key: cord-1017358-7ro9qb2m authors: Ochs, Matthias; Timm, Sara; Elezkurtaj, Sefer; Horst, David; Meinhardt, Jenny; Heppner, Frank L.; Weber-Carstens, Steffen; Hocke, Andreas C.; Witzenrath, Martin title: Collapse induration of alveoli is an ultrastructural finding in a COVID-19 patient date: 2021-01-14 journal: Eur Respir J DOI: 10.1183/13993003.04165-2020 sha: 9451c7b45cded0cc5c9cd31115ad090d153eaa95 doc_id: 1017358 cord_uid: 7ro9qb2m Electron microscopy reveals collapse induration with alveolar epithelial cell death, basal lamina denudation, collapse and sealing of alveoli in COVID-19. This indicates surfactant dysfunction and alveolar instability as factors for initiation of fibrosis. Electron microscopy reveals collapse induration with alveolar epithelial cell death, basal lamina denudation, collapse and sealing of alveoli in COVID-19. This indicates surfactant dysfunction and alveolar instability as factors for initiation of fibrosis. The delicate alveolar blood-air barrier is a primary target in Coronavirus disease 2019 (COVID-19). Its micro-architecture consists of an alveolar epithelium composed of type I and type II cells and covered with surfactant, a thin interstitium and a capillary endothelium. Of particular relevance for the pathogenesis of severe COVID-19 is the infection of type II alveolar epithelial cells [1] . Based on their dual function as producers of surfactant and as precursors for both epithelial cell types, surfactant alterations and aberrant epithelial regeneration can be expected. Although there has been some discussion whether the pathophysiology of COVID-19 is a typical form of acute respiratory distress syndrome (ARDS) or a distinct form of lung injury, diffuse alveolar damage (DAD) as the histopathological correlate of acute lung injury and ARDS is a regular finding in COVID-19 [2] [3] [4] . DAD includes an early exudative (or acute) phase with alveolar epithelial injury, edema formation and surfactant alterations, and a late proliferative (or organizing) phase with cuboidal metaplasia of type II alveolar epithelial cells and thickening of inter-alveolar septa, finally resulting either in restoration or in fibrosis [5] . In the context of DAD, the concept of collapse (or atelectatic) induration as an important event in the progression of lung injury towards fibrosis has been emphasized [6; for review and further references, see 7, 8 ]. An initial alveolar epithelial injury with surfactant alterations leads to alveolar instability and collapse, which becomes irreversible due to "glueing" of infolded and denuded alveolar epithelial basal laminae and re-epithelialization over infolded alveoli. Morphological evidence for collapse induration, in particular the tracing of the thin alveolar epithelial basal lamina, requires a resolution, which can only be provided by transmission electron microscopy (TEM) [8] . Here, we report alterations at the TEM level demonstrating collapse induration in postmortem samples from a COVID-19 patient. The 62 years old male patient tested positive for SARS-CoV-2, was admitted to hospital due to newly onset of dyspnea and fever, and intubated one day later. He received catecholamines, antibiotics, renal replacement therapy but no anti-viral or anti-inflammatory therapy. Under lung protective mechanical ventilation (PEEP 16-21 mbar; tidal volume ca. 6 ml/kg bodyweight; PaO 2 /FiO 2 = 120 -150) and intermittent prone positioning, lung infiltrates partially resolved. After 13 days of mechanical ventilation, the patient deceased due to sudden onset septic shock. Autopsy was performed on the legal basis of §1 SRegG BE of the autopsy act of Berlin and §25(4) of the German Infection Protection Act. This study was approved by the Ethics Committee of the Charite (EA 1/144/927 13) and was in compliance with the Declaration of Helsinki. To inactivate potential infectivity of the virus, 2 larger tissue samples from the right lower lobe were fixed with 3% formaldehyde (prepared from freshly depolymerized paraformaldehyde) in 0.1 M cacodylate buffer for 12 h followed by changing the fixative for another 12 h Ultrastructural findings are illustrated in Figure 1 . Alveolar septa were thickened due to the formation of interstitial edema. The alveolar epithelium had an increased coverage by type II cells, indicating hyperplasia as an attempt to repair areas of epithelial injury. In places, type II cells were seen in transition towards a type I cell phenotype. Alveolar epithelial cells were partly swollen or necrotic, with isolated cells or connected cell layers desquamated into the alveolar lumen. As a consequence, the alveolar epithelial basal lamina was to a large extent denuded. In all of the 5 TEM blocks studied, infoldings of denuded basal laminae could be observed. These infoldings bordered collapsed alveolar lumina. At their entrance, these infoldings were partly covered with proliferating alveolar epithelial cells. The post-mortem ultrastructural findings from a COVID-19 patient presented here are similar to previous descriptions in DAD [6] and idiopathic bronchiolitis obliterans-organizing pneumonia [9] as well as to findings in the bleomycin injury model in rats and in explants from patients with idiopathic pulmonary fibrosis that underwent lung transplantation [8, 10] , thus indicating that they are a common reaction pattern to alveolar epithelial injury. The bleomycin model is of particular value in the context of COVID-19 because it induces a fibrotic response following acute lung injury, which can be followed in a pursuable, reproducible and revisable manner [11] . The findings can be interpreted as a sequence of events: Damage of alveolar epithelial cells leads to surfactant dysfunction and alveolar instability with subsequent micro-atelectasis. The denuded basal laminae of collapsed alveoli coalesce, and the entrances into collapsed alveoli are sealed by proliferating alveolar epithelial cells, thus forming thickened septa. The consequences are a loss of alveolar airspace volume and a loss of alveolar gas exchange surface. The ultrastructural alterations found in this post-mortem patient sample provide morphological evidence for a possible transition into lung fibrosis after COVID-19 lung injury. Although the mechanical ventilation of the patient may have contributed to the progression of DAD [12] , acute DAD is also seen in COVID-19 pneumonia independent of mechanical ventilation [2, 4] . Without the development of septic shock this patient might have survived but developed fibrosis. In the bleomycin animal model of lung injury and fibrosis, this transition towards lung fibrosis could be attenuated by exogeneous surfactant therapy [13] . This suggests that restoration of the surfactant system, e.g. by exogeneous surfactant therapy, might be considered as an early therapeutic intervention for COVID-19 patients. A similar concept has also been put forward by others [14, 15] and is in line with the general idea of preventive anti-fibrotic therapy in COVID-19 [11] . 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The expert technical assistance by Petra Schrade (Core Facility for