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.     

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.     

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.     

Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study

BACKGROUND: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis. HYPOTHESIS: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis. ANIMALS: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5). METHODS: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2. RESULTS: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.     

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis

Reasons for performing study: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis‐related laminitis than the black walnut extract (BWE) model. Objectives: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature >38.9°C (DEV group, n = 8) or at onset of Obel grade 1 lameness (OG1 group, n = 8). Control horses (CON group, n = 8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time‐quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. Results: Increased mRNA concentrations (P<0.05) for IL‐1β, IL‐6, IL‐12p35, COX‐2, E‐selectin and ICAM‐1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL‐2, IL‐4, IL‐10, TNF‐α, IFN‐γ or COX‐1 at either the DEV or OG1 time points. Conclusions: There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. Potential relevance: The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.     

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.     

Laminar leukocyte accumulation in horses with carbohydrate overload-induced laminitis

Background: While there is evidence of laminar leukocyte infiltration in black walnut extract (BWE)‐induced laminitis, there is no such evidence for carbohydrate overload (CHO) laminitis. Objective: To assess presence of leukocytes and signs of epidermal stress/injury in the laminar tissue from horses with CHO‐induced laminitis. Animals: Twenty‐four adult horses. Methods: Immunohistochemistry for myeloid cell markers calprotectin (CP) and monocyte‐specific marker (CD163) was performed on laminar sections obtained from 2 groups of horses in the CHO model: the developmental time point (DTP) group (n = 6) and the onset of lameness (LAM) group (n = 6), and a control (CON) group (n = 8). Results: DTP was characterized by an increase in CP⁺ leukocytes (7.8‐fold increase versus CON, P < .001), and LAM time point was characterized by a more marked increase in laminar CP⁺ (108.5‐fold, P < .001) and mild increase in CD163⁺ (1.9‐fold, P= .007) cell counts. Increased CP epidermal signal (indicating epidermal stress or injury) occurred consistently at the LAM time point, although histological evidence of basement membrane (BM) detachment was minor, only being present in 3/6 horses. Conclusions and Clinical Relevance: Maximal laminar leukocyte infiltration and epithelial stress occurred at the onset of lameness in the CHO model showing a different temporal pattern from the BWE model, where maximal leukocyte infiltration clearly precedes epithelial stress. Leukocyte infiltration before major histological changes in the CHO model indicates that leukocyte infiltration can be a cause of and not a reaction to BM degradation and structural failure.     

Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response

REASONS FOR PERFORMING STUDY: Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. OBJECTIVES: To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the black walnut extract (BWE) and oligofructose (OF) models of laminitis. METHODS: Real-time qPCR was used to assess lamellar mRNA expression of interleukins-1beta, 2, 4, 6, 8, 10, 12 and 18, and tumour necrosis factor alpha and interferon gamma at the developmental stage and at the onset of lameness. RESULTS: Significantly increased lamellar mRNA expression of cytokines important in the innate immune response were present at the developmental stage of the BWE model, and at the onset of acute lameness in both the BWE model and OF model. Of the cytokines characteristic of the Th1 and Th2 arms of the adaptive immune response, a mixed response was noted at the onset of acute lameness in the BWE model, whereas the response was skewed towards a Th1 response at the onset of lameness in the OF model. CONCLUSIONS: Lamellar inflammation is characterised by strong innate immune response in the developmental stages of laminitis; and a mixture of innate and adaptive immune responses at the onset of lameness. POTENTIAL RELEVANCE: These results indicate that anti-inflammatory treatment of early stage laminitis (and the horse at risk of laminitis) should include not only therapeutic drugs that address prostanoid activity, but should also address the marked increases in lamellar cytokine expression.     

In Vivo and In Vitro Evidence of the Involvement of CXCL1, a Keratinocyte-Derived Chemokine, in Equine Laminitis

Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans.
Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis.
Animals: Twenty adult horses.
Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression.
Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [ P = .0001]) and DTP groups (21-fold [ P = .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases ( P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H₂O₂ (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA ( P = .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes.
Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.     

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.     

Laminar xanthine oxidase, superoxide dismutase and catalase activities in the prodromal stage of black-walnut induced equine laminitis

REASONS FOR STUDY: Xanthine oxidase (XO)-dependent production of superoxide anion and hydrogen peroxide, a characteristic of ischaemia-reperfusion injury, may contribute to the development of equine laminitis. OBJECTIVE: To determine the levels of XO and antioxidant enzymes (catalase, superoxide dismutase [SOD]) in the digital laminae of normal horses (CON) and horses in the developmental stage of laminitis using the black walnut extract (BWE) model. METHODS: Healthy horses (n = 12) were administered BWE (BWE group, n = 6), or water (CON group, n = 6) through a nasogastric tube. At the onset of leucopenia in the BWE-treated animals, all horses were anaesthetised, digital laminae and other samples collected rapidly and flash frozen, and the animals subjected to euthanasia. Extracts of the frozen tissues were assayed for the 2 conformational forms of xanthine: oxygen oxidoreductase (XOR), namely, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), as well as the antioxidant enzymes, SOD and catalase. RESULTS: Extracts of liver, lungs and skin, but not digital laminae, from either CON or BWE-treated horses had endogenous SOD, whereas all had endogenous XO and catalase. The levels of XDH, XO and catalase were similar in extracts of laminae from CON and BWE-treated horses as was the ratio of XDH to XO in extracts. CONCLUSIONS AND POTENTIAL RELEVANCE: The absence of increased XO activity suggest against the involvement of this reactive oxygen intermediate-generating system in the development of laminar pathology in BWE-treated horses. Conversely, the absence of SOD from extracts of equine digital laminae, but not other tissues, suggests that the equine digital laminae are highly susceptible to damage by superoxide anion, produced, for example, by emigrant inflammatory leucocytes.     

Expression of the cyclooxygenase isoforms in the prodromal stage of black walnut-induced laminitis in horses

OBJECTIVE: To compare the levels of mRNA expression of cycooxygenase (COX)-1 and COX-2 in the digital laminae of normal horses and horses in the developmental stages of laminitis experimentally induced by administration of black walnut extract (BWE). SAMPLE POPULATION: Samples of mRNA extracted from the digital laminae of 5 control horses and 5 horses at the onset of leukopenia after administration of BWE. PROCEDURE: Specimens of laminae were collected from anesthetized horses prior to euthanasia. Expression of COX-1 and COX-2 mRNA in laminae of control and affected horses was evaluated via real-time quantitative polymerase chain reaction techniques. RESULTS: Expression of COX-2 mRNA was significantly increased in the BWE-treated group, compared with that in control horses. In contrast to COX-2 regulation, COX-1 mRNA expression was not significantly different between groups. Interestingly, despite consistent clinical signs such as leukopenia in all BWE-treated horses, distinct differences in COX-2 mRNA expression were detected among those 5 horses (compared with values for control horses, the increase in COX-2 mRNA expression ranged from no increase to a 30-fold increase). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there was a significant upregulation of COX-2 mRNA expression during the developmental stages of laminitis, with no significant change in expression of the COX-1 isoform. These data appear to provide support for aggressive use of nonsteroidal anti-inflammatory drugs in horses at risk for laminitis; further investigation into the clinical value of selective COX-2 inhibitors for treatment of laminitis in horses appears to be warranted.     

Early laminar events involving endothelial activation in horses with black walnut- induced laminitis

OBJECTIVE: To determine proinflammatory gene expression, endothelial adhesion molecule gene expression, and matrix metalloproteinase (MMP) concentrations in laminar specimens at 1.5 hours after administration of black walnut extract (BWE) and to compare these values with later time points. ANIMALS: 25 horses. PROCEDURES: After nasogastric administration of BWE, anesthesia was induced at 1.5 hours in early time point (ETP) horses (n = 5), between 3 and 4 hours in developmental time point horses (5), and between 9 and 10 hours in acute onset of lameness time point horses (5). Anesthesia was induced at 3 and 10 hours after nasogastric administration of water in 2 groups of control horses (3-hour control group, n = 5; 10-hour control group, 5). Real-time quantitative PCR assay was performed on laminar specimens from control and ETP horses for cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6, IL-8, IL-10, MMP-2, and MMP-9 gene expression; and on laminar specimens from all groups for endothelial adhesion molecules, intercellular adhesion molecule (ICAM)-1, and E-selectin gene expression. Leukocyte emigration was assessed via CD13 immunohistochemistry, and gelatinase accumulation was determined by gelatin zymography. RESULTS: Laminar concentrations of IL-1beta, IL-6, IL-8, COX-2, ICAM-1, and E-selectin mRNA were significantly increased in ETP horses, compared with control horses. Concentrations of IL-1beta, IL-8, ICAM-1, and E-selectin mRNA peaked at 1.5 hours. In ETP horses, leukocyte emigration was present in 3 of 5 horses and pro-MMP-9 was detected in 2 of 5 horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that endothelial activation and laminar inflammation are early events in laminitis; MMP accumulation likely is a downstream event.     

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.     

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.     

Increased expression of MAIL, a cytokine-associated nuclear protein, in the prodromal stage of black walnut-induced laminitis

REASONS FOR PERFORMING STUDY: The mediators and signalling cascades important in the initiation of laminitis remain unclear. We therefore wanted to explore the genes and overall signalling mechanisms that play an important role in the developmental stage of laminitis. OBJECTIVE: To use a broad genomic screening technique to identify novel genes that are differentially regulated in the equine lamellae during the developmental period of laminitis. METHODS: Differential mRNA display (DRD) was performed to discover regulated genes, and real-time quantitative polymerase chain reaction (RT-qPCR) was then used to evaluate lamellar mRNA levels of a regulated gene (MAIL) and mediators related to that gene (IL-1beta and IL-6) in control horses (n = 5) and horses administered black walnut extract (BWE; n = 5). RESULTS: Using DRD, MAIL was identified as a regulated gene. RT-qPCR indicated a 4-fold increase in expression of the MAIL mRNA in BWE lamellae compared to controls. A 30-fold increase in IL-1beta, and a 160-fold difference in IL-6 mRNA expression was present in BWE lamellae. Differences in MAIL, IL-1beta and IL-6 mRNA expression were statistically significant between groups (P < 0.05). CONCLUSIONS AND POTENTIAL RELEVANCE: The data strongly support a role for inflammatory cytokines in the developmental stages of laminitis, possibly inducing the vascular and metabolic alterations reported to occur in the affected digit. These results potentially support the use of anti-inflammatory drugs in horses at risk of laminitis, and warrant further investigation of the link between systemic disease processes associated with laminitis and the reported digital inflammation.     

Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance

We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of the same tissues were screened for SDS-renaturable and native MMP-9 activities by denaturing and non-denaturing gelatin zymography. Samples were also screened for MMP-2 and MMP-9 gene expression by RT-qPCR. Control laminae were devoid of both MMP-9 and neutrophils, whereas neutrophils and SDS-renaturable MMP-9 activity were detected in laminae from BWE-treated horses and were strongly correlated at the acute stage of the disease at which time laminar MMP-9 gene expression was significantly (15-fold) elevated. In contrast, BWE-treatment did not significantly elevate MMP-2 gene or protein expression in the laminae. Interestingly, MMP-9 that was present in extracts of laminae from BWE-treated horses at both the prodromal and acute stages of the disease was mainly in the zymogen form, suggesting that the accumulation of the MMP did not contribute to pathology during these stages. However, elevated presence of the MMP-9 zymogen in the tissue would predispose it to catastrophic damage should conditions arise that cleave the regulatory propeptide domain.     

Expression of interleukin-1beta in the digital laminae of horses in the prodromal stage of experimentally induced laminitis

OBJECTIVE: To study expression of interleukin-1beta (IL-1beta) in the digital laminae of horses in the prodromal stage of experimentally induced laminitis. ANIMALS: 8 healthy adult horses with no signs of laminitis. PROCEDURES: Black walnut extract was administered via nasogastric tube to 4 horses, and water was administered to the remaining 4 (controls). Complete blood counts and physical examinations were performed every 30 minutes after administration of black walnut extract or water. General anesthesia was induced when total WBC count decreased by 30% in horses given the black walnut extract and 3 hours after water administration in control horses. The left forefoot was perfusion fixed with neutral-buffered 10% formalin, and paraffin-embedded sections of the digit were used for in situ hybridization with an equine-specific IL-1beta probe. RESULTS: IL-1beta mRNA expression was observed in perivascular cells of the small laminar venules and capillaries in all 4 horses given black walnut extract and in interstitial cells remote from the microvasculature in 1 of the 4. Other cellular components of the laminar tissue and cellular components of the digital arterioles and veins did not exhibit IL-1beta mRNA expression. Expression of IL-1beta mRNA was not detected in laminae from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that IL-1beta mRNA is expressed by perivascular cells in the laminar tissues of horses in the prodromal stage of experimentally induced laminitis. This provides evidence of an inflammatory process during the prodromal stage of laminitis, indicating that local digital proinflammatory cytokine expression may be an initiating factor in laminitis.     

A scanning electron microscopical study of the dermal microcirculation of the equine foot

The microcirculation of the dermal laminae and papillae of the equine foot from seven clinically normal Australian ponies was studied using an improved microvascular casting corrosion technique and scanning electron microscopy. Casts of veins, arteries, capillaries and arteriovenous anastomoses (AVAs) were readily identified by their characteristic surface morphology. Arteries entered the laminar circulation axially, between pairs of axial veins, and were connected to each other by smaller calibre interconnecting arteries. Short abaxial branches of the axial interconnecting arteries gave rise to tufts of predominantly, proximodistally orientated, capillaries arranged abaxially in rows. The laminar veins anastomosed with each other extensively (the axial venous plexus) and formed most of the vascular skeleton of casts of the dermal laminae. AVAs were found throughout the laminar circulation but the largest and longest (40 mu diameter) were found clustered close to the origin of the axial arteries. The density of the laminar AVAs was estimated to be 500 AVAs/cm2. Blood vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog shared a basic structural organisation. The cast of each papillary unit consisted of a central artery and vein enmeshed in a sheath of fine capillaries. At intervals along the length of the central artery were short branches which gave rise to tufts of capillaries. The capillaries formed a tortuous anastomosing plexus which encircled the papillary unit and drained into the central vein at intervals along its length. AVAs were always present at the base of the papillary units and anastomoses connected the central artery and vein. AVAs are important components of the dermal microcirculation of the equine foot and their distribution and density is compatible with their proposed role in the pathophysiology of equine laminitis.     

Characterisation and distribution of epidermal growth factor receptors in equine hoof wall laminar tissue: comparison of normal horses and horses affected with chronic laminitis

Epidermal growth factor (EGF) receptors were detected in plasma membrane preparations of equine hoof wall laminar tissue at concentrations comparable to that of equine liver. Scatchard analysis of the equilibrium binding data suggested the presence of two classes of EGF binding sites in most of the controls (plasma membranes from clinically normal horses); a high-affinity class and a more numerous low-affinity class. The dissociation constant of the low-affinity class of EGF-specific receptors (KD = 1 x 10(-9)M) is in reasonable agreement with other values established for the EGF receptor. The variability between individual estimates for the KD of the high-affinity receptor class precluded an accurate estimate for those sites. A possible explanation is discussed. The high-affinity binding sites were uniformly absent in plasma membranes prepared from horses affected by chronic laminitis. Autoradiographic analysis localised the EGF receptors primarily to the secondary epidermal laminae, with an apparent greater density over the proliferative basal keratinocytes. Little label was associated with the dermal or the keratinised primary epidermal laminae. Tissue from horses with chronic laminitis had EGF receptors located uniformly over the hyperplastic epidermal keratinocytes. These data suggest that an EGF-mediated response may be involved in the hyperproliferative response that is characteristic of chronic laminitis.