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Ann Thorac Surg 2000;69:1439-1444
© 2000 The Society of Thoracic Surgeons

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Original articles: Cardiovascular

Impairment of aortal tone by no flow–reflow conditions and its partial amelioration by mannitol

^a Department of Anesthesiology, Intensive Care and Post Anesthesia Care Units, Tel Aviv–Sourasky Medical Center and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
^b Division of Trauma, Tel Aviv–Sourasky Medical Center and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Address reprint requests to Dr Weinbroum, Post Anesthesia Care Unit and Department of Anesthesiology, Tel Aviv Sourasky Medical Center, 6 Weizman St, Tel Aviv 64239, Israel
e-mail: draviw{at}tasmc.health.gov.il

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Although postischemic cardiac or pulmonary dysfunction can relate to the impact of remotely generated oxygen stress mediators on the heart, their direct effect on the vascular bed remains unresolved. Thus, we tested these remote effects in an ex-vivo double organ model.

Methods. After stabilization With Krebs-Henseleit solution, isolated rat livers were either perfused or made ischemic for 2 hours. Aortic rings were stabilized, immersed in postischemic liver perfusates and their functions were tested. Some organs originated from donors fed with tungstate, whereas others had mannitol (0.25 g/kg) in the buffer.

Results. Incubation of aortic rings with postischemic hepatic effluent resulted in protracted contraction. Spasm was slightly lesser when the livers were pretreated with tungstate or exposed to mannitol, but worse in pretreated rings. The return to basal tone was abrupt in all ischemia–reperfusion aortae. The response of the rings to phenylephrine under the influence of the ischemia–reperfusion hepatic effluent was deficient. Mannitol prevented most abnormal responses.

Conclusions. Aortal tone impairment can occur by direct influence of the ischemia–reperfusion liver. It cannot be attributed entirely to xanthine oxidase, but also to other hepatic-released factors.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
A growing number of surgical procedures, such as clamping–declamping of the vasculature, or coronary artery bypass grafting, result in no flow–reflow conditions [1–3]. The local and systemic effects of the resumption of circulation (reperfusion) have been studied by different techniques and in several clinical models [4], and attempts have been made to attenuate these harmful consequences [5–7].

The effect of the postischemic insult in one organ on the cardiovascular performance is multiform: the velocities of contraction and relaxation of the isolated beating heart decrease significantly when exposed to postischemic perfusate, and the pulmonary arterial pressure increases under similar conditions [8]. We and other investigators [4, 8, 9] have previously discussed the possible role of various circulating mediators, such as radical oxygen species (ROS) that are generated or potentiated by either xanthine oxidase (XO) polymorphonucleates, arachidonic acid derivatives, transition metals (eg, univalent copper or divalent iron), or nitric oxide in reperfusion injury. Although part of the cardiovascular depression can be related to the impact of these remotely released mediators on the heart, their effect on the vascular bed has never been established. Indeed, the influence on the tone of the great vessels, of the same elements that depress the heart after remote flow–reflow situations, remains unsettled.

We hypothesize that XO, and subsequently the ROS that are generated during the reflow through the postischemic liver, could be detrimental to the aortal tone. This model of exposure of the isolated aortal ring to no flow–reflow insult, simulates the clinical conditions of uncompensated hemodynamics that requires frequent administration of vascular drugs, such as phenylephrine, to reestablish it. Therefore, in the present study we tested the vascular behavior upon exposure to effluents of hepatic ischemia and reperfusion (IR) and its response to this drug under these circumstances.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
All chemicals were of reagent grade or better and had been purchased from Sigma Chemical Co (St. Louis, MO), unless otherwise specified.

All experiments were carried out in accordance with guidelines established by the Institutional Animal Care and Use Committee at the Tel-Aviv Medical Center and the University of Tel-Aviv. Organs were donated by adult male Wistar rats (n = 168) weighing 320 to 380 g that were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). Two donors, one for each organ, were used for each experiment.

Isolated liver preparation
Liver isolation and perfusion were accomplished as previously described [6]. Briefly, an abdominal incision was performed, and combined perfusion and pressure monitoring cannulas (14 gauge) were placed in the portal vein and in the suprahepatic inferior vena cava. The organ was then placed in an environmental chamber designed to control temperature (at 37.5°C) and to minimize evaporation. A pressure transducer, thermometer, and a bubble trap were set up immediately before the isolated liver, and sample ports for the collection of aliquots were positioned at the entrance and exit of the liver.

Organs were perfused with freshly prepared hemoglobin-free, modified Krebs-Henseleit (MKH) solution of osmolality of 300 to 305 mOsm. Flow was of 3 to 5 mL · min^-1 · g^-1 liver weight, and was readjusted during stabilization to maintain an exit flow of physiologic pH and partial pressure of carbon dioxide. The effluent drainage pressure in the vena cava was 0 cm H₂O. The incoming perfusate had a constant temperature of 37°C and was equilibrated with 95%O₂–5%CO₂ to achieve an influent partial pressure of oxygen of 300 mm Hg or higher, partial pressure of carbon dioxide of 34 mm Hg, and pH of 7.34 to 7.46.

Isolated aortic ring preparation
In other anesthetized rats, the thorax was opened to quickly identify the descending thoracic aorta, which was excised and part of it was placed in a beaker containing oxygenated, warm MKH. After the removal of the surrounding tissue and fat, the aorta was cut to make a ring of about 2.5 mm wide. It was then mounted on an electromechanical transducer (FT-03; Grass Instrument Co, Quincy, MA) placed within a jacketed organ bath of 20 mL of working capacity, and changes in the ring tone were continuously recorded. The buffer was constantly warmed to 37°C and equilibrated with 95%O₂–5%CO₂.

Because aortic ring tone can change after surgical manipulation, a period of 30 minutes or more was allowed to pass until the ring gained plateau tone. The resting (isometric) tone of the ring was then standardized by stretching the ring with 2.0 g weight tension. Forty minutes or more were usually necessary for the ring to reequilibrate its resting tone. Then, 10 µg of phenylephrine were added to the solution to standardize the contracting potency of the ring. The ring was allowed to equilibrate again, and acetylcholine (10^-6 mol/L) was added to ensure the endothelial integrity. As a rule, after a plateau value was reached, fresh MKH replaced the ring’s solution and the time of return to baseline was recorded; only then was the ring ready for the double-organ experiment. Rings deficient in their drug response, or unable to relax to the original tone, were excluded from the study.

Specific study groups
Figure 1 depicts the perfusion system and its timetable. After liver stabilization phase (30 minutes), the livers (12 per group) were treated following one of seven protocols: groups 1, 2, and 3 were perfused for 125 minutes with MKH, as during stabilization. The donor rats of group 2 had been provided for 14 days with normal chow containing 0.07% wt/wt sodium tungstate, and with demineralized water. Such treatment was shown to significantly reduce body XO activity [10]. Mannitol (0.25 g/kg body weight), a known hydroxyl radical scavenger, was added to the MKH perfusing group 3.

View larger version (14K): [in this window] [in a new window]   Fig 1. Scheme of the experimental timetable.

Rings were exposed to the posthepatic solutions for 15 minutes, or longer, as required for the ring to reach steady state, and then phenylephrine (10 µg) was added to the solution of half of each group, and saline to the other half. The solution was refreshed when the effect of drugs gained steady state, and the time required to return to baseline tone under the new MKH was recorded in the saline subgroup.

Variations in the tone of the rings were expressed in absolute force change (g). At the end of the experiment the rings were examined histochemically by a vascular pathologist who was blinded to the protocols.

Assessment of hepatic damage
Hepatic lactate dehydrogenase activity was measured to assess hepatocellular damage [6]. Biochemical analyses were performed on 1.5-mL aliquots of the ring’s bathing solution, sampled at various times and kept on ice until processed. Lactate dehydrogenase, copper, and zinc concentrations were determined in the perfusate in duplicates, using commercial kits (Roche–Boehringer Mannheim, Mannheim, Germany) and an Hitachi 747 analyzer. Total XO activity (both reduced and oxidized enzyme forms) was analyzed following the procedure of Hashimoto [11].

Statistical analysis
The data variables are summarized as mean (standard error of the mean). At each time point, analysis of variance, with posthoc comparisons between groups by the Student’s Newman-Keuls test or the Fisher’s protected least significant different test were performed. To compare group values at different time points two-way repeated measured analysis of variance was performed followed by Bonferroni multiple comparison test. Significance level was set at probability value less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Hepatic function
Prefeeding rats with sodium tungstate or the addition of mannitol to the perfusion buffer did not affect the control or ischemic hepatic variables, except for the lowering of total XO activity in the liver perfusate of animals pretreated by tungstate (Table 1). Total XO in the IR perfusates was significantly (p < 0.01) higher than in any control liver group, except for IR livers treated only with tungstate, where the activity was as low as in the controls (Table 1). The levels of lactate dehydrogenase, representing hepatocellular damage, are also reported in Table 1. There was a clear distinction between the low lactate dehydrogenase activity in the controls and the high activity in IR livers perfusates. In the IR groups, the activity of both enzymes decayed during the reperfusion.

Aortal tone
All aortal rings, regardless of tungsten pretreatment or the presence of mannitol, had a similar response of contraction when standardized with phenylephrine and required similar periods to return to baseline (Fig 2). Acetylcholine reduced the tone to a similar degree in all the rings (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig 2. Normal aortal ring response to 10 µg of phenylephrine (top) and after contraction relaxation (bottom). The response of rings not exposed to hepatic effluent. (Control =untreated; Tungstate = from donors fed with sodium tungstate; Mannitol = the modified Krebs-Henseleit buffer contained 0.25 g/kg of mannitol.) All values are expressed as mean ± standard error of the mean.

View larger version (23K): [in this window] [in a new window]   Fig 3. Changes in aortal tone upon exposure to posthepatic perfusates (mean ± standard error of the mean). Controls are the average of all control sets, both treated and untreated. **p < 0.01 between all ischemia and reperfusion (IR) groups and the controls; #p < 0.01 between aortae exposed to effluents of ischemia and reperfusion untreated or ischemia and reperfusion tungstate-treated livers (groups 4 or 5) and the ischemia and reperfusion tungstate-treated aortae (group 6) or ischemia and reperfusion mannitol-treated (group 7).

The time necessary to return to basal tone in the saline-added rings was not only much shorter in all the IR groups, compared to controls, but it was without the gradual relaxation observed in control rings after phenylephrine treatment was replaced with fresh buffer. Thus, during stabilization the normal rings required 41 ± 1 minute to return to baseline (p < 0.001 versus all IR groups), but the IR rings required 13 ± 2 minutes, IR tungstate-treated livers (group 5) 16 ± 1 minute, group 6 (tungstate-treated rings) 10 ± 1, and the mannitol group 6.5 ± 1 minute.

Figure 4 depicts the rings’ response to phenylephrine while under the influence of the IR hepatic effluent, showing abnormal contractions in all the IR rings (p < 0.01 for all versus control). Specifically, 10 µg of phenylephrine generated a lesser contraction of the IR rings compared to the control potential: the worst response to phenylephrine was obtained in the group with the aorta pretreated with tungstate (group 6), whereas the mannitol (group 7) and the rings perfused with the livers that had been treated with tungstate (group 5) responded halfway between the controls and group 6 (Fig 4).

View larger version (27K): [in this window] [in a new window]   Fig 4. Aortic response to phenylephrine (mean ± standard error of the mean) after exposure to hepatic perfusates. Controls are the average of all control sets, both treated and untreated. **p < 0.01 between all ischemia and reperfusion (IR) groups and the corresponding controls; #p < 0.01 between the ischemia and reperfusion rings, untreated or tungsten pretreated, and the rings bathing in effluents of ischemia and reperfusion livers pretreated with tungstate, or rings treated with mannitol.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
The hypothesis that IR in one area of the body, such as the liver, directly contributes to the development of functional vascular impairment was confirmed in the present study. The incubation of a normal aortal ring in a blood-free buffer that exited from the postischemic liver resulted in an acute, abnormal, and prolonged contraction and in an aberrant return to basal tone. When subjected to phenylephrine, these aortae contracted less potently than those bathed in nonischemic hepatic effluents. Furthermore, the tungsten pretreatment of the ring donor rats, but not the liver donor rats, further worsened the aortal dysfunction throughout the experiment. Contrarily, partial protection of the aortal functionality was obtained when incubated in hepatic perfusates containing mannitol.

The prospective of severe abnormal changes in aortal tone and in other vascular areas is clinically very significant. The advent of a spastic contraction for the duration of the exposure to the reperfusate, and the perpetual loss of normal response to vasoactive drugs during that period, and even after the possible culprits were removed from the circulation, may detriment the cardiovascular compensation of patients after IR events. Although it is risky to extrapolate from an asanguinous ex-vivo isolated perfused organ model to events that take place in the living organism, some of the principal detrimental factors and the vascular response to the vasoactive substances, are identifiable in both processes. Indeed, this ex-vivo model is especially suitable for the investigation of mechanisms of damage to the aortic ring because of the absence of blood circulating elements, and because the confounding sympathetic readjustment process is not present in this model.

By maintaining normal values of partial pressure of carbon dioxide and pH in the ring’s buffer, the possibility that these could modulate the endothelial behavior, and from it the entire process of prolonged muscular spasm, was practically excluded. We also negated the possible endothelial damage by the observed normal vascular histology, and by the normal acetylcholine test found after the preparation of the rings. Thus, the greatly modified aortic responses could confirm the temporary modulatory effect of toxic compounds released from the liver, rather than an abnormally functioning endothelium.

It is now accepted that a cascade of events can start after damage to a vascular tree or to other body areas, even if followed by volume and pharmacological resuscitation. Events such as limb ischemia [12] and its reperfusion [1, 2], aortic cross-clamping, and aneurysmectomy [5, 13] result in the release of xanthine dehydrogenase and XO into the circulation. Our recent study demonstrated an association between XO and damage to the pulmonary airways with a simultaneous deterioration in alveolocapillary integrity [6]. An almost complete abrogation of these phenomena was obtained by the prior administration of the XO inhibitor allopurinol to the organ donor animals and its addition to the circulation.

In the present study we contended that the substitution of molybdenum, which is an essential element for the activity of XO, with tungsten [10], should minimize the deleterious effects of IR to the vascular tissue. Our second hypothesis was that the aorta could also be damaged by stress metabolites (ie, oxidants such as superoxide anion [O₂^-], hydrogen peroxide [H₂O₂], and hydroxyl radical [ · OH]) that are generated by XO when abundant purine substrates and free molecular oxygen are present [4, 8]. Hydrogen peroxide was proven to promptly induce and sustain pulmonary microvascular lesions [14], and the toxicity of the hydroxyl and superoxide radicals, despite their limited reactivity in biological systems, could account for remote aortic damage. This mechanism requires a continuous buildup of ROS, and should respond to appropriate scavengers.

Our first hypothesis was not supported by the collected data, because of the minimal attenuation of the functional damage when livers originated from tungsten-fed rats. The damage to the rings by perfusates of tungsten-treated livers could also suggest that even if XO is involved in the process, other factors affect the rings directly. This can be associated with the study by Svensson and colleagues [15], where the consequences of IR injury from cross-clamping of the thoracic aorta could not be prevented by allopurinol.

Humeral factors that are released from the ischemic and reperfused liver were blamed for an increase in arterial blood pressure in an animal transfused with blood from an IR animal [16]. The activity of such compounds is short lasting and could explain the initial brief, ubiquitous contraction seen in all IR rings. Catecholamines that can be released from Kupffer cells upon reperfusion have such properties [17]. They could interact with the aortic constituents and modulate the aorta muscular layer, independently of XO. Finally, the aorta might have also been disturbed by liver-released prostaglandins and anaphylotoxins [4]; however, their effect in this specific isolated perfused liver–aorta model is still unclear.

An indisputable source of interference with the normal aorta tone is nitric oxide. It must be produced sufficiently and released promptly from the endothelium to modulate the vascular tone. In a similar model of isolated aorta preparation [18], the presence of considerable amounts of ROS reduced the availability of nitric oxide through aortal endothelial dysfunction. This could support our second hypothesis, that is, that specific ROS in the bathing solution might have caused changes in aortal tone. Indeed, exogenous superoxide radical was demonstrated to cause loss of endothelium-dependent canine arterial relaxation [19]. Finally, Beckman and colleagues [20] have implicated a peroxynitrite radical-mediated mechanism for endothelial damage after ischemia–reperfusion.

The addition of mannitol to the rings’ bathing solution reduced in part their abnormal response to the IR solution. Mannitol is a hydroxyl radical (OH ·) scavenger and as such has been used clinically in various doses, and proven effective in attenuating ROS-induced injury to the kidney [21] and the heart [22], and was moderately effective in preventing pulmonary dysfunction after aortic cross-clamping in humans [7]. Our finding of partial attenuation of the aortal dysfunction would thus sustain the contention that hydroxyl radicals, as well as other IR-related mediators, are associated with this complex event.

In conclusion, this study documented the damaged functionality of rat aortae after ischemia and reperfusion in a remote organ. The impairment of the aortal tone can be reduced by mannitol, and therefore, future studies will examine more specific doses or different modalities of its administration, and other compounds that could mitigate these damaging remote effects.

	Acknowledgments

 
This study was partially supported by the "Anonymous Italian Family Foundation."

	References

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