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.     

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.     

Antagonism in isolated equine digital vessels of contraction induced by epinephrine in the presence of hydrocortisone and an aqueous extract of black walnut (Juglans nigra)

Prazosin, isoxsuprine, and nifedipine were screened for ability to reverse contraction of isolated equine digital vascular strips produced by epinephrine (Epi) in the presence of hydrocortisone (Hc) and an aqueous extract of black walnut (Juglans nigra) (BW). Two arteries and two veins from each of three horses for each drug (n = 9) were maintained in isolated tissue baths in Krebs' bicarbonate buffer with 95% oxygen at 37 degrees C. Six-point Epi concentration-response (C-R) curves were obtained for each vessel in the presence of Hc, BW, and the appropriate vehicle. This was repeated for each vessel using one of two concentrations of one of the three test drugs. Each drug and concentration combination was tested on a total of three arteries and three veins. Prazosin produced a concentration-dependent shift of the Epi C-R curve to the right but the curve maintained the same maximum height and slope, which is consistent with competitive alpha 1 adrenergic blockade. Isoxsuprine exhibited similar behavior, although the precise mechanism of action for isoxsuprine is unknown. Conversely, nifedipine did not shift the curve but did depress maximum contraction, suggesting a non-competitive interaction consistent with its mechanism of calcium-channel blockade.     

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.     

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‐1β, 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‐1β, 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.     

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.     

Vascular distribution of contrast medium during intraosseous regional perfusion of the distal portion of the equine forelimb

OBJECTIVE: To describe the vascular distribution pattern of contrast medium during intraosseous regional perfusion (IORP) of the distal portion of the equine forelimb. SAMPLE POPULATION: 13 cadaveric forelimbs from 12 horses without forelimb diseases. PROCEDURES: Serial lateromedial radiographic views were taken of the distal portion of 10 heparinized cadaveric forelimbs at 0, 1, 2, 6, 15, and 30 minutes during IORP of the third metacarpal bone (MCIII) by use of iodinated contrast medium and a tourniquet placed over the proximal portion of MCIII. Vascular regions of interest (ROI) were created for each radiograph. Reviewers identified the presence or absence of contrast medium-induced opacified vessels in all ROI on radiographs. This information was summarized to identify vessel-filling patterns over time. Vessel identification was verified by use of computed tomography angiography and latex perfusion studies on the distal portion of separate cadaveric forelimbs. RESULTS: During IORP, contrast medium filled the medullary cavity of the MCIII; exited via transcortical vessels; and diffused distally to the remaining arteries and veins of the forelimb, distal to the tourniquet. Maximum vessel and soft tissue opacification occurred in most specimens at 6 and 30 minutes, respectively. Serial radiography vessel patterns matched those of computed tomography images and dissected specimens. CONCLUSIONS AND CLINICAL RELEVANCE: IORP provides a repeatable pattern of vascular distribution in the distal portion of the equine forelimb. To our knowledge, our study provides the first documentation of arterial perfusion by use of IORP; results of previous reports indicate that IORP delivers medications to only the venous vessels of the perfused forelimb.     

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.     

Doppler sonographic measurements of arterial blood flow and their repeatability in the equine foot during weight bearing and non-weight bearing

In six normal adult horses, pulsed-wave Doppler sonographic inspections were made of the left and right lateral palmar proper digital artery (LPPDA) of each thoracic limb. Using a two-period, crossover design with repeated measures, each LPPDA was inspected on four occasions throughout the day over a 7 hour period and, on each occasion, with the limb weight bearing and non-weight bearing. In comparison with the observations made during weight bearing, there were significant increases (P<0.001) in blood-flow velocities when the limb was non-weight bearing. There was no relationship between blood velocities and the time of day, the order in which the inspections were made, resting heart rate, age of the horse, or hoof angle. In both weight bearing and nonweight bearing, the Doppler waveform demonstrated a systolic peak followed by at least two diastolic peaks and an end diastolic plateau. Horses that moved least during sonographic inspections had the lowest co-efficients variation.     

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.     

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.     

Laminar microvascular flow, measured by means of laser Doppler flowmetry, during the prodromal stages of black walnut-induced laminitis in horses

OBJECTIVE: To measure changes in laminar microvascular blood flow (LMBF) over time in healthy horses and horses in the prodromal stage of black walnut-induced laminitis and to determine the effects of glyceryl trinitrate application on LMBF in horses with acute laminitis. ANIMALS: 10 healthy adult horses. PROCEDURE: Laser Doppler flowmetry was used to measure LMBF Baseline measurements were obtained, horses were given deionized water via a nasogastric tube, and measurements were obtained hourly for 12 hours. Twenty-four hours later, baseline measurements were again obtained, and horses were given black walnut extract. Measurements were obtained hourly for 12 hours or until development of Obel grade-3 laminitis. At this time, 5 horses were treated with phenylbutazone, and the other 5 were treated with phenylbutazone and glyceryl trinitrate, and measurements were obtained hourly for an additional 12 hours. RESULTS: LMBF was significantly decreased 1 and 2 hours after administration of the black walnut extract but then returned to near-baseline values for the next 6 hours. Eight hours after extract administration, there was a second significant decrease in LMBF that persisted until the end of the study. Glyceryl trinitrate had no effect on LMBF. Clinical signs of laminitis developed 8 to 12 hours after extract administration. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that in horses with black walnut-induced laminitis, there is an early decrease in LMBF followed by reperfusion prior to onset of clinical signs. Treatment with glyceryl trinitrate after development of clinical signs of laminitis did not have a significant effect on LMBF.     

Risk factors for development of acute laminitis in horses during hospitalization: 73 cases (1997-2004)

OBJECTIVE: To identify risk factors for development of acute laminitis in horses during hospitalization for illness or injury. DESIGN: Retrospective case-control study. ANIMALS: 73 horses that developed laminitis (case horses) and 146 horses that did not develop laminitis (control horses) during hospitalization. PROCEDURES: Case and control horses were matched in a 2:1 ratio by the date on which each horse was evaluated. Potential risk factors investigated included age, breed, and sex; highest and lowest values recorded during hospitalization for fibrinogen concentration, WBC count, PCV, and total solids concentration; and comorbid disease states, including pneumonia, endotoxemia, diarrhea, medically treated colic, surgically treated colic, pituitary adenoma, retained placenta or metritis, forelimb lameness, hind limb lameness, acute renal failure, and vascular abnormalities. A univariate screening of all potential risk factors was performed to determine which variables should be selected for further analysis. All factors found to be associated with development of laminitis were included in a multivariate conditional logistic regression model. RESULTS: Development of laminitis was marginally associated with lowest and highest fibrinogen concentrations, highest PCV, and lowest total solids concentration and significantly associated with pneumonia, endotoxemia, diarrhea, abdominal surgery for colic, and vascular abnormalities. In the multivariate analysis, only endotoxemia was significantly associated with laminitis. CONCLUSIONS AND CLINICAL RELEVANCE: Endotoxemia is an important risk factor for development of acute laminitis in horses during hospitalization for medical or surgical conditions. Early recognition of endotoxemia, or the potential for it to develop in certain disease states, and initiation of treatment directed at endotoxemia or its consequences may help prevent laminitis in horses during hospitalization.     

Evaluation of hoof wall surface temperature as an index of digital vascular perfusion during the prodromal and acute phases of carbohydrate-induced laminitis in horses.

OBJECTIVE: To evaluate the use of hoof wall surface temperature (HWST) as an indirect indicator of digital perfusion and to describe HWST patterns during the prodromal and acute phases of carbohydrate-induced laminitis in horses. ANIMALS: 30 adult horses without foot abnormalities. PROCEDURES: Three experiments were performed. In the first, HWST was measured in 2 groups of horses acclimatized to hot (n = 6), or cold (6) environments and exposed to cold (15 C) ambient temperature. In the second experiment, HWST were measured in both forefeet of 6 horses before and after application of a tourniquet to 1 forefoot to induce vascular occlusion. In the third experiment, HWST were recorded in 12 horses before and during the prodromal and acute phases of carbohydrate-induced laminitis. RESULTS: Mean HWST of hot-acclimatized cold-challenged horses was significantly less than that of cold-acclimatized cold-challenged horses at all times. Transient episodes of high HWST were observed during prolonged cold-induced vasoconstriction. Hoof wall surface temperature significantly decreased during arterial occlusion and increased during reperfusion. Digital hypothermia was observed during the prodromal phase of carbohydrate-induced laminitis. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of HWST is a valid technique to evaluate digital perfusion under appropriate controlled conditions in horses. Digital hypothermia detected during the prodromal phase of laminitis is consistent with decreased digital vascular perfusion or metabolic activity. If administered to horses during the prodromal phase, agents that enhance digital perfusion may prevent development of laminitis.