Effect of an aqueous extract of black walnut (Juglans nigra) on isolated equine digital vessels

An aqueous extract was made from black walnut (Juglans nigra) heartwood obtained in the fall of the year. Ten hours after nasogastric administration of 5 L of the extract, a 550-kg, 13-yr-old Quarter Horse gelding experienced Obel grade-3 laminitis. The effect of aqueous extract of black walnut on vascular contractility was then tested, using isolated equine digital arteries and veins. The vessels were maintained in Krebs bicarbonate buffer with 95% oxygen at 37 C. The extract did not induce a direct contractile effect. It did, however, reversibly enhance the vasoconstriction induced in the isolated vessels by administration of epinephrine potentiated with hydrocortisone. In contrast, aqueous extracts made, using the same techniques, from the shavings of eastern white pine (Pinus strobus), eastern red cedar (Juniperus virginiana), and pin oak (Quercus palustrus) had no effect on epinephrine-induced digital vessel contractions.     

Gamma scintigraphic analysis of the distribution of perfusion of blood in the equine foot during black walnut (Juglans nigra)-induced laminitis

Twelve horses, with acute laminitis (primarily in the forefeet) at 12 hours after intragastric dosing with an aqueous extract of black walnut (Juglans nigra) heart-wood, were studied. The distribution of perfusion of blood to the foot and to outlined regions within the foot was quantified, using gamma scintigraphy of regionally infused 99mTc-labeled macroaggregated albumin, before and 12 hours after extract administration. Horses 1 to 3 were not studied further. Perfusion was quantified again for horses 4 to 12 at 84 hours after extract administration. At the onset of acute laminitis, horses 7 to 12 were administered a single dose of prazosin (0.025 mg/kg of body weight, IV) immediately after scintigraphy of the right forelimb and before scintigraphy of the left forelimb. When compared with baseline images, perfusion to the forefoot of horses after the development of acute laminitis was quantitatively decreased vs perfusion to the entire distal portion of the forelimb. Also with the onset of laminitis, perfusion also decreased to the dorsal laminar and coronary corium regions vs the distal portion of the forelimb. The acute laminitis-associated deficit in perfusion to the dorsal laminitis-associated deficit in perfusion deficit in perfusion to either the coronary corium or the entire forefoot. Equivalent deficits in the distribution of perfusion were not detected in forelimbs from horses with acute laminitis and which had been treated with prazosin. When compared with baseline images, perfusion to the dorsal lamina was increased in relation to perfusion to the distal portion of the limb at postdosing hour 84. Prazosin treatment did not influence that increase in perfusion to the dorsal lamina.     

Dynamic changes in circulating leukocytes during the induction of equine laminitis with black walnut extract

Administration of black walnut heartwood extract (BWHE) via nasogastric tube induces acute laminitis in horses. However, the processes responsible for the development of laminitis, including laminitis induced with BWHE, remain unclear. The results of recent studies indicate that administration of BWHE initiates an inflammatory response in the laminar tissues and that this response may be due to extravasation of activated leukocytes from the circulation. This study examines the effects of BWHE administration on the dynamics of circulating neutrophils and monocytes, and the capacity of blood leukocytes to produce radical oxygen species (ROS) over the time period from administration of BWHE to the development of lameness consistent with Obel grade I laminitis.

Individual horses, free of pre-existing musculoskeletal disease, were administered either 6 l of BWHE or an equal volume of water at time 0 (T = 0). Blood samples were collected prior to dosing and at 1, 2, 3, 4, 6, 8, 10 and 12 h after dosing, or until the onset of Obel grade I laminitis. For each sample, total leukocyte counts were determined followed by collection of buffy coats and removal of erythrocytes by hypotonic lysis. Leukocytes were either fixed for flow cytometric assessment of differential counts or maintained in culture to measure endogenous and phorbol ester-induced production of ROS. At each sample time, the number of cells recovered and the flow cytometric differential counts were compared with corresponding total leukocyte counts determined by the Clinical Pathology laboratory.

Horses administered BWHE had a significant reduction in circulating leukocytes at 3–4 h relative to values for horses administered the same volume of water. Horses that developed Obel grade I laminitis had a significant reduction in circulating leukocytes when compared to values for horses administered BWHE that did not become lame. Flow cytometric analysis revealed a consistent decrease in the total number of monocytes obtained from horses that developed laminitis. In these same horses, the endogenous level of ROS production was significantly higher at T = 0 than for horses that did not become lame. Furthermore, production of ROS by leukocytes from horses that developed laminitis increased significantly and coincided with the decrease in circulating leukocytes.

Collectively, these findings support a role for systemic activation of leukocytes and induction of inflammation by BWHE as a factor in the early pathogenesis of acute laminitis. Because laminitis often develops as a sequel to diseases characterized by systemic inflammatory events, activation and emigration of neutrophils and monocytes may be important factors in the early pathogenesis of laminitis in clinical cases.     

Investigation of the effect of black walnut extract on in vitro ion transport and structure of equine colonic mucosa

OBJECTIVE: To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). ANIMALS: 14 adult horses. PROCEDURE: Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. RESULTS: Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation.     

Presence of mononuclear cells in normal and affected laminae from the black walnut extract model of laminitis

Reasons for performing study: There is increasing evidence of involvement of inflammatory cells in acute laminitis. Objective: To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)‐induced laminitis. Methods: Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recognise lymphocytes (T and B) and monocytes/macrophages, respectively. Results: Mononuclear cells were present in laminar tissue of normal horses. The majority of CD3‐ and CD20‐positive lymphocytes were localised around the deep dermal vessels but were also evident around vessels of the primary dermal laminae. CD163‐positive macrophages were primarily perivascular in deep dermis or in dermal laminae. No changes in the number of laminar B or T lymphocytes occurred at any time point post BWE administration. However, increases (P = 0.0016) in laminar CD163‐positive cells occurred in the secondary dermal laminae (SDL) in the 1.5H DTP and 3H DTP groups, returning to basal values in LAM group. Conclusions: Lymphocyte and macrophage populations are present in the laminar tissue of clinically normal horses and BWE administration induces an increase in CD163‐positive macrophages in SDL. Potential relevance: Both the host tissue population of mononuclear cells and the influx of monocytes may play an important role in the pathophysiological changes leading to laminar injury.     

Effects of monoamines formed in the cecum of horses on equine digital blood vessels and platelets

OBJECTIVE: To determine in vitro vasoactive potency of monoamines formed in the cecum and found in the systemic circulation of horses. SAMPLE POPULATION: Segments of digital blood vessels obtained from 6 healthy mixed-breed horses and ponies euthanatized at an abattoir and platelets isolated from 4 healthy ponies. PROCEDURE: Paired rings of digital artery and vein from the same horse were examined, and isometric tension was recorded. Concentration-response curves for tryptamine (TRP), tyramine (TYR), phenylethylamine (PEA), isoamylamine (IAA), and isobutylamine (IBA) were obtained. Vasoconstrictor mechanisms were investigated for TRP and TYR by the use of antagonists. Washed platelets loaded with [3H]-5-hydroxytryptamine (5-HT) were incubated with monoamines; the amount of radioactivity displaced after 30 minutes was estimated. RESULTS: TRP, TYR, and PEA were potent constrictors of arteries and veins, with TRP and TYR being more potent in veins than arteries. Constrictions induced by TYR were inhibited by benextramine (alpha-antagonist) and nisoxetine (neuronal-uptake blocker), whereas TRP responses were inhibited by ketanserin (5-HT receptor antagonist). All 5 amines displaced 5-HT from platelets with the order of potency being TYR > TRP > PEA > IAA > IBA. CONCLUSIONS AND CLINICAL RELEVANCE: Amines from the equine cecum cause digital vasoconstriction. The most potent (TRP and TYR) cause selective venoconstriction. Tyrosine activates predominantly alpha-adrenoceptors through the release of neuronal norepinephrine, whereas TRP activates 5-HT receptors. All amines tested released 5-HT from platelets. Amines formed in the cecum and released into the systemic circulation warrant additional investigation as trigger factors for digital ischemia and subsequent laminitis.     

紫竹根水提物对小鼠体内外抗氧化作用的研究

为了研究紫竹根水提物(PNR)对动物体内外抗氧化的作用,本试验体外调查了PNR对二苯基苦基苯肼(DPPH)自由基、羟自由基、超氧阴离子的清除能力,并测定了其总还原力;以免疫抑制小鼠为试验对象,测定心、肝、肾匀浆液中蛋白、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)的活性、总抗氧化能力(T-AOC)和丙二醛(MDA)水平。结果显示,PNR在体外抗氧化的4个指标中,尤其是清除超氧阴离子试验中,表现出良好的抗氧化能力。在小鼠体内试验中,与模型对照组相比,PNR高剂量组的SOD、CAT、GSH-Px和TAOC活性显著增加(P0.05),所有剂量组的MDA水平显著降低(P0.05)。试验结果表明,PNR可以有效地清除自由基,具有较强的抗氧化活性。     

黄花草木樨水浸提液中潜在化感物质的分离、鉴定

本研究旨在分离、鉴定黄花草木樨水浸提液中的主要化感物质。采用色谱分离、薄层分析和生物活性检测相结合的方法分离出了活性较强的化感物质组分,并通过气质联用(GC-MS)对潜在的化感物质进行了定性分析。结果表明,黄花草木樨水浸提液的乙酸乙酯相中含有多种化感物质,相对含量最高的为香豆素,相对含量较高的有6-(3-Hydroxy-but-1-enyl)-1,5,5-trimethyl-7-oxabicyclo[4,1,0]heptan-2-ol、二氢苯并吡喃酮、2,6-二叔丁基对甲酚、二溴三甲基环丙烷、磷酸三甲酯等物质;将乙酸乙酯相进一步分离得到3层混合物,上层混合物中含量相对最高的为香豆素;中层混合物中含量相对较高的有二溴三甲基环丙烷、6-(3-Hydroxy-but-1-enyl)-1,5,5-trimethy-7-oxabicyclo[4,1,0]heptan-2-ol、2,6-二叔丁基对甲酚、甲基环戊醇等物质;下层混合物中含量相对较高的有二溴三甲基环丙烷、甲基环戊醇、环己基溴化膦等物质。乙酸乙酯相及其分离后的各层混合物处理对多花黑麦草种子萌发和幼苗生长均具有一定的化感作用,其中,乙酸乙酯相处理对多花黑麦草化感抑制作用最强,其处理对多花黑麦草种子萌发和幼苗生长的抑制作用显著强于其分层后的各层混合物处理(P<0.05);其次是上层混合物处理,其处理对多花黑麦草种子萌发和幼苗生长的抑制作用显著强于其他2层(P<0.05);下层混合物处理对多花黑麦草的种子萌发和幼苗生长则具有明显的促进作用(P>0.05)。以上结果提示,黄花草木樨水浸提液中化感物质种类非常多,香豆素的含量最高,且香豆素含量高的组分处理对黑麦草的抑制作用就强,推测香豆素应为黄花草木樨水浸提液中的主要化感物质之一。     

Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s)

Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses.     

Ultrasonographic diagnosis of injuries to the deep digital flexor tendon and associated structures in the equine foot (suprasesamoidean area)

Foot pain is the most common cause of lameness in horses. In sport horses, podotrochlear syndrome (‘navicular syndrome’) is reported to be the most frequent condition affecting the front foot. Ultrasonography has the potential to detect damage to the soft tissues as well as the bone surfaces; in some clinics it has become the technique of choice for the identification and documentation of many podotrochlear injuries. The purpose of this paper is to review the main pathological conditions of the proximal part of the podotrochlear apparatus (PTA) that can be diagnosed ultrasonographically, focusing on the deep digital flexor tendon (DDFT), podotrochlear bursa (PTB) and distal digital annular ligament (DDAL). Potentially significant ultrasonographic findings of the DDFT include thickening of one or both lobes, longitudinal tears, focal or diffuse changes in echogenicity, irregularities of the dorsal border and adhesions between the DDFT and the proximal sesamoidean ligament and/or distal sesamoid bone. Deep digital flexor tendon injuries are often associated with concurrent lesions of the PTB (acute to chronic bursitis) and of the DDAL (desmopathy). Both feet should be routinely examined as lesions of the PTA are often bilateral. We currently consider that ultrasonography should be routinely employed as the primary diagnostic procedure to complement radiography of the equine foot.     

Blood lead levels of wild Steller's eiders (Polysticta stelleri) and black scoters (Melanitta nigra) in Alaska using a portable blood lead analyzer.

Sea duck populations are declining in Alaska. The reasons for the decline are not known; environmental lead exposure is one suspected cause. Thirty wild Steller's eider ducks (Polysticta stelleri) and 40 wild black scoter ducks (Melanitta nigra) were tested for blood lead levels using a portable blood lead analyzer (LeadCare; ESA, Inc., Chelmsford, Massachusetts 01824, USA). Sixty-seven and one-tenth percent of the sea ducks had undetectable blood lead levels, 30.0% had values indicating normal or background lead exposure, and 2.9% had values indicating lead exposure. None of the birds had values indicating lead toxicity, and no birds demonstrated clinical signs of toxicity. Birds in areas with higher human population density had higher blood lead levels than those in less densely populated areas. This is the first time a portable blood lead analyzer has been utilized with sea ducks in a field setting. Because it provides immediate results, it is valuable as a screening tool for investigators carrying out surgical procedures on birds in the field as well as establishing baseline blood lead data on sea ducks. Lead exposure does occur in wild sea ducks, and the study indicates that additional research is needed in order to determine the role environmental lead plays in declining sea duck populations.     

Amplification and differentiation of the DNA of an abortigenic (type 1) and a respiratory (type 4) strain of equine herpesvirus by the polymerase chain reaction

Unpurified DNA derived from cultures of equine fetal kidney cells infected with either equine herpesvirus type 1 or equine herpesvirus type 4 was amplified by the polymerase chain reaction using one pair of oligonucleotide primers. Restriction endonuclease digestion of the amplified segments with PvuII, followed by electrophoresis, revealed restriction fragment length polymorphisms which enabled the two virus types to be differentiated.     

Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain

The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAV_FDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAV_UK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAV_FDDV13 compared with eMDM infected with EIAV_UK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAV_FDDV13 and was correlated with enhanced TLR3, sELR1 and IFNβ expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAV_FDDV13. These results indicate that the induction of restraining infection by EIAV_FDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

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