Immunophenotypic analysis of the chronological events of tissue repair in aortic medial dissections

Highlights

• For medico-legal reasons it can be important to estimate as accurately as possible the consecutive steps of tissue repair in aortic medial dissections.

• Immunohistochemistry of resident vascular wall cells and inflammatory cells is a useful adjunct in grading the time dependent changes in aortic dissections.

• Not only the dissected artery itself, but also adjacent perivascular fat appears to be a site for histopathological grading of repair processes.

• Netosis is a form of cell death which is prominently involved in the repair of medial dissections in a time dependent manner.

Abstract

Acute medial dissection of aorta can occur in the context of a sudden and unexpected death. For medico-legal reasons it is important to estimate as accurately the histological age of dissections. We evaluated the additional value of a systematic application of immunohistochemistry, compared with conventional histology only, in determining chronological steps of injury and repair. Thirty two paraffin embedded specimens of aortic dissection were retrospectively allocated to one of four defined stages: acute (I), subacute (II), early organizing (III) and scarring (IV) using Hematoxylin and Eosin and Elastica van Gieson stained sections. Subsequent immunohistochemically staining was performed with the following markers: (myeloperoxidase (neutrophils), citrullinated-Histone 3 (neutrophil extracellular traps), CD68 (macrophages), CD3 (T-cells), CD31 and CD34 (endothelial cells), and smooth muscle actin. Immune stained sections were scored semi-quantitatively. Histologically, five cases were identified as stage I, 16 as II, 7 as III and 4 as IV. Additional immunostaining for smooth muscle cells and endothelial cells altered the classification in 25% of cases (all in groups II and III). Immunostaining and semi-quantitative grading of involvement of neutrophils, macrophages and NETs also provided specific distribution patterns over the 4 age categories, including unexpected involvement of the peri adventitial fat tissue. In conclusion, it appears that semi-quantitative immunohistochemistry of resident vascular wall cells, inflammatory cells and NETS represents a useful adjunct in detailed histopathological grading of the chronological age of aortic dissections.

Introduction

Acute aortic dissection is a life-threatening condition, whose global death rate is still increasing [1]. The classical clinical presentation of aortic dissection is a sudden onset severe back pain, but symptoms can vary greatly, sometimes being mild or even clinically silent until the moment of collapse due to hemorrhage. In other instances, the clinical presentation of aortic dissection can mimic an acute coronary syndrome, leading to delayed diagnosis and inappropriate antithrombotic treatment [2], [3].

Most recent research efforts have focused on the pathological conditions underlying the aortic dissection, such as atherosclerosis, chronic auto-immune aortitis or cystic medial degeneration [4]. However, despite the currently increasing interest and evolving insights in underlying pathologies, data on the histopathological features of the dissection process itself are less well documented and rely mostly on histomorphology. Yet, and especially in forensic context, it can be important to evaluate not only the underlying pathological substrate, but also the time interval that elapsed between initiation of a dissection and death. Usually, an estimation of the age of a dissection is based on the histomorphological features of tissue injury and subsequent repair in the hematoma and adjacent aortic wall, which change markedly over time.

Xu et al. reported four distinct histologically defined stages in the healing process of a dissection, which were based on correlations with the clinically documented time of onset of symptoms [5]. They showed that during the process of tissue repair of a dissecting hematoma, numbers of different sets of inflammatory cells initially increase and subsequently decline, not only in the hematoma itself, but also in adjacent media and adventitia. In coronary thrombectomy specimens that were obtained from patients with acute myocardial infarction during life, a similar histological pattern of chronologic repair of the clot has been described [6], [7]. In all these studies, especially neutrophilic granulocytes appeared to play a principal role in the remodeling of thrombus over time.

Upon activation, neutrophils can be initiated to undergo netosis, a specific form of cell death [8]. During netosis, thread-like extensions of chromatin are released by neutrophils, which are covered with the contents of their cytoplasmic granules [8]. These neutrophil extracellular traps (NETs) play a regulatory role in many different pathological processes, such as autoimmune diseases, infection, and thrombosis [9], [10]. In case of thrombosis, neutrophils are considered to modulate thrombus stability by forming a scaffold for fibrin fibers and pro- inflammatory proteins along their extending NETs [11]. In the coronary thrombi of patients with acute myocardial infarction, occurrence of netosis has shown to be related to the histological age of a thrombus, with significantly larger amounts of NETs being detected in lytic (older) thrombi compared to fresh thrombi or the very old fibrocellularly organized thrombi [12]. Therefore, we hypothesize that not only neutrophils but also generated NETs may provide additional information about the histological age of dissecting hematomas.

The aim of this study is to provide a detailed immunophenotypical profile of the subsequent episodes of repair of acute aortic dissections, superimposed on previously described histopathological features of clot aging. For this purpose, a major emphasis was put on the presence and extent of formation of NETS, in combination with markers for fibrocellular organization and angiogenesis.