[Implantation strategy of tissue-engineered liver based on decellularized spleen matrix in rats].

OBJECTIVE: To explore the optimal implantation strategy of tissue-engineered liver (TEL) constructed based on decellularized spleen matrix (DSM) in rats. METHODS: DSM was prepared by freeze-thawing and perfusion with sodium dodecyl sulfate (SDS) of the spleen of healthy SD rats. Primary rat hepatocytes isolated using modified Seglen 2-step perfusion method were implanted into the DSM to construct the TEL. The advantages and disadvantages were evaluated of 4 transplant strategies of the TEL, namely ectopic vascular anastomosis, liver cross-section suture transplantation, intrahepatic insertion and mesenteric transplantation. RESULTS: The planting rate of hepatocytes in the DSM was (74.5∓7.7)%. HE staining and scanning electron microscopy showed satisfactory cell status, and immunofluorescence staining confirmed the normal expression of ALB and G6Pc in the cells. For TEL implantation, ectopic vascular anastomosis was difficult and resulted in a mortality rate of 33.3% perioperatively and massive thrombus formation in the matrix within 6 h. Hepatic cross-section suture failed to rapidly establish sufficient blood supply, and no viable graft was observed 3 days after the operation. With intrahepatic insertion method, the hepatocytes in the DSM could survive as long as 14 days. Mesenteric transplantation resulted in a hepatocyte survival rate of (38.3+7.1)% at 14 days after implantation. CONCLUSION: TEL constructed based on DSM can perform liver-specific functions with a good cytological bioactivity. Mesenteric transplantation of the TEL, which is simple, safe and effective, is currently the optimal transplantation strategy.

SNAI1, an endothelial-mesenchymal transition transcription factor, promotes the early phase of ocular neovascularization.

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target.

Macrophages promote vasculogenesis of retinal neovascularization in an oxygen-induced retinopathy model in mice.

To investigate the role of macrophages in oxygen-induced retinal neovascularization (NV) in mice, particularly the involvement of bone marrow-derived cells (BMCs) and the underlying mechanisms, BMCs from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P) 1 mice after irradiation. The mice were exposed to 75 % oxygen from P7 to P12 to initiate oxygen-induced retinopathy (OIR). The macrophages were depleted by injection of clodronate-liposomes (lip) intraperitoneally. The eyes were collected at P12 and P17. Retinal flatmounts and histopathological cross-sections were performed to analyze the severity of retinal NV and BMC recruitment. BMCs immunopositive for CD31 (PECAM-1; endothelial cell marker) and α-SMA (smooth muscle cell marker) antigens were detected using a confocal microscope. Expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) mRNA was detected by RT-PCR. The VEGF, SDF-1, CXCR4 and CD45 protein expression was detected by western blot examination. The retinal avascular area in OIR mice at P12 was unaffected after macrophage depletion carried out twice (38.27 ± 1.92 % reduction) using clodronate-lip. The retinal avascular area and the NV area at P17 were reduced after macrophage depletion four times (79.53 ± 1.02 % reduction); these findings were supported by retinal flatmounts and histopathological cross-sections. Macrophage depletion led to significant inhibition of BMC recruitment into the NV tufts at P17, with decreased expression of retinal VEGF, SDF-1, CXCR4 and CD45. The recruited BMCs differentiated primarily into CD31-positive endothelial cells (ECs) and α-SMA-positive smooth muscle cells (SMCs). This study suggested that macrophages promoted the vasculogenesis of retinal NV, particularly the contribution of BMCs in the mouse OIR model, which might be triggered by VEGF and SDF-1 production.

In-vivo investigation of laser-induced choroidal neovascularization in rat using spectral-domain optical coherence tomography (SD-OCT).

PURPOSE: This study investigated the in-vivo formation process of laser-induced choroidal neovascularization (CNV) in rat using high-resolution spectral-domain optical coherence tomography (SD-OCT), and compared the results to histological methods. METHODS: Brown Norway rats (n = 60, 6-8 weeks of age) received 532-nm diode laser photocoagulation. SD-OCT and fluorescein angiography (FA) were performed in vivo 2, 5, 7, 14, and 21 days post-laser application. Haematoxylin and eosin (H&E) staining and immunohistochemistry for CD31, phosphorylated vascular endothelial factor receptor 2 (pVEGFR2) were conducted at each time point to observe the CNV in vitro. Choroidal flatmount preparations were observed using a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM). RESULTS: SD-OCT monitored the longitudinal morphological changes of laser-induced CNV. CNV reached its maximal size on day 7, and began a gradual reduction on day 14. FA revealed similar dynamic changes in leakage. CNV thickness, as assessed by SD-OCT, was consistent with H&E-stained sections at each time point. CLSM and SEM revealed the details of the fibrovascular membrane. CD31 and pVEGFR2 expression supported the results of SD-OCT and histology. CONCLUSIONS: SD-OCT was a convenient and reliable tool for the imaging of the CNV formation process and quantification of the lesion size in vivo.