacrificed at regular time points, plasma and liver samples were collected, liver mRNA extracted, and liver microsomes prepared. FCA injection led to the development of an acute inflammatory response evidenced by paw edema and increased alpha-1-acid glycoprotein (AGP) and total-nitrite (NOx) plasma concentrations. Plasma IL-6 levels were significantly higher in FCA-treated rats than in controls at 8 h post-FCA. Within 24 h, these changes were accompanied by a rapid decrease in total P450 contents in FCA-treated rat liver and the selective downregulation of specific CYP isoforms, as illustrated by decreased mRNA levels (CYP2B, CYP2CI1, CYP3A1, and CYP2E1), protein contents (CYP2B, CYP2C11, and CYP2E1) or catalytic activities (CYP2C6, CYP2C11, and CYP2E1). CYP3A1 mRNA levels were severely decreased by FCA administration, whereas CYP3A2 mRNA and protein levels remained unchanged. These early biochemical and metabolic modifications may have pharmacokinetic and pharmacodynamic consequences when hepatically cleared drugs are administered to FCA-treated rats, especially within the first 24-72 h post-FCA.

Daily variations of amino acid concentration in mediobasal hypothalamus, in rats injected with Freund's adjuvant. Effect of cyclosporine

Although the existence of central responses to inflammatory injuries was already reported, the existence of hypothalamic amino acid responses has been less explored. The present study was designed to characterize the 24-h changes in mediobasal hypothalamic excitatory and inhibitory amino acid neurotransmitter contents and to analyze the effect of Freund's complete adjuvant administration on these patterns. Also the effects of the immunosuppressant drug Cyclosporine was studied. The content of aspartate, glutamate, glutamine, GABA and taurine was measured by HPLC with fluorimetric detection. The results show the existence of specific daily rhythms of aspartate, glutamate, glutamine, GABA and taurine contents in the mediobasal hypothalamus of control rats. Maxima for these amino acids was found at midnight, although another peak of lesser magnitude, occurred during the light phase of the photoperiod, except for TAU in which both peaks were of similar magnitude. Freund's complete adjuvant administration did not modify the 24-h pattern of any amino acid studied. It reduced the midnight peak of glutamate, glutamine and GABA and increased that of taurine. Moreover, it increased and extended the midday peak of glutamate. Besides, Freund's adjuvant did not modify aspartate content at any time point studied. Cyclosporine pretreatment did not prevent the inhibitory effects of Freund's complete adjuvant on glutamate, glutamine and GABA midnight peaks. However, the drug blocked the increase in the content of taurine at midnight and increased its midday peak. Moreover, cyclosporine administration abolished the variations of ASP during the scotophase, as compared to control animals and shift delayed both peaks of glutamate. The results indicate the existence of a significant effect of immune-mediated inflammatory response of the mediobasal hypothalamic amino acids studied, at an early phase after Freund's adjuvant administration, and that these changes were partially sensitive to the immunosuppression induced by cyclosporine.

Humoral and peritoneal cell responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin, Vibrio anguillarum and Freund's complete adjuvant following intraperitoneal and bath immunisation

ELISA methods were used to evaluate the humoral immune responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin and Vibrio anguillarum. Antibody responses to ovalbumin administered intraperitoneally (i.p.) were inconsistent even when Freund's complete adjuvant (FCA) was used and the induction phase (4-6 weeks) of the response was longer compared with the response to V. anguillarum (<4 weeks). Significant elevation in antibody level was noted 3 weeks after bath vaccination with V. anguillarum but levels decreased thereafter. Humoral responses of greatest magnitude occurred where V. anguillarum was given in an emulsion with Freund's complete adjuvant (FCA). However, i.p. administration of FCA alone 3 weeks prior to i.p. immunisation with non-adjuvanted V. anguillarum resulted at 5 weeks in similar elevations in antibody to those in fish given V. anguillarum and FCA concurrently, suggesting that the effects of FCA were not limited to creation of an antigen depot. Proliferation of peritoneal inflammatory cell populations which included macrophages and plasma cells was detected histologically within 3 weeks of administration of FCA, but changes were not detected in the spleen or haematopoietic kidney.

Pre treatment with MK-801, a non-competitive NMDA antagonist, prevents development of mechanical hyperalgesia in a rat model of chronic neuropathy, but not in a model of chronic inflammation

In the rat, loose ligation of the sciatic nerve results in behavioural signs of hyperalgesia reminiscent of neuropathy in man. A further rat model, of chronic inflammatory hyperalgesia, is produced by intraplantar administration of Freund's complete adjuvant (FCA). We report here that pre-emptive administration of a non-competitive antagonist of the glutamate N- methyl- d-aspartate (NMDA) receptor, MK-801 (0.3 mg kg −1, s.c.) 30 min prior to and twice daily for a further 8 days following loose ligation of the sciatic nerve, blocks the development of mechanical hyperalgesia measured 27 days later. In contrast, MK-801 administration using the same dosing regimen did not significantly inhibit the hyperalgesia apparent 15 days following i.pl. administration of FCA. Our results suggest that the mechanisms responsible for the development of mechanical hyperalgesia associated with chronic nerve injury and chronic inflammation differ.

Induction of Bl receptors In vivo in a model of persistent inflammatory mechanical hyperalgesia in the rat

The induction of mechanical hyperalgesia by B1 and B2 receptor agonists following an inflammatory insult has been studied in the rat. Intra-articular injection of bradykinin, but not desArg 9 bradykinin, into naïve joints induced mechanical hyperalgesia with a reduction of up to 38% in load tolerated by the treated leg. Following intra-articular injection of Freund's complete adjuvant (FCA) the load tolerated by the treated leg was depressed for three days. The development of FCA-induced hyperalgesia was reduced by desArg 9Leu 8BK or Hoe 140. Bradykinin, desArg 9BK and the metabolically stable B1 receptor agonist SarDPhe 8desArg 9BK were all hyperalgesic upon intra-articular administration 3 days after FCA administration. The hyperalgesia induced by desArg 9 BK and SarDPhe 8desArg 9BK was prevented by co-administration of desArg 9Leu 8BK but not by Hoe 140. At low doses (< 10 nmol) bradykinin-induced hyperalgesia was presented by Hoe 140 but not by des Arg 9 Leu 8 BK. At higher doses of bradykinin (> 100 nmol) the hyperalgesia was reduced by both Hoe 140 and desArg 9 Leu 8 BK. These data suggest that following an inflammatory insult both bradykinin B1 and B2 receptor mechanisms are involved in the accompanying hyperalgesia.

Non-specific resistance of sheep against Haemonchus conforms with Freund's complete adjuvant

Six groups of four ovines each were injected intraperitoneally with Freund's complete adjuvant (FCA), at various times before, during, or after infection with 10 000 larvae (L3) of Haemonchus contortus per sheep. Animals in groups A and B received FCA on days 14 and 7, respectively, before infection. Ovines in group C were treated with FCA on the same day of infection; while sheep in groups D, E, and F were injected with the adjuvant on days 7, 14, and 21, respectively, after infection. Significant reductions in adult worm numbers compared with non-treated controls, as determined 42 days after infection, were of 30%, 34%, 45%, 52%, 56%, and 55% in groups A, B, C, D, E, and F, respectively. A significant correlation, between the time of FCA administration (referred to the day of infection) and the number of worms recovered at necropsy, was also observed.

Repopulation of Macrophages in Popliteal Lymph Nodes of Mice After Liposome-Mediated Depletion

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes. No effect of liposome-entrapped Cl2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo. This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine. These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.

Pain threshold changes in adjuvant-induced inflammation: A possible model of chronic pain in the mouse

A chronic hyperalgesic condition was induced in mice by the injection of Freund's complete adjuvant (FCA) into the lower lumbar region or directly into the hind footpads. Although little or no visible inflammation was observed after a single intradermal injection of FCA into the lower lumbar area of rats or mice, significant alterations in nociceptive thresholds occurred in each species as determined by decreases in response latency in tail-flick and hot-plate assays. Unilateral intraplantar administration of FCA in mice resulted in visible inflammation in the area of the tibiotarsal (ankle) joint. Changes in the response latency to a noxious stimulus in the areas surrounding the inflamed joint were similar to those observed in non-inflamed limbs, suggesting that changes in sensitivity to noxious stimuli were not merely the result of local hypersensitivity of the inflamed tissue, but may also be due to alterations in nociception at the level of the central nervous system (CNS). When the chronic inflammatory condition induced in the mouse tibiotarsal joint was evaluated by histological and morphological techniques, it was found to have the same characteristics as described in the early stages of FCA-induced arthritis in rats. The similarities between the response to FCA in rat and mouse suggest that injection of FCA in mice may prove to be a useful model for the study of chronic pain in mice as well as in rats.

Modulatory Effects of Freund’s Adjuvant Treatment on Mast Cell Histamíne Release and Homocytotropic Antibody Synthesis

The present study examines the influence of Freund's complete adjuvant (FCA) injections on sensitized PVG rats with respect to serum levels of IgE and IgG2 alpha antibodies and total IgE (all assessed by radioimmunoassays) and the capacity of serosal mast cells to release histamine on challenge in vitro with 'immunological' secretagogues (specific antigen, anti-IgE, concanavalin A) or with compound 48/80. The rats were immunized with 10 micrograms ovalbumin (OA); alum, Bordetella pertussis vaccine, or silica gel were employed as adjuvants. Treatment with FCA was performed by single intraperitoneal injections 3, 2, or 1 week(s) before or 1 or 2 weeks after sensitization. Tests were conducted 3 weeks after sensitization. The results show that the effect of FCA treatment varied reproducibly with the adjuvant employed for sensitization and with the timing of the FCA administration. FCA treatment could either increase, fail to affect, or decrease total serum IgE and OA-IgG2 alpha antibody levels as well as serosal mast cell responsiveness, whereas OA-IgE antibody responses were decreased or not affected. Moreover, serum levels of OA-IgE and OA-IgG2 alpha antibodies and total IgE were affected by FCA treatment independently of each other. Finally, serosal mast cell responsiveness to a given secretagogue could be influenced by the FCA treatment apparently independently of that to other secretagogues. A salient finding was that effects of FCA treatment on mast cell responsiveness did not necessarily conform to effects on antibody synthesis. Collectively, these data support the opinion that the mechanisms of action of the IgE-promoting adjuvants employed differ and suggest that the expression of serosal mast cell responsiveness to each examined secretagogue can be regulated separately. They also suggest that the serosal mast cell sensitizing capacity of homocytotropic antibodies may not be adequately quantified by immunochemical methods employing reagents prepared against IgE and IgG2 alpha protein.

Enzymatic changes in various organs of guinea pigs caused by the administration of Freund's complete adjuvant.

Although our previous studies demonstrated marked changes in the activities of hydrolytic enzymes in guinea pigs afflicted with experimental allergic encephalomyelitis (EAE), it is possible that at least some parts of them were due to the effects of Freund's complete adjuvant (FCA) per se used for immunization. Thus we tried to find the property of the enzymatic changes in the tissues of limb muscle, heart muscle, spleen, liver, kidney, brain, spinal cord and serum induced by the administration of FCA alone to the guinea pigs. FCA caused rather intensive enzymatic changes in every organ tested. When the patterns of these enzymatic changes were compared with those of the animals afflicted with EAE, a significant correlation was seen only in forelimb muscle at 1 week after the injection of the agents, whereas at 2 weeks significant correlations were seen in heart and liver. Although these results indicate that in some organs there are some similarities between the two experimental conditions, the degree of determination for this phenomenon was not high, being 28% at the highest. This means that most of the enzymatic changes seen in the animals afflicted with EAE could not be explained by the effects of FCA. It was concluded that FCA causes unique and systemic enzymatic changes when administered to the animals.