Neuroprotective Autophagic Flux Induced by Hyperbaric Oxygen Preconditioning is Mediated by Cystatin C / 神经科学通报·英文版

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.

Human tissue kallikrein-1 protects against the development of erectile dysfunction in a rat model of hyperhomocysteinemia / 亚洲男科学杂志(英文版)

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.

Direct modification of spermatogonial stem cells using lentivirus vectors in vivo leads to efficient generation of transgenic rats / 亚洲男科学杂志(英文版)

Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.

Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Enhanced Green Fluorescent Protein-transgenic Rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To isolate, culture, and identify bone marrow mesenchymal stem cells (BMSCs) from enhanced green fluorescent protein (EGFP)-transgenic rats in vitro.</p><p><b>METHODS</b>Bone marrows were isolated from tibia and femur of healthy EGFP-transgenic rats of specific pathogen free (SPF) grade. Then,the whole bone marrow adherent method was used for isolation,culture,and purification of BMSCs. The morphological change was noted by continuous observation under inverted fluorescence microscope. The growth curve of cells was drawn through the method of CCK-8 and the proliferation compared with wild type BMSCs. The surface markers of BMSCs were detected by flow cytometry. The BMSCs were induced to differentiate into osteoblasts, adipocytes, and chondrocytes lineages. The EGFP-BMSCs were transplanted into the rats intravenously, and the expression of GFP was detected.</p><p><b>RESULTS</b>BMSCs stably expressing EGFP gene were obtained successfully, with the fusiform-shaped appearance and the forming of circinate cell colonies. The growth curve of EGFP-MSCs showed the characteristic of active proliferation, showing no significant difference compared with the wild-type BMSCs. The expression rates of the surface markers of BMSCs CD29, CD90, CD34, CD49d, and CD45 were 99.4%, 96.4%, 0.171%, 0.049%, and 0.038%. The GFP were detected in lung 3 days after transplantation. After osteogenic, adipogenic, and chondrogenic induction, oil red-O and alizarin red positive signals and toluidine blue positive cells were detected.</p><p><b>CONCLUSIONS</b>High-purity BMSCs stably expressing green fluorescent protein gene can be cultured using the whole bone marrow adherent method. EGFP does not affect the stem cell properties and expresses stably after transplantation. The cells can be used as seed cells for subsequent research.</p>

Animal Models of Spondyloarthritis

The clinical spectrum of spondyloarthritis is included various diagnostic entities that share clinical, genetic and pathological characteristics. As human tissue specimens of the sacroiliac joints are very difficult to obtain, most of the new concepts have emerged from different animal models of disease. Animal models are available for the study of several different aspects of spondyloarthritis. The models include human leukocyte antigen (HLA) B-27 based on transgenic rat and mouse models, inflammation-driven models, and models of ankylosing enthesitis. Areas of investigation to which these models contribute include the role of HLA B-27, process of spinal and peripheral joint inflammation and calcification, immune responses to candidate antigens and the role of tumor necrosis factor.

Suppression of prostate cancer growth by resveratrol in the transgenic rat for adenocarcinoma of prostate (TRAP) model.

Research into actions of resveratrol, abundantly present in red grape skin, has been greatly stimulated by its reported beneficial health influence. Since it was recently proposed as a potential prostate cancer chemopreventive agent, we here performed an in vivo experiment to explore its effect in the Transgenic Rat for Adenocarcinoma of Prostate (TRAP) model, featuring the rat probasin promoter/SV 40 T antigen. Resveratrol suppressed prostate cancer growth and induction of apoptosis through androgen receptor (AR) down-regulation, without any sign of toxicity. Resveratrol not only downregulated androgen receptor (AR) expression but also suppressed the androgen responsive glandular kallikrein 11 (Gk11), known to be an ortholog of the human prostate specific antigen (PSA), at the mRNA level. The data provide a mechanistic basis for resveratrol chemopreventive efficacy against prostate cancer.