Supercritical Carbon Dioxide Decellularization of Porcine Nerve Matrix for Regenerative Medicine.

Tissue engineering scaffolds are often made from the decellularization of tissues. The decellularization of tissues caused by prolonged contact with aqueous detergents might harm the microstructure and leave cytotoxic residues. In this research, we developed a new technique to use supercritical carbon dioxide (Sc-CO2)-based decellularization for porcine nerve tissue. The effect of decellularization was analyzed by histological examination, including Hematoxylin and Eosin, Masson's Trichrome staining, and 4',6-diamidino-2-phenylindole staining. Moreover, biochemical analysis of the decellularized tissues was also performed by measuring DNA content, amount of collagen, and glycosaminoglycans (GAGs) after decellularization. The results showed that the tissue structure was preserved, cells were removed, and the essential components of extracellular matrix, such as collagen fibers, elastin fibers, and GAG fibers, remained after decellularization. In addition, the DNA content was decreased compared with native tissue, and the concentration of collagen and GAGs in the decellularized nerve tissue was the same as in native tissue. The in vivo experiment in the rat model showed that after 6 months of decellularized nerve implantation, the sciatic function index was confirmed to recover in decellularized nerve. Morphological analysis displayed a range of infiltrated cells in the decellularized nerve, similar to that in native tissue, and the number of Schwann cells that play essential for motor function and sensory in the decellularized nerve was confirmed. These findings indicate that tissue decellularization using Sc-CO2 has been successfully used in tissue engineering.

Effect of a differential training paradigm with varying frequencies and amplitudes on adaptation of vestibulo-ocular reflex in mice.

The vestibulo-ocular reflex (VOR) functions to maintain eye stability during head movement, and VOR gain can be dynamically increased or decreased by gain-up or gain-down adaptation. In this study, we investigated the impact of a differential training paradigm with varying frequencies and amplitudes on the level of VOR adaptation in mice. Training for gain-up (out of phase) or gain-down (in phase) VOR adaptation was applied for 60 min using two protocols: (1) oscillation of a drum and turntable with fixed frequency and differing amplitudes (0.5 Hz/2.5°, 0.5 Hz/5° and 0.5 Hz/10°). (2) Oscillation of a drum and turntable with fixed amplitude and a differing frequency (0.25 Hz/5°, 0.5 Hz/5° and 1 Hz/5°). VOR adaptation occurred distinctively in gain-up and gain-down learning. In gain-up VOR adaptation, the learned increase in VOR gain was greatest when trained with the same frequency and amplitude as the test stimulation, and VOR gain decreased after gain-up training with too high a frequency or amplitude. In gain-down VOR adaptation, the decrease in VOR gain increased as the training frequency or amplitude increased. These results suggest that different mechanisms are, at least in part, involved in gain-up and gain-down VOR adaptation.

Adaptation of Optokinetic Reflex by Training with Different Frequency and Amplitude.

BACKGROUND: Although the occurrence of optokinetic reflex (OKR) adaptation after OKR training is well established, the dynamic properties of OKR adaptation has not been fully studied. This study aimed to examine the difference in the amount of OKR adaptation according to OKR training protocols which have different frequency or amplitude of drum oscillation. METHODS: Using C57BL/6N male mice, we induced OKR adaptation by 3 different categories of learning paradigm as follows: (1) Optokinetic drum oscillation for 60 min with same amplitude and different frequency. (2) Optokinetic drum oscillation for 60 min with same frequency and different amplitude. (3) Training with serial combination of different frequency or amplitude. RESULTS: The results show that the amount of OKR adaptation was greater after OKR training with lower frequency or amplitude than that with higher frequency or amplitude. CONCLUSIONS: This finding may suggest that the retinal slip signal with lower-velocity OKR stimulation serves as more precise instructive signal for learning, leading to induction of more efficient training effect. Another interesting finding was that the OKR gain increase tended to be greater after training composed of sequential combination of decreasing frequency or amplitude than that composed of sequential combination of increasing frequency or amplitude. Furthermore, the OKR training with high frequency or amplitude eliminated a part of learning effects which have already formed by previous training. We postulate that the stimulation during training with high frequency or amplitude may implement a disturbing instruction for OKR learning when it is conducted in mice with increased OKR gain after previous OKR training.

NeoPep S: A New Generation of AIMP1-derived Peptide (AdP) Effects on Wound Healing In Vivo.

BACKGROUND/AIM: The skin plays an important role in protecting the body from mechanical damage, microbial infection, ultraviolet radiation, and extreme temperatures. Many products as well as ongoing studies have focused on skin injury and repair; however, unlimited challenges are still being faced. Furthermore, the drugs that are currently on the market are not adequate to meet the increasing medical needs. This study aimed to discover whether our new product can efficiently promote wound repair and skin restoration. MATERIALS AND METHODS: in this study, we applied a new AIMP1-derived peptide (AdP), NeoPep S, administered in two dose types (1 ppm and 3 ppm), and determined their effect on skin wound repair in rat models. Cell proliferation and inflammatory responses were assessed using immunofluorescence (IF) staining and ELISA assay. RESULTS: As expected, our results showed more rapid and satisfactory progress in wound closure upon treatment with NeoPep S 3 ppm than with NeoPep S 1 ppm. The 3 ppm peptide derived from AIMP1 protein, harmoniously interacted with the wound to promote re-epithelialization and collagen regeneration, as well as the down-regulation of several types of cytokines and chemokines, such as TNF-α, IL-6, IL-8, IL-lß, MCP-1, and F4/80. Moreover, it was demonstrated to promote fibroblast proliferation, migration, and differentiation by TGF-ßl and TGF-ß3 modulation, as well as nitrite and reactive oxygen species scavenging. CONCLUSION: The novel peptide NeoPep S 3 ppm showed high effectiveness and safety in wound healing.

Adaptation of vestibulo-ocular and optokinetic reflexes after massed and spaced vestibulo-ocular motor learning.

Although the superiority of spaced training over massed training has been established in many forms of learning, the learning efficacy between the two with respect to time efficiency may not be simply compared because a longer total duration of learning is required in spaced training than massed training due to spacing intervals intervening between training sessions in the former. The purpose of the present study was to evaluate the differences in the adaptation of the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) after visuo-vestibular training, and to investigate the efficacy of spaced and massed training in mice. Associative visuo-vestibular stimulation was applied to induce VOR and OKR motor learning. Training paradigms were categorized into five groups according to the duration of the spacing interval, keeping the total training time including spacing equal in all training paradigms. Both gain-up VOR training, which increased VOR gain and gain-down VOR training, which decreased VOR gain, increased OKR gain in the massed and spaced learning paradigms. While the increment in OKR gain after gain-up and gain-down training was maintained at 48 h after the end of the last training session, the change in VOR gain by gain-up or gain-down training recovered gradually after training. The OKR adaptation was still in progress during the spacing interval, and the amount of gain increase was greater with longer spacing interval. On the other hand, the VOR gain change after gain-up and gain-down training substantially recovered during the spacing interval. In conclusion, the present study, using learning paradigms with same total duration of training, demonstrated that the spacing effect was more robust in the adaptation of OKR than that of VOR, and the learning effect was maintained longer in OKR than in VOR. These differences in the adaptation of VOR and OKR following identical training conditions suggest that multiple plasticity mechanisms may be differentially involved in the gaze stabilization circuitry.

Efficacy of spaced learning in adaptation of optokinetic response.

INTRODUCTION: The superiority of spaced training, in which repeated training sessions are given with resting intervals, over massed training in learning efficacy has been well established. However, longer duration of total training time has been required for spaced training than massed training because spacing intervals intervene between training sessions in spaced training. Thus, the learning efficacy may not be simply compared between spaced and massed training in terms of "time efficiency." The aim of the present study was to investigate the efficacy of spaced and massed training using adaptation of horizontal optokinetic reflex (hOKR) in mice. METHODS: Training paradigms were categorized into seven groups according to the duration of spacing interval, keeping total duration of hOKR training including spacing almost equal in all training paradigms. RESULTS: The amount of short-term hOKR gain increase immediately after the 60 min hOKR training was not significantly different among seven training paradigms. The hOKR adaptation was still in progress during a spacing interval, and the increment in hOKR gain tended to be greater with the longer spacing interval. The increase in hOKR gain was maintained until 48 hr after the end of training in both massed and spaced training. CONCLUSION: The short-term learning effect was not significantly different among training paradigms regardless of spacing interval in hOKR adaptation, which suggests that the spacing effect is robust enough to overcome the shortage of optokinetic training cycles in hOKR adaptation.

Differential effects of inferior olive lesion on vestibulo-ocular and optokinetic motor learning.

The combined operation of optokinetic reflex (OKR) and vestibular-ocular reflex (VOR) is essential for image stability during self-motion. Retinal slip signals, which provide neural substrate for OKR and VOR plasticity, are delivered to the inferior olive. Although it has been assumed that the neural circuitry and mechanisms underlying OKR and VOR plasticity are shared, differential role of the inferior olive in the plasticity of OKR and VOR has not been clearly established. To investigate the differential effect of inferior olive lesion on OKR and VOR plasticity, we examined the change of OKR and VOR gains after gain-up and gain-down VOR training. The results demonstrated that inferior olive-lesion differentially affected cerebellum-dependent motor learning. In control mice, OKR gain increased after both gain-up and gain-down VOR training, and VOR gain increased after gain-up VOR training and decreased after gain-down VOR training. In inferior olive-lesioned mice, OKR gain decreased after both gain-up and gain-down VOR training, and while VOR gain did not significantly change after gain-up VOR training, VOR gain decreased after gain-down VOR training. We suggest that multiple mechanisms of plasticity are differentially involved in VOR and OKR adaptation, and gain-up and gain-down VOR learning rely on different plasticity mechanisms.

Density difference between perilymph and endolymph: A new hypothesis for light cupula phenomenon.

Light cupula is an emerging concept accounting for positional nystagmus. It can be diagnosed when persistent geotropic direction-changing positional nystagmus (PG DCPN) is observed in a head-roll test. Although hypotheses explaining light cupula phenomenon such as "light debris", "lighter cupula", and "heavier endolymph" have been proposed, the mechanism underlying light cupula has not been clearly elucidated yet. In the present study, we proposed a new hypothesis accounting for light cupula, i.e., density difference between perilymph and endolymph could elicit characteristic PG DCPN in a head-roll test. We also discussed the mechanism how membranous canal containing endolymph became buoyant within the perilymphatic space under constant influence of gravity when the density of perilymph was higher than that of endolymph.

The Light Cupula: An Emerging New Concept for Positional Vertigo.

Benign paroxysmal positional vertigo (BPPV) is the most common type of positional vertigo. A canalolithiasis-type of BPPV involving the lateral semicircular canal (LSCC) shows a characteristic direction-changing positional nystagmus (DCPN) which beats towards the lower ear (geotropic) on turning the head to either side in a supine position. Because geotropic DCPN in LSCC canalolithiasis is transient with a latency of a few seconds, the diagnosis can be challenging if geotropic DCPN is persistent without latency. The concept of "light cupula" has been introduced to explain persistent geotropic DCPN, although the mechanism behind it requires further elucidation. In this review, we describe the characteristics of the nystagmic pattern in light cupula and discuss the current evidence for possible mechanisms explaining the phenomenon.

Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors.

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg(+2), ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 µM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.