Antiepileptic drugs lamotrigine and valproate differentially affect neuronal maturation in the developing chick embryo, yet with PAX6 as a potential common mediator.

Exposing the immature nervous system to specific antiepileptic drugs (AEDs) during pregnancy is linked to neurodevelopmental disorders such as autism spectrum disorder (ASD). Newer AEDs like lamotrigine (LTG) are hailed as safer, but recent epidemiological data suggest that even LTG carries a risk, although much lower than that associated with valproic acid (VPA), an older AED, which is also known to cause morphological alterations in the developing brain. Increasing evidence highlights cerebellar abnormalities as important in ASD pathophysiology. Transcription factor PAX6 is a key activity-dependent mediator and regulates crucial processes during cerebellar development. The chicken cerebellum recapitulates important characteristics of human cerebellar development, and may thus be suitable for the assessment of interventions aiming to modify maturation and cerebellar development. In the present study, exposure of chicken on embryonic day 16 (E16) to LTG or VPA resulted in decreased cerebellar mass and level of proliferating nuclear cell antigen (PCNA) for clinically relevant concentrations of VPA. However, both AEDs reduced cerebellar protein levels of PAX6 and MMP-9 at E17. Furthermore, PAX6 immunohistochemical staining of coronal sections of chicken cerebellum showed a significant reduction in PAX6-positive cell density and changes in cerebellar cortex thickness, mostly caused by the change in IGL-layer thickness. In conclusion, prenatal exposure to LTG or VPA provoked differential maturational changes in the developing cerebellum that may reflect some of the underlying molecular mechanisms for the observed human ASD pathology after AEDs exposure during pregnancy.

A woman in her sixties with a knee injury after a fall. / En kvinne i 60-årene med kneskade etter fall.

BACKGROUND: The diagnosis of necrotising soft tissue infections (NSTIs, necrotising fasciitis, myositis and cellulitis) may be clinically challenging, and can result in fatal outcomes. CASE PRESENTATION: A previously healthy woman in her sixties fell and cut her right patella. The wound was complicated by localised infection, which subsequently developed into a bacterial bursitis. She responded to intravenous antibiotics and was followed up at the outpatient clinic. Nineteen days later she was admitted with the same symptoms and clinical presentation as at the previous admission. She was started on the same antibiotics based on the last prepatellar bursal fluid culture. This time, however, she became systemically impaired and septic. Many differential diagnoses were suspected, and she was repeatedly examined with the aid of blood samples, blood cultures, knee joint and prepatellar bursal punctures, and ultrasound scans. The patient's right lower extremity became swollen and was further examined with a CT scan, giving rise to suspicion of an NSTI. Ultimately four surgical revisions were performed (fasciotomy) in addition to continuous administration of antibiotics, fluid and pressor treatment. Biopsies of the fascia, muscle and fatty tissue were secured for microscopy, culture and histology. Unfortunately the patient died and histology confirmed necrotising fasciitis. INTERPRETATION: NSTIs are aggressive infections with dismal outcomes. This case illustrates the importance of clinical suspicion of this diagnosis, also in healthy patients. Immediate treatment with surgical debridement and intravenous antibiotics is crucial.

Hampered Lung Maturation in Methimazole-Induced Hypothyroidism in Fetal Chicken: Morphological and Molecular Correlates to Human Fetal Development.

BACKGROUND: Molecular understanding of lung development is crucial for developing therapies and diagnostic tools. Animal models with altered thyroid hormone signaling provide mechanistic insight into thyroid-dependent neonatal lung disease. Repression of Klf2 (Krüppel-like factor 2), a suggested T3 target gene, is associated with disrupted lung development in mice. Klf2 is proposed to be specifically involved in type I pneumocyte differentiation. OBJECTIVES: To explore mechanisms of thyroid-dependent lung disease, we studied developing chicken fetuses with experimentally induced hypothyroidism. METHODS: Morphology and the expression of a panel of molecules linked to Klf2 were assessed using histology, immunohistochemistry, Western blot and qPCR. RESULTS: Methimazole injections at E14 hampered lung maturation. The effects of methimazole were evident in several tissue compartments, and impacted on both pneumocyte and vascular differentiation, suggesting cellular and molecular pleiotropy. CONCLUSIONS: Concomitant expression changes in a panel of selected microRNAs regulated by Klf2 suggest importance in lung development. These microRNAs may thus represent potential clinical targets and diagnostic and prognostic tools in thyroid-dependent lung disease.

Cracking the Egg: Potential of the Developing Chicken as a Model System for Nonclinical Safety Studies of Pharmaceuticals.

The advance of perinatal medicine has improved the survival of extremely premature babies, thereby creating a new and heterogeneous patient group with limited information on appropriate treatment regimens. The developing fetus and neonate have traditionally been ignored populations with regard to safety studies of drugs, making medication during pregnancy and in newborns a significant safety concern. Recent initiatives of the Food and Drug Administration and European Medicines Agency have been passed with the objective of expanding the safe pharmacological treatment options in these patients. There is a consensus that neonates should be included in clinical trials. Prior to these trials, drug leads are tested in toxicity and pharmacology studies, as governed by several guidelines summarized in the multidisciplinary International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use M3 (R2). Pharmacology studies must be performed in the major organ systems: cardiovascular, respiratory, and central nervous system. The chicken embryo and fetus have features that make the chicken a convenient animal model for nonclinical safety studies in which effects on all of these organ systems can be tested. The developing chicken is inexpensive, accessible, and nutritionally self-sufficient with a short incubation time and is ideal for drug-screening purposes. Other high-throughput models have been implemented. However, many of these have limitations, including difficulty in mimicking natural tissue architecture and function (human stem cells) and obvious differences from mammals regarding the respiratory organ system and certain aspects of central nervous system development (Caenorhabditis elegans, zebrafish).This minireview outlines the potential and limitations of the developing chicken as an additional model for the early exploratory phase of development of new pharmaceuticals.

Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5.

Pressure overload-induced TGF-ß signaling activates cardiac fibroblasts (CFB) and leads to increased extracellular matrix (ECM) protein synthesis including fibrosis. Excessive ECM accumulation may in turn affect cardiac function contributing to development of heart failure. The aim of this study was to examine the effects of SM16, an orally active small molecular inhibitor of ALK5, on pressure overload-induced cardiac fibrosis. One week after aortic banding (AB), C57Bl/6J mice were randomized to standard chow or chow with SM16. Sham operated animals served as controls. Following 4 weeks AB, mice were characterized by echocardiography and cardiovascular magnetic resonance before sacrifice. SM16 abolished phosphorylation of SMAD2 induced by AB in vivo and by TGF-ß in CFB in vitro. Interestingly, Masson Trichrome and Picrosirius Red stained myocardial left ventricular tissue revealed reduced development of fibrosis and collagen cross-linking following AB in the SM16 treated group, which was confirmed by reduced hydroxyproline incorporation. Furthermore, treatment with SM16 attenuated mRNA expression following induction of AB in vivo and stimulation with TGF-ß in CFB in vitro of Col1a2, the cross-linking enzyme LOX, and the pro-fibrotic glycoproteins SPARC and osteopontin. Reduced ECM synthesis by CFB and a reduction in myocardial stiffness due to attenuated development of fibrosis and collagen cross-linking might have contributed to the improved diastolic function and cardiac output seen in vivo, in combination with reduced lung weight and ANP expression by treatment with SM16. Despite these beneficial effects on cardiac function and development of heart failure, mice treated with SM16 exhibited increased mortality, increased LV dilatation and inflammatory heart valve lesions that may limit the use of SM16 and possibly also other small molecular inhibitors of ALK5, as future therapeutic drugs.

Type I and II pneumocyte differentiation in the developing fetal chicken lung: conservation of pivotal proteins from birds to human in the struggle for life at birth.

BACKGROUND: Antenatal corticosteroids and surfactant replacement therapy have dramatically reduced mortality caused by lung disease in premature babies. Knowledge about mechanisms regulating epithelial differentiation of the respiratory membrane is limited, as are effects of pharmacological interventions. The chicken fetus is a valuable model for exploring pharmacological actions on developing organs. However, more precise information about the timing of developmental events in the chicken lung is needed for human correlation. OBJECTIVES: Characterization of morphological development and protein expression in the respiratory membrane of the developing chicken lung to create a platform for pharmacological testing in a human context. METHODS: Fetal chicken lungs, embryonic days (E) 7-20, were characterized by morphology and protein expression of epithelial differentiation markers. This was compared with publications on the same processes during human lung development. RESULTS: The respiratory membranes of developing chicken and human lungs show basic similarities. In chicken, surfactant protein B is expressed in cuboidal type II epithelial cells from E17. Aquaporin 5 is expressed in the epithelium from E7 and selectively in type I pneumocytes from E17. The type I pneumocyte and endothelial marker, caveolin 1, is expressed in the endothelium from E7 to E20. CONCLUSION: Despite phylogenetic distance, central aspects of cellular development in the chicken and human lung are similar. The fetal chicken model has important additional advantages to mammalian models, including fetal independence and short incubation, and is thus well suited for in vivo studies of lung maturation relevant to human development.

Differential regulation of extracellular matrix constituents in myocardial remodeling with and without heart failure following pressure overload.

Patients with aortic stenosis develop various degrees of myocardial hypertrophy and heart failure (HF) despite comparable transvalvular gradients. An important element in the transition from compensated hypertrophy to HF is dilatation of the left ventricle (LV). The molecular pathology associated with LV dilatation and development of HF is not known. Thus, we examined potential differences in the regulation of myocardial extracellular matrix (ECM) constituents in mice with hypertrophy only (ABnonHF) and with HF (ABHF) as response to comparable pressure overload. The ascending aorta was banded, or left loose in sham-operated mice. Increased lung weight and left atrial diameter indicating pulmonary congestion were used to identify ABHF mice. Cardiac function and geometry were evaluated by echocardiography. Despite comparable pressure gradients and cardiac output, ABHF had reduced fractional shortening (23%), reduced systolic (28%) and diastolic (32%) tissue velocity and increased LV internal dimension in diastole (10%) and systole (17%) (LVIDd/s) compared to ABnonHF (p≤0.05). Microarray analyses identified 120 differently regulated genes related to ECM in ABHF compared to ABnonHF (p≤0.05). Interestingly, in ABHF, we found a 24% (p≤0.05) reduction of the LV collagen VIII protein levels despite increased levels of LV total collagen by 23% (p≤0.05). LV collagen VIII correlated negatively with LVIDd (R=0.55, p=0.03) and LVIDs (R=0.72, p=0.002). As this protein may function as a "sealant" binding collagen fibrils together, reduction of collagen VIII could potentially contribute to LV dilatation and development of HF.