Combination of BMSCs-laden acellular nerve xenografts transplantation and G-CSF administration promotes sciatic nerve regeneration.

Peripheral nerve gaps often lead to interrupted innervation, manifesting as severe sensory and motor dysfunctions. The repairs of the nerve injuries have not achieved satisfactory curative effects in clinic. The transplantation of bone marrow stromal cells (BMSCs)-laden acellular nerve xenografts (ANX) has been proven more effective than the acellular nerve allografting. Besides, granulocyte colony-stimulating factor (G-CSF) can inhibit inflammation and apoptosis, and thus is conducive to the microenvironmental improvement of axonal regeneration. This study aims to investigate the joint effect of BMSCs-seeded ANX grafting and G-CSF administration, and explore the relevant mechanisms. Adult SD rats were divided into five groups randomly: ANX group, ANX combined with G-CSF group, BMSCs-laden ANX group, BMSCs-laden ANX combined with G-CSF group, and autograft group. Eight weeks after transplantation, the detection of praxiology and neuroelectrophysiology was conducted, and then the morphology of the regenerated nerves was analyzed. The inflammatory response and apoptosis in the nerve grafts as well as the expression of the growth-promoting factors in the regenerated tissues were further assayed. G-CSF intervention and BMSCs implanting synergistically promoted peripheral nerve regeneration and functional recovery following ANX bridging, and the restoration effect was matchable with that of the autologous nerve grafting. Moreover, local inflammation was alleviated, the apoptosis of the seeded BMSCs was decreased, and the levels of the neuromodulatory factors were elevated. In conclusion, the union application of BMSCs-implanted ANX and G-CSF ameliorated the niche of neurotization and advanced nerve regeneration substantially. The strategy achieved the favorable effectiveness as an alternative to the autotransplantation.

Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways.

Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of Grb10 expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmt1 overexpression dramatically reversed Grb10 up-regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that Grb10 expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future.

Synergistic effects of G-CSF and bone marrow stromal cells on nerve regeneration with acellular nerve xenografts.

Peripheral nerve defects result in severe denervation presenting sensory and motor functional incapacitation. Currently, a satisfactory therapeutic treatment promoting the repair of injured nerves is not available. As shown in our previous study, acellular nerve xenografts (ANX) implanted with bone marrow stromal cells (BMSCs) replaced allografts and promoted nerve regeneration. Additionally, granulocyte-colony stimulating factor (G-CSF) has been proven to mobilize supplemental cells and enhance vascularization in the niche. Thus, the study aimed to explore whether the combination of G-CSF and BMSC-laden ANX exhibited a synergistic effect. Adult Sprague-Dawley (SD) rats were randomly divided into five groups: ANX group, ANX combined with G-CSF group, BMSCs-laden ANX group, BMSCs-laden ANX combined with G-CSF group and autograft group. Electrophysiological parameters and weight ratios of tibialis anterior muscles were detected at 8 weeks post-transplantation. The morphology of the regenerated nerves was assayed, and growth-promoting factors present in the nerve grafts following G-CSF administration or BMSCs seeding were also investigated. Nerve regeneration and functional rehabilitation induced by the combination therapy were significantly advanced, and the rehabilitation efficacy was comparable with autografting. Moreover, the expression of Schwann cell markers, neurotrophic factors and neovessel markers in the nerve grafts was substantially increased. In conclusion, G-CSF administration and BMSCs transplantation synergistically promoted the regeneration of ANX-bridged nerves, which offers a superior strategy to replace autografts in repairing peripheral nerve injuries.

The Catalytically Lignan-Activation-Based Approach for the Synthesis of (epi)-Podophyllotoxin Derivatives.

Under the effect of a catalytic amount of Au(I) complex, 4-O-(2-cyclopropylethynyl)benzoyl-(epi)-podophyllotoxins, easily prepared via dehydrative condensation between (epi)-podophyllotoxin and ortho-cyclopropylethynylbenzoic acid, could efficiently couple with a variety of nucleophiles including alcohol, phenol, aniline, and carbon nucleophiles, all to provide (epi)-podophyllotoxin derivatives. Thus, the first catalytic and lignan-activation-based approach for (epi)-podophyllotoxin derivatization was established. Based on the new methodology, as well as the judicious choice of N3, AZMB, and Cbz protecting groups, an efficient approach forward was set. NK-611, an antitumoral agent at a phase II clinical trial was established, featuring an in situ anomerization of the hemiacetal OHs in the critical condensation step. Commencing from easily available starting material, the target molecule was obtained using the longest linear sequence of six steps and a 38% overall yield.

Expression of NR1 and apoptosis levels in the hippocampal cells of mice treated with MK­801.

The aim of the present study was to investigate the characteristics of N­methyl­D­aspartate receptor R1 (NR1) expression and apoptosis in the nerve cells of the hippocampus in schizophrenia­like mice. C57BL/6 mice were randomly allocated to the following groups: i) Blank group; ii) MK­801 group; iii) MK­801+NMDA group, according to body weight. The NMDAR antagonist, MK­801 (0.6 mg/kg/d) was intraperitoneally injected daily for 14 days to induce a schizophrenia­like phenotype mouse model, and the effect of the NMDA injection via the lateral ventricle was observed. The results demonstrated that the number of NR1 positive cells in the MK­801 group increased in the CA1 and DG regions, indicating that NMDA may reverse this change. The level of damage decreased in the MK­801 treated group when compared with the blank group in the CA3 region. The protein expression of NR1 increased however, at the mRNA expression level, NR1 was lower in the MK­801 treated group when compared to the blank group; NMDA also reversed this change. In addition, early and total apoptosis detected in the hippocampal nerve cells was significantly increased in the MK­801 group when compared with the blank group, which was reversible following treatment with NMDA. These results indicated that NMDA may regulate the expression of NR1 and suppress apoptosis in hippocampal nerve cells in schizophrenia­like mice. Thus, NR1 may be a promising therapeutic target for the treatment of schizophrenia.

Neurochemical features of endomorphin-2-containing neurons in the submucosal plexus of the rat colon.

AIM: To investigate the distribution and neurochemical phenotype of endomorphin-2 (EM-2)-containing neurons in the submucosal plexus of the rat colon. METHODS: The mid-colons between the right and left flexures were removed from rats, and transferred into Kreb's solution. For whole-mount preparations, the mucosal, outer longitudinal muscle and inner circular muscle layers of the tissues were separated from the submucosal layer attached to the submucosal plexus. The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin-coated glass slides, and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), nitric oxide synthetase (NOS), neuron-specific enolase (NSE), substance P (SP) and vasoactive intestinal peptide (VIP). After staining, all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGRP, ChAT, NOS, NSE, SP and VIP, ganglia, which have a clear boundary and neuronal cell outline, were randomly selected from each specimen for this analysis. RESULTS: In the submucosal plexus of the mid-colon, many EM-2-immunoreactive (IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM-2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for ChAT, SP, VIP or NOS. EM-2-IR nerve fibers coursed near ChAT-IR neurons, and some of these fibers were even distributed around ChAT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers, but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for ChAT, SP, VIP or NOS were approximately 91% ± 2.6%, 36% ± 2.4%, 44% ± 2.5% and 44% ± 4.7%, respectively, but EM-2 did not co-localize with CGRP. CONCLUSION: EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.

Neurochemical phenotype and function of endomorphin 2-immunopositive neurons in the myenteric plexus of the rat colon.

The distribution and activity of endomorphins (EMs), which are endogenous µ-opioid receptor (MOR) ligands in the gastrointestinal tract (GI), are yet to be elucidated. The current study aimed to shed light on this topic. EM2 was expressed in the enteric neurons in the myenteric plexus of the mid-colon. Of the EM2-immunoreactive (EM2-IR) neurons, 53 ± 4.6%, 26 ± 4.5%, 26 ± 2.8% and 49 ± 4.2% displayed immunopositive staining for choline acetyl transferase (ChAT), substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide synthetase (NOS), respectively. A bath application of EM2 (2 µM) enhanced spontaneous contractile amplitude and tension, which were reversed by ß-FNA (an antagonist of MOR) but not NG-nitro-L-arginine methyl ether (L-NAME, a non-selective inhibitor of NOS) or VIP6-28 (an antagonist of the VIP receptor) in the colonic strips. EM2 significantly suppressed inhibitory junction potentials (IJPs) in 14 of the 17 examined circular muscle cells, and this effect was not antagonized by preincubation in L-NAME. EM2 was widely expressed in interneurons and motor neurons in the myenteric plexus and presynaptically inhibited fast IJPs, thereby enhancing spontaneous contraction and tension in the colonic smooth muscle.