The Journal of Bone and Joint Surgery 79:647-55 (1997)
© 1997 The Journal of Bone and Joint Surgery, Inc.
Effect of the Duration of Room-Temperature Ischemia on Function of the Vascular Endothelium: The Role of Adrenomedullin in Reperfusion Injury*
TEIJI KATO, M.D. ,
ALLEN T. BISHOP, M.D.,
YUAN-KUN TU, M.D. and
MICHAEL B. WOOD, M.D., ROCHESTER, MINNESOTA
Investigation performed at the Department of Orthopedics, Mayo Clinic and Mayo Foundation, Rochester
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Abstract
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The function of the vascular endothelium after storage at room temperature (24 degrees Celsius) for four, eight, and twenty-four hours was investigated with use of an ex vivo canine tibial perfusion model. Function was assessed in terms of changes in perfusion pressure and changes in the concentration of endothelin-1 in the venous effluent of the perfused tibiae. Endothelin-1 is a potent vasoconstrictor that is produced in low concentrations by normal endothelial cells and in increased concentrations by injured vascular endothelial cells.
The mean perfusion pressures at flow rates of 1.0 and 1.5 milliliters per minute were significantly higher in the tibiae that had been stored for eight hours than in the tibiae that had been stored for four hours (p < 0.05), and they were significantly higher in the tibiae that had been stored for twenty-four hours than in the tibiae that had been stored for four or eight hours (p < 0.05). The increase in perfusion pressure with increasing duration of storage was associated with an increase in production of endothelin-1. The production of endothelin-1 in the tibiae that had been stored for eight hours (10.6 ± 0.46 picograms per milliliter) was approximately ten times greater than that in the tibiae that had been stored for four hours (1.1 ± 0.29 picograms per milliliter). The tibiae that had been stored for twenty-four hours had 19.1 ± 1.5 picograms of endothelin-1 per milliliter, nearly twice that produced in the tibiae that had been stored for eight hours.
Injection of acetylcholine demonstrated muscarinic receptor-mediated vasodilation in the tibiae that had been stored for four hours. In contrast, the tibiae that had been stored for eight and twenty-four hours had no evidence of acetylcholine-induced vasodilation of baseline perfusion vascular smooth-muscle tone. However, there was some preservation of endothelium-dependent vascular smooth-muscle relaxation in the tibiae that had been stored for eight and twenty-four hours, as norepinephrine-induced vascular smooth-muscle contraction was significantly greater in the presence of NG-monomethyl-L-arginine acetate (p < 0.05). Moreover, in the second phase of the study, a bolus injection of calcium ionophore A23187 in tibiae that had been stored for twenty-four hours relaxed vascular smooth muscle.
Adrenomedullin, a novel peptide with known vasodilator properties, relaxed vascular smooth muscle in all three groups and also attenuated the pressor response to norepinephrine.
In conclusion, the function of the vascular endothelium was impaired after storage at room temperature for four hours. However, the vascular endothelium in the tibiae that had been stored for twenty-four hours maintained some function with regard to the production of nitric oxide. The effect of adrenomedullin as a potent vasodilator was observed in the tibiae that had been stored for four, eight, and twenty-four hours.
CLINICAL RELEVANCE: In the clinical setting, some replantation and microvascular free-tissue transfer procedures may involve ischemic periods of more than four hours. On rare occasions, this period may be as long as twenty-four hours in the absence of hypothermic cooling, particularly in instances of operative re-exploration after thrombosis of a site of vascular anastomosis with microvascular bone transfer. An understanding of time-dependent changes in the function of the vascular endothelium of bone with warm ischemia is important. We have demonstrated that adrenomedullin, a relatively newly identified and potent vasodilator peptide, may have value for the mitigation of these ischemic changes.
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Introduction
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Vascularized bone transfer is a well accepted technique for the reconstruction of large defects in bone after resection of a tumor, débridement for osteomyelitis, or traumatic bone loss. Despite sustained vascular patency, blood flow within the grafted bone is predictably less than that within normal bone25. A variety of factors may be responsible, including intracellular edema, damage of the endothelial cells, thrombosis, impaired venous drainage, or the so-called no-reflow phenomenon (reperfusion injury)19. With any organ transplantation, damage of the vascular endothelium may occur, particularly if there is a prolonged ischemic interval. Dysfunction of the endothelial cells may result in a decrease in the production of vasodilator substances and a concomitant increase in the production of vasoconstrictor substances7,9,22 such as endothelin-1. Endothelin-1 is one of the most potent endogenous vasoconstrictors produced by endothelial cells27. The plasma level of endothelin-1 is known to be elevated in disorders of cardiovascular function. Endothelin-1 has been proposed as a key mediator of cerebral vasospasm after subarachnoid hemorrhage14.
With an absent or dysfunctional vascular endothelium, physiological regulation of the local blood supply is impaired. Under clinical circumstances, some free-tissue transfer procedures may involve periods of room-temperature ischemia of nearly four hours or, occasionally, longer. The ischemic period could approach twenty-four hours if a postoperative thrombosis at the site of a microvascular anastomosis has escaped early detection. These time-dependent changes in the function of endothelial cells with ischemia at warm temperatures have not been investigated thoroughly in bone, as far as we know, and they were the focus of this study.
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Materials and Methods
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Isolation of the Canine Tibiae
Thirty-two male and female adult mongrel dogs that weighed twenty-three to twenty-eight kilograms were anesthetized with thirty milligrams of pentobarbital per kilogram of body weight and were killed. Both tibiae were excised. After removal of the fibula, the tibial nutrient artery was identified on the posterolateral surface of the proximal tibial metaphysis. The nutrient artery was separated from the accompanying nutrient vein and nutrient nerve close to the nutrient foramen. The nutrient artery was then cannulated with a polyethylene cannula with an external diameter of 0.965 millimeter (Clay Adams, Parsippany, New Jersey) with the aid of magnification and microsurgical instruments2-5,12,20. All periosteal and muscular branches of the nutrient artery were ligated, and both tibiae were isolated in an extraperiosteal manner from soft-tissue attachments. Both bones were then wrapped in towels that had been moistened with Krebs-Ringer solution and were kept on the counter at room temperature (24 degrees Celsius).
Ex Vivo Perfusion Apparatus
Ex vivo perfusion was performed with a previously described technique2-5,12,20. Each tibia was placed in a humidifier. The cannula in the nutrient artery was connected to a constant-flow roller pump (Minipuls 2; Gilson Medical Electronics, Middleton, Wisconsin) and was perfused with modified Krebs-Ringer solution at 37 degrees Celsius. The Krebs-Ringer solution was prepared daily and aerated (in 95 per cent O2 and 5 per cent CO2) to produce a pH of 7.45. The perfusion pressure was measured with a strain-gauge pressure transducer (Viggo-Spectramed DTX/Plus; Oxnard, California) and recorded with a pen recorder (model 2125E; Allen Datagraph, Salem, New Hampshire), as described previously2-5,12,20.
Flow Rate
The rate of flow through the nutrient artery initially was set at 0.5 milliliter per minute and was increased gradually to 3.0 milliliters per minute in the tibiae that had been stored for four or eight hours. In the tibiae that had been stored for twenty-four hours, the peripheral resistance to perfusion was extremely high, necessitating an initial flow rate of 0.2 milliliter per minute, which was increased gradually to 1.5 milliliters per minute. Faster flow rates could not be obtained in the tibiae that had been stored for twenty-four hours because of the extremely high perfusion pressure.
Selection of the Drugs
The endothelium synthesizes a variety of substances that affect vascular smooth-muscle tone and the coagulation of blood. To evaluate this endothelial exocrine function after various periods of ischemic injury, we assayed concentrations of endothelin in the perfusate effluent of the tibiae after perfusion with or without adrenomedullin, acetylcholine, NG-monomethyl-L-arginine acetate, calcium ionophore A23187, and norepinephrine. Acetylcholine is a muscarinic receptor-mediated agonist that induces the production of nitric oxide from vascular endothelial cells26. Nitric oxide is a relaxing agent for vascular smooth-muscle cells. Acetylcholine is also known to stimulate muscarinic receptors on vascular smooth-muscle cells, which results in vasoconstriction in the absence of a normally functioning vascular endothelium1,6. NG-monomethyl-L-arginine acetate inhibits the synthesis of nitric oxide in the vascular endothelium21. Calcium ionophore A23187 activates the synthesis and production of nitric oxide and is an endothelium-dependent but receptor-independent vasorelaxant15,23.
Endothelin is known to be a potent vasoconstrictor and is produced by the endothelium, particularly if the endothelium is dysfunctional. Norepinephrine acts at both  1-adrenoreceptors and 2-adrenoreceptors and is known to be a powerful smooth-muscle contractile agent in the canine tibia. Adrenomedullin is a peptide that was originally discovered in human pheochromocytoma and is known to be one of the most potent vasodilators in vivo13. We have previously observed and reported a role for adrenomedullin as a long-lasting vasodilator that attenuates the pressor response to norepinephrine in an ex vivo canine tibial perfusion model12.
Endothelin-1,2 [125I] Assay
The venous effluent from each perfused canine tibia was collected into tubes on ice for defined periods of time. A one-milliliter aliquot of the effluent was stored at -20 degrees Celsius until the assay was performed. Endothelin was assayed with the endothelin-1,2[125I] radioimmunoassay system (Amersham, Arlington Heights, Illinois) with use of a gamma-scintillation counter (Beckman Instruments, Palo Alto, California).
Preparation of the Drugs
Adrenomedullin solution (Phoenix Pharmaceutical, Mountain View, California) was prepared once every two days in Krebs-Ringer solution. Norepinephrine, acetylcholine, calcium ionophore A23187 (all from Sigma Chemical, St. Louis, Missouri), and NG-monomethyl-L-arginine acetate (Calbiochem-Novabiochem, La Jolla, California) were used throughout the study and were prepared daily. Norepinephrine and calcium ionophore A23187 were dissolved in distilled water. Solutions of acetylcholine and NG-monomethyl-L-arginine acetate were prepared in Krebs-Ringer solution.
Experimental Design
The right and left tibiae from eight dogs were stored for four hours and those from another eight were stored for eight hours; the tibiae from sixteen dogs were stored for twenty-four hours, and these were divided into two groups (phase 1 and phase 2). For each dog, one tibia was selected at random to serve as the control and the other served as the experimental bone.
Storage at Room Temperature for Four or Eight Hours
After the baseline perfusion pressure stabilized at a flow rate of 3.0 milliliters per minute, effluent from the bone was collected for twenty minutes to assay the levels of endothelin-1. A standard norepinephrine dose-response curve for doses of sixty-four, 128, and 256 picomoles was generated with use of 0.1-milliliter bolus injections into the perfusate (Fig. 1). Then, 0.1 milliliter of 10-5-molar acetylcholine solution was injected into the perfusate of both tibiae and the subsequent change in baseline perfusion pressure was observed for twenty minutes (Fig. 1). Next, a 0.1-milliliter bolus of Krebs-Ringer solution was injected into the control tibia and a 0.1-milliliter bolus of 10-5-molar adrenomedullin solution was injected into the corresponding experimental tibia. For the tibiae that had been stored for four hours, repetitive norepinephrine dose-response curves with use of the same three doses of norepinephrine as those used at the start of the experiment were generated at twenty, fifty, eighty, and 110 minutes after injection of the adrenomedullin or Krebs-Ringer solution to determine the time-dependent effects of adrenomedullin on pressor response to norepinephrine. These time-periods were selected on the basis of previous studies of freshly perfused tibiae, in which adrenomedullin had a relaxation effect on vascular smooth muscle that lasted 100 minutes12. For the tibiae that had been stored for eight hours, a standard norepinephrine dose-response curve was generated at twenty, fifty, and eighty minutes after injection of the adrenomedullin or Krebs-Ringer solution. In both groups, a second twenty-minute sample of the effluent was obtained immediately after injection of either the adrenomedullin or the Krebs-Ringer solution.
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Fig. 1 The time course of the methods. t0 and T0 indicate the time at which the initial norepinephrine dose-response curve was generated. t1 through t4 and T1 indicate thirty-minute intervals, beginning at twenty minutes after injection of the adrenomedullin (AM), Krebs-Ringer (KR), or NG-monomethyl-L-arginine acetate (L-NMMA) solution. The transverse arrows indicate twenty-minute periods. ET-1 = endothelin-1, ACh = acetylcholine, and A23187 = calcium ionophore A23187.
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Storage at Room Temperature for Twenty-four Hours
Phase 1: After the baseline perfusion pressure stabilized at the maximum flow rate of 1.5 milliliters per minute, a standard norepinephrine dose-response curve was generated for doses of sixty-four, 128, and 256 picomoles with use of 0.1-milliliter bolus injections into the perfusate. Then, 0.1 milliliter of 10-5-molar acetylcholine solution was injected into the perfusate of both tibiae, and the change in baseline perfusion pressure was observed for twenty minutes. Next, 0.1 milliliter of Krebs-Ringer solution was injected into the control tibia and 0.1 milliliter of 10-5-molar adrenomedullin solution was injected into the experimental tibia. A norepinephrine dose-response curve was generated twenty and fifty minutes later (Fig. 1).
Phase 2: After stabilization of the baseline perfusion pressure at the flow rate of 1.5 milliliters per minute, effluent from the bone was collected for twenty minutes to assay the levels of endothelin-1. A norepinephrine dose-response curve was generated for doses of sixty-four, 128, and 256 picomoles with use of 0.1-milliliter bolus injections into the perfusate. One tibia was perfused continuously with 0.1 milliliter of 2 x 10-4-molar NG-monomethyl-L-arginine acetate solution per minute and the other, with 0.1 milliliter of Krebs-Ringer solution per minute as a control. Twenty minutes later, a second norepinephrine dose-response curve was generated with use of the same doses of norepinephrine as before. Thereafter, the infusions of the NG-monomethyl-L-arginine acetate and Krebs-Ringer solutions were discontinued. At least twenty minutes later, a 0.1-milliliter bolus of Krebs-Ringer solution was injected into the perfusate of both tibiae and the change in perfusion pressure was observed for twenty minutes. Finally, a 0.1-milliliter bolus of 10-5-molar calcium ionophore A23187 solution was injected into the perfusate of both tibiae.
Analysis of the Data
For the purposes of this investigation, a variety of quantitative parameters of endothelial exocrine function were evaluated. The baseline perfusion pressure at each flow rate for the three groups, the change in baseline perfusion pressure after the injection of acetylcholine in each group, and the change in baseline perfusion pressure resulting from injection of a 0.1-milliliter bolus of 10-5-molar adrenomedullin solution as compared with that produced by injection of Krebs-Ringer solution were evaluated. The levels of endothelin-1 were quantitated by their concentration (picograms per milliliter). The change in each norepinephrine dose-response curve after injection of adrenomedullin or Krebs-Ringer solution was expressed as a percentage of the initial dose-response curve (the initial norepinephrine dose-response curve was arbitrarily defined as maximum [100 per cent] norepinephrine-induced smooth-muscle contractive response).
In the phase-2 tibiae that had been stored for twenty-four hours, the change in baseline perfusion pressure resulting from infusion of NG-monomethyl-L-arginine acetate was compared with that resulting from infusion of Krebs-Ringer solution. The norepinephrine dose-response curve immediately before infusion of NG-monomethyl-L-arginine acetate was arbitrarily interpreted as a maximum (100 per cent) contraction. The magnitude of subsequent norepinephrine dose-response curves in the presence or absence of NG-monomethyl-L-arginine acetate perfusion was expressed as a percentage of the initial response curve. The change in baseline perfusion pressure caused by a 0.1-milliliter bolus injection of 10-5-molar calcium ionophore A23187 solution as compared with the pressure in the control tibia was evaluated with the paired two-tailed Student t test.
Since, at times, the pair of tibiae from the same animal demonstrated quantitatively different responses to vasoactive agents, statistical evaluation of the data was performed with the unpaired two-tailed Student t test, even though paired tibiae were used in the experimental design. However, in the experiments in which pressure responses within the same bone were compared (the calcium ionophore A23187 study), a paired Student t test was used. P values of less than 0.05 were considered significant. All study protocols had been reviewed and had received previous approval from the Mayo Foundation Institutional Animal Care and Use Committee.
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Results
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One pair of tibiae that had been stored for four hours was excluded from the results because one of the bones had a very unstable baseline perfusion pressure.
Baseline Perfusion Pressure
Baseline perfusion pressures at each flow rate were compared among the three groups (storage for four, eight, and twenty-four hours) (Fig. 2). The mean baseline perfusion pressures (and the standard error of the mean) at flow rates of 1.0 and 1.5 milliliters per minute were significantly lower in the tibiae that had been stored for four or eight hours than in the tibiae that had been stored for twenty-four hours (p < 0.05), and they were significantly higher in the tibiae that had been stored for eight hours than in the tibiae that had been stored for four hours (p < 0.05). However, there was no significant difference between the four and eight-hour groups at flow rates of 2.0 and 3.0 milliliters per minute.
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Fig. 2 Graph of the mean perfusion pressure (and the standard error of the mean) of the tibiae that had been stored at room temperature for four (circles), eight (squares), and twenty-four (diamonds) hours. * = significantly different from the tibiae that had been stored for four hours (p < 0.05), and ** = significantly different from the tibiae that had been stored for four or eight hours (p < 0.05). One millimeter of mercury is equivalent to 0.13 kilopascal.
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Production of Endothelin-1 and Effect of Adrenomedullin
In the tibiae that had been stored for four hours, the mean concentration of endothelin-1 was 1.1 ± 0.29 picograms per milliliter (Fig. 3-A). Nine of the fourteen tibiae that had been stored for four hours had less than 0.5 picogram per milliliter of endothelin-1. Because less than 0.5 picogram per milliliter was considered non-measurable, the effect of adrenomedullin on the production of endothelin-1 could not be quantitated with accuracy. For purposes of statistical analysis, the production of endothelin-1 in these bones was assigned the value of 0.5 picogram per milliliter.
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Fig. 3-A Graph of the mean production (and the standard error of the mean) of endothelin-1 (ET-1) in the tibiae that had been stored at room temperature for four, eight, and twenty-four hours.
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The concentration of endothelin-1 in the tibiae that had been stored for eight hours ranged from 7.3 to 14.1 picograms per milliliter, with a mean of 10.6 ± 0.46 picograms per milliliter, before the injection of adrenomedullin or Krebs-Ringer solution. Injection of adrenomedullin solution resulted in a mean concentration of 10.9 ± 0.75 picograms per milliliter, whereas injection of Krebs-Ringer solution resulted in a mean concentration of 10.4 ± 0.56 picograms per milliliter (Fig. 3-B). The mean production of endothelin-1 in the tibiae that had been stored for eight hours was approximately ten times greater than that in the tibiae that had been stored for four hours. The level of endothelin-1 did not change significantly following injection with either adrenomedullin or Krebs-Ringer solution (Fig. 3-B), and, with the numbers available, there was no significant difference between the tibiae injected with adrenomedullin solution and the control tibiae.
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Fig. 3-B Graph of the effect of injection of adrenomedullin solution (AM) or Krebs-Ringer solution (KR) (controls) on the mean production (and the standard error of the mean) of endothelin-1 (ET-1) in the tibiae that had been stored for eight hours.
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The mean concentration of endothelin-1 in the tibiae that had been stored for twenty-four hours was 19.1 ± 1.5 picograms per milliliter.
Response to Acetylcholine
All of the tibiae that had been stored for four hours demonstrated a vascular smooth-muscle relaxation response to acetylcholine (Fig. 4). Seven pairs of tibiae that had been stored for eight hours demonstrated a constrictor response to acetylcholine (Fig. 4). Six of the eight pairs of tibiae that had been stored for twenty-four hours, and used for phase 1, demonstrated no response to acetylcholine, and the remaining two pairs demonstrated a slight constrictor response. The maximum perfusion-pressure response three minutes after injection of the acetylcholine solution was significantly different among the three groups (p < 0.05).
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Fig. 4 Graph of the changes in the mean baseline perfusion pressure (and the standard error of the mean) following injection of acetylcholine solution in the tibiae that had been stored at room temperature for four (circles), eight (squares), and twenty-four (diamonds) hours. The maximum perfusion-pressure response three minutes after injection of the acetylcholine solution was significantly different among the three groups (p < 0.05). One millimeter of mercury is equivalent to 0.13 kilopascal.
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Effect of Adrenomedullin on Perfusion Pressure
Five minutes after a 0.1-milliliter bolus injection of 10-5-molar adrenomedullin solution, the baseline perfusion pressure decreased by a mean of 9.4 ± 3.3, 9.7 ± 4.8, and 11.0 ± 2.5 millimeters of mercury (1.25 ± 0.440, 1.29 ± 0.640, and 1.47 ± 0.333 kilopascals) in the tibiae stored for four, eight, and twenty-four hours, respectively (Fig. 5). In all three groups, these decreases in perfusion pressure persisted and were significantly different from those in the control specimens for a duration of twenty minutes after injection of the adrenomedullin solution (p < 0.05).
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Fig. 5 Graph of the changes in the mean baseline perfusion pressure (and the standard error of the mean) after injection of adrenomedullin solution (AM) or Krebs-Ringer solution (KR) (controls). One millimeter of mercury is equivalent to 0.13 kilopascal.
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Effect of Adrenomedullin on Pressor Response to Norepinephrine
Adrenomedullin significantly attenuated the pressor response to norepinephrine, as compared with the response in the control tibiae, for a duration of eighty minutes in the tibiae that had been stored for four hours (Fig. 6-A) (p < 0.05) and for a duration of twenty minutes in the tibiae that had been stored for eight (Fig. 6-B) or twenty-four hours (Fig. 6-C) (p < 0.05).
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Figs. 6-A, 6-B, and 6-C: Graphs of the changes in the mean percentage residual response (and the standard error of the mean) to norepinephrine compared with the initial value (t0). t1 = twenty minutes after injection of adrenomedullin solution (AM) or Krebs-Ringer solution (KR) (controls), t2 = fifty minutes after injection, t3 = eighty minutes after injection, and t4 = 110 minutes after injection. * = significantly different (p < 0.05) from the controls.
Fig. 6-A: The tibiae that had been stored at room temperature for four hours.
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Effect of NG-Monomethyl-L-Arginine Acetate on the Phase-2 Tibiae That Had Been Stored for Twenty-four Hours
The infusion of NG-monomethyl-L-arginine acetate solution after twenty-four hours of storage had no significant effect on the baseline perfusion pressure. However, the second norepinephrine dose-response curves in the presence of NG-monomethyl-L-arginine acetate and Krebs-Ringer solution were 180 ± 10.0 per cent and 102 ± 7.1 per cent of the initial values, respectively. This difference was significant (p < 0.05).
Effect of Calcium Ionophore A23187 on Perfusion Pressure in the Phase-2 Tibiae That Had Been Stored for Twenty-four Hours
Twenty minutes after a 0.1-milliliter bolus injection of 10-5-molar calcium ionophore A23187 solution, the baseline perfusion pressure was significantly lower than that in the controls (Fig. 7) (p < 0.05). This indicated that endothelial-derived synthesis of nitric oxide was still present in these tibiae.
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Fig. 7 Graph of the changes in the mean baseline perfusion pressure (and the standard error of the mean) after injection with calcium ionophore A23187 (solid squares) or Krebs-Ringer solution (open squares; controls) in the phase-2 tibiae that had been stored for twenty-four hours. One millimeter of mercury is equivalent to 0.13 kilopascal.
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Discussion
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Since Furchgott and Zawadzki reported the essential role of the endothelium as a mediator of vascular relaxation, there has been great interest in the investigation of the exocrine function of the endothelium. Several potent vasoactive factors, including nitric oxide, prostaglandins, endothelin, and adrenomedullin, have recently been identified as products of the vascular endothelium. Nitric oxide and adrenomedullin have been well documented as smooth-muscle relaxing agents6,13, whereas endothelin has been reported as a powerful constrictor of smooth muscle10,26. A variety of prostanoid agents of endothelial origin may have either smooth-muscle relaxing or smooth-muscle constricting properties24. For example, prostacyclin is a documented smooth-muscle relaxing agent and thromboxane A2 is a well known constrictor. Thus, the vascular endothelium is now known to have an important role not only as a passive barrier between the body and the bloodstream but also as a functioning exocrine tissue that regulates arterial tone as well as perfusion.
In clinical replantations and transplantations, the warm ischemic period rarely exceeds eight hours and, more typically, is less than four hours. The results of this study suggest that the storage of tibiae at room temperature for four hours is associated with low-level production of endothelin-1 and preservation of muscarinic receptor-mediated vasodilation under resting tone. Our data also demonstrate that, after storage at room temperature for eight hours, there is a marked increase in baseline perfusion pressure, a much higher production of endothelin-1, and a loss of muscarinic receptor-mediated vasodilation under resting tone. Dysfunction of endothelial cells has been shown to be associated with increased vascular smooth-muscle constriction2. We suggest that at least one mechanism is the ischemia-induced augmentation of endothelin release. It seems logical, therefore, that strategies to retard the deterioration of normal function of the vascular endothelium are desirable when warm ischemia exceeds four hours. The most common techniques for this are hypothermic storage and certain perfusion protocols. Davis and Wood demonstrated the release of nitric oxide from the endothelium after as much as forty-eight hours of hypothermic ischemia2. Moran et al. found that endothelial exocrine function was preserved by cold storage for as long as five days after a washout with the University of Wisconsin solution. These methods may be expected to improve the operative results of replantation and transplantation.
In general, a twenty-four-hour period of ischemia is thought to exceed the limitations for successful replantation or vascularized transplantation of virtually any organ or tissue, including bone17,18. One explanation for this may be tissue necrosis and vascular obstruction by endothelial cellular debris, edema leading to obstruction of the lumen of the vessel, or intravascular coagulation of blood components7. An additional explanation may be an ischemia-induced alteration in the function of the vascular endothelium favoring enhanced synthesis of vascular smooth-muscle contractile factors (endothelin) and impaired function of vascular smooth-muscle relaxing factors (nitric oxide). The results of the present study of increasing periods of storage at room temperature are consistent with this last explanation. However, our results also suggest some preservation of the endothelium-mediated synthesis of nitric oxide, as the contractile response to norepinephrine was significantly greater in the presence of NG-monomethyl-L-arginine acetate, even in the tibiae that had been stored for twenty-four hours. Furthermore, calcium ionophore A23187 (an endothelium-dependent and receptor-independent relaxing agent) decreased the baseline perfusion pressure in the phase-2 tibiae that had been stored for twenty-four hours. This chemical substance causes the production of nitric oxide in the vascular endothelium as long as the cell function of the endothelium is maintained even after receptor-mediated production of nitric oxide has been lost. We therefore hypothesized that the exceedingly high vascular resistance in the tibiae stored for twenty-four hours could be diminished if appropriate vasoactive agents were applied.
Adrenomedullin is known to be one of the most potent vasodilator drugs in vivo13. This drug decreases total peripheral resistance and blood pressure without affecting cardiac output or heart rate8,11. In the present study, a 0.1-milliliter bolus injection of 10-5-molar adrenomedullin solution attenuated the pressor response to norepinephrine for at least twenty minutes and relaxed vascular smooth muscle in the tibiae that had been stored for eight or twenty-four hours. Since cardiac output and heart rate are not altered during the marked systemic vasodepressor response to adrenomedullin and since adrenomedullin has a direct mechanism to relax vascular smooth muscle, activation of the adrenomedullin vasodilator mechanism may offer a therapeutic alternative in the clinical treatment of the so-called no-reflow phenomenon (reperfusion injury)19 known to occur in some clinical states of excessive ischemia.
Endothelin has three known isoforms (endothelin-1, endothelin-2, and endothelin-3). Endothelin-1 is produced by the vascular endothelium and is the most potent known vasoconstrictor27. It is also the most important member of the endothelin family found in the peripheral vascular bed. The circulating concentration of endothelin has been found to be increased in patients who have essential hypertension and atherosclerotic vascular disease as well as in the presence of reperfusion injury10,16. Moreover, the potent vascular smooth-muscle relaxing agent nitric oxide is known to be functionally impaired with prolonged ischemia and certain situations of reperfusion injury9. Thus, increased production of endothelin and decreased production or effectiveness of nitric oxide strongly favor increased vascular tone and vessel spasm23.
In conclusion, the exocrine function of the vascular endothelium in bone is altered in the intervals from four to eight and from eight to twenty-four hours of storage at room temperature. When practical, tactics that minimize the ischemic injury to the vascular endothelium should be employed. When ischemia-induced injury is established but not complete, adrenomedullin may be of some value in minimizing the factors that lead to sustained vascular smooth-muscle contraction and markedly elevated peripheral vascular resistance.
NOTE: The authors thank Mrs. Mary L. Adams and Mrs. Denise M. Heublein for their technical help.
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Footnotes
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*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were a fellowship from Kumamoto Kinou Hospital, Kumamoto, Japan (Dr. Kato), and Research Grant AR38671 from the National Institutes of Health.
Department of Orthopedics, Mayo Clinic, 200 First Street S.W., Rochester, Minnesota 55905. Please address requests for reprints to Dr. Wood.
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References
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T. KATO, A. T. BISHOP, Y.-K. TU, and M. B. WOOD
Function of the Vascular Endothelium after Hypothermic Storage at Four Degrees Celsius in a Canine Tibial Perfusion Model. The Role of Adrenomedullin in Reperfusion Injury
J. Bone Joint Surg. Am.,
September 1, 1998;
80(9):
1341 - 1348.
[Abstract]
[Full Text]
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Abstract
Introduction
