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Published in final edited form as: Ultrasound Med Biol. 2024 Oct 18;51(1):26–32. doi: 10.1016/j.ultrasmedbio.2024.08.013

Contrast Enhanced Ultrasound of Mouse Models of Hindlimb Ischemia Reveals Persistent Perfusion Deficits and Distinctive Muscle Perfusion Patterns

Alyssa B Becker 1, Lanlin Chen 1, John A Hossack 1, Alexander L Klibanov 1,2, Brian H Annex 1,2,3, Brent A French 1,2
PMCID: PMC12370005  NIHMSID: NIHMS2018268  PMID: 39426845

Abstract

Objective:

Mouse models of hindlimb ischemia are used to study peripheral arterial disease and evaluate novel therapies. Contrast enhanced ultrasound (CEUS) is a non-invasive perfusion measurement technique that is increasingly being employed in these models. The objective of this study was to evaluate two models of severe hindlimb ischemia by CEUS to characterize perfusion recovery and muscle perfusion patterns.

Methods:

Mice undergoing double femoral artery ligation were measured by CEUS and laser Doppler perfusion imaging (LDPI) at baseline and 1–150 days post-surgery. A second group undergoing femoral artery ligation and excision were measured 1–28 days post-surgery.

Results:

By LDPI, both surgeries showed robust perfusion recovery by 14 days post-surgery. However, by CEUS only a ~40% perfusion recovery plateau was reached in either group. These results are consistent with our previous work, employing a less severe single femoral artery ligation, that showed perfusion in the ischemic limb does not return to normal by 150 days post-surgery. Cluster analysis of muscle perfusion patterns indicated 3–5 different patterns at day 1 post-surgery. The double ligation model yielded significantly less variable perfusion patterns, suggesting that it can provide more reproducible results.

Conclusions:

Contrary to LDPI, perfusion as measured by CEUS never fully recovers after hindlimb surgery, even when followed 28–150 days post-surgery. Individual mice can manifest different patterns of muscle perfusion to the same surgery, but these patterns are conserved within and between different surgical techniques. These results may have significant implications for the evaluation of novel therapeutics to treat PAD in mice.

Keywords: perfusion imaging, ultrasound, ischemia, peripheral arterial disease

Introduction

Peripheral arterial disease (PAD) is the lack of perfusion to the limbs due to obstructive atherosclerosis1–4. PAD has various presentations including asymptomatic, intermittent claudication, and the most severe form of the disease, critical limb ischemia, where gangrene may be present necessitating amputation 5,6. Very few therapies exist for PAD, and mouse models of hindlimb ischemia play important roles in providing mechanistic insight into disease pathology and in evaluating novel therapeutic approaches for potential clinical translation 7–9.

Mouse models of PAD are most often hindlimb ischemia (HLI) models where unilateral femoral artery ligation(s) and/or an excision of the femoral artery is performed10. Most commonly, laser Doppler perfusion imaging (LDPI) is used to measure perfusion recovery over time. However, we have recently shown that LDPI does not correlate with fluorescent microsphere, histological, or photoacoustic microscopy measurements of perfusion late after HLI 11. We demonstrated contrast enhanced ultrasound (CEUS) does correlate well with these methods in an HLI model employing a single femoral ligation. This notwithstanding, it is unknown how the results of CEUS and LDPI analysis might compare in more severe HLI models that seek to emulate more advanced forms of PAD.

Another advantage of using CEUS is that perfusion can be visualized in individual muscle groups. Spatial information about perfusion is lacking in the literature, as the techniques that can provide spatial information are typically destructive or limited to anatomical vascular structure rather than functional perfusion information. For example, micro-CT and angiography can provide good anatomic structure, but limited functional perfusion information 12. Other imaging techniques such as MRI or PET can provide spatial and perfusion information, but are much more costly and can suffer from poor resolution in small animals with low flow rates 13,14. Compared to ultrasound, these techniques often require more resources including a dedicated support staff.

Here, we evaluate perfusion recovery in two mouse models of severe HLI using both CEUS and LDPI. We also evaluate muscle perfusion patterns after surgery in the three most common HLI models, and apply k-means clustering to explore variation within and between surgical techniques.

Materials and Methods

Mice

Male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were provided with water and standard chow ad libitum. Mice were housed 1–4 animals per cage and in a room with a 12 hour light cycle. Cages had standard corn cob bedding except post-surgery when Iso-pad bedding (Envigo, Indianapolis, IN, USA) was used until the mice were ambulating normally. The Institutional Animal Care and Use Committee of the University of Virginia approved all animal experiments.

Mouse Models of Hindlimb Ischemia

Mice (10–12 weeks old) were anesthetized with ketamine (90mg/kg) and xylazine (10 mg/kg). The left medial leg was shaved and depilated and the area was prepared for aseptic surgery. A heating lamp was used to maintain body temperature during surgery. An incision was made over the femoral artery and the following procedures for each type of surgery were followed:

Single ligation- 6–0 silk sutures were used to ligate the femoral artery proximal to the lateral circumflex femoral artery 15.

Double ligation- 6–0 silk sutures were used to ligate the femoral artery proximal to the iliacofemoral artery and the popliteal/saphenous bifurcation 15.

Ligation/excision- 6–0 silk sutures were used to ligate the femoral artery proximal to the pudendoepigastric trunk and the popliteal/saphenous bifurcation 10,15. The segment between the sutures was then excised. Care was taken to not damage nearby structures such as the femoral nerve and vein. Buprenorphine-SR (0.5 mg/kg) was administered subcutaneously after surgery and re-administered as needed every 48h or as recommended by veterinary staff based on facial grimace score and burrowing behavior. Mice were euthanized and excluded if necrosis reached the heel.

Microbubble Preparation

Microbubbles were prepared as previously described 11,16. Briefly, microbubbles were formed by dispersion in saline and have a decafluorobutane gas core and a lipid monolayer shell composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and PEG stearate. The presence of any microbubbles larger than 7 μm was minimized by flotation at normal gravity. The concentration of microbubble stocks was measured approximately every two weeks (Beckman Coulter, Indianapolis, Indiana, USA). Microbubbles were diluted with sterile saline directly before administration to a concentration of 1×109 MBs/ml and infused at 10 μl/min.

Contrast Enhanced Ultrasound

Imaging was performed on an Acuson Sequoia C512 system with a 15L8W transducer (Siemens, Munich, Germany) as previously described 11. Briefly, mice were anesthetized with 1–2% isoflurane in air, placed prone on a heated stage with legs extended, and the feet were secured. Mice were anesthetized with isoflurane in air rather than oxygen because the use of air as the carrier gas limits the circulation lifetime of MB contrast agents 17. A 27G catheter filled with heparinized saline (100 units/ml) was inserted in the tail vein and secured. Ultrasound gel was put on the calves and the transducer was positioned perpendicular to the calves. Previously optimized scanning parameters were used (frequency: 7 MHz, dynamic range: 100 dB, gain: −10, imaging mechanical index: 0.2, burst mechanical index: 1.9, and burst time: one second) 11.

Video of the ultrasound imaging was captured in real time. The microbubble solution (1×107 MB/min) was infused via tail vein catheter. After reaching steady state, burst pulses were applied once per minute for 10–15 minutes for data acquisition. The burst pulses destroy only the microbubbles within the field of view, while microbubbles from upstream continue to flow into the scan plane thus enabling perfusion to be quantified18. CEUS measurements were always taken after LDPI measurements because it has previously been reported that ultrasound-mediated microbubble cavitation can transiently increase hindlimb perfusion in mice for up to 24h 19. However, we did not observe this phenomenon during our initial test/retest study of CEUS performed at 3–4 day intervals over the course of 11 days 11.

A custom MATLAB (Mathworks, Natick, Massachusetts, USA) script was used for analysis as previously described 11. The image intensity over time of each flash-replenishment sequence was fit to y=A*(1-e-βt) where A represents blood volume, beta represents blood velocity, and A*β represents blood flow 18. An average ischemic/control hindlimb A*β ratio was used as the CEUS output metric.

Perfusion Pattern Analysis

CEUS imaging data from day 1 post-surgery was used for perfusion pattern analysis. 46 mice (17 single ligation, 18 double ligation, and 11 excision/ligation) were analyzed. Raw data for the single ligation surgery was from a previous study 11, however a perfusion pattern analysis was not performed at that time. These data were retrospectively analyzed and included in this analysis for comparison with the other two models. A B-mode image, a contrast mode image before microbubble infusion (background), and a contrast mode image during steady state microbubble infusion were extracted from the video data for each mouse. Data were analyzed using a custom MATLAB (Mathworks, Natick, Massachusetts, USA) script. The background image was subtracted from the contrast image to ensure any artifacts were removed before quantification. The B-mode image was used to determine the region of interest in the same way as was done with the video data. The region of interest was further broken into four major muscle groups plus the skin based on published microscopy images of the mouse hindlimb (Figure 1, Figure 2) 20. This segmentation scheme grouped some of the smaller muscles with larger ones because the small muscles are difficult to correctly segment and contribute minimally to the total signal. The average image intensity in each major muscle region was then quantified. k-means clustering was performed on these muscle region intensities for two to ten clusters 21,22. The number of clusters was optimized using silhouette analysis 23.

Figure 1:

Figure 1:

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Example images of a mouse one day after ligation/excision surgery. The B-mode images were used to define the muscle segmentation shown in Fig. 2. The contrast mode shows that there is almost no flow in the ischemic limb.

Figure 2:

Figure 2:

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Schematic of the muscle segmentation mask for the left mouse hindlimb used for cluster analysis. Only the major muscle groups were segmented because smaller muscles were in some cases difficult to identify individually and were therefore grouped with larger muscles.

Laser Doppler Perfusion Imaging

LDPI was performed as previously described using the PeriCam PSI (Perimed, Sweden) 11. Mice were anesthetized with 1–2% isoflurane in oxygen, and calves were shaved and depilated. Mice were placed prone and the feet were secured in place. Three to five measurements were taken and data were analyzed using the included PimSoft analysis software. Regions of interest (ROIs) were selected around the feet and the image intensity was averaged over all measurements. A left/right (ischemic/control) foot ratio was calculated.

Histological Analysis

Mice were anesthetized with ketamine (90mg/kg) and xylazine (10 mg/kg). Mice were then perfused through the left ventricle with heparinized saline (10 units/ml) followed by 4% paraformaldehyde. Skeletal muscles of the calf were harvested and incubated in 4% paraformaldehyde at 4 °C for 48–72 hours. The tissue was then rinsed three times in saline and placed in 15% sucrose overnight followed by 30% sucrose overnight. The resulting tissue was flash frozen in OCT and 10 μm sections were cut and stained with H&E.

Statistical Analysis

Power analysis for sample size was performed using standard deviation values from our previous study that employed a similar experimental design 11. Animals were randomly assigned to experimental groups. All results are expressed as the mean ± standard error of the mean. A linear mixed model with Bonferroni correction was used to analyze the longitudinal LDPI and CEUS data using SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA). A p value <0.05 was considered significant.

Results

Surgical Mortality and Exclusions

A total of 32 mice were enrolled in these studies, but 3 mice succumbed to complications of surgery prior to imaging on day 1 and were therefore excluded from data analysis. Mortality due to surgery was thus <10% and all mice that survived for 24h post-surgery successfully completed their assigned imaging studies. However, we were unable to analyze images from two mice at day 1 after double-ligation surgery due to file corruption, and 8 of the 18 doubly-ligated mice in the 0–14 day study were part of an extended study that lasted 150 days. Tables summarizing the number of mice analyzed per time point and associated degrees of freedom for each study are appended as Supplementary Data.

Double Ligation Surgery Results in Sustained Perfusion Deficit

To evaluate perfusion recovery over time in the double ligation HLI model, mice were imaged at baseline and days 1, 4, 7, 14, 28, 60, 90, and 150 after surgery by LDPI and CEUS (Figure 3). By LDPI, perfusion in the ischemic limb was reduced to 35 ± 3% of the control limb day 1 post-surgery. Perfusion in the ischemic limb then increased approximately linearly until day 14 post-surgery, at which time it returned to pre-surgery levels and remained steady through day 150 post-surgery. By CEUS, perfusion in the ischemic limb was reduced to 6 ± 2% of the control limb on day 1 post-surgery. In contrast to LDPI, perfusion by CEUS in the ischemic limb modestly peaked at ~52% on days 7 and 14 post-surgery and then plateaued at ~35% of the control limb through the end of the study. Measurements by LDPI and CEUS are significantly different at all time points (p<0.005, n=18 for baseline, n=16 for 1 day, n=18 for 4 to 14 day, and n=8 for 28–150 day time points).

Figure 3:

Figure 3:

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The ratio of microvascular blood flow in the ischemic/control limbs as measured by LDPI and CEUS at baseline and days 1, 4, 7, 14, 28, 60, 90, and 150 after double ligation surgery. Flow by LDPI in the ischemic limb returns to normal within 14 days post-surgery. In contrast, flow by CEUS modestly peaks at days 7 and 14 and then plateaus at ~35% of the control limb through the end of the study. Measurements by LDPI and CEUS are significantly different at all time points (*p<0.005 by paired analysis; n=18 for baseline, n=16 for 1 day, n=18 for 4 to 14 day, and n=8 for 28–150 day time points).

Ligation/excision Surgery Results in Sustained Perfusion Deficit

To evaluate perfusion recovery over time in the ligation/excision HLI model, mice were imaged days 1, 4, 7, 14, and 28 after surgery by LDPI and CEUS (Figure 4). Baseline data were not acquired for this group of mice since baseline data (acquired in normal mice prior to surgery) were consistent in all 35 mice previously studied (17 single ligation and 18 double ligation mice). We therefore used the previously acquired average baseline data from those 35 mice as a reference for the ligation/excision group. When assessed by LDPI, perfusion in the ischemic limb was reduced to 36 ± 3% of the control limb day 1 post-surgery. Perfusion in the ischemic limb then increased approximately linearly until day 14 post-surgery, at which time it returned to pre-surgery levels and remained steady until day 150 post-surgery. By CEUS, perfusion in the ischemic limb was reduced to 7 ± 1% of the control limb on day 1 post-surgery. In contrast to LDPI, perfusion by CEUS in the ischemic limb modestly peaked at 54 ± 10% on day 7 post-surgery and then plateaued at ~41% of the control limb through the end of the study. Measurements by LDPI and CEUS were significantly different at days 1, 14, and 28 post-surgery (*p<0.05, n=11 at all time points).

Figure 4:

Figure 4:

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The ratio of microvascular blood flow in ischemic/control limbs as measured by LDPI and CEUS at days 1, 4, 7, 14, and 28 after ligation/excision surgery. Flow by LDPI returns to normal within 14 days post-surgery. However, flow by CEUS increases until day 7 post-surgery, after which a modest decline was observed to a level that was 32% that of the control limb at 28 days post-surgery. Measurements by LDPI and CEUS are significantly different at days 1, 14, and 28 post-surgery (*p<0.05 by paired analysis, n=11 at all time points).

Pathological Muscle Morphology

H&E staining was used to evaluate muscle morphology in control and ischemic limbs at day 150 post-surgery for the double ligation surgery and at day 28 post-surgery for the ligation/excision surgery (Figure 5). Ischemic limb muscles from both surgeries show irregular fiber structure and fibers with centralized nuclei. In control limbs from both surgeries, there is more consistent fiber organization and most muscle fibers have peripheral nuclei.

Figure 5:

Figure 5:

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H&E staining of mouse hindlimb muscle 150 days after double ligation surgery and 28 days after ligation/excision surgery at 200x. Representative control limb muscle shows peripheral nuclei and uniform myofiber architecture. Representative ischemic limb muscle from either type of surgery shows irregular myofibers and many myofibers with centralized nuclei.

CEUS is Well Suited for Regional Muscle Analysis

Example B-mode and contrast-mode images of a mouse one day after ligation/excision surgery are shown in Figure 1. Based on microscopy images of the mouse calf, the locations of different muscle groups in the ultrasound images could be segmented (Figure 2) 20. Several small muscles were at times difficult to place individually and were therefore grouped with larger muscles.

Perfusion Patterns are Conserved Across HLI Models

During CEUS imaging on day 1 post-surgery, it became immediately apparent that not every mouse responded the same way to a given surgery. While all mice had significant decreases in perfusion, individual mice had different regions of the limb that were more or less perfused compared to other mice. Additional rounds of CEUS imaging also revealed that there were conserved patterns of perfusion within and between surgeries. To quantitatively assess these distinct perfusion patterns, k-means clustering was applied (Figure 6). The optimal number of clusters was determined to be five by silhouette analysis 23. In the initial analysis of all 46 mice undergoing the 3 different surgical protocols, Cluster 1 was relatively rare (4%) and corresponds to no perfusion in the tibialis anterior alone. Cluster 2 was observed 13% of the time and corresponds to perfusion in all muscles. Cluster 3 was observed 17% of the time and corresponds to perfusion only to the gastrocnemius. Cluster 4 was observed 24% of the time and corresponds to no perfusion in the tibialis anterior, flexor digitorum longus, tibialis posterior, and flexor hallucis longus. Cluster 5 was observed 41% of the time and corresponds to little to no perfusion in any muscle and minimal perfusion to the skin. The single ligation surgery contained all five of the clusters (1, 2, 3, 4, 5). The double ligation surgery contained three of the clusters (2, 3, 5). The ligation/excision surgery contained a different but overlapping set of three clusters (3, 4, 5). This data is summarized in Table 1.

Figure 6:

Figure 6:

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A) Scatterplot visualizing the outcome of cluster analysis, each column represents one mouse. Each data point is the mean signal intensity of a single muscle grouping identified in Figure 2. There are five data points per mouse and the pattern of muscle group intensities for each mouse determines what cluster it is in, each color represents a different cluster. B) A representative contrast mode image and percentage of mice in each of the five clusters. C) The distribution of clusters within each type of surgery. Raw data for the Single Ligation model was from a previously reported study 11, but this is the first cluster analysis of that model.

Table 1:

Summary of the frequency, surgery type, and muscle groups with perfusion in each cluster.

Cluster Mice per Cluster (%) Surgery Types Muscle Groups with Perfusion
Single Double Ligation/Excision Gastrocnemius Soleus and Plantaris Flexor digitorum longus, Tibialis posterior, and Flexor hallucis longus Tibialis anterior
1 4 ✔ ✔ ✔ ✔
2 13 ✔ ✔ ✔ ✔ ✔ ✔
3 17 ✔ ✔ ✔ ✔
4 24 ✔ ✔ ✔ ✔
5 41 ✔ ✔ ✔
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Discussion

In this study, we sought to evaluate double ligation and ligation/excision mouse models of hindlimb ischemia, continuing our work in exploring the use of CEUS to study PAD. Previously, we validated CEUS in a single ligation HLI model with fluorescent microspheres, histopathology and photoacoustic microscopy. In that study, we reported that the results from LDPI were inconsistent with the results obtained from CEUS and the three validation techniques 11. In this paper we measured perfusion recovery in two more severe models of HLI, and again compared LDPI with CEUS.

Assessment of both severe models of HLI using LDPI shows perfusion in the ischemic limb decreases to approximately the same extent relative to the control limb (35% for double ligation vs. 36% for ligation/excision) on day 1 post-surgery. Perfusion by LDPI subsequently increases until day 14 post-surgery, at which point it recovers to pre-surgery levels in both models. Our previous LDPI results in the single ligation model showed that perfusion in the ischemic limb decreases to only 72% of the control limb on day 1 post-surgery, indicating the initial injury in the double ligation and excision models was indeed more severe. Interestingly, the time required for perfusion recovery by LDPI was also 14 days for the single ligation surgery. These results are consistent with those reported by other groups using similar models 13,24–28.

In contrast, perfusion by CEUS in the ischemic limb decreases to 6% for the double ligation surgery and 7% for the ligation/excision surgery on day 1 post-surgery. Perfusion then makes a modest recovery at days 7–14 post-surgery, then ultimately achieves a plateau at about 40% of the control limb, never returning to normal in either the double ligation or ligation/excision models. Using CEUS, we were able to detect a modest peak in perfusion recovery in the ischemic limb that reached 52–54% of the control limb by 7–14 days post-surgery, but then waned to a level of 35–41% of control by 28 days post-surgery in both models. A hypothesis for this transient peak in perfusion recovery is that there is overgrowth and then pruning of the vasculature after surgery, as reported in similar models by Landázuri and colleagues 29. By micro-CT, they found that vascular volume, density and connectivity peak at day 7 post-surgery and then decline. These results are largely consistent with the patterns of recovery measured by LDPI and CEUS in our previous report using a single ligation model of HLI11.

It is noteworthy that at baseline the perfusion ratio was greater than 1 by CEUS, as observed in our previous study as well 11. Quality control studies were done to evaluate CEUS instrumentation and procedures, but no defects were found. We used a hydrophone to measure the output across three points on the transducer; no differences were seen. We also excluded transducer orientation as a potential source of bias by rotating the transducer 180 degrees halfway through the in vivo imaging study. Both orientations resulted in the same perfusion ratio. Having excluded these potential technical sources of bias, we turned to the literature and discovered that mean differences in limb perfusion of 10% have been reported in humans as measured by MRI flowmetry 30. We therefore propose that the baseline difference in perfusion ratio detected by CEUS may be attributable to natural flow asymmetry between limbs, as opposed to any defect in CEUS instrumentation or procedures.

The discrepancy between the results of LDPI and CEUS are consistent with those reported in our previous study of a single ligation model. LDPI primarily measures skin perfusion since, as an optical technique, it is inherently depth-limited 31,32. CEUS primarily measures muscle perfusion, and it is therefore not surprising that these techniques could produce different results. We previously validated CEUS against a gold standard for perfusion measurement, fluorescent microspheres, to provide support for the contention that CEUS results are truly reflective of muscle perfusion in mouse models of HLI 11. Additionally, CEUS perfusion data demonstrating incomplete recovery is consistent with our histopathology results showing irregular myofibers and centralized nuclei persist for months after HLI. Consistent with these results, abnormal muscle morphology at late time-points after HLI surgery has also been reported by other groups 14,33,34. With the addition of the double ligation model and the ligation/excision model, we have covered the range of severity that is typical of studies employing HLI models. This further extends the impact of our findings as being broadly applicable in more severe models of HLI employing unilateral femoral artery ligation and excision techniques.

One of the advantages to the CEUS strategy used here is that when the transducer is positioned across the calves, the differential perfusion to individual muscle groups can easily be measured. This is especially prominent when imaging 1 day post-surgery. k-means cluster analysis of all day 1 post-surgery images for the three surgeries reveals 5 predominant patterns, most of which occur in multiple surgery conditions. While it is consistent with anatomic variation of the vasculature, it is not widely appreciated that the same surgery can affect different muscle groups in different mice. We hypothesize that this variation is due to anatomic differences in arterial branch points, and perhaps to differential levels of collateralization that enable some mice to recover better levels of muscle perfusion after surgery 15. Other groups have shown that different mouse strains recover from HLI differently based on innate collateralization 25,35,36. It is therefore possible that similar mechanisms operate on a per-mouse basis within the same strain to a lesser extent. Our results also have implications for HLI studies that employ biochemical and immunohistochemical assays that require the sampling of individual muscles, as it is impossible to know whether the sampled muscle was truly ischemic without CEUS (or some other method capable characterizing perfusion on a per-muscle basis). Importantly, the surgery with the least pattern variability was the double ligation model, where about 80% of the mice had the same pattern of perfusion (Cluster 5) characterized by minimal perfusion to any muscle or skin. It follows that the variability inherent in mouse models of HLI can be significantly reduced by applying CEUS in the double ligation model and by excluding the ~20% of mice that retain perfused muscles in the ischemic lower hindlimb at day 1 post-surgery. This strategy would solve the conundrum of sampling error and would ultimately require fewer mice to obtain statistical significance and reproducible results.

As with any study, there are a number of limitations to consider. CEUS-related limitations have previously been elaborated in detail 11, and include imaging in a single plane and the possibility that CEUS itself might have a transient influence on muscle perfusion. These limitations are unlikely to significantly impact the central conclusions of this study, although additional validation studies are certainly warranted to further establish the accuracy and reproducibility of CEUS in mouse models of HLI. For pattern detection and analysis, system resolution limited our ability to identify smaller muscles. While higher resolution would be helpful in identifying these small muscles, the CEUS technique described here nevertheless represents a significant improvement over LDPI with regard to the accurate assessment of hindlimb muscle perfusion. Finally, this study did not establish the underlying mechanism responsible for the different perfusion patterns that were observed. It is reasonable to hypothesize that these are due to innate differences in the positioning of arterial branch points as determined by genetics during embryonic development, and perhaps influenced by differential collateralization after the induction of ischemia 15,25,35,36. However, it is also possible that subtle differences in surgical technique might also contribute to the variable perfusion patterns. The widespread prevalence of PAD, along with the significant morbidity and mortality that it carries, warrants future work in this area to further improve the predictive power of surgically-induced mouse models of PAD.

Conclusions

To our knowledge, this is the first study to use CEUS to directly compare different HLI surgeries. We believe that this is also the first study to demonstrate that different patterns of muscle perfusion can result from different HLI surgical techniques. In conclusion, this study aimed to evaluate two severe models of hindlimb ischemia with CEUS. It showed that, contrary to LDPI, perfusion as measured by CEUS never fully recovers after surgery, even when followed out to 28–150 days post-surgery. It also showed that individual mice can manifest dramatically different patterns of muscle perfusion to the same surgery and that these patterns are conserved within and between different surgical techniques. The surgical technique with the least pattern variability was the double ligation model, which may be the preferred choice for future studies to minimize variability. The results of this study have significant implications for the evaluation of therapeutics to treat PAD, and more research is needed to elucidate the mechanistic basis underlying the variable responses to the different surgical techniques.

Supplementary Material

1

Acknowledgements

The authors thank Jim Patrie, MS, in the University of Virginia Department of Public Health Sciences for his contribution to the statistical analysis of the time course of the recovery data set.

This work was supported by American Heart Association Grant 15PRE25100003 (A.B.B.) and National Institutes of Health/National Heart, Lung and Blood Institute (NIH/NHLBI) Grants R01HL116455 (MPI to B.H.A. and B.A.F.), R01HL150003 (B.H.A), and R01HL148590 (B.H.A).

Footnotes

Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

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Data Availability

The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2018268-supplement-1.docx (16.3KB, docx)

Data Availability Statement

The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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