[An improved method for in vivo electroporation of morpholinos into the adult zebrafish retina].

In vivo electroporation of morpholinos (MOs) into the retina of adult zebrafish is an efficient method to study gene function related to retinal disease and regeneration. However, the currently reported methods are complicated with low MO transfer efficiency and high probability to cause collateral damage. The present study was aimed to optimize the existing MO electroporation methods. Two major changes were made to MO electroporation procedure in zebrafish retina. One was to coat the inner side of the electrode with ultrasonic gel. The other was to replace the commonly used round electrode with novel rectangular one. The results showed that the use of ultrasonic gel reduced collateral damage caused by retinal electroporation and simplified the experimental procedure. The rectangular electrode significantly increased transfection efficiency of MO electroporation. In particular, knocking down the expression of Ascl1a in the retina by using our method significantly inhibited the generation of retinal progenitor cells. These results suggest our method is the optimization of the current MO electroporation methods and may be a better alternative for relevant researchers.

New Iridoid Glycosides with Antidepressant Activity Isolated from Cyperus rotundus.

Based on bioactive screening results, two new iridoid glycosides, named rotunduside G (1) and rotunduside H (2), were isolated from the rhizomes of Cyperus rotundus, together with four known ones, negundoside (3), nishindaside (4), isooleuropein (5) and neonuezhenide (6). Their structures were elucidated on the basis of spectroscopic methods and from literature values. In mice models of despair, 1 and 2 showed significant antidepressant activity.

Association of genetic polymorphisms at 1q22 but not 10q23 with gastric cancer in a southern Chinese population.

OBJECTIVE: Data from a recent genome-wide association studiesy of gastric cancer (GC) and oesophageal squamous cell carcinoma in Chinese living in the Taihang Mountains of north-central China suggest that 1q22 and 10q23 are susceptibility-associated regions for GC. However, this has not been confirmed in southern Chinese populations. The aim of this study was to investigate whether these polymorphisms at 1q22 and 10q23 are associated with the risk of GC in a southern Chinese population. METHODS: We selected seven top significant associated single nucleotide polymorphisms (SNPs) at 1q22 and 10q23 and conducted a population-based case- control study in a southern Chinese population. Genotypes were determined using MassARRAYTM system (Sequenome, San Diego, CA). RESULTS: Two SNPs at 1q22, rs4072037 and rs4460629, were significantly associated with a reduced risk of GC, best fitting the dominant genetic model. Logistic regression models adjusted for age and sex showed that rs4072037 AG and GG (OR=0.64, P=0.017, compared with AA) and rs4460629 CT and TT (OR=0.54, P=0.0016, compared with TT) significantly reduced the risk of GC. However, no significant results for the five SNPs at 10q23 were obtained in this study. CONCLUSION: These outcomes indicate that 1q22 is associated with GC susceptibility in this southern Chinese population, while an association for the locus at 10q23 was not confirmed.

Transplanted neural stem cells promote nerve regeneration in acute peripheral nerve traction injury: assessment using MRI.

OBJECTIVE: The purpose of our study was to monitor neural stem cells (NSCs) transplanted in acute peripheral nerve traction injury and to use MRI to assess the ability of NSCs to promote nerve regeneration. MATERIALS AND METHODS: After labeling with gadolinium-diethylene triamine pentaacetic acid (gadopentetate dimeglumine) and fluorescent dye (PKH26), 5 × 10(5) NSCs were grafted to acutely distracted sciatic nerves in 21 New Zealand White rabbits. In addition, 5 × 10(5) unlabeled NSCs (n = 21) and vehicle alone (n = 21) subjects were injected as a control. Serial MRI was performed with a 1.5-T scanner to determine the distribution of grafted cells. Sequential T1 and T2 values of the nerves and functional recovery were measured over a 70-day follow-up period, with histologic assessments performed at regular intervals. RESULTS: The distribution and migration of labeled NSCs could be tracked with MRI until 10 days after transplantation. Compared with vehicle control, nerves grafted with labeled or unlabeled NSCs had better functional recovery and showed improved nerve regeneration but exhibited a sustained increase of T1 and T2 values during the phase of regeneration. CONCLUSION: Gadopentetate dimeglumine-based labeling allowed short-term in vivo MRI tracking of NSCs grafted in injured nerves. NSCs transplantation could promote nerve regeneration in acute peripheral nerve traction injury as shown by a prolonged increase of nerve T1 and T2 values.

Formulation, characterization and hypersensitivity evaluation of an intravenous emulsion loaded with a paclitaxel-cholesterol complex.

The objective of this paper was to develop a novel Cremophor-free, autoclave stable, intravenous emulsion for paclitaxel (PACE). A paclitaxel-cholesterol complex was used as the drug carrier to improve the solubility of paclitaxel in the oil phase of emulsions. The complex and PACE were prepared by rotary evaporation and high-pressure homogenization, respectively. Effects of oil phases, emulsifiers and pH values on the characteristics of PACE were investigated. PACE was characterized with regard to its appearance, morphology, osmolality, pH value, particle size, zeta potential, encapsulation efficiency and stability. Hypersensitivity was evaluated by guinea pig hypersensitivity reaction. The final formulation was composed of the complex, soybean oil, medium-chain triglyceridel, soybean lecithin, poloxamer 188 and glycerol. The resulting PACE had an encapsulation efficiency of 97.3% with a particle size of 135 nm and a zeta potential of -38.3 mV. Osmolality and pH of the formulation were 383 mOsmol/kg and 4.5, respectively. The formulation survived autoclaving at 115 °C for 30 min and remained stable for at least 12 months at 6 °C. PACE also exhibited a better tolerance than an equal dose of Cremophor-based paclitaxel injection in guinea pigs, as no obvious hypersensitivity reaction was observed. These results suggested that PACE has a great potential for industrial-scale production and clinical applications.

In vivo MR imaging tracking of transplanted mesenchymal stem cells in a rabbit model of acute peripheral nerve traction injury.

PURPOSE: To investigate in vivo MRI tracking mesenchymal stem cells (MSCs) in peripheral nerve injures using a clinically available paramagnetic contrast agent (Gd-DTPA) and commercially available rhodamine-incorporated transfection reagents (PEI-FluoR). MATERIALS AND METHODS: After bone marrow MSCs were labeled with Gd-DTPA and PEI-FluoR complex, the labeling efficacy and longevity of Gd-DTPA maintenance were measured and cell viability, proliferation, and apoptosis were assessed. Thirty-six rabbits with acute sciatic nerve traction injury randomly received 1 × 10(6) labeled (n = 12) or unlabeled MSCs (n = 12) or vehicle alone injection. The distribution and migration of implanted cells was followed by MRI and correlated with histology. The relative signal intensity (RSL) of the grafts was measured. RESULTS: The labeling efficiency was 76 ± 4.7% and the labeling procedure did not influence cell viability, proliferation, and apoptosis. A persistent higher RSL in grafts was found in the labeled group compared with the unlabeled and vehicle groups until 10 days after transplantation (P < 0.05). The distribution and migration of labeled cells could be tracked by MRI until 10 days after transplantation. Transplanted MSCs were not found to transdifferentiate into Schwann-like cells within 14-day follow-up. CONCLUSION: Labeling MSCs with the dual agents may enable cellular MRI of the engraftment in the experimental peripheral nerve injury.

Transdermal delivery of insulin using microneedle rollers in vivo.

This study characterizes skin perforation by commercially available microneedle rollers and evaluates the efficacy of transdermal delivery of insulin to diabetic rats. Three different needle lengths, 250, 500 and 1000 microm, were used in this work. Creation and resealing of the skin holes that were produced by the needles were observed by Evan's blue (EB) staining and transepidermal water loss (TEWL) measurements. EB clearly showed that microchannels were formed in the skin and that the pores created by the longest microneedle (1000 microm) persisted no longer than 8h, while the hypodermic injury was still observed 24h later. TEWL significantly increased after the application of the needles and then decreased with time, which explains the recovery of skin barrier function and agrees well with EB results. The extent of permeation was demonstrated by insulin delivery in vivo. The rapid reduction of blood glucose levels in 1h was caused by the increased permeability of the skin to insulin after applying microneedle rollers. The reduced decrease after 1h is closely associated with hole recovery. In conclusion, microneedle rollers with 500-microm or shorter lengths are safe and useful in transdermal delivery of insulin in vivo.

MR neurography: T1 and T2 measurements in acute peripheral nerve traction injury in rabbits.

PURPOSE: To prospectively evaluate magnetic resonance (MR) signal abnormalities and the time course of T1 and T2 values in a rabbit model of acute nerve traction injury with histologic and functional recovery correlation. MATERIALS AND METHODS: All experimental protocols were approved by the institutional animal use and care committee. Acute traction injury was produced in the sciatic nerve of one hind limb in each of 28 rabbits. The contralateral sham-operated nerves served as controls. Sequential MR imaging and T1 and T2 measurements, as well as measurements of functional changes, were obtained over a 70-day follow-up period, with histologic assessments performed at regular intervals. Signal abnormalities and the time course of T1 and T2 values were observed in the proximal, traction, and distal portions of the injured nerves and the sham-operated nerves, and were compared with each other. RESULTS: Nerves with acute traction injury showed visible hyperintense signals on T2-weighted images and had prolonged T1 and T2 values. Differences of T1 and T2 values were dependent on the sites along the same injured nerve, with the most pronounced and prolonged phase of T1 and T2 increases (peak values of 1333 msec +/- 46 and 79 msec +/- 3.7, respectively) observed in the most severely damaged portion of the injured nerve. T1 and T2 values and functional changes after nerve injury showed a similar time course. A return of T1 and T2 signals to normal values correlated with functional improvement. CONCLUSION: MR imaging could be used to help predict the degree of nerve damage and monitor the process of nerve recovery in acute peripheral nerve traction injury. (c) RSNA, 2010.

Magnetic resonance microneurography of rabbit sciatic nerve on a 1.5-T clinical MR system correlated with gross anatomy.

This study was performed to investigate the feasibility and accuracy of magnetic resonance (MR) microneurography of the rabbit sciatic nerve on a 1.5-T clinical MR system by correlation with the gross anatomy. The 3D T2-weighted imaging (3D-T2WI), 3D T2-weighted imaging plus spectral presaturation with inversion recovery (SPIR), and T1-weighted imaging (T1WI) of the sciatic nerve in ten rabbits were performed on a 1.5-T MR system. The radiological anatomy of the sciatic nerve was observed and correlated with the gross anatomy. The anterior-posterior diameter of the sciatic nerve trunk was measured on 3D T2WI and on gross anatomy. The T1 and T2 relaxation times were also measured with multiecho spin echo and mixed sequence, respectively. The tibial nerve and peroneal nerve in the sciatic nerve trunk in all ten rabbits could be clearly displayed on T2WI and T2WI. The fine branches of the gastrocnemius nerve, posterior femoral cutaneous nerve, and the posterior gluteal nerve could be clearly depicted on T2WI. The T1 and T2 relaxation times of the sciatic nerves were 915 and 40 ms, respectively. The anterior-posterior diameter of sciatic nerve trunk was measured grossly, and on T2WI was 3.17 +/- 0.21 mm and 3.15 +/- 0.19 mm, respectively. There was no statistically significant difference (t = 0.768, P = 0.462). With the 1.5-T clinical MR system, the microneurography of the sciatic nerve could be revisualized, and the finer structure of the sciatic nerve trunk could be clearly and accurately delineated.