Ultrastructural changes in acute lung allograft rejection: novel insights from an animal study.

BACKGROUND: Acute rejection (AR) episodes after lung transplantation (Tx) are orchestrated by cells of the innate and adaptive immune system targeting the engrafted organ. The assessment and classification of pathologic changes of AR relies essentially on conventional histology. Herein we apply the technique of scanning electron microscopy (SEM) to identify and characterize ultrastructural changes of the pulmonary graft after lung Tx. METHODS: Orthotopic single-lung Tx was performed between BALB/c (donor) and C57BL/6 (recipient) mice. At Day 5 after Tx, lung allografts were recovered for SEM and for histologic analysis. RESULTS: Upon Tx, high numbers of leukocytes and thrombocytes were found, showing an activated surface pattern and a change of their cell body shape. These cells adhered and partly transmigrated through the endothelium of vessels. Larger vessels were more affected than smaller vessels and the endothelium was roughened in its surface texture throughout. As a phenomenon, airways were partly covered by activated dendritic cells. Numerous thrombocytes and macrophages accumulated on the endothelium of the cuff anastomosis region exposing this area to a particularly higher risk of thrombosis. CONCLUSIONS: SEM allows for detection of morphologic changes during pulmonary allograft rejection and adds important data to conventional histology when making the diagnosis of acute rejection.

Ischaemia-reperfusion injury in orthotopic mouse lung transplants - a scanning electron microscopy study.

Lung ischaemia-reperfusion (I/R) injury remains a major cause of graft failure in lung transplantation (Tx). With the implementation of orthotopic lung Tx in mice, a physiological model on the base of a perfused and ventilated graft became available for the investigation of I/R injury. Using the scanning electron microscopy (SEM) technique, we here present an analysis of early and late morphological changes of pulmonary I/R injury. Syngeneic lungs were orthotopically transplanted between C57BL/6 mice. Grafts were exposed to 2 h of cold ischaemia. Transplants and right lungs were examined by SEM with corresponding haematoxylin-eosin histology 30 min and 4 h after reperfusion. Thirty minutes after reperfusion, the alveolar surface of transplants showed a discontinued lining of surfactant, while the lining of the non-transplanted lung was normal. Within the graft, leucocytes displayed an irregular surface with development of pseudopodia, and microvilli were detected on the membrane of pneumocytes. At 4 h after reperfusion, leucocytes significantly increased in numbers within the alveolar space. Also, the number of microvilli on pneumocytes increased significantly. Similar to these, the endothelium of vessels increasingly developed microvilli from 30 min towards 4 h after reperfusion. The airways of transplanted grafts showed mild changes with thickening of the bronchial epithelium and a destruction of kinocilia. Taken together, SEM detects pathological events of I/R that are previously not described in normal histology. These findings may influence the interpretation of studies investigating the I/R injury in the mouse model of lung Tx.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model.

OBJECTIVE: The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. STUDY DESIGN: Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. RESULTS: Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). CONCLUSION: Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

Prevention of bacterial leakage through instrumented root canals by bioactive glass S53P4 and calcium hydroxide suspensions in vitro.

OBJECTIVE: The capacities of bioactive glass S53P4 and calcium hydroxide suspensions to prevent microbial leakage through instrumented root canals were compared. STUDY DESIGN: Root canals of caries-free extracted human mandibular premolars were instrumented and randomly filled with calcium hydroxide or S53P4 (n = 14 each). Crowns were exposed to Enterococcus faecalis ATCC 29212 suspensions in a test setup with 2 broth chambers, and leakage was checked daily. Event times were compared using log rank test. After 50 days, teeth were fractured and inspected in a scanning electron microscope (SEM). RESULTS: Calcium hydroxide prevented leakage significantly better than S53P4 (P < .05). Estimated mean days to leakage were 26 for calcium hydroxide and 4 days for S53P4. The SEM observations revealed substantial calcification of the glass material in the root canal but also gaps containing clusters of coccoidal bacteria. CONCLUSION: The bioactive glass material under investigation could not prevent contamination of instrumented root canals.