Evolution of Excitation-Inhibition Ratio in Cortical Cultures Exposed to Hypoxia.

In the core of a brain infarct, neuronal death occurs within minutes after loss of perfusion. In the penumbra, a surrounding area with some residual perfusion, neurons initially remain structurally intact, but hypoxia-induced synaptic failure impedes neuronal activity. Penumbral activity may recover or further deteriorate, reflecting cell death. Mechanisms leading to either outcome remain ill-understood, but may involve changes in the excitation to inhibition (E/I) ratio. The E/I ratio is determined by structural (relative densities of excitatory and inhibitory synapses) and functional factors (synaptic strengths). Clinical studies demonstrated excitability alterations in regions surrounding the infarct core. These may be related to structural E/I changes, but the effects of hypoxia /ischemia on structural connectivity have not yet been investigated, and the role of structural connectivity changes in excitability alterations remains unclear. We investigated the evolution of the structural E/I ratio and associated network excitability in cortical cultures exposed to severe hypoxia of varying duration. 6-12 h of hypoxia reduced the total synaptic density. In particular, the inhibitory synaptic density dropped significantly, resulting in an elevated E/I ratio. Initially, this does not lead to increased excitability due to hypoxia-induced synaptic failure. Increased excitability becomes apparent upon reoxygenation after 6 or 12 h, but not after 24 h. After 24 h of hypoxia, structural patterns of vesicular glutamate stainings change. This possibly reflects disassembly of excitatory synapses, and may account for the irreversible reduction of activity and stimulus responses seen after 24 h.

Endothelial dysfunction in pulmonary arterial hypertension: loss of cilia length regulation upon cytokine stimulation.

Pulmonary arterial hypertension (PAH) is a syndrome characterized by progressive lung vascular remodelling, endothelial cell (EC) dysfunction, and excessive inflammation. The primary cilium is a sensory antenna that integrates signalling and fine tunes EC responses to various stimuli. Yet, cilia function in the context of deregulated immunity in PAH remains obscure. We hypothesized that cilia function is impaired in ECs from patients with PAH due to their inflammatory status and tested whether cilia length changes in response to cytokines. Primary human pulmonary and mouse embryonic EC were exposed to pro- (TNFα, IL1ß, and IFNγ) and/or anti-inflammatory (IL-10) cytokines and cilia length was quantified. Chronic treatment with all tested inflammatory cytokines led to a significant elongation of cilia in both control human and mouse EC (by ∼1 µm, P < 0.001). This structural response was PKA/PKC dependent. Intriguingly, withdrawal of the inflammatory stimulus did not reduce cilia length. IL-10, on the other hand, blocked and reversed the pro-inflammatory cytokine-induced cilia elongation in healthy ECs, but did not influence basal length. Conversely, primary cilia of ECs from PAH patients were significantly longer under basal conditions compared to controls (1.86 ± 0.02 vs. 2.43 ± 0.08 µm, P = 0.002). These cilia did not elongate further upon pro-inflammatory stimulation and anti-inflammatory treatment did not impact cilia length. The missing length modulation was specific to cytokine stimulation, as application of fluid shear stress led to increased cilia length in the PAH endothelium. We identified loss of cilia length regulation upon cytokine stimulation as part of the endothelial dysfunction in PAH.

Measuring the primary cilium length: improved method for unbiased high-throughput analysis.

BACKGROUND: Primary cilia are cellular protrusions involved in mechanic and chemical sensing on almost all cells of our body. Important signaling pathways, including Hedgehog, TGFß, and Ca(2+), are linked to cilia and/or cilia function. Cilia can vary in length, which has functional implications. To measure these lengths correctly, a standardized method with high reliability and throughput is required. To date, methods for length measurements in cultured cells after fluorescent staining for ciliary components are error prone with a possible human selection bias, primarily caused by the orientation of cilia with respect of the imaging plane. In tissue sections, accurate measurements become an even larger challenge due to additional random sectioning plane. Cilia can be reconstructed in 3D and measured one by one, but this is a labor-intensive procedure. Therefore, we developed a new, high-throughput method with less selection bias. RESULTS: To identify the optimal type of measurement of straight and relatively short cilia, three methods were compared. The first method is based on maximum intensity projection (MIP), the second method is based on the Pythagorean theorem (PyT), and the third is based on 3D alternative angled slicing (DAAS). We investigated whether cilia visible in the plane of focus ('flat cilia'), and the ones that are angled with respect to the plane of focus are represented differently among the various methods. To test the agreement between the methods, intraclass correlations are calculated. To measure flat cilia, MIP and DAAS provided representative results, with the MIP method allowing for higher throughput. However, when measuring the angled cilia with MIP, the actual cilium length is overtly underestimated. DAAS and PyT are exchangeable methods for length measurements of the angled cilia, while PyT exhibits higher throughput and is therefore the preferred method for measuring the length of an angled cilium. CONCLUSION: PyT is a universal measuring method to measure straight cilia, without selection bias. MIP provides similar results for flat cilia, but underestimates the length of angled cilia. In addition, PyT facilitates high-throughput length measurements. Manual tracking or reconstruction will be the method of choice to measure irregularly shaped cilia.