Attenuation of carrageenan-induced hind paw edema and plasma TNF-α level by Philippine stingless bee (Tetragonula biroi Friese) propolis.

Despite decades-long existence of the Philippine stingless bee industry, the biological activity of propolis from this native bee species (Tetragonula biroi Friese) remains poorly understood and sparingly investigated. Herein, we examined the potential anti-inflammatory efficacy of Philippine stingless bee propolis using the lambda (λ)-carrageenan-induced mice model of hind paw edema. Thirty (30), six-week-old, male ICR mice were randomly assigned into three treatment groups (n=10/group) as follows: distilled water group, diclofenac sodium group (10 mg/kg), and propolis group (100 mg/kg). All treatment were administered an hour prior to the injection of the phlogistic agent. As observed at 3 h post-injection, λ-carrageenan remarkably evoked the classical signs of hind paw edema exemplified grossly by swelling and hyperemia. The ameliorative effect of propolis became apparent at the onset of 6 h post-injection with a statistically significant finding evident at the 24-h period. This gross attenuation histologically correlated to a considerable and specific reduction of the dermal edema, which mirrored those of the diclofenac sodium group. Furthermore, both propolis and diclofenac sodium significantly attenuated the λ-carrageenan-induced increase in the protein expression levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) depicting more than two-fold decrement relative to the distilled water group. Altogether, these suggest that Philippine stingless bee propolis also exhibited a promising in vivo anti-inflammatory property, which can be partly mediated through the inhibition of TNF-α.

Anti-inflammatory and analgesic activity of total flavone of Cunninghamia lanceolata.

The present study was undertaken to investigate the anti-inflammatory and analgesic activity of total flavone of branches and leaves of Cunninghamia lanceolata (TFC) to provide a scientific basis for its clinical use and resource development. TFC was evaluated for anti-inflammatory and analgesic activity in mice or rats using chemical and thermal models of nociception, including acetic acid-induced writhing test, hot plate latency test, formalin test and carrageenan induced paw oedema test. Results showed that TFC given orally can significantly attenuate acetic acid-induced writhing in mice in a dose-dependent manner. In the hot plate latency test, TFC showed common activity in prolonging duration time only at the highest dose (400 mg/kg). Each dose of TFC could not significantly inhibit the first phase but was active in the later phase of formalin-induced pain, whereas morphine showed notable activity in the two phases. In the carrageenan-induced paw oedema model, TFC could significantly and dose-dependently reduce the carrageenan-induced paw edema at the third and fifth hour, and decrease the content of PEG(2) in paw edema tissue and that of COX-2 in blood serum. It may be concluded that TFC showed both anti-inflammatory and analgesic effects, showing that it can be of importance in drug development, especially in the field of pain and inflammation.

Detection of calprotectin and apoptotic activity within the equine colon from horses with black walnut extract-induced laminitis.

The black walnut extract (BWE) model of equine laminitis is associated with a systemic inflammatory response manifest by increased expression of inflammatory cytokines in the lungs and liver as well as the laminae. The specific role of the gastrointestinal tract in development of this response is unclear and is of utmost importance, as gastrointestinal disease and laminitis are intimately related. We investigated calprotectin expression and epithelial and endothelial apoptosis in the colon of horses exposed to orally administered BWE. Sections of colon from 19 horses including 7 controls not exposed to BWE, 6 horses at the developmental time-point of leukopenia (DTP) and 6 at the onset of Obel grade 1 laminitis (LAM) after BWE-administration were histologically examined. Immunohistochemical evaluation for calprotectin expression with MAC 387 antibody was performed along with assessment of epithelial and endothelial apoptosis with caspase-3 active antibody. Calprotectin expression and percentage of apoptotic cells were compared between controls and the two treatment groups and presence of a correlation between calprotectin expression and apoptosis was evaluated. Histological findings from BWE-treated horses included eosinophil and lymphocyte epitheliotropism. The DTP group had a higher (p<0.01) calprotectin score with respect to the control group, while there was no significant difference in percentage of epithelial and endothelial apoptotic cells between groups (p=0.08 and p=0.48 respectively). No significant correlation was found between calprotectin score and epithelial or endothelial apoptosis (p=0.69 and p=0.29 respectively). There is preliminary evidence that exposure of horses to BWE results in an early inflammatory response in the colon. Further studies are needed to characterize the nature of the colonic injury in BWE-exposed horses and the link to the development of laminitis.

Evaluation of the in vitro effects of aqueous black walnut extract on equine mononuclear cells.

OBJECTIVE: To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. SAMPLE: Mononuclear cells separated from blood samples from 8 horses. PROCEDURES: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activities of caspases 3 and 7, nitric oxide production, procoagulant activity, and tumor necrosis factor-α production. The effect of LPS on cellular responses induced by BWE was assessed by coincubation with 13 U of polymyxin B/mL; mononuclear cells incubated with LPS were used as a reference. RESULTS: BWE did not cause loss of cell membrane integrity in mononuclear cells but did induce a dose-dependent increase in activities of caspases 3 and 7. Neither BWE nor LPS significantly induced production of nitric oxide. Both BWE and LPS induced comparable amounts of procoagulant activity and tumor necrosis factor-α production; coincubation with polymyxin B reduced the activity for BWE and LPS by 50% and approximately 100%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Addition of BWE induced inflammatory activation of equine mononuclear cells, a portion of which was independent of the effects of LPS. Furthermore, BWE and LPS may work in concert to induce systemic inflammatory responses that contribute to the development of acute laminitis in horses.

Role of oxidative tissue injury in the pathophysiology of experimentally induced equine laminitis: a comparison of 2 models.

BACKGROUND: Oxidative stress reportedly plays a role in sepsis-induced organ dysfunction and failure in many species. In septic horses, laminae are targeted; evidence of laminar oxidative stress has been reported experimentally in the black walnut extract (BWE) model. Carbohydrate (CHO)-induced laminitis may be more similar to clinical sepsis-related laminitis than the BWE model in that animals with CHO-induced disease commonly develop laminar failure. The role of oxidative stress in the CHO model remains unknown. HYPOTHESIS/OBJECTIVES: Markers of oxidative stress will be increased in laminae from horses with BWE- and CHO-induced laminitis. ANIMALS: Banked laminar tissue from various time points from animals subjected to BWE (n = 15) and CHO (n = 20) protocols. METHODS: Laminar 4-hydroxynonenal (4-HNE) and protein carbonyl content were evaluated by slot blot analysis. Laminar 3-nitrotyrosine (3-NT) immunohistochemistry was performed. RESULTS: The number of laminar 3-NT (+) cells was increased at developmental and Obel grade 1 (OG1) time points in the BWE model (versus control [CON]; P= .013) and lower in OG1 tissues than CON in the CHO model (P = .04). No change in 4-HNE content was observed in the CHO model, and no increase in laminar protein carbonyl content was present in either model (P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: These results do not support a prominent role for oxidative stress at examined time points in CHO-overload laminitis and support transient oxidative stress in the BWE model. Tissue oxidation does not appear to be a central early pathophysiologic event in CHO-associated laminitis.

In vivo priming and ex vivo activation of equine neutrophils in black walnut extract-induced equine laminitis is not attenuated by systemic lidocaine administration.

Laminitis is a crippling disease of horses characterized by an inflammatory response in the tissue that suspends the axial skeleton within the hoof. Pain is a common feature of laminitic pathology and its management is an important component of the treatment regime for this disease. Systemic lidocaine administration is commonly utilized to manage pain in equine laminitis; however, the potential anti-inflammatory effects of this drug during the treatment of equine laminitis have not been investigated. Here, we sought to determine if lidocaine concentrations achieved in the plasma (therapeutic concentrations) of horses systemically administered lidocaine are capable of attenuating neutrophil activation and associated inflammation. To identify markers of activation, purified neutrophils were stimulated in vitro with LPS or recombinant equine IL-8 (reqIL-8) and surface expression of CD13 and CD18 was ascertained by immunofluorescent staining. Activation with LPS or reqIL-8 in vitro induced an elevated expression of CD13 as well as a putative conformational change in CD18 detected by elevated staining with a sub-saturating concentration of anti-CD18 mAb. Lidocaine attenuated the activation-induced changes in CD13 and CD18 expression only when used at 30-70 times therapeutic concentrations. For in vivo analyses, horses were administered black walnut extract (BWE) to induce laminitis and either systemic lidocaine (n=6) or saline (n=6) as a control. Whole blood was collected and incubated with or without reqIL-8. Following which, leukocytes were stained for CD13 and CD18. Protein was extracted from laminar tissue and subjected to gelatin zymography to measure matrix metalloproteinase-9 (MMP-9) accumulation. Results obtained show that changes in neutrophil size, granularity/complexity, CD13 surface expression and CD18 staining intensity occurred over time post BWE administration irrespective of lidocaine treatment in response to incubation alone or with 100 ng/ml of reqIL-8. The mean fluorescence intensities of neutrophils stained for either CD13 or CD18 did not differ between lidocaine treated and saline controls, nor did lamellar MMP-9 content measured by gelatin zymography. Thus, using changes in surface expression of CD13 and CD18 as markers of neutrophil activation in the horse we have shown that BWE treatment activates neutrophils in vivo and this is not affected by systemic administration of lidocaine at levels used to manage pain.

Effect of intravenous lidocaine administration on laminar inflammation in the black walnut extract model of laminitis.

REASONS FOR PERFORMING STUDY: Laminitis is a serious complication of horses suffering from sepsis/endotoxaemia-related events. Laminitis in horses and organ injury in human sepsis are both reported to involve inflammatory injury to the laminae/organs including early activation of endothelium and leucocytes leading to emigration of neutrophils into the tissue interstitium. In the black walnut extract (BWE) model, systemic inflammatory events coincide with marked increase in laminar mRNA concentrations of inflammatory genes including proinflammatory cytokines (i.e. IL-1beta, IL-6), COX-2, chemokines (i.e. IL-8) and endothelial adhesion molecules (i.e. ICAM-1 and E-selectin). In models of human sepsis, i.v. lidocaine has been reported to decrease leucocyte and endothelial activation, and the expression of proinflammatory cytokines and chemokines. OBJECTIVES: To evaluate the effect of i.v. lidocaine therapy on the inflammatory processes documented to occur in the BWE model of laminitis. METHODS: Twelve horses were administered BWE and treated immediately with either lidocaine (1.3 mg/kg bwt bolus, followed by 0.05 mg/kg bwt/min CRI, n=6) or saline (n=6) for 10 h. At 10 h post BWE administration, laminar samples were obtained under general anaesthesia for assessment of proinflammatory gene expression (using RT-qPCR) and leucocyte emigration (via CD13 immunohistochemistry). At 0, 3 and 10 h post BWE administration, skin samples were obtained for assessment of leucocyte emigration (via calprotectin immunohistochemistry). RESULTS: No significant differences between groups were noted for inflammatory gene mRNA concentrations (IL-1beta, IL-6, IL-8, COX-2) or for number of leucocytes present within the laminar interstitium or skin dermis. Increased (P<0.05) laminar E-selectin mRNA concentrations were present in the LD group (vs. SAL group). CONCLUSIONS: Continuous administration of i.v. lidocaine does not inhibit inflammatory events in either the laminae or skin in the horse administered black walnut extract. POTENTIAL RELEVANCE: This work questions the use of continuous i.v. administration of lidocaine as an effective anti-inflammatory therapy for systemic inflammation.

Black walnut extract: an inflammatory model.

The black walnut extract (BWE) model was developed after the discovery that horses bedded on shavings from black walnut trees commonly developed laminitis. The first investigators that consistently induced laminitis with black walnut shavings established that it was only the heartwood of the tree that induced laminitis. The BWE model of laminitis has allowed investigators to determine many of the early pathologic signaling events likely to occur in the developmental and acute clinical stages of the disease process, and has brought inflammatory injury to the forefront of laminitis research. These events must also be assessed in the carbohydrate overload models, the models that more closely reflect the clinical case of laminitis.

Evaluation of the possible role of prostaglandin F(2 alpha) in laminitis induced in horses by nasogastric administration of black walnut heartwood extract.

OBJECTIVE: To provide insights into the role of prostaglandin F(2 alpha) (PGF(2 alpha)) in the developmental stages of laminitis induced in horses by ingestion of black walnut heartwood extract (BWHE). SAMPLE POPULATION: 10 adult mixed-breed horses. PROCEDURES: Horses were separated into 2 groups and were euthanatized at 12 hours after placebo (water) administration (control horses) or after BWHE administration and development of Obel grade 1 laminitis. Blood samples were obtained to determine plasma PGF(2 alpha) concentrations hourly for the first 4 hours and subsequently every 2 hours after substance administration. Laminar arteries and veins were isolated, and responses to increasing concentrations of PGF(2 alpha) were measured before and after preincubation of blood vessels with prostanoid and thromboxane receptor antagonists SQ 29,548, SC-19220, and AH 6809. RESULTS: Plasma PGF(2 alpha) concentrations increased in horses given BWHE; the WBC count decreased concurrently. In control horses, PGF(2 alpha) was a potent contractile agonist for laminar veins but not for laminar arteries. In horses given BWHE, PGF(2 alpha) was similarly selective for laminar veins; however, the magnitude of PGF(2 alpha)-induced venoconstriction was less than that in control horses. After preincubation with SQ 29,548, laminar veins from control horses responded to PGF(2 alpha) with a small degree of dilation, whereas laminar veins from horses given BWHE did not. CONCLUSIONS AND CLINICAL RELEVANCE: PGF(2 alpha) may play a role in the inflammatory and vascular dysfunction associated with the prodromal stages of laminitis. Prostanoids such as PGF(2 alpha) may be viable targets for the prevention of acute laminitis in horses.

Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract.

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5-15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5h (n=5), 3h (n=6) or 12h (n=10) after BWHE administration. Control horses (n=7) were euthanized 12h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3h BWHE horses, in 6 of the 12h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p<0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8h compared to the control group and at 8 and 10h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.

Comparison of characteristics and enzymatic products of leukocytes in the skin and laminar tissues of horses administered black walnut heartwood extract or lipopolysaccharide.

OBJECTIVE: To compare characteristics and enzymatic products of leukocytes detected in the skin and laminar tissues of horses administered black walnut heartwood extract (BWHE) and horses administered purified lipopolysaccharide (LPS). ANIMALS: 25 healthy 5- to 15-year-old horses. PROCEDURES: Horses were randomly assigned to receive LPS (20 ng of O55:B5 Escherichia coli endotoxin/kg; n = 5) IV or 6 L of BWHE (10) or water (control group; 10) via nasogastric intubation. Horses were euthanatized 12 hours after treatment or at onset of Obel grade 1 lameness. Laminar tissue samples and skin samples from the middle region of the neck were harvested at the time of euthanasia. Leukocyte emigration (determined via CD13 immunohistochemical analysis) and matrix metalloproteinase (MMP)-2 and MMP-9 gene expressions and activities (determined via reverse transcription PCR assay and gelatin zymography, respectively) were measured in skin and laminar tissue samples. RESULTS: Tissues of horses receiving BWHE contained significantly higher numbers of CD13-positive cells and increased MMP-9 gene expression and activity, compared with findings in the other 2 groups. Values for laminar tissue and skin from LPS-treated horses were not increased, compared with findings in the control group, in any experiment. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that BWHE administration causes increases in CD13-positive leukocyte numbers and MMP-9 expression and activity in laminar tissue and skin in horses; similar effects were not detected following LPS administration. Leukocyte emigration in horses with experimentally induced endotoxemia and in horses administered BWHE differed markedly, thereby providing additional evidence that the development of laminitis involves more complex mechanisms than endotoxemia-induced leukocyte activation alone.

Indices of inflammation in the lung and liver in the early stages of the black walnut extract model of equine laminitis.

The liver and lung are not only described as "target organs" in sepsis in most species, but are purported to be sources of circulating inflammatory mediators central to the systemic inflammatory response syndrome (SIRS). As we have recently reported an inflammatory response in the laminar tissue in laminitis similar to that described in "target organs" in human sepsis, we investigated the inflammatory response of the lung and liver in the black walnut extract (BWE) model of equine laminitis to determine (1) if a similar systemic inflammatory response occurs in this laminitis model as described for these organs in human sepsis, and (2) if these organs may be an important source of the inflammatory mediators leading to laminar inflammation. Real-time quantitative PCR (RT-qPCR) was used to measure hepatic and pulmonary mRNA concentrations of IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha, COX-1 and COX-2. Hepatic samples were assessed from two time points in the developmental/prodromal period: (1) 1.5h post-BWE administration (BWE-1.5H, n = 5), and (2) the "developmental time point" (onset of leukopenia, approximately 3h post-BWE administration, BWE-DEV, n = 5). Pulmonary samples were only assessed for the BWE-DEV group. One control group (CON-3H, n = 5) was used for both the 1.5H and DEV groups. Finally, CD13 immunohistochemistry was performed to assess leukocyte emigration into hepatic and pulmonary parenchyma. Hepatic and pulmonary mRNA concentrations of the proinflammatory cytokines IL-6, IL-8 and TNF-alpha were significantly increased (P < 0.05) in BWE-1.5H and BWE-DEV groups compared to the control group; IL-1beta mRNA concentrations were only increased in the lung. The "anti-inflammatory" cytokines, IL-10 and IL-4, underwent transient decreases at different time points. Significant increases in parenchymal leukocyte numbers occurred in both the lung and liver at the BWE-DEV time point. Hepatic and pulmonary proinflammatory cytokine expression differ from that previously reported for the laminae in that TNF-alpha was increased in the hepatic and pulmonary tissues, the increases in expression of IL-6 and IL-8 are dramatically smaller for the liver and lung compared to those reported for the laminae, and the peak changes appear to occur later in the disease process in the liver than in the laminae (BWE-DEV in liver vs. 1.5H in the laminae).

Temporal aspects of laminar gene expression during the developmental stages of equine laminitis.

The results of recent studies indicate that inflammatory responses occurring in the early stages of equine laminitis lead to downstream events that eventually result in failure of the bond between the hoof wall and the distal phalanx. In order to gain further insights into the molecular mechanisms involved in the development of laminitis, an equine-specific cDNA microarray consisting of transcripts for more that 3000 genes was used to assess temporal changes in gene expression in laminar tissues at 1.5, 3 and 12 h after administration of either a laminitis-inducing agent (black walnut heartwood extract; BWHE) or an equal volume of water (control). As early as 1.5 h after BWHE administration, pro-inflammatory genes associated with leukocyte activation and emigration, including MCP-3/CCL7, MCP-1/CCL2, IP-10/CXCL10 and ICAM-1 were up-regulated. At both 1.5 and 3h after administration of BWHE, expression of B-cell specific transcripts (e.g., Ig-gamma 3, Ig-gamma 1 and lambda-light chain) were decreased in the laminar tissues. At the onset of Obel grade 1 lameness in horses administered BWHE, other genes involved in inflammatory processes (e.g., serum amyloid A, calgranulin C and NFAT-activation molecule 1), regulation of inflammation (e.g., inter-alpha-trypsin inhibitor, BiP/GRP78 [Ig binding protein], L-plastin, serpin and nexin-1), antioxidant responses (e.g., superoxide dismutase), matrix turnover (e.g., MMP-9 and TIMP-1), and anti-microbial responses (e.g., serotransferrin, beta-defensin-1 and elafin) were up-regulated. These results provide convincing evidence that genes associated with inflammation, activation and extravasation of leukocytes, antimicrobial activities, and destruction of the lamellar basement membrane are induced during the early stages of development of laminitis in response to administration of BWHE.

Calprotectin in myeloid and epithelial cells of laminae from horses with black walnut extract-induced laminitis.

BACKGROUND: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin. HYPOTHESIS: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis. ANIMALS: Twenty adult horses. METHODS: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed. RESULTS: Laminar epidermal CP signal was significantly increased (P= .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups (P= .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.

Tissue concentrations of 4-HNE in the black walnut extract model of laminitis: indication of oxidant stress in affected laminae.

In the septic horse prone to laminitis, a similar activation of the innate immune system appears to occur as reported in the septic human prone to organ failure. Because oxidant injury plays a central role in organ failure occurring due to an overzealous innate immune response in human sepsis, this study was performed to determine whether there was evidence of oxidant stress in the laminar tissue in the early stages of laminitis. 4-Hydroxy-2-nonenal (4-HNE), a lipid aldehyde that forms due to lipid peroxidation occurring during episodes of oxidant stress, readily forms adducts with cellular proteins; these adducts can be assessed as a marker of oxidant stress in the form of lipid peroxidation. In this study, a slot blot technique was used to assess 4-HNE adduct concentrations in the laminae, lung, liver, and intestinal tract in the black walnut extract (BWE) model of laminitis. Significant increases in laminar 4-HNE adduct concentrations were identified at two early stages in the BWE model, in the absence of such changes in the other tissues. These data indicate that oxidant stress may play an important role in the laminar failure in laminitis, and further support the concept that a poor antioxidant response in the laminae relative to other equine tissues may be responsible for failure of the laminae in the septic horse. In contrast, tissues such as the lung and liver that undergo oxidant injury in human sepsis appear to be relatively protected in horses.

Thromboxane and isoprostanes as inflammatory and vasoactive mediators in black walnut heartwood extract induced equine laminitis.

Inflammation and vascular dysfunction occur concurrently during the prodromal stages of equine laminitis. The aim of this study was to provide insights into the role that thromboxane and isoprostanes may play in the development of black walnut heartwood extract (BWHE)-induced laminitis. Horses were divided into two groups, either control or BWHE-administered horses. Plasma concentrations of thromboxane increased transiently after administration of BWHE and coincided with the nadir in white blood cell counts, whereas plasma concentrations of iso-prostaglandin PGF(2alpha) (iso-PGF(2alpha)) did not change in either group. At 12h (for the control group) or Obel grade 1 laminitis (for the BWHE group) the horses were euthanized and laminar tissue collected. Laminar arteries and veins were used in functional studies with vasoconstrictor substances and tissue samples were used for the determination of laminar iso-PGF(2alpha) concentrations. Laminar tissue concentrations of iso-PGF(2alpha) were significantly greater in BWHE horses when compared to control horses. In parallel studies concentrations of iso-PGF(2alpha) in laminar tissue samples obtained 1.5 and 3h after administration of BWHE were indistinguishable from those for control horses at 3 or 12h after administration of an equal volume of water. Laminar vessel constrictor responses to either a thromboxane mimetic (U46619), iso-prostaglandin PGE(2) (iso-PGE(2)) or iso-PGF(2alpha) were determined using small vessel myographs. In some vessels, the effects of putative prostanoid and thromboxane receptor antagonists, SQ 29,548, SC-19220 and AH 6809, upon contractile responses were determined. In control horses, U46619, iso-PGF(2alpha) and iso-PGE(2) more potently and efficaciously constricted laminar veins when compared to laminar arteries. Responses of laminar veins from BWHE horses to iso-PGE(2) were similar to those of laminar veins from control horses, whereas iso-PGF(2alpha) elicited significantly greater responses in laminar veins from BWHE horses when compared to controls. In contrast, responses to U46619 were smaller in laminar veins isolated from BWHE horses when compared to those in laminar veins from control horses. In the presence of SQ 29,548, iso-PGF(2alpha) elicited a small dilation in laminar veins from control horses, which was not apparent in laminar veins from BWHE horses. These results are consistent with both systemic and local inflammatory events occurring during the prodromal stages of BWHE-induced laminitis. Because laminar veins are sensitive to thromboxane and isoprostanes, these substances may act as conduits between the inflammatory and vascular events occurring in laminitis and may be therapeutic targets for this crippling condition.

Anti-inflammatory effects of leaf and twig of Tripterygium wilfordii on paw edema in mice.

The root of Tripterygium wilfordii (TWH) is a traditional Chinese herb used to treat the immune-related diseases such as rheumatoid arthritis, whereas the leaf and twig of TWH was considered useless and discarded. We performed a study on the anti-inflammatory effects on the leaf and twig portion agent using carrageenan- and adjuvant-induced paw edema in rats. They showed a marked inhibitory effect on edema in both models of inflammation in rats, at the dose of 50, 100 and 200 mg/kg, especially on secondary immunological arthritis. Based on this study, we confirmed that the leaf and twig of TWH is a potentially useful drug suitable for further evaluation for rheumatoid arthritis and can replace root of TWH.

Anti-inflammatory effects of low-level laser therapy (LLLT) with two different red wavelengths (660 nm and 684 nm) in carrageenan-induced rat paw edema.

It has been suggested that low-level laser therapy (LLLT) can modulate inflammatory processes. The aim of this experiment was to investigate what effects red laser irradiation with two different wavelengths (660 nm and 684 nm) on carrageenan-induced rat paw edema and histology. Thirty two male Wistar rats were randomly divided into four groups. One group received a sterile saline injection, while inflammation was induced by a sub-plantar injection of carrageenan (1 mg/paw) in the three other groups. After 1 h, LLLT was administered to the paw in two of the carrageenan-injected groups. Continuous wave 660 nm and 684 nm red lasers respectively with mean optical outputs of 30 mW and doses of 7.5 J/cm(2) were used. The 660 nm and 684 nm laser groups developed significantly (p<0.01) less edema (0.58 ml [SE+/-0.17] ml and 0.76 ml [SE+/-0.10] respectively) than the control group (1.67 ml [SE+/-0.19]) at 4h after injections. Similarly, both laser groups showed a significantly lower number of inflammatory cells in the muscular and conjunctive sub-plantar tissues than the control group. We conclude that both 660 nm and 684 nm red wavelengths of LLLT are effective in reducing edema formation and inflammatory cell migration when a dose of 7.5 J/cm(2) is used.

In vivo study of the anti-inflammatory effect of Rhus toxicodendron.

BACKGROUND: Homeopathic Rhus toxicodendron (Rhus tox) is used in various inflammatory conditions. We screened its effect compared to succussed ethanol controls and appropriate active controls. METHOD: We initially experimented with Rhus tox 6, 12, 30 and 200 cH, using carrageenan-induced paw oedema in rats. The 6 cH dilution appeared most effective and was used in subsequent assays. We used pre-treatment and single treatment regimes in Wistar rats, and mice. RESULTS: We found significant reductions compared to control in carrageenan-induced paw oedema, vascular permeability, writhing induced by intraperitoneal acetic acid and stress induced gastric lesions. CONCLUSIONS: Rhus tox in homeopathic dilution appears to interfere with inflammatory processes involving histamine, prostaglandins and other inflammatory mediators.