Tree shrew neural stem cell transplantation promotes functional recovery of tree shrews with a hemi­sectioned spinal cord injury by upregulating nerve growth factor expression.

The aim of the present study was to determine the effect of implanted neural stem cells (NSCs) on the functional recovery of tree shrews (TSs) subjected to hemi­sectioned spinal cord injury (hSCI), and to investigate the possible mechanism involved. NSCs (passage 2), derived from the hippocampus of TSs (embryonic day 20), were labeled with Hoechst 33342 and transplanted intraspinally into the hSC of TSs at thoracic level 10 in the acute (immediately after injury) and chronic (day 9 post­injury) stages. The Basso­Beattie­Bresnahan (BBB) score was recorded from days 1 to 16 post­injury, and the survival, migration, differentiation and neurotrophic factor (NTF) expression in vivo were detected. In vitro and in vivo, the expanded NSCs were able to differentiate into neurons and astrocytes, and secreted a variety of NTFs, including ciliary NTF, transforming growth factor­ß1, glial cell line­derived NTF, nerve growth factor (NGF), brain­derived NTF and insulin­like growth factor. Following transplantation, the BBB score in the TSs with chronic­stage transplantation exhibited a statistically significant increase, while there was no significant difference in the acute group, compared with the control group. This corresponded with the marked upregulation of NGF indicated by reverse transcription­quantitative polymerase chain reaction. In conclusion, the transplantation of NSCs into the hSC in the chronic phase, but not the acute stage, of hSCI in non­human primate TSs is effective and associated with upregulated NGF expression. These findings may provide novel strategies for the treatment of SCI in clinical patients.

[Isolation and identification of Tupaia orthoreovirus].

Pathogenic viruses can harm acutely the life and health of laboratory tree shrews acutely; however, few papers exist regarding natural pathogenic virus infection in this species. Six fecal samples obtained from dead tree shrews were collected. The fecal supernatant infected Vero cell line resulted in cytopathic effects (CPE) after 72 h. The CPE included granulating, shrinking, rounding, seining and falling off. Electron microscopy showed the isolation was spherical, double-layered capsid, and about 75 nm in diameter. The purified isolation genome was 10 segments in a typical 3:3:4 arrangements, as shown by polyacrylamide gel electrophoresis (PAGE). The isolation was confirmed by RT-PCR assays targeting the conserved region of the L1 gene, sequence analysis and reconstruction of a phylogenetic tree. The isolation was a Tupaia Orthoreovirus (TRV), belonging to Mammalian Orthoreovirus (MRV). The obtained strain had the closest phylogenetic relationship to the MRV strain T3/Bat/Germany/342/08. As a zoonotic virus, the novel TRV strain was first isolated from wild tree shrews, which is significant for promoting tree shrew standardization and providing scientific data for preventing zoonotic tree shrew-to-human transmission.

[Experimental study on the rhesus monkey corneal endothelial cells substituted by the allogeneic vascular endothelial cells cultivated in vitro].

OBJECTIVE: To Explore the feasibility of rhesus monkey vascular endothelial cell(RMVEC) transplantation to substitute the allogeneic corneal endothelial cells(CEC), through observe the morphologic and functional change of the vascular endothelial cell which was transplanted to the inner surface of cornea without Descemet's membrane. METHODS: It was an experimental study. The rhesus monkey vascular endothelial cell was cultivated to proliferation and marked by BrdU in vitro. The experimented monkeys are divided into 2 groups: experimental group (6 monkeys), control group (3 monkey). In experimental group: the cultured RMVEC, marked by BrdU, were transplanted onto the posterior surface of rhesus monkey cornea without Descemet's membrane though centrifugal sedimentation, then sew back the corneal graft. In control group: detach the corneal Descemet's Membrane of rhesus monkey but without cultured RMVEC transplantation. Corneal transparency of every monkey was frequently observed postoperation. On 30, 60, 90 postoperative days, corneal graft were respectively detached to observe the distribution, appearance and ultrastructures morphological structure of RMVEC on the inner surface of cornea, by pathological section, anti BrdU monoclonal antibody immunohistochemistry, scanning electronic microscope(SEM) and transmission election microscopy(TEM). RESULTS: Corneal transparency: In the experimental observation period (three months), the corneal graft of experimental group had better transparency than control group and without corneal neovascularization and bullous keratopathy. Pathological section: A layer of cells with BrdU staining positive was found on the posterior surface of cornea in experimental group, indicated the cells are RMVEC. And no cell-like structure was found in control group. SEM: Experimental group showed that RMVEC with irregular shape uniformly distributed on the inner surface of cornea and growing well, a small amount of white blood cells can be seen between RMVEC, and part of cellular debris exist in the trabecular meshwork. Control group showed a fiber material without RMVEC. TEM: The cultured RMVEC and which in posterior surface of cornea was irregular oblateness, a large number of desmosomes link were been seen between RMVEC. Abundant organelles and characteristic WPBs appear in cytoplasm, which suggest the characteristics and vitality of vascular endothelial cells in vivo, and no cell structure in the control group. CONCLUSIONS: Rhesus monkey endothelial cells can growth on the posterior surface of cornea without Descemet's membrane. The cultured cells, with similar ultrastructure to RMVEC in vivo, can play a role of barrier to keep the corneal dehydration and transparency to some extent.

[Vaccination of rhesus monkeys with recombinant antigen fragments and protection from hepatitis E virus infection].

OBJECTIVE: To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta). METHODS: Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR. RESULTS: Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus. CONCLUSIONS: The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.