Comparison of the oral and rectal mucosal and colonic serosal microcirculations of healthy, anesthetized horses

Abstract

The objectives of the study were to: i) determine baseline microvascular perfusion indices (MPI) and assess their repeatability in healthy horses under general anesthesia, and ii) compare the MPIs of 3 microvascular beds (oral mucosa, colonic serosa, and rectal mucosa). Healthy adult horses were anesthetized and sidestream dark field microscopy was used to collect video loops of the oral mucosa, rectal mucosa, and colonic serosa under normotensive conditions without cardiovascular support drugs; videos were later analyzed to produce MPIs. Baseline MPI values were determined for each site, which included the total vessel density (TVD), perfused vessel density (PVD), portion perfused vessels (PPV), and microcirculatory flow index (MFI). Differences in MPIs between microvascular beds were not statistically significant. Repeatability of the measurements varied for each MPI. In particular, the site of sampling had a profound effect on the repeatability of the PPV measurements and should be considered in future studies.

Introduction

Microcirculatory regulation is highly complex and includes autoregulation, pressure, neuronal, hormonal, metabolic, and other influences (1,2). This regulation is observed in both homeostasis and states of disease (1,3–11). Inadequate perfusion or blood flow to the microcirculation may result in cellular hypoxia and eventually dysfunction and failure (12). Clinically, microcirculatory dysfunction may be present despite normal macrovascular perfusion indices (MaPIs) such as blood pressure (7,10). In some conditions, the increase in peripheral vascular resistance required to maintain normal blood pressure may result in decreased microcirculatory flow. Disease states and processes that result in a systemic inflammatory response syndrome, hypovolemia, abdominal hypertension, and/or hypoxemia, can result in substantial dysfunction of the microcirculation (3,5,6,10,11). In equine patients undergoing colic surgery, many of these processes may be occurring concurrently.

Assessment of the microcirculation of a tissue allows for detection of tissue specific trends and provides information regarding the overall perfusion status of the patient. In vivo evaluation of tissue microcirculation and perfusion can be performed with sidestream dark field microscopy. The commercial system (Microscan; MicroVision Medical, Amsterdam, The Netherlands) used here consists of a videomicroscope, which records video loops of the superficial microcirculation of a mucosal or serosal surface. Analysis is semi-automated and performed post-collection with provided software (Automated Vascular Analysis, Version 3.2; MicroVision Medical) yielding 4 main microvascular perfusion indices (MPIs): total vessel density (TVD), perfused vessel density (PVD), proportion perfused vessel (PPV), and microvascular flow index (MFI). While microcirculation is considered to reflect the arterioles, venules, and capillaries with a cross-sectional area < 300 μm, darkfield microscopic evaluation primarily evaluates vessels with a cross-section < 20 μm.

In human medicine, sidestream dark field microscopy has been used to evaluate the microcirculation of various regions in normal and disease states, such as sepsis and neonatal intensive care, as well as the response of the microcirculation to various interventions, such as fluid therapy, cardiac surgery, and pharmacological interventions (3–7,10–15). In veterinary medicine, sidestream dark field microscopy and orthogonal spectral imaging (a similar technology) have been successfully applied in canine, feline, and equine patients as well as experimentally in other veterinary species (8,9,13,16–19). Specific sites of microcirculatory evaluation are selected based on clinical interest and ease of access to a suitable mucosal surface. The sublingual microcirculation has been the most popular region for study in humans and small animals (3,4,8,10,17–20). In horses, oral and rectal mucosa and colonic serosa have been evaluated in both the awake and anesthetized patient, though not all in the same study (9,16). While these studies demonstrate the feasibility of using sidestream dark field microscopy, direct comparisons between the different sites cannot be made. Therefore, to date, it has not been determined if regions of the gastrointestinal tract that are readily available for assessment (oral and rectal mucosa) are reflective of the microcirculation throughout the gastrointestinal tract (colonic serosa). This study was designed to allow comparison of MPIs between colonic serosa, oral mucosa, and rectal mucosa in a cohort of anesthetized horses.

The objectives of this study were to: i) determine baseline MPIs and assess their repeatability in healthy horses under general anesthesia, and ii) compare the MPIs of 3 microvascular beds (oral mucosa, colonic serosa, and rectal mucosa). Our hypotheses were that in healthy horses under general anesthesia, microvascular measurements would be repeatable within each site and the MPI parameters would be quantitatively similar between sites.

Materials and methods

Animals, anesthesia, and monitoring

Six healthy adult (average age: 17 y, range: 10 to 29 y) horses (3 mares and 3 geldings) were included in the study (average weight: 468 kg, range: 418 to 575 kg). Each horse underwent a physical examination and was deemed systemically healthy before inclusion in the study. Horses were pre-medicated with intravenous (IV) xylazine hydrochloride [1.1 mg/kg body weight (BW)] and anesthesia was induced with IV diazepam (0.05 mg/kg BW) and ketamine hydrochloride (2.2 mg/kg BW). Once anesthetized, the horses were intubated orotracheally and placed into dorsal recumbency. Isoflurane was delivered in 100% oxygen and mechanical ventilation was used to maintain end-tidal CO₂ between 35 to 45 mmHg. Intravenous polyionic fluids were delivered initially at 10 mL/kg per hour and adjusted at the discretion of the anesthesiologist. Adjustment of the percentage of inhaled isoflurane at the discretion of the anesthetist was used to maintain normotension. Direct cannulation of the facial artery for invasive blood pressure monitoring was performed and maintained throughout the study period. In addition to blood pressure, vital parameters, electrocardiography, end-tidal CO₂, oxygen flow rate, inspired oxygen concentration, tidal volume, exhaled isoflurane concentration, and peak inspiratory pressure were collected every 5 min. Electrolyte concentrations, blood gas analysis, and cardiac index (21), taken in duplicate via lithium dilution, were obtained at the time of collection of the microcirculatory variables.

A ventral midline celiotomy was carried out to access the pelvic flexure. The abdomen was clipped and aseptically prepared and a sterile drape was placed. A 20-cm long ventral midline incision was made with a scalpel. The pelvic flexure was exteriorized onto an enterotomy (colon) tray to allow for image collection. The tray was positioned downward at a gentle angle from the incision considered clinically appropriate to the horse’s body size and conformation, allowing the colon to rest without tension. The angle was subjectively based on the clinical experience of PK, KE, and JW.

The probe was stabilized for collection using sandbags placed on the surgical table, surgical cart, or mayo stand. The probe was placed perpendicularly on the sandbags, providing complete support of the unit. Once the unit was placed, the operator was able to remove their hands from the unit during collection in most cases. Warmed sterile isotonic fluids (0.9% NaCL) were applied as needed to the tissue and abdomen at the discretion of the investigators to maintain tissue integrity and image quality. Gross fecal material was digitally removed from the rectum if necessary and lavaged away.

For videos obtained from the rectum, the probe was manipulated to be inserted at least half the distance of the disposable protective cover (approximately 1 to 2 cm in length). The operator then used the table as a reference to angulate the probe on the mucosa as ideally as possible for image collection. The probe was placed on gingival mucosa and stabilized using sandbags for videos obtained from the oral mucosa. Video loops were collected around the controlled respiration of the horse to minimize motion.

Measurement of microvascular perfusion indices

Microcirculation videos were obtained from the oral mucosa, rectal mucosa, and the serosa of the pelvic flexure region of the large colon. Videos were collected according to a previously described protocol (22). The videos were obtained when the patients were in a normotensive state [mean arterial pressure (MAP) 70 to 90 mmHg] without the administration of any cardiovascular support drugs. A minimum of 3 videos, 20 s long, were taken from each location. Videos were obtained by the same unit. Videos for each site were obtained sequentially and the order of site acquisition was randomized. The probe was moved between each video to adjacent but similar sites for subsequent image collection. Following completion of data collection for this study, additional data were collected from the subjects for other projects and subjects were then humanely euthanized while under general anesthesia (Beuthanasia-D Special; Merck Animal Health, Madison, New Jersey, USA). The study was approved by the University of Georgia Institutional Animal Care and Use Committee.

Analysis was performed in accordance with the De Backer et al (22) roundtable. Three videos of at least 50 frames were produced from the full videos for each site. Video selection was based off clarity, stability, and acceptable image quality. Videos were then blinded by one investigator (JMW). Analysis of the blinded videos was performed by one investigator (PJK) using manufacturer provided software (Automated Vascular Analysis, Version 3.2).

Data analysis

Normality of the data was assessed based on examination of histograms and normal Q-Q plots of the residuals as well as the Shapiro-Wilk test. Variance of the data was assessed by plotting residuals against predicted values and using Levene’s test. Comparison of each MPI between sites (colon, oral, or rectal) was performed using linear marginal models for repeated measurements with unstructured covariance. To assess the repeatability of measurement of MPIs, the coefficient of variation (CV) of triplicate measurements was calculated for each horse, site (colon, oral, or rectal) and MPI. The CV was transformed to the natural logarithm for analysis. The effects of site, MPI, and interaction between site and microvascular variable on the LnCV was evaluated using a linear mixed-effects model with horse modeled as a random effect and site, MPI, and their interaction modeled as fixed nominal effects. Model fit was assessed using the Akaike information criterion values. Multiple pairwise comparisons were done using the method of Sidak. For all analyses, P < 0.05 was considered significant. All statistical analyses were performed using SPSS.

Results

The average time from induction of anesthesia to the start of collection of videos averaged 37 min. Collection of all videos from all sites averaged 43 min. This time includes the moving of the device between sites. Also included in the collection time is the exteriorization prior to obtaining videos and subsequent return of the colon to the abdomen following video collection.

The means and standard deviations for MaPI at the time of video collection were: heart rate = 35.00 ± 3.46 beats/min, MAP = 81.08 ± 4.76 mmHg, cardiac index = 46.44 ± 4.69 L/min/kg, and lactate concentration = 0.97 ± 0.34 mmol/L. There were no significant differences in any MPI between sites (Figures 1A to 1D). There was a significant effect of site (P = 0.004) and MPI (P = 0.044) on the CV as well as a significant interaction between site and MPI (P = 0.006). The CV of PPV measured on the oral mucosa was significantly higher than that of the colon (P = 0.020) and rectal mucosa (P = 0.030) (Figure 2). The CV of other MPI was not significantly different between sites. The CV of PPV measured on the oral mucosa was significantly higher than that of TVD (P = 0.018) measured at the same site (Figure 2). Differences in CV between MPI indices measured on the rectal mucosa or colon were not significant. No significant differences were present in macrovascular parameters between subjects.

Figure 1.

A — Total Vessel Density (TVD) (mm/mm²). Comparison of the mean and standard deviation (error bars) of the total vessel density of the oral mucosa, rectal mucosa, and colonic serosa under normotensive conditions. B — Perfused Vessel Density (PVD) (mm/mm²). Comparison of the mean and standard deviation (error bars) of the perfused vessel density of the oral mucosa, rectal mucosa, and colonic serosa under normotensive conditions. C — Proportion Perfused Vessels (PPV) (%). Comparison of the mean and standard deviation (error bars) of the proportion of perfused vessels of the oral mucosa, rectal mucosa, and colonic serosa under normotensive conditions. D — Microvascular Flow Indices (MFI). A comparison of the mean and standard deviation (error bars) of the microvascular flow index of the oral mucosa, rectal mucosa, and colonic serosa under normotensive conditions.

Figure 2.

Coefficient of variations for the microvascular perfusion indices (MPIs). Comparison of the mean coefficients of variation for the MPIs at each site. Error bars represent standard deviation from the mean.

Discussion

In healthy normotensive anesthetized horses, the MPIs did not significantly differ between oral mucosa, rectal mucosa, and colonic serosa. These values were consistently measured and repeatable with the exception of PPV measured on oral mucosa compared with that of rectal mucosa, colonic serosa, and TVD of oral mucosa. Thus, the hypotheses that MPIs would be measurable, repeatable, and not significantly different among the sites is supported, except for the aforementioned individual comparisons of repeatability. However, the lack of a significant difference between sites must be interpreted with caution due to the small sample size and resulting low statistical power.

The previous work by Hurcombe et al (9) included a control population of normal horses. A higher colonic TVD was present herein (21.55 versus 15.6 ± 2.95) than was present in the Hurcombe et al (9) study. The PVD was similar between the studies; however, the PPV and the MFI were lower in this study (9). The MaPI of the horses in both studies were similar, with the horses in this study having a slightly higher mean arterial pressure (81 ± 4.7 versus 76 ± 11) (9). The reason for the difference in the PPV is due to the differences in TVD reported between the 2 studies. With a relatively small sample size in both studies, the differences in TVD seen between the 2 studies could be a natural variation observed within a larger population as a whole. Some portion of the discrepancy in MFI value, which is the least objective of the 4 MPIs, reflects the inherent limitations of the technology; though the lower PVD likely affected the MFI score (24).

It is not immediately clear why the CV for PPV of the oral mucosa was significantly higher than that of the other sites. A noticeable anatomic difference between the oral mucosa compared with the rectal mucosa and colonic serosa is the presence of a bone deep to the oral mucosa versus that of strictly soft tissue deep to the other locations. This anatomic difference may have made this location more susceptible to pressure variations and alterations in the proportion of vessels that are not perfused from external occlusion. Also, it is possible that the inherent architecture and orientation of the vessels in this location, which tend to be oriented with the long axis of the capillary loops directed perpendicular to the surface, make this area less consistent to interpretation (13).

The establishment of baseline values and assessment of repeatability are the first steps to allow application of the sidestream dark field microscopy technology to clinical cases. However, the baseline values determined in this study were established in healthy anesthetized horses maintained in normotension, without pharmacologic support. Thus, these values are most relevant and clinically applicable to a similar population, such as patients undergoing elective surgical procedures.

Microcirculatory dysfunction is more likely in horses with diseases resulting in more severe systemic compromise. Thus, the experimental effects of hypotension and disease states on the MPIs at these measured sites must be evaluated to allow application across a broader population. Ultimately, the understanding of microcirculatory changes associated with a given scenario of macrocirculatory changes could help to evaluate and guide pharmacologic intervention and success in improving both hypotension and perfusion in the anesthetized patient.

Comparison of MPIs between microvascular beds in healthy human subjects is lacking. However, multiple studies have shown a dissociation between microcirculatory units of septic patients. Both Boerma et al (4) and Edul et al (25) showed that in patients experiencing abdominal sepsis and/or septic shock, the MPIs for intestinal and sublingual sites were not comparable. This is in contrast to the findings in pigs subjected to an Escherichia coli sepsis model, where correlation has been found between microvascular sites (sublingual, jejunal, rectal, conjunctival) (26,27).

Of note in this study is the use of gingival and not sublingual mucosa. This discrepancy was chosen based on ease to obtain a gingival recording versus accessing the sublingual mucosa and for the potential for comparison to the awake horse. Image quality is completely dependent on the patient being still; therefore, the oral gingiva represented an easier location to access with minimal movement. Image collection of the gingival mucosa was not difficult. However, due to the firm resistance of the rostral maxilla deep to the mucosa, microvascular occlusion from compression was noted and adjustments were made during collection to eliminate this during image capture.

The microvasculature is a complex and dynamic system. Complete control of all aspects of regulation is impossible and this inherent limitation should be acknowledged in all studies examining the microcirculation, particularly in the anesthetized patient. This study attempted to normalize these regulatory features of the macrocirculation by measuring and accounting for normal blood pressure, heart rate, cardiac output, and systemic lactate levels. The authors acknowledge that the injectable tranquilizers and inhalant anesthetic utilized in this study precipitate documented effects on vascular tone in horses (27–29). In addition, ketamine has been documented to reduce peripheral vascular resistance in dogs (30). In an effort to account for this variable, the anesthetic protocol was standardized to minimize inter-individual variability of any effects of the pre-medication or induction agents on the microcirculation. As with so many other studies, sample size is a limitation that must be recognized. The sample size of 6 was chosen based on a balance between financial and ethical concerns and to provide accurate data.

In summary, for anesthetized adult horses with normal macrovascular parameters, microvascular perfusion variables of the oral mucosa, rectal mucosa, and colonic serosa were not significantly different. This study adds to a growing collection of literature in the veterinary field supporting the feasibility of microcirculatory assessment for future clinical and research applications.