TdT-mediated dUTP-biotin Nick End Research Articles

Resveratrol Inhibits Diabetic-Induced Müller Cells Apoptosis through MicroRNA-29b/Specificity Protein 1 Pathway.

The aim of this study was to evaluate the anti-apoptosis effects of resveratrol (RSV) on diabetic rats retinal Müller cells in vivo and in vitro and to further investigate the roles of microRNA-29b (miR-29b)/specificity protein 1 (SP1) in the anti-apoptosis mechanism of RSV. Retina was obtained from normal and diabetic rats with or without RSV (5 and 10mg/kg/day) treatments at 1-7months. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and Annexin V/PI staining were used to detect apoptosis. Immunofluorescence was used to assess distribution of SP1 in retina. MiR-29b and SP1 messenger RNA (mRNA) expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). SP1, Bax, and bcl-2 protein expression was evaluated by western blotting. Caspase-3 activity was detected by assay kit. Our study showed that the TUNEL-positive cells were mainly localized in the inner nuclear layer (INL) of retina and RSV administration effectively suppressed streptozotocin (STZ)-induced apoptosis of retinal cells in INL in vivo (P < 0.001). Our study also showed that RSV administration effectively suppressed high glucose (HG)-induced retinal Müller cells' apoptosis in vitro (P < 0.001). Furthermore, our study revealed that the diabetes-induced downregulated expression of miR-29b and upregulated expression of SP1 could be rescued by RSV in vivo and in vitro (P < 0.05). The anti-apoptosis effect and downregulated SP1 expression effect of RSV was prevented by miR-29b inhibitor (P < 0.05). MiR-29b mimic increased the above-mentioned effects of RSV (P < 0.001). These findings indicate that RSV is a potential therapeutic option for diabetic retinopathy (DR) and that miR-29b/SP1 pathway play roles in the anti-apoptosis mechanism of RSV.

A Novel and Sensitive Approach for the Evaluation of Liver Ischemia-Reperfusion Injury After Liver Transplantation.

The purpose of our study was to evaluate the potential of x-ray propagation-based phase-contrast imaging (PCI) computed tomography (CT) for the detection and characterization of early changes after ischemia-reperfusion (IR) in a standardized rat liver transplantation (LTx) model. Syngeneic orthotopic liver transplantation was performed in male Lewis rats. Ischemia-reperfusion injury (IRI)-induced changes of liver parenchyma were investigated in a time-dependent manner (2, 16, 24, and 32 hours). X-ray phase-contrast images of formalin-fixated liver specimens were acquired in CT mode by using a voxel size of 8 × 8 × 8 μm. Necrapoptotic cell death was visualized with the TdT-mediated dUTP-biotin nick end labeling technique, and alterations of liver graft microhemodynamics, that is, acinar and sinusoidal perfusion failure, were evaluated by in vivo fluorescence microscopy. Acquired and reconstructed PCI-CT images showed an increase in necrotic liver parenchyma dependent on cold storage time, measuring 5.7% ± 1.6% after 2 hours (comparable to 2.6% ± 0.4% for sham livers), 11.5% ± 2.1% (16 hours; P < 0.05 vs control), 23.0% ± 0.5% (24 hours; P < 0.001 vs control), and 31.3% ± 2.2% (32 hours; P < 0.001 vs control). There were a significant lower number of perfused acini in dependence on increasing cold storage time. The acinar perfusion index reached 0.970 ± 0.006 after 2 hours of cold ischemia (comparable to 0.960 ± 0.009 for sham livers) and declined continuously after 16, 24, and 32 hours cold ischemia (0.58 ± 0.03, 0.49 ± 0.02, 0.41 ± 0.03, each P < 0.0001 vs controls). Comparable results were found for sinusoidal perfusion, reaching 1.8% ± 0.4% of nonperfused sinusoids for 2 hours of cold ischemia and 8.2% ± 0.8% after 16 hours, 18.8% ± 1.4% after 24 hours, and 39.0% ± 2.4% after 32 hours (each P < 0.0001 vs controls). Prolonged cold ischemia was associated with an increasing number of TdT-mediated dUTP-biotin nick end labeling-positive cells (hepatocytes and sinusoidal lining cells), reaching 0.4 ± 0.1 (sham), 0.7 ± 0.4 (2 hours), 6.4 ± 1.1 (16 hours), 2.1 ± 0.3 (24 hours), and 14.7 ± 3.5 (32 hours; P = 0.002) for hepatocytes. X-ray PCI of histological liver specimens can detect IR-induced tissue necrosis and can provide detailed complementary 3-dimensional information to standard histopathologic findings.

Abstract TP258: Enoph1-ADI1-MT-MMP-1 Pathway Contributes to Ischemic Brain Microvascular Endothelial Cell Apoptosis in vivo and in vitro

Enolase-Phosphatase 1 (Enoph1), a newly discovered enzyme of the methionine salvage pathway, has been identified as a candidate gene involved in the modulation of stress responses. In the present study, we investigated the effect of Enoph1 on ischemic blood brain barrier (BBB) injury on an in vitro model of oxygen-glucose deprivation (OGD) and an in vivo rat model of middle cerebral artery occlusion (MCAO). Cell death and apoptosis were assessed by measuring 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide formation, lactate dehydrogenase release and TdT-mediated dUTP-biotin nick end labeling staining. BBB disruption was evaluated based on fluorescein-labeled albumin (FITC-albumin) extravasation. ROS generation was assessed by DCFH staining. Real time-PCR and western blot were used to measure mRNA and protein expression, respectively. CRISPR-activated plasmid and siRNA were applied to manipulate Enoph1 expression in brain microvascular endothelial cells (BMVECs). Exposure of BMVECs to OGD for 6 hours markedly increased Enoph1 expression and decreased the protein levels of its substrate aci-reductone dioxygenase 1 (ADI1) protein, which was accompanied by membrane type 1 matrix metalloproteinase (MT1-MMP) upregulation, increased apoptotic cell death and ROS generation. Increased Enoph1 expression was also observed in ischemic cerebromicrovassels where FITC-albumin extravasation was observed, indicating the co-localization of BBB disruption and increased Enoph1 expression. Knockdown of Enoph1 expression with siRNA significantly inhibited OGD-induce apoptosis, ROS generation, ADI1 protein reduction and MT1-MMP upregulation in BMVECs, while overexpression of Enoph1 CRISPR-activated plasmids exacerbated OGD-induced cell death and ROS generation. Of note, overexpression of ADI1 or knockdown of MT1-MMP with siRNA significantly blocked OGD-induced BMVECs apoptosis. In conclusion, our data demonstrate for the first time that cerebral ischemia activates Enoph1-ADI1-MT1-MMP axis to mediate BMVEC apoptosis and BBB disruption, and Enoph1 may represent a new therapeutic target for ischemic stroke.

Intraarticular Injection of Allogenic Mesenchymal Stem Cells has a Protective Role for the Osteoarthritis.

Background:Researchers initially proposed the substitution of apoptotic chondrocytes in the superficial cartilage by injecting mesenchymal stem cells (MSCs) intraarticularly. This effect was termed as bio-resurfacing. Little evidence supporting the treatment of osteoarthritis (OA) by the delivery of a MSC suspension exists. The aim of this study was to investigate the effects of injecting allogenic MSCs intraarticularly in a rat OA model and to evaluate the influence of immobility on the effects of this treatment.Methods:We established a rat knee OA model after 4 and 6 weeks and cultured primary bone marrow MSCs. A MSC suspension was injected into the articular space once per week for 3 weeks. A subgroup of knee joints was immobilized for 3 days after each injection, while the remaining joints were nonimmobilized. We used toluidine blue staining, Mankin scores, and TdT-mediated dUTP-biotin nick end labeling staining to evaluate the therapeutic effect of the injections. Comparisons between the therapy side and the control side of the knee joint were made using paired t-test, and comparisons between the immobilized and nonimmobilized subgroups were made using the unpaired t-test. A P value < 0.05 was considered significant.Results:The three investigative approaches revealed less degeneration on the therapy sides of the knee joints than the control sides in both the 4- and 6-week groups (P < 0.05), regardless of immobilization. No significant differences were observed between the immobilized and nonimmobilized subgroups (P > 0.05).Conclusions:Therapy involving the intraarticular injection of allogenic MSCs promoted cartilage repair in a rat arthritis model, and 3-day immobility after injection had little effect on this therapy.

DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells.

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2′-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

Protective effects of probiotic Lactobacillus plantarum BJ0021 on liver and kidney oxidative stress and apoptosis induced by endosulfan in pregnant rats

Endosulfan (EDS) is one of the most widely organochlorine insecticide used in many parts of the world, although it is currently banned or severely restricted in use in some countries. EDS causes a variety of negative effects in non-target species including humans. Therefore, the aim of the present study was to investigate the possible protective effects of Lactobacillus plantarum BJ0021 on toxicity, oxidative stress, and apoptosis induced by EDS intoxication on liver and kidneys of pregnant rats. This pesticide induced a significant increase in total cholesterol, alanine-amino transferase (ALAT), aspartate-amino transferase (ASAT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), urea and creatinine in serum, while urinary urea and creatinine were lower than those of the control group. In the liver and kidney, lipid peroxidation increased significantly, the antioxidant levels, such as superoxide dismutase (SOD) and catalase (CAT) were markedly depressed and TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) revealed more apoptotic cells. In contrast, co-administration of L. plantarum BJ0021 to EDS-treated animals ameliorated most of these biochemical parameters, but the activity of antioxidant enzymes (SOD, CAT) did not modify and the number of apoptotic nuclei remained significantly raised in kidney compared to control. In conclusion, the administration of L. plantarum BJ0021 decreased apoptosis and might play a protective role in reducing toxicity of EDS in pregnant rats.

Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways.

Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3) both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg). Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE) cells, primary retinal cells, and the cone photoreceptor (PRC) cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1) was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

Neuroprotective effect of geniposide on Parkinson's disease model mice

Objective To investigate the neuroprotective effect of geniposide on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced Parkinson's disease (PD) model mice and possible mechanism. Methods A total of 48 male C57BL/6 mice were randomly divided into control, model, geniposide and MPTP + geniposide groups. The behaviors of C57BL/6 mice were assessed by using open field test, the tyrosine hydroxylase (TH) and Bcl-2 positive neurons in the midbrain substantia nigra of mice were detected by immunohistochemistry and the number of apoptosis neurons were observed with TdT-mediated dUTP-biotin nick end labeling (TUNEL). Results The number of mobile grid [(76.33 ± 8.59) times/5 min], standing [(19.58 ± 3.97) times/5 min], TH-positive neurons [(12.83 ± 2.32)/HPF] and Bcl-2-positive neurons [(10.83 ± 2.23)/HPF] in model group were significantly lower than those in the control group [(142.50 ± 11.65) times/5 min, (39.17 ± 4.75) times/5 min, (35.67 ± 1.75)/HPF, (20.67 ± 1.75)/HPF; P = 0.000, for all]. The apoptosis neurons in model group [(20.33 ± 2.58)/HPF] were significantly higher than that in control group [(3.83 ± 1.67) /HPF, P = 0.000). The number of mobile grid [(97.67 ± 13.15) times/5 min, P = 0.000], standing [(29.33 ± 2.90) times/5 min, P = 0.000], TH-positive neurons [(17.50 ± 2.07)/HPF, P = 0.002] and Bcl-2-positive neurons [(15.17 ± 2.79) /HPF, P = 0.003] in MPTP + geniposide group were significantly higher than those in model group. The number of apoptosis neurons [(14.67 ± 3.08) /HPF] in MPTP + geniposide group was significantly lower than that in model group ( P = 0.001). Conclusions Geniposide can protect dopaminergic neurons in MPTP-induced neurodegeneration and the mechanism may be associated with the inhibition of neuronal apoptosis. DOI: 10.3969/j.issn.1672-6731.2015.06.012

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.

Oestrous cycle-dependent expression of Fas and Bcl2 family gene products in normal canine endometrium.

During the oestrous cycle canine endometrium undergoes cyclical cellular proliferation, apoptosis and differentiation. To study the regulation of endometrial apoptosis and proliferation events the expression of apoptosis-related genes was analysed by real-time polymerase chain reaction and cellular expression of their proteins was identified through immunohistochemistry. Cellular apoptosis and proliferation events were detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and proliferation marker Ki67 immunostaining, respectively. The highest proliferative index was observed in the follicular phase (all endometrial cellular components) and at early dioestrus (basal glands). This was associated with a low apoptotic index and a strong expression of anti- (Bcl2) and pro-apoptotic proteins (Fas, FasL, Bax). Subsequently (Days 11-45 of dioestrus), basal glandular epithelium experienced the highest apoptotic index, coincidental with a decrease of Bcl2 expression and a low ratio of Bcl2/Bax transcription. An increase in the apoptotic index of crypts, stromal and endothelial cells was observed at late dioestrus and the beginning of anoestrus. These results indicate that pro- and anti-apoptotic proteins regulate the balance between cell proliferation and death in the canine endometrium during the oestrous cycle. High Bcl2 expression in both the follicular and early dioestrous phases stimulate glandular proliferation and prevent apoptosis but, in the non-pregnant uterus, a decrease in Bcl2 expression together with an increase in pro-apoptotic proteins induces apoptosis of basal glandular epithelium cells.

New drug delivery system for liver sinusoidal endothelial cells for ischemia-reperfusion injury.

To investigate the cytoprotective effects in hepatic ischemia-reperfusion injury, we developed a new formulation of hyaluronic acid (HA) and sphingosine 1-phophate. We divided Sprague-Dawley rats into 4 groups: control, HA, sphingosine 1-phosphate (S1P), and HA-S1P. After the administration of each agent, we subjected the rat livers to total ischemia followed by reperfusion. After reperfusion, we performed the following investigations: alanine aminotransferase (ALT), histological findings, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, and transmission electron microscopy (TEM). We also investigated the expression of proteins associated with apoptosis, hepatoprotection, and S1P accumulation. S1P accumulated in the HA-S1P group livers more than S1P group livers. Serum ALT levels, TUNEL-positive hepatocytes, and expression of cleaved caspase-3 expression, were significantly decreased in the HA-S1P group. TEM revealed that the liver sinusoidal endothelial cell (LSEC) lining was preserved in the HA-S1P group. Moreover, the HA-S1P group showed a greater increase in the HO-1 protein levels compared to the S1P group. Our results suggest that HA-S1P exhibits cytoprotective effects in the liver through the inhibition of LSEC apoptosis. HA-S1P is an effective agent for hepatic ischemia/reperfusion injury.

Histologic evaluation of human benign prostatic hyperplasia treated by dutasteride: a study by xenograft model with improved severe combined immunodeficient mice.

To evaluate histologic change in human prostate samples treated with dutasteride and to elucidate direct effects of dutasteride on human prostate tissue, the present study was conducted by using a xenograft model with improved severe combined immunodeficient (super-SCID) mice, although it is well known that dutasteride reduces prostate volume. After establishment of a xenograft model of human benign prostatic hyperplasia in morphology and function, samples implanted into super-SCID mice with and without dutasteride were evaluated pathohistologically at 2 and 6 months after initiation of dutasteride administration. The proliferative index evaluated by Ki-67 staining was significantly lower in the dutasteride group than the control at 2 and 6 months after administration. Apoptotic index evaluated by the terminal transferase TdT-mediated dUTP-biotin nick end labeling staining was higher in the dutasteride group than the control at 2 and 6 months after administration. Quick scores in the dutasteride group for staining of both cyclooxygenase-2 (Cox-2) and Ras homolog gene family, member A (RhoA) were significantly lower than those in the control group at 2 and 6 months after administration. Dutasteride inhibits cell proliferation and induces apoptosis of prostatic cells, causing a reduced prostate volume. Furthermore, decreased expression of Cox-2 and RhoA within benign prostatic hyperplasia tissue by dutasteride may induce an early effect on improvement of lower urinary tract symptoms, probably by attenuating inflammation reaction of the prostate and decreasing intraurethral pressure, other than the mechanism of reduced prostate volume.

Roles of cytochrome c, caspase-9, and caspase-3 in pentavalent vanadium-induced neuronal apoptosis

To investigate the roles of cytochrome c (Cyt-c), caspase-9, and caspase-3 in pentavalent vanadium-induced neuronal apoptosis and to provide a basis for mechanism research. Neurons from rats aged 1-3 days were cultured and treated with vanadium pentoxide (V2O5) at 5, 10, or 20 mmol/L. Neuronal apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL). The protein levels of Cyt-c, caspase-9, and caspase-3 were determined by Western blot. Apoptosis bodies were detected in the nuclei of neurons by TUNEL. The number of neurons with apoptosis bodies increased with increasing dose of V2O5 The apoptosis index (AI) was significantly higher in the 10 and 20 mm/L exposure groups than in the control group (P < 0.05 or P < 0.01). Western blot showed that the protein expression levels of Cyt-c and caspase-3 significantly increased in the 5 mmol/L exposure group as compared with the control group (P < 0.05). In the 10 and 20 mmol/L exposure groups, the protein expression of Cyt-c, caspase-9, and caspase-3 all increased as compared with the control group (P < 0.01). Neuronal AI was positively correlated with Cyt-c, caspase-9, and caspase-3 (r = 0.954, P < 0.01; r = 0.938, P < 0.01; r = 0.943, P < 0.01). Pentavalent vanadium may induce neuronal apoptosis. The protein expression of Cyt-c, caspase-9, and caspase-3 may play an important role in neuronal apoptosis induced by pentavalent vanadium.

Dipeptidyl Peptidase IV Inhibitor Protects Against Tacrolimus-Induced Pancreatic and Renal Injury.

Background: Dipeptidyl peptidase IV (DPP IV) inhibitors had a protective effect in various disease models. This study was performed to evaluate whether sitagliptin, a DPP IV inhibitor, is effective against tacrolimus-induced pancreatic and renal injury. Method: Tacrolimus (TAC, 1.5mg/kg/day, s.c.) with or without sitagliptin (10, 20 mg/kg/day, gavage) were administered to rats for 4 weeks. We evaluated the effect of sitagliptin on DPP IV activity and GLP-1 level in tacrolimus-treated rats, and assessed the protective effect of sitagliptin in an experimental model of chronic tacrolimus-induced nephropathy and pancreatic injury. Oxidative stress and oxidative resistance were evaluated by measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG) and heme oxygenase-1 (HO-1). The protective mechanism of sitagliptin was also tested based on the glucagon like peptide-1 (GLP-1)/ (forkhead box O) FoxO pathway. Results: Tacrolimus treatment for 4 weeks caused a 2-fold increase in plasma DPP4 activity and a 5-fold decrease in serum active GLP-1 levels; however, concomitant treatment with sitagliptin reversed these effects. Sitagliptin treatment attenuated tacrolimus-induced pancreatic islet and renal dysfunction, with improvement of islet size and renal tubulointerstitial fibrosis. Tacrolimus treatment increased oxidative stress marker, 8-OHdG and decreased oxidative resistance gene (reduced HO-1), and its underlying pathways (FoxO1 activation and FoxO3 deactivation), whereas sitagliptin treatment reversed these changes. Treatment of primary rat islet and HK-2 cells with GLP-1 also decreased the oxidative injury caused by tacrolimus. Consequently, sitagliptin upregulated antioxidant enzymes e.g. manganese superoxide dismutase, and downregulated proapoptotic proteins e.g. B-cell lymphoma 2-interacting mediator of cell death and apoptotic cell death assessed by the number of TDT-mediated dUTP-biotin nick end labeling-positive cells. Conclusion:DPP IV inhibition by sitagliptin attenuated TAC-induced pancreatic islet and renal dysfunction and enhanced the GLP-1 signaling pathway, which may be associated with a decrease in oxidative injury and apoptotic cell death. These beneficial effects of the DPP IV inhibitor may be helpful in delaying the onset of TAC-induced diabetes and nephropathy in renal transplant patients.

Expression and roles of aquaporin 1 in hippocampus of mice model with traumatic brain injury

Background The "secondary brain insult" including ischemia, hypoxia and edema after primary traumatic brain injury (TBI) may deteriorate the brain damages and greatly influence the prognosis. As a selective vulnerable region, the hippocampus is especially sensitive to ischemia, hypoxia or edema and yields irreversible sequelae. Aquaporin 1 (AQP1) has been reported to be related to cerebral edema, but the expression and role of AQP1 in hippocampal edema after TBI have seldomly been investigated. In this study, we established BALB/c mouse closed craniocerebral injury models and investigated the changes of AQP1 expression in hippocampus of mouse models after TBI, thereby discussing its effects on relevant pathophysiological processes. Methods Seventy-five BALB/c mice were used to establish experimental closed TBI models with a free-falling weight drop device, and the equal numbers of mice were subject to sham operation and categorized as sham group. The neurological function of each mouse in either TBI group or sham group was scored at different time points (1, 6, 24 and 72 h) after TBI or sham operation, and brain edema formation of the mice in both groups was also evaluated accordingly at 6, 24 and 72 h. The apoptotic hippocampal cells were stained in situ and detected using TdT-mediated dUTP-biotin nick end labeling (TUNEL) method at different time points (6, 24 and 72 h), then AQP1 expression in hippocampus was also correspondingly detected using immunohistochemistry and Western blotting. All the data were finally compared with those in sham operation group and analyzed. Results Experimental TBI models were successfully established and confirmed by the neurological function score and hippocampal edema evaluation. Six hours after craniocerebral injury, the apoptotic cells increased significantly in the hippocampus of mice in TBI group compared with those in sham group [(44.26 ± 15.18)% vs (8.61 ± 8.25)% , t = - 9.676, P = 0.002]. The apoptotic rate increased gradually with the prolonging of time and was highest at 72 h [(61.62 ± 26.55)% vs (10.17 ± 6.08)%; t = - 5.018, P = 0.015]. Determined at different time points by immunohistochemistry and Western blotting assays, the AQP1 expression in mouse hippocampus was constantly higher in TBI group than in sham group ( P < 0.05), and reached the peak at 24 h (0.69 ± 0.32 vs 0.15 ± 0.07, t = - 4.335, P = 0.023 and 0.46 ± 0.19 vs 0.14 ± 0.04, t = - 4.113, P = 0.004, respectively). Conclusions The up-regulation of AQP1 in mouse hippocampus after TBI perhaps participates in edema formation and delayed apoptosis of hippocampus, and AQP1 may be a new therapeutic target to protect hippocampus from secondary injury after TBI. doi: 10.3969/j.issn.1672-6731.2014.03.016

The protective effect of selenium in cisplatin-related retinotoxicity

Purpose: The aim of this study is to evaluate the retinal toxicity of cisplatin and neuroprotective effect of selenium in cisplatin-related retinal toxicity. Methods: Eighteen adult Wistar-Albino rats were divided into three groups. Group 1 (n = 6) received intraperitoneal (i.p.) injection of 2.5 ml physiologic saline for three days, group 2 (n = 6) received i.p. 16 mg/kg cisplatin for three days and group 3 (n = 6) received i.p. 16 mg/kg cisplatin for three days and 1.5 mg/kg twice daily selenium via gavage five days prior to cisplatin injection and for three days concomitantly with cisplatin injections. The total retinal thickness, outer nuclear layer (ONL), inner nuclear layer (INL) and inner plexiform layer (IPL) thicknesses were measured in hematoxylin/eosin and apoptotic index (AI) of ganglion cell layer (GCL) and INL was evaluated in TdT-mediated dUTP-biotin nick end labeling (TUNEL)-stained retina sections. Results: Selenium statistically succeeded to reduce total retinal thickness in cisplatin-toxicated retinas: from 210.17 ± 23.40 to 173.55 ± 20.43, ONL: 49.79 ± 5.32 to 41.87 ± 6.30, INL: 33.72 ± 7.93 to 25.06 ± 5.73 and IPL: 53.61 ± 8.63 to 45.61 ± 6.92 µm in hematoxylin/eosin-stained retina sections. The AI was also reduced in INL (30.10 ± 12.02 to 19.48 ± 12.99) and in GCL (37.59 ± 17.70 to 33.15 ± 13.78). However, statistical significance was present in only AI values of INL. Conclusions: Selenium limited edema due to the toxicity and reduced the retinal thickness and showed neuroprotection in cisplatin-induced retinotoxicity.

Role of receptor interacting protein (RIP)1 on apoptosis-inducing factor-mediated necroptosis during acetaminophen-evoked acute liver failure in mice

Acetaminophen (APAP) overdose induces apoptosis-inducing factor (AIF)-dependent necroptosis, but the mechanism remains obscure. The present study investigated the role of receptor interacting protein (RIP)1, a critical mediator of necroptosis, on AIF-dependent necroptosis during APAP-induced acute liver failure. Mice were intraperitoneally injected with APAP (300mg/kg). As expected, hepatic RIP1 was activated as early as 1h after APAP, which is earlier than APAP-induced hepatic RIP3 upregulation. APAP-evoked RIP1 activation is associated with hepatic glutathione (GSH) depletion. Either pretreatment or post-treatment with Nec-1, a selective inhibitor of RIP1, significantly alleviated APAP-induced acute liver failure. Moreover, Nec-1 improved the survival and prevented APAP-induced necroptosis, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that Nec-1 significantly inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. In addition, Nec-1 blocked APAP-induced translocation of AIF from the mitochondria to the nucleus. Of interest, no changes were induced by Nec-1 on hepatic CYP2E1 expression. In addition, Nec-1 had little effect on APAP-induced hepatic GSH depletion at early stage. Taken together, these results suggest that RIP1 is involved in APAP-induced necroptosis. Nec-1 is an effective antidote for APAP-induced acute liver failure.

The effects of vincristine sulfate on expression of galectin-3, Bcl-2, and carbohydrate structures specific for EEL, GSL-1, and RCA-1 lectins in bitches with naturally occurring canine transmissible venereal tumor

Vincristine sulfate is one of the most effective chemical agents used in canine transmissible venereal tumor (CTVT) chemotherapy. Its therapeutic effectiveness is pronounced and significant at the beginning of treatment. The present study was designed to investigate vincristine sulfate treatment and the relationship between CTVT tumor regression and apoptosis [estimated by TdT-mediated dUTP-biotin nick end labeling (TUNEL)], galectin-3 (H-160), Bcl-2 (N-9), and lectin [EEL (B-135), GSL I (L-1100), and RCA I (BA-0084)] expression using immunohistochemical staining in 20 bitches with naturally occurring CTVTs. Evaluation was semiquantitatively performed by HSCORE values using light microscopy. All observed staining was intracytoplasmic and increased endothelial staining was noted with the Bcl-2 marker in tumor vessels. Apoptosis, TUNEL, and lectin scoring was higher in vincristine sulfate-treated tumors than in untreated tumors, while expression of antiapoptotic factors galectin-3 and Bcl-2 were lower in treated than in untreated CTVTs. These results show that galectin-3, Bcl-2, and lectins (EEL, GSL-1, and RCA-1) are suitable indicators/markers for CTVT tumor regression after treatment with vincristine sulfate.

Differential effects of antihypertensive treatments on apoptosis, oxidative stress, and expression of angiotensin receptors in the cerebral cortex from the onset of prehypertension and hypertension in stroke-prone spontaneous hypertensive rats

To investigate the effects of losartan and amlodipine on cell apoptosis in the cerebral cortex of stroke-prone spontaneously hypertensive rats (SHRSP) from the onset of prehypertension or hypertension. SHRSP were randomly divided into five experimental groups that were administered losartan, amlodipine (n=8 in each group; 4 weeks old or 10 weeks old), or vehicle, respectively. Wistar-Kyoto rats were used as control animals. Systolic blood pressure was measured using the tail-cuff method every 2 weeks. At 20 weeks of age, apoptosis was analyzed by TdT-mediated dUTP-biotin nick end labeling, and the level of angiotensin II was measured by radioimmunoassay. Protein expressions of gp91(phox), superoxide dismutase, and angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors in the cerebral cortex were detected by western blot. Losartan and amlodipine effectively delayed the progression of systolic blood pressure elevation, especially from the onset of prehypertension, and they had no obvious effects on the level of angiotensin II. In addition, treatment with losartan or amlodipine significantly decreased cell apoptosis, downregulated the protein expression of gp91(phox), and upregulated the protein expression of superoxide dismutase. The protein expressions of AT1R and AT2R were decreased by the administration of both drugs. No difference was found in the expression of AT1R among the drug treatment groups, whereas the expression of AT2R was increased in rats with increased blood pressure. Amlodipine, especially from the onset of prehypertension, was more effective than losartan in reducing apoptosis in the cerebral cortex in SHRSP. This may be related to the antioxidative stress properties of amlodipine.

Morphological Differences in Adolescent Idiopathic Scoliosis

Histological and ultrastructural evaluation of cell morphologies at the concave and convex side of apical intervertebral discs (IVD) of adolescent idiopathic scoliosis (AIS). To determine changes in cell morphology, viability, and cell death after asymmetric disc loading in AIS and to compare the findings with the tilt angles. The reaction of cells to loading stimuli in the IVD seems to be specific. Although dynamic loads are more beneficial to the disc cells and maintain the matrix biosynthesis, static compressive loads suppress gene expression. Apical IVDs (Th8-Th9 to L1-L2) from 10 patients with AIS were studied histologically (including TUNEL [TdT-mediated dUTP-biotin nick end labeling] staining to identify disc cell death by apoptosis) and ultrastructurally for matrix evaluations and to quantify healthy, balloon, chondroptotic, apoptotic, and necrotic cells on the concave and convex sides. Patients' spines were classified according to the Lenke classification. Degeneration was assessed according to the Pfirrmann grading system. Two groups were established; group 1 (G1) with a tilt of 5° to 9° and group 2 (G2) with a tilt of 10° to 19°. Balloon cells were found in significantly higher numbers at the concave side (G1-annulus fibrosus [AF]: mean 16%), with almost none found at the convex side. Mean numbers of healthy cells did not show differences comparing both sides. Significantly higher numbers of healthy cells were found with increasing tilt angle at the concave side. Necrosis (mean, 47%) increased toward the center of the disc but did not differ between the sides of the IVDs. The fibrils found in the outer AF on the convex side were 30% thinner. This study was able to show significant differences in cell morphologies in the AF on both sides and in correlation to the different tilt angles. The type and magnitude of load seem to influence disc cells. Further studies are required to provide more information on disc and cell changes in scoliosis.