Systematic Interrogation of Angiogenesis in the Ischemic Mouse Hind Limb: Vulnerabilities and Quality Assurance

Abstract

Objective: There has been little success in translating preclinical studies of mouse hind limb ischemia into benefit for patients with peripheral artery disease. Using systematic strategies, we sought to define the injury and angiogenesis landscapes in mice subjected to hind limb ischemia and ascertain whether published studies to date have used an analysis strategy concordant with these data. Approach and Results: Maps of ischemic injury were generated from 22 different hind limb muscles and 33 muscle territories in 12-week-old C57BL/6 mice, based on loss or centralization of myofiber nuclei. Angiogenesis was similarly mapped based on CD (cluster of differentiation) 31-positive capillary content. Only 10 of 33 muscle territories displayed consistent muscle injury, with the distal anterior hind limb muscles most reliably injured. Angiogenesis was patchy and exclusively associated with zones of regenerated muscle (central nuclei). Angiogenesis was not observed in normal appearing muscle, necrotic muscle, or injury border zones. Systematic review of mouse hind limb angiogenesis studies identified 5147 unique publications, of which 509 met eligibility criteria for analysis. Only 7% of these analyzed manuscripts evaluated angiogenesis in distal anterior hind limb muscles and only 15% consistently examined for angiogenesis in zones of muscle regeneration.

Conclusions: In 12-week C57BL/6 mice, angiogenesis postfemoral artery excision proceeds exclusively in zones of muscle regeneration. Only a minority of studies to date have analyzed angiogenesis in regions of demonstrably regenerating muscle or in high-likelihood territories. Quality assurance standards, informed by the atlas and mapping data herein, could augment data reliability and potentially help translate mouse hind limb ischemia studies to patient care.

Keywords: animals; femoral artery; ischemia; lower extremity; muscle, skeletal.

Figure 1.

Atlas of the hind limb skeletal muscles of C57BL/6J mice subjected to femoral artery excision. A–C, Maps depicting the skeletal muscles in the proximal (A), mid- (B), and distal mouse hind limb (C). Adjacent to each map is a corresponding hematoxylin and eosin–stained full cross section of the hind limb, 10 d after femoral artery excision. Varying intensities of muscle eosinophilia can be seen; less intense staining is present in territories of ischemic injury. D, High-magnification images, corresponding to the outlined zones within the proximal and distal hind limb, depicting normal (left) and injured (right) regions of the same muscle sections. The uninjured regions have peripheral myofiber nuclei (arrowheads). The injured/regenerating regions have pale myofibers with centralized nuclei (arrowheads).

Figure 2.

Hind limb injury maps for C57BL/6 mice subjected to femoral artery excision. A, Maps depicting the sites of muscle injury in proximal (left), mid- (middle), and distal (right) hind limb of C57BL/6J mice, 10 d after femoral artery excision. Areas of injury/regeneration are shaded yellow or green, with the respective consistency of injury denoted by the shade. Dark green zones are those with the most consistent injury/regeneration. Areas of injury/necrosis are shaded orange. Map data are based on n=9 mice. B, Maps depicting the sites and consistency of muscle injury in C57BL/6J mice, 28 d after femoral artery excision. Map data are based on n=5 mice. C, Maps depicting the sites and consistency of muscle injury, 10 d after femoral artery excision in the C57BL/6N substrain (Charles River). Map data are based on n=5 mice. D, Graph depicting the extent of muscle injury in each of the proximal, mid, and distal hind limb for the different cohorts of mice analyzed. The proportion of injured distal hind limb muscles is greater than that for the proximal or mid-hind limb (P<0.001) but, for a given hind limb zone, there are no differences among the mouse substrains or time of assessment post-injury (P=0.648). Data are mean±SEM. See Tables I through III in the Data Supplement for individual data values.

Figure 3.

Hind limb angiogenesis maps for C57BL/6 mice subjected to femoral artery excision. A, Maps depicting the sites of angiogenesis and capillary loss in proximal (left), mid- (middle), and distal (right) hind limb of C57BL/6J mice, 10 d after femoral artery excision. Angiogenesis and capillary loss were determined based on a statistically significant increase or decrease in capillary density, relative to the corresponding region in the contralateral control muscle, as determined by immunostaining for CD (cluster of differentiation) 31. Injured/regenerating and injured/necrotic zones are overlaid in dashed lines. Dashed lines surrounding unshaded areas correspond to territories of muscle injury in which a statistically significant increase in capillary density was not found. Map data are based on n=9 mice. B, Maps depicting the sites of angiogenesis and capillary loss in the hind limb in C57BL/6J mice, 28 d after femoral artery excision. n=5 mice. C, Maps depicting the sites of angiogenesis and capillary loss in the hind limb in C57BL/6N mice, 10 d after femoral artery excision. Map data are based on n=5 mice. D, Graph of capillary densities in histologically defined muscle zones for the different cohorts of mice analyzed (mean±SEM).

Figure 4.

Angiogenesis following femoral artery excision occurs exclusively in injured/regenerating (Regen) muscle zones. A, Contingency table relating the presence of angiogenesis with histologically defined muscle zones. Data from a total of 87 zones from 9 C57BL/6J mice subjected to femoral artery excision and harvested 10 d later are depicted. P<0.0001. B, Micrographs of gastrocnemius muscle sections from a C57BL/6J mouse subjected to hind limb ischemia and harvested 10 d later, depicting the transition from injured to noninjured muscle. Top, Capillary content, based on immunostaining for CD (cluster of differentiation) 31 (green) with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). Bottom, Near-adjacent section stained with hematoxylin and eosin, illustrating the abrupt transition from centralized to peripheral myofiber nuclei. C, Capillary densities in defined muscle zones of the injured gastrocnemius muscle. *P<0.0001. D, Fluorescence micrographs of regions in the gastrocnemius muscle 10 d after surgery, immunostained for CD31 (green) and Ki-67 (pink). Nuclei are visualized with DAPI. Bord indicates border zone; Cont, control; and Uninj, uninjured zone.

Figure 5.

Systematic literature search strategy and manuscript yield. Flowchart indicating Medical Subject Headings search terms, exclusions, and resulting yield of studies included for analysis. WOS indicates Web of Science.

Figure 6.

Methodological metrics in published literature evaluating angiogenesis following hind limb ischemia. A, Graph showing the distribution of manuscripts based on the sex of mice used for angiogenesis assessment. B, Graph showing the distribution of manuscripts based on the vendor from which mice were procured. C, Distribution of manuscripts according to the time points at which angiogenesis analysis following femoral artery excision was undertaken.

Figure 7.

Breakdown of muscles analyzed in the published literature evaluating postischemia angiogenesis. A, Graph showing the prevalence of the specific muscles used for histological angiogenesis evaluation and quantitation, among all manuscripts analyzed (n=509 manuscripts). B, Graph showing the prevalence of the specific muscles used for histological angiogenesis evaluation, after author-level adjustment to account for multiple manuscripts from the same research group (n=283 manuscripts). EDL indicates extensor digitorum longus.

Figure 8.

Analysis of injured muscle zones depicted in the published literature evaluating postischemia angiogenesis. A, Pie charts showing the distribution of manuscripts based on the state of the skeletal myofibers evaluated for postinjury angiogenesis, as depicted in the representative histology images. Chart on the left is for all 509 manuscripts; chart on the right is for the 283 unique senior author manuscripts. B, Depiction of the probability of there being centralized nuclei in histological images of tissues from mice subjected to femoral artery excision, depending on the specific muscles analyzed. Data on right indicate the odds ratio (OR) and 95% CIs for there being central nuclei present in the images.