Aligned Schwann cells within 3D tissue-like gels provide guidance to regenerating neurites

There is a clinical demand to shorten the delay of reinnervation and improve functional recovery after peripheral nerve injury. A peripheral nerve repair device with the ability to direct and promote cellular growth across a lesion would be a promising alternative to nerve autograft repair, which is the current gold standard treatment. The growth of axons across a lesion is most effective when supported by columns of aligned Schwann cells, as found in an autograft. Here we report a technique to generate aligned Schwann cells within a stable and robust 3D collagen matrix, providing a cellular biomaterial that confers alignment on regenerating neurons. Collagen gels containing F7 Schwann cells were tethered for 24 h to permit cellular self-alignment and then plastic compressed by the rapid removal of the interstitial fluid from fully hydrated gels. This process generates stable tissue-like gels with cells situated within a dense, strong, three-dimensional matrix. Cell alignment was monitored before and after plastic compression using CellTracker dye and confocal image analysis. Dissociated dorsal root ganglia (DRG) cells were cultured on the surface of the material for 3 days and neurite growth was quantified using immunostaining and confocal microscopy. Chains of aligned Schwann cells were formed within the collagen matrix and persisted following plastic compression. This robust, aligned cellular biomaterial promotes and guides neuronal growth in a manner that mimics a nerve autograft. The next stage of this work is to integrate this cellular material into a repair device. Plastic compressed gels containing aligned Schwann cells have been rolled into columns which can then be packed together. In vitro testing of this engineered endoneurium, within a silicone outer tube, demonstrates the potential of such a device to function as an implantable conduit for peripheral nerve repair

Fast, quantitative in situ hybridization of rare mRNAs using 14C-standards and phosphorus imaging

The use of radiolabelled probes for in situ hybridization (ISH) bears the advantage of high sensitivity and quantifiability. The crucial disadvantages are laborious hybridization protocols, exposition of hybridized sections to film for up to several weeks and the time consuming need to prepare tissue standards with relatively short-lived isotopes like (33)P or (35)S for each experiment. The quantification of rare mRNAs like those encoding for subunits of neurotransmitter receptors is therefore a challenge in ISH. Here, we describe a method for fast, quantitative in situ hybridization (qISH) of mRNAs using (33)P-labelled oligonucleotides together with (14)C-polymer standards (Microscales, Amersham Biosciences) and a phosphorus imaging system (BAS 5000 BioImage Analyzer, Raytest-Fuji). It enables a complete analysis of rare mRNAs by ISH. The preparation of short-lived (33)P-standards for each experiment was replaced by co-exposition and calibration of long-lived (14)C-standards together with (33)P-labelled brain paste standards. The use of a phosphorus imaging system allowed a reduction of exposition time following hybridization from several weeks to a few hours or days. We used this approach as an example for applications to quantify the expression of GluR1 and GluR2 subunit mRNAs of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the hippocampus of untreated rats, and after intraperitoneal application of the organo-arsenic compound dimethyl arsenic acid

Interictal-like network activity and receptor expression in the epileptic human lateral amygdala

While the amygdala is considered to play a critical role in temporal lobe epilepsy, conclusions on underlying pathophysiological mechanisms have been derived largely from experimental animal studies. Therefore, the present study aimed to characterize synaptic network interactions, focusing on spontaneous interictal-like activity, and the expression profile of transmitter receptors in the human lateral amygdala in relation to temporal lobe epilepsy. Electrophysiological recordings, obtained intra-operatively in vivo in patients with medically intractable temporal lobe epilepsy, revealed the existence of interictal activity in amygdala and hippocampus. For in vitro analyses, slices were prepared from surgically resected specimens, and sections from individual specimens were used for electrophysiological recordings, receptor autoradiographic analyses and histological visualization of major amygdaloid nuclei for verification of recording sites. In the lateral amygdala, interictal-like activity appeared as spontaneous slow rhythmic field potentials at an average frequency of 0.39 Hz, which occurred at different sites with various degrees of synchronization in 33.3% of the tested slices. Pharmacological blockade of glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, but not N-methyl-D-aspartate receptors, abolished interictal-like activity, while the γ-aminobutyric acid A-type receptor antagonist bicuculline resulted in a dampening of activity, followed by highly synchronous patterns of slow rhythmic activity during washout. Receptor autoradiographic analysis revealed significantly higher α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, metabotropic glutamate type 2/3, muscarinic type 2 and adrenoceptor α(1) densities, whereas muscarinergic type 3 and serotonergic type 1A receptor densities were lower in the lateral amygdala from epileptic patients in comparison to autopsy controls. Concerning γ-aminobutyric acid A-type receptors, agonist binding was unaltered whereas antagonist binding sites were downregulated in the epileptic lateral amygdala, suggesting an altered high/low-affinity state ratio and concomitant reduced pool of total γ-aminobutyric acid A-type receptors. Together these data indicate an abnormal pattern of receptor densities and synaptic function in the lateral nucleus of the amygdala in epileptic patients, involving critical alterations in glutamate and γ-aminobutyric acid receptors, which may give rise to domains of spontaneous interictal discharges contributing to seizure activity in the amygdala