Number Of Caspase-3 Positive Cells Research Articles

Evaluation of the protective and therapeutic effects of extra virgin olive oil rich in phenol in experimental model of neonatal necrotizing enterocolitis by clinical disease score, ınflammation, apoptosis, and oxidative stress markers.

Necrotizing Enterocolitis (NEC) is an inflammation-associated ischemic necrosis of the intestine. To investigate the effects of extra virgin olive oil (EVOO) on inflammation, oxidative stress, apoptosis, and histological changes in NEC-induced newborn rats. 24 rats were randomly divided into three groups: control, NEC and NEC + EVOO. NEC induction was performed using hypoxia-hyperoxia, formula feeding, and cold stress. The NEC + EVOO group received 2ml/kg EVOO with high phenolic content by gavage twice a day for 3days. 3cm of bowel including terminal ileum, cecum, and proximal colon was excised. Weight gain and clinical disease scores were significantly higher in the NEC + EVOO group than in the NEC group (p < 0.001). EVOO treatment caused significant decreases in IL1β, IL6 levels (p = 0.016, p = 0.029 respectively) and EGF, MDA levels (p = 0.032, p = 0.013 respectively) compared to NEC group. Significant decreases were observed in IL6 gene expression in the NEC + EVOO group compared to the NEC group (p = 0.002). In the group NEC + EVOO, the number of Caspase-3 positive cells was found to be significantly reduced (p < 0.001) and histopathological examination revealed minimal changes and significantly lower histopathological scores (p < 0.001). Phenol-rich EVOO prevents intestinal damage caused by NEC by inhibiting inflammation, oxidative stress, apoptosis.

Effect of sugammadex, rocuronium and sevoflurane on oxidative stress and apoptosis in cerebral ischemia reperfusion model in rats.

Cerebral ischemia-reperfusion (I/R) injury causes neurological dysfunction and cell death. Sugammadex, as a large molecule, is normally difficult to pass through the blood-brain barrier (BBB). In ischemia, molecules can pass into the brain tissue. In this study, we aimed to evaluate the effect of sugammadex in the presence of cerebral I/R damage in rats with a general anesthesia model with sevoflurane and rocuronium. Rats were divided into 7 groups; Group 1 (Control), Group 2 (Sham), Group 3 (Sevoflurane), Group 4 (Sugammadex), Group 5 (Sevoflurane + Rocuronium), Group 6 (Sevoflurane + Sugammadex), Group 7 (Sevoflurane + Rocuronium + Sugammadex). Brain tissues of rats with cerebral I/R damage with bilateral carotid occlusion were removed. Tissue Malondialdehyde (MDA), Myeloperoxidase (MPO), and Superoxide dismutase (SOD) levels were examined with ELISA and apoptosis was examined by Caspase-3. The number of caspase-3 positive cells decreased the most in Group 4 compared to the other groups. Group 4's mean MDA and MPO levels were lower than Group 2. There was no significant difference in terms of SOD levels. The apoptotic effect of sugammadex was lowest compared to other agent groups, and it did not increase oxidative damage as much as the other groups.

Stimulation of functional recovery via neurorepair mechanisms by the traditional Japanese Kampo medicine, Ninjin'yoeito, and physical exercise in a rat ischemic stroke model

Ethnopharmacological relevanceNinjin'yoeito (NYT), a traditional Japanese Kampo medicine consisting of 12 herbs, has been reported to improve cognitive dysfunction, depression, and neurological recovery in patients with neurovascular diseases such as Alzheimer's disease and stroke. Several studies have reported that the NYT components exert neurotrophic, neurogenic, and neuroprotective effects. In addition, exercise enhances neuroprotection and functional recovery after stroke. Rehabilitative exercises and pharmacological agents induce neurophysiological plasticity, leading to functional recovery in stroke patients. These reports indicate that NYT treatment and exercise may promote functional recovery following stroke through their beneficial effects. However, no study has determined the effects of NYT and the possible mechanisms of neurorepair and functional recovery after stroke. Aim of the studyThis study aimed to investigate the combined effects of NYT and exercise on neuroprotection and functional recovery and the underlying mechanisms in a rat ischemic stroke model. Materials and methodsStroke was induced with 60-min middle cerebral artery occlusion (MCAO) followed by reperfusion in adult male Sprague-Dawley rats. After stroke, the rats were assigned to four groups: ischemia reperfusion (IR), NYT, exercise (Ex), and NYT + Ex. NYT-treated rats were fed a diet containing 1% NYT one day after stroke. Exercise was performed using a motorized treadmill for 5 days a week (8–15 m/min, 20 min/day), starting 3 days after stroke. The NYT treatment and exercise were continued for 4 weeks after the stroke. Infarct volume, neurological deficits, sensorimotor functions, expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase A (TrkA) and B (TrkB), caspase-3 activity, and the p-Akt/Akt ratio were examined by immunohistochemistry and western blotting. ResultsCompared to the IR group, all treated groups indicated reduced infarct volumes. The NYT + Ex group showed significantly improved waking time and beam walking score compared with the IR group. The expression of NGF/TrkA/p-TrkA and BDNF/TrkB was significantly increased in the NYT + Ex group compared with those in the IR group, whereas the number of caspase-3 positive cells around the lesion was significantly lower in the NYT + Ex group than in the IR group. In addition, the ratio of p-Akt/Akt was significantly higher in the NYT + Ex group than in the IR group. ConclusionsThis study suggests that NYT in combination with exercise provides neuroprotective effects and improves sensorimotor function by stimulating NGF/TrkA and BDNF/TrkB, and by activating the Akt pathway in ischemic stroke of rats. NYT may be an effective adjunctive agent in post-stroke rehabilitation.

Compatibility of ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) and their protective effects on cerebral ischemia-reperfusion injury in rats.

Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) are two drugs commonly prescribed together, which are often used in the treatment of cerebrovascular diseases in China. Due to the complexity of the ingredients of Danhong, the present study focused on performing the orthogonal compatibility method on the primary effective molecules of this drug: Tanshinol, salvianolic acid A, salvianolic acid B and hydroxysafflor yellow A. These four molecules were studied to determine their protective effects and to screen for the most compatible ingredients to improve cerebral ischemia-reperfusion injury (IR) in rats. Focal middle cerebral artery occlusion was performed to establish the cerebral IR model in rats. Male Sprague-Dawley rats were randomly divided into sham operation group, IR group and nine orthogonal administration groups with different ratios of Danhong effective ingredients and Danhong injection group. Neurological deficit score and cerebral infarction volume were measured postoperatively. Morphological pathological alterations were observed via H&E staining. Bcl-2 and Bax were quantified using ELISA. Immunohistochemistry was conducted to analyze the expression of caspase-3 in the hippocampus. The expression levels of cytochrome c, apoptotic peptidase activating factor 1 (apaf-1), caspase-9, caspase-3 and p53 mRNA in the hippocampus were assessed via reverse transcription-quantitative PCR. The results demonstrated that different compatibility groups significantly reduced the neurological function score and decreased the volume of cerebral infarct compared with the IR group. These groups were also indicated to improve the pathological damage to the brain tissue. In addition, certain compatibility groups significantly decreased the number of caspase-3 positive cells in the hippocampus and the expression levels of cytochrome c, apaf-1, caspase-9, caspase-3 and p53 mRNA in the brain tissue. Orthogonal group 4 (30 mg/kg tanshinol; 2.5 mg/kg salvianolic acid A; 16 mg/kg salvianolic acid B; 8 mg/kg hydroxysafflor yellow A) was indicated to be the most effective. The four effective ingredients of Danhong exhibited a protective effect on rats with cerebral IR injury, potentially through the inhibition of apoptosis via the downregulation of key targets upstream of the caspase-3 pathway. In addition, the present study provided novel insights for the continued study of the drug compatibility rules of TCM.

The Possible Protective Role of Echinacea and Ginger and both of them on the Lung of Diabetic Male Rats: Histological and Immunohistochemical Study

Diabetes is a systemic disease characterized by a state of hyperglycemia. Several changes in the respiratory functions had been detected in diabetic patients. Aim of the work: the current study estimated the efficiency of two herbal extract (ginger and echinacea) on the changes in the lung of diabetic animal model.Materials and methods: 50 adult male albino rats were used. They were divided into five groups. Control group (group I) received regular diet. Group II: diabetic group (diabetes induced by a single intraperitoneal injection of streptozotocin 60 mg/kg). Group III: diabetic rats treated with 100mg/kg echinacea orally for 30 days. Group IV: diabetic rats treated with 400mg/kg ginger orally for 30 days. Group V: diabetic rats treated with both 100mg/kg echinacea and 400mg/kg ginger orally for 30 days. Paraffin blocks were prepared for histological and immunohistochemical examinations. Morphometric study and statistical analysis were done for the thickness of the interalveolar septum, the area percentage of collagen fibers and number of positive caspase -3 cells and CD68 positive macrophages. Ultrathin sections were prepared for electron microscopic examination. Results: Diabetes caused a significant increase in the thickness of the interalveolar septa, the area percentage of collagen fibers, the number of caspase-3 positive cells as well as the number of CD68 positive alveolar macrophages compared to the control group. Ultrastructurally, pneumocyte type II appeared with denser nucleus, numerous vacuoles and disorganized lamellar bodies. Groups treated with echinacea or ginger or both showed a significant decrease in the thickness of the interalveolar septa, the area percentage of collagen fibers, the number of caspase-3 positive cells as well as the number of CD68 positive alveolar macrophages compared to the diabetic group. These groups also showed that pneumocyte type II restored their normal appearance

New Psychoactive Substance 5-MeO-MiPT In vivo Acute Toxicity and Hystotoxicological Study.

The hallucinogenic tryptamine analog 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT) causes social problems worldwide. There are several studies on the metabolism; however, not more studies were found in the literature on acute toxicity. To report the acute toxicity of 5-MeO-MiPT in mice, followed by quantitative toxicological analysis of blood and organs, hystotoxicological and immunohistochemical analysis of tissues and cells. Animal experiment Methods: In vivo experiments were performed using CD1 adult female mice (n=26). Animals were caged in 4 groups randomly. First group was a control (n=3). Second group was vehicle control (n=3) and injected 150 μL of blank solution (50% dimethyl sulfoxide in saline /0.9% of NaCl). While for acute toxicity experiments, 5-MeO-MiPT was added to a blank solution in order to obtain a dose of 0.27 mg/kg in 150 μL injection (n=10) and the last group were injected 2.7 mg/kg 5-MeO-MiPT in a 150 μL injection (n=10). Quantitative toxicological analysis, hystotoxicological and immunohistochemical analysis were performed. In the toxicological analysis, 5-MeO-MiPT was found negative in biological samples which were control, vehicle control, and 0.27 mg/kg dose mice groups. 5-MeO-MiPT was found 2.7-13.4 ng/mL in blood, 11-29 ng/g in kidney, 15.2-108.3 ng/g in liver, and 1.5-40.6 ng/g in the brain in 2,7 mg/kg injected group. In a low dose of the 5-MeO-MiPT liver section, compared with normal tissues, the difference in staining was statistically significant (p<0.0001). In high-dose of 5-MeO-MiPT, H-score showed that the increase in the number of Caspase-3 positive cells was significant compared to the control (p<0.05). In high-dose of 5-MeO-MiPT, intense Caspase-3 immunoreactivity was observed and the increase in the number of Caspase-3 positive cells compared to the control was statistically significant (p<0.05). In brain section, the statistics of the results obtained from the H-score showed that the increase in the number of Caspase-3 positive cells was significant compared to the control (p=0.0183). In vehicle control liver section, there were few Caspase-8 positive cells characterized by a light brown appearance (p=0.0117). In the high-dose 5-MeO-MiPT group, the numbers of positive cells at low and high doses of 5-MeO-MiPT group were statistically significant compared to the control (p<0.05). In the high-dose 5-MeO-MiPT group, Caspase-8 immunoreactivity was detected in the glomerular structures. Compared to control, the increase in Caspase-8 immunoreactivity was found to be statistically significant (p<0.05). Low-dose 5-MeO-MiPT did not cause any serious histopathological effects on the liver, kidney, and brain. High doses induce apoptotic cell death through caspase activity.

Non-additive effects of adjunct erythropoietin therapy with therapeutic hypothermia after global cerebral ischaemia in near-term fetal sheep.

Recombinant human erythropoietin (rEpo) is neuroprotective in immature animals, but it is unclear whether the combination of high-dose rEpo therapy with therapeutic hypothermia can further improve outcomes. Hypothermia and rEpo independently improved neuronal survival, with greater improvement with hypothermia, and similarly reduced numbers of caspase-3 positive cells and reactive microglia after 7days recovery. Hypothermia, but not rEpo, was associated with markedly improved EEG power, whereas both interventions improved recovery of EEG frequency. There was no significant improvement in any outcome after combined rEpo and hypothermia compared with hypothermia alone, and of concern, the combination was associated with increased numbers of cortical caspase-3-positive cells compared with ischaemia-hypothermia. These data suggest that the mechanisms of neuroprotection with hypothermia and rEpo overlap and, thus, high-dose rEpo infusion does not appear to be an effective adjunct therapy for therapeutic hypothermia. Therapeutic hypothermia for hypoxic-ischaemic encephalopathy (HIE) provides incomplete neuroprotection. Recombinant human erythropoietin (rEpo) is neuroprotective in immature animals, but it is unclear whether adjunct rEpo therapy with therapeutic hypothermia can further improve outcomes. Near-term fetal sheep received sham-ischaemia (n=9) or global cerebral ischaemia for 30min (ischaemia-vehicle, n=8), followed by intravenous infusion of rEpo (ischaemia-Epo, n=8; 5000U/kg loading dose, then 833.3U/kg/h), cerebral hypothermia (ischaemia-hypothermia, n=8), or rEpo plus hypothermia (ischaemia-Epo-hypothermia, n=8), from 3 to 72h post ischaemia. Fetal brains were collected 7days after cerebral ischaemia. Cerebral ischaemia was associated with severe neuronal loss and microglial induction in the parasagittal cortex and subcortical regions. Hypothermia reduced overall neuronal loss, cortical caspase-3 and reactive microglia in the striatum and cortex, with greater recovery of electroencephalographic (EEG) power and spectral edge (SEF) from 48h onwards. rEpo independently improved neuronal survival in the parasagittal cortex, hippocampal CA4 and thalamus, and reduced cortical caspase-3 and activated microglia in striatal and cortical areas, with greater SEF from 120h onwards. However, ischaemia-Epo-hypothermia did not further improve outcomes compared with ischaemia-hypothermia and was associated with increased numbers of cortical caspase-3-positive cells. These findings suggest that although delayed, prolonged treatment with both hypothermia and rEpo are independently neuroprotective, they have overlapping anti-inflammatory and anti-apoptotic mechanisms, such that the delayed, high-dose rEpo infusion for 3days did not materially augment neuroprotection with therapeutic hypothermia.

Pelvic Radiation-Induced Testicular Damage: An Experimental Study at 1 Gray

ABSTRACTTherapeutic radiation of the pelvic region has been shown to cause damage to testicular germ cells. In this study we aimed to evaluate the effects of a low therapeutic dose of 1 Gy on the induction of cellular and histological damage in early-stage testicular germ cells and the impact of this radiation on offspring sex ratio. Unirradiated and irradiated male rats were mated with unirradiated female rats. Female rats were followed and the sex of the offspring was determined. The male rats were sacrificed at the end of the second week, and the testicular germ cells were subjected to genetic analysis along with cytological and histopathological examination. Sperm DNA was amplified with primers specific to testis-specific Y-linked protein, rat actin beta and testis-specific X-linked genes. The resulting products were separated by capillary electrophoresis. Histopathological changes were investigated by light microscopy along with the TUNEL assay and immunohistochemical staining for caspase-3. There was no significant difference between the two groups for sex ratio and size of offspring. The number of sperm cells bearing X or Y chromosomes’ did not differ significantly between these two groups. However, a 1 Gy dose of radiation caused significant cytopathological and histopathological changes in the testicular tissue. In the irradiated group, edematous regions were evident. The number of caspase-3 positive cells in the germinal epithelium of the seminiferous tubules was also significantly higher in the irradiated group. Our results showed that low-dose radiation induced apoptosis and caused significant cyto- and histopathological changes in the testicular tissue. Further research is required to fully elucidate their contribution to apoptosis and if low-dose radiation may potentially lead to long-term effects in the offspring. These results may also lead us to develop a new technique using the caspase-3 staining to monitor the susceptibility to low dose radiation.

Antitumor and anti-inflammatory effects of oligosaccharides from Cistanche deserticola extract on spinal cord injury

In the present study, the pharmacological effects of oligosaccharides from Cistanche deserticola extract on inflammation, oxidative stress, and apoptosis in male albino rats with spinal cord injury were investigated. Lipid peroxidation, GSH, catalase, superoxide dismutase, acetylcholine esterase, GPx, ROS, and nitric acid were significantly altered in the rats with spinal cord injury. The mRNA expression levels of IL-6, TNF-α, cyclooxygenase-2, iNOS, p53, caspase-3, bax, and pro-NGF were reduced by >20% following extract supplementation. Protein expression levels of caspase-3 and pro-NGF were also reduced by >20%. The number of p53 positive cells was 1, 79, 54, 33, and 19 in groups GI–GV, respectively, and the corresponding numbers of caspase-3 positive cells were 2, 87, 51, 23, and 14. Based on the present results, the use of oligosaccharides from Cistanche deserticola extract was effective against inflammation, oxidative stress, and apoptosis in spinal cord injury male albino rats.

Efficacy of phosphocreatine pre-administration on XIAP and Smac in ischemic penumbra of rats with focal cerebral ischemia reperfusion injury.

To observe the efficacy of phosphocreatine pre-administration (PCr-PA) on X-linked inhibitor of apoptosis protein (XIAP), the second mitochondia-derived activator of caspase (Smac) and apoptosis in the ischemic penumbra of rats with focal cerebral ischemia-reperfusion injury (CIRI). A total of 60 healthy male Sprague Dawley (SD) rats were randomly divided into three groups (n=20): group A (the sham operation group), group B &lt;intraperitoneally injected with 20 mg/kg (10 mg/ml) of saline before preparing the ischemia-reperfusion (IR) model&gt;, and group C &lt;intraperitoneally injected with 20 mg/kg (10 mg/ml) of PCr immediately before preparing the IR model&gt;. After 24 h for reperfusion, the neurological function was evaluated and the tissue was sampled to detect expression of XIAP, Smac and caspase-3 positive cells in the ischemic penumbra so as to observe the apoptosis. Compared with group B, neurological deficit scores, numbers of apoptotic cells, expression of Smac,caspase-9 and the numbers of Caspase-3 positive cells were decreased while expression of XIAP were increased in the ischemic penumbra of group C. Phosphocreatine pre-administration may elicit neuroprotective effects in the brain by increasing expression of X-linked inhibitor of apoptosis protein, reducing expression of second mitochondia-derived activator of caspase, and inhibiting the apoptosis in the ischemic penumbra.

Naringin attenuates thioacetamide-induced liver fibrosis in rats through modulation of the PI3K/Akt pathway

AimsNaringin (NR) is a flavanone glycoside extracted from grapefruits and citrus fruits. The aim of this study is to investigate the antifibrotic efficacy of NR in thioacetamide (TAA)-induced hepatic fibrosis in rats through evaluating NR effect on the PI3K/Akt pathway. Main methodsHepatic fibrosis was induced in rats by intraperitoneal injection of TAA (200mg/kg) twice per week for 6weeks. Simultaneously, NR (40mg/kg/day, p.o.) was given along with TAA injection. The ratio of P-Akt/Akt was assessed in hepatic homogenate as well as antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx)) and lipid peroxidation marker, malondialdehyde (MDA). Serum level of interleukin (IL)-6 were measured using ELISA. Hepatic tissues were examined histopathologically using hematoxylin and eosin (H&E) and Masson trichome staining. Tissue expression of alpha smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1), caspase-3 and fibronectin were scored immunohistochemically. Finally, the mRNA level of cytokine genes (IL-1β, IL-6, IL-10, interferon gamma (IFN-γ)), caspase-3, TGF-β1 and fibronectin were quantified using qPCR. Key findingsNR significantly suppressed Akt phosphorylation associated with increased number of caspase-3 positive cells especially in the fibrotic areas. Liver tissues of treated rats showed restoration of normal liver histology and decrease in collagen and fibronectin deposition. Furthermore, NR treatment ameliorated oxidative stress and inflammatory cytokine production. SignificanceNR alleviated experimental liver fibrosis through inhibition of PI3K/Akt pathway beside its anti-inflammatory and antioxidant effects. Therefore, NR is a promising therapeutic candidate for hepatic fibrosis.

Electric injury-induced Purkinje cell apoptosis in rat cerebellum: Histological and immunohistochemical study

IntroductionElectrical injury is a prominent problem in low income countries with increased morbidity and mortality rate. Nervous system is one of the most susceptible systems to electrical current because of its low resistance. There were different studies demonstrated electrocution effect on the nervous system, however little was made on the cerebellum. AimThis study was conducted to produce an experimental suggestion concerning injury of the nervous system through evaluating Purkinje cell apoptosis and number in rat cerebellum by fatal and non-fatal electric current using histological and immunohistochemical study. Also to support the diagnosis of electrocution as a probable cause of death and delayed neurological damage as well as disability. Materials & methodsFifty male Wistar rats were divided into five groups (10 rats each); control group: normal rats that were sacrificed without exposure to electric current, groups 1–3 (non-fatal electrocution groups): rats were exposed to alternating electric current (220v, 50Hz) for one minute then were sacrificed immediately, after 2h, and after 4h respectively, and group 4 (fatal electrocution group): rats were sacrificed after being electrified up to death (153±27s). Sections from cerebellum were processed for histological and caspase-3 immunohistochemical study. ResultsPurkinje cells showed marked histopathological changes in the form of shrunken dark stained cells with significant reduction of their number in H &E stained sections when compared to control, widespread argyrophilia, and degenerated organelles along with shrunken irregular nuclei. For caspase-3 staining, there was significantly increased number of caspase-3 positive cells in groups 1–3 (non-fatal electrocution groups) and markedly increased in group 4 (fatal electrocution group) in comparison to control group. These changes were gradually increased with the increased duration after exposure to the electric current. ConclusionApoptosis and loss of Purkinje cells were involved in the pathogenesis of immediate and long term effect of electrical injury on Purkinje cells, which will be an aid to the forensic pathologist to determine the cause of death and residual damage as well as disability after electric shock.

Chitooligosaccharide promotes immune organ development in broiler chickens and reduces serum lipid levels.

This study investigated the effects of chitooligosaccharide on lipid metabolism, immune organ development, and lymphocyte apoptosis in broiler chickens. A total of 480 one-day-old broiler chickens (Arbor Acres) were randomly and evenly assigned to control group and experimental groups I, II, and III. The control group was given a basic diet, while experimental groups I, II, and III were given basic diets for 42 days, supplemented with 50 mg/kg chlortetracycline, 20, and 40 mg/kg chitooligosaccharide, respectively. We found levels of serum triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) to be significantly reduced in experimental group II after 21 days, while the periarterial lymphatic sheath area of the spleens and the average number of bursa of Fabricius nodes were markedly increased. The serum total protein (TP) and high density lipoprotein cholesterol (HDL-C) levels, bursa of Fabricius index, and bursa of Fabricius lobule areas were additionally increased in experimental group III. After 42 days, the serum TP content had also increased and the bursa of Fabricius lobule area was augmented as well in experimental group II. Moreover, the splenic periarterial lymphatic sheat areas and the average numbers of bursa of Fabricius nodes were significantly increased in experimental group III. At both 21 and 42 days, numbers of Caspase 3-positive cells in spleen and bursa of Fabricius were significantly decreased in experimental groups II and III. Our results show that appropriate supplementation of chitooligosaccharide may improve lipid metabolism, promote immune organ development, and inhibit lymphocyte apoptosis in broilers.

A Hydrogel-Based Epirubicin Delivery System for Intravesical Chemotherapy.

This study aimed to examine the efficacy of epirubicin-loaded gelatin hydrogel (EPI-H) in the treatment of superficial urothelium carcinoma. Hydrogel was prepared by Schiff base-crosslinking of gelatin with glutaraldehyde. EPI-H exhibited high entrapment efficiency (59.87% ± 0.51%). EPI-H also increased epirubicin accumulation in AY-27 cells when compared with the effect of aqueous solutions of epirubicin (EPI-AQ); respective epirubicin-positive cell counts were 69.0% ± 7.6% and 38.3% ± 5.8%. EPI-H also exhibited greater cytotoxicity against AY-27 cells than that of EPI-AQ; IC50 values were 13.1 ± 1.1 and 7.5 ± 0.3 μg/mL, respectively. Cystometrograms showed that EPI-H reduced peak micturition, threshold pressures, and micturition duration, and that it increased bladder compliance more so than EPI-AQ. EPI-H enhanced epirubicin penetration into basal cells of urothelium in vivo, whereas EPI-AQ did so only to the umbrella cells. EPI-H inhibited tumor growth upon intravesical instillation to tumor-bearing bladder of F344 rats, inducing higher levels of caspase-3 expression than that observed with EPI-AQ treatment; the number of caspase-3 positive cells in treated urothelium carcinoma was 13.9% ± 4.0% (EPI-AQ) and 34.1% ± 1.0%, (EPI-H). EPI-H has value as an improved means to administer epirubicin in intravesical instillation treatments for bladder cancer.

Effects of Pretreatment with Warfarin or Rivaroxaban on Neurovascular Unit Dissociation after Tissue Plasminogen Activator Thrombolysis in Ischemic Rat Brain

Warfarin and rivaroxaban are highly effective in reducing stroke risk in patients with atrial fibrillation (AF). However, their effects on anticoagulation and neurovascular unit (NVU) change remain elusive. In this study, we assessed the risks and benefits of pre-treatment with warfarin or rivaroxaban after tissue-type plasminogen activator (tPA) thrombolysis in ischemic rat brain. Pre-treatment with warfarin (.2 mg/kg/day), low dose rivaroxaban (60 mg/kg/day), high dose rivaroxaban (120 mg/kg/day) or vehicle was performed for 2 weeks, transient middle cerebral artery occlusion (tMCAO) was induced for 90 min, then followed by reperfusion with tPA. At 24 hours (h) after reperfusion, we observed the changes of matrix metalloproteinase-9 (MMP-9), tissue factor, caspase 3 and NVU dissociation. Prothrombin time (PT) was significantly prolonged in the warfarin and rivaroxaban pretreated groups. MMP-9 expression greatly increased in the warfarin group, and this was reduced in the rivaroxaban groups compared with the vehicle group. Tissue factor expression remarkably decreased in the warfarin and rivaroxaban groups. The number of caspase 3-positive cells had no difference among all the groups. Marked dissociations between astrocyte foot processes and the basal lamina or pericytes were observed in the warfarin pretreated group, but such dissociations were improved in the rivaroxaban groups. Our present study shows that pre-treatment with rivaroxaban was noninferior to warfarin in the anticoagulation, but a lower risk of NVU dysfunction and dissociation after tPA treatment in rivaroxaban. This finding could partly explain the mechanism of reducing hemorrhagic complications by rivaroxaban in clinical studies.

Superficial zone cellularity is deficient in mice lacking lubricin: a stereoscopic analysis.

BackgroundLubricin, a mucinous glycoprotein secreted by synoviocytes and chondrocytes plays an important role in reducing the coefficient of friction in mammalian joints. Elevated cartilage surface friction is thought to cause chondrocyte loss; however, its quantification and methodological approaches have not been reported. We adapted a stereological method and incorporated vital cell staining to assess cellular loss in superficial and upper intermediate zones in lubricin deficient mouse cartilage.MethodsThe femoral condyle cartilage of the intact knees from lubricin wild type (Prg4 +/+), heterozygote (Prg4 +/-), and knockout (Prg4 -/-) mice was imaged using fluorescein diacetate (FDA), propidium iodide (PI), and Hoechst staining, and confocal microscopy. Three dimensional reconstructions of confocal images to a depth of 14 μm were analyzed using Matlab to determine the volume fraction occupied by chondrocytes in cartilage of both medial and lateral femoral condyles. Living chondrocyte volume fraction was defined as FDA stained chondrocyte volume/total volume of superficial + upper intermediate zone. Living and dead (total) chondrocyte volume fraction was defined as FDA + PI stained chondrocyte volume/total volume of superficial + upper intermediate zone. MicroCT provided an orthogonal measure of cartilage thickness. Immunohistology for activated caspase-3 and TUNEL staining were performed to evaluate the presence of apoptotic chondrocytes in Prg4 mutant mice.ResultsLiving chondrocyte volume fraction of the medial femoral condyle was significantly lower in Prg4 -/- mice compared to Prg4 +/+ (p = 0.002) and Prg4 +/- (p = 0.002) littermates. There was no significant difference in medial condyle chondrocyte volume fraction between Prg4 +/+ and Prg4 +/- mice (p = 0.82). No significant differences were observed for the chondrocyte volume fraction for the lateral condyle (p > 0.26). Cartilage thickness increased in the medial condyle for Prg4 -/- mice compared to Prg4 +/+ (p = 0.02) and Prg4 +/- (p = 0.03) littermates, and the lateral condyle for Prg4 -/- mice compared to Prg4 +/+ (p < 0.0001) and Prg4 +/- (p < 0.0001) littermates, indicating that a multi-dimensional increase in cartilage volume did not artifactually lower the chondrocyte volume fraction in the medial condyle. Significantly higher number of caspase-3 positive cells were observed in the superficial and upper intermediate zone cartilage of the medial femoral condyle of Prg4 -/- mice compared to Prg4 +/+ (p = 0.01) and Prg4 +/- (p = 0.04) littermates, and the lateral femoral condyle of Prg4 -/- mice compared to Prg4 +/+ (p = 0.02) and Prg4 +/- (p = 0.02) littermates. There were no significant differences in TUNEL staining among different Prg4 genotypes in both condyles (p > 0.05 for all comparisons).ConclusionsIncreased Caspase-3 activation is observed in Prg4 deficient mice compared to Prg4 sufficient littermates. Absence of Prg4 induces loss of chondrocytes in the superficial and upper intermediate zone of mouse cartilage that is quantifiable by a novel image processing technique.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0967-4) contains supplementary material, which is available to authorized users.

Effects of total flavone of hawthorn leaf on expression of p38MAPK signaling pathway and inflammation factors in rats brain with chronic cerebral ischemia

Objective To investigate the therapeutic effect and mechanism of total flavone of hawthorn leaf (TFHL) on p38MAPK signaling pathway and inflammation factors in rats brain with chronic cerebral ischemia. Methods SPF class healthy male SD rats were randomly divided into sham group, model group, TFHL group and Ginkgo leaf group(12 rats in each group). Permanent bilateral carotid artery ligation was used to prepare chronic cerebral ischemia model. Morris water maze method was used to evaluate learning and memory abilities of rats. Immunohistochemistry and Western blot methods were used to measure the expression of caspase-3 and p38MAPK proteins. ELISA method was used to measure the amounts of TNF-α and IL-1β in hippocampal tissue. Results Compared with the model group, TFHL treatment (36 d) can improve learning and memory capabilities of vascular dementia rats, shorten the escape latency (TFHL group (10.01±2.85) s vs Model group (19.54±6.12) s, P<0.05) and the course of searching platform(TFHL group(2.6044±0.3219)m vs model group(3.3502±0.6231)m, P<0.05), increase the numbers of crossing the platform (TFHL group(5.17±2.12) times vs Model group (3.96±1.34) times, P<0.05) and the platform quadrant swimming distance percentage (TFHL group(48.22±7.39)% vs model group (33.42±5.32) %, P<0.01). The number of caspase-3 positive cells in the hippocampus significantly reduced (TFHL group(1.677±0.164) vs Model group (2.387±0.171), P<0.05), the expression level of P38MAPK protein (TFHL group(0.0161±0.0003) vs Model group (0.0254±0.0018), P<0.05), TNF-α(TFHL group(19.61±3.61) ng/10 mg vs Model group (27.82±6.57) ng/10 mg, P<0.01)and IL-1β(TFHL group(24.41±2.56) ng/10 mg vs Model group (29.43±5.26) ng/10 mg, P<0.05) were significantly decreased. Conclusion TFHL plays a protective role in nerve function of the chronic cerebral ischemia rats. The mechanism of its anti-apoptosis might be associated with the activation of P38MAPK signaling pathway, inflammation and the apoptosis of neurons in the brain. Key words: Total flavone of hawthorn leaf; Chronic cerebral ischemia; Learning and memory; p38MAPK; TNF-α; IL-1β

Apoptosis and cell proliferation in the mouse model of embryonic death induced by Tritrichomonas foetus infection

Bovine tritrichomonosis is a sexually transmitted disease caused by the protozoon Tritrichomonas foetus and characterised by embryonic-death and abortion. During pregnancy, the processes of cell proliferation and death play a crucial role for blastocyst implantation and the subsequent maintenance of early pregnancy, and their misbalance may lead to the abortion. In this study, we aimed to investigate whether cell proliferation and death may be altered during tritrichomonosis. For this purpose, we used pregnant BALB/c mice as an alternative experimental animal model that has successfully reproduced the infection. We analysed the immunohistochemical expression of active caspase-3 and proliferating cell nuclear (PCNA) antigens in the endometrium of infected mice. We found an increase in the number of caspase-3 positive cells in infected mice that were not pregnant at the necropsy. Besides, the number of positive proliferating cells increased in the uterine luminal epithelium of infected animals killed at 5–7 days post coitum (dpc). Pregnant infected mice killed at 8–11 dpc showed higher proliferation than control animals. We suggest that the cytopathic effect induced by T. foetus in the uteri of infected mice may induce the apoptosis of the epithelial cells and, as a result, promote a compensatory proliferative response. The information described here will be helpful to further study the pathogenesis of the bovine tritrichomonosis.

Systemic physiology and neuroapoptotic profiles in young and adult rats exposed to surgery: A randomized controlled study comprising four different anaesthetic techniques

BackgroundExperimental evidence indicates that general anaesthetics can induce apoptotic neurodegeneration in the developing brain. The majority of these studies have been performed in the absence of surgery and it currently remains unclear how the presence of surgical stimuli would influence neuroapoptosis as well as systemic homeostasis. Here we explored this possibility by performing dorsal skin flap surgery in young and adult rats under four distinct currently used anaesthesia regimens. MethodsYoung (21-days) and adult (2 months) male Sprague–Dawley rats were randomized to 150min exposure to one of four anaesthetics regimens: (i) sevoflurane/dexmedetomidine, (ii) sevoflurane/fentanyl; (iii) propofol/dexmedetomidine, and (iv) propofol/fentanyl. Animals underwent a dorsal skin flap procedure while physiologic, metabolic and biochemical parameters were closely monitored. Neuroapoptotic profiles were evaluated in the cortex, thalamus and hippocampus (CA1 and CA3) at the end of the procedure in each experimental group. ResultsSignificant perturbations of systemic homeostasis were found under all anaesthetic regimens. Hyperglycemia and decreased heart rate were particularly relevant in experimental groups receiving dexmedetomidine, while propofol administration was associated with increased systemic lactate levels and metabolic acidosis. A substantial difference in anaesthesia/surgery-induced neuroapoptosis was found between young and adult rats in several brain regions. Combination of sevoflurane and dexmedetomidine resulted in the highest number of caspase-3 positive cells, although the extent of cell death remained relatively low in all experimental groups. ConclusionCombination of anaesthesia and surgery induces significant perturbations of physiological parameters in both young and adult spontaneously breathing rats undergoing surgery. These observations further enlighten the need for detailed physiological monitoring under these experimental conditions. Although some statistically significant differences in activated caspase-3 profiles were detected between experimental groups, the overall extent of neuronal cell death remained very low under all conditions questioning, thereby, the physiological significance of apoptotic neurodegeneration in the context of anaesthesia and surgery.

Proliferation, apoptosis and expression of matrix metalloproteinase-9 in human fetal lung

Expression pattern of the Ki-67, caspase-3 and matrix metalloproteinases-9 (MMP-9) factors were immunohistochemically analyzed in 48 human fetal lungs from 12 to 40 weeks of gestation. The number of Ki-67 positive cells in the epithelium of canaliculare (88cells/mm2) and sacculare stage (93cells/mm2) were significantly higher than in the epithelium of pseudoglandular stage (12cells/mm2) (p=0.0008 vs. p=0.003). The number of Ki-67 positive cells in the mesenchyme of canaliculare stage (132cells/mm2) was significantly higher than in the mesenchyme of pseudoglandular stage (37cells/mm2) (p=0.001). The proliferation of mesenchymal cells was higher than the epithelial cells in all developmental stages, especially in the canaliculare stage (p=0.007). Similarly, the number of caspase-3 positive cells in the epithelium of canalicular stage (13cells/mm2) was significantly higher than in the epithelium of pseudoglandular stage (6cells/mm2) (p=0.002) with peaks in the conductive epithelium of canalicular stage. The number of caspase-3 positive cells in the mesenchyme of canaliculare stage (3cells/mm2) was significantly higher than in the mesenchyme of saccular stage (0cells/mm2) (p=0.05). There were no caspase-3 positive cells in the mesenchyme of pseudoglandular stage. However, unlike the Ki-67 expression, mesenchymal cells in comparison to epithelial cells express substantially less caspase-3 in all developmental stages. Up to the saccular stage, the expression of MMP-9 in mesenchymal cells showed a linear increase with most pronounced expression in that stage. The number of MMP-9 positive cells in the mesenchyme of canaliculare (20cells/mm2) and sacculare (39cells/mm2) stage were significantly higher than in the mesenchyme of pseudoglandular stage (12cells/mm2) (p=0.04 vs. p=0.004). The first epithelial cells that express MMP-9 were present only at the alveolar stage. Increased proliferation and apoptosis of the mesenchymal cells of canalicular stage is important for formation of definite structures within the stroma of the lung parenchyma. Although apoptosis in the epithelium is not pronounced as proliferation, it is important for thinning of the epithelium and consequent spread of respiratory tract. However in the saccular stage when mesenchyme disappears, MMP-9 expression is more important for primitive alveoli differentiation.