Pancreatic cancer orthotopic graft in a murine model

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with increasing incidence. Even if progress have been made, the five-year overall survival remains lower than 10%. There is a desperate need in therapeutic improvements. In the last two decades, new in-vitro models have been developed and improved, including tridimensional-culture spheroids and organoids. However, animal studies remain mandatory in the upscaling before clinical studies. Orthotopic and syngeneic grafting is a robust model to test a drug efficiency in a tumor and its microenvironment. Methods: We described a method for orthotopic and syngeneic graft of KRAS mutated, p53 wildtype, 8305 cells in a C57BL/6J mouse model. Results: With this microsurgical method, 30 mice were grafted, 24 by a junior and six by a senior, resulting in 95,8 and 100% of (partial and total) successful tumoral implantation, respectively. Twenty mice underwent ultrasound follow-up. It was an efficient method for the tumoral growth evaluation. At day 16 after grafting, 85% of the tumors were detectable by ultrasound, and at day 22 all tumors were detected. Conclusions: The presented method appears to be a robust and reliable method for pre-clinical studies. A junior master student can provide positive results using this technique, which can be improved with training.

Ulinastatin alleviates early brain injury after traumatic brain injury by inhibiting oxidative stress and apoptosis

Purpose: Traumatic brain injury (TBI) remains a major public health problem and cause of death. Ulinastatin (UTI), a serine protease inhibitor, has been reported to have an anti-inflammatory effect and play a role in immunoregulation and organ protection by reducing reactive oxygen species (ROS) production, oxidative stress and inflammation. However, the neuroprotective of UTI in TBI has not been confirmed. Therefore, this study aimed to investigate the neuroprotection and potential molecular mechanisms of UTI in TBI-induced EBI in a C57BL/6 mouse model. Methods: The neurological score and brain water content were evaluated. Enzyme-linked immunosorbent assay was used to detect neuroinflammatory cytokine levels, ROS and malondialdehyde detection to evaluate oxidative stress levels, and TUNEL staining and western blotting to examine neuronal damages and their related mechanisms. Results: Treatment with UTI markedly increased the neurological score; alleviated brain oedema; decreased the inflammatory cytokine tumour necrosis factor a, interleukin-1ß (IL-1ß), IL-6 and nuclear factor kappa B (NF-kB) levels; inhibited oxidative stress; decreased caspase-3 and Bax protein expressions; and increased the Bcl-2 levels, indicating that UTI-mediated inhibition of neuroinflammation, oxidative stress and apoptosis ameliorated neuronal death after TBI. The neuroprotective capacity of UTI is partly dependent on the TLR4/NF-kB/p65 signalling pathway. Conclusions: Therefore, this study reveals that UTI improves neurological outcomes in mice and reduces neuronal death by protecting against neural neuroinflammation, oxidative stress and apoptosis.

Immunomodulatory effects of Blaps rynchopetera extract

Purpose: To explore the potential immunomodulatory effects of total extract and different polar parts from Blaps rynchopetera Fairmaire. Methods: Phagocytic activity was evaluated by neutral red assay, and the effect of the immune function was investigated by normal and immunocompromised mice models. Results: In vitro, total extract, as well as chloroform, ethyl acetate, n-butanol and water fractions could individually enhance the phagocytic ability of mouse peritoneal macrophages; in addition, chloroform and ethyl acetate fractions had an increasing tendency when combined stimulation with lipopolysaccharide (LPS). In vivo, ethyl acetate fraction (EAF) could enhance the immune organ index, increase the serum hemolysin level and peripheral blood immune cells of immunocompromised mice, while for normal mice, the effect was inconspicuous. Conclusions: Blaps rynchopetera extracts had noteworthy immunomodulatory effect, especially for individuals with immune disorders.

Jatrorrhizine reduces myocardial infarction-induced apoptosis and fibrosis through inhibiting p53 and TGF-ß1/Smad2/3 pathways in mice

Purpose: To explore the mechanism of jatrorrhizine on apoptosis and fibrosis induced by myocardial infarction (MI) in an animal model. Methods: The left anterior descending branch of coronary artery was surgically ligated to duplicate the mouse model of MI. The sham and infarcted mice were treated with normal saline once a day, while mice in experimental groups received low-dose (LD) and high-dose (HD) jatrorrhizine once a day respectively. Two weeks later, cardiac function was detected by echocardiography, and histopathological examination was performed using hematoxylin and eosin (H&E) and Masson staining. The expressions of p53, TGF-ß1, Smad/2/3, Bax, Bcl-2, collagen I and collagen III were quantified using qRT-PCR and western blot assays. Results: Jatrorrhizine significantly improved left ventricular ejection fraction (LVEF) and left ventricle end-systolic (LVES) in mice. Histopathological, administration of jatrorrhizine weakened infiltration of inflammatory cells and cardiac fibrosis in myocardium of mice caused by MI. Additionally, jatrorrhizine suppressed cardiomyocyte apoptosis exhibited as its capability to reverse changes of Bax and Bcl-2 levels in myocardium caused by MI. Jatrorrhizine statistically significantly downregulated expression of collagen I and collagen III, as well as TGF-ß1, Smad2/3 and p53. Conclusions: Jatrorrhizine reduce cardiomyocyte apoptosis and fibrosis through inhibiting p53/Bax/Bcl-2 and TGF-ß1/Smad2/3 signaling pathways.

Protective effect of polysaccharides isolated from the seeds of Cuscuta chinensis Lam. on 5-fluorouracil-induced intestinal mucositis in mice

Purpose: To evaluate the protective effect of Cuscuta chinensis Lam. polysaccharides (PCCL) on 5-fluorouracil-(5-FU)-induced intestinal mucositis (IM) in mice. Methods: PCCL was orally administered at a dose of 20 mg·kg­1 for 7 days and its protective effect on 5-FU-induced IM (5-FU, 50 mg·kg­1 for 5 days) was evaluated by monitoring changes in body weight, degree of diarrhea, levels of tissue inflammatory factors (tumor necrosis factor α, interleukin 6, and interleukin 1ß levels), apoptosis rates, and the expression levels of caspase-3, Bax and Bcl-2. Results: The severity of mucosal injury (as reflected by body weight changes, degree of diarrhea, height of villi, and damage to crypts) was significantly attenuated by PCCL administration. PCCL also reduced the levels of tissue inflammatory factors, the apoptosis rate, and the expression of caspase-3 and Bax, and increased Bcl-2 expression. Conclusions: PCCL administration may be significantly protective against 5-FU-induced IM by inhibiting apoptosis and regulating the abnormal inflammation associated with it.

Ulinastatin alleviates early brain injury after intracerebral hemorrhage by inhibiting oxidative stress and neuroinflammation via ROS/MAPK/Nrf2 signaling pathway

Purpose: Spontaneous intracerebral hemorrhage (ICH) is still a major public health problem, with high mortality and disability. Ulinastatin (UTI) was purified from human urine and has been reported to be anti-inflammatory, organ protective, and antioxidative stress. However, the neuroprotection of UTI in ICH has not been confirmed, and the potential mechanism is unclear. In the present study, we aimed to investigate the neuroprotection and potential molecular mechanisms of UTI in ICH-induced early brain injury in a C57BL/6 mouse model. Methods: The neurological score, brain water content, neuroinflammatory cytokine levels, oxidative stress levels, and neuronal damage were evaluated. Results: UTI treatment markedly increased the neurological score, alleviated brain edema, decreased the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and NF-κB, decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and upregulated the levels of glutathione (GSH), superoxide dismutase (SOD), and Nrf2. This finding indicated that UTI-mediated inhibition of neuroinflammation and oxidative stress alleviated neuronal damage after ICH. The neuroprotective capacity of UTI is partly dependent on the ROS/MAPK/Nrf2 signaling pathway. Conclusions: UTI improves neurological outcomes in mice and reduces neuronal death by protecting against neural neuroinflammation and oxidative stress.

Effects of systemic inflammation due to hepatic ischemia-reperfusion injury upon lean or obese visceral adipose tissue

Purpose: To evaluate how the induction of liver damage by ischemia and reperfusion affects the adipose tissue of lean and obese mice. Methods: Lean and diet-induced obese mice were subjected to liver ischemia (30 min) followed by 6 h of reperfusion. The vascular stromal fraction of visceral adipose tissue was analyzed by cytometry, and gene expression was evaluated by an Array assay and by RT-qPCR. Intestinal permeability was assessed by oral administration of fluorescein isothiocyanate (FITC)-dextran and endotoxemia by serum endotoxin measurements using a limulus amebocyte lysate assay. Results: It was found that, after liver ischemia and reperfusion, there is an infiltration of neutrophils, monocytes, and lymphocytes, as well as an increase in the gene expression that encode cytokines, chemokines and their receptors in the visceral adipose tissue of lean mice. This inflammatory response was associated with the presence of endotoxemia in lean mice. However, these changes were not observed in the visceral adipose tissue of obese mice. Conclusions: Liver ischemia and reperfusion induce an acute inflammatory response in adipose tissue of lean mice characterized by an intense chemokine induction and leukocyte infiltration; however, inflammatory alterations are already present at baseline in the obese adipose tissue and liver ischemia and reperfusion do not injure further.

Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Distribution of commensal rodents in some shops of three districts in Malakand region, Pakistan

Abstract Using wire mesh live traps distribution pattern of the Rattus rattus and Mus musculus in different shops of three districts of Malakand region, Pakistan were recorded from September 2014 to October 2015. Over all 103 rodents (Rattus rattus 86 and Mus musculus 17) were caught during in 0.04 trap success (2448 trap nights). Regression of daily captures on cumulative captures revealed an estimate of 103 rodents from all the sampled structures with an average of 3.55 rodents per shop. R. rattus; 83.4% of captures were numerically dominant in almost all types of shops sampled, and were significantly different than Mus musculus; 16.5% of captures. Both species were found together in some shops while they were mostly trapped from the separate shops. Male rodents outnumbered the females.

Resumo Usando o padrão de distribuição de armadilhas vivas de malha de arame do Rattus rattus e Mus musculus em diferentes lojas de três distritos da região de Malakand, o Paquistão foi registrado de setembro de 2014 a outubro de 2015. No total, 103 roedores (Rattus rattus 86 e Mus musculus 17) foram pegos durante em 0,04 armadilha de sucesso (2448 noites de armadilha). A regressão das capturas diárias em capturas cumulativas revelou uma estimativa de 103 roedores de todas as estruturas amostradas, com uma média de 3,55 roedores por loja. R. rattus; 83,4% das capturas foram numericamente dominantes em quase todos os tipos de lojas da amostra e foram significativamente diferentes do Mus musculus; 16,5% das capturas. Ambas as espécies foram encontradas juntas em algumas lojas, enquanto estavam na maior parte presas em lojas separadas. Os roedores machos eram mais numerosos do que as fêmeas.

Eficácia do método de graham para o diagnóstico de Syphacia obvelata (Rudolphi, 1802) no monitoramento sanitário de camundongos Swiss Webster de um biotério de criação / Effectiveness of the graham method for Syphacia obvelata (Rudolphi, 1802) diagnosis in the health monitoring of Swiss Webster mice in a breeding colony

A infecção por Oxyuridae constitui um problema nas barreiras sanitárias em colônias de roedores, tendo em vista que esta é a família de parasito mais encontrada em instalações de animais de laboratório, especialmente a espécie Syphacia obvelata. Camundongos outbred stock Swiss Webster apresentam sinais clínicos quando associados com elevada carga parasitária e, geralmente, é observado um aumento no número de animais com prolapso retal. O controle desse parasito e outros patógenos é realizado mediante monitoramento sanitário trimestral, conforme as recomendações da Federação das Associações Europeias de Ciência de Animais de Laboratório (FELASA). O diagnóstico pelo método de Graham é determinante para a pesquisa dos ovos através de impressão de fita adesiva na região perianal do animal no início do ciclo do oxiurídeo. Foi realizada uma análise comparativa entre os métodos de Graham e o exame direto da mucosa intestinal para diagnóstico de S. obvelata de 60 camundongos sentinelas fêmeas outbred stock Swiss webster, procedentes de instalações de roedores de um biotério de criação. Os resultados mostraram uma diferença significativa entre o método de Graham e o exame direto da mucosa intestinal (p > 0,001 a 95%, teste de Fisher exato), com uma prevalência de 50% de ovos do parasito pela técnica de Graham e 8,33% pelo exame direto da mucosa intestinal, que detectou todas as formas do parasito. Pelo exame direto, além de ovos, foram identificadas outras formas do parasito, como: larvas, fêmeas adultas e machos adultos. Concluiu-se que a técnica de Graham é capaz de ampliar o espectro de identificação de S. obvelata, pois detecta com maior precisão a presença de ovos do parasita, uma vez que a fita adesiva demonstra que as fêmeas adultas grávidas migram para o ânus antes de morrer, depositando seus ovos na região perianal do hospedeiro. Sendo assim, o método de Graham deve ser adicionado ao programa de monitoramento sanitário, associado aos outros rotineiramente empregados no controle da qualidade de animais de laboratório, contribuindo com as condutas de manejo da criação animal em prol do controle e erradicação do agente em questão.

Infection by Oxyuridae is a problem in sanitary barriers in rodent colonies, considering that this is the parasite family most commonly found in laboratory animal facilities, especially the species Syphacia obvelata. Outbred stock Swiss Webster mice show clinical signs when associated with a high parasite load and, generally, an increase in the number of animals with rectal prolapse is observed. The control of this parasite and other pathogens is carried out through quarterly sanitary monitoring, in accordance with the recommendations of the Federation of European Associations of Laboratory Animal Science (FELASA). Diagnosis by Graham's method is crucial for the search for eggs by printing adhesive tape in the animal's perianal region at the beginning of the oxyuride cycle. A comparative analysis was performed between Graham's methods and direct examination of the intestinal mucosa for the diagnosis of S. obvelata in 60 female sentinel Swiss outbred stock Swiss webster mice, from rodent installations in a breeding animal facility. Results showed a significant difference between Graham's method and direct examination of the intestinal mucosa (p > 0.001 at 95%, Fisher's exact test), with a prevalence of 50% of parasite eggs by Graham's technique and 8.33% by direct examination of the intestinal mucosa, which detected the whole parasite forms. By direct examination, in addition to eggs, other forms of the parasite were identified, such as: larvae, adult females and adult males. It was concluded that Graham's technique is capable of expanding the identification spectrum of S. obvelata, as it detects the presence of parasite eggs more accurately, since the adhesive tape demonstrates that adult pregnant females migrate to the anus before dying, depositing its eggs in the perianal region of the host. Therefore, Graham's method should be added to the health monitoring program, associated with others routinely used in the quality control of laboratory animals, contributing to the management of animal husbandry in favor of the control and eradication of the agent in question.

Morphological, biochemical and histological effects of aqueous extracts of peanut (Arachis hypogaea) on swiss mice in different diets

ABSTRACT Purpose: To evaluate the morphological, biochemical, and histological effects of aqueous extracts of peanut (skinless and added to 1% skin) in Swiss mice submitted to a high-fat diet. Methods: Forty male Swiss mice were divided into four groups (n=10 per group): GI) normocaloric diet; GII) high-fat diet; GIII) high-fat diet + 0.5 mL of peanut extract; GIV) high-fat diet + 0.5 mL of peanut extract + 1% peanut skin. The animals were weighed weekly and euthanized after 12 weeks for histopathological and biochemical analyses. The study was approved by the Animal Use Ethics Committee. Results: The animals in the GIV group had higher body weight when compared to the other ones. Increase in total cholesterol in GIII, increase in blood glucose in groups GII, GIII and GIV, decrease in serum low-density lipoprotein (LDL) concentration in groups GI and GIV and increase in serum concentration of C-reactive protein in GII were seen. The presence of vacuolar fat deposits was found in animal livers from GII. Conclusions: The extracts improved the plasma concentrations of animals that received a high-fat diet, including preventing morphological damage to liver tissue. These benefits were enhanced by the association of peanut shells with the extract.

Changes in ovarian morphology and hormone concentrations associated with reproductive seasonality in wild large Japanese field mice (Apodemus speciosus)

Abstract Wild large Japanese field mice (Apodemus speciosus) responses to cyclic seasonal changes are associated with physiological and behavioral changes. However, the detailed regulation of oogenesis in the ovary during the seasonal reproductive cycle in wild large Japanese field mice has not been studied. We assessed the dynamics and changes in ovarian morphology and hormone concentrations associated with reproductive seasonality throughout the year. The stages of the ovarian morphological breeding cycle of wild large Japanese field mice were classified as breeding, transition, and non-breeding periods during the annual reproductive cycle. Measurement of blood estradiol concentrations throughout the year showed that the levels in September and October were higher than those in other months. It is presumed that follicle development starts from a blood estradiol concentration of 38.4 ± 27.1 pg/mL, which marks a shift from the transitional season to the breeding season, followed by the transition to the non-breeding season at 26.1 ± 11.6 pg/mL. These results suggest that seasonal follicle development in wild rodents is correlated with estradiol regulation. We consider this species to be an alternative animal model for studying seasonal reproductive changes and the effects of environmental changes.

Blood metabolic and hematology parameters and survivorship in mice after application of the rabies challenge virus standard in vaccine potency test

Background: Rabies virus can cause intensive and lethal infection of the central nervous system (CNS) in animals andhumans. Metabolic examinations are conducted at the cerebrospinal fluid (CSF), and it has been found that many metabolicchanges occur during RABV infection. However, although it is a neurotropic virus, it can cause damage to extraneuraltissues - lungs, heart, kidneys and liver. This study aimed to determine differences in metabolic, endocrinology and hematologic parameters in blood of mice after application of rabies challenge with virus standard 27 strain (CVS-27).Materials, Methods & Results: This study included 30 survived, and 30 dead mice that were part of the standard procedureof NIH (National Institute of Health) test in Pasteur Institute in Novi Sad. Tests were performed in the following order: twogroups of mice were vaccinated in a 7 day period with different dilutions of standard vaccine and the examined vaccine.Seven days after the last vaccination, immunized animals and animals in the control group received test virus CVS-27.Blood samples were collected from a heart puncture. Differences in hematologic and biochemical parameters were determined by t-test. Due to a high number of blood parameters, we performed a joint analysis of multiple dependent variables.Higher pH value and higher concentrations of glucose, cholesterol, lactate dehydrogenase (LDH), creatine kinase (CK),albumin, urea, creatinine, α-amylase, magnesium (Mg), nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHB)and lactate were noted in dead mice. Higher granulocytes and mean platelet volume (MPV) were noted in mice whichdied, but also reduced lymphocytes, erythrocytes, haemoglobin, hematocrit and platelets count. Higher values of insulin,cortisol and HOMA-IR (homeostatic model assessment insulin resistance) were noted in the group of dead mice comparedto the surviving one. Reduced QUICKI...

Changes in ovarian morphology and hormone concentrations associated with reproductive seasonality in wild large Japanese field mice (Apodemus speciosus)

Wild large Japanese field mice (Apodemus speciosus) responses to cyclic seasonal changes are associated with physiological and behavioral changes. However, the detailed regulation of oogenesis in the ovary during the seasonal reproductive cycle in wild large Japanese field mice has not been studied. We assessed the dynamics and changes in ovarian morphology and hormone concentrations associated with reproductive seasonality throughout the year. The stages of the ovarian morphological breeding cycle of wild large Japanese field mice were classified as breeding, transition, and non-breeding periods during the annual reproductive cycle. Measurement of blood estradiol concentrations throughout the year showed that the levels in September and October were higher than those in other months. It is presumed that follicle development starts from a blood estradiol concentration of 38.4 ± 27.1 pg/mL, which marks a shift from the transitional season to the breeding season, followed by the transition to the non-breeding season at 26.1 ± 11.6 pg/mL. These results suggest that seasonal follicle development in wild rodents is correlated with estradiol regulation. We consider this species to be an alternative animal model for studying seasonal reproductive changes and the effects of environmental changes.(AU)

Blood metabolic and hematology parameters and survivorship in mice after application of the rabies challenge virus standard in vaccine potency test

Background: Rabies virus can cause intensive and lethal infection of the central nervous system (CNS) in animals andhumans. Metabolic examinations are conducted at the cerebrospinal fluid (CSF), and it has been found that many metabolicchanges occur during RABV infection. However, although it is a neurotropic virus, it can cause damage to extraneuraltissues - lungs, heart, kidneys and liver. This study aimed to determine differences in metabolic, endocrinology and hematologic parameters in blood of mice after application of rabies challenge with virus standard 27 strain (CVS-27).Materials, Methods & Results: This study included 30 survived, and 30 dead mice that were part of the standard procedureof NIH (National Institute of Health) test in Pasteur Institute in Novi Sad. Tests were performed in the following order: twogroups of mice were vaccinated in a 7 day period with different dilutions of standard vaccine and the examined vaccine.Seven days after the last vaccination, immunized animals and animals in the control group received test virus CVS-27.Blood samples were collected from a heart puncture. Differences in hematologic and biochemical parameters were determined by t-test. Due to a high number of blood parameters, we performed a joint analysis of multiple dependent variables.Higher pH value and higher concentrations of glucose, cholesterol, lactate dehydrogenase (LDH), creatine kinase (CK),albumin, urea, creatinine, α-amylase, magnesium (Mg), nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHB)and lactate were noted in dead mice. Higher granulocytes and mean platelet volume (MPV) were noted in mice whichdied, but also reduced lymphocytes, erythrocytes, haemoglobin, hematocrit and platelets count. Higher values of insulin,cortisol and HOMA-IR (homeostatic model assessment insulin resistance) were noted in the group of dead mice comparedto the surviving one. Reduced QUICKI...(AU)

Inhibition of CXCR2 alleviates the development of abdominal aortic aneurysm in Apo E-/- mice

Purpose To investigate the relationship between atherosclerotic abdominal aortic aneurysm (AAA) and CXC chemokine receptor type 2 (CXCR2). Methods Mouse AAA model was established by embedding angiotensin-II pump (1000 ng/kg/min) in ApoE-/- mice. Mice were received SB225002, a selective CXCR2 antagonist, for treatment. Blood pressure was recorded, and CXCR2+ macrophages were examined by flow cytometry analysis. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was performed to detect cell apoptosis of abdominal aortic aneurysms. Macrophages were isolated from ApoE-/- mice and treated with Ang II and/or SB225002. Dihydroethidium staining was carried out to determine reactive oxygen species (ROS) activity. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the production of IL-1 and TNF-. The corresponding gene expressions were measured using real-time polymerase chain reaction (PCR), western blot, and immunohistochemistry staining. Results We found that Ang II activated the expression of CXCR2 in monocytes during the formation of AAA. Inhibition of CXCR2 significantly reduced the size of AAA, attenuated inflammation and phenotypic changes in blood vessels. Ang II-induced macrophages exhibited elevated ROS activity, and elevated levels of 1 and TNF-, which were then partly abolished by SB225002. Conclusions CXCR2 plays an important role in AAA, suggesting that inhibiting CXCR2 may be a new treatment for AAA.(AU)

Techniques of fragile renal organoids transplantation in mice

Purpose: This study aimed to develop a microsurgical technique to transplant extremely fragile renal organoids in vivo, created by in-vitro reaggregation of metanephros from fetal mice. These organoids in reaggregation and development were examined histologically after transplantation under the renal capsule. Methods: Initially, metanephros from fetal mice were enzymatically treated to form single cells, and spheroids were generated in vitro. Under a microscope, the renal capsule was detached to avoid bleeding, and the outer cylinder of the indwelling needle was inserted to detach the renal parenchyma from the renal capsule using water pressure. The reaggregated spheroid was aspirated from the culture plate using a syringe with an indwelling needle outer cylinder and carefully extruded under the capsule. Pathological analysis was performed to evaluate changes in reaggregated spheroids over time and the effects of co-culture of spinal cord and subcapsular implantation on maturation. Results: In vitro, the formation of luminal structures became clearer on day 5. These fragile organoids were successfully implanted without tissue crapes under the renal capsule and formed glomerular. The effect of spinal cord co-transplant was not obvious histrionically. Conclusions: A simple and easy method to transplant fragile spheroids and renal under the renal capsule without damage was developed.

Gynura segetum induces hepatic sinusoidal obstruction syndrome in mice by impairing autophagy

ABSTRACT Purpose: To investigate the underlying mechanism of hepatic sinusoidal obstruction syndrome (HSOS) induced by Gynura segetum by measuring autophagy in mouse models. Methods: The model group was administered G. segetum (30 g/kg/d) by gavage, while the normal control group was administered an equal volume of saline daily for five weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic histopathological examinations, and Masson staining were performed to evaluate liver injury. Liver intercellular adhesion molecule-1 (ICAM-1) and P-selectin were evaluated by immunohistochemistry. Hepatocellular apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Protein expression levels of autophagy markers were measured using Western blot analysis. Results: Gynura segetum was found to significantly induce liver injury compared with control mice, as evidenced by the increase of serum transaminases, a decrease in triglyceride levels, and histopathological changes in mice. Gynura segetum remarkably induced hepatocellular apoptosis and upregulated the expressions of ICAM-1 and P-selectin and also downregulated the protein expression levels of LC3, Atg12 and cytoplasmic polyadenylation element binding protein. Conclusions: Our results suggested that G. segetum induced liver injury with HSOS, and it was partly due to its ability to impair the autophagy pathway.

Atractylenolide III alleviates sepsis-mediated lung injury via inhibition of FoxO1 and VNN1 protein

ABSTRACT Purpose: To evaluate the influence of atractylenolide (Atr) III on sepsis-induced lung damage. Methods: We constructed a mouse sepsis model through cecal ligation and puncture. These mice were allocated to the normal, sepsis, sepsis + Atr III-L (2 mg/kg), as well as Atr III-H (8 mg/kg) group. Lung injury and pulmonary fibrosis were accessed via hematoxylin-eosin (HE) and Masson's staining. We used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and flow cytometry for detecting sepsis-induced lung cell apoptosis. The contents of the inflammatory cytokines in lung tissue were measured via enzyme-linked immunosorbent assay (ELISA). Results: Atr III-H did not only reduce sepsis-induced lung injury and apoptosis level, but also curbed the secretion of inflammatory factors. Atr III-H substantially ameliorated lung function and raised Bcl-2 expression. Atr III-H eased the pulmonary fibrosis damage and Bax, caspase-3, Vanin-1 (VNN1), as well as Forkhead Box Protein O1 (FoxO1) expression. Conclusions: Atr III alleviates sepsis-mediated lung injury via inhibition of FoxO1 and VNN1 protein.

The neuroprotection of cerebrolysin after spontaneous intracerebral hemorrhage through regulates necroptosis via Akt/ GSK3B signaling pathway

ABSTRACT Purpose: Spontaneous intracerebral hemorrhage (ICH) is a major cause of death and disability with a huge economic burden worldwide. Cerebrolysin (CBL) has been previously used as a nootropic drug. Necroptosis is a programmed cell death mechanism that plays a vital role in neuronal cell death after ICH. However, the precise role of necroptosis in CBL neuroprotection following ICH has not been confirmed. Methods: In the present study, we aimed to investigate the neuroprotective effects and potential molecular mechanisms of CBL in ICH-induced early brain injury (EBI) by regulating neural necroptosis in the C57BL/6 mice model. Mortality, neurological score, brain water content, and neuronal death were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Evans blue extravasation, Western blotting, and quantitative real-time polymerase chain reaction (PCR). Results: The results show that CBL treatment markedly increased the survival rate, neurological score, and neuron survival, and downregulated the protein expression of RIP1 and RIP3, which indicated that CBL-mediated inhibition of necroptosis, and ameliorated neuronal death after ICH. The neuroprotective capacity of CBL is partly dependent on the Akt/GSK3β signaling pathway. Conclusions: CBL improves neurological outcomes in mice and reduces neuronal death by protecting against neural necroptosis.