• View in gallery
    Figure 1—

    Mean ± SEM intensity of MMP-2 (A and B) and MMP-9 (C and D) zymogen bands (determined by use of SDS-PAGE gelatin zymography) in laminar tissue (A and C) and skin (B and D) samples obtained from horses administered 6 L of water via nasogastric intubation (control group [Con]; n = 10), LPS (20 ng of endotoxin/kg; 5) IV, or 6 L of BWHE (10) via nasogastric intubation. Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. *Value is significantly (P < 0.05) higher than the corresponding value for the control group. ODu = Arbitrary units of optical density.

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    Figure 2—

    Representative results of western blot analysis of CD13 expression in laminar tissue and skin samples obtained from 3 horses administered either 6 L of water via nasogastric intubation (control treatment [Con]), LPS (20 ng of endotoxin/kg) IV, or 6 L of BWHE (10) via nasogastric intubation. Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. The intensity of CD13 expression in the BWHE-treated horse is greater than that detected in the control or LPS-treated horse. + = Positive control sample.

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    Figure 3—

    Representative photomicrographs of sections of laminar tissue (A, C, and E) and skin (B, D, and F) samples obtained from 3 horses administered either 6 L of water via nasogastric intubation (control treatment [Con]; A and B), LPS (20 ng of endotoxin/kg) IV (C and D), or 6 L of BWHE (E and F) via nasogastric intubation to illustrate the presence and distribution of CD13-positive myeloid cells. In the laminar tissue sections, most CD13-positive cells were located within the primary dermal laminae (PDL). Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. Laminar tissue from the control and LPS-treated horses (A and C) contains occasional extravascular CD13-positive cells that are closely associated with laminar vessels (V); skin from the control and LPS-treated horses (B and D) contains low numbers of both intravascular (open arrow) and extravascular (closed arrow) CD13-positive cells. Laminar tissue from the BWHE-treated horse (E) contains large numbers of extravascular CD13-positive cells, commonly in the PDL; skin from the BWHE-treated horse (F) contains marked numbers of extravascular CD13-positive cells (arrows). Bars in panels A and B = 100 μm; bars in panels C through F = 50.2 μm.

  • View in gallery
    Figure 4—

    Mean ± SEM number of CD13-positive leukocytes (determined via immunohistochemical analysis) in laminar tissue (A) and skin (B) samples obtained from horses administered 6 L of water via nasogastric intubation (control group [Con]; n = 10), LPS (20 ng of endotoxin/kg; 5) IV, or 6 L of BWHE (10) via nasogastric intubation. Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. See Figure 1 for key.

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

Laura M. RiggsDepartment of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Thomas M. KrunkoskyDepartment of Anatomy and Radiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Erik NoschkaDepartment of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Lindsay A. BoozerDepartment of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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James N. MooreDepartment of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Thomas P. RobertsonDepartment of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Abstract

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.

Abstract

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.

The integument, as an organ system, is composed of the avascular epidermis and the vascular dermis, which are intimately connected at the basement membrane. The hoof of horses is a specialized modification of the integumentary system and includes interdigitations of the dermis and epidermis that are termed laminae. These interdigitations dramatically increase surface area, dissipate the load, and increase the weight-bearing strength of the hoof.1,2 The findings of previous studies3,4 of acute laminitis in horses indicate that enzymatic degradation of the basement membrane of the hoof results in dermal-epidermal separation and the subsequent loss of the integrity of laminar interdigitations, which consequently compromises weight bearing.

Proteases such as MMP-2 and MMP-9 have been implicated in the disruption of the basement membrane that occurs during episodes of laminitis in horses.5–8 The MMPs are secreted in a latent (pro-MMP) form and require an activation step before they gain full enzymatic activity. Matrix metalloproteinase-2 is normally present in laminar tissue (constitutively produced by cells such as keratinocytes and fibroblasts) and is involved in growth and remodeling of the hoof. Matrix metalloproteinase-9 is produced by neutrophils, stored in tertiary granules, and released upon cellular activation.9 In recent studies10,11 of acute laminitis in horses, the presence of MMP-2 and MMP-9 in laminar tissue varies depending on whether the disease is naturally occurring or experimentally induced by means of carbohydrate overload or BWHE administration. Compared with findings in horses without laminitis, increases in MMP-2 and MMP-9 activities were detected in laminar tissue obtained from several horses with naturally occurring acute or chronic laminitis10 and in 2 horses with carbohydrate overload–induced laminitis.11 Tissues obtained from the latter 2 horses also had increased expression of MMP-2, although the authors did not indicate the Obel grade of lameness in the horses at the time that the samples were collected.12 In contrast, laminar tissue obtained from horses after administration of BWHE had increased expression and activity of MMP-9 but not MMP-2, compared with controls.6

Recent studies6,13,14 have revealed that activation of peripheral blood leukocytes is involved in the development of acute laminitis. Experimental induction of laminitis in horses via administration of BWHE has been associated with peripheral leukopenia13 followed by increases in myeloperoxidase and MMP-9 activities in both skin and laminar tissue.6,14 Both enzymes are produced in neutrophils, stored in cytoplasmic granules within those cells, and released upon cellular activation. Although changes in activities of those enzymes have been detected in horses following experimental induction of laminitis, changes associated with endotoxemia in horses have not been assessed, to our knowledge. Endotoxemia, a condition that is commonly associated with the development of laminitis under clinical conditions,15 is characterized by activation and emigration of leukocytes from the peripheral circulation.16

Because horses with diseases that are characterized by clinical evidence of endotoxemia (eg, colitis, enteritis of the proximal portion of the small intestine, and intestinal strangulation obstruction) appear to be at increased risk for development of laminitis17 and endotoxin has been detected in horses that develop laminitis subsequent to carbohydrate overload,18 there has been considerable interest in defining the relationship, if any, between endotoxemia and the development of laminitis. The purpose of the study reported here was to compare the characteristics and enzymatic products of leukocytes detected in the skin and laminar tissues of horses administered BWHE and horses administered purified LPS (endotoxin). Because experimentally induced endotoxemia and administration of BWHE result in leukopenia of similar severity and duration but only the latter induces clinical signs of laminitis, we hypothesized that there would be significant differences in the distribution and enzyme products of activated leukocytes in horses treated with BWHE or LPS. Consequently, we compared leukocyte emigration and MMP-2 and MMP-9 gene expressions and activities during the period in which horses developed Obel grade 1 lameness following administration of BWHE or LPS.

Materials and Methods

Animals—Twenty-five healthy horses (age, 5 to 15 years old) were used in the study. All horses were apparently not lame and lacked clinical evidence of systemic inflammatory disease. No evidence of preexisting laminitis was evident on lateral or dorsopalmar radiographic views of the forelimb digits. Prior to the start of the study, the hair on the middle region of the neck was clipped and an IV catheter was placed in the left jugular vein of each horse. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Georgia.

BWHE preparation—For each treatment, a BWHE was prepared as described previously.19 Briefly, 1 kg of black walnut heartwood shavings was agitated in 7 L of water at room temperature (approx 22°C) for 24 hours. An aqueous filtrate was then obtained by filtering the solution through cheesecloth. Six liters of the resulting extract was administered via nasogastric intubation to a horse that was assigned to the BWHE treatment group.

Experimental design—Horses were randomly assigned to a control group, a BWHE treatment group, and an LPS treatment group. Horses in the control group (n = 10) received 6 L of water via nasogastric intubation. Horses in the BWHE treatment group (n = 10) received 6 L of BWHE via nasogastric intubation. Horses in the LPS treatment group (n = 5) received 20 ng of O55:B5 Escherichia coli endotoxin/kg in 500 mL of saline (0.9% NaCl) solution via the jugular vein catheter over a 30-minute period. The time of treatment administration was designated as 0 hours.

Each horse was evaluated immediately prior to the start of the study (baseline assessment) and every hour thereafter for attitude, heart rate, respiratory rate, capillary refill time, hoof temperature, digital pulse presence, and evidence of Obel grade 1 laminitis. Horses were considered to have Obel grade 1 lameness when ≥ 2 of the following were detected: weight shifting, increased warmth of the hooves, and strong digital pulses without evidence of lameness while walking. Blood samples were collected at 2-hour intervals for WBC count determination.

Horses in the control group were euthanatized 12 hours after treatment. Horses in the BWHE treatment group were euthanatized at the onset of Obel grade 1 lameness or 12 hours after administration of BWHE if signs of Obel grade 1 lameness had not developed by that time. Horses in the LPS treatment group were euthanatized 12 hours after administration of LPS. All horses were euthanatized by use of a penetrating captive bolt.

Collection of laminar tissue and skin samples—Both forelimbs of each horse were disarticulated at the level of the metacarpophalangeal joint, and the hooves were cut into sections with a band saw. During the procedure, thermal damage was minimized by constant irrigation of the tissues with ice-cold physiologic saline solution (118mM NaCl, 24mM NaHCO3, 1mM MgSO4, 0.435mM NaH2PO4, 5.56mM glucose, 1.8mM CaCl2, and 4mM KCl). Two full-thickness segments from each forelimb foot were then placed in the ice-cold physiologic saline solution. The hard keratinized portion of the hoof and the distal phalanx of each forelimb were removed, and specimens of laminar tissue were collected via sharp dissection. Concurrently, full-thickness sections (6 × 6 cm) of skin from the middle region of the neck were collected. These specimens were also placed in the ice-cold physiologic saline solution. Half of the laminar and skin tissue samples were rapidly frozen in liquid nitrogen and stored at 80°C until assayed; the remaining tissue samples were fixed in neutral-buffered 10% formalin for immunohistochemical analysis.

Real-time PCR assay—To prepare the samples, the frozen tissues were first shaved with a razor blade and then homogenized by use of a motorized mortar and pestle.a Gene expression was quantified (on the basis of mRNA concentration) in a 2-step RT PCR assay. Total RNA extraction was performed by use of a total RNA purification kitb according to the manufacturer's protocol. Reverse transcription for production of cDNA was performed by use of a high-capacity cDNA RT kit.c Oligo nucleotide primers and probesd were designed by use of computer software,e and equine MMP-2 and MMP-9 were sequences obtained from GenBank.f The quantitative real-time PCR assay was performed in duplicate for each sample by use of a sequence detection systemg; the reference gene 18S rRNAh was used as an endogenous control.

Gelatin zymography—Frozen tissue samples were homogenized by use of a tissue homogenizeri in western lysis buffer (50mM HEPES, 150mM NaCl, 1% Triton X-100, 1mM egtazic acid, 6mM sodium deoxycholate, 1mM Na3VO4, 1mM NaF, 1mM phenylmethylsulphonyl fluoride, aprotinin [20 mg/mL], and leupeptin [20 mg/mL]). After homogenization, the samples were sonicated and centrifuged at 14,000 × g for 15 minutes at 4°C. A colorimetric protein assay (Bradford assay) was performed on the supernatant; aliquots of the supernatant were stored frozen at −80°C.

By use of a 4% stacking gel, 8% SDS-PAGE gels containing 0.1% gelatin were prepared. Samples were loaded onto the gels (20 μg of protein/lane) and electrophoresed at 100 V for 120 minutes. An MMP-2–MMP-9 positive control sample was included on each gel. After electrophoresis, each gel was washed twice for 30 minutes in 2.5% Triton X-100 on a rocking platform to remove the SDS. Gels were then incubated for 16 hours in 100mM Tris-HCl (pH, 8.0), 5mM CaCl2, 0.005% Brij-35, and 0.001% NaN3 at 37°C and subsequently stainedj for 60 minutes. Gels were destained on a rocking platform in 5% methanol and 7.5% acetic acid for 2 hours. Digital images of the gels were recorded by use of a quantitative chemiluminescence imaging system.k The specificity of the enzymatic activity was validated by incubating samples in a solution that lacked calcium, a requirement for gelatinase enzymatic activity; this resulted in zero enzymatic activity for the gelatin substrate (data not shown).

Western immunoblot analysis—Samples containing 20 μg of protein from previously homogenized tissue were separated via 12% SDS-PAGE, electrophoretically transferred to a membrane, and incubated with anti-equine CD13 monoclonal antibody.l At the end of that incubation period, the blots were incubated with horseradish peroxidase–conjugated secondary antibody.m Blots were then treated with chemiluminescence substrate solutionn and analyzed by use of the quantitative chemiluminescence imaging system.k A standard quantity of leukocyte-rich equine plasma was included on each immunoblot as a comparative positive control sample.

Immunohistochemical analysis—Full-thickness formalin-fixed tissues were paraffin-embedded and sectioned for immunohistochemical analysis with antiequine CD13 monoclonal antibody.l Sections were deparaffinized, and endogenous peroxidase activity was blocked by use of 1% hydrogen peroxide in water. Heat-induced epitope retrieval with sodium citrate buffer was performed followed by blocking with 10% goat serum prior to incubation with antibody. Sections were incubated with primary antibody for 16 hours at 4°C, followed by incubation with biotinylated papGIII goat anti-mouse secondary antibodyo and avidin-biotin-peroxidase complex at room temperature for 30 and 45 minutes, respectively. Staining was performed with 3,3-diaminobendidine as the peroxidase substrate, and sections were counterstained with hematoxylin. Sections were evaluated microscopically for the presence and number of CD13-positive cells by 1 investigator (LMR), who was unaware of the treatment group from which the samples were derived. The evaluation was performed by centering the field of view (40X objective lens) on a dermal vessel in the area of the primary dermal lamina and counting all CD13-positive cells in that field. One sample of both laminar tissue and skin was evaluated from each horse. For each tissue sample, 5 fields were evaluated and the mean number of CD13-positive cells was calculated.

Data and statistical analyses—Initially, the BWHE-treated horses were subdivided into 2 groups on the basis of whether they were euthanatized at the onset of Obel grade 1 lameness (n = 8) or at 12 hours after administration of the extract (without development of Obel grade 1 lameness; 2). Statistical analysis of data from these 2 subsets revealed no significant differences; furthermore, data from each subset did not differ from the control group data. Therefore, the subsets were recombined and were designated as the BWHE treatment group.

Gene expression data for MMP-2 and MMP-9 were normalized to 18S rRNA expression and analyzed relative to control tissue findings by use of the 2−ΔΔCT method.20 For RT PCR data, a 2-tailed t test was performed to analyze data from each treatment group for MMP-2 and MMP-9 expressions in skin and laminar tissues. For all comparisons, a value of P ≤ 0.05 was considered significant.

Following gelatin zymography, MMP-2 and MMP-9 bands were analyzed by use of quantitation software.p Volume intensity was measured for the MMP-2 zymogen band (72 kDa) and the MMP-9 zymogen band (92 kDa), adjusted for background stain, and normalized to findings obtained for an MMP-2–MMP-9 positive control sample that was included on each gel. Results were reported as arbitrary units of optical density. The MMP-2 and MMP-9 data for skin and laminar tissues from control, BWHE-treated, and LPS-treated horses were compared by use of a 1-way ANOVA with a Tukey-Kramer posttest.q A value of P ≤ 0.05 was considered significant. Correlation between skin and laminar tissue data was determined by use of the Pearson product-moment correlation coefficient.

The numbers of CD13-positive cells in skin and laminar tissue samples from control, BWHE-treated, and LPS-treated horses were normally distributed, as determined by use of a D'Agostino-Pearson normality test. The data from each group were compared by use of a 1-way ANOVA with a Tukey-Kramer posttest. A value of P ≤ 0.05 was considered significant.

Results

None of the horses in the control or LPS treatment groups developed signs of acute laminitis. Eight of the 10 horses in the BWHE treatment group developed clinical signs consistent with Obel grade 1 lameness and were euthanatized at the time that these signs were detected (9 to 12 hours after administration of the extract); the other 2 BWHE-treated horses were euthanatized 12 hours after administration of the extract. White blood cell counts in blood samples from horses in the LPS and BWHE treatment groups revealed similar decreases (> 30%) in total leukocyte counts. Leukocyte counts were lowest in the LPS- and BWHE-treated horses at the 2- and 4-hour time points, respectively. Circulating leukocyte counts returned to values indistinguishable from baseline values at the time of euthanasia in both groups.

MMP-2 and MMP-9 mRNA RT PCR assays—On the basis of detected MMP mRNA concentrations, gene expression of MMP-9 was significantly increased in both laminar tissue and skin from BWHE-treated horses, compared with findings in the control horses (P < 0.001 and P = 0.034, respectively; Table 1). A significant increase in MMP-2 gene expression was detected in the laminar tissue samples (P < 0.001) but not in the skin samples from horses in the BWHE treatment group (P = 0.096), compared with findings in the corresponding tissue samples obtained from horses in the control group. Gene expressions of MMP-2 and MMP-9 were significantly less in the laminar tissue and skin samples from the LPS treatment group, compared with control group data (all values of P < 0.001).

Table 1—

Concentrations of MMP-2 and MMP-9 mRNAs (ie, gene expression) in laminar tissue and skin samples obtained from horses administered LPS (20 ng of endotoxin/kg; n = 5) IV or 6 L of BWHE (10) via nasogastric intubation expressed as fold changes relative to corresponding concentrations in tissue samples obtained from horses administered 6 L of water via nasogastric intubation (control group [10]).

VariableTreatment groupLaminar tissueSkin
MMP-2LPS−6.67*−10.0*
BWHE+5.54*NS
MMP-9LPS−2.94*−3.34*
BWHE−46.3*+2.3*

Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. Fold changes representing increased and decreased concentrations, compared with the control group findings, are designated as + and − values, respectively.

Concentration was significantly (P < 0.05) different from the corresponding control group value.

NS = Fold change not reported because MMP concentration was not significantly different from the control group value.

Gelatin zymography—Mean MMP-2 zymogen activity in laminar and skin samples among the control, LPS treatment, and BWHE treatment groups did not differ significantly (P = 0.068 and P = 0.195, respectively; Figure 1). There was a very weak correlation (r2 = 0.18) for MMP-2 zymogen activity between skin and laminar samples from individual animals.

Figure 1—
Figure 1—

Mean ± SEM intensity of MMP-2 (A and B) and MMP-9 (C and D) zymogen bands (determined by use of SDS-PAGE gelatin zymography) in laminar tissue (A and C) and skin (B and D) samples obtained from horses administered 6 L of water via nasogastric intubation (control group [Con]; n = 10), LPS (20 ng of endotoxin/kg; 5) IV, or 6 L of BWHE (10) via nasogastric intubation. Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. *Value is significantly (P < 0.05) higher than the corresponding value for the control group. ODu = Arbitrary units of optical density.

Citation: American Journal of Veterinary Research 70, 11; 10.2460/ajvr.70.11.1383

Mean MMP-9 zymogen activities in laminar tissue and skin samples from horses in the control group were not significantly (P = 0.337) different (Figure 1). However, there was a significant increase in MMP-9 zymogen activity in the laminar and skin samples from the BWHE-treated horses, compared with the corresponding activities of the control horses (both P < 0.001). For the laminar tissue and skin samples, mean MMP-9 zymogen activity was not significantly (P = 0.347 and P = 0.980, respectively) increased in the LPS treatment group, compared with control group data. A weak correlation (r2 = 0.42) was identified between skin and laminar samples for MMP-9 zymogen activity from individual animals.

CD13 western immunoblot analysis—In all tissues from all treatment groups, CD13 was present at visibly detectable levels (Figure 2). A single band consistent in molecular weight (150 kDa) with that identified for leukocyte-rich equine plasma (positive control sample) was present for each individual horse sample. Band intensity was subjectively increased in skin and laminar tissue samples from the BWHE treatment group, compared with findings for samples from the LPS treatment and control groups.

Figure 2—
Figure 2—

Representative results of western blot analysis of CD13 expression in laminar tissue and skin samples obtained from 3 horses administered either 6 L of water via nasogastric intubation (control treatment [Con]), LPS (20 ng of endotoxin/kg) IV, or 6 L of BWHE (10) via nasogastric intubation. Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. The intensity of CD13 expression in the BWHE-treated horse is greater than that detected in the control or LPS-treated horse. + = Positive control sample.

Citation: American Journal of Veterinary Research 70, 11; 10.2460/ajvr.70.11.1383

CD13 immunohistochemical analysis—Analysis revealed that CD13-positive cells were present in sections of laminar tissue and skin samples obtained from horses in all 3 treatment groups. The CD13-positive cells in laminar tissue appeared to be exclusively of myeloid origin and were present only in the laminar dermis; the laminar epidermis contained no CD13-positive cells. The skin samples, like the laminar tissues, contained CD13-positive myeloid cells as well as CD13-positive cells within the sebaceous glands, dermal root sheath of hair follicles, and stratum basale of the epidermis. The distribution of CD13-positive nonmyeloid cells did not vary among treatment groups, whereas the distribution of CD13-positive myeloid cells within the tissues varied between intravascular and extravascular locations depending on the treatment group (Figure 3). The CD13-positive leukocytes were consistent with a mixed population of neutrophils and monocytes. All skin sections evaluated contained both intra- and extravascularly located CD13-positive myeloid cells, but the number of those cells varied depending on treatment group. Similarly, CD13-positive cells were detected in intra- and extravascular locations in laminar tissue sections, but such cells were rarely seen in the control group samples.

Figure 3—
Figure 3—

Representative photomicrographs of sections of laminar tissue (A, C, and E) and skin (B, D, and F) samples obtained from 3 horses administered either 6 L of water via nasogastric intubation (control treatment [Con]; A and B), LPS (20 ng of endotoxin/kg) IV (C and D), or 6 L of BWHE (E and F) via nasogastric intubation to illustrate the presence and distribution of CD13-positive myeloid cells. In the laminar tissue sections, most CD13-positive cells were located within the primary dermal laminae (PDL). Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. Laminar tissue from the control and LPS-treated horses (A and C) contains occasional extravascular CD13-positive cells that are closely associated with laminar vessels (V); skin from the control and LPS-treated horses (B and D) contains low numbers of both intravascular (open arrow) and extravascular (closed arrow) CD13-positive cells. Laminar tissue from the BWHE-treated horse (E) contains large numbers of extravascular CD13-positive cells, commonly in the PDL; skin from the BWHE-treated horse (F) contains marked numbers of extravascular CD13-positive cells (arrows). Bars in panels A and B = 100 μm; bars in panels C through F = 50.2 μm.

Citation: American Journal of Veterinary Research 70, 11; 10.2460/ajvr.70.11.1383

In laminar tissue samples obtained from the control horses, CD13-positive myeloid cells were evident within the dermal vasculature and rarely in extravascular locations (approx 0 to 2 cells/tissue section). In skin samples, CD13-positive cells were present in low numbers; these cells were located in the superficial dermis at the level of sebaceous glands and hair follicles along with intravascular myeloid cells.

In laminar tissue samples obtained from the LPS-treated horses, occasional intravascular CD13-positive myeloid cells were detected; CD13-positive cells were rarely identified in extravascular locations (approx 0 to 2 cells/tissue section). Skin samples from the LPS-treated horses also contained low numbers of CD13-positive cells, which were similar in distribution and number to those identified in skin samples from the control horses. The numbers of CD13-positive cells identified in tissue samples from LPS-treated horses were not significantly (P = 0.497) greater than the numbers of those cells in the corresponding tissue samples from control horses (Figure 4).

Figure 4—
Figure 4—

Mean ± SEM number of CD13-positive leukocytes (determined via immunohistochemical analysis) in laminar tissue (A) and skin (B) samples obtained from horses administered 6 L of water via nasogastric intubation (control group [Con]; n = 10), LPS (20 ng of endotoxin/kg; 5) IV, or 6 L of BWHE (10) via nasogastric intubation. Samples were collected after horses were euthanatized at 12 hours after treatment or at onset of Obel grade 1 lameness. See Figure 1 for key.

Citation: American Journal of Veterinary Research 70, 11; 10.2460/ajvr.70.11.1383

In laminar tissue and skin samples obtained from the BWHE-treated horses, large numbers of CD13-positive myeloid cells were evident both within and surrounding the dermal vessels. In laminar tissue sections, CD13-positive cells were located predominately along the vessels of the primary dermal laminae and in deeper sections of the dermis. Similar to findings in skin samples from horses in the other 2 groups, CD13-positive myeloid cells (along with intravascular myeloid cells) were located in the superficial dermis at the level of sebaceous glands and hair follicles in skin samples from BWHE-treated horses. The number of CD13-positive cells in tissues from horses in the BWHE treatment group was significantly increased, compared with numbers of cells in both the control and LPS treatment groups (both P < 0.001; Figure 4).

Discussion

Results of the present study have provided evidence that biochemical abnormalities develop in integumentary tissues, specifically, both the skin and laminar tissues of horses during the developmental phase of BWHE-induced laminitis. Several studies8,11,21 have revealed that MMP-9 expression and activity in laminar tissue of horses increase during development of laminitis. In the present study, MMP-9 gene expression and zymogen protein concentration were also increased in skin samples obtained from horses with experimentally induced laminitis. Unlike the results of another study6 in which MMP-9 zymogen was not present in laminar tissues from horses that were administered water, a low basal concentration of MMP-9 zymogen was detected in both skin and laminar tissues of healthy control horses in the present study. The difference between results of these 2 studies may be attributable to slight differences in zymography protocols that may have resulted in increased sensitivity for MMP-9 zymogen detection in our investigation.

Gene expressions and activities of MMP-9 in skin and laminar tissue samples obtained from horses in the control group in the present study were low, which is consistent with other data22,23 that indicate that few neutrophils are present in these tissues from healthy horses. The paucity of neutrophils in normal integument is also consistent with the low numbers of CD13-positive cells identified in tissue samples from horses in the control group. Although there was only a moderate correlation (r2 = 0.420) of MMP-9 zymogen concentration between skin and laminar tissue samples, there does appear to be a prominent difference in MMP-9 zymogen concentrations in both skin and laminar tissues from control and BWHE-treated horses.

Horses in the developmental phase of laminitis become leukopenic at the same time that an increase in neutrophils can be detected in the integument.13,14,22 Metalloproteinase-9, myeloperoxidase, and other tissue-degrading enzymes are produced by and stored in neutrophils. These enzymes are markers of neutrophil presence and may contribute to the development of laminitis. Although these enzymes are present at increased concentrations in the skin of horses at the time of onset of laminitis, there is no clinically apparent evidence of disease in these tissues. The reason that pathological changes occur selectively in the laminar tissues, and not in the skin, most likely relates to key differences between these types of integument. For example, unlike the skin, laminar tissue is subjected to extreme biomechanical loads. As an adaptation to those stresses, laminar tissue has developed complex dermal-epidermal interdigitations that are not present in skin.24,25 Moreover, the vasoactive properties of the laminar microvasculature appear to be unique; the predisposition of laminar veins to vasoconstriction and the increase in digital postcapillary resistance that develops after administration of BWHE are strongly suggestive of the contribution of venous disturbances to the disease process.26–29

The importance of the changes in the integumentary system that occur during the development of laminitis is highlighted by the data obtained from the LPS-treated horses in the present study. In a recent multivariate analysis of clinical risk factors for development of acute laminitis in horses,15 endotoxemia was the only factor significantly associated with the disease. This association was identified despite the fact that IV administration of LPS at doses 20 to 60 times as great as the dose used in the present study did not induce signs of laminitis, even when the horses were monitored for days to weeks.16 In the present study, LPS was administered at a dose of 20 ng of E coli endotoxin/kg. This dose consistently resulted in the degree of leukopenia evident following BWHE administration30,31; also, at this dose, the clinical signs associated with administration of higher doses of LPS were minimized because those effects do not develop following BWHE administration.14 The administration of LPS in the present study caused leukopenia but did not result in emigration of leukocytes into the integument, which did occur following BWHE administration. The large numbers of CD13-positive myeloid cells surrounding dermal vessels in the skin and laminar tissue samples obtained from BWHE-treated horses were not detected in the samples obtained from control or LPS-treated horses. One possible reason for the lack of emigration of leukocytes into the tissues of horses administered LPS may be that the delivery of leukocytes to the laminar capillary beds was impeded as a result of the increased precapillary resistance that develops in LPS-treated horses.31,32 Alternatively, the chemoattractants (eg, complement factor 5a, leukotriene B4, platelet-activating factor, and chemokines) that induce leukocytes to move from the vasculature and into the tissues32 may not be present in similar amounts in tissues of horses administered LPS or BWHE. Thus, it appears that the emigration of CD13-positive leukocytes and the increases in MMP-9 concentration and activity in horses with BWHE-induced laminitis are events that are distinct from those associated with acute endotoxemia.

Data from the present study and previous investigations14,22 support the concept that a generalized disruption of physiologic processes in the integument of horses occurs during the developmental phase of laminitis. To understand the naturally occurring disease process, it will be essential to determine which early pathological changes are related to the development of laminitis rather than the development of endotoxemia. This information and the ease by which skin biopsy samples can be collected from horses suggest that histologic evaluation of skin samples may become a useful tool for identifying generalized alterations in the integument of horses at risk for the development of naturally occurring laminitis. The early identification of at-risk patients would allow more accurate prognosis and timely administration of aggressive interventional treatments. Furthermore, these samples may provide a means with which the progression of changes in the integument of horses with acute laminitis can be evaluated.

ABBREVIATIONS

BWHE

Black walnut heartwood extract

LPS

Lipopolysaccharide

MMP

Matrix metalloproteinase

RT

Reverse transcription

a.

Pellet pestle, Kimble/Kontes, Vineland, NJ.

b.

Versagene total RNA purification kit, Gentra Systems, Minneapolis, Minn.

c.

High-capacity cDNA archive kit, Applied Biosystems, Foster City, Calif.

d.

Taqman Custom Probe, Applied Biosystems, Foster City, Calif.

e.

Primer Express software, Perkin-Elmer Biosystems, Waltham, Mass.

f.

NCBI. Equine MMP-2/9 nucleotide sequence. Available at: www.ncbi.nlm.nih.gov/sites/gquery. Accessed March 8, 2008.

g.

ABI Prism 7700 sequence detection system, Applied Biosystems, Foster City, Calif.

h.

TaqMan Ribosomal RNA Control Reagent, Applied Biosystems, Foster City, Calif.

i.

Polytron tissue homogenizer, Glen Mills Inc, Clifton, NJ.

j.

Coomassie brilliant blue R-250 staining solution, Bio-Rad Laboratories, Richmond, Calif.

k.

Bio-Rad Fluor-S MAX 2 MultiImager, Bio-Rad Laboratories, Richmond, Calif.

l.

Mouse anti-horse CD13 monoclonal antibody, AbD Serotec, Raleigh, NC.

m.

HRP-conjugated goat anti-mouse IgG, Pierce Biotechnology, Rockford, Ill.

n.

SuperSignal West Dura extended duration chemiluminescence substrate solution, Pierce Biotechnology, Rockford, Ill.

o.

Goat anti-mouse IgG, biotin conjugated, AbD Serotec, Raleigh, NC.

p.

Quantity One quantitation software, Bio-Rad Laboratories, Richmond, Calif.

q.

GraphPad Prism, version 3.03, GraphPad Software, San Diego, Calif.

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Contributor Notes

Dr. Riggs' present address is Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803.

Dr. Noschka's present address is Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

Dr. Boozer's present address is Red Bank Veterinary Hospital, 197 Hance Ave, Tinton Falls, NJ 07724.

Supported by an American Quarter Horse Association research grant and the White Fox Farm fund.

Presented in abstract form at the British Equine Veterinary Association Colic Research Symposium, Liverpool, England, June 2008.

Address correspondence to Dr. Riggs (lriggs@vetmed.lsu.edu).