Chronic Rejection Pathology after Orthotopic Lung Transplantation in Mice: The Development of a Murine BOS Model and Its Drawbacks

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50–75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25–50% of the mice from 4–12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV0.1, TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options

Brain perfusion fixation in male pigs using a safer closed system

Tissue fixation methods are well established for rodents, but not for large animals. We present a simple technique for in situ brain perfusion fixation in a male porcine model, using cervical vessels for inflow and outflow and achieving a closed system. Thirty-four pigs, aged 4.7 ± 0.6 months and weighing 60.7 ± 10.9 kg, were anaesthetised and mechanically ventilated. The ipsilateral common carotid artery and external jugular vein were dissected and constituted the inflow and outflow access, respectively. The brains were perfused and fixed in situ with heparinised saline followed by buffered formaldehyde. Then, specimens (brain, cerebellum and brainstem) were extracted and processed for histology. Fixative fluid leakage was avoided, achieving a closed system. This technique minimises the exposure to toxic chemicals such as formaldehyde and associated hazards (inherent toxicity, eye irritation), thereby increasing operators' safety. Perfusion was performed with a peristaltic pump for 20-30 minutes at an optimum rate of 0.20 l/min and required only 5 litres of the fixative. The specimens were sufficiently hardened to be extracted. High-quality tissues were available for histology analysis. This technique offers a user-friendly closed system for brain perfusion fixation which can be adapted for other tissues of the head, face and neck.status: accepte

Right ventricular support for pulmonary arterial hypertension: an acute feasibility study in sheep

status: accepte

Neutrophilic Reversible Airways Dysfunction After Liver Transplantation: A Case Report

In the present case report we have described a 46-year-old female patient who underwent a liver transplantation in 1998 for polycystic disease and developed a syndrome of increasing dyspnea, with sputum production and a progressive decline in pulmonary function [forced expiratory volume in one second (FEV(1)) (decreased from 153% predicted to 87% predicted). Further examination revealed an impressive tree in a bud pattern with diffuse peribronchiolar infiltrates on computed axial tomographic scan of the thorax. Sputum cultures remained negative. Bronchoscopic central airway biopsy specimens showed lymphocytic bronchitis; sputum induction showed 92% neutrophils. This condition was similar to the bronchiolitis obliterans syndrome after lung transplantation, although the specific neutrophilic phenotype of bronchiolitis obliterans syndrome has recently been renamed as neutrophilic reversible allograft/airway dysfunction, based on a progressive decline in FEV(1), neutrophilic airway inflammation and its response to neomacrolides. Additional azithromycin treatment resulted in complete recovery in our patient, with normalization of FEV(1) and computed axial tomographic scan of the thorax at 3 months after initiation. This case report suggests that neutrophilic reversible allograft airway dysfunction can no longer be diagnosed only after lung transplantation. Moreover, it demonstrates that this condition is not always related to allograft rejection, but rather may be induced by non-immunologic factors, which remain to be further investigated.status: publishe

Skeletal muscle contractile and fatigue properties in smoking mice

status: publishe

Heterogeneity of chronic lung allograft dysfunction: insights from protein expression in broncho alveolar lavage

BACKGROUND: Chronic lung allograft dysfunction (CLAD) remains a major risk factor for death after lung transplantation. Previous data suggested that within CLAD at least 2 phenotypes are present: a neutrophilic type (nCLAD or neutrophilic reversible allograft dysfunction [NRAD]), reversible with azithromycin therapy, vs a low neutrophilic type, non-responsive to azithromycin (fibrotic bronchiolitis obliterans syndrome [fBOS]). We aimed to further characterize this dichotomy by measuring multiple proteins in the bronchoalveolar lavage (BAL) fluid of 28 lung recipients. METHODS: Patients were retrospectively subdivided by the absence or presence of CLAD and subsequently by their response to azithromycin, resulting in 3 groups: 10 stable, 9 responsive (nCLAD/NRAD), and 9 non-responsive (fBOS). Enzyme-linked immunosorbent assay was used to measure 32 different proteins. RESULTS: Protein variations were predominantly present in the nCLAD/NRAD group, whereas no differences were observed in the fBOS group compared with control. MCP-1 (p < 0.01), RANTES (p < 0.05), IL-1β (p < 0.01), IL-8 (p < 0.01), TIMP-1 (p < 0.01), MMP-8 (p < 0.01), MMP-9 (p < 0.01), HGF (p < 0.001), MPO (p < 0.01), and bile acid (p < 0.05) concentrations were upregulated in nCLAD/NRAD compared with fBOS, whereas PDGF-AA (p < 0.05) was downregulated. CONCLUSIONS: These data provide further evidence that within CLAD there is a heterogeneity of phenotypes with different mechanisms involved. Further investigation is warranted to unravel the pathophysiology of both phenotypes.status: publishe

Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research

Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(©) - FlexiVent or Buxco(©) - Forced Pulmonary Maneuvers(®) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.status: publishe

Bronchoalveolar lavage neutrophilia in acute lung allograft rejection and lymphocytic bronchiolitis

BACKGROUND: Acute cellular rejection and lymphocytic bronchiolitis can impair allograft function after lung transplant (LTx). Both may be refractory to corticosteroid treatment. We hypothesized that bronchoalveolar lavage (BAL) neutrophilia may be increased in either acute rejection or lymphocytic bronchiolitis or may increase with increasing histologic severity. METHODS: All consecutive BAL with subsequent transbronchial biopsy (TBB) specimens, performed in 339 LTx recipients from 2001 to 2008, were retrospectively analyzed. TBB specimens were classified according to histologic grade with analysis of BAL total cell count and cell differentials. RESULTS: The analysis included 768 TBB specimens. After adjustment for possible confounders, BAL total cell count significantly increased both with grade A or B severity (p < 0.0001). A higher A grade was characterized by a significant increase in BAL lymphocytosis and neutrophilia (p < 0.0001), whereas for higher B grades, only a more prominent BAL neutrophilia was seen (p < 0.0001). CONCLUSIONS: Higher grade A, but, particularly, higher grade B severity scores are characterized by increased BAL neutrophilia.status: publishe