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    An SDF video microscope with a sterile cover. The black cable connects via USB to a computer for video storage and analysis.

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Evaluation of jejunal microvasculature of healthy anesthetized dogs with sidestream dark field video microscopy

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  • 1 1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.

Abstract

OBJECTIVE

To determine the feasibility of sidestream dark field (SDF) video microscopy for the evaluation of the jejunal microvasculature of healthy dogs.

ANIMALS

30 healthy sexually intact female shelter dogs anesthetized for ovariohysterectomy.

PROCEDURES

Preoperative physical and clinicopathologic assessments were performed to confirm health status. Then healthy dogs were anesthetized, and the abdomen was incised at the ventral midline for ovariohysterectomy and jejunal microvasculature evaluation. An SDF video microscope imaged the microvasculature of 2 sites of a portion of the jejunum, and recorded videos were analyzed with software capable of quantitating parameters of microvascular health. Macrovascular parameters (heart rate, respiratory rate, and hemoglobin oxygen saturation) were also recorded during anesthesia.

RESULTS

Quantified jejunal microvascular parameters included valid microvascular density (mean ± SD, 251.72 ± 97.10 μm/mm), RBC-filling percentage (66.96 ± 8.00%), RBC column width (7.11 ± 0.72 μm), and perfused boundary region (2.17 ± 0.42 μm). The perfused boundary region and RBC-filling percentage had a significant negative correlation. Strong to weak positive correlations were noted among the perfused boundary regions of small-, medium-, and large-sized microvessels. No significant correlations were identified between microvascular parameters and age, body weight, preoperative clinicopathologic results, or macrovascular parameters.

CONCLUSIONS AND CLINICAL RELEVANCE

Interrogation of the jejunal microvasculature of healthy dogs with SDF video microscopy was feasible. Results of this study indicated that SDF video microscopy is worth additional investigation, including interrogation of diseased small intestine in dogs.

Abstract

OBJECTIVE

To determine the feasibility of sidestream dark field (SDF) video microscopy for the evaluation of the jejunal microvasculature of healthy dogs.

ANIMALS

30 healthy sexually intact female shelter dogs anesthetized for ovariohysterectomy.

PROCEDURES

Preoperative physical and clinicopathologic assessments were performed to confirm health status. Then healthy dogs were anesthetized, and the abdomen was incised at the ventral midline for ovariohysterectomy and jejunal microvasculature evaluation. An SDF video microscope imaged the microvasculature of 2 sites of a portion of the jejunum, and recorded videos were analyzed with software capable of quantitating parameters of microvascular health. Macrovascular parameters (heart rate, respiratory rate, and hemoglobin oxygen saturation) were also recorded during anesthesia.

RESULTS

Quantified jejunal microvascular parameters included valid microvascular density (mean ± SD, 251.72 ± 97.10 μm/mm), RBC-filling percentage (66.96 ± 8.00%), RBC column width (7.11 ± 0.72 μm), and perfused boundary region (2.17 ± 0.42 μm). The perfused boundary region and RBC-filling percentage had a significant negative correlation. Strong to weak positive correlations were noted among the perfused boundary regions of small-, medium-, and large-sized microvessels. No significant correlations were identified between microvascular parameters and age, body weight, preoperative clinicopathologic results, or macrovascular parameters.

CONCLUSIONS AND CLINICAL RELEVANCE

Interrogation of the jejunal microvasculature of healthy dogs with SDF video microscopy was feasible. Results of this study indicated that SDF video microscopy is worth additional investigation, including interrogation of diseased small intestine in dogs.

Failure to resect devitalized small intestine can result in devastating consequences, including surgical site dehiscence, intestinal perforation, septic peritonitis, and death. However, intestinal resection can also result in adverse consequences, including short-bowel syndrome, and may necessitate rerouting of pancreatic and biliary ducts.1,2 Therefore, a surgeon often avoids resecting portions of intestine unless determined necessary on the basis of their intraoperative appearance. For decision-making, surgeons evaluate intestinal color and thickness and the presence of peristalsis, pulsations, and bleeding with incision of the intestinal wall. However, evaluation of intestinal viability is highly subjective, with important intra- and interobserver variability.3–5

Alternative methods described3,5–11 to enhance a surgeon's subjective assessment of intestinal viability include angiographic studies, infusion of arterial fluorescein dye, trans-serosal pulse oximetry, pulsed-wave Doppler ultrasonography, and laser Doppler flowmetry. However, each alternative has low sensitivity, specificity, and predictive values, and the lack of comparative data for healthy and unhealthy intestine precludes meaningful interpretation of the previous studies’ results.5–8,12–14

Microvascular damage of tissue precedes macrovascular damage,15 and studies have shown that markers of macrovascular perfusion, such as tissue color and pulse quality, are not always predictive of microvascular perfusion.16–18 Thus, assessment of microvascular perfusion may be more sensitive for detecting devitalized tissue. Indeed, microvascular damage is considered the most sensitive indicator of cardiovascular failure in people.15

The microvasculature comprises microvessels, each with a single layer of endothelial cells protected by the EG, an intraluminal biopolymer layer that has an inner (luminal) portion permeable to circulating cells, and an outer (endothelial) portion impermeable to circulating cells.19,20 The EG regulates endothelial permeability and microvascular perfusion and protects the endothelium against adhesion of inflammatory cells and platelets.15,21,22 When the EG is damaged, its normal homeostatic functions are disrupted.15 Dysregulation of parenchymal perfusion and heterogenous capillary blood flow results, leading to regional tissue hypoxia in affected organs, despite unchanged arterial and venous blood flow.23–26

Knowledge of the central role the microvasculature and its associated EG has in regulating organ perfusion has resulted in its extensive study, including quantification of the EG. The quantity of EG permeable to circulating cells reliably indicates the microvascular health of people in critical care.24 Proprietary software that analyzes videos captured through SDF video microscopy for RBC flow at the capillary level has been validated as a rapid, bedside, user-friendly, unbiased tool to assess the EG in people.15,24,27 The results of the assessment allow for improved prognostication and formulation of individualized therapies for people in critical care.28

After video acquisition by the SDF video microscope, the proprietary software calculates Dperf, RBCW, RBC% (the average time that each perfused vascular segment contained a detectable RBC), valid microvascular density (the number of valid [high-quality] vascular segments per area of interrogated tissue), and, most importantly, PBR. The PBR is the portion of the EG that is permeable to RBCs and is calculated from Dperf and RBCW (ie, [Dperf – RBCW]/2).29 As the EG becomes more permeable (ie, the permeable portion of EG enlarges, and the impermeable portion lessens), the PBR increases, indicating poor microvascular perfusion.

Recently, safe and repeatable interrogation of the sublingual vasculature with an SDF video microscope has been reported for dogsa and cats,b and results were similar to those reported for people.15,21,27 However, veterinary use of SDF video microscopy for the evaluation of the small intestinal microvasculature has not been reported. Therefore, the objectives of the study presented here were to determine the feasibility of SDF video microscopy and to report the results of analysis of recorded videos with software capable of assessing the small intestinal microvasculature of healthy dogs. We speculated that interrogation of the small intestinal microvasculature would be feasible.

Materials and Methods

Animals

Thirty young to middle-aged (range, 1 to 5 years) 15- to 30-kg sexually intact female dogs from an animal shelter anesthetized for elective ovariohysterectomy and previously identified as healthy on the basis of history, physical examination by a veterinarian, and results for PCV and total plasma protein, blood glucose, and BUN concentrations (clinicopathologic parameters) were enrolled in this study. Dogs were excluded if they weighed < 10 kg (because of size restrictions outlined in Institutional Animal Care and Use Committee protocol No. 201809002, which permitted ovariohysterectomy in the dogs of the present study) or had grossly abnormal small intestines. The present study was approved by the Institutional Animal Care and Use Committee of the University of Florida (protocol No. 201910557).

Anesthesia and monitoring

All dogs were premedicated with acepromazinec (0.05 mg/kg, IM) and long-acting buprenorphined (0.2 mg/kg, SC) prior to placement of an IV catheter. Anesthesia was induced with propofole (2 to 5 mg/kg, IV) to achieve orotracheal intubation and was maintained with isoflurane (1% to 2%) in oxygen (2 L/min). For the duration of anesthesia, all dogs received lactated Ringer solutionf (5 mL/kg/h, IV), and heart rate, respiratory rate, and hemoglobin oxygen saturation (macrovascular parameters) were monitored by percutaneous and esophageal auscultation with a stethoscope and pulse oximetry,g respectively. Anesthetic induction and monitoring were supervised by a board-certified anesthesiologist.

Glycocalyx evaluation

After a stable plane of anesthesia was achieved and the abdomen was prepared for surgery, the abdomen was incised on the ventral midline for routine open ovariohysterectomy. Then, a random jejunal segment was isolated. A sterile hard plastic cover that was replaced after the evaluation of each dog was placed over the tip of a handheld SDF video microscopeh (Figure 1). The SDF video microscope camera, connected to a computer with softwarei capable of analyzing the recorded videos for microvasculature perfusion, was applied sequentially to 2 sites of the isolated jejunal segment for video acquisition (40 frames/s; Supplemental Video S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.81.11.888). The video microscope operator used a sterile technique to stabilize the tip of the video microscope perpendicular to the antimesenteric border of the jejunal segment with gentle but firm pressure while an assistant focused the camera and operated the computer with the software. To prevent bubble artifact, sterile dry gauze was used to blot the serosal surface of the jejunal segment to remove excess moisture. Data acquisition automatically started once the software identified optimal tissue motion, illumination intensity, and focus.

Figure 1—
Figure 1—

An SDF video microscope with a sterile cover. The black cable connects via USB to a computer for video storage and analysis.

Citation: American Journal of Veterinary Research 81, 11; 10.2460/ajvr.81.11.888

The camera interrogated perfused microvessels with diameters of 5 to 25 μm and subdivided each microvessel along its length into 10-μm vascular segments.30,31 After video acquisition, the software categorized vascular segments on the basis of data quality (valid vs invalid). For each valid vascular segment, which the software reported as the number of recording sites, the software determined Dperf, RBCW, and RBC%. The PBR, an indirect measure of perfusion, was calculated from Dperf and RBCW. Additionally, valid microvascular density, a direct measure of perfusion, was determined.15 Data collection was considered complete when 3,000 vascular segments had been interrogated. When poor camera focus or impaired perfusion precluded identification of 3,000 vascular segments, data collection ceased when 50 videos were recorded or the assistant manually stopped the recording.32

Statistical analysis

Descriptive statistics were used to report the continuous variables. The Spearman rank correlation coefficient test was used to assess for associations among continuous variables (age, body weight, and clinicopathologic, macrovascular, and microvascular parameters). Statistical analyses were performed with commercial software.j Values of P ≤ 0.05 were considered significant.

Results

A total of 60 videos (2 sets of videos for each of 30 dogs) of the jejunal microvasculature were analyzed. No dogs were excluded, and SDF video microscopy was easily repeated at all interrogation sites without complication. Age, body weight, and measured macrovascular and clinicopathologic parameters were summarized (Table 1).

Table 1—

Age, body weight, and macrovascular and clinicopathologic parameters for 30 healthy sexually intact female dogs from an animal shelter anesthetized for elective ovariohysterectomy and evaluation of the jejunal microvasculature with SDF video microscopy.

ParameterMean ± SDRange
Age (approx [mo])22.1 ± 12.48.0-60.0
Body weight (kg)19.9 ± 4.112.3-27.5
Heart rate (beats/min)90.7 ± 17.665.0-135.0
Respiratory rate (breaths/min)9.7 ± 6.05.0-28.0
Spo2 (%)96.8 ± 1.792.5-97.5
PCV (%)44.9 ± 4.634.0-53.0
Total plasma protein (g/dL)7.4 ± 0.66.4-8.6
BUN (mmol/L)14.6 ± 6.37.5-20.0

Spo2 = Hemoglobin oxygen saturation.

Jejunal microvasculature parameters were summarized (Table 2). Strong negative correlations were evident between valid microvascular density and the number of video recordings (r2 = 0.90; P < 0.001), PBR of microvessels with diameters of 5 to 25 μm and RBC% (r2 = 0.57; P < 0.001), and PBR of microvessels with diameters of 10 to 19 μm and RBC% (r2 = 0.66; P < 0.001). Strong positive correlations were evident between PBR of microvessels with diameters of 10 to 19 μm and 5 to 25 μm (r2 = 0.78; P < 0.001), PBR of microvessels with diameters of 20 to 25 μm and 5 to 25 μm (r2 = 0.65; P < 0.001), and PBR of microvessels with diameters of 10 to 19 μm and 20 to 25 μm (r2 = 0.61; P < 0.001). Weak positive correlations were evident between valid microvascular density and number of recording sites (r2 = 0.28; P = 0.011), PBR of microvessels with diameters of 5 to 9 μm and 5 to 25 μm (r2 = 0.26; P = 0.004), and PBR of microvessels with diameters of 5 to 9 μm and 10 to 19 μm (r2 = 0.23; P = 0.007).

Table 2—

Jejunal microvascular parameters obtained through analysis of SDF video microscopic videos for the dogs of Table 1.

ParameterMean ± SDRange
Video recordings (No.)37.98 ± 11.4215.00-60.50
Recording sites (No.)2,385.10 ± 302.911,506.50-2,870.33
Valid microvascular density (μm/mm)251.72 ± 97.10107.50-164.33
RBC% (%)66.96 ± 8.0052.23-81.05
RBCW (μm)7.11 ± 0.725.92-8.83
PBR of microvessels  
5-25 μm (μm)2.17 ± 0.421.50-3.00
5-9 μm (μm)1.12 ± 0.160.69-1.61
10-19 μm (μm)2.54 ± 0.491.62-3.94
20-25 μm (μm)3.02 ± 0.921.39-5.20

No significant associations were identified between microvascular parameters and clinicopathologic results or macrovascular parameters and between clinicopathologic results and macrovascular parameters.

Discussion

The results of the present study supported the use of SDF video microscopy as a repeatable, unbiased, and quantitative technique for assessing the jejunal microvasculature of healthy dogs. Assessment was possible for all dogs, and consistent measurements were obtained for both interrogated sites of a jejunal segment for each dog. Interrogation of the ileal microvasculature of people with ileostomies has been reported.33 Yet to our knowledge, interrogation of the small intestinal microvasculature of dogs with an SDF video microscope has not been previously reported.

Evaluation of sublingual microvasculature with SDF video microscopy has been described for healthy dogs30,a and cats.34,b Mean ± SD PBR for sublingual microvessels with diameters of 5 to 25 μm was 2.04 ± 0.31 μm for dogsa and 2.39 ± 0.26 μm for cats.b These results are similar to that reported for people (2.14 ± 0.25 μm)15 and that noted for the jejunal microvasculature of dogs in the present study (2.17 ± 0.42 μm). Because the PBR for the jejunal microvasculature of dogs in the present study was comparable to that reported for the sublingual microvasculature of dogs in a previous study,a the PBR of the sublingual microvasculature may be a surrogate for the PBR of the jejunal microvasculature. However, further studies are necessary to confirm whether an association exists between sublingual and small intestinal PBR determined simultaneously in healthy dogs, as suspected on the basis of the above data. If the PBR of the sublingual microvasculature can be a surrogate of the PBR of the small intestinal microvasculature, studies are then necessary to determine whether the same association is maintained between the PBR of the sublingual and small intestinal microvasculature determined simultaneously in dogs with compromised small intestinal microvasculature. It is important to first uncover whether any significant differences exist between the small intestinal microvascular parameters in dogs with and without compromised small intestinal microvasculature. However, guidelines to differentiate healthy from diseased small intestinal microvasculature in people have yet to be established33; therefore, differences in the microvasculature of healthy and diseased small intestine in people cannot be used to predict any expected differences in dogs.

Prior to the availability of software capable of analyzing videos of the microvasculature captured with SDF video microscopy, analysis required manual calculation by highly trained personnel.15,30 Therefore, data analysis was labor intensive, which precluded bedside analysis and limited the clinical applicability of SDF video microscopy. With the coupling of SDF video microscopy and software, however, more rapid quantification of microvascular health is possible. For example, video acquisition of the sublingual microvasculature of healthy dogs with an SDF video microscope only takes 1.41 min/interrogated site,a and automated analysis quickly follows. Although the time to record 3,000 vascular segments or 50 videos was not recorded for the present study, data acquisition was rapid and calibration for tissue motion, illumination intensity, and focus was easily performed with minimal preprocedural training.

Higher microvascular density for healthy (vs unhealthy) small intestine should allow for fewer video recordings because a greater number of valid microvascular segments should be present in each video. This supposition was supported by the results of the present study, in which a significant negative correlation was identified between valid microvascular density and number of recordings; therefore, fewer videos were required to achieve the study end point (3,000 valid vascular segments) when the valid microvascular density was high at an interrogated site. Furthermore, a significant positive correlation was identified between valid microvascular density and number of recording sites, indicating a greater number of valid vascular segments at recording sites with greater valid microvascular density.

In an animal with systemic disease or focal tissue devitalization, high PBR indicates damage to the EG and subsequent microvascular dysfunction.15 Therefore, direct measures of microvascular perfusion (valid microvascular density, RBC%, and RBCW) should reflect impaired microvascular perfusion when the PBR is high.15 Indeed, in the present study, a significant negative correlation was identified between PBR and RBC% for the complete group of microvessels with diameters of 5 to 25 μm and the subgroup with diameters of 10 to 19 μm. As the EG is damaged and microvascular dysfunction results, blood inflow through each of these microvessels is reduced (represented by reduced RBC%), resulting in an overall decrease in microvascular perfusion. As the EG endures further damage, becoming increasingly more permeable to inflammatory cells, RBCs, and platelets, the RBCW is expected to increase. Additionally, the valid microvascular density is expected to decrease as blood inflow completely halts in an increasing proportion of microvessels. Although no correlations were significant between PBR and RBCW and between PBR and valid microvascular density in the present study, interrogation of grossly normal small intestine was performed, and significant correlations may have been identified with interrogation of diseased small intestine.

The PBRs for all sizes of small intestinal microvessels are expected to simultaneously increase with small intestinal devitalization. The microvessel walls are composed of a single layer of endothelial cells with an underlying basement membrane. Therefore, variation in wall thickness is minimal, and microvessels, regardless of their diameter, are equally susceptible to collapse and microvascular dysfunction secondary to high intraluminal small intestinal pressures.35 Indeed, in the present study, positive correlations were noted among the PBRs of all sizes of evaluated microvessels.

The major limitation of the present study was the inability to directly measure the EG. The software used was capable of indirectly assessing the EG by calculating the PBR from other microvasculature parameters. Linear regression analysis indicates a negative correlation between PBR and EG thickness and between PBR and microvascular perfusion.15 Despite possible inaccuracies that may arise with indirect measurement of EG, SDF video microscopy is a minimally invasive intraoperative technique that allows for rapid data collection and interpretation, making this technique ideal for clinical use. Moreover, the software is able to provide direct measures (eg, valid microvascular density) of microvascular perfusion, which is most applicable for determining small intestinal viability.

Another limitation of this study was that only the jejunum was interrogated. Thus, the reported results may not be applicable to other segments of the gastrointestinal tract. The jejunum was selected because it is the most common portion of the small intestine obstructed by foreign bodies in dogs.2 For a dog requiring surgery because of a foreign body, a surgeon must decide whether to perform an enterotomy to remove the foreign body or an intestinal resection and anastomosis, and evaluation of the microvasculature of the affected portion of the jejunum could inform the surgeon's decision. Moreover, the present study was limited by the sole use of sexually intact female dogs and the failure to perform additional clinicopathologic diagnostic tests (eg, CBC and serum biochemical analyses) prior to surgery to better ensure those dogs were healthy.

Furthermore, systemic arterial blood pressure was not measured during surgery; therefore, whether any dog was hypotensive at the time of interrogation of the jejunal microvasculature was unknown. Systemic hypotension may have reduced microvascular perfusion and therefore may have affected the reported results. Acepromazine was administered as a premedication, and it may have induced systemic hypotension. Although the effects of acepromazine on the jejunal microvasculature could not be independently assessed because a second group of dogs that did not receive acepromazine was not included in the present study, the results of the present study closely mirrored those previously reporteda when the sublingual microvasculature of healthy dogs that did not receive acepromazine was assessed. All dogs in the present study received lactated Ringer solution IV during surgery in an attempt to maintain normotension.

In conclusion, SDF video microscopy of the jejunal microvasculature of healthy dogs was feasible, and results obtained through analysis of the recorded videos with software were consistent. Thus, intraoperative indirect EG assessment of the small intestinal microvasculature of dogs undergoing small intestinal surgery could be valuable because a reliable, quantitative technique to assess small intestinal viability has not been available. Results obtained may serve as a baseline for future comparative studies of the small intestinal microvasculature of dogs.

Acknowledgments

No external funding was used in this study. The authors declare that there were no conflicts of interest.

The authors thank James Colee for help with statistical analysis.

ABBREVIATIONS

Dperf

RBC-perfused luminal diameter

EG

Endothelial glycocalyx

PBR

Perfused boundary region

RBC%

RBC-filling percentage

RBCW

RBC column width

SDF

Sidestream dark field

Footnotes

a.

Londoño LA, Bowen CM, Buckley GJ. Evaluation of the endothelial glycocalyx in healthy anesthetized dogs using rapid, patient-side GlycoCheck analysis software (abstr), in Proceedings. Int Vet Emerg Crit Care Symp 2018;28:S7.

b.

Millar KK, Londoño LA, Monday JS, et al. Evaluation of the endothelial glycocalyx in healthy anesthetized cats using rapid, patient-side Glycocheck analysis software (abstr), in Proceedings. Int Vet Emerg Crit Care Symp 2019;29:S11.

c.

LGM Pharma, Erlanger, Ky.

d.

Simbadol, Zoetis, Parsippany, NJ.

e.

Propofol, Pfizer, New York, NY.

f.

Hospira, Lake Forest, Ill.

g.

Nellcor Oximax N-65 pulse oximeter, Medtronic, Minneapolis, Minn.

h.

CapiScope HVCS Handheld Video Capillaroscopy System, KK Technology, Devon, England.

i.

GlycoCheck BV, Maastricht, Netherlands.

j.

SAS, version 9.4, SAS Institute Inc, Cary, NC.

References

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    • Search Google Scholar
    • Export Citation
  • 2. Giuffrida MA, Brown DC. Small intestine. In: Tobias KM, Johnston SA, eds. Veterinary surgery: small animal. St Louis: Saunders, 2012;17321760.

    • Search Google Scholar
    • Export Citation
  • 3. Erikoglu M, Kaynak A, Beyatli EA, et al. Intraoperative determination of intestinal viability: a comparison with transserosal pulse oximetry and histopathological examination. J Surg Res 2005;128:6669.

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Supplementary Materials

Contributor Notes

Drs. Regier and Londoño contributed equally to this work.

Address correspondence to Dr. Regier (pregier@ufl.edu).