Vein Of Rats Research Articles

In Vivo Bioluminescence Imaging for Targeting Acute Hypoxic/Ischemic Small Intestine with Engineered Salmonella typhimurium

This study aimed at investigating the feasibility of bioluminescence imaging (BLI) with engineered Salmonella typhimurium (ΔppGpp S. typhimurium) for visualizing acute hypoxic/ischemic bowels. At the start of 12- or 24-h reperfusion, ΔppGpp S. typhimurium was injected into the lateral tail veins of rats in which three segments of the small intestine were respectively subjected to 2, 3, and 4 h of ischemia. BLI and magnetic resonance imaging were performed at each reperfusion time point. Bioluminescence was exclusively detected in the hypoxic/ischemic segment of the intestine, showing the ability of ΔppGpp S. typhimurium to specifically target and proliferate in a hypoxic/ischemic area. Serial monitoring of these rat models revealed a progressive increase in bacterial bioluminescence in the ischemic intestines in conjunction with viable bacterial counts. The viable bacterial counts were positively correlated with lactate dehydrogenase levels after 24 h of reperfusion following 3 or 4 h of ischemia as well as interleukin-6 levels after 24 h of reperfusion following 4 h of ischemia. Our findings demonstrated that BLI was able to detect the acute hypoxic/ischemic bowel via monitoring of the distribution, internalization, and activity of administered ΔppGpp S. typhimurium. These findings may be useful for the early diagnosis of ischemic bowel disease.

The effect of intravenous administration to rats of magnetite nanoparticles with various shells on the functional state and morphology of the endothelium and on antioxidant status

Background . Today, the development of nanoparticles for targeted drug delivery is underway. A separate place is occupied by magnetic nanoparticles based on iron oxides (MNPs) due to their chemical stability, relatively high biocompatibility, and comparative ease of preparation. Unmodified MNPs can easily be oxidized, that can to lead to toxic effects and a loss of magnetic and colloidal stability, in order to avoid these effects, it is necessary to coat their surface, then to study of their effect on vascular endothelium and antioxidant status. Objective . The aim of the work was to study the effect of intravenous injection to rats of magnetic nanoparticles of iron oxide with various shells on the functional condition and morphology of the endothelium and on antioxidant status. Design and methods . In this work, we used colloidal solutions of magnetite nanoparticles (MNP) in a 0.9 % NaCl solution. MNPs were coated with albumin (MNP + albumin), polylactide (MNP + polylactide) and polysaccharide (MNP + polysaccharide). The study was performed on male rats of the Wistar SPF-category. MNP were injected once into the lateral tail vein of rats. Vascular morphology was studied by histological methods. The functional activity of the endothelium was studied by wire myography method. The concentration of nitric oxide and the concentration of lipid peroxidation products were evaluated by enzyme immunoassay. Total antioxidant activity and total antioxidant capacity of plasma were evaluated by using the method of luminol-activated chemiluminescence. Results . A single intravenous injection to rats of MNP + albumin and MNP + polylactide does not provide changes in vascular reactivity, however, the introduction of MNP + polysaccharide causes a decrease in endothelium-dependent relaxation while maintaining the ability to reduce. The studied types of magnetic nanoparticles do not affect the metabolic activity of the endothelium and vascular morphology. It was found that the introduction of the studied agents causes a decrease in the level of lipid peroxidation. MNP + albumin, MNP + polylactide, and MNP + polysaccharide, when it injected once intravenously, provide a similar effect, but different mechanisms of realization on the antioxidant status. Conclusion . The obtained data indicate that the studied species of MNPs have equal biocompatibility when they are once administered intravenously to rats.

Cirrhotic Endothelial Progenitor Cells Enhance Liver Angiogenesis and Fibrosis and Aggravate Portal Hypertension in Bile Duct-Ligated Cirrhotic Rats.

BackgroundCirculating cirrhotic endothelial progenitor cells (EPC) interact with both liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC) and promote angiogenesis in vitro. This study evaluated the effect of cirrhotic and control EPCs on hepatic angiogenesis, microcirculation, and fibrosis in vivo in rat models of cirrhosis.MethodologyAnimal models of cirrhosis were prepared by bile duct ligation (BDL). Circulating EPCs isolated from healthy human and cirrhotic blood were characterized by flow cytometry, cultured and administered through the tail vein in BDL rats after 2 weeks of ligation. The cells were given thrice a week for 2 weeks. The untreated group of BDL rats received only saline. Fibrosis was evaluated by Masson’s trichrome staining. Dedifferentiated LSECs were identified by the expression of CD31, and activated HSCs were marked as alpha-SMA-positive cells and were studied by immunohistochemistry and western blotting in saline-, healthy EPC-, and cirrhotic EPC-treated rats. In vivo, hepatic and systemic hemodynamic parameters were evaluated. Liver functions were evaluated.ResultsIn comparison to controls, BDL rats revealed an increase of fibrosis and angiogenesis. Among the treated rats, cirrhotic EPC-treated rats had increased fibrosis grade as compared to healthy EPC-treated and saline-treated rats. There was an increase of both fibrosis and angiogenesis markers, alpha-SMA and CD31 in cirrhotic EPC-treated rats as compared to healthy EPC-treated and saline-treated rats in immunohistochemistry and western blot studies. Cirrhotic EPC-treated BDL rats had high portal pressure and portal blood flow with significantly elevated hepatic vascular resistance in comparison with healthy EPC- and saline-treated BDL animals, without significant differences in mean arterial pressure. Cirrhotic EPC-treated BDL rats also showed a substantial increase in the hepatic expression of angiogenic receptors, VEGFR2 and CXCR4 in comparison with saline-treated rats.ConclusionThe study suggests that transplantation of cirrhotic EPCs enhances LSEC differentiation and angiogenesis, activates HSCs and worsens fibrosis, thus resulting in hepatic hemodynamic derangements in BDL-induced cirrhosis.

Rodent Leukocyte Isolation and Radiolabeling for Inflammation Imaging Study.

The objective of this study was to describe to develop methods of rodent leukocyte isolation and radiolabeling for in vivo inflammation imaging. Thigh muscle inflammation was induced by injection of collagenase. Blood was collected from the jugular vein and separated by Histopaque. The collected cells were incubated in a 37°C CO2 incubator for 1~2h. After incubation, 99mTc-HMPAO and 18F-FDG were used to treat leukocytes followed by incubation for 30min. 99mTc-HMPAO and 18F-FDG labeled autologous leukocytes were injected into the tail veins of rats. The images were then acquired at various time points. Image-based lesion to normal muscle ratio was compared. After Histopaque separation, the proportion of lymphocytes was higher than that of other cell types. After CO2 incubation, the collected leukocytes were viable, while room temperature exposed leukocytes without CO2 incubation were non-viable. Granulocytes, especially, were more quickly influenced by various conditions than the mononuclear cells. Labeling efficiencies of 99mTc-HMPAO and 18F-FDG were 4.00 ± 2.06 and 1.8%, respectively. 99mTc-HMPAO- and 18F-FDG-labeled leukocytes targeted well the inflamed lesion. 99mTc-HMPAO-labeled leukocytes, but not 18F-FDG-labeled leukocytes, were found in the abdomen activity. Inflamed lesions of rats were well visualized using autologous radiolabeled leukocytes. This method might provide good information for understanding inflammatory diseases.

The effect of intravenous human adipose-derived stem cells (hADSC) on transforming growth factor β1 (TGF-β1), collagen type 1, and kidney histopathological features in the unilateral ureteropelvic junction obstruction model of wistar rats.

The fibrotic process of kidney resulting in glomerulosclerosis was found in patients with ureteropelvic junction obstruction (UPJO) who underwent renal biopsy during pyeloplasty. Transforming growth factor β1 (TGF-β1) plays a role in collagen accumulation, resulting in fibrosis. Adipose tissue-derived stem cells (ADSCs) have an anti-apoptotic effect on target cells and enhance the kidney function recovery. We will further investigate the use of ADSC in the prevention of kidney fibrosis in the unilateral UPJO model of Wistar rats. A total of twenty-two 12-week-old Wistar rats were divided into three groups. We made the UPJO models using nylon 6-0 inside the left ureter and tied the ureter with nylon 6-0, creating partial ureteral obstruction. The treatment group was then injected with 1.0 × 106 cells of human ADSC via the tail vein of rats. All rats were euthanized after 2 and 4 weeks of treatment. The left kidney used hematoxylin-eosin for histopathological examination. Statistical analysis using one-way analysis of variance (ANOVA) was done with SPSS version 21.0. TGF-β1 concentration in the treatment group was significantly lower in the 4th week of observation (p4=0.0001), as well as collagen type 1, which was also significantly lower in the 4th week (p4=0.0001). There was a significant difference in the glomerulus count between the control group and the human ADSC (hADSC) group therapy in week 2 and week 4 (p2=0.0001 and p4=0.026). Administration of hADSC therapy reduces TGF-β1 and collagen type 1 levels and then improves the histopathological features in the process of renal fibrosis in the UPJO model.

Evidence of Accumulated Endothelial Progenitor Cells in the Lungs of Rats with Pulmonary Arterial Hypertension by 89Zr-oxine PET Imaging

Endothelial progenitor cells (EPCs) play a major role in regulating pulmonary vascular remodeling during pulmonary arterial hypertension (PAH) development. Several preclinical and clinical trials of EPCs transplantation have been performed for the treatment of PAH. However, there is no reliable method to monitor real-time cell trafficking and quantify transplanted EPCs. Here in this paper we isolated EPCs from human peripheral blood, identified their functional integrity, and efficiently labeled the EPCs with 89Zr-oxine and DiO. Labeled EPCs were injected into the tail vein of normal and PAH rats to be tracked in vivo. From the microPET/CT images, we found EPCs were distributed primarily in the lung at 1 h and then migrated to the liver and spleen. We could observe the 3,3′ dioctadecyloxacarbocyanine perchlorate (DiO)-labeled EPCs binding in the pulmonary vasculature by CellVizio confocal. The result of quantitative analysis revealed significantly higher accumulation of EPCs in the lungs of PAH rats than in those of healthy rats. The distribution and higher accumulation of EPCs in the lungs of PAH rats could help to evaluate the safety and provide evidence of effectiveness of EPC therapy.

MiR-379 relieves myocardial injury after acute myocardial infarction by regulating tumor necrosis factor-α-induced protein 8.

Acute myocardial infarction (AMI) is the myocardial avascular necrosis syndrome caused by coronary atherosclerotic plaque rupture, thrombosis or coronary artery occlusion. Therefore, it is of great significance to find new targets for the treatment of myocardial infarction. The purpose of this study was to investigate the effect of microRNA-379 (miR-379) on AMI and its mechanism. MiR-379 mimic was used to transfect H9c2 cells and we determined the protective effect of miR-379 on H9c2 by detecting the level of apoptosis. TargetScan software was used to detect miR-379's downstream targets. We constructed siRNA to analyze the effect of miR-379's downstream targets on H9c2 cells. In addition, we used miR-379 agomir to inject the tail vein of AMI rats to verify the effect of miR-379 on rat cardiomyocytes. TargetScan detected that miR-379 and Tumor necrosis factor-α-induced protein 8 (TNFAIP8) may have binding sites and the dual luciferase reporter assay found that miR-379 binds to TNFAIP8 and inhibits its activity. MiR-379 mimic was found to reduce the expression of caspase3 and caspase9 in H9c2 cells and thereby reduce H<inf>2</inf>O<inf>2</inf>-induced cell damage. Inhibition of TNFAIP8 also significantly reduced apoptosis level and inhibited the NF-κB signaling pathway in H9c2 cells. Finally, miR-379 agomir was used to inject the tail vein of AMI rats and verified the protective effect of miR-379 in the heart in vivo. MiR-379 has a binding site with TNFAIP8 and can inhibit its activity by binding to TNFAIP8 mRNA. SiRNA-TNFAIP8 can inhibit the NF-κB signaling pathway and protect myocardial cells from AMI-induced myocardial damage by reducing the apoptosis level of myocardial cells.

CTLA4-Ig Abatacept Ameliorates Proteinuria by Regulating Circulating Treg/IL-17 in Adriamycin-Induced Nephropathy Rats

Objective This study is aimed at investigating the efficacy of CTLA4-Ig abatacept in normalizing proteinuria and its possible mechanism in adriamycin-induced nephropathy (AIN) rats. Methods A total of 32 healthy male Sprague-Dawley rats were randomly divided into a normal group, an AIN group, an abatacept group, and a prednisone group. Adriamycin (6.5 mg/kg) was injected once via the tail vein of rats to induce nephrotic syndrome. After adriamycin treatment, the abatacept group rats were given abatacept (0.5 mg/kg) once by intraperitoneal injection on day 14. In addition, the prednisone group rats were given prednisone (12.5 mg/kg) daily consecutively by gavage from day 14 to day 21. Blood, urine, and kidney tissue specimens were collected when sacrificed on day 21. The 24-hour urinary protein, serum albumin, cholesterol, creatinine, and urea nitrogen were then detected. An enzyme-linked immunosorbent assay was used to determine the level of urine CD80 and serum IL-17. Flow cytometry was used to investigate the prevalence of circulating Treg. Hematoxylin-eosin staining and electron microscopy were used for a renal histological study. Immunofluorescence staining was performed to confirm the CD80 expression of renal tissue. Results The 24-hour urinary protein of the abatacept group was significantly lower than that of the prednisone group and the AIN group. The level of urine CD80 of the abatacept group was significantly lower than that of the AIN group. Compared with the AIN group and the prednisone group, the circulating Treg prevalence of the abatacept group was significantly higher, while the level of serum IL-17 was lower. A negative kidney staining of CD80 expression was demonstrated in each group in this study. The 24-hour urinary protein had a negative correlation with the circulating Treg prevalence and Treg/IL-17 and a positive correlation with the urine CD80 and serum IL-17. Urinary CD80 had a positive correlation with serum IL-17 and no correlation with the circulating Treg prevalence. Conclusions CTLA4-Ig abatacept can reduce proteinuria of adriamycin-induced nephropathy rats, possibly at least partially as a result of regulating circulating Treg/IL-17. CTLA4-Ig abatacept could be a promising regimen for idiopathic nephrotic syndrome.

Vascular smooth muscle cell phenotypic transition regulates gap junctions of cardiomyocyte

Atrial fibrillation (AF) is one of the most prevalent arrhythmias. Myocardial sleeves of the pulmonary vein are critical in the occurrence of AF. Our study aims to investigate the effect of synthetic vascular smooth muscle cells (SMCs) on gap junction proteins in cardiomyocytes. (1) Extraction of vascular SMCs from the pulmonary veins of Norway rats. TGF-β1 was used to induce the vascular SMCs switching to the synthetic phenotype and 18-α-GA was used to inhibit gap junctions of SMCs. The contractile and synthetic phenotype vascular SMCs were cocultured with HL-1 cells; (2) Western blotting was used to detect the expression of Cx43, Cx40 and Cx45 in HL-1 cells, and RT-PCR to test microRNA 27b in vascular SMCs or in HL-1 cells; (3) Lucifer yellow dye transfer experiment was used to detect the function of gap junctions. (1) TGF- β1 induced the vascular SMCs switching to synthetic phenotype; (2) Cx43 was significantly increased, and Cx40 and Cx45 were decreased in HL-1 cocultured with synthetic SMCs; (3) The fluorescence intensity of Lucifer yellow was higher in HL-1 cocultured with synthetic SMCs than that in the cells cocultured with contractile SMCs, which was inhibited by18-α-GA; (4) the expression of microRNA 27b was increased in HL-1 cocultured with synthetic SMCs, which was attenuated markedly by 18-α-GA. (5) the expression of ZFHX3 was decreased in HL-1 cocultured with synthetic SMCs, which was reversed by 18-α-GA. The gap junction proteins of HL-1 were regulated by pulmonary venous SMCs undergoing phenotypic transition in this study, accompanied with the up-regulation of microRNA 27b and the down-regulation of ZFHX3 in HL-1 cells, which was associated with heterocellular gap junctions between HL-1 and pulmonary venous SMCs.

Role of Thioredoxin‐interacting Protein in Diabetic Myocardial Ischemia‐Reperfusion Injury

BackgroundMyocardial ischemia‐reperfusion injury is a major threat to human health, causing great cardiac morbidity and mortality worldwide. Yet, it’s even more severe among diabetic patients. Thioredoxin‐interacting protein (TXNIP), a critical regulator with its inhibitory effect against antioxidation, is a novel target during myocardial ischemia‐reperfusion injury. TXNIP is also found to be highly expressed in hyperglycemic conditions, but its role in diabetic myocardial ischemia‐reperfusion injury is unclear. The current research aimed to study the role of TXNIP in myocardial ischemia‐reperfusion injury in diabetes mellitus.MethodsDiabetes was induced by streptozotocin injection via tail vein (65mg/kg) in Sprague‐Dawley rats. In vivo model of myocardial ischemia‐reperfusion injury was established as 30 minutes occlusion of the left anterior descending coronary artery and then 2 hours of reperfusion. In vitro hypoxia/re‐oxygenation (H/R) model was established as 6 hours of hypoxia and 12 hours of re‐oxygenation of H9C2 cells. Also, glucose was added to the medium at a concentration of 33 mmol/L to mimic the condition of diabetes. TXNIP small interference RNA (siRNA) was introduced to knock down the expression of TXNIP.ResultsThe expression of TXNIP significantly increased in the diabetic rat group, together with much greater infarct size and higher release of CK‐MB after myocardial ischemia‐reperfusion injury, compared to non‐diabetic group. In vitro study showed that TXNIP expression was significantly higher in high glucose (HG) group with greater cell injury and much higher LDH release after H/R treatment, compared to normal glucose (NG) group. However, TXNIP knock‐down via siRNA could alleviate this cellular injury with lower LDH release and higher cell viability. The expression of cleaved caspase‐3 significantly decreased even after H/R treatment in the TXNIP knock‐down group. Also, down‐regulation of NF‐kB and less phosphorylation of IkBα were detected after H/R in the TXNIP knock‐down group. Further study found higher conversion of LC3II from LC3I and lower expression of p62, indicating the activation of autophagy via TXNIP knock‐down. Finally, quantitative polymerase chain reaction test revealed significantly lower gene expression of TNF‐α and IL‐6.ConclusionTXNIP is highly expressed in diabetes after myocardial ischemia‐reperfusion injury. TXNIP knock‐down can alleviate myocardial ischemia‐reperfusion injury, possibly via activating autophagy and also via inhibiting inflammation. It is reasonable to believe that TXNIP would be a potential target for the treatment of diabetic myocardial ischemia‐reperfusion injury.Support or Funding InformationSupported by RGC (17118619) and NSFC (81970247)

Experimental study on the relationship between traumatic stress and tumor growth, proliferation, and metastasis*

Abstract Objective This study aimed to investigate the relationship between traumatic stress and tumor growth, proliferation, and metastasis. Methods A scalding method was used as an injurious factor to induce traumatic stress in Wistar rats. The rats were randomly divided into three groups—the control group, mild-scald group, and severe-scald group, with 14 rats in each group. Wistar rats were used to subculture the Walker-256 cell line for the generation of tumor ascites. Tumor cells from the ascites were cultured and used to establish a rat subcutaneous xenograft model. After 7 days, the mild-burn group and the severe-burn group were subjected burns to 10% and 15% of their backs, respectively. Blood was taken from the tail vein of rats at different times to detect changes in blood cortisol, IL-1β, and TNF-α levels. Pathological specimens were collected 14 days later, and immunohistochemistry was performed to examine vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), E-cadherin, and vimentin. Results Cortisol, IL-1β and TNF-α levels were significantly higher in the scalding groups than in the control group. Tumor examination was performed after 14 days. The changes in tumor size showed that the tumor volume in the control group (0.593 ± 0.195 cm3) and the mild-scald group (0.782 ± 0.344 cm3) were not significantly different. However, the tumor volume was significantly larger in the severe-burn group (1.806 ± 0.838 cm3) than in the control and the mild-burn groups(P &lt; 0.05). Tumor tissue immunohistochemistry showed that the percentage of cells expressing PCNA in the control group, mild-scald group, and severe-scald group was 57.1%, 71.4% and 85.7%, respectively, and the differences among the groups were statistically significant. The number of VEGF-positive cells in the mild- and severe-scald groups was significantly higher than those of the control group (P &lt; 0.05). The number of E-cadherin-positive cells in the tumor tissues was significantly lower in the severe-scald group than that in the control and mild-scald groups. Vimentin showed the opposite trend in the tumor tissue, and the differences were statistically significant (P &lt; 0.05). Conclusion Different degrees of a traumatic response in tissues caused by scalding can cause a corresponding stress response in the body. The release of inflammatory mediators; increase in VEGF, PCNA and vimentin in the tumor tissue; and decrease in E-cadherin lead to a change in tumor tissue growth and metastasis. Traumatic stress is associated with tumor growth, proliferation, and metastasis.

Compromised Ileal Mucus Barrier Due to Impaired Epithelial Homeostasis Caused by Notch1 Signaling in Cirrhotic Rats.

In liver cirrhosis, intestinal mucus barrier is rarely studied. This study aimed to investigate whether mucus barrier in ileum is altered in cirrhotic rats and its underlying mechanisms. Thioacetamide was injected to induce liver cirrhosis in rats. Serum from portal vein blood, and ileum and liver tissues were obtained for further analysis. Goblet cell-like Ls174T cells were cultured for in vitro experiments. The ileal mucus was thin, loose, and porous with small bubbles in cirrhotic rats. mRNA expressions of Muc2 and TFF3 were also down-regulated in cirrhotic rats. Bacteria located near to crypts and LPS were increased in the serum from portal vein in cirrhotic rats. Smaller theca area and few goblet cells were found in cirrhotic rats compared with control. Increased proliferation of ileal epithelia was observed in cirrhotic rats. Notch1, Dll1, and Hes1 expressions were enhanced, and KLF4 expression was suppressed in ileum of cirrhotic rats. In Ls174T cells, EDTA and NICD plasmid induced NICD and Hes1 expression and suppressed KLF4 concomitantly, and mucus expression almost vanished in these cells. NICD plasmid induced more proliferation in Ls174T cells. Oppositely, after DBZ treatment, NICD and Hes1 were inhibited along with augmentation of KLF4 and increased mucous expression in Ls174T cells, while proliferation of the cells was suppressed. In cirrhotic rats, mucus barrier was impaired. This might be attributed to increased proliferation and decreased differentiation of epithelia, which might be mediated by Notch1-Hes1-KLF4 signaling.

Comparison between the therapeutic effects of differentiated and undifferentiated Wharton's jelly mesenchymal stem cells in rats with streptozotocin-induced diabetes

Despite the availability of current therapies, including oral antidiabetic drugs and insulin, for controlling the symptoms caused by high blood glucose, it is difficult to cure diabetes mellitus, especially type 1 diabetes mellitus. Cell therapies using mesenchymal stem cells (MSCs) may be a promising option. However, the therapeutic mechanisms by which MSCs exert their effects, such as whether they can differentiate into insulin-producing cells (IPCs) before transplantation, are uncertain. In this study, we used three types of differentiation media over 10 d to generate IPCs from human Wharton's jelly MSCs (hWJ-MSCs). We further transplanted the undifferentiated hWJ-MSCs and differentiated IPCs derived from them into the portal vein of rats with streptozotocin-induced diabetes, and recorded the physiological and pathological changes. Using fluorescent staining and C-peptide enzyme-linked immunoassay, we were able to successfully induce the differentiation of hWJ-MSCs into IPCs. Transplantation of both IPCs derived from hWJ-MSCs and undifferentiated hWJ-MSCs had the therapeutic effect of ameliorating blood glucose levels and improving intraperitoneal glucose tolerance tests. The transplanted IPCs homed to the pancreas and functionally survived for at least 8 wk after transplantation, whereas the undifferentiated hWJ-MSCs were able to improve the insulitis and ameliorate the serum inflammatory cytokine in streptozotocin-induced diabetic rats. Differentiated IPCs can significantly improve blood glucose levels in diabetic rats due to the continuous secretion of insulin by transplanted cells that survive in the islets of diabetic rats. Transplantation of undifferentiated hWJ-MSCs can significantly improve insulitis and re-balance the inflammatory condition in diabetic rats with only a slight improvement in blood glucose levels.

MiR-424 Inhibits neuronal apoptosis in rats with cerebral infarction through regulating TGF-β1/Smad3 signaling pathway.

It has been reported that micro ribonucleic acid (miR)-424 is an important molecule in cerebral ischemia. However, the precise mechanism of action and biological effects of miR-424 remain to be further explored. miR-424 mimic and miR-424 inhibitor were injected via the caudal vein in rats, and the effect of miR-424 expression on brain tissue damage induced by middle cerebral artery occlusion (MCAO) was detected. The miR-424 mimic-induced changes in genomic levels were detected via the gene chip assay, and the signaling pathways regulated by miR-424 and its potential targets were explored combined with target prediction. Then the effect of miR-424 mimic on apoptosis of PC12 cells induced by oxygen-glucose deprivation (OGD) was determined using Annexin V/PI assay. Finally, drosophila mothers against decapentaplegic protein 7 (Smad7) was overexpressed to further verify the mechanism of action of miR-424 mimic. Compared with that in the sham group, the expression of miR-424 in brain tissues significantly declined in the model group. The results of 2,3,5-triphenyltetrazolium chloride (TTC) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that the miR-424 mimic obviously reduced the cerebral infarction area and apoptosis level of brain tissues, while the miR-424 inhibitor obviously increased the cerebral infarction area and apoptosis level of brain tissues. It was found, using bioinformatics and KEGG enrichment analysis, that differentially expressed genes induced by miR-424 were significantly enriched in the transforming growth factor-β (TGF-β) signaling pathway. According to the results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, the miR-424 mimic could evidently lower the expression of Smad7, thus activating the TGF-β1/Smad3 signaling pathway. Overexpression of Smad7 could weaken the protective effect of miR-424 mimic on ischemic-hypoxic cells. Increasing the expression of miR-424 can inhibit Smad7 to activate the TGF-β1/Smad3 signaling pathway, thereby exerting a protective effect against the brain tissue damage induced by MCAO.

GLP-2 Acutely Prevents Endotoxin-Related Increased Intestinal Paracellular Permeability in Rats.

Circulating endotoxin (lipopolysaccharide, LPS) increases the gut paracellular permeability. We hypothesized that glucagon-like peptide-2 (GLP-2) acutely reduces LPS-related increased intestinal paracellular permeability by a mechanism unrelated to its intestinotrophic effect. We assessed small intestinal paracellular permeability in vivo by measuring the appearance of intraduodenally perfused FITC-dextran 4000 (FD4) into the portal vein (PV) in rats 1-24h after LPS treatment (5mg/kg, ip). We also examined the effect of a stable GLP-2 analog teduglutide (TDG) on FD4 permeability. FD4 movement into the PV was increased 6h, but not 1 or 3h after LPS treatment, with increased PV GLP-2 levels and increased mRNA expressions of proinflammatory cytokines and proglucagon in the ileal mucosa. Co-treatment with a GLP-2 receptor antagonist enhanced PV FD4 concentrations. PV FD4 concentrations 24h after LPS were higher than FD4 concentrations 6h after LPS, reduced by exogenous GLP-2 treatment given 6 or 12h after LPS treatment. FD4 uptake measured 6h after LPS was reduced by TDG 3 or 6h after LPS treatment. TDG-associated reduced FD4 uptake was reversed by the VPAC1 antagonist PG97-269 or L-NAME, not by EGF or IGF1 receptor inhibitors. Systemic LPS releases endogenous GLP-2, reducing LPS-related increased permeability. The therapeutic window of exogenous GLP-2 administration is at minimum within 6-12h after LPS treatment. Exogenous GLP-2 treatment is of value in the prevention of increased paracellular permeability associated with endotoxemia.

Effects of Human Amnion-Derived Mesenchymal Stem Cell (hAD-MSC) Transplantation In Situ on Primary Ovarian Insufficiency in SD Rats.

Human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced primary ovarian insufficiency (POI). However, ensuring that stem cells home to the ovary to improve their effects on ovarian injury is challenging. This research aimed to directly inject ovarian tissue with hAD-MSCs and improve the homing of stem cells to the ovary. The animals were divided into POI, hAD-MSC (tail vein) treatment, hAD-MSC (in situ) treatment, and control groups. POI rat models were established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS). The hAD-MSCs isolated from the amnion were injected into the tail vein or ovary of POI rats. The estrous cycle, serum sex hormone levels, follicle counts, ovarian pathological changes, and proteome of the ovaries were evaluated. hAD-MSCs were successfully isolated and cultured from the amnion. Both hAD-MSC (tail vein) and hAD-MSC (in situ) transplantation increased body weight, improved the AMH levels and follicle numbers, and reduced reproductive organ injuries in POI rats. Transplantation of hAD-MSCs (in situ) upregulated 24 proteins and downregulated 4 proteins. Both hAD-MSC (tail vein) and hAD-MSC (in situ) transplantations can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced POI. The paracrine proteome of hAD-MSCs in the ovarian microenvironment can protect against chemotherapy-induced damage by reducing apoptosis and promoting angiogenesis, cell proliferation, and gene expression.

A combination of Semen Cuscutae and Fructus Lycii improves testicular cell proliferation and inhibits their apoptosis in rats with spermatogenic dysfunction by regulating the SCF/c-kit--PI3K--Bcl-2 pathway

Semen Cuscutae is the seed of Cuscuta japonica Choisy, and Fructus Lycii is the mature fruit of Lycium barbarum L. (Solanaceae). Semen Cuscutae and Fructus Lycii (SC-FL) are well-known Chinese medicine which have been used to tonify the kidney and replenish the essence for thousands of years. Chinese physicians prefer to prescribe them for treating male infertility. Recent studies have found that SC-FL repair spermatogenic dysfunction, however, the therapeutic mechanism has yet to be clearly elucidated. This study aimed at evaluating the therapeutic effect of SC-FL in glucosides of Tripterygium wilfordii Hook. f (GTW)-induced dyszoospermia rats and elucidating the underlying molecular mechanism. Seventy-eight Sprague-Dauley (SD) rats were randomly divided into five groups: normal control (treated with saline), GTW (treated with saline), GTW+levocarnitine (treated with levocarnitine), GTW+SCFL (treated with SC-FL), and LY (LY294002, the PI3K inhibitor) +SCFL (treated with SC-FL). GTW (40mg/kg/d) was intragastrically administered for 4 weeks to establish dyszoospermia model. From the start of the study, LY was additionally injected into the tail vein of rats of the LY+SCFL group once a week. After 8 weeks, semen quality and organ coefficient were determined and sex hormone, inhibin B, and epididymal carnitine levels were measured. Testicular tissue and its ultrastructure were observed using H&E (hematoxylin-eosin) staining and electron microscopy. Immunohistochemistry, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to determine the protein and mRNA expression of SCF, c-kit, PI3K, p-Akt, Bad, Bcl-2, and Bax in rat testis. Compared with the GTW group, semen quality, the organ coefficient, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and epididymal carnitine levels were significantly improved in the GTW+SCFL group (P<0.05 or P<0.01). Histomorphology and testicular ultrastructural evaluation showed that in the GTW+SCFL group, the structure and arrangement of seminiferous tubules were better, the amount of spermatogenic cells increased significantly, the morphology of spermatogenic cells improved, and the mitochondria increased, compared to those in the GTW group. Immunohistochemistry, western blotting, and qRT-PCR results showed that compared with the GTW group, the expression of SCF, c-kit, PI3K, p-Akt, and Bcl-2 in the GTW+SCFL group was increased, while that of Bax and Bad was decreased. The expression of p-Akt and Bcl-2 decreased, while that of Bad and Bax increased in the LY+SCFL group compared with the SCFL group. SC-FL can effectively inhibit spermatogenic cell apoptosis and promote their proliferation, and the mechanism may be related to the regulation of the SCF/c-kit--PI3K--Bcl-2 pathway.

Lack of toxicological influences by microsampling (50 µL) from jugular vein of rats in a collaborative 28-day study.

Due to finalization of the ICH S3A Q&A focusing on microsampling, application of microsampling technique to regular non-clinical animal studies is expected for non-clinical safety assessment of pharmaceuticals. In Europe, microsampling from the tail vein or saphenous vein has often been used, whereas sampling from the jugular vein is thought to be more common for non-clinical studies in Japan. Therefore, we assessed the toxicological effects of serial microsampling from the jugular vein of SD rats in a common 28-day study at 4 independent organizations. Fifty microliter sampling was performed at 6 timepoints on day 1 to 2 and 7 timepoints on day 27 to 28 and its toxicological influences on body weight, food consumption, hematological and clinical chemistry parameters, and organ weights (on day 29 for 3 and day 28 for 1 organizations) were evaluated. The serial microsampling was shown to have no or minimal influences on the assessed parameters. The observed statistical differences for the 18 parameters were sporadic and did not appear to be systemically associated with microsampling. However, the sporadic changes were more often observed in females (14/18 parameters) than in males (6/18), suggesting the possibility that female rats were more susceptible to treatment-based influences. The current results indicate that serial 50 μL sampling from the jugular vein of SD rats had no or very slight toxicological effects, suggesting that this microsampling condition is applicable for toxicokinetic evaluation of non-clinical rat toxicity studies.

Microvenous anastomosis: The rat tail model alternative

Background: The rat femoral vein and dorsal penile vein have been have been well described as microvenous models. We report the use of the rat tail lateral vein as model for microvenous anastomosis training. Methods: Careful anatomic dissections were made of the rat tail noting the basic gross anatomy in 3 rats. 20 albino rats were anaesthesized via intraperitoneal ketamine injection, lateral vein of the tail is then transected about 5 cm distal to the tail base. Immediate repair was carried out by standard interrupted microanastomosis suture technique with 10/0 nylon suture. Patency was assessed at 30 minutes and 1 week. Results: 40 consecutive anastomosis of the lateral tail vein was done in 20 rats . The mean diameter of the rat tail lateral vein in the study rats was 0.8mm SD± 0.02mm.The mean anastomosis time was 22.6 minutes SD±4.3 minutes from clamp application to removal. There was a 90% patency rate at 30 minutes and 81 % at 1 week. Conclusion: The rat lateral tail vein appears to be a viable alternate model for microvenous anastomosis. The study identifies the ease of access, duality, potentially long length and the absence of accompanying artery as some of the unique features of this microvenous anastomosis practice model.

Effects of serial cervical or tail blood sampling on toxicity and toxicokinetic evaluation in rats.

To assess the influences of blood sampling volumes or sites on toxicological and toxicokinetic (TK) evaluations, 4-week duration animal studies and a single-dose TK study of imipramine were conducted. In the toxicological evaluation, six-week-old Sprague-Dawley rats were divided into no blood and blood sampling groups. Fifty microliters (microsampling) or 100 μL (larger sampling) of blood/time point was collected from the jugular vein (50 μL of data was reported previously as Yokoyama et al., 2020) or the tail vein 6 to 7 times on days 1/2 and in week 4. Although no parameters were affected by the 100 μL sample from the tail vein, the 100 μL jugular vein sampling decreased the red blood cell parameters in females, possibly due to hemorrhage at the sampling site. Regarding the TK assessment, 50 μL of blood/site/time point was collected at 6 time points from the tail and jugular vein of the same male rats after single oral administration of 10 or 100 mg/kg imipramine, which was selected as a representative drug with high distribution volume. Although there were no differences in the AUC0-24hr and Cmax values between the sites, the plasma concentrations at the early time points were significantly lower from the tail vein than the jugular vein. From our studies, 50 μL of jugular and tail vein microsampling did not affect the toxicity parameters or AUC/Cmax. However, appropriate toxicity considerations and/or selection of the blood sampling site may be important in the case of larger sampling volumes or blood concentration assessment.