Endothelial cells and angiogenesis in the horse in health and disease-A review.

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.

Pododermal angioarchitecture in the equine hoof wall: A light and scanning electron microscopic study of the wall proper.

BACKGROUND: Blood supply is an important factor for the normal function of the equine hoof, but earlier studies present conflicting data on functional characteristics of its angioarchitecture. OBJECTIVE: Emphasis was laid on demonstration of the microvascularisation in the different hoof wall regions, aiming at assessment of specialised vascular structures, e.g. vascular sphincter mechanisms and arteriovenous anastomoses. METHODS: The angioarchitecture of the adult pododerma in the equine hoof wall was examined by scanning electron microscopy of micro-corrosion casts assisted by exemplary histological and immuno-histochemical characterisation of the pododermal vasculature. RESULTS: The microvasculature of the lamellae and terminal papillae in all hoof wall regions was described in detail. Focal dilations and microvascular sphincters were a common feature. In contrast to former investigations, true arteriovenous anastomoses were detected at the base of the primary lamellae and the terminal papillae only, while thoroughfare channels proved a regular element within the microvasculature of the wall proper. Bicuspid venous valves were detected as regular feature. For the first time, the alpha-smooth muscle actin-reactivity of the microvascularisation in the hoof wall was systematically assessed, verifying its specialised vasomotor devices. CONCLUSIONS: The vasculature of the hoof wall displays specific angio-adaptations to high pressure and tensile load.

Evaluation of arterial digital blood flow in dairy cows with claw horn disruption lesions using Doppler ultrasonography.

Vascular changes play an important role in the pathogenesis of claw horn disruption lesions in cattle. The aim of the study was to measure arterial blood flow in the hind limbs of German Holstein cows with claw horn disruption lesions. A 10-MHz linear transducer was used to assess blood flow in the interdigital artery in the dorsal pastern region in the hind limbs of 11 non-lame and 33 lame German Holstein cows in which lameness was scored clinically. Qualitative and quantitative blood flow parameters were compared in affected limbs and unaffected contralateral hind limbs in lame cows and in the hind limbs of lame cows and non-lame cows. A pulsed-wave Doppler signal suitable for analysis was obtained in 78 of 88 limbs (33 affected and contralateral limbs, 22 limbs of control cows). Blood flow curve types 1 and 2 were predominant in the hind limbs of lame cows. Vessel diameter, end-diastolic velocity, and blood flow rate were significantly greater in lame cows than in non-lame cows and were numerically greater in moderately lame cows than in mildly lame cows. The differences in the qualitative and quantitative parameters between lame and non-lame cows were most likely caused by inflammation of the pododerm. The role of weight distribution between the paired hind limbs and the existence of claw horn disruption appeared to have an effect on the differences in local circulation in the affected and unaffected contralateral hind limbs in lame cows.

Endothelium-dependent relaxation mechanisms involve nitric oxide and prostanoids in the isolated bovine digital vein.

Endothelial dysfunction contributes to the development of ungulate's laminitis. Although extensively studied in equines, the endothelial function is not fully examined in bovine digital veins (BDVs). BDVs were studied under isometric conditions to describe the acetylcholine (ACh) endothelium-dependent relaxation. Concentration-response curves were constructed to phenylephrine, ACh, and sodium nitroprusside (SNP). Relaxation responses were evaluated using either phenylephrine or depolarizing high-potassium Krebs solution (DKS) as precontraction agents. Endothelium denudation and incubation with L-NAME (300 µM), indomethacin (10 µM) or both were used to explore endothelial-mediated mechanisms. Endothelium denudation did not modify phenylephrine and SNP responses, however, significantly (p < 0.05) converted a relaxation (63.2 ± 5%) response to ACh into a contraction (30.3±9%). The ACh-evoked relaxation was significantly (p < 0.05) reduced in the presence of indomethacin (37.5 ± 6%) and L-NAME (6.40 ± 2%). The presence of both inhibitors abolished the ACh-evoked relaxation. Although DKS caused a higher precontraction than phenylephrine, ACh-evoked relaxation (22.4 ± 3.4%) was still observed and was reduced by the combination of inhibitors (7.0 ± 1.0%). The ACh endothelium-dependent relaxation in BDVs is essentially mediated by nitric oxide and endothelium-derived prostanoids. The BDV endothelium function is a dynamic component in the control of the bovine digital blood flow, particularly under endothelial dysfunction conditions when venoconstriction might lead to postcapillary resistance increase.

Sole haemorrhages in Danish bull calves: Prevalence and risk factors.

To evaluate the prevalence of sole haemorrhages among Danish dairy breed bull calves and associated risk factors, 730 calves were hoof trimmed shortly before slaughter and the presence of sole haemorrhages was recorded. Associations between the outcomes sole haemorrhages and severe sole haemorrhages, and the explanatory variables daily weight gain, liver abscesses and flooring, were analysed. Of the 730 calves, 545 (74.7%) had sole haemorrhages and 119 (16.3%) had severe sole haemorrhages. Calves housed on slatted concrete floors with cubicles had higher odds of sole haemorrhages (odds ratio, OR, 2.69, 95% confidence interval, CI, 1.81-4.00; P<0.001) and severe sole haemorrhages (OR 2.07, 95% CI 1.36-3.14; P<0.001) than calves housed on deep litter straw. The odds of severe sole haemorrhages increased with increasing daily weight gain (OR 1.28, 95% CI 1.07-1.54 for an increase of 100g/day; P=0.008).

Claw disorders in dairy cattle - an unexpected association between energy metabolism and sole haemorrhages.

The present study investigated whether changes of energy metabolism post-partum (pp) are associated with claw health. For this purpose, back-fat-thickness (BFT) was measured and blood samples were taken from 146 cows at four examination times. The serum levels of free fatty acids (FFA), ß-hydroxybutyrate (BHB) and glucose were measured. Furthermore, in the first week postpartum (pp) and at 8 weeks pp, a claw trimming was done and the presence and extent of sole haemorrhages (SH) was recorded. Animals with high BFT at calving and therefore high fat mobilisation and whose FFA and BHB levels in the first week pp exceeded the reference values had fewer pathological changes of the claws than thinner animals whose FFA and BHB levels stayed within reference ranges. The body condition before calving, represented in this study by BFT, plays an important role in non-infectious claw disorders. Poorer body condition was found to be associated with the SH that develop in the first 2 months of lactation.

Técnica venográfica contrastada in vivo dos dígitos de ovinos e caprinos / Contrast venography technique in vivo of the digits of sheep and goat

Foram utilizados 10 membros torácicos e pélvicos de ovinos e caprinos hígidos, de raça e sexo variados, com idade entre dois e quatro anos e peso corpóreo médio de 53kg. A porção distal dos membros torácicos e pélvicos foi submetida à técnica venográfica contrastada e à contagem vascular venosa da mesma região, em ambas as espécies. Não foram observadas diferenças numéricas em relação às veias entre machos e fêmeas, nem entre os membros direito e esquerdo da mesma espécie. Os ovinos possuem maior número de veias em relação aos caprinos. A técnica venográfica distal anterógrada de ambos os membros em ovinos e caprinos se mostrou aplicável revelando a vasculatura distal e podal, a comunicação entre os vasos venosos e a quantidade dos vasos presentes na região.

Ten forelimbs and hindlimbs of healthy sheep and goats, of varied breeds and gender with ages ranging between two and four years and an average body weight of 53kg were used in the study. The forelimbs and hindlimbs underwent a contrasted venography of the distal region. No numerical differences were observed in relation to veins between males and females and between the left and right members of the same species. Sheep had more veins than goats. The antiretrograde venography technique of both limbs in sheep and goats was proved to be applicable, showing the vasculazation of the distal region of the foot, the communication between the vessels and the quantity of vessels.

Evaluation of treatments for claw horn lesions in dairy cows in a randomized controlled trial.

Lameness is one of the most significant endemic disease problems facing the dairy industry. Claw horn lesions (principally sole hemorrhage, sole ulcer, and white line disease) are some of the most prevalent conditions. Despite the fact that thousands of animals are treated for these conditions every year, experimental evidence is limited on the most effective treatment protocols. A randomized, positively controlled clinical trial was conducted to test the recovery of newly lame cows with claw horn lesions. Animals on 5 farms were locomotion scored every 2wk. Cows were eligible for recruitment if they had 2 nonlame scores followed by a lame score and had a claw horn lesion on a single claw of a single foot. Following a therapeutic trim, enrolled cows were randomly allocated to 1 of 4 treatments: treatment 1-no further treatment (positive control; TRM), treatment 2-trim plus a block on the sound claw (TB), treatment 3-trim plus a 3-d course of the nonsteroidal anti-inflammatory drug (NSAID) ketoprofen (TN), treatment 4-trim plus a block plus ketoprofen (TBN). The primary outcome measure was locomotion score 35d after treatment, by an observer blind to treatment group. Descriptive statistics suggested that treatment groups were balanced at the time of enrollment, that is, randomization was successful. Based on a sound locomotion score (score 0) 35d after treatment, the number of cures was 11 of 45 (24.4%) for TRM, 14 of 39 (35.9%) for TB, 12 of 42 (28.6%) for TN, and 23 of 41 (56.1%) for TBN. The difference between TBN and TRM was significant. To test for confounding imbalances between treatment groups, logistic regression models were built with 2 outcomes, either sound (score 0) or nonlame (score 0 or 1) 35d after treatment. Compared with TRM, animals that received TBN were significantly more likely to cure to a sound outcome. Farm, treatment season, lesion diagnosis, limb affected, treatment operator, and stage of lactation were included in the final models. Our work suggests that lameness cure is maximized with NSAID treatment in addition to the common practices of therapeutic trimming and elevation of the diseased claw using a block when cows are newly and predominantly mildly lame.

Scintigraphic tracking of mesenchymal stem cells after intravenous regional limb perfusion and subcutaneous administration in the standing horse.

OBJECTIVE: To assess distribution, uptake, and persistence of radiolabeled mesenchymal stem cells (MSC) using scintigraphy after intravenous regional limb perfusion (RLP) and subcutaneous injections in standing, sedated horses. STUDY DESIGN: Experimental study. ANIMALS: Horses (n = 12). METHODS: Six horses had RLP performed through the cephalic vein on 1 limb and subcutaneous injection in the metacarpal area in the opposite limb. The other 6 horses had RLP performed through the lateral palmar digital vein and subcutaneous injection in the coronary band. A pneumatic tourniquet was used for the RLP. MSC were labeled with technetium-HMPAO. Scintigraphic images were obtained at the time of injection, 1, 6, and 24 hours later. Results of RLP were compared with results from previous studies where similar injections were performed in anesthetized horses. RESULTS: Both RLP techniques led to greater variability, lower uptake, lower persistence, and poorer distribution when compared to results previously reported for horses under general anesthesia. The subcutaneous injections in the metacarpal area and coronary band resulted in MSC loss to the general circulation but no evidence of local migration. CONCLUSION: Due to partial or complete failure of the tourniquet, RLP performed in the standing horse as described is less efficient than performed under general anesthesia. Further work is needed to optimize the use of tourniquets to perform RLP for MSC administration in standing patients. The subcutaneous injections did not result in local migration in these normal horses.

Comparison of Hematologic and Biochemical Test Results in Blood Samples Obtained by Jugular Venipuncture Versus Nail Clip in Moluccan Cockatoos (Cacatua moluccensis).

In birds, blood samples are often collected from the jugular, medial metatarsal, and basilic vein. Samples are sometimes collected by toe nail clip, but concerns to avoid drawing blood from the nail include pain after nail clips for blood collection, potential differences in complete blood count (CBC) results, and potential contamination with uric acid values. To compare differences in biochemical and hematologic values in blood samples obtained by jugular venipuncture versus toenail clip, blood samples were collected from Moluccan cockatoos (Cacatua moluccensis) (N = 23) and sent to a commercial laboratory for routine CBCs and serum biochemical analysis. Results showed good agreement between venipuncture and nail clip blood samples in red blood cell count, packed cell volume, heterophil count and percentage, lymphocyte count and percentage, aspartate aminotransferase, chloride, creatine phosphokinase, glucose, lactate dehydrogenase, total protein, and uric acid values. Constant bias was found in values of bile acids, cholesterol, and hemoglobin. Proportional bias toward higher values in the jugular sample were found in total white blood cell (WBC) count and inorganic phosphorus. Serum calcium plots revealed a proportional bias toward higher values in the toe nail blood when values were increased. Results suggest some differences in WBC count, bile acids, calcium, cholesterol, hemoglobin, and phosphorus values between blood samples collected by jugular venipuncture and samples collected by toe nail clip, but the differences are mostly minor and, with the possible exception of inorganic phosphorus and marginally elevated or very low WBC counts, are unlikely to affect the use or interpretation of the avian blood panel.

The microvasculature in the equine distal phalanx: implications for fracture healing.

OBJECTIVES: To describe the intra-osseous microvasculature of the distal phalanx of the equine forelimb with regard to its potential clinical relevance. METHODS: Eleven clinically normal equine forelimbs were used from six adult horses (range: 4 to 18 years old) euthanatized for reasons unrelated to lameness. In each limb the median artery was catheterized at the level of the carpus and India ink was injected under constant manual pressure. The limbs were frozen and 5 mm thick sections of the foot were cut in the sagittal, coronal, or transverse planes on a band saw. The sections were fixed in 10% formalin and cleared using a modified Spalteholz technique. Once cleared, the sections were photographed and the microvascular anatomy identified. RESULTS: The vascular injections revealed a rich intra-osseous microvascular supply of the distal phalanx originating from the medial and lateral palmar digital arteries. In addition, numerous smaller vessels from the terminal arch, formed by anastomosis of the medial and lateral palmar digital arteries, could be seen branching into the distal aspects of the distal phalanx. This distal portion of the distal phalanx appeared more densely vascularized than the proximal part in all specimens examined. CLINICAL SIGNIFICANCE: The increased vascularity demonstrated in the distal portion of the distal phalanx appears to correlate with improved fracture healing reported in this area. This may also explain why healing fractures which involve both the distal and proximal portions of the distal phalanx have been described as progressing from distal-to-proximal.

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia.

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X1-5, 7) was detected in all sampled equine tissues, whereas P2X6 receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X1-3, 7 antibodies, and these were used in immunohistochemistry studies. P2X1-3, 7 receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X3 subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X2 and P2X3 receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X1-3 receptor subunits, whereas glial cells were positive for P2X7 and P2X1 and 2 receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.

Seasonal variation in maintenance of phenylephrine-induced tone in isolated equine digital arteries under hypoxic or hyperoxic conditions in vitro.

Digital vasoconstriction, ischaemia and hypoxia may predispose to acute laminitis. Laminitis incidence varies seasonally, peaking in spring and summer. Direct seasonal influences on equine digital artery (EDA) contractility have not been investigated. This study assessed seasonal variation in maintenance of phenylephrine (PHE)-induced tone in isolated EDAs under hypoxic (95% nitrogen) and hyperoxic (95% oxygen) conditions. The objective was to measure change in arterial tone over time after constriction to a plateau with PHE. Tone was measured at plateau and over time and percentage change calculated. Hyperoxic EDAs maintained PHE-induced tone over 1 h with no seasonal variation. Hypoxic EDAs relaxed in fall (median [inter-quartile range] 59% [44-77%] decrease from plateau; P=0.008), contracted in spring (65% [20-192%] increase from plateau; P=0.03) and did not significantly change tone in winter (18% [0-28%] decrease; P=0.13). Continued contraction under hypoxic conditions in spring may contribute to digital vasoconstriction.

Short-term incubation of equine laminar veins with cortisol and insulin alters contractility in vitro: possible implications for the pathogenesis of equine laminitis.

This study investigated the effects of cortisol and insulin, hormones that affect both glycaemic status and vascular function, on the in vitro contractility of isolated healthy equine small laminar veins. Small veins (150-500 µm) draining the digital laminae from healthy horses or ponies were investigated by wire myography. Concentration response curves were constructed for noradrenaline (NA), phenylephrine (PE), endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) in the presence of either cortisol (10(-6 ) m) or insulin (1000 µIU/mL). Cortisol significantly increased the maximum contractility of laminar veins to the vasoconstrictors NA and 5-HT but decreased the maximal contraction to ET-1. Insulin decreased the contractility of vessels to PE and ET-1. It is possible that short-term cortisol excess could enhance venoconstrictor responses to 5-HT and NA in laminar veins in vivo, thereby predisposing to laminitis. Additionally, a reduction in the ability of insulin to counteract alpha-adrenoreceptor and ET-1-mediated contraction, likely to occur in subjects with insulin resistance, may further exacerbate venoconstriction in animals prone to laminitis. These mechanisms may also predispose horses with disorders such as equine Cushing's disease and equine metabolic syndrome to laminitis.

Effects of the addition of endotoxin during perfusion of isolated forelimbs of equine cadavers.

OBJECTIVE: To examine the effect of endotoxins on metabolism and histopathologic changes of isolated perfused equine forelimbs. SAMPLE: Forelimbs (comprising the metacarpus and digit) were collected from cadavers of 12 healthy adult horses after slaughter at an abattoir (14 limbs; 1 forelimb of 10 horses and both forelimbs of 2 horses). PROCEDURES: Forelimbs were perfused for 10 hours with autologous blood, with and without the addition of endotoxin (80 ng of lipopolysaccharide [LPS]/L). Two limbs of the endotoxin exposure group and 2 nonperfused limbs were loaded to failure of the suspensory apparatus of the pedal bone to evaluate the effect of body weight. Metabolic and histologic variables were evaluated. RESULTS: Blood pressure increased during the first hour and did not differ between groups. Lactate dehydrogenase activity was similar in both groups and increased significantly during the 10-hour period; glucose consumption at 5 hours and lactate concentration at 8 hours were significantly higher in limbs exposed to endotoxin. The width of secondary epidermal lamellae was greater in LPS limbs. In the primary dermal lamellae of LPS limbs, there were significantly more vessels with an open lumen and aggregates of intravascular neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: In the blood-perfused isolated forelimbs of equine cadavers, exposure to LPS led to significant changes in the laminar tissue as well as to metabolic changes. Therefore, endotoxin should be considered as a causative factor for laminitis and not merely as a risk factor.

The impact of endophyte-infected fescue consumption on digital circulation and lameness in the distal thoracic limb of the horse.

This experiment was conducted to evaluate if consumption of endophyte-infected fescue alters digital circulation in the distal thoracic limb of the horse and to assess if soundness of the hooves of horses is affected by consumption of endophyte-infected fescue. Twelve American Quarter Horses (mean initial BW 459 ± 31 kg), 6 mares and 6 geldings, were used in this 90-d study that comprised high-endophyte (E+) and low-endophyte (E-) treatment groups. Fescue seed was integrated into the E+ diet at a rate sufficient to bring total ergovaline to 200 µg/kg, and endophyte-free fescue seed was incorporated into the E- diet from d 0 to 90. From d 30 to 60, native prairie hay was replaced with high- or low-endophyte fescue hay, bringing total dietary ergovaline to 280 µg/kg (E+) and 18 µg/kg (E-). From d 61 to 90, fescue seed was ground to decrease particle size. On d 0, 30, 60, and 90, Doppler ultrasonography and thermographic imaging were used to measure the diam. of the medial palmar artery, velocity of blood flow, and surface temperature of the hoof as indicators of digital circulation. Lameness examinations were conducted on the same days. There were no consistent treatment differences observed when evaluating measurements of digital circulation. On d 60, horses in the E+ treatment group showed increased hoof sensitivity in the left limb (P = 0.02). These horses tended to have increased hoof sensitivity when both thoracic limbs were averaged (P = 0.06), and they demonstrated increased lameness during longeing (P = 0.08). Data indicated that mares may have increased digital circulation, regardless of treatment, compared with geldings (P ≤ 0.05). Heavier horses also had greater arterial diam., velocity of blood flow, and hoof temperature than lighter BW horses (P ≤ 0.05) on d 30, 60, and 90 at time points that ranged from 90 to 180 min after feeding. Although horses consuming the E+ diet demonstrated increased lameness, especially on d 60, compared with horses consuming the E- diet, the measures of digital circulation did not support the hypothesis that digital circulation was reduced. Because of observed lameness issues, limiting the access of horses to endophyte-infected fescue may be prudent.

Evaluation of coronary band temperatures in healthy horses.

OBJECTIVE: To measure coronary band temperature (CBT) in healthy horses fed high-fructan or low-carbohydrate diets and to analyze the association of CBT with diet, time of day, and ambient temperature. ANIMALS: 6 healthy horses. PROCEDURES: Horses were fed 3 diets (treatment 1, 1 g of fructan/kg fed daily in the morning; treatment 2, 1 g of fructan/kg fed daily in the afternoon; and treatment 3, a low-carbohydrate [7.2%] diet) in a 3 × 3 Latin square study design. For each horse, the CBT of all 4 limbs as well as rectal and ambient temperatures were recorded by use of infrared thermometry and standard thermometers hourly from 8 am to 10 pm for 4 consecutive days after the initiation of each diet. Each horse received each diet, and there was a 10-day washout period between each diet change. Data were analyzed by use of a mixed linear model. RESULTS: 4,320 CBTs were obtained from the 6 horses. The CBT ranged from 9.6° to 35.5°C. Coronary band temperature followed a diurnal pattern and was positively associated with ambient temperature but was not associated with diet. CONCLUSIONS AND CLINICAL RELEVANCE: CBT of healthy horses varied significantly during the day and among limbs. These results should be considered whenever increased CBT is used as an indication of incipient laminitis or in other clinical investigations.

Effects of endotoxin and influence of cyclooxygenase-2 on ß-adrenergic mediated relaxation in isolated equine digital artery.

The effects of endotoxin on ß-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA ß-adrenoceptor functionality was also evaluated. Endothelium-intact (e(+)) and/or -denuded (e(-)) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS+NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e(-)) rings acutely exposed to NS398. Fresh and incubated (e(-)) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of ß-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e(-)) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA ß-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.

The relationship between digital perfusion pressure and hoof lamellar blood flow in isoflurane-anesthetized horses.

Digital perfusion pressure (DPP) equals mean arterial pressure (MAP) at the hoof coronet minus digital interstitial pressure (DIP) within the hoof. To test whether lamellar blood flow (LBF) changes proportionately to DPP, anesthesia was induced and maintained with isoflurane in six horses to target a MAP of 60 mmHg. Arterial, venous, and hoof interstitial pressures were measured in each pelvic limb. LBF was measured using fluorescent microspheres during dobutamine infusions targeting either 60 (low), 80 (medium), or 100 (high) mmHg MAP. Following euthanasia, hoof lamina was collected for microsphere isolation. To reduce intra-individual variability, medium and high pressures and flows were divided by their respective low pressure and flow baseline values, yielding indexed variables of ΔLBF and ΔDPP. The ΔLBF correlated negatively with the ΔDPP. We conclude that LBF was not solely determined by passive pressure-flow relationships and that systemic hypertension may not effectively increase dermal LBF in horses.

The effect of cooling on the contractility of equine digital small lamellar arteries: modulating role of the endothelium.

The equine hoof displays thermoregulatory functions, and the blood vessels lying under the hoof wall are temperature sensitive. The aim of this study was to investigate the effect of cooling on the contractile responses to α-adrenoceptor and 5-HT receptor stimulation in equine small lamellar arteries using wire myography. The role of the endothelium in the response mediated by 5-HT was also evaluated. Moderate cooling caused a reduction of the contraction induced by depolarizing Krebs solution (DKS, containing 118 mm KCl) and the maximal contraction caused by UK-14304 (α(2)-adrenoceptor agonist). The potency of methoxamine (α(1)-adrenoceptor agonist) was reduced by cooling [pD(2) (-log EC(50)) at 22°C, 5.7 (5.5-6.0) versus 30°C, 5.9 (5.7-6.1)]; however, the efficacy was maintained. The contractions evoked by sumatripan and α-methyl 5-HT (5-HT receptor agonists) were not modified by moderate cooling. In contrast, a cooling-enhanced contraction was observed in response to 5-HT [maximum response (E(max)) at 22°C, 213.2 ± 13% DKS versus 30°C, 179.9 ± 11% DKS]. Furthermore, this effect was more evident in endothelium-denuded lamellar arteries (E(max) at 22°C, 270.2 ± 26% DKS versus 30°C, 172.2 ± 20% DKS), suggesting a potential modulating role of the endothelium. The L-NAME/ibuprofen-resistant relaxation in response to carbachol was reduced by cooling in small lamellar arteries precontracted with 5-HT but not phenylephrine. Therefore, a moderate reduction of temperature modulates the reactivity of small lamellar arteries by enhancing the 5-HT-mediated contraction, but inhibits the α-adrenoceptor-mediated response. Furthermore, the endothelium of these blood vessels may play an important role in preventing excessive vasoconstriction in response to 5-HT and maintaining digital blood flow in cool environmental temperatures.