Acute laminitis is a severely debilitating and often life-threatening or career-ending disease of the sensitive and insensitive laminae of the digit of horses; horses with acute laminitis show signs of excruciating pain. Histologic studies of the hooves of horses with early laminitis have revealed pathologic changes in the basement membrane without epidermal necrosis at 48 hours after CHO-induced lameness develops,1 epidermal changes without pathologic changes in the basement membrane or edema at 4 to 8 hours after onset of lameness,2 and edema of the primary and secondary lamellae at 12 hours after onset of lameness.3,4 Thrombosis within the microvasculature accompanies these changes.5,6 To the authors' knowledge, histologic studies of the feet of horses during the first 24 hours after CHO have not been performed. Garner et al7 introduced the hypothesis that the predominant cause of laminitis was a disturbance in digital blood flow that occurred during the onset of the syndrome after CHO of the gastrointestinal tract. By use of contrast radiography, reduced perfusion in the terminal vasculature of the foot after the onset of lameness has been identified.8 It has also been determined that the changes in digital perfusion are associated with a decrease in right atrial pressure.9
The mechanisms responsible for digital hypoperfusion were first evaluated by tracing radioactive albumin particles through the foot during the development of laminitis.10 A reduction in laminar capillary perfusion and shunting of blood at the level of the coronary band suggest the presence of arteriovenous anastomoses that open during the development of laminitis, resulting in hypoperfusion of the digital microcirculation.10 Furthermore, Kirker-Head et al11 and Hood et al12 independently determined that this hypoperfusion was accompanied by decreased oxygen delivery to the digital tissues and decreased hoof wall surface temperature, respectively. However, Pollitt et al13 failed to detect this early-stage decrease in digital blood flow but detected a later increase in hoof temperature. A low ambient temperature may have precluded the ability to detect the prodromal digital hypothermia.12
In studies14,15 involving isolated perfused digits, changes in digital microvascular hemodynamics and Starling forces in horses with early laminitis that developed subsequent to administration of CHO or black walnut extract include decreased blood flow and precapillary-to-postcapillary resistance ratio and increased capillary and laminar interstitial pressures. This imbalance promotes the flux of fluid across the capillary bed within the foot, resulting in laminar edema.14,15 Increased tissue pressure could exceed the critical closing pressure of the capillary bed and contribute to poor capillary perfusion,14 or alternatively, the interstitial edema could contribute to degradation of extracellular matrix.16 Therefore, the predominant hemodynamic alteration in experimentally induced laminitis is development of increased postcapillary resistance subsequent to increased venomotor tone.
Endothelin-1 is a potent vasoconstrictor peptide produced by endothelial cells, vascular smooth muscle cells, and macrophages.17 It not only induces prolonged vasoconstriction in arteries and arterioles, but also causes profound venoconstriction in the systemic and pulmonary circulations.17 High concentrations of ET-1 have been measured in jugular and mesenteric venous blood during endotoxemia.18 These increased ET-1 concentrations correlate with the severity of intestinal mucosal damage during endotoxemia in rats.18 Most of the ET-1 produced by endothelial cells is released locally into the vascular wall, allowing direct contact with vascular smooth muscle cells and resulting in intense vasoconstriction.17 Although high concentrations of ET-1 directly cause vasoconstriction, lower concentrations can enhance the vasoconstrictor action of other vasoactive mediators, such as epinephrine, serotonin, and histamine,19 which exert potent vasoconstrictor effects in normal and diseased digital vessels of horses.20–23 Endothelin synthesis is stimulated by epinephrine and cytokines.17 The circulating concentrations of epinephrine and cytokines are increased during many diseases in horses that are characterized by an inflammatory response (eg, pleuropneumonia, endometritis, intestinal ischemia, enterocolitis, and duodenitis-jejunitis), and these substances are empirically linked to the development of laminitis.
Recently, increased amounts of ET-1 in laminar connective tissues of horses with experimentally induced and naturally occurring laminitis were detected by use of immunohistochemistry.24 Furthermore, endotoxin-induced tissue ischemia and necrosis are accompanied by increases in concentrations of ET-1 in venous blood.18 Tissue damage and ischemia are reversed by local infusion of an ET receptor antagonist after administration of endotoxin.18 Additionally, circulating ET-like immunoreactivity is increased in horses with naturally acquired gastrointestinal tract disease, especially those with enterocolitis, strangulating obstruction, and peritonitis25; horses with these diseases are highly predisposed to the development of laminitis. Endothelin-1 is a likely candidate to be involved in the hemodynamic changes in the digit of horses with laminitis because of its ability to induce profound, sustained vasoconstriction (especially venoconstriction), the fact that its production and release are induced by mediators (eg, epinephrine and cytokines) and processes (eg, ischemia and endotoxemia) proposed to be involved in the pathophysiology of laminitis, and its potentiation of numerous vasoactive agents possibly involved in laminitis.
In blood vessels, biosynthesis of ET-1 occurs in the endothelium, and receptors for ET-1 are located on endothelial and vascular smooth muscle cells.26 In these cells, the 2 principal receptor types that respond to ET-1 are ETA and ETB receptors. Vascular ETB receptors are further subclassified into the ETB1 and ETB2, which are located on endothelial cells and vascular smooth muscle cells, respectively. The ETA receptors are predominantly located on vascular smooth muscle cells; through several signal transduction mechanisms, ET-1 binding to these receptors results in slowly developing, but sustained, vasoconstriction.27 Vascular ETB1 receptors are located principally on endothelial cells, and it is thought that during normal physiologic conditions these receptors trigger the release of the endothelial-derived relaxing factor, nitric oxide (NO).27 Through these mechanisms, ET-1 and NO contribute to maintenance and homeostasis of vasomotor tone. The actions of 2 antagonists of both ETA and ETB receptors (PD145065 and PD142893) have been evaluated in equine digital and colonic vessels.28,29 Of the 2 antagonists, PD145065 is more effective and, at a concentration of 10−5M, completely inhibits the in vitro contractile effects of ET-1 in palmar digital veins and arteries.29 These findings are supported by results of a study30 involving rabbit femoral and pulmonary arteries, which indicated that PD145065 is 10-fold as potent as PD142893 in binding to ETA and ETB receptors.
Nitric oxide is a potent vasodilatory substance that mediates relaxation of the vasculature induced by acetylcholine to maintain perfusion of vital tissues.31 Nitric oxide is synthesized by the enzyme, NO synthase, from the dietary amino acid L-arginine and oxygen with NADPH as a cofactor.32 Different types of NO synthase have been discovered and are distinguished by differences in their functional expression and by the amount and duration of NO release.33,34 Endothelial cells contain constitutive NO synthase, which is a potent regulator of vascular tone via release of small, intermittent, pulsatile quantities of NO.31 In horses with CHO-induced laminitis, acetylcholine-mediated relaxations of digital vessels in vitro are decreased, suggesting that the NO-producing capacity of the digital vascular endothelium is reduced, thereby rendering the vessels more sensitive or vulnerable to vasoconstrictive agents.21 Injection of nitroglycerin increases NO concentration independent of NO synthase activity. Therefore, nitroglycerin may improve microvascular function during the prodromal stages of laminitis.
The purpose of the study reported here was to evaluate changes in digital vascular function in horses with CHO-induced laminitis and determine the effects of an ET receptor antagonist and nitroglycerin on laminitis-associated vascular dysfunction. We hypothesized that during the prodromal stages of CHO-induced laminitis, there is digital vascular endothelial dysfunction resulting in decreased digital vascular endothelial production of NO and increased digital vascular smooth muscle concentrations of ET-1 that lead to intense venoconstriction and subsequent decreases in digital blood flow. Consequently, this study was undertaken to measure digital hemodynamic and vascular alterations in horses with CHO-induced laminitis after local intra-arterial infusion of an ET receptor antagonist and NO donor into the digital circulation and compare those data with findings in horses treated with saline (0.9% NaCl) solution alone.
Materials and Methods
Animals—All procedures were approved by the Louisiana State University Institutional Animal Care and Use Committee. Twenty adult horses of various breeds (age range, 5 to 15 years) were determined to be free of laminitis on the basis of findings of physical and lameness examinations and radiographic evaluation. A CBC and serum biochemical analyses were performed, and plasma fibrinogen concentration was assessed to rule out inflammatory or systemic disease.
Fourteen days prior to experimentation, the horses underwent surgery to place a probe for digital arterial blood flow measurement and isolate the medial palmar artery. Food was withheld from horses 6 hours before surgery. A catheter was placed IV for administration of penicillin G potassium (22,000 U/kg, IV, q 6 h), gentamicin (6.6 mg/kg, IV, q 24 h), and phenylbutazone (2.2 mg/kg, PO, q 12 h) beginning before surgery and continuing for 3 days. Anesthesia was induced with xylazine hydrochloride (1.1 mg/kg, IV), guaifenesin (50 mg/kg, IV), and ketamine hydrochloride (2.2 mg/kg, IV) and was maintained with halothane (1% to 3%) in oxygen.
Surgery—During anesthesia, a calibrated 3-mm perivascular ultrasonic flow probea was placed around the medial palmar digital artery of the right forelimb for measuring digital arterial blood flow. Additionally, the medial palmar artery (at the level of the midmetacarpal region) was isolated and elevated, and the fascia was sutured beneath it to hold the vessel in a more accessible subcutaneous position. The skin was sutured over the vessel, and a sterile dressing and bandage were applied to the limb. Horses were each kept in a box stall and fed a completed pelleted feed for 14 days after surgery.
Experimental and temporal design—Carbohydrate overload was induced 7 days after flow probe implantation and medial palmar artery elevation. Twenty horses were randomly allocated to 1 of 4 groups. All horses were administered a laminitis-inducing ration (85% corn starch and 15% wood flour) at the rate of 17.6 g of starch/kg of body weight via a nasogastric tube.6,9,24 Hemodynamic variables were recorded before (baseline) and hourly after all horses were administered a CHO ration via nasogastric tube.
All catheters were placed percutaneously following routine aseptic preparation of the skin and SC administration of a local anesthetic. A 14-gauge, 13.3-cm polytef catheter was inserted into the left jugular vein for administration of anesthetics. Polyethylene tubing was passed into the right atrium via a catheter introducer placed in the right jugular vein. The ET receptor antagonist or saline (0.9% NaCl) solution (control treatment) was administered via a 20-gauge, 3.9-cm polytef catheter placed in the medial palmar artery. The position of each catheter was confirmed by observation of typical pressure waveforms.
In group 1, an ET receptor antagonistb was administered to each horse at 4 and 8 hours after administration of the laminitis-inducing ration, whereas in group 3, an ET receptor antagonist was administered to each horse at 4, 10, and 16 hours after receiving the laminitis-inducing ration. In groups 2 and 4, an equivalent volume of saline solution was administered to each horse at times similar to those used in groups 1 and 3, respectively. For the horses receiving the ET receptor antagonist, the amount needed to yield a 10−5M concentration in digital arterial blood (based on the digital arterial blood flow) was calculated and this dose was infused over a 2-minute period.
Digital microvascular assessments were made at the onset (approx 8 hours) and plateau (approx 16 hours) of changes in right atrial pressure.14 At 8 (groups 1 and 2) and 16 hours (groups 3 and 4) after receiving the laminitis-inducing ration, anesthesia was induced in the horses by use of thiopental sodium (10 mg/kg, IV) and pentobarbital sodium (7.5 mg/kg, IV) and maintained with pentobarbital sodium (5 to 15 mg/kg/h, IV). Horses were positioned in right lateral recumbency and ventilated with positive pressure and 100% oxygen. Techniques previously used to measure digital hemodynamic variables were applied.14,15 Briefly, horses were administered 500 units of heparin sodium/kg,IV, immediately before cannulation. The medial palmar artery was isolated distal to the portion elevated previously in the midmetacarpal region and cannulated with polyethylene tubing (PE320) and connected to a peristaltic pump. The medial and lateral palmar digital arteries and veins were isolated at the level of the mid portion of the proximal phalanx. The outflow limb from the perfusion pump was connected to a Y connector, with the 2 free limbs of the connector coupled to arterial cannulas (PE240) inserted in the medial and lateral palmar digital arteries. The digital veins were cannulated and connected to a Y connector, so that the effluent emptied into an extracorporeal reservoir. Blood from the reservoir was returned to the horse via the medial palmar vein by use of a second peristaltic pump. The medial and lateral palmar arteries and veins were ligated and transected at the level of the metacarpophalangeal joint. The digit was disarticulated at the level of the metacarpophalangeal joint via electrocautery. Any residual hemorrhage after disarticulation was controlled by use of electrocoagulation and ligation. The digit was placed on a wire grid and suspended from a sensitive force displacement transducerc calibrated to yield approximately 30 mm of deflection/g to permit accurate assessment of changes in the weight of the digit. Vascular flows and pressures were adjusted to maintain the digit in an isogravimetric condition. The preparation of disarticulated digits and measurement of capillary pressure were performed by the same investigators in each experiment (SCE).
Digital arterial and venous pressures were recorded continuously via pressure transducers interposed into the venous and arterial loops close to the Y connectors. The rate of blood flow through the digit was monitored periodically via timed collection into a graduated cylinder. Digital arterial pressure was maintained at approximately 100 mm Hg, and venous pressure was set at approximately 30 mm Hg by adjusting the arterial pump and the height of the venous reservoir, respectively.14,35 Capillary pressure was determined by the venous occlusion technique14,35 previously used in the equine digit.
Total vascular resistance, precapillary resistance, and postcapillary resistance were determined by dividing the arterial-to-venous, arterial-to-capillary, and capillary-tovenous pressure gradients by the rate of blood flow, respectively.14,35 The precapillary-to-postcapillary resistance ratio was determined by use of the calculated mean values. The relative contribution of precapillary and postcapillary resistances to the total vascular resistance was determined by dividing the appropriate mean value by the total vascular resistance.14,35
Vascular compliance and capillary filtration coefficients were determined as previously described.14 Capillary pressure was determined while the digit was in an isogravimetric condition. Then, venous pressure was rapidly increased by 15 mm Hg by raising the venous reservoir. The increase in venous pressure caused a biphasic increase in the weight of the digit. The first increase in weight was rapid and was attributed to expansion of the digital vasculature. Vascular compliance was calculated by dividing the weight gain (volume gain) during this phase by the increase in venous pressure that produced this weight gain. The second, slower increase in weight was a result of capillary filtration. The rate of filtration was used to determine the capillary filtration (unit density of the filtrate was assumed, and a second capillary pressure determination was made after the weight reached a new plateau).
After completion of the digital vascular assessment in the disarticulated digits of all horses, nitroglycerind was infused into the digital arterial circuit at a concentration and rate calculated to produce a 10−5M concentration of nitroglycerin in the digital arterial blood (based on digital arterial blood flow). Maximum rate of fluid infusion was < 0.5 mL/min; the infusion was maintained for 12 minutes. Values for digital arterial pressure reached a new steady state by 5 minutes after initiation of nitroglycerin infusion. Digital vascular assessment was repeated beginning 6 minutes after initiation of nitroglycerin infusion.
Horses were euthanatized with an overdose (100 mg/kg, IV) of pentobarbital sodium at the conclusion of the microcirculatory assessment performed at 8 or 16 hours after administration of the laminitis-inducing ration. The digit undergoing microcirculatory assessment was weighed before being boiled to remove soft tissues. The amount of richly perfused tissues was determined by subtracting the weight of the remaining bone, hoof, and sole from the initial total weight of the digit. To eliminate variability in foot size, all data were expressed as mean ± SEM per 100 g of richly perfused tissue.
Data analysis—Values for right atrial pressure, capillary pressure, capillary filtration coefficient, digital blood flow, preand postcapillary resistances, and total vascular resistance were tested for normality by use of the Shapiro-Wilk method with the null hypothesis rejected at α = 0.05. If unequal variances were identified for normally distributed variables, the data were transformed. All normally distributed and transformed data were analyzed by use of a mixedeffect general linear model (with repeated measures and horse considered a random variable). Predetermined post hoc comparisons were made by use of a least square mean with a Bonferroni correction. A value of P < 0.05 was considered significant for all tests. All statistical analyses were performed with a statistical software package.e
Results
Compared with baseline values, right atrial pressure began to decrease at approximately 6 hours after CHO and continued to decrease until it reached a plateau at 16 hours (Figure 1). Right atrial pressure was used to determine time to anesthesia. When right atrial pressure began its initial decline (ie, 2 consecutive decreasing right atrial pressure measurements were obtained), horses in groups 1 and 2 were anesthetized; time to anesthesia was 7 hours in 4 horses, 8 hours in 5 horses, and 9 hours in 1 horse. In groups 3 and 4, horses were anesthetized after 2 consecutive right atrial pressure measurements indicated right atrial pressure had reached the nadir; time to anesthesia was 14 hours in 2 horses, 15 hours in 3 horses, and 16 hours in 5 horses. Treatment group did not have a significant (P = 0.891) effect on right atrial pressure.
Right atrial pressure (mean ± SEM) in horses that had received CHO (corn starch-wood flour gruel [17.6 g/kg body weight]) and had been treated with saline (0.9% NaCl) solution (solid circles) or an ET receptor antagonist (open circles). These treatments had no significant effect on right atrial pressure. *Data pooled for groups 1 through 4 (n = 20 for data collected at baseline to 8 hours; n = 10 for data collected at 10 to 16 hours) were significantly (P < 0.05) different from the pretreatment baseline (time 0) value.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1204
Right atrial pressure (mean ± SEM) in horses that had received CHO (corn starch-wood flour gruel [17.6 g/kg body weight]) and had been treated with saline (0.9% NaCl) solution (solid circles) or an ET receptor antagonist (open circles). These treatments had no significant effect on right atrial pressure. *Data pooled for groups 1 through 4 (n = 20 for data collected at baseline to 8 hours; n = 10 for data collected at 10 to 16 hours) were significantly (P < 0.05) different from the pretreatment baseline (time 0) value.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1204
Right atrial pressure (mean ± SEM) in horses that had received CHO (corn starch-wood flour gruel [17.6 g/kg body weight]) and had been treated with saline (0.9% NaCl) solution (solid circles) or an ET receptor antagonist (open circles). These treatments had no significant effect on right atrial pressure. *Data pooled for groups 1 through 4 (n = 20 for data collected at baseline to 8 hours; n = 10 for data collected at 10 to 16 hours) were significantly (P < 0.05) different from the pretreatment baseline (time 0) value.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1204
Digital arterial blood flow in horses treated with saline solution was quite low by 8 hours after carbohydrate administration (3.8 ± 0.3 mL/min/100 g) and was significantly (P = 0.048) decreased further at 16 hours (1.6 ± 0.2 mL/min/100 g). The low digital blood flow in saline-treated horses (Figure 2) was accompanied by extremely high total digital vascular resistance (24.7 ± 10.1 mm Hg/mL/min/100 g at 8 hours, which was significantly [P = 0.008] different from 93 ± 21 mm Hg/mL/min/100 g at 16 hours; Table 1). Most of the increase in resistance was attributable to an increase in precapillary resistance at 8 hours, which was significantly (P = 0.006) greater at 16 hours. However, postcapillary resistance at 16 hours was significantly (P = 0.016) greater than the value at 8 hours.
Digital vascular pressures and resistances measured before and after administration of nitroglycerin (a donor of nitric oxide) in horses that were anesthetized 8 and 16 hours after administration of CHO (corn starch-wood flour gruel [17.6 g/kg body weight]) and that had been treated with either saline (0.9% NaCl) solution or an ET receptor antagonist.
| Variable | Treatment conditions | |||||||
|---|---|---|---|---|---|---|---|---|
| Saline solution | ETRA | Saline solution and NG | ETRA and NG | Saline solution | ETRA | Saline solution and NG | ETRA and NG | |
| Time after CHO (h) | 8 | 8 | 8 | 8 | 16 | 16 | 16 | 16 |
| Digital arterial pressure (mm Hg) | 143.2±18.4 | 131.5±14.1 | 146.8±18.1 | 133.0±14.5 | 148.6±10.2 | 106.2±12.1† | 124.5±17.6 | 106.8±11.2† |
| Digital venous pressure (mm Hg) | 30.4±0.2 | 30.4±0.34 | 31.4±0.8 | 30.5±0.25 | 30.3±0.2 | 30.6±0.5 | 30.5±0.5 | 30.2±0.4 |
| Capillary pressure (mm Hg) | 37.0±1.2 | 39.4±0.8 | 48.4±5.4 | 45.3±1.6 | 38.0±3.1 | 39.8±3.1 | 46.5±4.6 | 40.8±2.1 |
| Total capillary resistance (mm Hg/mL/min/100 g) | 37.0±12.0 | 25.0±11.5 | 13.0±6.6 | 13.8±4.5 | 93.1±10.8 | 24.0±6.6 | 36.2±17.0 | 7.9±2.2 |
| Precapillary resistance (mm Hg/mL/min/100 g) | 35.3±12.2 | 22.7±10.2 | 11.5±2* | 11.4±6.2* | 94.2±6.2 | 21.6±6.2 | 31.3±18.4 | 12.4±4.2† |
| Postcapillary resistance (mm Hg/mL/min/100 g) | 1.9±0.5 | 1.9±0.7 | 1.9±0.6 | 1.5±0.5 | 5.5±1.2 | 2.0±0.2† | 4.2±0.6 | 1.6±0.3† |
| Precapillary-to-postcapillary resistance ratio | 20.8±6.1 | 8.4±3.0* | 8.8±3.4* | 6.5±1.6* | 18.5±3 | 12.1±5.1 | 9.0±2.8† | 7.4±4.8† |
Values represent data from 5 horses/group.
Value significantly (P 0.05) different from the value of this variable after treatment with saline solution alone at 8 hours after CHO.
Value significantly (P 0.05) different from the value of this variable after treatment with saline solution alone at 16 hours after CHO.
ETRA = ET receptor antagonist. NG = Nitroglycerin.
Isolated digital blood flow (mean ± SEM) measured via timed collection from the digital venous catheter placed in horses anesthetized 8 (A) and 16 hours (B) after administration of CHO and treated with either saline solution (Sal) or an ET receptor antagonist (ETRA) before and after administration of nitroglycerine (NG). Values represent data from 5 horses/group. *Significantly (P < 0.05) different from value obtained after saline treatment alone. †Significantly (P < 0.05) different from values marked with an asterisk and those obtained after treatment with saline solution alone in both panels.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1204
Isolated digital blood flow (mean ± SEM) measured via timed collection from the digital venous catheter placed in horses anesthetized 8 (A) and 16 hours (B) after administration of CHO and treated with either saline solution (Sal) or an ET receptor antagonist (ETRA) before and after administration of nitroglycerine (NG). Values represent data from 5 horses/group. *Significantly (P < 0.05) different from value obtained after saline treatment alone. †Significantly (P < 0.05) different from values marked with an asterisk and those obtained after treatment with saline solution alone in both panels.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1204
Isolated digital blood flow (mean ± SEM) measured via timed collection from the digital venous catheter placed in horses anesthetized 8 (A) and 16 hours (B) after administration of CHO and treated with either saline solution (Sal) or an ET receptor antagonist (ETRA) before and after administration of nitroglycerine (NG). Values represent data from 5 horses/group. *Significantly (P < 0.05) different from value obtained after saline treatment alone. †Significantly (P < 0.05) different from values marked with an asterisk and those obtained after treatment with saline solution alone in both panels.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1204
Treatment with the ET receptor antagonist and nitroglycerin caused significant decreases in total resistance, compared with treatment with saline solution alone. Combined treatment with the ET receptor antagonist and nitroglycerin caused the greatest decrease in total resistance (P = 0.008). Treatment with the ET antagonist caused a significant (P = 0.006) reduction in postcapillary resistance at 16 hours, whereas treatment with nitroglycerin caused a significant decrease in precapillary resistance without affecting postcapillary resistance (P = 0.452). There were no significant differences in capillary pressure (P = 0.106; Table 1), capillary filtration coefficient (P = 0.353), or vascular compliance (P = 0.892) among the treatment groups. With regard to the capillary filtration coefficient, the value at 8 hours after CHO and after treatment with saline solution or ET receptor antagonist alone was 0.009 ± 0.003 mL/min/mm Hg/100 g and 0.015 ± 0.003 mL/min/mm Hg/100 g, respectively; for horses treated with saline solution or ET receptor antagonist and nitroglycerin, the value was 0.008 ± 0.002 mL/min/mm Hg/100 g and 0.008 ± 0.001 mL/min/mm Hg/100 g, respectively. The capillary filtration coefficient at 16 hours after CHO and after treatment with saline solution or ET receptor antagonist alone was 0.008 ± 0.002 mL/min/mm Hg/100 g and 0.009 ± 0.001 mL/min/mm Hg/100 g, respectively; for horses treated with saline solution or ET receptor antagonist and nitroglycerin, the value was 0.008 ± 0.002 mL/min/mm Hg/100 g and 0.009 ± 0.002 mL/min/mm Hg/100 g, respectively. With regard to vascular compliance, the value at 8 hours after CHO and after treatment with saline solution or ET receptor antagonist alone were each 0.03 ± 0.02 mL/mm Hg; for horses treated with saline solution or ET receptor antagonist and nitroglycerin, the value was 0.03 ± 0.02 mL/mm Hg and 0.03 ± 0.01 mL/mm Hg, respectively. The vascular compliance at 16 hours after CHO and after treatment with saline solution or ET receptor antagonist alone was 0.03 ± 0.01 mL/mm Hg and 0.05 ± 0.02 mL/mm Hg, respectively; the values were the same for each treatment when nitroglycerine was administered.
Discussion
Administration of an ET receptor antagonist resulted in significant improvement in vascular resistance in isolated perfused digits of anesthetized horses after CHO. Interpretation of these data would be easier via comparison with control horses not receiving CHO. However, studies have already determined digital Starling forces for control horses so that repetition of these studies would have been wasteful of horses.36,37
Specifically, at 8 hours after CHO, there was a decrease in digital blood flow with a significant increase in digital vascular resistance attributable to an increase in total resistance, compared with findings in healthy horses.14,36,37 The ET receptor antagonist caused an approximate doubling of digital blood flow with decreases in total and precapillary sphincter resistance that were not significant. However, the ET receptor antagonist significantly improved the precapillary-topostcapillary resistance ratio. Nitroglycerin caused a significant reduction in pre- and postcapillary resistances and blood flow to near normal values in horses evaluated 8 hours after CHO.14 The greatest increase in digital blood flow occurred after treatment with both the ET receptor antagonist and nitroglycerin.
At 16 hours after CHO, horses treated with saline solution had a decrease in digital blood flow because of an increase in postcapillary resistance along with an increase in precapillary resistance, compared with healthy horses.14 Administration of the ET receptor antagonist alone resulted in a significant decrease in both the pre- and postcapillary resistances with a significant increase in digital blood flow. In contrast, treatment with nitroglycerin improved total and precapillary resistances without a significant effect on postcapillary resistance. Combination treatment with the ET receptor antagonist and nitroglycerin significantly decreased total, precapillary, and postcapillary resistances and digital blood flow to values near normal.14 It is possible that both ET and NO are involved in the pathogenesis of the early vascular changes during CHO with NO having a greater effect (involving precapillary sphincters) at 8 hours and ET having a greater effect (involving pre- and postcapillary sphincters) at 16 hours.
The dose of ET receptor antagonist was chosen on the basis of the concentration that completely inhibited the in vitro contraction of palmar digital arteries and veins from horses without laminitis29 and that subsequently was shown to prevent or reverse the in vivo vasoconstrictor effects (decreased digital blood flow) of ET-1 infused into the digital artery of healthy horses.f In that previous studyf, ET-1 infused into the digital artery caused a dose-related reduction in digital arterial blood flow that lasted for 1 to 2 hours after treatment. In the previousf and present study, the dose of the ET receptor antagonist was calculated on the basis of the amount needed to yield a 10−5M concentration in digital arterial blood (based on the digital arterial blood flow). The infusion rate and volumes were constant, and infusion of saline solution at a similar rate and volume did not alter digital hemodynamics. In the in vivo study,f infusion of the ET receptor antagonist at this dose prevented the effects of ET-1 on digital blood flow. Similarly, in that previous study,f nitroglycerin infused at a concentration and rate yielding a 10−5M concentration in digital arterial blood (based on the digital arterial blood flow) increased digital blood flow in horses with ET-1–induced vasoconstriction.
In another study14 of microvascular changes in horses after CHO, decreased digital arterial blood flow, increased total resistance, increased precapillary resistance, increased postcapillary resistance, decreased precapillary-to-postcapillary resistance ratio, and increased capillary pressure were detected at 10 to 16 hours after administration of the laminitis-inducing ration. In the present study, microvascular changes in horses 8 hours after CHO included dramatic increases in total vascular and precapillary resistances with more modest increases in postcapillary resistance, thereby resulting in an increased precapillary-to-postcapillary resistance ratio. In horses anesthetized at 16 hours, there were marked increases in postcapillary resistance and dramatic increases in precapillary resistance. In the previous study,14 anesthesia was induced for microvascular assessment when right atrial pressure decreased by at least 20% at 10 to 16 hours after CHO administration. For each treatment assessed in the present study (ET receptor antagonist or saline solution), anesthesia was induced in 1 group of horses at 6 to 8 hours and in the second group at 14 to 16 hours; the relative differences in pre- and postcapillary resistances between these horses resulted from differences in time after CHO and reflected the natural course of the disease. That is, in the early hours after CHO, the relative balance of vasoactive mediators results in constriction of precapillary sphincters, thereby increasing precapillary sphincter resistance. Over time, increased quantities of other vasoactive substances constrict the venous vasculature causing an increase in postcapillary resistance that increases the capillary pressure, which could eventually cause edema formation and subsequent increases in laminar interstitial pressure. Increases in tissue pressure can exceed the critical closing pressure of capillaries, thus contributing to later increases in precapillary resistance as detected at 16 hours in the horses of our study.
The present study failed to detect increases in capillary pressure, unlike previous studies14,15 of CHO-induced laminitis in horses. In the present study, increases in postcapillary pressure were accompanied by increases in precapillary pressure, thereby limiting capillary perfusion and increases in capillary pressure. The preparation of disarticulated digits and measurement of capillary pressure in the present study were performed by individuals (SCE and RMM) who were involved in the previous studies,14,15 which limited the likelihood of variations in techniques. The ability of the administered vasoactive modulators (ET-1 receptor antagonist and NO) to decrease pre- and postcapillary resistances to normal levels reduces the likelihood that blood flow and resistances were limited by the resistance within the preparation and tubing. Therefore, it is likely that the aforementioned differences in resistance are attributable to variation in the response of horses in the present study to the carbohydrate ration and differences in the time periods evaluated. Postcapillary resistance increased and digital blood flow decreased similarly in the previous studies14,15 and in the study reported here, creating a situation where- by digital ischemia and edema are likely to occur.
In the present study, the capillary filtration coefficient was increased from that in healthy horses14 and was similar to that found in the prodromal stages of black walnut extract–induced laminitis.15 The capillary filtration coefficient is determined by measuring the weight gain in the digit during rapid elevation of venous pressure.37,38 The initial rapid increase in weight is attributable to vascular volume change, which is dependent on venous compliance.38 The second phase is a slower, more prolonged component that represents capillary filtration.38 Two distinct slopes were detected in the present study, thereby reducing the likelihood of error during calculation of the capillary filtration coefficient.37,38 Because the second phase of the weight gain occurs 30 to 60 seconds after the first phase, it is unlikely that the second phase represents delayed vascular compliance rather than an increased filtration.37,38
The capillary filtration coefficient is a measure of the hydraulic conductance of the capillary bed and is influenced by the size and number of capillaries perfused.15 Increased capillary perfusion results from relaxation of precapillary sphincters and is commonly accompanied by an increase in capillary pressure.39 Under normal conditions, a large number of capillaries are not perfused at any given time.39 Consequently, the increase in the capillary filtration coefficient detected in the present study could be explained in part by recruitment of additional capillaries; however, it is unlikely that recruitment of additional capillaries could be sufficient to account for the 4-fold increase in the capillary filtration coefficient.40 Increases in capillary filtration of this magnitude are usually caused by increases in vascular permeability.40 Capillary permeability, as measured by the osmotic reflection coefficient, did not increase in previous studies of CHO- or black walnut-induced laminitis.14,15 However, an increase in the radius of small pores in capillary beds can increase the capillary filtration coefficient without increasing the permeability to protein.41 Pore stripping analysis is needed to detect changes in these small pores.41
Histologic studies to investigate whether increased capillary filtration results in demonstrable edema are lacking. To the authors' knowledge, laminar histopathologic findings during the first 24 hours after CHO administration have not been reported. Histologic evidence of mild edematous changes in the laminae 8 to 10 hours after development of lameness (approx 33 hours after CHO) has been reported.3,4 The predominant lesion at 4 and 6 hours after CHO is hydropic swelling of the epithelial cells.2 However, failure to confirm edema by use of routine histologic techniques does not preclude that venous pressure changes cause notable alteration of capillary filtration and tissue pressure. For example, despite the use of a validated and calibrated optical system in studies42–44 of pulmonary and skin microvascular and perivascular interstitial geometry, only minimal changes in interstitial thickness could be identified during increases in interstitial volume and pressure caused by intra-arterial infusion of saline solution. The degree of widening of the interstitial spaces is dependent on lymphatic drainage and the compliance of the extracellular matrix.16 As the tissue pressure and volume increase, extracellular matrix degradation develops, thereby increasing its compliance so that in the severe edematous states gross widening of the interstitial space occurs.16 It remains to be determined whether the increased venous resistance measured in the present study increases fluid filtration and tissue pressure sufficiently to widen interstitial spaces and contribute to the degradation of the extracellular matrix.
Alternatively, if increased tissue pressure is the predominant consequence of increased capillary filtration, the critical closing pressure of the capillary bed could be exceeded leading to collapse and poor tissue perfusion.14 In a previous study14 of Starling forces in the equine digit during laminitis, tissue pressure was increased substantially. Because the osmotic reflection coefficient was not measured, tissue pressure was not calculated in the present study. Micropuncture assessments of tissue pressure provide more accurate assessment of moment-to-moment changes in tissue pressure42 but are precluded by the keratinized hoof. If the interstitial pressure increases contribute to poor laminar perfusion, we must next consider whether this resolves prior to a subsequent phase of hyperperfusion as suggested by results of studies12,13 of hoof wall surface temperature. The increased tissue pressure could reach a point at which further capillary filtration is limited hydraulically.16 Excessive tissue fluid can be removed, although slowly, via the digital lymphatic system to restore tissue pressure over time.16 However, the existence of hyperperfusion during the acute phase remains at question. Surface temperature of the hoof wall has limitations as a measure of capillary perfusion. Hoof wall surface temperature is determined by tissue metabolism, flow through the capillary bed, and flow through AVAs. In the connective tissue of the hoof, changes in metabolism probably change surface temperature very little.12 It is proposed that in the limb of sheep, flow through capillary beds has a greater effect on skin surface temperature than flow through AVAs, but this has not been determined in horses.45 Flow through AVAs warms the hoof under several physiologic conditions.13 The opening of AVAs in the equine digit at rest at an ambient temperature of 25°C suggests that their function in temperature and pressure control may be more complicated than that of the AVAs in sheep limbs.46 Because AVAs are able to warm the hoof when the ambient temperature is at freezing point, blood flow through these shunts would clearly impact hoof wall surface temperature,13 thus obscuring the ability of hoof wall surface temperature measurements to differentiate flow through AVAs and flow through capillary beds.
Techniques used in the present study are not able to characterize the anatomy of the digital circulation. The pressures and resistances measured in this extracorporeal system represent the mean values for all the vasculature from the lateral and medial palmar digital arteries and drained by the same veins, including circulation occurring in series and parallel. Within this context, preferential flow could be diverted from the laminar circulation and channeled to other tissues such as bone or vice versa, resulting in greater oxygen deprivation. Such is the case in the intestinal circulation during ischemia, in which flow is diverted from the more metabolically active mucosa to the submucosa.40 The extracorporeal technique employed in the present study does not differentiate the location of flow and necessitates the use of other techniques such as microsphere studies.40
In the study of this report, treatment with an ET receptor antagonist resulted in a substantial improvement in vascular resistance in the isolated perfused digit of anesthetized horses after CHO. The ET receptor antagonist significantly reduced postcapillary resistance at 16 hours after CHO. Nitroglycerin caused significant improvement in total resistance in blood flow, but did not alter postcapillary resistance. The greatest improvement in vascular resistance and blood flow occurred after treatment with both the ET receptor antagonist and nitroglycerin. Endothelin synthesis could increase in the digital vasculature during laminitis as a result of several stimuli including cytokines, sympathomimetic agonists, or shear stress caused by primary digital ischemia. Although treatment with the ET receptor antagonist improved vascular function in horses with CHO, further study is needed to identify whether endogenous ET-1 production is responsible for the vascular dysfunction in horses with laminitis.
ABBREVIATIONS
| CHO | Carbohydrate overload |
| ET | Endothelin |
| AVA | Arteriovenous anastomosis |
Transonics Systems Inc, Ithaca, NY.
PD145065, American Peptide Co, Sunnyvale, Calif.
FT03, Grass Medical Instruments, Quincy, Mass.
Nitroglycerin, American Reagent Laboratories Inc, Shirley, NY.
Proc Mixed, SAS Institute Inc, Cary, NC.
Stokes AM. Role of endothelin in the pathogenesis of acute laminitis in horses. PhD dissertation, Department of Physiology and Pharmacology, Louisiana State University. Baton Rouge, La, 2003.
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