A Comprehensive Review on the Surgical Aspect of Lung Transplant Models in Mice and Rats

Lung transplantation improves the outcome and quality of life of patients with end-stage pulmonary disease. However, the procedure is still hampered by the lack of suitable donors, the complexity of the surgery, and the risk of developing chronic lung allograft dysfunction. Over the past decades, translational experiments in animal models have led to a better understanding of physiology and immunopathology following the lung transplant procedure. Small animal models (e.g., rats and mice) are mostly used in experiments regarding immunology and pathobiology and are preferred over large animal models due to the ethical aspects, the cost-benefit balance, and the high throughput possibility. In this comprehensive review, we summarize the reported surgical techniques for lung transplantation in rodent models and the management of perioperative complications. Furthermore, we propose a guide to help identify the appropriate species for a given experiment and discuss recent experimental findings in small animal lung transplant models

Myeloid-Derived Suppressor Cells in Lung Transplantation

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immune cells from the myeloid lineage. MDSCs expand in pathological situations, such as chronic infection, cancer, autoimmunity, and allograft rejection. As chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation (LTx), MDSCs may play a role in its pathophysiology. We assessed phenotype and frequency of MDSCs in peripheral blood from lung transplant recipients and its relationship to post-transplant complications and immunosuppression. Granulocytic (G)-MDSC were identified and quantified by flow cytometry of blood from 4 control subjects and 20 lung transplant patients (stable n = 6, infection n = 5; CLAD n = 9). G-MDSC functionality was assessed in vitro by their capability to block CD4 and CD8 T cell proliferation. More G-MDSC could be assessed using EDTA tubes compared to heparin tubes (p = 0.004). G-MDSC were increased in stable lung transplant recipients vs. non-transplant controls (52.1% vs. 9.4%; p = 0.0095). The infection or CLAD groups had lower G-MDSCs vs. stable recipients (28.2%p = 0.041 and 33.0%; p = 0.088, respectively), but were not different among CLAD phenotypes. G-MDSC tended to correlate with cyclosporine A and tacrolimus levels (r2 = 0.18; r2 = 0.17). CD4 and CD8 cells proliferation decreased by 50 and 80% if co-cultured with MDSCs (1:6 and 1:2 MDSC:T-cell ratio, respectively). In conclusion, circulating MDSCs are measurable, functional and have a G-MDSC phenotype in lung transplant patients. Their frequency is increased in stable patients, decreased during post-transplant complications, and related to level of immunosuppression. This study may pave the way for further investigations of MDSC in the context of lung transplantation

Connective Tissue Growth Factor Is Overexpressed in Explant Lung Tissue and Broncho-Alveolar Lavage in Transplant-Related Pulmonary Fibrosis

Background: Connective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD). Materials and Methods: CTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients. Results: qPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74). Conclusions: Lung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD

Real-time investigation of Lysozyme crystallization kinetics: A neutron diffraction study

The isothermal crystallization kinetics of hen-egg-white Lysozyme has been investigatedby means of a time-resolved neutron diffraction experiment for almost 3 days,starting from a supersaturated solution of Lysozyme (30 mg/ml, 3 wt % NaCl, pD 4.75at 298 K) until the growth of crystals, in order to have complementary informationabout the crystallization kinetics. The temporal evolution of the intensity of the Braggpeaks, observed in the neutron diffraction images when a single crystal appears, hasbeen studied. Simultaneously, the analysis of the small angle neutron scattering curvesfrom the Lysozyme solution, during the crystallization process, has been performed.A correlated behaviour between the decrease of the Lysozyme concentration and theincrease of the crystallization fraction was observed. The crystallization kinetics wasdescribed by means of the Johnson-Mehl-Avrami-Kolmogorov model and parameterscompatible with our previous Lysozyme crystallization study have been found.The Lysozyme crystallization under constant neutron flux underlines the known strengthof neutrons in studying biological samples without causing radiation damage

Change of Fractal Dimension during the early stages of Lysozyme Crystallization

In this study we focussed on the question of how to grow crystals as large as possible in light of their use as samples for neutron protein crystallography. We investigated the early stages of the crystallisation process where the directions are set between the growth of many small crystals or few large ones. We used lysozyme since it is considered as a model system for crystal growth. Small angle neutron scattering was used in combination with static light scattering in order to realize an extended q-range. In situ dynamic light scattering at the neutron scattering sample cell was used to obtain an overview of all sizes present in the crystallisation process. We could observe a fractal growth of the crystal seeds with a change in the fractal dimension from 1.0 to 1.7 in the first 90 min. This can be interpreted that at first a branched crystal seed is formed which grows more in a linearly. Later, the space between the arms is filled to cross over to a more densely packed fractal

In-situ light scattering at neutron beam lines - experiences made and challenges ahead

What is often well established at many synchrotron beam lines is still in the development phase at neutron beam lines: In-situ light scattering techniques. The science case for in-situ light scattering at neutron instruments lies mostly in the limited reproducibility of sample preparation and stability of the samples over time. Whereas many soft matter or hard matter samples are not transparent for light, many biological samples often show a sufficiently broad spectral range where light absorption does not play a dominant role. Natural candidates for neutron instruments to be equipped with in-situ light scattering techniques are small angle scattering, spin echo, time-of-flight and backscattering beam lines. We routinely supply in-situ dynamic light scattering with one fixed scattering angle at the instrument KWS-2 at MLZ to users who would like to control their sample during the neutron measurement. Recently, we have successfully tested a three angle dynamic light scattering set-up at KWS-2. For the Jülich neutron spin echo spectrometer we are currently developing a prototype sample environment which includes two scattering angles and a transmission detector. The transmission detector reports on a change in turbidity with a very high time resolution. This is especially attractive to thermoresponsive soft matter samples with a very narrow transition from a swollen to a more compact micellar state

In-situ light scattering at neutron beam lines - experiences made and challenges ahead

What is often well established at many synchrotron beam lines is still in the development phase at neutron beam lines: In-situ light scattering techniques. The science case for in-situ light scattering at neutron instruments lies mostly in the limited reproducibility of sample preparation and stability of the samples over time. Whereas many soft matter or hard matter samples are not transparent for light, many biological samples often show a sufficiently broad spectral range where light absorption does not play a dominant role. Natural candidates for neutron instruments to be equipped with in-situ light scattering techniques are small angle scattering, spin echo, time-of-flight and backscattering beam lines. We routinely supply in-situ dynamic light scattering with one fixed scattering angle at the instrument KWS-2 at MLZ to users who would like to control their sample during the neutron measurement. Recently, we have successfully tested a three angle dynamic light scattering set-up at KWS-2. For the Jülich neutron spin echo spectrometer we are currently developing a prototype sample environment which includes two scattering angles and a transmission detector. The transmission detector reports on a change in turbidity with a very high time resolution. This is especially attractive to thermoresponsive soft matter samples with a very narrow transition from a swollen to a more compact micellar state

Local nebulization of 1α,25(OH) 2 D 3 attenuates LPS-induced acute lung inflammation

peer reviewedBackground: Evidence supports a critical role of vitamin D status on exacerbation in chronic obstructive pulmonary disease, indicating the need to avoid vitamin D deficiency in these patients. However, oral vitamin D supplementation is limited by the potential risk for hypercalcemia. In this study, we investigated if local delivery of vitamin D to the lungs improves vitamin D-mediated anti-inflammatory action in response to acute inflammation without inducing hypercalcemia. Methods: We studied vitamin D sufficient (VDS) or deficient (VDD) mice in whom 1α,25(OH) 2 D 3 (0.2 μg/kg) or a vehicle followed by lipopolysaccharide (LPS 25 µg) were delivered to the lung as a micro-spray. Results: Local 1α,25(OH) 2 D 3 reduced LPS-induced inflammatory cells in bronchoalveolar lavage (BAL) in VDS (absolute number of cells: − 57% and neutrophils − 51% p < 0.01) and tended to diminish LPS-increased CXCL5 BAL levels in VDS (− 40%, p = 0.05) while it had no effect on CXCL1 and CXCL2 in BAL and mRNA in lung of VDS and VDD. It also significantly attenuated the increased IL-13 in BAL and lung, especially in VDD mice (− 41 and − 75%, respectively). mRNA expression of Claudin-18 in lung was significantly lower in VDS mice with local 1α,25(OH) 2 D 3 while Claudin-3,-5 and-8 mRNA levels remained unchanged. Finally, in VDD mice only, LPS reduced lung mRNA expression of adhesion junction Zona-occludens-1, in addition to increasing uric acid and total protein in BAL, which both were prevented by local 1α,25(OH) 2 D 3. Conclusion: Under normal levels of vitamin D, local 1α,25(OH) 2 D 3 nebulization into the lung efficiently reduced LPS induction of inflammatory cells in BAL and slightly attenuated LPS-increase in CXCL5. In case of severe vitamin D deficiency, although local 1α,25(OH) 2 D 3 nebulization failed to significantly minimize cellular inflammation in BAL at this dose, it prevented epithelial barrier leakage and damage in lung. Additional research is needed to determine the potential long-term beneficial effects of local 1α,25(OH)2D3 nebulization on lung inflammation