Visualizing the 3D Architecture of Multiple Erythrocytes Infected with Plasmodium at Nanoscale by Focused Ion Beam-Scanning Electron Microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures

Immunopathological changes in a uraemic rat model for peritoneal dialysis

BACKGROUND: Peritoneal dialysis (PD) is a treatment modality for patients with renal failure. Both the uraemic state of these patients and chronic exposure to PD fluid are associated with the development of functional and structural alterations of the peritoneal membrane. In a well-established chronic PD rat model, we compared rats with normal renal function with subtotal nephrectomized rats that developed uraemia. METHODS: Uraemic and control rats received daily 10 ml conventional glucose containing PD fluid, via peritoneal catheters during a 6 week period. Uraemic and control rats receiving no PD fluid served as controls. Parameters relevant for peritoneal defence and serosal healing responses were analyzed. RESULTS: Uraemic animals were characterized by 2-3-fold increased serum urea and creatinine levels, accompanied by a significantly reduced haematocrit. Uraemia (without PD fluid exposure) induced new blood vessels in different peritoneal tissues, accompanied by increased accumulation of advanced glycation end products (AGEs) and elevated levels of angiogenic factors such as vascular endothelial growth factor and monocyte chemoattractant protein-1 (MCP-1) in peritoneal lavage fluid. A much stronger peritoneal response was observed upon PD fluid exposure in non-uraemic rats. This included the induction of angiogenesis and fibrosis in various peritoneal tissues, accumulation of AGEs, immunological activation of the omentum, damage to the mesothelial cell layer, focal formation of granulation tissues and increased MCP-1 and hyaluronan levels in peritoneal lavage fluid. Finally, chronic PD fluid instillation in uraemic rats did not induce an additional peritoneal response compared to PD fluid exposure in non-uraemic rats, except for the degree of AGE accumulation. CONCLUSIONS: Both uraemia and PD fluid exposure result in pathological alterations of the peritoneum. However, uraemia did not induce major additive effects to PD fluid-induced injury. These results substantially contribute to the understanding of the pathobiology of the peritoneum under PD conditions

Beneficial effects of aminoguanidine on peritoneal microcirculation and tissue remodelling in a rat model of PD

BACKGROUND: The formation of glucose degradation products (GDPs) and accumulation of advanced glycation end products (AGEs) partly contribute to the bioincompatibility of peritoneal dialysis fluids (PDF). Aminoguanidine (AG) scavenges GDPs and prevents the formation of AGEs. METHODS: In a peritoneal dialysis (PD) rat model, we evaluated the effects of the addition of AG to the PDF on microcirculation and morphology of the peritoneum, by intravital microscopy and quantitative morphometric analysis. RESULTS: AG-bicarbonate effectively scavenged different GDPs from PDF. Daily exposure to PDF for 5 weeks resulted in a significant increase in leucocyte rolling in mesenteric venules, which could be reduced for approximately 50% by addition of AG-bicarbonate (P<0.02). Vascular leakage was found in rats treated with PDF/AG-bicarbonate, but not with PDF alone. Evaluation of visceral and parietal peritoneum showed the induction of angiogenesis and fibrosis after PDF instillation. PDF/AG-bicarbonate significantly reduced vessel density in omentum and parietal peritoneum (P<0.04), but not in mesentery. PDF-induced fibrosis was significantly reduced by AG (P<0.02). PDF instillation led to AGE accumulation in mesentery, which was inhibited by supplementation of AG. Since addition of AG-bicarbonate to PDF raised pH from 5.2 to 8.5, a similar experiment was performed with AG-hydrochloride that did not change the fluid acidity. We could reproduce most of the results obtained with AG-bicarbonate; however, AG-hydrochloride induced no microvascular leakage and had a minor effect on angiogenesis. CONCLUSION: The supplementation of either AG reduced a number of PDF-induced alterations in our model, emphasizing the involvement of GDPs and/or AGEs in the PDF-induced peritoneal injury