Laser nano-neurosurgery from gentle manipulation to nano-incision of neuronal cells and scaffolds: an advanced neurotechnology tool

Current optical approaches are progressing far beyond the scope of monitoring the structure and function of living matter, and they are becoming widely recognized as extremely precise, minimally-invasive, contact-free handling tools. Laser manipulation of living tissues, single cells, or even single-molecules is becoming a well-established methodology, thus founding the onset of new experimental paradigms and research fields. Indeed, a tightly focused pulsed laser source permits complex tasks such as developing engineered bioscaffolds, applying calibrated forces, transfecting, stimulating, or even ablating single cells with subcellular precision, and operating intracellular surgical protocols at the level of single organelles. In the present review, we report the state of the art of laser manipulation in neuroscience, to inspire future applications of light-assisted tools in nano-neurosurgery

Volumetric Lissajous confocal microscopy with tunable spatiotemporal resolution

Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are rigid in adapting the acquisition parameters over time, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Such lack of tunability forces a user to provide scan parameters, which may not be optimal, based on the best assumption before an acquisition starts. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, the volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with 3D acquisition speeds of 30 Hz and frame rates up to 5 kHz

Bioresorbable insertion aids for brain implantable flexible probes: a comparative study on silk fibroin, alginate, and disaccharides

Miniaturized, flexible, and biocompatible neural probes have the potential to circumvent the brain's foreign body response, but the problem of surgical implantation remains. Herein, a probe intended for implantation in the rat hippocampus is coated in four bioresorbable stiffeners to determine which is most effective in aiding insertion. The stiffeners (sucrose, maltose, silk fibroin, and alginate) are evaluated through mechanical, chemical, and dissolution tests. After coating with silk fibroin, the buckling force of the neural probe increases from 0.31 to 75.99 mN. This goes in accordance with subsequent successful insertion tests. Fourier transform infrared spectroscopy results demonstrate the increase in β-sheet content of silk fibroin samples after treatment (e.g., water annealing) and show relevant changes due to dehydration of the alginate hydrogel. Both qualitative and quantitative dissolution studies in artificial cerebrospinal fluid illustrate that alginate and silk fibroin outlasts the disaccharide stiffeners. In this work, a variety of multidisciplinary analyses are carried out to find the best bioresorbable stiffener for deep brain implantable devices with the highest buckling force, longest dissolution time, and the most tunable structure. For the first time, an alginate hydrogel is used as a stiffener to aid insertion, expanding its usefulness beyond neural tissue engineering

The Flavonoid Isoquercitrin Promotes Neurite Elongation by Reducing RhoA Activity

<div><h3>Background</h3><p>Neurite formation and synaptic patterning are fundamental to the development of a functional nervous system. Flavonoids are natural molecules known for having beneficial effects on brain health through diverse molecular pathways. Cytoskeletal changes occurring during neuritogenesis and synapse formation often involve Rho GTPases. Here we hypothesized that the flavonoid isoquercitrin promotes neuronal differentiation through Rho signalling.</p> <h3>Methodology/Principal Findings</h3><p>We performed time lapse imaging of NG108-15 cells during incubation with/without isoquercitrin. Isoquercitrin stimulated extensive neurites enriched in the synaptic vesicle protein synaptotagmin-1. Neurite extension was augmented by the ROCK inhibitor Y-27632 suggesting an inactivation of RhoA/Rho kinase as the mechanism. To test this, we first measured the dose-dependent effect of isoquercitrin on RhoA activity and found a 47% reduction in RhoA activity at concentrations which induced neurites (≥40 µM). Secondly, we tested the ability of isoquercitrin to rescue the neural phenotype in a model of RhoA-induced neurite retraction and found that 40 µM isoquercitrin added to cultures previously treated with the RhoA activator calpeptin produced significantly more neurite length/cell than calpeptin alone. Finally, we tested the hypothesis that isoquercitrin may affect RhoA localization preventing the translocation to the plasma membrane. Unexpectedly, immunolocalization studies showed that RhoA was present in nuclear compartments of control NG108-cells, but underwent translocation to the cytoplasm upon treatment with isoquercitrin. DNA microarrays and reverse transcription - quantitative PCR (RT-qPCR) revealed differences in global gene expression of Rho GTPase family members. These data taken together indicate that isoquercitrin is a potential stimulator of neuronal differentiation, through multiple Rho GTPase mediated mechanisms.</p> <h3>Conclusions/Significance</h3><p>As several members of the Rho GTPase family are implicated in human neurological disorders/injuries, our results suggest that isoquercitrin could be used in the treatment of these pathological states through its effect on this family of molecular switches.</p> </div

Proteomic profiling of vesicles released by 8701-bc cells

8701-BC cells were shown to release “membrane vesicles” playing a role in tumor progression mechanisms. On the other hand, production of “exosomes”, smaller vesicles known to be involved in immune response activation, had not been revealed. The first goal of this study was to separate different vesicle populations from 8701-BC cell conditioned medium. To this aim, the medium was differentially centrifuged. Western analysis revealed that the 15,000xg pelletted fraction contains β1-integrin, which had been shown to be clustered in membrane vesicles shed by 8701-BC cells, but not Hsc70, a protein found in exosomes. On the contrary, Hsc70 is detectable while β1-integrin is not present in the fraction obtained by a further centrifugation at 100,000xg from 15,000xg supernatant. Moreover, the absence of Cytochrom C in both fractions excludes the contamination with apoptotic vesicles.These results suggested that 8701-BC cells release both membrane vesicles and exosomes and that their separation can be achieved by differential centrifugation.Then, to analyze the whole protein content of the vesicle preparations, a proteomic approach was chosen. Protein 2D-PAGE analysis was performed and the gel images were analyzed in silico, using ImageMaster 2D Platinum software. The preliminary comparative proteomic analysis revealed a set of protein spots differently abundant in the vesicle fractions. These data strongly encourage for further investigation using 2D-PAGE coupled with MS-MALDI-TOF analysis which could highlight physiological roles of the two different kinds of vesicles

Isoquercitrin is able to restore the neural phenotype in calpeptin stimulated cells.

<p>The fluorescence images show 6 hr cultures stained with anti-neurofilament antibody. Green = neurofilament protein, blue = nuclei. Scale bar = 50 micron. <b>Bottom right</b>, The double y-axis graph shows: 1) the neurite length (micron)/cell (black bars) measured on the fluorescence images as described in M&M (n = 10 images, one-way ANOVA ****p<0.0001); 2) RhoA activation state (grey bars) evaluated through the Rho G LISA assay (n = 3, one-way ANOVA *p<0.05). Data are shown as means ± s.e.m.</p