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Temporal and concentration effects of isoflurane anaesthesia on intestinal tissue oxygenation and perfusion in horses K. Hopster a,*, C. Hopster-Iversen a, F. Geburek a, K. Rohn b, S.B.R. Kästner a,c a b c
Equine Clinic, University of Veterinary Medicine Hanover, Foundation, Bünteweg 9, 30559 Hanover, Germany Institute of Biometry and Information Processing, University of Veterinary Medicine Hanover, Foundation, Bünteweg 17, 30559 Hanover, Germany Centre for Systems Neuroscience Hanover, University of Veterinary Medicine Hanover, Foundation, Bünteweg 17, 30559 Hanover, Germany
A R T I C L E
I N F O
Article history: Accepted 20 April 2015 Keywords: Gastrointestinal tract Laser Doppler flowmetry Microperfusion White-light spectrophotometry
A B S T R A C T
The aim of this study was to assess the effect of duration of anaesthesia and concentration of isoflurane on global perfusion as well as intestinal microperfusion and oxygenation. Nine Warmblood horses were premedicated with xylazine; anaesthesia was induced with midazolam and ketamine, and maintained with isoflurane. Horses were ventilated to normocapnia. During 7 h of anaesthesia, mean arterial blood pressures (MAP), heart rate, central venous pressure, pulmonary artery pressure, expiratory isoflurane concentration (ETIso) and cardiac output using lithium dilution were measured; cardiac index (CI) was calculated. Intestinal microperfusion and oxygenation were measured using laser Doppler flowmetry and white-light spectrophotometry. Surface probes were placed via median laparotomy on the serosal and mucosal site of the jejunum and the pelvic flexion of the colon. After 3 h of constant ETIso (1.4%), ETIso was increased in 0.2% increments up to 2.4%, followed by a decrease to 1.2% and an increase to 1.4%. The CI and MAP decreased continuously with increasing ETIso to 40 ± 5 mL/kg/min and 52 ± 8 mmHg, respectively. Microperfusion and oxygenation remained unchanged until an ETIso of 2.0% resulted in CI and MAP of 48 ± 5 mL/kg/min and 62 ± 6 mmHg, respectively, and then decreased rapidly. When ETIso decreased back to baseline, CI, MAP, microperfusion and oxygenation recovered to baseline. Isoflurane concentration but not duration of isoflurane anaesthesia influenced central and intestinal oxygenation and perfusion in healthy horses. Under isoflurane, intestinal perfusion appeared to be preserved until a threshold MAP or blood flow was reached. © 2015 Elsevier Ltd. All rights reserved.
Introduction In anaesthetised horses, the physiological function of the gastrointestinal tract is compromised, but information on the effect of general anaesthesia on gastrointestinal microperfusion is limited. In humans, splanchnic perfusion and oxygenation are impaired early in the course of reduced systemic O2 transport, which might occur during anaesthesia (Gelman and Mushlin, 2004; Giglio et al., 2009), and impairment of splanchnic perfusion and/or oxygenation can contribute to alterations in intestinal motility (Buell and Harding, 1989) as well as disruption of the intestinal mucosal barrier (Fink et al., 1991), leading to septicaemia and ileus. Laser Doppler flowmetry and radionuclide-labelled microspheres have been used to study relative changes in microvascular blood flow in the skeletal muscle of anaesthetised horses (Raisis et al., 2000; Edner et al., 2002). These studies demonstrated that inhalant anaesthesia resulted in a dose-independent decrease in skeletal muscle blood flow. However, none of the studies investigated the effects
* Corresponding author. Tel.: +49 511 953 6613. E-mail address: [email protected] (K. Hopster).
of anaesthesia on perfusion of other peripheral organs. Spectrophotometry provides information about tissue oxygenation and is used as an indirect measure of perfusion. The technique has been used to measure haemodynamic variables and oxygenation in the hooves of conscious horses (Hinckley et al., 1995) and in the skeletal muscle of anaesthetised horses (Pringle et al., 2000). Using these techniques, muscle ischaemia could be differentiated from hypoxaemia, and muscle deoxygenation associated with clinically relevant hypoxaemia was reported in anaesthetised horses. The aim of the present study was to evaluate the effects of prolonged isoflurane anaesthesia at various concentrations on global perfusion, oxygenation, microperfusion and oxygenation of the gastrointestinal tract, using surface lightguide tissue spectrophotometry combined with laser Doppler flowmetry. Materials and methods Animals The study was reviewed by the Ethics Committee for Animal Experiments of Lower Saxony, and approved (approval number 33.14-42502-04-11/0572; date of approval 1 October 2011) according to Article 8, German Animal Welfare Act (Tierschutzgesetz).
http://dx.doi.org/10.1016/j.tvjl.2015.04.030 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
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Nine research horses weighing 546 ± 27 kg (mean ± standard deviation, SD) and aged 11 ± 5 years were used in this study. All horses had chronic, unresolved orthopaedic diseases that were refractory to treatment. Owners were informed about the study and provided their consent. General anaesthesia was induced before euthanasia. The horses were kept in box stalls and had free excess to hay and water. Eight hours before the induction of anaesthesia, horses were allowed access to water but not food. All horses were part of a terminal, experimental surgery study and were euthanased with pentobarbital 70 mg/kg (Euthadorm 400, CP-Pharma). Anaesthesia Premedication was with xylazine 0.8–1.1 mg/kg IV (Xylapan, Vetoquinol) and anaesthesia induced with a combination of intravenous (IV) midazolam 0.05 mg/kg (Midazolam-ratiopharm 15 mg/3 mL, Ratiopharm) and ketamine 2.2 mg/kg (Narketan, Vetoquinol). Anaesthesia was maintained with isoflurane (Isofluran, CP-Pharma) in 100% O2. Lactated Ringer’s solution (Ringer–Laktat–Lösung, B. Braun) was administered at 10 mL/kg/h. Following the induction of anaesthesia and endotracheal intubation, horses were positioned in dorsal recumbency and ventilated immediately with a pressure limited and pressure cycled large animal ventilator (Vet.-Tec. Model JAVC 2000 J.D. Medical Distributing). Horses were ventilated using intermittent positive pressure ventilation with peak inspiratory pressure (PIP) of 25 cm H2O. Respiratory rate (fR) was adjusted to maintain arterial partial carbon dioxide pressure (PaCO 2 ) at 40– 45 mmHg (5.3–6 kPa). Instrumentation Before anaesthesia, catheters were placed in both jugular veins, the right atrium and the pulmonary artery. The skin over the right and left jugular veins was clipped and aseptically prepared. After infiltration of the skin with mepivacaine hydrochloride (Scandicain 2%, AstraZeneca), a 12 G catheter (EquiCath Fastflow, B. Braun) was placed into the left jugular vein for drug administration. Two introduction ports were also placed into the right jugular vein under local anaesthesia, one at the midcervical region and the other close to the superior thoracic aperture. A balloontipped catheter (Balloon Wedge Pressure Catheter, Arrow) with a length of 160 mm was placed via the right jugular vein into the pulmonary artery to measure pulmonary artery pressure (PAP). A second balloon-tipped catheter was placed via the right jugular vein into the right atrium to measure central venous pressure (CVP). Correct placement of these catheters was verified by pressure curves as well as by transthoracic ultrasound. After induction of anaesthesia and during the instrumentation period, the transverse facial artery was cannulated with a 20G catheter (Venocan IV Catheter, Kruuse) for invasive blood pressure monitoring and arterial blood sampling. Catheters were connected to calibrated pressure transducers (Gould Statham Transducer, PD 23 ID) via fluid-filled low compliance extension lines. The pressure transducers were positioned at the level of the sternal manubrium. Combined spectrophotometry and laser-Doppler flow probes of a micro-lightguide spectrophotometer O2C (Oxygen to See, LEA Medizintechnik) were placed via median laparotomy on the serosal surface and were inserted into the lumen on the mucosal site of the jejunum and the pelvic flexion of the colon. Probes with a penetration depth of 2.5 mm (LF2, LEA Medizintechnik) were used on the serosal site. A probe with a penetration depth of 1 mm (LF5, LEA Medizintechnik) was inserted into the lumen of the small and large intestine and placed on the mucosal site. Measured variables Recording and evaluation of the data started 60 min after induction of anaesthesia. Mean arterial blood pressures (MAP), PAP, CVP, heart rate (HR), fR and expiratory isoflurane concentration (ETIso) were measured continuously with the Cardiocap 5-monitor (Datex-Ohmeda) and recorded.
Cardiac output (CO) measurements were performed by lithium dilution (LiDCOplus Hemodynamic Monitor, LiDCO). Software to accommodate LiDCOplus for measurements in large animals (LiDCOplus V4 Vet Configuration) was installed. Blood haemoglobin and plasma Na concentration were entered into the LiDCOplus monitor. A bolus of 2.25 mmol of LiCl was delivered manually through the drug catheter into the jugular vein. The LiCl was injected 5 s after initiating measurement to allow 12 s of stable baseline measurement, as required for accurate CO calculation. During anaesthesia, arterial blood samples were taken every 20 min and arterial pH, arterial partial O2 pressure (PaO2, mmHg) and PaCO2, as well as haemoglobin concentrations and arterial O2 saturation, were measured immediately after sampling (AVL995, AVL Medizintechnik). The O2 content of arterial blood (CaO2; mL/ 100 mL blood) and O2 delivery to peripheral tissue (DO2, mL/min) were calculated using standard equations:
CaO2 = (1.34 × [Hb] × SaO2 ) + (0.003 × PaO2 ) DO2 = CaO2 × CO where [Hb] is haemoglobin concentration (g/100 mL blood), SaO2 is % saturation of Hb with O2, and CO is expressed in mL/min. Tissue oxygenation and blood flow Tissue oxygenation (sO2 in %) and blood flow (flow) were measured by a micro-lightguide spectrophotometer (O2C) and data were sampled with 20 Hz. Measurements were performed every 20 min for at least five consecutive breaths over at least 40–50 s. Tissue depths at relevant sampling sites were confirmed by ultrasound investigation (Logiq E9, Fa. GE Healthcare; ultrasonic probe 6–15 MHz). Experimental protocol After 60 min of equilibration and instrumentation, six baseline measurements were performed at a stable plane of anaesthesia with E T Iso of 1.4% every 20 min over 2 h. Thereafter, isoflurane concentration was increased in 0.2% steps up to 2.4%, followed by decreases to 1.8%, 1.6% and 1.4%. Measurements were performed at the end of a 20 min equilibration period at each target isoflurane concentration. After another 60 min with constant E T Iso of 1.4% and three baseline measurements, isoflurane was reduced to 1.2% for two measurements (40 min), followed by a third period of 1 h and three measurements at ETIso of 1.4% (Fig. 1). The following parameters were measured every 20 min, after the target isoflurane concentration was reached: CO, MAP, CVP, PAP and HR, PaO2 and PaCO2, tissue oxygenation and tissue blood flow at the stomach, jejunum and colon (Fig. 1). Statistical analysis Statistical significance was set at P < 0.05. Analyses were carried out with commercially available statistical software (SAS, version 9.1.3; GraphPad Prism 5, GraphPad Software). For analysis of the linear model, the procedure MIXED was used. Normal distribution of model residuals of dependent variables was confirmed by visual assessment of q–q plots and by Shapiro–Wilks tests. Data are presented as means ± SD. Two-way ANOVA and Tukey’s post-hoc tests were used to compare measured parameters by period of time (repeated measurements). Correlations between parameters MAP, CI, ETIso and tissue flow were tested using Pearson correlation testing. Non-linear curve fitting was used to construct the curve that had the best fit to the data points (Fig. 4), to demonstrate correlations between tissue flow and MAP and tissue flow and CI.
Fig. 1. Time-line of the experimental protocol. Sixty minutes after induction of anaesthesia, isoflurane concentration is increased and decreased stepwise with baseline measurements in between. The red arrows indicate time points when following parameters were measured: CO, MAP, CVP, PAP and HR, PaO2 and PaCO2, tissue oxygenation and tissue blood flow. Equilib, equilibration period.
Please cite this article in press as: K. Hopster, C. Hopster-Iversen, F. Geburek, K. Rohn, S.B.R. Kästner, Temporal and concentration effects of isoflurane anaesthesia on intestinal tissue oxygenation and perfusion in horses, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.04.030
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Haemodynamic and respiratory variables There were no significant changes in HR over time; mean HR ranged from 38 to 43 beats/min. Increasing ETIso resulted in decreased MAP and CI, whereas PAP (range, 10.2–22.7 mmHg) and CVP (range, 3.6–9.1 mmHg) did not change significantly (Fig. 2). MAP and CI returned to baseline when ETIso reduced to baseline (Fig. 2). There was a close inverse correlation of ETIso with MAP and CI, with correlation coefficients of r2 = 0.96 (P < 0.0001) and r2 = 0.95 (P < 0.0001), respectively. Respiratory rate was adjusted (5–8 breaths/min) and a PIP of 25 cm H2O resulted in tidal volumes of 12–14 mL/kg. The PaO2 decreased over time from 226 ± 42 mmHg (30.1 ± 5.6 kPa) to 109 ± 42 mmHg (14.5 ± 5.6 kPa), whereas PaCO2 and SaO2 did not change. There was a decrease in DO2 with increasing ETIso, reaching statistical significance at ETIso ≥ 2.0% (Table 1). Oxygen content remained constant over time (Table 1). Tissue blood flow and oxygenation Perfusion (flow) of the muscular layer of the colon and jejunum remained constant over time and up to an ETIso of 2.0%. Above an E T Iso of 2.0% (CI and MAP lower at 48 ± 5 mL/kg/min and 62 ± 6 mmHg, respectively), a significant decrease of blood flow in the muscular layer of jejunum and colon occurred (Figs. 3 and 4). There was substantial variation between horses in mucosal perfusion of the jejunum and the colon; no significant changes were detected with changes in ETIso. There were no significant changes in tissue O2 saturation (sO2) of the muscular layer of the jejunum or the colon over time, or when changing isoflurane concentrations (Fig. 5). There was a significant drop in mucosal oxygenation of the jejunum and the colon when ETIso reached values of ≥2.2% and 2.4%, respectively. Discussion Fig. 2. Mean ± standard deviation of (A) mean arterial blood pressure (MAP) and (B) cardiac index (CI) over time with different isoflurane concentrations. *Statistically different from baseline.
This study demonstrated that plane but not duration of isoflurane anaesthesia influenced gastrointestinal perfusion and oxygenation in horses. In contrast to concentration of isoflurane, duration
Table 1 Mean ± standard deviation arterial O2 partial pressure (PaO2), O2 content of arterial blood (CaO2) O2 delivery (DO2), and mean cardiac output (CO) during isoflurane anaesthesia over time (min after 1 h of equilibration) with different concentrations of expired isoflurane concentrations (ETISO). Time points are minutes after 1 h of equilibration. Time
ETIso
PaO2 (mmHg)
PaO2 (kPa)
CaO2 (mL/dL)
DO2 (mL/min)
CO (L/min)
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
1.4 1.4 1.4 1.4 1.4 1.4 1.6 1.8 2.0 2.2 2.4 1.8 1.6 1.4 1.4 1.4 1.2 1.2 1.4 1.4 1.4
226 ± 42 216 ± 31 211 ± 28 147 ± 26 162 ± 29 132 ± 28a 138 ± 17a 149 ± 21a 115 ± 25a 142 ± 32a 125 ± 27a 141 ± 34a 130 ± 36a 122 ± 33a 127 ± 38a 122 ± 33a 119 ± 34a 132 ± 29a 121 ± 43a 112 ± 39a 109 ± 42a
30.1 ± 5.6 28.8 ± 4.1 28.1 ± 3.5 19.6 ± 3.5 21.6 ± 3.9 17.6 ± 3.7a 18.4 ± 2.3a 19.9 ± 2.8a 15.3 ± 3.3a 18.9 ± 4.3a 16.7 ± 3.6a 18.8 ± 4.5a 17.3 ± 4.8a 16.3 ± 4.4a 16.9 ± 5.1a 16.3 ± 4.4a 15.9 ± 4.5a 17.6 ± 3.9a 16.1 ± 5.7a 14.9 ± 5.2a 14.5 ± 5.6a
18.8 ± 0.4 18.8 ± 0.3 18.7 ± 0.2 18.6 ± 0.3 18.6 ± 0.2 18.5 ± 0.2 18.6 ± 0.3 18.6 ± 0.2 18.5 ± 0.3 18.6 ± 0.2 18.5 ± 0.3 18.6 ± 0.2 18.5 ± 0.4 18.5 ± 0.2 18.5 ± 0.4 18.5 ± 0.3 18.5 ± 0.3 18.5 ± 0.3 18.5 ± 0.4 18.5 ± 0.4 18.4 ± 0.4
6968 ± 523 6769 ± 612 7515 ± 584 7439 ± 624 7084 ± 632 7235 ± 559 6685 ± 526 5953 ± 578 4810 ± 398a 4645 ± 416a 4076 ± 361a 5388 ± 468 6491 ± 712 7038 ± 674 6873 ± 429 6952 ± 478 7326 ± 349 6824 ± 562 6743 ± 436 6625 ± 585 6743 ± 498
37 ± 5 36 ± 4 40 ± 6 40 ± 5 38 ± 6 39 ± 4 36 ± 3 32 ± 5a 26 ± 4a 25 ± 5a 22 ± 3a 29 ± 6a 35 ± 7 38 ± 6 37 ± 3 38 ± 4 40 ± 3 37 ± 5 36 ± 3 34 ± 6 35 ± 4
a
Significantly (P < 0.05) different from time point 0.
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Fig. 3. Mean ± standard deviation of the perfusion (flow in AU, arbitrary units) of the lamina muscularis of (a) the jejunum and (b) the colon and the lamina mucosa of (c) jejunum and (d) colon over time with different isoflurane concentrations. *Statistically different from baseline.
of anaesthesia within the observation period of this study had no effect on global and gastrointestinal perfusion. This was demonstrated by the repeated baseline measurements with constant isoflurane concentrations and by the return to the initial values after the isoflurane challenge after several hours of anaesthesia. There was a close inverse correlation of MAP and cardiac index with isoflurane concentrations as indices of global perfusion. Dosedependent cardiovascular depression by isoflurane has been well described (Steffey and Howland, 1980; Serteyn et al., 1987). Decreased cardiac index is a direct effect of isoflurane on myocardial contractility, leading to a reduction in stroke volume and consequent reduction in MAP (Steffey et al., 1987). High doses of isoflurane could also reduce vasomotor tone and systemic vascular resistance, further contributing to hypotension (Driessen et al., 2006). Perfusion of the muscle layers of the colon and jejunum remained unchanged until isoflurane concentrations reached ≥2.0%, resulting in cardiac index and MAP values of about 50 mL/kg/min and 60 mmHg, respectively. We used healthy, normovolaemic horses, but the effects of inadequate depth of anaesthesia in hypovolaemic horses (e.g. those with colic) could have more detrimental effects on cardiovascular function. In these horses, vigilant monitoring and meticulous adaption of depth of anaesthesia and isoflurane concentrations is even more important if gastrointestinal perfusion is to be adequately maintained.
In dogs, pigs and rats it has been demonstrated that during hypovolaemia, perfusion of the skin and gastrointestinal tract is disproportionally reduced compared to the muscle tissue (Nielsen and Secher, 1970; Lundeen et al., 1983; Mellström et al., 2009). In humans, the organs of the gastrointestinal tract (e.g. the pancreas) appeared to undergo considerable loss of blood flow with only small reductions in blood volume, with stabilisation at low levels despite further haemorrhage (Gosain et al., 1991). Other organs, notably the kidney, appeared to be relatively unaffected by substantial loss of blood volume (20–40%), after which their blood flow quite abruptly became sensitive to further hypovolaemia (Gosain et al., 1991). A sudden drop in perfusion was also observed in the intestinal muscularis in our study. We assume that in horses under isoflurane anaesthesia, intestinal perfusion is sustained for relatively long periods during anaesthesia-induced reduction in central perfusion parameters. One explanation could be the differential regulation of large and small vessels. Large vessels are under the influence of humoral (catecholamines, angiotensin II, vasopressin, serotonin) and neuronal (sympathetic and parasympathetic tone) mediators, whereas the capillaries of the gastrointestinal tract are mainly regulated by paracrine (endothelin-1, prostacyclin) and metabolic (pH, PaO2, PaCO2) mediators (Chou, 1992). It is possible that in our study, despite reduced blood flow in large vessels, local microperfusion
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Fig. 4. Scatter plots showing correlation between the colon (blue circles) and jejunum (green squares) muscularis tissue perfusion (flow in AU, arbitrary units) and (a) mean arterial blood pressure (MAP) or (b) the cardiac index (CI). The blue sigmoidal lines indicate the nonlinear regression between colon muscularis flow and MAP (a) or CI (b); the turning point (log IC50) is MAP, 65 mmHg and CI, 51 mL/kg/min. The green sigmoidal lines indicate the nonlinear regression between jejunum muscularis flow and MAP (a) or CI (b); the turning point (log IC50) is MAP, 63 mmHg and CI, 49 mL/kg/min.
of gastrointestinal organs was maintained for a wider range of increasing isoflurane concentrations. In contrast to gastrointestinal muscle layer perfusion, there were no significant changes in mucosal perfusion. A retrospective power analysis calculated that at the given variability, 170 horses would be required to demonstrate a statistically significant difference in mucosal perfusion between baseline and the highest isoflurane concentrations (P < 0.05; statistical power, 80%). It is possible that local autoregulation of mucosal blood flow is functional at lower ranges of MAP and CI than we achieved by increasing isofluraneconcentrations. Several studies suggest a pronounced metabolic regulation of blood flow supply to the gastrointestinal mucosal layer (Kiel et al., 1987; Yamaguchi, 1990; Chou, 1992). This autoregulation occurs under conditions of low flow, hypotension and tissue hypoxia, so that temporal tissue perfusion maintains adequate nutritional blood flow in tissues under conditions of limited O2 supply (Pajk et al., 2002). However, it is also possible that perfusion of the mucosal layer was already impaired at baseline, leaving no potential for further decreases. When comparing the mucosal and muscular blood flow of the jejunum and the colon, it was striking that mucosal values were much lower than muscularis values. In a porcine study comparing the mucosal and muscularis metabolism of the small intestine, early blood loss was indicated by an immediate decrease in intestinal
intramucosal pH and an increase in PCO2, in contrast to muscularis measurements, which did not change until severe blood loss occurred, indicating an earlier change in mucosal blood flow (Mellström et al., 2009). Mucosal flow measurements in our horses were highly variable. This could be related to methodology, since mucosal measurements were performed from the luminal site, making them very sensitive to artefacts related to intestinal motility. Additionally, it is possible that there were differences in contact pressure that influenced focal perfusion. Another aim of our study was to investigate the effects of isoflurane anaesthesia on tissue oxygenation. There was a progressive decrease in PaO2 that was isoflurane concentration-independent, and which did not result in severe hypoxaemia (arterial O2 saturation >95%). Therefore, adequate O2 content was always provided. Increasing isoflurane concentrations resulted in decreased CO and therefore decreased O2 delivery. This decrease in delivery lowered tissue oxygenation of the lamina mucosa but not of the lamina muscularis. The intestinal mucosa of pigs is known to be vulnerable to local hypoxaemia soon after the induction of hypovolaemia and countercurrent diffusion of O2 between adjacent microvessels has been proposed as a possible mechanism for this (Mellström et al., 2009). In contrast to mucosal oxygenation, the oxygenation of the muscular layer was not significantly affected by decreased O2
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Fig. 5. Mean ± standard deviation of the tissue oxygenation of the lamina muscularis of (a) the jejunum and (b) the colon as well as the lamina mucosa of (c) the jejunum and (d) the colon over time with different isoflurane concentrations. *Statistically different from baseline.
delivery in our study, which is also comparable to findings in anaesthetised pigs (Mellström et al., 2009). This might be related to lower O2 requirements by the muscular layer than the mucosal cell layer during anaesthesia, since the mucosal cell layer requires high O2 concentrations because of high metabolic and resorptive activity (Phillis, 1976). To our knowledge, the present study is the first to use a combination of lightguide tissue spectrophotometry and laser Doppler flowmetry (O2C) to measure the impact of isoflurane anaesthesia on oxygenation and perfusion of the gastrointestinal tract in horses. An earlier study demonstrated that micro-lightguide tissue spectrophotometry was easy to use and provided reliable and reproducible information about microcirculation of the intestinal wall in horses (Reichert et al., 2014). In human medicine, the O2C is considered a reliable method to examine microperfusion and oxygenation (Forst et al., 2008). The major limitation of laser Doppler flowmetry for perfusion measurements is its inability to obtain quantitative flow measurements. Absolute calibration of the output voltage of a laser Doppler flowmeter is difficult, due to the small sample volume size, temporal variations in the microcirculation and variations in the optical properties of the tissues (Borgos, 1994). As a result, flow can only be expressed in arbitrary units. Laser Doppler flowmetry is limited to the measurement of relative flow at a single sample site during
a single study period, so to determine tissue oxygenation, lightguide tissue spectrophotometry was used. The O2C is calibrated for the absorption characteristics of human blood, which differs from equine blood. It is possible that absolute values measured by the O2C were different from real tissue oxygenation. Nonetheless, changes in oxygenation measured in our study were considered reliable because all measurements were performed over time with the same probe in the same animal. Potential advantages of the O2C technique include potential for continuous real-time measurements and the use of surface probes that mean the measurements are non-invasive, without local bleeding or other tissue irritation that could influence values. Conclusions In this equine study, the concentration of isoflurane, but not the duration of anaesthesia, influenced global gastrointestinal perfusion and oxygenation and microperfusion. Under isoflurane anaesthesia, gastrointestinal microperfusion appeared to be preserved until a threshold MAP of approximately 60 mmHg or CO < 50 mL/kg/min was reached. The intestinal mucosal layers desaturated earlier than the intestinal muscularis layers under a deep plane of general anaesthesia.
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Conflict of interest statement None of the authors has any financial or personal relationships with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements The authors wish to thank Dr. P. Conze, Dr. C. Reichert and A. Wogatzki for their technical assistance. Preliminary results were presented as an Abstract at the AVA Autumn Meeting, Moscow, Russia, 3–4 October 2013. References Borgos, J., 1994. Principles of instrumentation: Calibration and technical issues. In: Belcaro, G., Hoffman, U., Bollinger, A., Nicolaides, A. (Eds.), Laser Doppler. Med-Orion, London, UK, pp. 3–16. Buell, M.G., Harding, R.K., 1989. Effects of peptide YY on intestinal blood flow distribution and motility in the dog. Regulatory Peptides 24, 195–208. Chou, C.C., 1992. Intestinal blood flow regulation. In: Dulbecco, R. (Ed.), Encyclopedia of Human Biology. Academic Press, San Diego, CA, pp. 547–556. Driessen, B., Nann, L., Benton, R., Boston, R., 2006. Differences in need for hemodynamic support in horses anesthetized with sevoflurane as compared to isoflurane. Veterinary Anaesthesia and Analgesia 33, 356–367. Edner, A., Nyman, G., Essén-Gustavsson, B., 2002. The relationship of muscle perfusion and metabolism with cardiovascular variables before and after detomidine injection during propofol-ketamine anaesthesia in horses. Veterinary Anaesthesia and Analgesia 29, 182–199. Fink, M.P., Kaups, K.L., Wang, H.L., Rothschild, H.R., 1991. Maintenance of superior mesenteric arterial perfusion prevents increased intestinal mucosal permeability in endotoxic pigs. Surgery 110, 154–160. Forst, T., Hohberg, C., Tarakci, E., Forst, S., Kann, P., Pfützner, A., 2008. Reliability of Lightguide Spectrophotometry (O2C®) for the investigation of skin tissue microvascular blood flow and tissue oxygen supply in diabetic and nondiabetic subjects. Journal of Diabetes Science and Technology 2, 1151–1156. Gelman, S., Mushlin, P.S., 2004. Catecholamine-induced changes in the splanchnic circulation affecting systemic hemodynamics. Anesthesiology 100, 434–439. Giglio, M.T., Marucci, M., Testini, M., Brienza, N., 2009. Goal-directed haemodynamic therapy and gastrointestinal complications in major surgery: A meta-analysis of randomized controlled trials. British Journal of Anaesthesia 103, 637–646.
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Gosain, A., Rabkin, J., Reymond, J.P., Jensen, J.A., Hunt, T.K., Upton, R.A., 1991. Tissue oxygen tension and other indicators of blood loss or organ perfusion during graded hemorrhage. Surgery 109, 523–532. Hinckley, K.A., Fearn, S., Howard, B.R., Henderson, I.W., 1995. Near infrared spectroscopy of pedal haemodynamics and oxygenation in normal and laminitic horses. Equine Veterinary Journal 27, 465–470. Kiel, J.W., Riedel, G.L., Shepherd, A.P., 1987. Local control of canine gastric mucosal blood flow. Gastroenterology 93, 1041–1053. Lundeen, G., Manohar, M., Parks, C., 1983. Systemic distribution of blood flow in swine while awake and during 1.0 and 1.5 MAC isoflurane anesthesia with or without 50% nitrous oxide. Anesthesia and Analgesia 62, 499–512. Mellström, A., Månsson, P., Jonsson, K., Hartmann, M., 2009. Measurements of subcutaneous tissue PO2 reflect oxygen metabolism of the small intestinal mucosa during hemorrhage and resuscitation. An experimental study in pigs. European Surgical Research 42, 122–129. Nielsen, P.A., Secher, N.J., 1970. Blood flow in adipose tissue and skeletal muscle during hemorrhagic shock in heparinized dogs. Life Sciences. Pt. 1: Physiology and Pharmacology 15, 75–82. Pajk, W., Schwarz, B., Knotzer, H., Friesenecker, B., Mayr, A., Dünser, M., Hasibeder, W., 2002. Jejunal tissue oxygenation and microvascular flow motion during hemorrhage and resuscitation. American Journal of Physiology, Heart and Circulatory Physiology 283, 2511–2517. Phillis, J.W., 1976. The gastro-intestinal system. Functions of the gastro-intestinal tract. In: Phillis, J.W. (Ed.), Veterinary Physiology. Wright-Scientechnica, Bristol, UK. Pringle, J., Roberts, C., Art, T., Lekeux, P., 2000. Assessment of muscle oxygenation in the horse by near infrared spectroscopy. Equine Veterinary Journal 32, 59–64. Raisis, A.L., Young, L.E., Blissitt, K.J., Brearley, J.C., Meire, H.B., Taylor, P.M., Lekeux, P., 2000. A comparison of the haemodynamic effects of isoflurane and halothane anaesthesia in horses. Equine Veterinary Journal 32, 318–326. Reichert, C., Kästner, S.B., Hopster, K., Rohn, K., Rötting, A.K., 2014. Use of microlightguide spectrophotometry for evaluation of microcirculation in the small and large intestines of horses without gastrointestinal disease. American Journal of Veterinary Research 75, 990–996. Serteyn, D., Coppens, P., Mottart, E., Michelet, S., Micheels, J., Philippart, C., Lamy, M., 1987. Measurements of muscular microcirculation by laser Doppler flowmetry in isoflurane and halothane anaesthetised horses. Veterinary Record 121, 324–326. Steffey, E.P., Howland, D., 1980. Comparison of circulatory and respiratory effects of isoflurane and halothane anesthesia in horses. American Journal of Veterinary Research 41, 821–825. Steffey, E.P., Dunlop, C.I., Farver, T.B., Woliner, M.J., Schultz, L.J., 1987. Cardiovascular and respiratory measurements in awake and isoflurane-anesthetized horses. American Journal of Veterinary Research 48, 7–12. Yamaguchi, T., 1990. Relationship between gastric mucosal hemodynamics and gastric motility. Journal of Gastroenterology 25, 299–305.
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Temporal and concentration effects of isoflurane anaesthesia on intestinal tissue oxygenation and perfusion in horses K. Hopster a,*, C. Hopster-Iversen a, F. Geburek a, K. Rohn b, S.B.R. Kästner a,c a b c
Equine Clinic, University of Veterinary Medicine Hanover, Foundation, Bünteweg 9, 30559 Hanover, Germany Institute of Biometry and Information Processing, University of Veterinary Medicine Hanover, Foundation, Bünteweg 17, 30559 Hanover, Germany Centre for Systems Neuroscience Hanover, University of Veterinary Medicine Hanover, Foundation, Bünteweg 17, 30559 Hanover, Germany
A R T I C L E
I N F O
Article history: Accepted 20 April 2015 Keywords: Gastrointestinal tract Laser Doppler flowmetry Microperfusion White-light spectrophotometry
A B S T R A C T
The aim of this study was to assess the effect of duration of anaesthesia and concentration of isoflurane on global perfusion as well as intestinal microperfusion and oxygenation. Nine Warmblood horses were premedicated with xylazine; anaesthesia was induced with midazolam and ketamine, and maintained with isoflurane. Horses were ventilated to normocapnia. During 7 h of anaesthesia, mean arterial blood pressures (MAP), heart rate, central venous pressure, pulmonary artery pressure, expiratory isoflurane concentration (ETIso) and cardiac output using lithium dilution were measured; cardiac index (CI) was calculated. Intestinal microperfusion and oxygenation were measured using laser Doppler flowmetry and white-light spectrophotometry. Surface probes were placed via median laparotomy on the serosal and mucosal site of the jejunum and the pelvic flexion of the colon. After 3 h of constant ETIso (1.4%), ETIso was increased in 0.2% increments up to 2.4%, followed by a decrease to 1.2% and an increase to 1.4%. The CI and MAP decreased continuously with increasing ETIso to 40 ± 5 mL/kg/min and 52 ± 8 mmHg, respectively. Microperfusion and oxygenation remained unchanged until an ETIso of 2.0% resulted in CI and MAP of 48 ± 5 mL/kg/min and 62 ± 6 mmHg, respectively, and then decreased rapidly. When ETIso decreased back to baseline, CI, MAP, microperfusion and oxygenation recovered to baseline. Isoflurane concentration but not duration of isoflurane anaesthesia influenced central and intestinal oxygenation and perfusion in healthy horses. Under isoflurane, intestinal perfusion appeared to be preserved until a threshold MAP or blood flow was reached. © 2015 Elsevier Ltd. All rights reserved.
Introduction In anaesthetised horses, the physiological function of the gastrointestinal tract is compromised, but information on the effect of general anaesthesia on gastrointestinal microperfusion is limited. In humans, splanchnic perfusion and oxygenation are impaired early in the course of reduced systemic O2 transport, which might occur during anaesthesia (Gelman and Mushlin, 2004; Giglio et al., 2009), and impairment of splanchnic perfusion and/or oxygenation can contribute to alterations in intestinal motility (Buell and Harding, 1989) as well as disruption of the intestinal mucosal barrier (Fink et al., 1991), leading to septicaemia and ileus. Laser Doppler flowmetry and radionuclide-labelled microspheres have been used to study relative changes in microvascular blood flow in the skeletal muscle of anaesthetised horses (Raisis et al., 2000; Edner et al., 2002). These studies demonstrated that inhalant anaesthesia resulted in a dose-independent decrease in skeletal muscle blood flow. However, none of the studies investigated the effects
* Corresponding author. Tel.: +49 511 953 6613. E-mail address: [email protected] (K. Hopster).
of anaesthesia on perfusion of other peripheral organs. Spectrophotometry provides information about tissue oxygenation and is used as an indirect measure of perfusion. The technique has been used to measure haemodynamic variables and oxygenation in the hooves of conscious horses (Hinckley et al., 1995) and in the skeletal muscle of anaesthetised horses (Pringle et al., 2000). Using these techniques, muscle ischaemia could be differentiated from hypoxaemia, and muscle deoxygenation associated with clinically relevant hypoxaemia was reported in anaesthetised horses. The aim of the present study was to evaluate the effects of prolonged isoflurane anaesthesia at various concentrations on global perfusion, oxygenation, microperfusion and oxygenation of the gastrointestinal tract, using surface lightguide tissue spectrophotometry combined with laser Doppler flowmetry. Materials and methods Animals The study was reviewed by the Ethics Committee for Animal Experiments of Lower Saxony, and approved (approval number 33.14-42502-04-11/0572; date of approval 1 October 2011) according to Article 8, German Animal Welfare Act (Tierschutzgesetz).
http://dx.doi.org/10.1016/j.tvjl.2015.04.030 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
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Nine research horses weighing 546 ± 27 kg (mean ± standard deviation, SD) and aged 11 ± 5 years were used in this study. All horses had chronic, unresolved orthopaedic diseases that were refractory to treatment. Owners were informed about the study and provided their consent. General anaesthesia was induced before euthanasia. The horses were kept in box stalls and had free excess to hay and water. Eight hours before the induction of anaesthesia, horses were allowed access to water but not food. All horses were part of a terminal, experimental surgery study and were euthanased with pentobarbital 70 mg/kg (Euthadorm 400, CP-Pharma). Anaesthesia Premedication was with xylazine 0.8–1.1 mg/kg IV (Xylapan, Vetoquinol) and anaesthesia induced with a combination of intravenous (IV) midazolam 0.05 mg/kg (Midazolam-ratiopharm 15 mg/3 mL, Ratiopharm) and ketamine 2.2 mg/kg (Narketan, Vetoquinol). Anaesthesia was maintained with isoflurane (Isofluran, CP-Pharma) in 100% O2. Lactated Ringer’s solution (Ringer–Laktat–Lösung, B. Braun) was administered at 10 mL/kg/h. Following the induction of anaesthesia and endotracheal intubation, horses were positioned in dorsal recumbency and ventilated immediately with a pressure limited and pressure cycled large animal ventilator (Vet.-Tec. Model JAVC 2000 J.D. Medical Distributing). Horses were ventilated using intermittent positive pressure ventilation with peak inspiratory pressure (PIP) of 25 cm H2O. Respiratory rate (fR) was adjusted to maintain arterial partial carbon dioxide pressure (PaCO 2 ) at 40– 45 mmHg (5.3–6 kPa). Instrumentation Before anaesthesia, catheters were placed in both jugular veins, the right atrium and the pulmonary artery. The skin over the right and left jugular veins was clipped and aseptically prepared. After infiltration of the skin with mepivacaine hydrochloride (Scandicain 2%, AstraZeneca), a 12 G catheter (EquiCath Fastflow, B. Braun) was placed into the left jugular vein for drug administration. Two introduction ports were also placed into the right jugular vein under local anaesthesia, one at the midcervical region and the other close to the superior thoracic aperture. A balloontipped catheter (Balloon Wedge Pressure Catheter, Arrow) with a length of 160 mm was placed via the right jugular vein into the pulmonary artery to measure pulmonary artery pressure (PAP). A second balloon-tipped catheter was placed via the right jugular vein into the right atrium to measure central venous pressure (CVP). Correct placement of these catheters was verified by pressure curves as well as by transthoracic ultrasound. After induction of anaesthesia and during the instrumentation period, the transverse facial artery was cannulated with a 20G catheter (Venocan IV Catheter, Kruuse) for invasive blood pressure monitoring and arterial blood sampling. Catheters were connected to calibrated pressure transducers (Gould Statham Transducer, PD 23 ID) via fluid-filled low compliance extension lines. The pressure transducers were positioned at the level of the sternal manubrium. Combined spectrophotometry and laser-Doppler flow probes of a micro-lightguide spectrophotometer O2C (Oxygen to See, LEA Medizintechnik) were placed via median laparotomy on the serosal surface and were inserted into the lumen on the mucosal site of the jejunum and the pelvic flexion of the colon. Probes with a penetration depth of 2.5 mm (LF2, LEA Medizintechnik) were used on the serosal site. A probe with a penetration depth of 1 mm (LF5, LEA Medizintechnik) was inserted into the lumen of the small and large intestine and placed on the mucosal site. Measured variables Recording and evaluation of the data started 60 min after induction of anaesthesia. Mean arterial blood pressures (MAP), PAP, CVP, heart rate (HR), fR and expiratory isoflurane concentration (ETIso) were measured continuously with the Cardiocap 5-monitor (Datex-Ohmeda) and recorded.
Cardiac output (CO) measurements were performed by lithium dilution (LiDCOplus Hemodynamic Monitor, LiDCO). Software to accommodate LiDCOplus for measurements in large animals (LiDCOplus V4 Vet Configuration) was installed. Blood haemoglobin and plasma Na concentration were entered into the LiDCOplus monitor. A bolus of 2.25 mmol of LiCl was delivered manually through the drug catheter into the jugular vein. The LiCl was injected 5 s after initiating measurement to allow 12 s of stable baseline measurement, as required for accurate CO calculation. During anaesthesia, arterial blood samples were taken every 20 min and arterial pH, arterial partial O2 pressure (PaO2, mmHg) and PaCO2, as well as haemoglobin concentrations and arterial O2 saturation, were measured immediately after sampling (AVL995, AVL Medizintechnik). The O2 content of arterial blood (CaO2; mL/ 100 mL blood) and O2 delivery to peripheral tissue (DO2, mL/min) were calculated using standard equations:
CaO2 = (1.34 × [Hb] × SaO2 ) + (0.003 × PaO2 ) DO2 = CaO2 × CO where [Hb] is haemoglobin concentration (g/100 mL blood), SaO2 is % saturation of Hb with O2, and CO is expressed in mL/min. Tissue oxygenation and blood flow Tissue oxygenation (sO2 in %) and blood flow (flow) were measured by a micro-lightguide spectrophotometer (O2C) and data were sampled with 20 Hz. Measurements were performed every 20 min for at least five consecutive breaths over at least 40–50 s. Tissue depths at relevant sampling sites were confirmed by ultrasound investigation (Logiq E9, Fa. GE Healthcare; ultrasonic probe 6–15 MHz). Experimental protocol After 60 min of equilibration and instrumentation, six baseline measurements were performed at a stable plane of anaesthesia with E T Iso of 1.4% every 20 min over 2 h. Thereafter, isoflurane concentration was increased in 0.2% steps up to 2.4%, followed by decreases to 1.8%, 1.6% and 1.4%. Measurements were performed at the end of a 20 min equilibration period at each target isoflurane concentration. After another 60 min with constant E T Iso of 1.4% and three baseline measurements, isoflurane was reduced to 1.2% for two measurements (40 min), followed by a third period of 1 h and three measurements at ETIso of 1.4% (Fig. 1). The following parameters were measured every 20 min, after the target isoflurane concentration was reached: CO, MAP, CVP, PAP and HR, PaO2 and PaCO2, tissue oxygenation and tissue blood flow at the stomach, jejunum and colon (Fig. 1). Statistical analysis Statistical significance was set at P < 0.05. Analyses were carried out with commercially available statistical software (SAS, version 9.1.3; GraphPad Prism 5, GraphPad Software). For analysis of the linear model, the procedure MIXED was used. Normal distribution of model residuals of dependent variables was confirmed by visual assessment of q–q plots and by Shapiro–Wilks tests. Data are presented as means ± SD. Two-way ANOVA and Tukey’s post-hoc tests were used to compare measured parameters by period of time (repeated measurements). Correlations between parameters MAP, CI, ETIso and tissue flow were tested using Pearson correlation testing. Non-linear curve fitting was used to construct the curve that had the best fit to the data points (Fig. 4), to demonstrate correlations between tissue flow and MAP and tissue flow and CI.
Fig. 1. Time-line of the experimental protocol. Sixty minutes after induction of anaesthesia, isoflurane concentration is increased and decreased stepwise with baseline measurements in between. The red arrows indicate time points when following parameters were measured: CO, MAP, CVP, PAP and HR, PaO2 and PaCO2, tissue oxygenation and tissue blood flow. Equilib, equilibration period.
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Haemodynamic and respiratory variables There were no significant changes in HR over time; mean HR ranged from 38 to 43 beats/min. Increasing ETIso resulted in decreased MAP and CI, whereas PAP (range, 10.2–22.7 mmHg) and CVP (range, 3.6–9.1 mmHg) did not change significantly (Fig. 2). MAP and CI returned to baseline when ETIso reduced to baseline (Fig. 2). There was a close inverse correlation of ETIso with MAP and CI, with correlation coefficients of r2 = 0.96 (P < 0.0001) and r2 = 0.95 (P < 0.0001), respectively. Respiratory rate was adjusted (5–8 breaths/min) and a PIP of 25 cm H2O resulted in tidal volumes of 12–14 mL/kg. The PaO2 decreased over time from 226 ± 42 mmHg (30.1 ± 5.6 kPa) to 109 ± 42 mmHg (14.5 ± 5.6 kPa), whereas PaCO2 and SaO2 did not change. There was a decrease in DO2 with increasing ETIso, reaching statistical significance at ETIso ≥ 2.0% (Table 1). Oxygen content remained constant over time (Table 1). Tissue blood flow and oxygenation Perfusion (flow) of the muscular layer of the colon and jejunum remained constant over time and up to an ETIso of 2.0%. Above an E T Iso of 2.0% (CI and MAP lower at 48 ± 5 mL/kg/min and 62 ± 6 mmHg, respectively), a significant decrease of blood flow in the muscular layer of jejunum and colon occurred (Figs. 3 and 4). There was substantial variation between horses in mucosal perfusion of the jejunum and the colon; no significant changes were detected with changes in ETIso. There were no significant changes in tissue O2 saturation (sO2) of the muscular layer of the jejunum or the colon over time, or when changing isoflurane concentrations (Fig. 5). There was a significant drop in mucosal oxygenation of the jejunum and the colon when ETIso reached values of ≥2.2% and 2.4%, respectively. Discussion Fig. 2. Mean ± standard deviation of (A) mean arterial blood pressure (MAP) and (B) cardiac index (CI) over time with different isoflurane concentrations. *Statistically different from baseline.
This study demonstrated that plane but not duration of isoflurane anaesthesia influenced gastrointestinal perfusion and oxygenation in horses. In contrast to concentration of isoflurane, duration
Table 1 Mean ± standard deviation arterial O2 partial pressure (PaO2), O2 content of arterial blood (CaO2) O2 delivery (DO2), and mean cardiac output (CO) during isoflurane anaesthesia over time (min after 1 h of equilibration) with different concentrations of expired isoflurane concentrations (ETISO). Time points are minutes after 1 h of equilibration. Time
ETIso
PaO2 (mmHg)
PaO2 (kPa)
CaO2 (mL/dL)
DO2 (mL/min)
CO (L/min)
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
1.4 1.4 1.4 1.4 1.4 1.4 1.6 1.8 2.0 2.2 2.4 1.8 1.6 1.4 1.4 1.4 1.2 1.2 1.4 1.4 1.4
226 ± 42 216 ± 31 211 ± 28 147 ± 26 162 ± 29 132 ± 28a 138 ± 17a 149 ± 21a 115 ± 25a 142 ± 32a 125 ± 27a 141 ± 34a 130 ± 36a 122 ± 33a 127 ± 38a 122 ± 33a 119 ± 34a 132 ± 29a 121 ± 43a 112 ± 39a 109 ± 42a
30.1 ± 5.6 28.8 ± 4.1 28.1 ± 3.5 19.6 ± 3.5 21.6 ± 3.9 17.6 ± 3.7a 18.4 ± 2.3a 19.9 ± 2.8a 15.3 ± 3.3a 18.9 ± 4.3a 16.7 ± 3.6a 18.8 ± 4.5a 17.3 ± 4.8a 16.3 ± 4.4a 16.9 ± 5.1a 16.3 ± 4.4a 15.9 ± 4.5a 17.6 ± 3.9a 16.1 ± 5.7a 14.9 ± 5.2a 14.5 ± 5.6a
18.8 ± 0.4 18.8 ± 0.3 18.7 ± 0.2 18.6 ± 0.3 18.6 ± 0.2 18.5 ± 0.2 18.6 ± 0.3 18.6 ± 0.2 18.5 ± 0.3 18.6 ± 0.2 18.5 ± 0.3 18.6 ± 0.2 18.5 ± 0.4 18.5 ± 0.2 18.5 ± 0.4 18.5 ± 0.3 18.5 ± 0.3 18.5 ± 0.3 18.5 ± 0.4 18.5 ± 0.4 18.4 ± 0.4
6968 ± 523 6769 ± 612 7515 ± 584 7439 ± 624 7084 ± 632 7235 ± 559 6685 ± 526 5953 ± 578 4810 ± 398a 4645 ± 416a 4076 ± 361a 5388 ± 468 6491 ± 712 7038 ± 674 6873 ± 429 6952 ± 478 7326 ± 349 6824 ± 562 6743 ± 436 6625 ± 585 6743 ± 498
37 ± 5 36 ± 4 40 ± 6 40 ± 5 38 ± 6 39 ± 4 36 ± 3 32 ± 5a 26 ± 4a 25 ± 5a 22 ± 3a 29 ± 6a 35 ± 7 38 ± 6 37 ± 3 38 ± 4 40 ± 3 37 ± 5 36 ± 3 34 ± 6 35 ± 4
a
Significantly (P < 0.05) different from time point 0.
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Fig. 3. Mean ± standard deviation of the perfusion (flow in AU, arbitrary units) of the lamina muscularis of (a) the jejunum and (b) the colon and the lamina mucosa of (c) jejunum and (d) colon over time with different isoflurane concentrations. *Statistically different from baseline.
of anaesthesia within the observation period of this study had no effect on global and gastrointestinal perfusion. This was demonstrated by the repeated baseline measurements with constant isoflurane concentrations and by the return to the initial values after the isoflurane challenge after several hours of anaesthesia. There was a close inverse correlation of MAP and cardiac index with isoflurane concentrations as indices of global perfusion. Dosedependent cardiovascular depression by isoflurane has been well described (Steffey and Howland, 1980; Serteyn et al., 1987). Decreased cardiac index is a direct effect of isoflurane on myocardial contractility, leading to a reduction in stroke volume and consequent reduction in MAP (Steffey et al., 1987). High doses of isoflurane could also reduce vasomotor tone and systemic vascular resistance, further contributing to hypotension (Driessen et al., 2006). Perfusion of the muscle layers of the colon and jejunum remained unchanged until isoflurane concentrations reached ≥2.0%, resulting in cardiac index and MAP values of about 50 mL/kg/min and 60 mmHg, respectively. We used healthy, normovolaemic horses, but the effects of inadequate depth of anaesthesia in hypovolaemic horses (e.g. those with colic) could have more detrimental effects on cardiovascular function. In these horses, vigilant monitoring and meticulous adaption of depth of anaesthesia and isoflurane concentrations is even more important if gastrointestinal perfusion is to be adequately maintained.
In dogs, pigs and rats it has been demonstrated that during hypovolaemia, perfusion of the skin and gastrointestinal tract is disproportionally reduced compared to the muscle tissue (Nielsen and Secher, 1970; Lundeen et al., 1983; Mellström et al., 2009). In humans, the organs of the gastrointestinal tract (e.g. the pancreas) appeared to undergo considerable loss of blood flow with only small reductions in blood volume, with stabilisation at low levels despite further haemorrhage (Gosain et al., 1991). Other organs, notably the kidney, appeared to be relatively unaffected by substantial loss of blood volume (20–40%), after which their blood flow quite abruptly became sensitive to further hypovolaemia (Gosain et al., 1991). A sudden drop in perfusion was also observed in the intestinal muscularis in our study. We assume that in horses under isoflurane anaesthesia, intestinal perfusion is sustained for relatively long periods during anaesthesia-induced reduction in central perfusion parameters. One explanation could be the differential regulation of large and small vessels. Large vessels are under the influence of humoral (catecholamines, angiotensin II, vasopressin, serotonin) and neuronal (sympathetic and parasympathetic tone) mediators, whereas the capillaries of the gastrointestinal tract are mainly regulated by paracrine (endothelin-1, prostacyclin) and metabolic (pH, PaO2, PaCO2) mediators (Chou, 1992). It is possible that in our study, despite reduced blood flow in large vessels, local microperfusion
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Fig. 4. Scatter plots showing correlation between the colon (blue circles) and jejunum (green squares) muscularis tissue perfusion (flow in AU, arbitrary units) and (a) mean arterial blood pressure (MAP) or (b) the cardiac index (CI). The blue sigmoidal lines indicate the nonlinear regression between colon muscularis flow and MAP (a) or CI (b); the turning point (log IC50) is MAP, 65 mmHg and CI, 51 mL/kg/min. The green sigmoidal lines indicate the nonlinear regression between jejunum muscularis flow and MAP (a) or CI (b); the turning point (log IC50) is MAP, 63 mmHg and CI, 49 mL/kg/min.
of gastrointestinal organs was maintained for a wider range of increasing isoflurane concentrations. In contrast to gastrointestinal muscle layer perfusion, there were no significant changes in mucosal perfusion. A retrospective power analysis calculated that at the given variability, 170 horses would be required to demonstrate a statistically significant difference in mucosal perfusion between baseline and the highest isoflurane concentrations (P < 0.05; statistical power, 80%). It is possible that local autoregulation of mucosal blood flow is functional at lower ranges of MAP and CI than we achieved by increasing isofluraneconcentrations. Several studies suggest a pronounced metabolic regulation of blood flow supply to the gastrointestinal mucosal layer (Kiel et al., 1987; Yamaguchi, 1990; Chou, 1992). This autoregulation occurs under conditions of low flow, hypotension and tissue hypoxia, so that temporal tissue perfusion maintains adequate nutritional blood flow in tissues under conditions of limited O2 supply (Pajk et al., 2002). However, it is also possible that perfusion of the mucosal layer was already impaired at baseline, leaving no potential for further decreases. When comparing the mucosal and muscular blood flow of the jejunum and the colon, it was striking that mucosal values were much lower than muscularis values. In a porcine study comparing the mucosal and muscularis metabolism of the small intestine, early blood loss was indicated by an immediate decrease in intestinal
intramucosal pH and an increase in PCO2, in contrast to muscularis measurements, which did not change until severe blood loss occurred, indicating an earlier change in mucosal blood flow (Mellström et al., 2009). Mucosal flow measurements in our horses were highly variable. This could be related to methodology, since mucosal measurements were performed from the luminal site, making them very sensitive to artefacts related to intestinal motility. Additionally, it is possible that there were differences in contact pressure that influenced focal perfusion. Another aim of our study was to investigate the effects of isoflurane anaesthesia on tissue oxygenation. There was a progressive decrease in PaO2 that was isoflurane concentration-independent, and which did not result in severe hypoxaemia (arterial O2 saturation >95%). Therefore, adequate O2 content was always provided. Increasing isoflurane concentrations resulted in decreased CO and therefore decreased O2 delivery. This decrease in delivery lowered tissue oxygenation of the lamina mucosa but not of the lamina muscularis. The intestinal mucosa of pigs is known to be vulnerable to local hypoxaemia soon after the induction of hypovolaemia and countercurrent diffusion of O2 between adjacent microvessels has been proposed as a possible mechanism for this (Mellström et al., 2009). In contrast to mucosal oxygenation, the oxygenation of the muscular layer was not significantly affected by decreased O2
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Fig. 5. Mean ± standard deviation of the tissue oxygenation of the lamina muscularis of (a) the jejunum and (b) the colon as well as the lamina mucosa of (c) the jejunum and (d) the colon over time with different isoflurane concentrations. *Statistically different from baseline.
delivery in our study, which is also comparable to findings in anaesthetised pigs (Mellström et al., 2009). This might be related to lower O2 requirements by the muscular layer than the mucosal cell layer during anaesthesia, since the mucosal cell layer requires high O2 concentrations because of high metabolic and resorptive activity (Phillis, 1976). To our knowledge, the present study is the first to use a combination of lightguide tissue spectrophotometry and laser Doppler flowmetry (O2C) to measure the impact of isoflurane anaesthesia on oxygenation and perfusion of the gastrointestinal tract in horses. An earlier study demonstrated that micro-lightguide tissue spectrophotometry was easy to use and provided reliable and reproducible information about microcirculation of the intestinal wall in horses (Reichert et al., 2014). In human medicine, the O2C is considered a reliable method to examine microperfusion and oxygenation (Forst et al., 2008). The major limitation of laser Doppler flowmetry for perfusion measurements is its inability to obtain quantitative flow measurements. Absolute calibration of the output voltage of a laser Doppler flowmeter is difficult, due to the small sample volume size, temporal variations in the microcirculation and variations in the optical properties of the tissues (Borgos, 1994). As a result, flow can only be expressed in arbitrary units. Laser Doppler flowmetry is limited to the measurement of relative flow at a single sample site during
a single study period, so to determine tissue oxygenation, lightguide tissue spectrophotometry was used. The O2C is calibrated for the absorption characteristics of human blood, which differs from equine blood. It is possible that absolute values measured by the O2C were different from real tissue oxygenation. Nonetheless, changes in oxygenation measured in our study were considered reliable because all measurements were performed over time with the same probe in the same animal. Potential advantages of the O2C technique include potential for continuous real-time measurements and the use of surface probes that mean the measurements are non-invasive, without local bleeding or other tissue irritation that could influence values. Conclusions In this equine study, the concentration of isoflurane, but not the duration of anaesthesia, influenced global gastrointestinal perfusion and oxygenation and microperfusion. Under isoflurane anaesthesia, gastrointestinal microperfusion appeared to be preserved until a threshold MAP of approximately 60 mmHg or CO < 50 mL/kg/min was reached. The intestinal mucosal layers desaturated earlier than the intestinal muscularis layers under a deep plane of general anaesthesia.
Please cite this article in press as: K. Hopster, C. Hopster-Iversen, F. Geburek, K. Rohn, S.B.R. Kästner, Temporal and concentration effects of isoflurane anaesthesia on intestinal tissue oxygenation and perfusion in horses, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.04.030
ARTICLE IN PRESS K. Hopster et al./The Veterinary Journal ■■ (2015) ■■–■■
Conflict of interest statement None of the authors has any financial or personal relationships with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements The authors wish to thank Dr. P. Conze, Dr. C. Reichert and A. Wogatzki for their technical assistance. Preliminary results were presented as an Abstract at the AVA Autumn Meeting, Moscow, Russia, 3–4 October 2013. References Borgos, J., 1994. Principles of instrumentation: Calibration and technical issues. In: Belcaro, G., Hoffman, U., Bollinger, A., Nicolaides, A. (Eds.), Laser Doppler. Med-Orion, London, UK, pp. 3–16. Buell, M.G., Harding, R.K., 1989. Effects of peptide YY on intestinal blood flow distribution and motility in the dog. Regulatory Peptides 24, 195–208. Chou, C.C., 1992. Intestinal blood flow regulation. In: Dulbecco, R. (Ed.), Encyclopedia of Human Biology. Academic Press, San Diego, CA, pp. 547–556. Driessen, B., Nann, L., Benton, R., Boston, R., 2006. Differences in need for hemodynamic support in horses anesthetized with sevoflurane as compared to isoflurane. Veterinary Anaesthesia and Analgesia 33, 356–367. Edner, A., Nyman, G., Essén-Gustavsson, B., 2002. The relationship of muscle perfusion and metabolism with cardiovascular variables before and after detomidine injection during propofol-ketamine anaesthesia in horses. Veterinary Anaesthesia and Analgesia 29, 182–199. Fink, M.P., Kaups, K.L., Wang, H.L., Rothschild, H.R., 1991. Maintenance of superior mesenteric arterial perfusion prevents increased intestinal mucosal permeability in endotoxic pigs. Surgery 110, 154–160. Forst, T., Hohberg, C., Tarakci, E., Forst, S., Kann, P., Pfützner, A., 2008. Reliability of Lightguide Spectrophotometry (O2C®) for the investigation of skin tissue microvascular blood flow and tissue oxygen supply in diabetic and nondiabetic subjects. Journal of Diabetes Science and Technology 2, 1151–1156. Gelman, S., Mushlin, P.S., 2004. Catecholamine-induced changes in the splanchnic circulation affecting systemic hemodynamics. Anesthesiology 100, 434–439. Giglio, M.T., Marucci, M., Testini, M., Brienza, N., 2009. Goal-directed haemodynamic therapy and gastrointestinal complications in major surgery: A meta-analysis of randomized controlled trials. British Journal of Anaesthesia 103, 637–646.
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Please cite this article in press as: K. Hopster, C. Hopster-Iversen, F. Geburek, K. Rohn, S.B.R. Kästner, Temporal and concentration effects of isoflurane anaesthesia on intestinal tissue oxygenation and perfusion in horses, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.04.030
