Histologic and Biomolecular Similarities in Healing between Aneurysms and Cutaneous Skin Wounds

SUMMARY: The poorly understood mechanisms of aneurysm healing contribute substantially to the pressing medical problem of coiled aneurysm recanalization. Using an established saccular aneurysm model, we developed an animal model system in rabbits to study aneurysm and skin wound healing concurrently in the same animal. We found treated aneurysm healing to be similar to skin wound healing both histologically and in biomarker gene and protein expression, but in a delayed fashion.

D espite wide use and considerable safety of endovascular coiling of intracranial aneurysms, the recurrence rate is high even with second-generation coils, 1 partially due to a dearth of knowledge about healing mechanisms involved. 2 Conversely, cutaneous wound healing is well-established. The 4 distinct-yet-overlapping stages of cutaneous wound healing (hemostasis, inflammation, proliferation, and remodeling) have been thoroughly described. 3 Here we demonstrate that concurrent study, in the same research subjects, of endovascular-versus-cutaneous healing provides insight into deficiencies in the cellular, molecular, and humoral features in aneurysm healing, enabling elucidation of potential targets for improved outcomes.

Aneurysm Creation and Coil Embolization
All procedures were approved by the Institutional Animal Care and Use Committee before study initiation. Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery in 12 female New Zealand white rabbits. 4 At least 3 weeks later, aneurysms were treated with platinum coils. DSA was performed after embolization.

Cutaneous Ear Wound Creation
Immediately after aneurysm treatment, an ear skin defect was created on the ventral side of 1 ear using a 1-cm stainless steel punch, 5 with removal of skin down to the cartilage. Hemostasis was achieved with pressure, followed by occlusive dressing (TegaDerm; 3M, St. Paul, Minnesota). Ear wounds were assessed visually daily for healing and wound closure rate.

Follow-Up Imaging and Tissue Harvest
At days 1, 5, 14, and 30 (groups 1-4) following treatment, DSA was performed from a left femoral approach, and the animals were euthanized. The aneurysm, ear wound, and contralateral control tissues were harvested for histology or cut in 2 for biomolecular studies.

Angiographic Analysis
Aneurysm occlusions at follow-up were rated either stable or progressive, or recanalizated compared with posttreatment angiograms using the modified Raymond scale. 6

Histologic Analysis
Formalin-fixed aneurysms were evaluated for the degree of neck tissue coverage. 7 Following a modified histologic technique, aneurysms and ear wounds were sectioned and stained with hematoxylin-eosin (H&E) or Masson Trichrome for collagen. A semiquantitative scoring for thrombus organization, neoepithelialization, inflammation, cellularity, and collagen deposition was performed by a blinded reviewer. In addition, an ordinal grading system was used to evaluate histologic healing of aneurysms between groups.

RNA Extraction and Expression Analysis by Real-Time Polymerase Chain Reaction
Total RNA was isolated and converted to complementary DNA followed by real-time polymerase chain reaction with SYBR greenER (www.thermofisher.com). Target gene expression (Table) was normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and reported as a fold change relative to control tissues (contralateral tissues) using the 2 -⌬⌬Ct method.

Protein Extraction and Analysis by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blot
Soluble proteins were extracted from tissue samples with RIPA buffer (https://www.thermofisher.com/order/catalog/product/ 89900). Protein separation and western analysis were performed using a Wes system (ProteinSimple, San Jose, California). Protein expression data were normalized to control proteins (GAPDH or ␤-actin) using Compass Software (http://www.compasssoftware.de/en-us) and reported as fold change relative to control tissues.

Statistical Analysis
Continuous variables were compared using a Student t test, and categoric variables were compared using the Fisher exact test.

Histology
Histologic analysis revealed necrosis and inflammatory cell infiltration in both epidermal and dermal layers of ear wounds at both 24 hours and 5 days, whereas scab formation in the epidermis and diffuse, dense cellular proliferation of myofibroblasts within the dermal layer with minimal inflammatory cells was found by 14 days. Aneurysm features were consistent with previous findings, 8 with sparse myofibroblasts at the periphery of the aneurysm dome by 14 days, increasing dramatically by 30 days (Fig 1D, -F).

Biomolecules
Inflammatory marker gene expression was elevated at 5 days in ear wounds but reduced/delayed until 30 days in aneurysms (Fig  2A, -B). Protein expression, however, was only 2-fold for transforming growth factor ␤1 (TGF␤1) at 24 hours and 30 days in ear wounds (data not shown). Proliferation marker expressions in aneurysms were higher compared with ear wounds (Fig 2C, -D) and with inflammatory markers, overall. ␣ smoothmuscle actin (SMA1) remodeling marker expressions were relatively unremarkable, with a small increase at 14 days in ear wounds (Fig 2E). Macrophage analysis showed an overall M1 polarization, with peak CD86 levels at 24 hours in aneurysms and 5 days in ear wounds (data not shown).

DISCUSSION
Our results reveal similarities and differences in wound-healing mechanisms for these 2 disparate environments. Histologically, skin wounds at 2 weeks resemble aneurysms at 1 month following platinum coil treatment, 8 suggesting a roughly 2-week healing delay in coiled aneurysms relative to skin wounds. Previous studies 8 showed similar findings, supporting this issue of deficient immune cell infiltration and immunedriven healing mechanisms in platinum-coiled aneurysms. 2 The biomolecular profiles reflect this healing delay and confirm an M1 polarization in the macrophage profile, indicating a deficient prohealing phenotype. 9

CONCLUSIONS
This novel animal model enabling longitudinal study of platinum-coiled aneurysm healing in parallel with cutaneous wound healing provides an exceptional level of control and will help determine key signaling events necessary for proper arterial wound healing, promising development of improved treatments.