A novel histological occlusion classification for coiled aneurysms based on multiphoton microscopy

Abstract

ObjectiveIntracranial aneurysm (IA) coiling remains the most commonly used endovascular approach for ruptured and unruptured IA, and recanalization is a common drawback that impairs treatment success. Angiographic occlusion and aneurysm healing are not synonymous, and histological evaluation of embolized aneurysms remains a challenge. We propose here an experimental study of coil embolization in animal models by multiphoton microscopy (MPM) in comparison with conventional histological staining. The purpose of his work is to analyze coil healing process using histological sections of aneurysms.MethodsBased on a rabbit elastase model, 27 aneurysms were fixed, embedded in resin, and cut in thin histological sections 1 month after coils implantation and after angiographic control. Hematoxylin and eosin (H&S) staining were realized. Non-stained adjacent slices were imaged for multiphoton excited autofluorescence (AF) and second-harmonic generation (SHG) to construct three-dimensional (3D) projections of sequentially and axially acquired images.ResultsThe contrast provided by the combination of these two imaging modalities can be used to distinguish five levels of aneurysm healing, based on a combination of thrombus evolution and increased extracellular matrix (ECM) deposit.ConclusionRDPC:\Users\SHAHUL\RDP6|We have established a novel histological scale from a rabbit elastase aneurysm model after coiling with a classification of five different stages thanks to nonlinear microscopy. This classification is an actualized tool in order to obtain a more precise evaluation of occlusion device efficacy in the scope of new innovative microscopy for research.

Keywords: Aneurysm; classification; healing; histology; multiphoton microscopy.

Figure 1.

Adjacent sections from normal arterial wall (common carotid artery) is shown as reference for healthy vascular tissue with H&S labeling in bright field microscopy (a and c) and with no labeling in multiphoton microscopy (MPM) (b and d). The elastin, when present, is clearly distinguishable by its appearance of anastomosed ribs and its strong fluorescence in red in MPM. Fibrous structures of both type I and type III collagen are major constituents of the intima, media, and adventitia but the SHG signal in green is due to type I collagen mainly (in b and d). Scale bar in a and c = 100 µm, respectively for a and b and c and d.

Figure 2.

Bare metal coil (a, a’) and bioenhanced coil (b, b’) under multiphoton microscopy (MPM). Bare metal coils in a and a’ generate a signal in the red channel despite the metallic platinum nature (the latter is not supposed to be fluorescent). We assume that the emission in the red is due to the reflection of the femtosecond infrared (IR) laser, whose peak power is extremely high, and this probably allows a bundle to reach the detector despite the presence of dichroic mirrors < 650 nm. What may seem an artifact turns out to be useful as it provides an optical signature of the coils in the histological sections. An example of bioenhanced functionalized polysaccharides coils (in b and b’) still generates laser reflections toward red channel, but also SHG (green). We assume that acrylic coating of the coils is responsible for this signal SHG. Scale bar in a and b = 125 µm, a’ = 167 µm, arrow in b’ = 200 µm.

Figure 3.

Representation of histological section plane of coiled aneurysm (a). In b, the full height of an aneurysm can be visualized in the same section from the narrow neck to the bottom after H&S labeling. “c” represents the holes where coils have been sometimes unintentionally removed during tissue sectioning. Due to the heterogeneity of healing stages within the same aneurysm, our observations were conducted in three distinct areas of the aneurysm (c, d, and e). For each aneurysm, a distal area was defined corresponding to a deeper zone (c), an intermediate area (d), and an area proximal to the neck (e). Because SHG microscopy (in white in c, d, and e) is highly specific for fibrillar collagens (especially type I), we can easily observe the fibrosis in wounding and repair of the aneurysm: density of the fibers, orientation, and thickness in these three areas. Scale bar in b = 500 µm, in c, d, and e = 50 µm.

Figure 4.

A new histological classification for aneurysm healing is validated based on H&S histological section compared with multiphoton microscopy (MPM). Stage I: Erythrocytes (Er) are transparent in H&S and slightly generating SHG; Stage II: Platelet aggregations appear red autofluorescence (AF) with erythrocytes still present, but without any signal of SHG; Collagen fibers appear in green on SHG in stages III, IV, and V; Cells are red AF in stages IV and V. Scale bars indicated in a are applied to a and b and scale bars under the circle at left are applied to both circles magnified images.