Rat Model Of Degeneration Research Articles

Differential Expression of Kinin Receptors in Human Wet and Dry Age-Related Macular Degeneration Retinae.

Kinins are vasoactive peptides and mediators of inflammation, which signal through two G protein-coupled receptors, B1 and B2 receptors (B1R, B2R). Recent pre-clinical findings suggest a primary role for B1R in a rat model of wet age-related macular degeneration (AMD). The aim of the present study was to investigate whether kinin receptors are differentially expressed in human wet and dry AMD retinae. The cellular distribution of B1R and B2R was examined by immunofluorescence and in situ hybridization in post-mortem human AMD retinae. The association of B1R with inflammatory proteins (inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor A (VEGFA)), fibrosis markers and glial cells was also studied. While B2R mRNA and protein expression was not affected by AMD, a significant increase of B1R mRNA and immunoreactivity was measured in wet AMD retinae when compared to control and dry AMD retinae. B1R was expressed by Müller cells, astrocytes, microglia and endothelial/vascular smooth muscle cells, and colocalized with iNOS and fibrosis markers, but not with VEGFA. In conclusion, the induction and upregulation of the pro-inflammatory and pro-fibrotic kinin B1R in human wet AMD retinae support previous pre-clinical studies and provide a clinical proof-of-concept that B1R represents an attractive therapeutic target worth exploring in this retinal disease.

Technical experience and postoperative complications with repeat transperitoneal approach to the lumbar spine in female Lewis rats.

Rat models of lumbar intervertebral disc (IVD) degeneration are widely employed to characterize biologic-based therapeutics, but their anatomy and small size preclude consistent delivery of injectable therapeutics to the lumbar spine via the traditional posterolateral approach. Here, we describe our experience with a repeat ventral transperitoneal approach in female Lewis rats, enabling induction of IVD degeneration and later intervention via an injectable therapeutic. In the initial surgery, the ventral aspect of the L5/L6 IVD was accessed, and an annular defect was created using a #11 scalpel blade. Eight weeks after the initial surgery, follow-up surgery was performed via the same approach, and an injectable gelatin hydrogel was delivered using a 31G needle. A custom injection guard was developed to control injection depth, ensuring consistent delivery to the nucleus pulposus. Notable challenges associated with repeat surgery were increased tissue adhesion, intraoperative bleeding, and difficulty placing the injection guard due to mobile gastrointestinal tissues. Complication rates were 9.4% and 15.6% for the initial and repeat surgeries, respectively. The most frequent complications associated with repeat surgery were transient neuropraxia and significant intraoperative bleeding (6.3% each). The repeat transperitoneal approach is a reproducible method to facilitate both injury and later intervention in a female rat model of lumbar IVD degeneration.

AB0069 LORECIVIVINT (SM04690), AN INTRA-ARTICULAR, SMALL-MOLECULE CLK/DYRK1A INHIBITOR THAT MODULATES THE WNT PATHWAY, AS A POTENTIAL TREATMENT FOR MENISCAL INJURIES

Background:Meniscal injuries, associated with pain, stiffness, and localized swelling, are the most common pathology of the knee with a prevalence of 61 per 100,000.1Meniscal damage is a frequent finding on MRI images of knee osteoarthritis (OA)2; while a meniscal tear can lead to knee OA, knee OA can also lead to a spontaneous meniscal tear.3Efforts to repair meniscal damage have been largely unsuccessful and do not prevent the progression of degenerative changes that lead to knee OA.4The Wnt signaling pathway has been shown to be regulated during meniscal development,5,6suggesting that manipulation of this pathway may influence the regenerative capacity of the meniscus. Lorecivivint (LOR; SM04690) is an intra-articular (IA), small-molecule CLK/DYRK1A inhibitor that modulates the Wnt pathway.Objectives:LOR was evaluated in preclinical studies to determine its protective and anabolic effects in ex vivo explants and in a rat model of chemically induced inflammatory meniscus degeneration.Methods:Effects of LOR (30 nM) on expression of matrix metalloproteinases (MMPs) in cultured rat menisci treated with IL-1B were measured by qPCR. In vivo, LOR activity was evaluated in a rat model of monosodium iodoacetate (MIA) injection-induced inflammatory meniscus degeneration. A single IA injection of MIA was immediately followed by a single IA injection of LOR (0.3 ug) or vehicle. Knees were harvested on Days 1, 4, and 11 and menisci were isolated. Anti-inflammatory effects were evaluated by measuringTNFAandIL6expression by qPCR. Meniscus protection was evaluated by qPCR for MMPs and aggrecanase and anabolic effects by qPCR for collagens.Results:In ex vivo meniscal explants, LOR inhibited expression ofMMP1,MMP3, andMMP13compared to DMSO (P<0.01). In vivo, LOR significantly decreased expression of these MMPs and aggrecanase (P<0.05) compared to vehicle in the rat model of inflammatory meniscus degeneration at Day 4 after MIA injection. In addition, LOR reduced expression of inflammatory cytokinesTNFAandIL6at Day 4 compared to vehicle. Finally, LOR increased expression of collagen types I, II, and III at Day 11 after MIA injection.Conclusion:LOR exhibited protective effects in the meniscus ex vivo and in vivo by reducing the expression of catabolic enzymes compared to control. Anti-inflammatory effects of LOR were demonstrated by inhibition of inflammatory cytokine expression. Compared to vehicle, LOR increased expression of collagens in vivo, indicating potential meniscal anabolic effects. These data support further investigation of LOR as a potential disease-modifying therapy for meniscal injuries.

Gene Therapy in the Shaker Rat Model of Cerebellar Degeneration and Ataxia (2208)

Gene Therapy in the Shaker Rat Model of Cerebellar Degeneration and Ataxia (2208)

18F]-DPA-714 PET as a specific in vivo marker of early microglial activation in a rat model of progressive dopaminergic degeneration.

To study the feasibility of the in vivo [18F]-DPA-714 TSPO positron emission tomography (PET) to detect glial activation in a rat model of progressive parkinsonism induced by viral-mediated overexpression of A53T mutated human α-synuclein (hα-syn) in the substantia nigra pars compacta (SNpc). We conducted a cross-sectional study in a model of progressive parkinsonism. Bilateral intranigral injections with 2/9 adeno-associated viral vectors encoding either hα-syn (AAV-hα-syn) or green fluorescent protein (AAV-GFP) were performed in rats (n = 60). In vivo [18F]-DPA-714 PET imaging was performed at different time points after inoculation (p.i.) of the viral vector (24 and 72h and 1, 2, 3, and 16weeks). Images were analyzed to compute values of binding potential (BP) in the SNpc and striatum using a volume of interest (VOI) analysis. Immunohistochemistry of markers of dopaminergic degeneration (tyrosine hydroxylase (TH)), microglia (Iba-1), and astrocytes (GFAP) was carried out. Binding potential (BP) of [18F]-DPA-714 PET in the in vivo PET study was correlated with post-mortem histological markers. In the SNpc of AAV-hα-syn rats, there was higher in vivo [18F]-DPA-714 BP (p < 0.05) and increased number of post-mortem Iba-1+ cells (p < 0.05) from second week p.i. onwards, which were highly correlated (p < 0.05) between each other. These findings antedated the nigral reduction of TH+ cells that occurs since third week p.i. (p < 0.01). In addition, the [18F]-DPA-714 BP was inversely correlated (p < 0.05) with the TH+ cells. In contrast, GFAP+ cells only increased at 16weeks p.i. and did not correlate with the in vivo results. In the striatum, no changes in the number of Iba-1+ and GFAP+ cells were observed, but an increment in the [18F]-DPA-714 BP was found at 16weeks p.i. Our study showed that in vivo PET study with [18F]-DPA-714 is a selective and reliable biomarker of microglial activation and could be used to study preclinical stages of Parkinson's disease (PD) and to monitor the progression of the disease.

Duhuo jisheng decoction suppresses matrix degradation and apoptosis in human nucleus pulposus cells and ameliorates disc degeneration in a rat model.

The lower back pain (LBP) caused by intervertebral disc (IVD) degeneration brings a heavy burden to society. A classic treatment method of Chinese medicine, fangji-duhuo jisheng decoction (DHJSD), has been effective in the clinical treatment of LBP, although the underlying mechanism remains unknown. In this work, the main objective was to study the effects of DHJSD on in vitro IVD degeneration of human nucleus pulposus (NP) cells after pressure treatment and on an in vivo interrupted IVD degeneration rat model. The effects of DHJSD on the viability of NP cells were detected using Cell Counting Kit-8. RT-qPCR, western blotting, TUNEL assay, transmission electron microscopy, and immunofluorescence staining were performed to explore the molecular mechanism underlying protection against compression-induced matrix degradation and apoptosis in NP cells by DHJSD. Furthermore, the effects of DHJSD on IVD degeneration in a rat IDD model were also determined. We found that DHJSD increased the viability of NP cells in a concentration- and time-dependent manner. Furthermore, DHJSD significantly reduced compression-induced NP matrix degeneration and apoptosis, activated autophagy, and inhibited the p38/MAPK signaling pathway in NP cells subjected to compression. Autophagy inhibitor 3-MA and p38/MAPK signaling pathway activator anisomycin reversed the beneficial effects of DHJSD in NP cells, indicating that DHJSD protects against IVD degeneration by autophagy activation and P38/MAPK signaling pathway inhibition. Furthermore, DHJSD treatment effectively delayed IVD degeneration in a puncture-induced IDD rat model. DHJSD prevents compression-induced matrix degradation and cell apoptosis through regulating autophagy and the P38/MAPK signaling pathway. The mechanism underlying the effects of DHSJD elucidated in this study provides a new direction for LBP treatment.

Focused ultrasound-induced blood-brain barrier opening improves adult hippocampal neurogenesis and cognitive function in a cholinergic degeneration dementia rat model

BackgroundThe persistence of adult hippocampal neurogenesis (AHN) is sharply decreased in Alzheimer’s disease (AD). The neuropathologies of AD include the presence of amyloid-β deposition in plaques, tau hyperphosphorylation in neurofibrillary tangles, and cholinergic system degeneration. The focused ultrasound (FUS)-mediated blood-brain barrier opening modulates tau hyperphosphorylation, the accumulation of amyloid-β proteins, and increases in AHN. However, it remains unclear whether FUS can modulate AHN in cholinergic-deficient conditions. In this study, we investigated the effect of FUS on AHN in a cholinergic degeneration rat model of dementia.MethodsAdult male Sprague-Dawley rats (n = 48; 200–250 g) were divided into control (phosphate-buffered saline injection), 192 IgG-saporin (SAP), and SAP+FUS groups; in the two latter groups, SAP was injected bilaterally into the lateral ventricle. We applied FUS to the bilateral hippocampus with microbubbles. Immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting, 5-bromo-2′-deoxyuridine labeling, an acetylcholinesterase assay, and the Morris water maze test were performed to assess choline acetyltransferase, acetylcholinesterase activity, brain-derived neurotrophic factor expression, neural proliferation, and spatial memory, respectively. Statistical significance of differences in between groups was calculated using one-way and two-way analyses of variance followed by Tukey’s multiple comparison test to determine the individual and interactive effects of FUS on immunochemistry and behavioral analysis. P < 0.05 was considered significant.ResultsCholinergic degeneration in rats significantly decreased the number of choline acetyltransferase neurons (P < 0.05) in the basal forebrain, as well as AHN and spatial memory function. Rats that underwent FUS-mediated brain-blood barrier opening exhibited significant increases in brain-derived neurotrophic factor (BDNF; P < 0.05), early growth response protein 1 (EGR1) (P < 0.01), AHN (P < 0.01), and acetylcholinesterase activity in the frontal cortex (P < 0.05) and hippocampus (P < 0.01) and crossing over (P < 0.01) the platform in the Morris water maze relative to the SAP group after sonication.ConclusionsFUS treatment increased AHN and improved spatial memory. This improvement was mediated by increased hippocampal BDNF and EGR1. FUS treatment may also restore AHN and protect against neurodegeneration, providing a potentially powerful therapeutic strategy for AD.

Protective Effect of Polygonatum sibiricum Polysaccharides on Apoptosis, Inflammation, and Oxidative Stress in Nucleus Pulposus Cells of Rats with the Degeneration of the Intervertebral Disc

Objective. Polygonatum sibiricum polysaccharide (PSP) has antioxidant activity, immune enhancement, and other biological properties. However, the effect of PSP on intervertebral disc degeneration has not been reported. In this study, we mainly investigated the effect of PSP on the apoptosis, inflammation, and oxidative stress of nucleus pulposus cells (NPCs) during the process of intervertebral disc degeneration. Methods. A rat NPC model induced by H2O2 was constructed. The CCK8 method was used to measure the effects of PSP on the apoptosis of rat NPCs induced by H2O2. The effects on the activity of SOD and content of MDA were also determined. The rat model of intervertebral disc degeneration was treated with PSP for 1 month, and the mRNA expression levels of IL-1β, COX2, iNOS, Col2α1, Col10α1, and MMP3 were measured by qPCR in the tissue of intervertebral disc. NPCs from the degenerated intervertebral discs were separated, and the cell viability was measured by the CCK8 method. The contents of SOD and MDA in NPCs were determined as well. Results. PSP significantly reduced the apoptosis of NPCs induced by H2O2, significantly increased the SOD content, and decreased the content of MDA in H2O2-induced NPCs. The expression level of IL-1β, COX2, and iNOS in the rat model with intervertebral disc degeneration was significantly downregulated after 1 month of PSP treatment. PSP treatment increased the expression of Col2α1 type and significantly decreased the expression of Col10α1 type collagen and MMP3 in rats with disc degeneration. PSP treatment significantly reduced NPC apoptosis and increased its SOD content and reduced MDA content, which is consistent with the results from cell-level experiments. Conclusion. PSP can effectively reduce the apoptosis, inflammation, and oxidative stress of H2O2-induced NPCs in rats with intervertebral disc degeneration and mitigate the progression of intervertebral disc degeneration, which has the potential to be developed as new drugs for the treatment of intervertebral disc degeneration.

Detection of apoptosis and matrical degeneration within the intervertebral discs of rats due to passive cigarette smoking.

Although low-back pain is considered to be associated with cigarette smoking, the influence of cigarette smoking on the intervertebral discs (IVD) has not been confirmed. We established a rat model of passive cigarette smoking-induced IVD degeneration, and investigated the cytohistological changes in the IVD and the accompanying changes in gene expression. IVD from rats exposed to 8 weeks of passive cigarette smoking were stained with Elastica van Gieson, and exhibited marked destruction of the supportive structure of the reticular matrix in the nucleus pulposus (NP). Positive signals on safranin O, alcian blue, type II collagen and aggrecan staining were decreased in the destroyed structure. Safranin O and type II collagen signals were also decreased in the cartilage end-plate (CEP) after 4- and 8-weeks of cigarette smoking. In the CEP, the potential for apoptosis was increased significantly, as demonstrated by staining for single-strand DNA. However, there were no signs of apoptosis in the NP or annulus fibrosus cells. Based on these findings, we hypothesized that passive cigarette smoking-induced stress stimuli first affect the CEP through blood flow due to the histological proximity, thereby stimulating chondrocyte apoptosis and reduction of the extracellular matrix (ECM). This leads to reduction of the ECM in the NP, destroying the NP matrix, which can then progress to IVD degeneration.

L-dopa response pattern in a rat model of mild striatonigral degeneration.

BackgroundUnresponsiveness to dopaminergic therapies is a key feature in the diagnosis of multiple system atrophy (MSA) and a major unmet need in the treatment of MSA patients caused by combined striatonigral degeneration (SND). Transgenic, alpha-synuclein animal models do not recapitulate this lack of levodopa responsiveness. In order to preclinically study interventions including striatal cell grafts, models that feature SND are required. Most of the previous studies focused on extensive nigral and striatal lesions corresponding to advanced MSA-P/SND. The aim of the current study was to replicate mild stage MSA-P/SND with L-dopa failure.Methods and resultsTwo different striatal quinolinic acid (QA) lesions following a striatal 6-OHDA lesion replicating mild and severe MSA-P/SND, respectively, were investigated and compared to 6-OHDA lesioned animals. After the initial 6-OHDA lesion there was a significant improvement of motor performance after dopaminergic stimulation in the cylinder and stepping test (p<0.001). Response to L-dopa treatment declined in both MSA-P/SND groups reflecting striatal damage of lateral motor areas in contrast to the 6-OHDA only lesioned animals (p<0.01). The remaining striatal volume correlated strongly with contralateral apomorphine induced rotation behaviour and contralateral paw use during L-dopa treatment in cylinder and stepping test (p<0.001).ConclusionOur novel L-dopa response data suggest that L-dopa failure can be induced by restricted lateral striatal lesions combined with dopaminergic denervation. We propose that this sequential striatal double-lesion model replicates a mild stage of MSA-P/SND and is suitable to address neuro-regenerative therapies aimed at restoring dopaminergic responsiveness.

Retinal degeneration rat model: A study on the structural and functional changes in the retina following injection of sodium iodate

Retinal disorders account for a large proportion of ocular disorders that can lead to visual impairment or blindness, and yet our limited knowledge in the pathogenesis and choice of appropriate animal models for new treatment modalities may contribute to ineffective therapies. Although genetic in vivo models are favored, the variable expressivity and penetrance of these heterogeneous disorders can cause difficulties in assessing potential treatments against retinal degeneration. Hence, an attractive alternative is to develop a chemically-induced model that is both cost-friendly and standardizable. Sodium iodate is an oxidative chemical that is used to simulate late stage retinitis pigmentosa and age-related macular degeneration. In this study, retinal degeneration was induced through systemic administration of sodium iodate (NaIO3) at varying doses up to 80 mg/kg in Sprague-Dawley rats. An analysis on the visual response of the rats by electroretinography (ERG) showed a decrease in photoreceptor function with NaIO3 administration at a dose of 40 mg/kg or greater. The results correlated with the TUNEL assay, which revealed signs of DNA damage throughout the retina. Histomorphological analysis also revealed extensive structural lesions throughout the outer retina and parts of the inner retina. Our results provided a detailed view of NaIO3-induced retinal degeneration, and showed that the administration of 40 mg/kg NaIO3 was sufficient to generate disturbances in retinal function. The pathological findings in this model reveal a degenerating retina, and can be further utilized to develop effective therapies for RPE, photoreceptor, and bipolar cell regeneration.

Photo-crosslinked gelatin-hyaluronic acid methacrylate hydrogel-committed nucleus pulposus-like differentiation of adipose stromal cells for intervertebral disc repair.

Nucleus pulposus-like differentiation is always the challenge with application of stem cells for intervertebral disc repair. The combination of injectable biomaterials and stem cells may provide a resolution for this problem, as the transmembrane force can affect the intracellular environment through integrin αβ. In this study, we developed a strategy of photo-crosslinked gelatin-hyaluronic acid methacrylate (GelHA) hydrogel to commit the nucleus pulposus-like differentiation of adipose stromal cells (ASCs) for intervertebral disc repair. ASCs were isolated and cultured in GelHA hydrogel to evaluate nucleus pulposus-like differentiation. The function of integrin αvβ6 was investigated with neutralising antibody. The efficacy of ASCs with GelHA hydrogel for intervertebral disc repair was studied in a rat model of intervertebral disc degeneration. The results showed that GelHA hydrogel promoted ASCs nucleus pulposus-like differentiation and that integrin αvβ6 neutralising antibody prevented ASCs from expression of nucleus pulposus matrix in vitro. The combination of GelHA hydrogel and ASCs promoted quality intervertebral disc repair in rats with much more nucleus pulposus matrix and significantly higher disc height index. The findings have demonstrated that the combination of photo-crosslinked GelHA hydrogel and ASCs can commit ASCs to nucleus pulposus-like differentiation and improve the efficacy of ASCs for intervertebral disc repair. These findings suggest a promising stem cell-based strategy for intervertebral disc repair.

A novel rat model of fatty organ degeneration induced by poloxamer 407

Abstract Traditional methods of inducing hyperlipidemia in animal models are either costly (genetic manipulation) or it is difficult to control for the effects of other variables. For example, during high-fat feeding, the amount of high-fat diet intake per animal cannot be precisely controlled. The aim of this study was to develop an experimental model of fatty organ degeneration induced by poloxamer 407 (P-407). The study was conducted in 2-month-old, male Sprague-Dawley rats that were administered intraperitoneally with either 10% (w/w) P-407 (1 g/kg) or saline (10 mL/kg) for 4 months. Their lipid profile, organ degeneration due to fat deposition, and body mass were assessed. Intraperitoneal administration of P-407 resulted in significant increases in plasma triglycerides (P ≤ 0.001), total cholesterol (P &lt; 0.001), high-density lipoprotein-cholesterol (P ≤ 0.001), and low-density lipoprotein (P &lt; 0.001) cholesterol. In contrast to the control group, fatty tissue degeneration was observed in the liver, spleen, and kidneys of P-407-treated rats. Positive correlations between fatty tissue degeneration, and the atherogenic index of plasma (P &lt; 0.001) and the ratio of total cholesterol to high-density-lipoprotein (P &lt; 0.001) were identified. In addition, treatment with P-407 for 3 to 4 months caused a significant reduction in body mass relative to controls (P &lt; 0.001). Thus, this study describes the development of a cost-effective experimental rat model of organ degeneration, characterized by fat accumulation in the liver, spleen, and kidneys, which could be used for the study of steatosis and related diseases arising from sustained untreated dyslipidemia. Furthermore, both the atherogenic index of plasma and the ratio of total cholesterol to high-density lipoprotein-cholesterol can be used to predict the risk of fatty tissue degeneration in this model. The study was approval of the University of Jishou Biomedical Research Ethics Committee, China.

Treatment of Intervertebral Disk Disease by the Administration of mRNA Encoding a Cartilage-Anabolic Transcription Factor

Intervertebral disk (IVD) degeneration is often associated with severity of lower back pain. IVD core is an avascular, highly hydrated tissue composed of type II collagen, glycosaminoglycans, and proteoglycans. The disk degeneration is not only a destruction of IVD structure but also is related to a disorder of the turnover of the disk matrix, leading the jelly-like IVD core to be replaced by fibrous components. Here we present a disease-modifying strategy for IVD degenerative diseases by direct regulation of the cells in the IVD using mRNA medicine, to alter the misbalanced homeostasis during disk degeneration. When mRNA encoding a cartilage-anabolic transcription factor, runt-related transcription factor-1, was administered to a rat model of coccygeal disk degeneration using a polyplex nanomicelle composed of polyethylene glycol-polyamino acid block copolymers and mRNA, the disk height was maintained to a significantly higher extent (≈81%) compared to saline control (69%), with prevention of fibrosis in the disk tissue. In addition, the use of nanomicelles effectively prevented inflammation, which was observed by injection of naked mRNA into the disk. This proof-of-concept study revealed that mRNA medicine has a potential for treating IVD degenerative diseases by introducing a cartilage-anabolic factor into the host cells, proposing a new therapeutic strategy using mRNA medicine.

Establishing a rat model of intervertebral disc degeneration using three methods

Establishing a rat model of intervertebral disc degeneration using three methods

Experimental research on the effect of microRNA-21 inhibitor on a rat model of intervertebral disc degeneration.

Intervertebral disc degeneration is associated with angiogenesis and is the primary cause of disc-associated disease. Several studies have indicated the importance of microRNA (miR)-21 in angiogenesis. Thus, the present study aimed to validate the role and underlying mechanisms of miR-21 in a rat model of intervertebral disc degeneration. A total of 60 specific-pathogen-free Sprague-Dawley rats were used for in vivo experiments. A rat model of intervertebral disc degeneration was established and miR-21 inhibitor (antagomiR-21) was administered. The vertebral pulp and annulus fibrosus were isolated for immunohistochemical analysis of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) expression. Lumbar spine proteoglycan content was detected with the phloroglucinol method. Disc cell apoptosis was detected with terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining. It was revealed that antagomiR-21 treatment decreased the expression of HIF-1α and VEGF in the vertebral pulp and annulus fibrosus. Furthermore, antagomiR-21 treatment increased proteoglycan content and inhibited cell apoptosis in lumbar spines from model rats with intervertebral disc degeneration. In conclusion, antagomiR-21 treatment exerted a protective role in a rat model of intervertebral disc degeneration, which may provide the basis for a potential therapeutic approach in the treatment of disc-associated diseases.

Leptin regulates disc cartilage endplate degeneration and ossification through activation of the MAPK-ERK signalling pathway invivo and invitro.

Recent findings demonstrate that leptin plays a significant role in chondrocyte and osteoblast differentiation. However, the mechanisms by which leptin acts on cartilage endplate (CEP) cells to give rise to calcification are still unclear. The aim of this study was to evaluate the effects of leptin that induced mineralization of CEP cells in vitro and in vivo. We constructed a rat model of lumbar disc degeneration and determined that leptin was highly expressed in the presence of CEP calcification. Rat CEP cells treated with or without leptin were used for in vitro analysis using RT‐PCR and Western blotting to examine the expression of osteocalcin (OCN) and runt‐related transcription factor 2 (Runx2). Both OCN and Runx2 expression levels were significantly increased in a dose‐ and time‐dependent manner. Leptin activated ERK1/2 and STAT3 phosphorylation in a time‐dependent manner. Inhibition of phosphorylated ERK1/2 using targeted siRNA suppressed leptin‐induced OCN and Runx2 expression and blocked the formation of mineralized nodules in CEP cells. We further demonstrated that exogenous leptin induced matrix mineralization of CEP cells in vivo. We suggest that leptin promotes the osteoblastic differentiation of CEP cells via the MAPK/ERK signal transduction pathway and may be used to investigate the mechanisms of disc degeneration.

Transplantation of Hypoxic-Preconditioned Bone Mesenchymal Stem Cells Retards Intervertebral Disc Degeneration via Enhancing Implanted Cell Survival and Migration in Rats.

Objective Special hypoxic and hypertonic microenvironment in intervertebral discs (IVDs) decreases the treatment effect of cell transplantation. We investigated the hypothesis that hypoxic preconditioning (HP) could improve the therapeutic effect of bone mesenchymal stem cells (BMSCs) to IVD degeneration. Methods BMSCs from green fluorescent protein-transgenic rats were pretreated with cobalt chloride (CoCl2, 100 μM, 24 h) for hypoxic conditions in vitro. Apoptosis (related pathways of caspase-3 and bcl-2) and migration (related pathways of HIF-1α and CXCR4) were detected in BMSCs. In vivo, BMSCs and HP BMSCs (H-BMSCs) were injected into the rat model of IVD degeneration. The IVD height, survival, migration, and differentiation of transplanted BMSCs and matrix protein expression (collagen II, aggrecan, and MMP-13) were tested. Results H-BMSCs could extensively decrease IVD degeneration by increasing IVD height and collagen II and aggrecan expressions when compared with BMSCs. Significantly, more GFP-positive BMSCs were observed in the nucleus pulposus and annulus fibrosus regions of IVD. HP could significantly decrease BMSC apoptosis (activating bcl-2 and inhibiting caspase-3) and improve BMSC migration (increasing HIF-1α and CXCR4) in vitro. Conclusion HP could significantly enhance the capacity of BMSCs to repair DDD by increasing the survival and migration of implanted cells and increasing matrix protein expression.

Protection of Kaempferol on Oxidative Stress-Induced Retinal Pigment Epithelial Cell Damage.

The protection of retinal pigment epithelium (RPE) injury plays an important role in the prevention of or in delaying the pathological progress of retinal degeneration diseases, like age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa. Oxidative stress has been identified as a major inducer of RPE injury, which eventually could lead to a loss of vision. Kaempferol is a natural flavonoid widely distributed in many edible plants, fruits, and traditional medicines and has been reported to have antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. The present study demonstrates that the total antioxidant capacity of kaempferol is approximately two times stronger than that of lutein which is also a natural antioxidant that is widely used in the prevention or treatment of AMD. Our data indicates that kaempferol protects human RPE cells (ARPE-19) from hydrogen peroxide- (H2O2-) induced oxidative cell damage and apoptosis through the signaling pathways involving Bax/Bcl-2 and caspase-3 molecules proofed by real-time PCR and Western blot results. Kaempferol also inhibits the upregulated vascular endothelial growth factor (VEGF) mRNA expression levels induced by H2O2 in ARPE-19 cells and affects the oxidation and antioxidant imbalanced system in ARPE-19 cells treated by H2O2 through the regulations of both the activities of reactive oxygen species (ROS) and superoxide dismutase (SOD). Furthermore, our in vivo experimental results show that in sodium iodate-induced retinal degeneration rat model, kaempferol could protect sodium iodate-induced pathological changes of retina tissue and retinal cells apoptosis as well as the upregulated VEGF protein expression in RPE cells. In summary, these novel findings demonstrate that kaempferol could protect oxidative stressed-human RPE cell damage through its antioxidant activity and antiapoptosis function, suggesting that kaempferol has a potential role in the prevention and therapeutic treatment of AMD or other retinal diseases mediated by oxidative stress.

Transplanted olfactory ensheathing cells restore retinal function in a rat model of light-induced retinal damage by inhibiting oxidative stress.

There is still not an effective treatment for continuous retinal light exposure and subsequent photoreceptor degeneration. Olfactory ensheathing cell (OEC) transplantation has been shown to be neuroprotective in spinal cord, and optic nerve injury and retinitis pigmentosa. However, whether OECs protect rat photoreceptors against light-induced damage and how this may work is unclear. Thus, to elucidate this mechanism, purified rat OECs were grafted into the subretinal space of a Long-Evans rat model with light-induced photoreceptor damage. Light exposure decreased a- and b- wave amplitudes and outer nuclear layer (ONL) thickness, whereas the ONL of rats exposed to light for 24 h after having received OEC transplants in their subretinal space was thicker than the PBS control and untreated groups. A- and b- wave amplitudes from electroretinogram of OEC-transplanted rats were maintained until 8 weeks post OEC transplantation. Also, transplanted OECs inhibited formation of reactive oxygen species in retinas exposed to light. In vitro experiments showed that OECs had more total antioxidant capacity in a co-cultured 661W photoreceptor cell line, and cells were protected from damage induced by hydrogen-peroxide. Thus, transplanted OECs preserved retinal structure and function in a rat model of light-induced degeneration by suppressing retinal oxidative stress reactions.