Development of two-photon polymerised scaffolds for optical interrogation and neurite guidance of human iPSC-derived cortical neuronal networks.

Recent progress in the field of human induced pluripotent stem cells (iPSCs) has led to the efficient production of human neuronal cell models for in vitro study. This has the potential to enable the understanding of live human cellular and network function which is otherwise not possible. However, a major challenge is the generation of reproducible neural networks together with the ability to interrogate and record at the single cell level. A promising aid is the use of biomaterial scaffolds that would enable the development and guidance of neuronal networks in physiologically relevant architectures and dimensionality. The optimal scaffold material would need to be precisely fabricated with submicron resolution, be optically transparent, and biocompatible. Two-photon polymerisation (2PP) enables precise microfabrication of three-dimensional structures. In this study, we report the identification of two biomaterials that support the growth and differentiation of human iPSC-derived neural progenitors into functional neuronal networks. Furthermore, these materials can be patterned to induce alignment of neuronal processes and enable the optical interrogation of individual cells. 2PP scaffolds with tailored topographies therefore provide an effective method of producing defined in vitro human neural networks for application in influencing neurite guidance and complex network activity.

[Implant Design by Means of Multiphoton Polymerization]. / Implantatdesign mittels Multiphotonen-Polymerisation.

BACKGROUND: Additive manufacturing and 3D printing create new paths for the design and manufacturing of implants. Technologies with high resolution are required for the development of microstructured eye implants. In the present study, we demonstrate how these technologies can be used during the design development and manufacturing of a multifocal diffractive aspheric intraocular lens. MATERIAL AND METHODS: Multiphoton polymerisation (MPP) is used to manufacture a diffractive relief with resolution in the sub-micrometer range. The relief is applied to the moulded body of a refractive lens, forming a trifocal lens. Propagation of light behind the lens is visualised in water with fluorescein. RESULTS: Multifocal lenses were successfully manufactured with this approach. The optical design with three foci is confirmed by the light propagation images. The images even clearly demonstrate the impact of the refractive and diffractive elements and may provide information on artefacts and aberrations. CONCLUSIONS: Multiphoton polymerisation is an interesting tool for the flexible manufacturing of complex multifocal lenses. With future technological progress in 3D printing with MPP, this is a promising method for on-demand manufacturing of patient individual intraocular lenses.

[Optimisation of the visualisation technique for optical paths through intraocular lenses for characterisation of multifocal imaging properties of Fresnel-zone plates]. / Optimierung der Visualisierungstechnik für Strahlenverläufe durch Intraokularlinsen zur Charakterisierung multifokaler Abbildungseigenschaften von Fresnel-Zonenplatten.

The utilisation of the diffractive properties of Fresnel zone plates offers the possibility of intraocular lens designs with multiple foci. Such intraocular lenses can be manufactured by two-photon polymerisation (2PP). This paper explains the underlying concept and shows the principles for visualisation of the focus properties of such implants.

Mivazerol, a novel alpha2-agonist and potential anti-ischemic drug, inhibits KC1-stimulated neurotransmitter release in rat nervous tissue preparations.

In this study, we have investigated the effect of mivazerol, [3-(1H-imidazol-4-yl)methyl-1]-2-hydroxy-benzamide hydrochloride, a new alpha2-agonist lacking hypotensive properties and a potential anti-ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1-T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 x 0.2 x 0.2 mm3) sections and the resulting minces were used for this study. Exposure to KCl (10-75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S1 and S2 (for norepinephrine release) were conducted. The release of norepinephrine (+ 150% above baseline) was inhibited in a dose-dependent manner by mivazerol in hippocampus (IC50 = 1.5 x 10(-8) M), spinal cord (IC50 = 5 x 10(-8) M), rostrolateral ventricular medulla (IC50 = 10(-7) M), and nucleus tractus solitarii (IC50 = 7.5 X 10(-8) M), and by clonidine in hippocampus IC50 = 5 X 10(-8) M), spinal cord (IC50 = 4.5 x 10(-8) M), rostrolateral ventricular medulla (IC50 = 2.5 x 10( -7) M), and nucleus tractus solitarii (IC50 = 10(-7) M). This effect was counteracted by the selective alpha2-antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and + 135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 microM, had any significant effect on KCI-induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 microM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl-induced norepinephrine release by mivazerol is mediated by its action on alpha2-adrenergic receptors; (2) at concentrations selective for alpha2-adrenergic receptors, only mivazerol was effective in blocking the KCl-induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis.

Age-dependent effect of AF64A on cholinergic activity in the septo-hippocampal pathway of the rat brain: decreased responsiveness in aged rats.

Our previous studies have demonstrated that intracerebroventricular (icv) administration of low doses of ethylcholine mustard aziridinium (AF64A), up to 1.0 nmol/side, induces a reversible cholinergic deficit in the hippocampus, paralleled by a compensatory transient increase in choline acetyltransferase (ChAT) activity in the septum [El Tamer, A., Corey, J., Wülfert, E. and Hanin, I., Neuropharmacology, 31 (1992) 397-402]. In the present study we have addressed the question as to whether this effect might differ in old rats. AF64A (0.5 nmol/side) icv administered to three groups of rats aged 4, 12 and 22 months, respectively, induced a reduction of ChAT activity in the hippocampus to the same extent (-26%, -30.6% and -29.6%; P < 0.01) by 7 days post-icv injection. Acetylcholinesterase (AChE) was decreased to a similar extent in the 4 and 12 month old rats (-22% and -29%; P < 0.01), respectively, but remained unchanged in the 22 month old group. Whereas AChE activity remained unchanged in the septum in all three groups of rats, ChAT activity was increased significantly (+20% and +20.8%; P < 0.05 versus corresponding control group) in the 4 and 12 month old groups, respectively. No change in ChAT activity was measured in the septum of the 22 months old group. By 14 days post-icv injection of AF64A, ChAT and AChE activities were back to normal in all three groups and in both brain regions studied. These results demonstrate that a difference in AF64A's effect does exist between the 22 month old group and the youngest group. This might reflect a possible age-dependent change in the ability of the cholinergic system to respond to the cholinotoxicity of AF64A, as well as in the potential of the cell bodies, at the septal level, to respond to such an insult by a compensatory mechanism such as increasing ChAT activity.

Cholinotoxic effects on acetylcholinesterase gene expression are associated with brain-region specific alterations in G,C-rich transcripts.

To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.

ucb 11056, a new potential nootropic drug, amplifies induced cyclic AMP formation in rat brain tissue.

ucb 11056 [2-(4-morpholino-6-propyl-1,3,5-triazin-2-yl)aminoethanol] induced a significant (approximately 25%) increase in cyclic AMP levels in different brain areas following its intraperitoneal injection. This effect started as early as 2 min postinjection and lasted for 30 min, after which cyclic AMP levels returned to normal. In hippocampal slice preparations in vitro, ucb 11056 exerted a strong potentiation of cyclic AMP levels when it was combined with agents such as norepinephrine, forskolin, and isoproterenol. Only a slight effect on cyclic AMP levels was measured when ucb 11056 was incubated alone with hippocampal slices. The potentiating effect of ucb 11056 on norepinephrine-stimulated cyclic AMP formation was partially reduced when slices were pretreated with yohimbine and totally abolished when slices were treated with propranolol. These combined data indicate that (a) ucb 11056 rapidly increases cyclic AMP levels in the rat brain in vivo and (b) ucb 11056 potentiates stimulated cyclic AMP formation in vitro. The data also suggest that the central effect of ucb 11056 might be via the modulation of cyclic AMP generation, most probably mediated through adenylate cyclase activation mechanisms combined with a weak inhibitory activity on the cyclic nucleotide phosphodiesterase activity.

Sex differences and estrous cycle-variations in the AF64A-induced cholinergic deficit in the rat hippocampus.

The influence of gender and stage of the estrous cycle on the levels of acetylcholine, serotonin, and noradrenaline in the hippocampus and on the susceptibility of the cholinergic septo-hippocampal pathway to the neurotoxic effect of ethylcholine aziridinium (AF64A) was investigated in the rat. Levels of acetylcholine and serotonin were consistently higher in female rats during the stage of diestrus and proestrus than in age-matched male rats (p < 0.05). Across the estrous cycle the highest levels of acetylcholine and serotonin, coinciding with the lowest levels of noradrenaline, were measured on proestrus. Eight to 10 days after the bilateral intracerebroventricular injection of a submaximal dose of AF64A (1 nmol/ventricle) the decrease of acetylcholine in hippocampus was larger in females than in male rats. The reduction of acetylcholine was most pronounced in female rats that had received submaximal doses of AF64A on proestrus (42.7 +/- 3.4%), whereas in male rats, the corresponding decrease was 25.9 +/- 5.1% (p < 0.05). At a maximal dose of AF64A (2 nmole/ventricle), the sex-specific or cycle-dependent difference in the cholinotoxicity of AF64A vanished. The dose-dependent loss of acetylcholine was associated with a secondary dose-dependent decrease in the levels of serotonin and noradrenaline, but significant differences between male and female rats or stages of estrous cycle were not apparent. The present data provide evidence that adult female rats in general, and particularly females on proestrus, are more susceptible to the neurotoxic action of submaximal doses of AF64A than age-matched male rats.

Reversible cholinergic changes induced by AF64A in rat hippocampus and possible septal compensatory effect.

Ethylcholine aziridinium (AF64A) was injected intracerebroventricularly in rats, and the dose- and time-dependent effect of this treatment was measured on cholinergic markers in different areas of the brain. Choline acetyltransferase (ChAT) in the hippocampus was reduced by 0, 25, 40 and 50%, 4 days after administration of 0.25, 0.5, 1.0 and 2.0 nmol/side of AF64A. This effect was paralleled by a 0, 21, 38 and 48% increase in septal ChAT, at the same time and dose, respectively. Hippocampal and septal ChAT returned to normal by 14 days, following the two smaller doses of AF64A. Normal values of ChAT activity in the hippocampus were measured by 3 months post-1.0 nmol/side and by 12 months after 2.0 nmol AF64A/side. A transient secondary increase in septal ChAT was measured at 28 and 42 days after 1.0 nmol/side, after which no further changes were measured in this area. High affinity choline transport (HAChT) in the hippocampus showed a 35 and 25% decrease but only 14 and 7 days, after injection of 0.25 and 0.5 nmol AF64A/side, respectively. However, significant reductions by 45% at 14 days and by 65% at 4 days, were measured after the infusion of 1.0 and 2.0 nmol/side, respectively. This effect was significantly attenuated by 3 months and returned to normal by 12 months after treatment. Acetylcholinesterase (AChE) activity in the hippocampus was most severely affected by AF64A. Specifically, significant inhibition of 20% (by 7 days), 35% (by 7 days), 25% (by 2 days) and 30% (by 2 days) was observed at 0.25, 0.5, 1.0 and 2.0 nmol AF64A/side, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells.

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A2 and Staphylococcus aureus sphingomyelinase C and on phospholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A1 and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells.

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.