Phototoxicities Caused by Continuous Light Exposure Were Not Induced in Retinal Ganglion Cells Transduced by an Optogenetic Gene.

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.

Effects of Taxol on Regeneration in a Rat Sciatic Nerve Transection Model.

Recent studies describe taxol as a candidate treatment for promoting central nerve regeneration. However, taxol has serious side effects including peripheral neurotoxicity, and little information is known about the effect of taxol on peripheral nerve regeneration. We investigated the effects of taxol on regeneration in a rat sciatic nerve transection model. Rats were divided into four groups (n = 10): normal saline (i.p.) as the control, Cremophor EL vehicle, and 2 or 6 mg/kg of taxol in the Cremophor EL solution (four times in day-2, 4, 6, and 8), respectively. We evaluated neuronal electrophysiology, animal behaviour, neuronal connectivity, macrophage infiltration, location and expression levels of calcitonin gene-related peptide (CGRP), and expression levels of both nerve growth factors and immunoregulatory factors. In the high-dose taxol group (6 mg/kg), neuronal electrophysiological function was significantly impaired. Licking latencies were significantly changed while motor coordination was unaffected. Neuronal connectivity, macrophage density, and expression levels of CGRP was dramatically reduced. Expression levels of nerve growth factors and immunoregulatory factors was also reduced, while it was increased in the low-dose taxol group (2 mg/kg). These results indicate that taxol can modulate local inflammatory conditions, impair nerve regeneration, and impede recovery of a severe peripheral nerve injury.

A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury.

Spinal cord injury (SCI) causes permanent, often complete disruption of central nervous system (CNS) function below the damaged region, leaving patients without the ability to regenerate lost tissue. To engineer new CNS tissue, a unique spinal cord bridge is created to deliver stem cells and guide their organization and development with site-specifically immobilized growth factors. In this study, this bridge is tested, consisting of adult neural stem/progenitor cells contained within a methacrylamide chitosan (MAC) hydrogel and protected by a chitosan conduit. Interferon-γ (IFN-γ) and platelet-derived growth factor-AA (PDGF-AA) are recombinantly produced and tagged with an N-terminal biotin. They are immobilized to streptavidin-functionalized MAC to induce either neuronal or oligodendrocytic lineages, respectively. These bridges are tested in a rat hemisection model of SCI between T8 and T9. After eight weeks treatments including chitosan conduits result in a significant reduction in lesion area and macrophage infiltration around the lesion site (p < 0.0001). Importantly, neither immobilized IFN-γ nor PDGF-AA increased macrophage infiltration. Retrograde tracing demonstrates improved neuronal regeneration through the use of immobilized growth factors. Immunohistochemistry staining demonstrates that immobilized growth factors are effective in differentiating encapsulated cells into their anticipated lineages within the hydrogel, while qualitatively reducing glial fibrillary acid protein expression.

Efficacy of hyaluronic acid or steroid injections for the treatment of a rat model of rotator cuff injury.

This study evaluated dorsal root ganglia from C3-C7, analyzed gait, and compared the expression of calcitonin gene-related peptide (CGRP) which was a marker of inflammatory pain in a rat rotator cuff tear model in which the supraspinatus and infraspinatus tendons were detached; comparisons were made to a sham group in which only the tendons were exposed. Fluorogold was injected into the glenohumeral joint 21 days after surgery in both groups, and saline, steroids, or hyaluronic acid was injected into the glenohumeral joint in the rotator cuff tear group 26 days after surgery. The proportions of CGRP-immunoreactive neurons were higher and the gait parameters were impaired in the rotator cuff tear group compared to in the sham group. However, the CGRP expression was reduced and the gait was improved with steroid or hyaluronic acid injection compared to saline, suggesting that both hyaluronic acid and steroid injections suppressed of inflammation which thought to be provided pain relief. While there were no significant differences, the suppression of CGRP expression and the improved gait after hyaluronic acid and steroid injections suggested that both methods were effective for rat rotator cuff tear model.

Correlative Microscopy for Localization of Proteins In Situ: Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold, Gold Enhancement, and Low-Temperature Resin.

Immuno-electron microscopy (immuno-EM) is a technique that has been used widely to determine subcellular localization of proteins. Different approaches are available for immuno-EM: pre-embedding method, post-embedding, and cryosectioning (Tokuyasu "style"). Here we describe a pre-embedding technique that allows the labeling of a target protein in situ, retention of fluorescence signal in plastic, and its localization at the EM level in a given cellular context. The procedure can be technically challenging and labor intensive: it requires optimization of fixation protocols to better preserve the cellular morphology and screening of compatible antibodies. Nevertheless, immuno-EM can be a powerful localization tool.

Comparison of templating abilities of urea and thioruea during photodimerization of bipyridylethyelene and stilbazole crystals.

Photodimerization of cocrystals of four bispyridylethylenes and two stilbazoles with urea as a template in the solid state has been investigated following our success with thiourea. Four investigated olefins photodimerized quantitatively to a single dimer in the crystalline state only. The reactivity of urea-olefin crystals is understood on the basis of their packing arrangements in the crystalline state. In reactive crystals the adjacent reactive molecules are within 4.2 Å and parallel, whereas the unreactive ones have their adjacent molecules are farther than 4.6Å and nonparallel. Thus, with the knowledge of crystal packing the reactivity of urea-olefin crystals is predictable on the basis of Schmidt's topochemical postulates. The templating property of urea, similar to thiourea, derives from its ability to form hydrogen bonds with itself and the guest olefins. Despite the similarities in molecular structures of urea and thiourea their subtle electronic properties, yet to be fully understood, affect the crystal packing and consequently their reactivity in the crystalline state. Further work is needed to fully exploit the templating properties of urea.

Hypothalamic and basal ganglia projections to the posterior thalamus: possible role in modulation of migraine headache and photophobia.

Migraine attacks are typically described as unilateral, throbbing pain that is usually accompanied by nausea, vomiting, and exaggerated sensitivities to light, noise and smell. The headache phase of a migraine attack is mediated by activation of the trigeminovascular pathway; a nociceptive pathway that originates in the meninges and carries pain signals through meningeal nociceptors to the spinal trigeminal nucleus and from there to the cortex through relay neurons in the thalamus. Recent studies in our lab have identified a population of trigeminovascular neurons in the posterior (Po) and lateral posterior (LP) thalamic nuclei that may be involved in the perception of whole-body allodynia (abnormal skin sensitivity) and photophobia (abnormal sensitivity to light) during migraine. The purpose of the current study was to identify sub-cortical areas that are in position to directly regulate the activity of these thalamic trigeminovascular neurons. Such process begins with anatomical mapping of neuronal projections to the posterior thalamus of the rat by performing discrete injections of the retrograde tracer Fluorogold into the Po/LP region. Such injections yielded retrogradely labeled neurons in the nucleus of the diagonal band of Broca, the dopaminergic cells group A11/A13, the ventromedial and ventral tuberomammillary nuclei of the hypothalamus. We also found that some of these neurons contain acetylcholine, dopamine, cholecystokinin and histamine, respectively. Accordingly, we speculate that these forebrain/hypothalamic projections to Po and LP may play a role in those migraine attacks triggered by disrupted sleep, skipping meals and emotional reactions.

Targeting the lateral interactions of transmembrane domain 5 of Epstein-Barr virus latent membrane protein 1.

The lateral transmembrane protein-protein interaction has been regarded as "undruggable" despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound 1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound 1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound 1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein-protein interactions.

Retrograde labeling of retinal ganglion cells by application of fluoro-gold on the surface of superior colliculus.

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.

Projections of olfactory bulbs to the olfactory and vomeronasal cortices.

Projections from the olfactory bulbs have been traditionally described as 'nontopographically organized'. Olfactory and vomeronasal projections have been reported to reach nonoverlapping cortical areas. Four receptor expression zones have been described in the olfactory epithelium, maintained in the main olfactory bulb, but none in the olfactory cortex. Recent data have demonstrated convergence in the basal telencephalon of olfactory and vomeronasal projections. Injections of methanesulfonate hydroxystilbamidine (FluoroGold) in the chemosensory cortex were done to map retrograde labeling in the bulbs. Topography was not observed in the four zones of the main olfactory bulb. Areas of the rostral telencephalon were shown to receive simultaneous inputs from the main and accessory olfactory bulbs.

GABAergic neurons in the ventrolateral subnucleus of the nucleus tractus solitarius are in contact with Kölliker-Fuse nucleus neurons projecting to the rostral ventral respiratory group and phrenic nucleus in the rat.

After ipsilateral injections of biotinylated dextran amine (BDA) into the ventrolateral subnucleus of the nucleus tractus solitarius (vlNTS) and Fluoro-gold (FG) into the rostral ventral respiratory group (rVRG) region or into the phrenic nucleus (PhN) region in the rat, an overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the Kölliker-Fuse (KF) nucleus ipsilateral to the injection sites. Using retrograde tracing combined with immunohistochemistry for glutamic acid decarboxylase isoform 67 (GAD67), we indicated that as many as 40% of the vlNTS neurons projecting to the KF were immunoreactive for GAD67. Using a combination of anterograde and retrograde tracing techniques, and immunohistochemistry for GAD67, we further demonstrated that the vlNTS axon terminals with GAD67 immunoreactivity established close contact to the rVRG- or PhN-projecting KF neurons. The present results suggest that GABAergic vlNTS fibers may exert inhibitory influences on the rVRG- as well as PhN-projecting KF neurons and these circuits may be involved in the respiratory reflexes such as the Hering-Breuer reflex.

Fluoro-Gold: composition, and mechanism of uptake.

Determining the mechanism by which fluorescent retrograde neuronal tracers are taken up requires knowledge of their composition. It has been claimed that Fluoro-Gold, a retrogradely transported fluorescent neuronal tracer, is 2-hydroxy-4,4'-diamidinostilbene (hydroxystilbamidine), an amidine antibiotic. However, this appears questionable, since the fluorescence spectrograms reported for Fluoro-Gold differ markedly from the spectrograms previously reported for purified hydroxystilbamidine. To help clarify the mechanism by which Fluoro-Gold might be taken up, it was decided to examine its composition and determine whether hydroxystilbamidine was its active agent. Fluoro-Gold was found by mass spectrometry to contain a component with a molecular weight of 280 Da (identical to that of hydroxystilbamidine), and fluorescence spectroscopy demonstrated the existence of a substance with a fluorescence spectrum similar to that of purified hydroxystilbamidine. Although a major fluorescent impurity was also observed, chromatographic separation of different fluorescent components of Fluoro-Gold suggested that the fraction resembling hydroxystilbamidine was responsible for its retrograde labeling of cells. It is concluded that hydroxystilbamidine is the active constituent of Fluoro-Gold. Chemically, hydroxystilbamidine is a weak base. In this respect it resembles True blue, DAPI, Granular blue, bis-benzimide, Nuclear yellow, and several other retrogradely transported molecules. It is suggested that these agents cross cell membranes in their uncharged form and are trapped in lysosomes and endosomes by a favorable pH gradient. Thus, the uptake of this type of retrograde tracer may be an example of a well-understood process occurring widely throughout biological systems: the trapping of weak bases in acidic cellular compartments.