Abstract
Objective—To evaluate the use of a micro-lightguide tissue spectrophotometer for measurement of tissue oxygenation and blood flow in the small and large intestines of horses under anesthesia.
Animals—13 adult horses without gastrointestinal disease.
Procedures—Horses were anesthetized and placed in dorsal recumbency. Ventral midline laparotomy was performed. Intestinal segments were exteriorized to obtain measurements. Spectrophotometric measurements of tissue oxygenation and regional blood flow of the jejunum and pelvic flexure were obtained under various conditions that were considered to have a potential effect on measurement accuracy. In addition, arterial oxygen saturation at the measuring sites was determined by use of pulse oximetry.
Results—12,791 single measurements of oxygen saturation, relative amount of hemoglobin, and blood flow were obtained. Errors occurred in 381 of 12,791 (2.98%) measurements. Most measurement errors occurred when surgical lights were directed at the measuring site; covering the probe with the surgeon's hand did not eliminate this error source. No measurement errors were observed when the probe was positioned on the intestinal wall with room light, at the mesenteric side, or between the mesenteric and antimesenteric side. Values for blood flow had higher variability, and this was most likely caused by motion artifacts of the intestines.
Conclusions and Clinical Relevance—The micro-lightguide spectrophotometry system was easy to use on the small and large intestines of horses and provided rapid evaluation of the microcirculation. Results indicated that measurements should be performed with room light only and intestinal motion should be minimized.
Evaluation of intestinal viability is essential during colic surgery and includes the assessment of intestinal macro- and microcirculation. Visual evaluations of intestinal color, mesenteric pulsation, and intestinal motility remain the most commonly used methods but are also often subjective.1–3 Methods used to determine intestinal circulation in equids include surface oximetry,4,5 Doppler ultrasonography,6 sodium fluorescein dye fluorescence,6,7 and pulse oximetry.8
Doppler ultrasonography has variable efficacy in evaluation of intestinal circulation depending on the type of strangulation, being more useful for assessing tissue perfusion following venous strangulation and less useful than other techniques following arterial strangulation.6 Another limitation of Doppler ultrasonography is the inability to examine large areas of intestine.9 Also, Doppler ultrasonography can be used to assess macrocirculation but provides no quantitative assessment of microcirculation.10 Surface oximetry has a moderate to low sensitivity but high specificity for detecting nonviable intestines; horses with a surface oxygen tension < 20 mm Hg had a lower survival rate than those with higher surface oxygen tension.5 Pulse oximetry depends on the detection of pulsatile blood flow,11,12 and movement and environmental light can interfere with measurement accuracy.11
Micro-lightguide spectrophotometry represents a noninvasive method to determine tissue microperfusion and oxygen saturation.10 By combining Doppler fluxmetry and micro-lightguide spectrophotometry, microvascular blood flow and tissue oxygenation can be assessed with a single probe.13 Several systems of microlightguide spectrophotometry have been described. The micro-lightguide tissue spectrophotometer described by Frank et al14 and another commercially available micro-lightguide tissue spectrophotometer are the most commonly used in current experimental and clinical applications. Use of these methods has been described for human diabetic research10,13,15,16 and in microcirculatory examinations of the gastrointestinal tract of dogs,12 pigs,17,18 humans,19,20 and rabbits21; nervous system of humans22,23 and rats24; liver of mice25; lungs of rabbits26; eyes of rabbits27; wounds during healing of humans10,a; and tissues in transplantation medicine of rodents28 and humans.29 Reliability of measurements could be influenced by factors like external light sources30 and movement of the examined tissue.30
A commonly used commercially available microlightguide tissue spectrophotometer transmits monochromatic laser light (830 nm; 30 mW) and white light (500 to 800 nm; 20 W; resolution, 1 nm) into the tissue, where it is scattered, reflected, and detected again in the probe at the tissue surface. The collected light is first divided into its spectral components by a charge-coupled device array and afterward converted into an electrical signal.10,13
Laser light determines perfusion quantities in tissue (eg, the relative blood flow and the blood flow velocity).31 A laser Doppler shift is caused by the movement of erythrocytes.10 The principle of the Doppler effect is a change of the wavelength while being reflected from moving objects like cellular components of blood.9 This Doppler shift is analyzed and displayed as the blood flow velocity. The detected laser light also correlates with the number of moving erythrocytes.31 The product of the number of moving erythrocytes and the erythrocyte velocity is used for the calculation of relative blood flow.10,13
Hemoglobin variables (eg, oxygen saturation and the relative amount of hemoglobin) are detected with the use of white light. Blood color changes with the degree of hemoglobin saturation. By detecting the blood color, the degree of oxygen saturation of hemoglobin is calculated.31 The relative amount of hemoglobin is a variable of light absorption by the tissue.10,13 The greater the amount of blood in the measured volume, the more light is absorbed by hemoglobin and the less light will be detected by the sensor.31 Only vessels up to 100 μm in diameter are included in the measurements because during transit through larger vessels (> 100 μm), all light is absorbed and no light is reflected to the detector system in the probe.32
The purpose of the study reported here was to validate the use of micro-lightguide spectrophotometry in the evaluation of the microcirculation in the intestines of horses. We hypothesized that a micro-lightguide spectrophotometry system would be easy to use to quickly and reliably assess microcirculation in the intestinal tract of horses without gastrointestinal disease.
Materials and Methods
Horses—Thirteen horses were included in the study. Age ranged from 3 to 27 years old (mean ± SD, 17.5 ± 9.2 years old), and body weight ranged from 350 to 591 kg (mean ± SD, 518.1 ± 76.4 kg). The group consisted of 10 mares, 1 gelding, and 2 stallions of the following breeds: warmblood (n = 10), Quarter Horse (1), and Sorraia (2). All horses were scheduled to be euthanized for reasons other than gastrointestinal disease. The study was reviewed by the Ethics Committee for Animal Experiments of Lower Saxony, Germany, and approved according to §8 of the German Animal Welfare Act (Tierschutzgesetz). Measurements were performed with horses under general anesthesia.
Anesthesia—Food but not water was withheld for 6 hours before surgery. After horses were premedicated with xylazineb (1 mg/kg, IV), general anesthesia was induced with ketaminec (2.2 mg/kg, IV) and midazolamd (0.05 mg/kg, IV). Anesthesia was maintained with isofluranee in oxygen balanced with xylazineb in a constant rate infusion of 1 mg/kg/h. The horses were positioned in dorsal recumbency and ventilated by intermittent positive pressure ventilation. Cardiovascular function was supported by IV administration of lactated Ringer's solution at a rate of 10 mL/kg/h and dobutaminef given to effect to maintain mean arterial blood pressure > 70 mm Hg. After the trials, without regaining consciousness, horses were euthanized by IV administration of pentobarbitalg (60 mg/kg).
Probe of the micro-lightguide tissue photometer—Measurements were performed by use of a flat probeh with a transmittable wavelength ranging from 500 to 850 nm. Penetration depth was adjusted by the manufacturer to 2.5 mm (ie, the detected signal on the probe's surface was derived from the 2.5-mm distance through which the laser and white light were transmitted). The probe contained one source for white light and another for laser light and 2 detectors each for white and laser light.
Experimental setup—After being anesthetized, horses were placed in dorsal recumbency. The ventral midline and paramedian area were clipped of hair, and ventral midline laparotomy was performed. Intestinal segments were exteriorized and placed on surgical drapes during measurements. Spectrophotometric measurements were obtained on the small intestine (middle part of the jejunum) and on the large intestine (ascending colon and pelvic flexure). Measurements in the small intestine were performed in all 13 horses included in the study, whereas measurements in the large intestine were conducted in 12 of 13 horses. Measurements were performed under a variety of conditions that were considered to have a potential effect on the repeatability of the measurements (ie, influence of external light sources, positioning of the probe and tension on the mesentery; Appendix). All measurements were performed by the same surgeon (CR) and obtained 3 times for each measuring condition; each measurement was obtained during a period of approximately 3 breaths (respiratory rate, 6 to 8 breaths/min) to avoid influence of the respiratory cycle on oxygenation and blood flow. During the measurement time, a micro-lightguide spectrophotometry systemi measured and calculated all variables every 50 milliseconds and displayed mean results on a monitor every 2 seconds. All individual data points were recorded and stored for later evaluation. Oxygen saturation (%), relative amount of hemoglobin (arbitrary units), and regional microcirculatory blood flow (arbitrary units) were measured. Oxygen saturation > 100% and relative amount of hemoglobin < 0 or > 120 arbitrary units were interpreted as measurement errors, as per the manufacturer. The frequency of the occurrence of measurement errors was evaluated for the various measuring conditions to determine whether any particular condition should be avoided when the micro-lightguide tissue spectrophotometeri is used. All sets of measurements with ≥ 1 measurement error were consecutively excluded from statistical analysis of measuring results.
Arterial Spo2 at the measuring sites was determined by use of transmission pulse oximetry.j A single value was obtained for each repeated measurement during each measuring condition.
Statistical analysis—All data were analyzed by use of a statistics program package.k Model residuals were tested for normal distribution by use of the Kolmogorov-Smirnov test and visual assessment of Q-Q plots. For descriptive statistics, mean and SD were calculated for oxygen saturation, relative amount of hemoglobin, and blood flow for each measuring condition in all horses. Differences of these 3 variables between measuring conditions with no measurement errors in both small and large intestines were examined by use of a 1-way repeated-measures ANOVA and post hoc Tukey test for multiple pairwise comparisons.
The reproducibility of measurements within random measurement repetitions was tested for microlightguide spectrophotometric and pulse oximetry measurements by use of ANOVA components, and the ICC (2-way random, average measure, and absolute agreement) was calculated for each measuring condition. For this purpose, the mean for each of the 3 sets of measurements for each measuring condition was calculated. One set of measurements consisted of all single measurements during the period of 3 breaths (mean, 26.1 seconds). During this time, the micro-lightguide tissue spectrophotometer recorded measurements every 50 milliseconds, and the mean of these measurements was calculated. Then, the mean of these mean values for the 3 measurement sets for each measuring condition was calculated, resulting in 1 value for each measuring condition, and horses and measuring conditions were correlated. To evaluate whether measurement errors were more frequent when surgical lights were on, the number of measurement errors obtained during measuring conditions possibly influenced by external light (conditions SI-6, SI-7, LI-6, and LI-7; Appendix) was compared with the number of measurement errors for conditions when no surgical lights were on. This was performed for the small intestinal and large intestinal values grouped separately with the Fisher exact test. Values of P ≤ 0.05 were considered significant.
Results
To maintain an adequate depth of anesthesia, expiratory isoflurane concentration was adjusted between 1.4% and 1.6%. A mean ± SD dobutamine infusion rate of 0.6 ± 0.1 μg/kg/min was necessary to keep the mean of the mean arterial blood pressure within stable limits of 84 to 93 mm Hg.
After finishing measurements in the small intestine and before starting measurements in the large intestine, 1 horse developed signs of severe bronchospasm with typical alterations of the capnogram and development of severe hypoxemia. Because hypoxemia per se and therapeutic interventions will alter tissue oxygenation and vessel tone, this horse was excluded from further large intestinal investigations. No other horses developed signs of hypoxemia during all measurements, and arterial oxygen saturation (measured every 15 minutes by use of blood gas analysis) always was > 97%.
Mean ± SD micro-lightguide spectrophotometry measuring time was 26.1 ± 7.06 seconds. In total, 12,791 single measurements of oxygen saturation, relative amount of hemoglobin, and blood flow were obtained. Eight small intestinal and 5 large intestinal measuring conditions resulted in 381 single measurement errors (2.98% of all measurements).
The ICCs for each measuring condition in the small and large intestines were summarized (Tables 1 and 2). In the small intestine, all ICCs for oxygen saturation and relative amount of hemoglobin were higher than for blood flow; blood flow measurements had a lower overall intraindividual reproducibility. A higher overall intraindividual variability of blood flow was found in the small intestine, compared with that of the large intestine, under various measuring conditions (ICC of blood flow < 0.5 for 10/15 measuring conditions of the small intestine, compared with for 3/11 measuring conditions of the large intestine). The ICCs for oxygen saturation and relative amount of hemoglobin measurements mostly ranged between 0.5 and 0.9, and this in general would indicate moderate to good reliability33 for oxygen saturation and relative amount of hemoglobin measurements, whereas most blood flow measurements had lower reliability
Intraclass correlation coefficients for micro-lightguide spectrophotometry and pulse oximetry measurements of the small intestine (mid jejunum) in 13 anesthetized adult horses without gastrointestinal disease.
| Micro-lightguide spectrophotometry | ||||
|---|---|---|---|---|
| Condition | Oxygen saturation | Relative amount of hemoglobin | Blood flow | Spo2 |
| SI-1 | 0.5357 | 0.8240 | 0.3950 | 0.7921 |
| SI-2 | 0.6658 | 0.6881 | 0.5536 | 0.4953 |
| SI-3 | 0.8306 | 0.8455 | 0.8053 | 0.5124 |
| SI-4 | 0.7266 | 0.7177 | 0.6411 | 0.0011 |
| SI-5 | 0.6222 | 0.7908 | 0.4430 | 0.5360 |
| SI-6 | 0.6516 | 0.6549 | 0.3294 | 0.3644 |
| SI-7 | 0.7607 | 0.7352 | 0.2071 | 0.2775 |
| SI-8 | 0.8430 | 0.8816 | 0.3003 | 0 |
| SI-9 | 0.7447 | 0.8201 | 0.3156 | 0.1923 |
| SI-10 | 0.9183 | 0.7995 | 0.3373 | 0.4597 |
| SI-11 | 0.8410 | 0.8241 | 0.7074 | 0.3436 |
| SI-12 | 0.7933 | 0.8230 | 0.6358 | 0.3010 |
| SI-13 | 0.9040 | 0.6735 | 0.2937 | 0.5996 |
| SI-14 | 0.8747 | 0.8495 | 0.3523 | 0.5861 |
| SI-15 | 0.9350 | 0.7038 | 0.1850 | 0.5808 |
Potential values for ICC are 0 to 1. A high ICC signifies low intraobserver variability, indicating a reliable measuring technique (micro-lightguide spectrophotometry or pulse oximetry).
See Appendix for description of conditions.
Intraclass correlation coefficients for micro-lightguide spectrophotometry and pulse oximetry measurements of the large intestine (pelvic flexure) in the same horses of Table 1.
| Micro-lightguide spectrophotometry | ||||
|---|---|---|---|---|
| Condition | Oxygen saturation | Relative amount of hemoglobin | Blood flow | Spo2 |
| LI-1 | 0.8974 | 0.6700 | 0.6524 | 0.5327 |
| LI-2 | 0.7816 | 0.5324 | 0.5290 | 0.1321 |
| LI-3 | 0.7732 | 0.6792 | 0.7221 | 0.9350 |
| LI-4 | 0.8180 | 0.6937 | 0.6819 | 0.4639 |
| LI-5 | 0.8130 | 0.5405 | 0.4722 | 0.6521 |
| LI-6 | 0.4936 | 0.6928 | 0.5052 | 0.9393 |
| LI-7 | 0.5880 | 0.8454 | 0.5754 | 0.8245 |
| LI-8 | 0.5623 | 0.7359 | 0.4760 | 0 |
| LI-9 | 0.5154 | 0.6574 | 0.5323 | 0.6093 |
| LI-10 | 0.5815 | 0.8423 | 0.4117 | 0.1738 |
| LI-11 | 0.4732 | 0.7775 | 0.5623 | 0.0923 |
Surgical lights pointing at the measuring site resulted in 92.07% of all measurement errors of the small intestine (conditions SI-6 and SI-7) and 71.43% of all measurement errors of the large intestine (conditions LI-6 and LI-7). Significantly more measurements errors occurred when surgical lights were pointing at the measuring site (conditions SI-6 and LI-6), compared with the other measuring conditions that resulted in measurement errors. Covering the probe with the surgeon's hand (conditions SI-7 and LI-7) could not eliminate this error source. Significantly (P < 0.001) more measurements errors occurred during SI-7 and LI-7 conditions, compared with the other conditions that resulted in measurement errors, except for conditions SI-6 and LI-6. However, when comparing both measuring conditions with surgical lights pointing at the measuring site (condition SI-6 vs SI-7 and condition LI-6 vs LI-7), covering the measuring site with the surgeon's hand significantly reduced the number of measurement errors (condition SI-6 vs SI-7 [P < 0.001]; condition LI-6 vs LI-7 [P = 0.03]). No measurement errors in both the small and large intestine were observed when the probe was positioned on the intestinal wall under room light at the mesenteric side or between the mesenteric and antimesenteric side, with the surgical lights off or directed away from the surgical site (conditions SI-2, SI-4, SI-5, and SI-8 and LI-2, LI-4, LI-5, and LI-8).
Measuring conditions resulting in no measurement errors in the small and large intestines were tested for significant differences between their variables (Tukey-Kramer test). Condition SI-4 (probe positioned on the mesenteric side of the intestinal wall, room light) had a significantly (P = 0.026) higher relative amount of hemoglobin (73.24 ± 8.75), compared with that for condition SI-8 (probe positioned between the mesenteric and antimesenteric intestinal wall, room light, surgical lights off; 68.15 ± 11.74). Condition LI-4 (probe positioned on the mesenteric side of the intestinal wall, room light) had a significantly higher relative amount of hemoglobin (73.69 ± 10.29), compared with that for conditions LI-5 (probe positioned between the mesenteric and antimesenteric intestinal wall, room light, 65.53 ± 8.53; P = 0.046) and LI-8 (probe positioned between the mesenteric and antimesenteric intestinal wall, room light, surgical lights off; 63.17 ± 10.21; P = 0.005). Because of these findings of higher relative amounts of hemoglobin, results for conditions SI-4 and LI-4 were excluded from further analysis. Descriptive analysis of measuring results for conditions SI-2, SI-5, and SI-8 and LI-2, LI-5, and LI-8 was then performed because these conditions did not cause any measurement errors (Table 3).
Mean ± SD oxygen saturation, relative amount of hemoglobin, and blood flow for micro-lightguide spectrophotometric measurements at measuring conditions SI-2, SI-5, and SI-8 (mid jejunum) and LI-2, LI-5, and LI-8 (pelvic flexure) in the same horses as in Table 1.
| Variable | Jejunum | Pelvic flexure |
|---|---|---|
| Oxygen saturation (%) | 81.04 ± 12.62 | 83.44 ± 11.79 |
| Relative amount of hemoglobin (arbitrary units) | 68.41 ± 10.91 | 65.81 ± 9.29 |
| Blood flow (arbitrary units) | 246.1 ± 73.43 | 216.27 ± 67.20 |
See Appendix for description of measuring conditions SI-2, SI-5, SI-8, LI-2, LI-5, and LI-8.
Oxygen saturation for the measuring conditions SI-2, SI-5, and SI-8 and LI-2, LI-5, and LI-8 (probe positioned at the intestinal wall with room light between the mesenteric and antimesenteric side, with the surgical lights off or directed away from the surgical site) ranged between 34% and 100% (mean ± SD, 85.23 ± 10.72%) for the small intestine and 53% and 100% (86.43 ± 9.49%) for the large intestine. Measurements were successful in 90 of the 117 (76.92%) small intestinal and 54 of 108 (50%) large intestinal observations. The ICCs were calculated for measuring conditions in the small intestine (Table 1) and large intestine (Table 2).
Discussion
To the authors’ knowledge, the present study is the first in which investigators have evaluated the use of micro-lightguide spectrophotometry in horses. In human medicine, micro-lightguide spectrophotometry is considered a reliable and valid method to examine tissue microperfusion.10,13 The high frequency of measurements is a major advantage of this system. By averaging numerous single measurements, it is possible to gain more accurate data.10 In our experience, use of the micro-lightguide tissue spectrophotometeri was easy and could be learned quickly. Measurement results could be read on a monitor almost immediately, making it possible to assess the quality of measurements in real time and avoiding measurement errors caused by movement or external light.
A possible explanation for the lower overall intraindividual reproducibility of blood flow measurements, compared with those for measurements of oxygen saturation and relative amount of hemoglobin, is that oxygen saturation and relative amount of hemoglobin are variables of white light and blood flow is calculated by the use of laser light. Movement (intestinal motility and operator movement) could interfere with the Doppler shift of the laser light, leading to measurement errors. In the manufacturer's manual,30 movement between the probe and the tissue is described as a source for errors in blood flow measurements. In contrast, oxygen variables (oxygen saturation and relative amount of hemoglobin) are determined by the use of white light and represent values derived from the blood color. They are independent of the Doppler shift, so movement artifacts are less likely a problem. The lower reproducibility of blood flow measurements in the small intestine, compared with that of the large intestine, may be explained by movement artifacts caused by peristalsis, which are subjectively more intense in the small intestine than in the large intestine. In a study by Snyder et al,4 peristaltic movements, particularly in the small intestine, caused momentary alterations in surface oxygen tension. A prolonged measuring time could potentially increase the accuracy of blood flow measurements. Because micro-lightguide spectrophotometry currently is the only available technique to measure tissue microperfusion with almost immediate results, its use can be justified with the ICCs calculated in the present study, given that even a test with moderate reliability can add sufficient information for some purposes.33
To avoid measurement errors, we recommend positioning the probe on the intestinal wall between the mesenteric and antimesenteric side, with room light off and the surgical lights off or directed away from the intestine (conditions; SI-2, SI-5, and SI-8 and LI-2, LI-5, and LI-8). Covering the probe with the surgeon's hand was not sufficient to avoid measurement errors when the surgical light was pointing at the measuring site. The manufacturer of the micro-lightguide tissue spectrophotometeri has documented in its manual that lights can disturb the quality of the hemoglobin spectrum, thereby resulting in invalid measurements of oxygen saturation and relative amount of hemoglobin.30 The monitor of the system displays the white light absorption spectrum of oxyhemoglobin with its typical bimodal appearance,34 with a changing spectrum under the influence of external light. The correct appearance of the oxyhemoglobin light absorption spectrum can also be used to evaluate the correct application of the probe.30
Micro-lightguide spectrophotometry is described as a highly sensitive tool in many fields of human medicine.10,13 One major advantage is that alterations in local microcirculation can easily be monitored even in the absence of concomitant changes of oxygen variables in the systemic circulation.12 The correlation of remission spectroscopy with other established methods18,25 in the assessment of tissue microvascular perfusion variables has been shown.
Significantly higher relative amounts of hemoglobin were found when positioning the probe on the mesenteric side of the intestinal wall, compared with amounts for nonmesenteric sites. This finding may be caused by the larger diameter of vessels in this area and thus a higher intravascular volume than for areas at more antimesenterically located sites, resulting in more hemoglobin per volume of tissue. This argument can only be valid for vessels up to 100 μm in diameter because, in cases of larger vessels, all light is absorbed.32 However, Faleiros et al35 analyzed the distribution of tissue perfusion in the small colon and found higher tissue perfusion in the antimesenteric border, although these differences were not significant. The microvascular anatomy may differ among various segments of the equine intestine, and the reason for the higher relative amount of hemoglobin at the mesenteric side in the present study should be further evaluated.
Measuring depth of the probe was set to 2.5 mm. Mean total intestinal wall thickness, determined by transabdominal ultrasonography has been described as 0.3 ± 0.02 cm for the small intestine (duodenum and jejunum) and 0.37 ± 0.01 cm for the large intestine (ventral colon) in clinically normal adult Thoroughbreds.36 Klohnen et al37 reported a wall thickness of the small intestine < 3 mm in clinically normal horses. The purpose in adjusting the measuring depth of the probe to 2.5 mm in the present study was to evaluate the intestinal wall without including the lumen.
In horses with strangulating intestinal obstruction, venous obstruction often predominates and will lead to severe edema and hemorrhage into the intestinal wall. As a result, the thickness of the intestinal wall increases and can be > 10 mm,38 and in these tissues, the entire wall thickness cannot be measured with the probe used in the present study. Probes are available with a setting to allow measurement depths of up to 8 mm, according to the manufacturer, allowing for measurements to include more of the intestinal wall. Reflectance photometry has been used to evaluate microcirculation with the probe applied to both intestinal mucosal and serosal surfaces in humans12,19,20 and other animals.17,18,21 In horses with large intestine volvulus, an enterotomy is often performed and micro-lightguide spectrophotometry measurements could be obtained on both the serosal and mucosal surfaces to obtain more information. This could be particularly useful because, in these clinical cases, the serosal color can return to normal after tissue reperfusion even if the mucosa is not viable. When interpreting measurements obtained at a pelvic flexure enterotomy site, positioning of the large intestine on the enterotomy tray should be considered because it could alter tissue perfusion. The micro-lightguide tissue spectrophotometer can differentiate between arterial and venous strangulating obstruction with intestinal wall hemorrhage.39 In both arterial and venous obstruction, oxygen saturation and blood flow would be decreased. The relative amount of hemoglobin would be decreased in arterial strangulating obstructions (ischemia) and increased in venous strangulating obstructions (hemorrhage).39 The impact of edema on results needs to be further evaluated.
A limitation of this study was the lack of an available gold standard for measuring tissue microperfusion and oxygenation. A comparison was made by simultaneously measuring intestinal Spo2. Use of pulse oximetry to determine intestinal Spo2 in dogs was described by MacDonald et al.2 In the present study, we could only obtain pulse oximetric measurements for 76.9% and 50% of the measurments in the small intestine and large intestine, respectively. The ICCs for Spo2 had a wide range and were mostly approximately ≤ 0.5. This and the difficulty in obtaining Spo2 during the time of micro-lightguide spectrophotometry may be explained by the fact that pulse oximetry depends on the presence of a pulsatile blood flow,11,12 and a pulsatile flow may not easily be detected within the intestinal wall. Although mean ± SD oxygen saturation measurements were approximately in the same range, measurements with the pulse oximeter were often not successful and had a wider range of the ICCs.
One weakness of the present study was the potential heterogeneity in the group of horses. Although all study horses were free of gastrointestinal diseases, differences among horses in subclinical pathological intestinal changes or the situation surrounding anesthesia were possible, despite a homogenous protocol. To minimize operator influence, all measurements in all horses were obtained by the same surgeon (CR).
As suggested by Snyder et al,4 reduction of the intestinal temperature while being exposed may be a specific concern. A change of tissue temperature might affect regional blood flow as well as the affinity of hemoglobin for oxygen.4 In the present study, we did not account for possible changes in oxygen saturation caused by changes in intestinal temperature. Nevertheless, intestines were exteriorized for only approximately 15 minutes. Values of measuring conditions at the beginning of exteriorization (conditions SI-2 and LI-2) did not differ significantly from values toward the end of exteriorization (conditions SI-8 and LI-8); therefore, any effect of the intestinal temperature seemed to be negligible. To minimize alterations caused by possible differences in the architecture of the intestinal microvasculature,4 we always located the probe on nearly the same intestinal location between the mesenteric and the antimesenteric intestinal border (except for conditions SI-3 and SI-4 and LI-3 and LI-4).
We did not control the contact pressure of the probe when applied to the intestinal wall but chose to apply the probe with a slight and constant pressure. One surgeon obtained all measurements to minimize the effect of different pressures applied to the probe. Kram and Shoemaker40 have described probe application with gentle manual pressure for the use of miniature oxygen sensors (polarographic electrode with silver-silver chloride anode and platinum cathode) on organ surfaces. Care should be taken to avoid the use of excessive pressure, which could lead to decreased local tissue perfusion.40
In conclusion, the micro-lightguide tissue spectrophotometeri used in the present study was easy to use on the intestines of horses and provided rapid evaluation of the microcirculation. Blood flow was the variable with the highest variability. We were able to identify measuring conditions that can be recommended to achieve the most reliable results. In our opinion, the micro-lightguide tissue spectrophotometer can be a valuable tool in a research setting (eg, for the evaluation of intestinal ischemia and reperfusion). The present study was experimental and conducted in clinically normal horses. Further research and clinical investigation are necessary before this technology could be used for evaluating viability of intestines following intestinal strangulation in clinical patients.
ABBREVIATIONS
| ICC | Intraclass correlation coefficient |
| Spo2 | Oxygen saturation as measured by pulse oximetry |
Coerper S, Beckert S, Beckert HD. New method for measurement of ischemia in wounds—a pilot study (asbtr). 6th World Cong Trauma Shock Inflammation Sepsis Pathophysiol Immune Consequences Ther 2004;553.
Xylapan, Vetoquinol GmbH, Ravensburg, Germany.
Narketan, Vetoquinol GmbH, Ravensburg, Germany.
Midazolam ratiopharm (15 mg/3 mL), Ratiopharm GmbH, Ulm, Germany.
Isofluran CP, CP-Pharma GmbH, Burgdorf, Germany.
Dobutamin ratiopharm (250 mg), Trockensubstanz, Ratiopharm GmbH, Ulm, Germany.
Euthadorm 400, CP-Pharma GmbH, Burgdorf, Germany.
Flachsonde LF-2, LEA Medizintechnik GmbH, Gießen, Germany.
O2C, Oxygen to See, LEA Medizintechnik GmbH, Gießen, Germany.
Cardiocap/5, GE Healthcare, Helsinki, Finland.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
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Appendix
Protocol for spectrophotometric measurements (measuring conditions) in the small and large intestines.
| Condition | Description |
|---|---|
| SI-1 | Intestinal wall laid on the probe; room light |
| SI-2 | Probe positioned on the intestinal wall; room light |
| SI-3 | Probe positioned on the antimesenteric side of the intestinal wall; room light |
| SI-4 | Probe positioned on the mesenteric side of the intestinal wall; room light |
| SI-5 | Probe positioned between the mesenteric and antimesenteric intestinal walls; room light |
| SI-6 | Probe positioned as in SI-5; surgical lights pointing at the measuring site |
| SI-7 | Probe positioned as in SI-5; surgical lights pointing at the measuring site; probe covered by surgeon's hand |
| SI-8 | Probe positioned as in SI-5; surgical lights off |
| SI-9 | Probe positioned as in SI-5; intestine exteriorized just to the level of the laparotomy |
| SI-10 | Probe positioned as in SI-5; intestine exteriorized as far as possible |
| SI-11 | Probe positioned as in SI-5; intestine exteriorized without torsion |
| SI-12 | Probe positioned as in SI-5; intestine exteriorized with 180° torsion |
| SI-13 | Probe positioned as in SI-5; intestine returned to the abdomen and exteriorized again |
| SI-14 | Probe positioned as in SI-5; intestine returned to the abdomen and exteriorized again |
| SI-15 | Probe positioned as in SI-5; intestine returned to the abdomen and exteriorized again |
| LI-1 | Intestinal wall laid on the probe; room light |
| LI-2 | Probe positioned on the intestinal wall; room light |
| LI-3 | Probe positioned on the antimesenteric side of the intestinal wall; room light |
| LI-4 | Probe positioned on the mesenteric side of the intestinal wall; room light |
| LI-5 | Probe positioned between the mesenteric and antimesenteric intestinal walls; room light |
| LI-6 | Probe positioned as in LI-5; surgical lights pointing at measuring site |
| LI-7 | Probe positioned as in LI-5; surgical lights pointing at measuring site; probe covered by surgeon's hand |
| LI-8 | Probe positioned as in LI-5; surgical lights off |
| LI-9 | Probe positioned as in LI-5; intestine exteriorized as far as possible; enterotomy tray at 0° relative to floor |
| LI-10 | Probe positioned as in LI-5; intestine exteriorized as far as possible; enterotomy tray at 10° relative to floor |
| LI-11 | Probe positioned as in LI-5; intestine exteriorized as far as possible; enterotomy tray at 20° relative to floor |
LI = Large intestine. SI = Small intestine.
Room light was a measuring condition with the surgical lights on but directed away from the intestine.
