bluuebunny/crossref_metadata_2025 huggingface dataset:
The activation of the(Pro)renin receptor (PRR) stimulates fibrotic factors expression independent of b‐catenin signaling pathway in collecting duct cells
Authors: Cuevas Catherina , Gonzalez Alexis , Inestrosa Nibaldo , Vio Carlos , Prieto Minolfa
Published: June 2021

Binding of the PRR to renin and prorenin activates intracellular signals independent of angiotensin II. The PRR is a component of the Wnt signaling pathway working as an adaptor between Wnt receptor and the vacuolar H+‐ATPase. The expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals; however its role in renal damage remains unclear. In this study we tested the hypothesis that the PRR activation induces the expression of fibronectin and collagen I via the b‐catenin pathway in mouse collecting duct M‐1 cells. Human recombinant prorenin (hrPR) for 16 h in M‐1 cells increased the mRNA and protein levels of fibronectin (1.48±0.1 fold change), collagen I (1.53±0.1 fold‐change) as well as the b‐catenin target genes, cyclin D1 (1.9 ± 0.1 fold change) and c‐myc (2.1 ± 0.2 fold change). Inhibition of b‐catenin degradation with pyrvinium pamoate (10‐9 M) did not prevent the hrPR‐induced expression of all these genes. However, when the cells were transfected with a siRNA‐PRR, the hrPR‐dependent increases in fibronectin, collagen, cyclin D1 and c‐myc were prevented. Furthermore, hrPR treatment did not change the stationary protein level of β‐catenin or phosphorylation of GSK‐3β until 24h, but the level of pERK1/2 displayed a significant increase after 8h of treatment (2.9 ± 0.21 fold change, P<0.05).These results indicate that in collecting duct cells, PRR activation stimulates fibrotic genes independent of β‐catenin pathway.
Identification of the Vertebrate RIM2 Ortholog by Genetic Complementation for Heme
Authors: M. Cabañero Javier , Nguyen An , Huston Nicholas , Knight Simon , Dancis Andrew , Paw Barry
Published: June 2021

Heme is the critical cofactor in hemoglobin responsible for its oxygen‐carrying properties. Mfrn1 (SLC25A37), functions as the major iron importer into the mitochondria for heme synthesis. The function of Mfrn1 is highly conserved from vertebrates to yeasts given the universal requirement for heme in many biological processes. The over‐expression of a related mitochondrial solute carrier, RIM2, allowed yeast deficient for the Mfrn1 orthologs, MRS3/4, to grow on low‐iron media, thereby implicating a role for other iron transporters in heme synthesis.

Hypothesis

Two potential RIM2 vertebrate orthologs, SLC25A33 and SLC25A36, play a role in mitochondrial iron import to complement the hemoglobinization‐defect of Mfrn1‐deficiency in a zebrafish model.

Using protein homology search, two top candidates, SLC25A33 and SLC25A36, were identified as potential orthologs for yeast RIM2. Others recently showed that these 2 carriers complemented the pyrimidine‐deficiency phenotype of ΔRIM2 yeasts (Di Noia 2014 JBC); although, the iron‐auxotrophy was not investigated.

Specific Aim 1

The over‐expression of vertebrate RIM2 orthologs will be evaluated for ability to rescue the anemia in Mfrn1‐deficient zebrafish, frascati.

We will examine whether the over‐expression of the vertebrate RIM2 orthologs could supply sufficient iron to enable mitochondrial heme synthesis in absence of Mfrn1, thereby correcting the hemoglobin‐defect. For this purpose, we will microinject cRNA with either SLC25A33 or SLC25A36 into frascati zygotes and evaluate the restoration of hemoglobinized erythrocytes.

Acknowledgments

This work was supported by grants from the APS (JMC, AN) and the NIH (ABD, BHP).
Differential Placental Expression of the Receptor for Advanced Glycation End‐Products (RAGE) in Normal and Complicated Pregnancies
Authors: Alexander Kristen , Lewis Joshua , Mejia Camilo , Howell Brain , Reynolds Paul , Arroyo Juan
Published: June 2021

Receptor for Advanced Glycation End‐Products (RAGE) is a cell surface receptor that modulates inflammatory pathways. Inflammation is associated with the development of pregnancy pathologies such as preeclampsia (PE), intrauterine growth restriction (IUGR), gestational diabetes mellitus (GDM), and pre‐term labor (PTL) that can lead to maternal and fetal complications. Our objective was to examine placental RAGE expression to implicate RAGE signaling in these complications. We performed qPCR, western blotting and immunofluorescence to determine levels of RAGE gene activation, protein expression and RAGE localization in the placenta. An ELISA was used to determine TNF‐α secretion. First trimester trophoblast cells were used to examine RAGE expression and downstream signaling molecules after RAGE activation by the ligand AGEs. Compared to control placental samples we observed: 1) increased localization of RAGE to the PE placental trophoblast; 2) decreased RAGE localization in the IUGR placenta; 3) a 1.5 fold increase (p<0.05) in placental RAGE gene expression during IUGR; 3) a 2‐fold increase (p<0.03) of placental RAGE protein expression in PE/IUGR; and 3) a 2‐fold decrease (p<0.05) in placental RAGE protein expression during IUGR. In first trimester cell lines we observed: 1) an 8‐fold increase in p‐ERK expression after 25ug treatment of AGES (p<0.007) and 2) a 1.8‐fold increase (p<0.05) of TNF‐α secretion after 25ug treatment of AGEs, a common RAGE ligand. We conclude that placental RAGE is activated in PE/IUGR situations and that RAGE‐mediated inflammation in the trophoblast includes increased TNF‐α secretion through the activation of ERK signaling.
Effects of Mast Cell Chymase on Cardiac Fibroblast Function
Authors: Carver Wayne , Carver Amanda , Fix Charity , Goldsmith Edie
Published: June 2021

Mast cells are bone marrow‐derived effector cells that have been described historically for their role in mediating allergic reactions. More recently, they have received considerable attention due to their involvement in innate defense, tissue remodeling and other processes. Several studies have illustrated roles for mast cells in cardiovascular diseases including ventricular remodeling associated with myocardial infarction, hypertension and volume overload. Mast cells produce a variety of secretory components including cytokines, proteases, growth factors and fatty acid metabolites. The compliment of components synthesized by a given mast cell appears to be dictated by the tissue microenvironment giving these cells diverse roles in tissue homeostasis and disease. Mast cell proteases including tryptase and chymase are some of the most abundant proteins produced by mast cells. Several studies have illustrated that mast cell chymase plays a significant role in the formation of angiotensin II in the heart and thus this protease may indirectly promote a fibrotic response via angiotensin II‐induced TGF‐beta production. However, the direct effect of this protease on cardiac fibroblast function has not been evaluated. The present studies were performed to elucidate the effects of chymase on isolated cardiac fibroblasts. Fibroblasts were treated for 24 hours with varying doses of chymase (0 to 1000 ng/ml) and the effects on proliferation, collagen scaffold remodeling, myofibroblast formation and expression of extracellular matrix components assayed. Treatment of cardiac fibroblasts with chymase significantly reduced the conversion of quiescent fibroblasts to a myofibroblast phenotype. Chymase also impaired contraction of three‐dimensional collagen gels and the expression of specific extracellular matrix components by fibroblasts. These studies illustrate that treatment of isolated cardiac fibroblasts with mast cell chymase elicits an anti‐fibrotic effect, which may contribute to the transition to heart failure in vivo.
The Inhibitory G‐protein, Gz, Accelerates the Progression of Insulitis and Hyperglycemia in a Type 1 Diabetes Mouse Model
Authors: Cadena Mark , Truchan Nathan , Brill Allison , Kimple Michelle
Published: June 2021

In type 1 diabetes (T1D), an autoimmune insulitis leads to β‐cell death. The non‐obese diabetic (NOD) mouse model mimics these facets of the human disease. Our laboratory has previously shown that mice deficient in the alpha subunit of the heterotrimeric G protein, Gz (Gαz), were protected from developing chemically induced diabetes via decreased β‐cell apoptosis and increased replication. We aimed to confirm this in the NOD model, as well as delineate the mechanisms of any protection. Between 4‐16 weeks of age, weekly blood glucose levels were monitored in wild‐type and Gαz‐null NOD mice. As compared to wild‐type mice, Gαz‐null mice were completely protected from developing hyperglycemia. Pancreas histologic analysis showed that Gαz‐null pancreata had significantly lower insulitis scores at 16 weeks as compared to wild‐type mice. We hypothesized that decreased immune infiltration increased β‐cell survival. To test our hypothesis, we calculated beta‐cell fractional area, and found it was increased in Gαz‐null NOD mice. New cohorts of mice were sacrificed at 4, 8, and 12 weeks of age. At 4 weeks, immune infiltration was already observed in the wild‐type islets, correlating with apoptotic beta‐cells in islet regions immediately surrounding the invading immune cells. As the first step in delineating mechanism, we showed that islets from Gαz‐null NOD mice have significantly less IL‐1β and IL‐6 cytokine expression; pro‐inflammatory cytokines that have been linked with β‐cell death. Overall, our results further strengthen our claim that Gαz has a protective effect in maintaining β‐cell regeneration and survival, and that the Gαz signaling pathway may be a valid therapeutic target for T1D.
Increasing Nasal Mucus Sonic Hedgehog (Shh) Improves Human Olfaction
Authors: Henkin Robert , Hosein Suzanna , Stateman William , Knoppel Alexandra
Published: June 2021

Sonic hedgehog (Shh) has been shown to act as a cell signaling and growth factor moiety in many tissues in humans and animals. Shh has not been associated with smell function. We therefore wondered if Shh played a role in human smell function and, if so, if it were present in nasal mucus. Thus, we measured Shh in nasal mucus in 14 normal subjects and in 44 patients with smell loss (hyposmia) of several etiologies by use of a sensitive spectrophotometric ELISA assay. Shh was found in nasal mucus in both normal subjects [7538±1105 pg/ml (M±SEM)] and in hyposmic patients (149±2 pg/ml) with levels in patients significantly lower than in normal subjects. These changes occurred in patients with losses of smell function related to post‐influenza‐like infection, allergic rhinitis and head injury. Treatment with oral theophylline 200‐800 mg daily for 2‐10 months significantly increased Shh levels in nasal mucus in hyposmic patients (49191±1710 pg/ml) and increased smell function significantly in over 60% of the patients. This is the first demonstration of Shh in nasal mucus. Its stimulation by theophylline resulted in improved smell function among hyposmic patients of multiple etiologies.
Lysophosphatidic acid upregulates calreticulin expressionin PC‐3 human prostate cancer cells
Authors: Lu Kuan‐Ying , Lin Kuan‐Hung , Lin Chu cheng , Lin Yueh‐Chien , Yang Ya‐Jan , Chen Wei‐min , Hsia Hsin‐Yi , Lee Hsinyu
Published: June 2021

Prostate cancer is one of the most commonly diagnosed cancers in males, and PC‐3 is a popular cell model for investigating late stage prostate cancer. Lysophosphatidic acid (LPA) is a low molecular weight lipid demonstrated to stimulate cell migration, invasion and proliferation in prostate cancer cells. In our previous study, calreticulin (CRT), a multifunctional Ca2+‐binding chaperon, has been shown to associate with poor prognosis in gastric cancer and bladder cancer. However, the roles of CRT in prostate cancer remain elusive. It has been demonstrated that LPA evoked Ca2+ mobilization from the lumen of the endoplasmic reticulum (ER) via phospholipase C (PLC) pathway. On the other hand, depletion of Ca2+ from ER activated CRT promoter activity in NIH/3T3 cells. Based on these evidences, we hypothesized that LPA may regulate CRT expression. By using RT‐qPCR and Western Blotting, we found that CRT expression is up‐regulated both in mRNA and protein level after LPA treatment. Pharmacological blockade of LPA1/3 by Ki16425 inhibitsthe enhancement effect of LPA on CRT. In addition, CRT expression is lower in LPA1 and LPA3 stable knockdown PC‐3 cells. Furthermore, activation of LPA3 by OMPT enhances CRT expression. On the contrary, activation of LPA2 by MDP and GRI977143 impaired CRT expression. Furthermore, knockdown of CRT resulted in decreased cell adherent ability and decreased expression of vascular endothelial growth factor‐A (VEGF‐A). Taken together, these results suggested that,LPA1/3 and LPA2 inversely regulate CRT expression and subsequently regulate cell adhesion and VEGF‐A expression in PC‐3 cells.
Involvement of Multiple Cell Signaling Pathways in Cigarette Smoke induced Modulation of Ovarian Cancer Cell (OCC) Proliferation.
Authors: Sumanasekera Wasana , Ngo Dung , Tran Liem , Nguyen Tanya , Castle Marilin
Published: June 2021

Cigarette smoking is a known risk factor for many cancers but not the ovarian cancer. Our study examined whether cigarette smoke extract (CSE) affects ovarian cancer development using A2780 ovarian cancer cell (OCC) model. The major objective is to investigate the effects of CSE on OCC proliferation and unveil the responsible cell signaling mechanism. The specific aims are 1) to detect CSE induced OCC proliferation, 2) investigate estrogen's role and 3) unveil the responsible pathway.

Methods

OCCs were cultured using standard methods. CSE was created according to a published protocol. OCCs were pre‐treated with several cell signaling pathway inhibitors and estrogen prior to CSE treatment. After CSE treatments (5%, 10% and 16%), WST‐1 reagent was added to each well, incubated for 3 hours, and the production of WST‐1 formazan, which is an indicative of cell proliferation, was detected.

Results

CSE stimulated OCC proliferation and this stimulation is significant in estrogen treated cells. Estrogen receptor / β2 adrenergic receptor blockage using the antagonist, ICI 118.551, has increased OCC proliferation and this increase was attenuated by CSE exposure. Blocking ERK1/2 pathway, using the antagonist U0126 significantly reduced CS induced OCC proliferation in non‐estrogen treated cells.

Conclusion

CSE activated OCC proliferation. Estrogen and β2 adrenergic receptor pathways showed protection towards OCC proliferation and CSE attenuated the protective effects. Activation of ERK signaling contributes to the CSE induced OCC proliferation in non – estrogen treated cells. In estrogen treated cells CS may cause OCC proliferation via non ERK related signaling.
SIRT3 Blocks Doxorubicin Induced Cardiac Hypertrophic Response by Suppressing ROS Synthesis from Mitochondria
Authors: Pillai Vinodkumar , Samant Sadhana , Kim Gene , Gupta Mahesh
Published: June 2021

Background and Objective

Doxorubicin is widely used to treat different types of cancers. Most serious adverse effect of doxorubicin is cardiomyopathy. Here we investigated whether the mitochondrial deacetylase SIRT3 can attenuate doxorubicin induced cardiac hypertrophy in mice.

Methods and Results

Doxorubicin treatment induced increased ROS levels in cardiomyocytes, which was significantly reduced by the overexpression of SIRT3. Further, SIRT3 overexpression significantly decreased doxorubicin induced cardiomyocyte death. Doxorubicin also induced exacerbated mitochondrial DNA damage in SIRT3 knockout cardiac fibroblasts (CF) as well as in mouse embryonic fibroblasts (MEFs). These results suggest that SIRT3 is capable of reducing doxorubicin induced ROS synthesis from mitochondria, thereby blocking its deleterious effects. To examine the cardioprotective effects of SIRT3 from doxorubicin induced damage in vivo, we treated wild type and cardiac‐specific SIRT3 over‐expressing transgenic mice with a cumulative dose of 15mg/kg of doxorubicin. Mice expressing cardiac‐specific SIRT3 (Sirt3‐Tg) showed 33% reduced HW/BW ratio compared to control mice. Echocardiographic evaluation of hearts showed significantly reduced fractional shortening in control mice compared to Sirt3‐Tg mice (24.6 vs 34.7 %, P<0.05)). Further, Masson's trichrome staining showed significantly reduced fibrosis in doxorubicin treated Sirt3‐Tg mice, compared to controls, thus suggesting that SIRT3 is capable of blocking doxorubicin‐induced cardiac hypertrophic response.

Conclusion

Our study suggests that SIRT3 is an endogenous negative regulator of doxorubicin induced cardiac hypertrophic response.
Mitochondrial Topoisomerase Dysfunction Contributes to Myocardial Mitochondrial Dysfunction and Mitochondrial DNA (mtDNA) Degradation
Authors: Edwards John , Mitry Maria , Laurent Dimtri , Mathew Jamie
Published: June 2021

Mitochondrial dysfunction has a significant role in the development of many different cardiomyopathies. Mitochondrial dysfunction and mtDNA mutations are also associated with other nonmyocardial diseases including cancer and neurodegenerative diseases such as Leigh Syndrome. One pathophysiologic response includes a decline in mtDNA integrity. Although it is widely accepted that mitochondrial‐derived oxidant stress is the sole cause of mtDNA damage, our work in models of diabetes and alcoholism has found involvement of the mitochondrial topoisomerases. To date, three unique mitochondrial topoisomerases have been identified in the myocardium. In models of type II diabetes or alcoholism we have observed a significant decreased in left ventricle cytochrome oxidase activity as an indicator of mitochondrial dysfunction, concomitant with significant increases in mtDNA damage. Using purified myocardial mitochondria, we observed significant increases in mitochondrial topoisomerase dependent DNA cleavage in response to diabetes or alcoholism. Mitochondrial topoisomerase is regulated at different levels and we found that DNA cleavage was significantly exacerbated by acute exposure to H2O2 as well as the topoisomerase inhibitor sobuzoxane to significantly increase DNA damage. Separate from a direct impact of oxidative stress on mtDNA, our studies find that alteration of mitochondrial topoisomerase reaction mechanism accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases have important consequences for the myocardium and participate in the pathophysiology leading to the development of cardiomyopathy.
The effects of Receptor Interacting Protein 3 on necroptosis in RGC‐5 following elevated hydrostatic pressure
Authors: Shang Lei , Xiong Kun , Huang Jufang
Published: June 2021

Objective

RIP3 has been found to induce necroptosis. Previous study have demonstrate necroptosis is an important neuronal death mode in retinal ischemia, and RIP3 may relate to necrosis in our observation. The lack of RIP3 inhibitor limited our study in vivo, so we first identify necroptosis in RGC‐5 following EHP, and then detect the protein level of RIP3, the percentage of necrosis using the pressure chamber via rip3 RNAi. Finally, we try to elaborate the mechanism of necroptosis.

Methods

After 6 hr, 12 hr, 24 hr of recovery, we execute PI‐staining with different pressure values insults, then detected the level of RIP3 expression and the number of necrosis cells using western‐blotting, fluorescence‐staining. We analyzed the percentage of necrosis in 24 hr to identify the existence of necroptosis within necrostatin‐1. Finally rip3‐RNAi were carried out to get the converse evidence on necroptosis.

Results

PI‐staining in different pressure levels showed there was little necrosis cell under 60 mmHg and the percentage of cell necrosis increased following the pressure elevation. As pressure level elevated, the number of necrosis cells increased too. RIP3 expression was up‐regulated and there exist double‐labeling of some RIP3 enhance‐labeled and PI‐positives after 6 hr. The results of flow cytometry indicated the ratio of necrosis cells were decreased from 13% to 5% within necrostatin‐1, the percentage of necrosis cells decreased to nearly 7% using rip3‐RNAi.

Conclusion

RIP3 may participate in necroptosis following EHP.

This work was supported by National Natural Science Foundation of China (81371011),Wu Jie‐Ping Medical Foundation of the Minister of Health of China (320.6750.14118), Natural Science Foundation of Hunan Province (2015JJ2187)
Up‐regulation of Clusterin is Involved in MSC Overexpressing GATA‐4 Mediated Cell Protection
Authors: Yang Yueting , Yu Bin , Liu Huan , Wang Yi‐Gang , Ashraf Muhammad , Xu Meifeng
Published: June 2021

We have previously reported that GATA‐4 gene transfection in mesenchymal stem cells (MSC) prolongs MSC survival in the ischemic heart and promotes heart repair. Our preliminary studies show that clusterin was prominently upregulated in MSCs overexpressing GATA‐4 (MSCGATA‐4). In this study, we hypothesized that upregulation of clusterin was responsible for GATA‐4 mediated cell protection.

Methods and Results

MSCs were harvested from rat bone marrow and transduced with GATA‐4 by using the murine stem cell virus retroviral expression system. The expression of clusterin in MSCs was documented by real‐time PCR, immunofluorescence, and western blotting. The released clusterin from MSCs was estimated using ELISA. Cardiomyocytes (CM) from neonatal rat ventricles were cultured and exposed to hypoxia and anoxia to mimic ischemic injury. The cytoprotection was determined by measuring lactate dehydrogenase release, MTT intake, Annexin‐V staining, and mitochondrial membrane potential (ΔΨm). Compared to vector‐empty transduced control MSCs, the expression of clusterin was significantly up‐regulated in MSCGATA‐4 accompanied by its significant release from MSCGATA‐4. Direct administration of clusterin protected MSCs and CM against ischemic injury by increased cell survival, reduced apoptosis, and maintenance of ΔΨm. The cytoprotective effect of clusterin was partially abrogated by Akt inhibitor LY294002. Clusterin also significantly augmented expression of p‐Akt in MSCs and CM.

Conclusions

It is concluded that clusterin played a prominent role in MSCGATA‐4 mediated cytoprotection via Akt signaling.
Effects of Chemotherapy and Hormone Therapy on Biomarkers of Cardiac Injury and Oxidative Stress in Women with Breast Cancer
Authors: Silva Fabrício , Romero Walckiria , Carvalho Ana Lígia , Abreu Gláucia
Published: June 2021

The increase in deaths from cardiovascular disease in women submitted to antineoplastic treatments for breast cancer is a fact observed in many work and is most often associated with cardiotoxic effects of chemotherapy. It is believed that the cardiotoxicity of chemotherapeutic agents is associated with increased oxidative stress in the body, although not well understood. The detection of this damage underlies the aims of this present study to assess the effects of treatments against breast cancer on biomarkers of cardiac injury and oxidative stress. Thirty women divided according to the therapeutic protocol in women treated only with hormone therapy with tamoxifen (Tam group, n = 10), women treated only with chemotherapy (chemo group, n = 10), and women treated with chemotherapy and then with hormone therapy with tamoxifen (Chemo+Tam group, n = 10) were monitored for one year. Blood samples was collected before initiation of treatment, six, and twelve months after for analysis of cardiac troponin I (cTnI), advanced oxidation protein products (AOPP) and plasma activity of the antioxidant enzyme glutathione peroxidase (GPx). At the end of research we observed that women in the Chemo group showed increased levels of cTnI and AOPP and lower plasma activity of GPx when compared to women groups Tam and Chemo + Tam. These data reinforce the involvement hypothesis of oxidative stress in cardiovascular diseases in a chronic period after end of chemotherapy and highlighted a cardioprotective effect of hormone therapy with tamoxifen.
The Form of Energy‐containing Food Alters Satiety and fMRI Brain Responses in Humans
Authors: Soto Marion , Darcel Nicolas , Piedcoq Julien , Tomé Daniel , Fromentin Gilles , Nadkarni Nachiket
Published: June 2021

We examined the differences in satiety and brain activation after ingestion of apple in the form of juice, puree or slices.

25 normal‐weight men had to eat a breakfast consisting of one of three apple products (cross‐over design): apple juice, puree or slices, each with the same caloric content. After breakfast, a fMRI session was conducted as follows: a 'liking' run consisting of viewing images (food and objects) whilst rating their appeal value, and a 'choice' run in which participants had to choose between two images of different categories (high‐fat/low‐fat sweet/savory). Measures of appetite were obtained by using visual analog scales at different times.

Consumption of the three apple products led to differences in short‐term satiety, in the order slice > puree > juice. In the 'liking' run, the nucleus accumbens (NAcc), the orbitofrontal cortex (OFC) and the hypothalamus were more activated after consumption of apple juice vs. apple slices. Neural activity in the NAcc and OFC was positively correlated with hunger ratings after breakfast. In the 'choice' run, the OFC was more activated after the juice breakfast vs. slices, when subjects chose sweet images (rather than savory) and high‐fat images (rather than low‐fat). BOLD signal in the OFC measured when high‐fat foods were chosen was positively correlated with hunger ratings and ratings of 'expected pleasure to eat' reported after breakfast.

These findings suggest that food form elicits differences in short‐term satiety, and leads to different responses to food cues in limbic (NAcc and OFC) and homeostatic (hypothalamus) regions underlying food intake.
The Underwater Treadmill as a Physical Therapy Intervention for Children with Cerebral Palsy
Authors: Hill Michael , McAuliffe Kristen , Watkins Pamela , Wheeler April , Raynes Edilberto
Published: June 2021

Cerebral Palsy is a congenital and physical disability that leads into abnormal muscle tone, dyskinesia, and muscle spasticity, which can negatively impact functional gait patterns. Using the principles of activity based programs and repetitive task specific exercises involving a treadmill in physical therapy can replicate normal gait patterns, which can improve the quality of life. There is a scarcity of research that explores the effectiveness of underwater treadmill training as a form of physical therapy intervention. As such, the purpose of the study is to examine available research that studied the benefits of aquatic therapy, land based treadmill training, and underwater treadmill training on the gait patterns in children with cerebral palsy. The researchers conducted a review of literature that focuses on beneficial effects of physical therapy interventions such as aquatic exercise, resistance training, land‐based treadmill training, as well as underwater treadmill training; then ranked them using the Oxford Level of Evidence‐based Medicine. Based on the evidence gathered, we can conclude that the physiological benefits associated with aquatic therapy in combination with the repetitive task specific exercise used with treadmill training can improve mobility and functional gait patterns of children with cerebral palsy.
In vitro human cell models for probing functional deficits relevant to neuropsychiatric disorders
Authors: Kano Shin‐ichi , Cardarelli Ross , Higurashi Norimichi , Yuan Ming , Chang Daniel , Gaval‐Cruz Meriem , Hirose Shinichi , Okano Hideyuki , O'Donnell Patricio , Kai Mihoko , Sawa Akira
Published: June 2021

It is widely accepted that functional alterations of brain cells and neuronal circuitry cause neuropsychiatric disorders such as schizophrenia, but the underlying biological mechanisms are still unclear. Recent technical advances in human neuronal cell culture methods have provided opportunities to examine neuronal cells derived from accessible patient tissues/cells. Here we report the optimization and utilities of human cell models to probe functional deficits relevant to neuropsychiatric disorders, induced neuronal (iN) cells, and olfactory epithelium‐derived cells (olfactory cells). By using iN cells with feature of immature postmitotic glutamatergic neuronal cells, we have found a novel functional phenotype in Dravet syndrome, a form of severe myoclonic epilepsy in infancy, which was not suggested by previous studies of classical mouse models and human autopsied brains. By analyzing gene expression profiles of olfactory cells, we have revealed that altered susceptibility to cellular stress responses underlie schizophrenia. Taken together, these studies demonstrate that in vitro human cell models are useful for probing functional deficits relevant to neuropsychiatric disorders.
Gastrodin Promotes SGC7901 Apoptosis by Up‐regulating the ProBDNF Signaling Pathway
Authors: jianhui Guo , jinwei Yang , wei Ma , bin Du , jingru Cheng , liyan Li
Published: June 2021

Objective

To investigate the affect of Gastrodin on human gastric cancer cells (SGC7901) and its relationship with precursor brain‐derived neurotrophic factor proBDNF signal pathway.

Methods

SGC‐7901 was cutured in vitro and randomly divided into normal, control and experimental groups, and then, each group was divided into 6, 12, 24, 48 and 72 h groups. The experimental group cells were co‐cultured with Gastrodin at 2 mg/ml, but saline in control groups, and normal groups had no other treatment. After that, 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to detect the cell activity and terminal dexynucleotidyl transferase(TdT)‐mediated dUTP nick end labeling (TUNEL) was used to detect the apoptosis of the cells. Immunofluorescence was used to locate the proBDNF, p75 and Sortilin positive cells, and the expression of proBDNF, p75 and Sortilin mRNA was detected by Real time Polymerase Chain Reaction (PCR).

Results

1, SGC7901 was successfully cultured. 2, Cell viability in experimental groups was decreased compared with normal or control groups, but the apoptosis was increased. 3, proBDNF, p75 and Sortilin mRNA was up‐regulated in experimental groups.

Conclusion

Gastrodin can decrease the biological activity and increase the apoptosis of SGC7901, and the mechanism may be by increasing the proBDNF apoptosis signal pathway.

Corresponding author: Li‐yan Li.
Region‐Specific Expression of ProBDNF and MatureBDNF in Alcohol Preference
Authors: Li Liyan , Ma Wei , Yang Jinwei , Yang Yanlei , Wang Dongyan , Luo Tao , Cheng Jingru , Guo Jianhui
Published: June 2021

Brain‐derived neurotrophic factor precusor (proBDNF) plays different roles from mature BDNF through different receptors and pathways in the central nerve system (CNS), some research indicated that BDNF plays important roles in alcohol preference in murine, however, the region‐specific BDNF expression remains to be controversial. So we investigated the responsiveness of proBDNF, matureBDNF and their receptors to acute and chronic alcohol exposure in rat. On the one hand, western blot was used for detecting the expression level of proBDNF, TrkB and Sortilin proteins in cortex, hippocampus, amygdala and dorsal striatum after acute alcohol exposure; on the other hand, we successfully established psychomotor sensitization (PS) model for chronic alcohol exposure. RT‐PCR were performed for measuring the mRNA level of BDNF, TrkB, P75NTR and Sortilin in the cortex, hippocampus and dorsal striatum after chronic alcohol exposure. Our research found that mRNAs for BDNF, trkB, p75 and sortilin were detected in all brain regions in both control and alcohol groups. Chronic alcohol exposure upregulated BDNF mRNA level in prefrontal cortex, but decreased Sortilin mRNA level in dorsal striatum. No changes of these mRNAs were found in other brain regions between alcohol group and control group. We conclude that chronic alcohol exposure caused differential expression of BDNF and sortilin in different brain regions, but proBDNF and matureBDNF do not play key roles in alcohol preference.

*Corresponding author: Jian‐hui Guo.
Endothelial Nitric Oxide Synthase (eNOS) Blockade Within the Ventrolateral Medulla Differentially Modulates the Exercise Pressor Reflex in Stroked‐Rats
Authors: Ally Ahmmed , Towiwat Pasarapa , Phattanarudee Siripan , Maher Timothy
Published: June 2021

We have previously shown that blockade of endothelial nitric oxide synthase (eNOS) within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulates cardiovascular responses which was associated with changes in extracellular glutamate and GABA concentrations during static skeletal muscle contraction, the Exercise Pressor Reflex. In this study using left‐sided stroked‐rats we determined if microdialyzing a specific eNOS antagonist into the left RVLM and/or the left CVLM would alter cardiovascular responses during the Exercise Pressor Reflex. Rats were stroked by employing a transient (90‐minute) left‐sided middle cerebral artery occlusion (MCAO) followed by 24 hr reperfusion. In Protocol 1, microdialysis of a selective eNOS antagonist, L‐N(5)‐(1‐iminoethyl)ornithine (L‐NIO; 10 µM) for 120 minutes into the left RVLM significantly potentiated cardiovascular responses during a static muscle contraction compared to those observed in intact rats. In Protocol 2, application of L‐NIO into the left CVLM significantly attenuated the cardiovascular responses during muscle contraction. Finally, in Protocol 3, administration of L‐NIO simultaneously into both the left RVLM and left CVLM produced results similar to those observed with Protocol 1. These results demonstrate that blockade of eNOS within the two regions of the ventrolateral medulla differentially modulates cardiovascular responses during static exercise in stroked‐rats.
AT1a Receptor Upregulation in the MnPO Following CIH Leads to Sustained Hypertension
Authors: Shell Brent , Nedungadi T , Cunningham J
Published: June 2021

Angiotensin type 1 (AT1) receptors play a central role in many forms of neurogenic hypertension. Two distinct subtypes, At1a and AT1b, exist for AT1 receptors. Activation of the AT1a subtype is characterized by increased sympathetic nerve activity and hypertension. The Median Preoptic Nucleus (MnPO) expresses both subtypes of the angiotensin receptor and our lab has previously demonstrated that AT1a message is increased in MnPO brain punches following CIH. This study sought to determine if changes in expression of the angiotensin receptor subtypes play a role in the sustained hypertension that develops from chronic intermittent hypoxia (CIH). We hypothesized that AT1a receptor expression increased and AT1b receptor expression remained unchanged resulting in the sustained elevation in blood pressure from CIH. Rats were microinjected in MnPO with an adeno‐associated virus loaded with a shRNA construct to prevent AT1a expression (shAT1a) or a scramble construct (shSCR). After recovery, rats were exposed to a 7 day CIH or normoxic protocol. Laser Capture Microdissection and qRTPCR was performed to determine the receptor subtype expression in MnPO with ribosomal subunit S18 as the control. shSCR rats exposed to CIH had significantly more AT1a RNA than their normoxic counterparts (P<.05), but their expression of AT1b remained constant. shAT1a CIH rats did not exhibit the sustained component of hypertension found in shSCR CIH animals and their MnPO AT1a message was reduced compared to control. Overall this indicates that the pathophysiological blood pressure increase during CIH is in part mediated by increases in AT1a receptor subtype expression in the MnPO.
Stimulation of the Prorenin Receptor in the Paraventricular Nucleus Increases Sympathetic Outflow in Anesthetized Rat
Authors: Huber Michael , Basu Rupsa , Chen Qing‐Hui , Shan Zhiying
Published: June 2021

Previous studies indicate that activation of brain prorenin receptors (PRR) contributes to hypertension. However, the detailed mechanism underlying the role of brain PRR on blood pressure control is not well understood. Here we investigated the hypothesis that stimulation of PRR in the hypothalamic paraventricular nucleus (PVN) results in an increase in sympathetic nerve activity (SNA) and therefore contributes to neurogenic hypertension. In anaesthetized adult Sprague Dawley (SD) rats, bilateral PVN microinjection of prorenin (2 pmol/side) increased splanchnic SNA (SSNA) (71±15%, n=7) and renal SNA (RSNA) (21±10%, n=7). Pre‐injection of the prorenin handle region peptide, a PRR binding blocker (PRRB), into the PVN, significantly attenuated the elevation in the SSNA (32±5%; p<0.05 vs. vehicle control; n=6) but did not block the increase in RSNA (3±11%; p=0.24 vs vehicle control; n=7) evoked by prorenin injection. We further investigated the effect of PRR activation on downstream gene expression using cultured brain neurons from the hypothalamus containing the PVN of newborn SD rats. Incubation of cultured neurons with prorenin (100 nM) for 6 hours resulted in a time‐dependent increase in the mRNA levels of iNOS (210‐fold) and Fosl1 (11‐fold), a marker for neuronal activation. In addition, increasing the availability of PRR ligand, renin, dramatically increased ROS production. All these increases were not attenuated by the AT1 receptor antagonist, losartan, indicating these effects are not mediated by PRR‐mediated AngII generation. Our results suggest that PRR induced increase in sympathetic outflow maybe through activation of the iNOS and ROS signal pathway. (AHA 11SDG7420029 and AHA2640130)
Olanzapine Reduces Neuronal Activity in the Dorsal Motor Nucleus of the Vagus
Authors: Anwar Imran , Miyata Kayoko , Zsombok Andrea
Published: June 2021

Olanzapine, a widely prescribed atypical antipsychotic, relieves the symptoms of schizophrenia and bipolar disorder. Despite its positive impact on mental health, patients treated with olanzapine experience side effects related to weight gain and impaired glucose homeostasis. A variety of mechanisms and brain areas underlying the metabolic side effects have been proposed; however, the involvement of the brainstem remained to be elucidated. Parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) set the vagal tone and thus control visceral functions including feeding, digestion, and glucose homeostasis. In this study, whole‐cell patch‐clamp recordings were performed to test the hypothesis that olanzapine modulates neuronal activity in the DMV of mice. Acute application of olanzapine (10 µM) hyperpolarized the majority (~70 %) of DMV neurons in male mice by an average of ‐7.77 ± 1.17 mV (n=20). This effect was concentration‐dependent (EC50 = 16.02 ± 1.78 µM), and was associated with a reduction of input resistance. Similarly, hyperpolarization was observed in female mice (n=9). The hyperpolarization also occurred in the presence of tetrodotoxin, suggesting a postsynaptic mechanism of action. Next, stomach‐ and liver‐projecting DMV neurons were identified with transsynaptic retrograde viral labeling. Acute olanzapine application resulted in hyperpolarization of the majority of both liver‐ and stomach‐related DMV neurons. In summary, our data demonstrate that in slices olanzapine administration hyperpolarizes the majority of DMV neurons via postsynaptic mechanisms. These data suggest that olanzapine has the potential to modulate vagal outflow to subdiaphragmatic organs.

Supported by: Pilot Project Grant Tulane University School of Medicine
NPY is Co‐localized with TH in Ventrolateral Medullary Neurons Projecting to the Hypothalamus in Mice
Authors: Wenker Ian , Inglis M , Guyenet Patrice , Stornetta Ruth
Published: June 2021

The catecholamine (CA) neurons of the ventrolateral medulla (VLM) are activated by many visceral stressors, including hypovolemia, hypoxia, hypoglycemia and infection and their activation results in equally diverse physiological output, including elevated BP, ventilation, arousal and neuroendocrine activation. In the rat, we previously described two subsets of VLM‐CA neurons: spinal‐ and hypothalamic‐projecting. In the current study we have identified the same subsets in mice. We distinguished between two VLM‐CA subtypes by rostral‐caudal location and neuropeptide Y (NPY) expression. In mice, Fluorogold (FG) was injected into either the spinal cord or paraventricular nucleus of the hypothalamus to identify spinal‐projecting and hypothalamic‐projecting VLM‐CA neurons, respectively. After allowing 5‐7 days for retrograde transport, the mice were perfused and brains processed for combined immunohistochemistry and in situ hybridization (ISH) to visualize VLM‐CA neuronal somata (TH‐ir) containing FG and NPY (ISH). We found that more than half (66.1 ± 2.5 %, n = 8) of the VLM‐CA neurons are positive for NPY mRNA. Spinal‐projecting VLM‐CA neurons generally resided more rostral, just behind and medial to the facial nucleus, and did not express NPY mRNA (0.5 ± 0.3 %, n = 4).Conversely, hypothalamic‐projecting VLM‐CA neurons were located slightly more caudal and most expressed NPY mRNA (63.8 ± 19.6 %, n = 3). In addition, over 90 % (n = 8) of NPY‐positive neurons co‐expressed TH, indicating that, within the mouse VLM, NPY is predominantly expressed by VLM‐CA neurons. NPY expression is a selective marker for hypothalamic‐projecting VLM‐CA neurons in mice as well as rats.
Tempol Intracerebroventricular Injection Abolishes the Hypertension and Mortality Arising from the Central Action of Scorpion Tityustoxin
Authors: Silva Fernanda , Guerra Joyce , de Menezes Rodrigo , Guidine Patricia , Chianca Deoclecio
Published: June 2021

Studies address the high sensitivity of the central nervous system (CNS) to Tityustoxin action (TsTX, α‐toxin of the T.serrulatus scorpion venom). TsTX increases the sympathetic modulation due to masssive release of the excitatory aminoacids (EAA) in CNS. The EAA extracellular accumulation causing brain oxidative insults. Considering that central sympathetic activation is one of the mechanisms underlying hypertension, in which reactive oxygen species (ROS) is involved, we aimed to assess if the tempol (superoxide dismutase mimetic) injections in the left lateral ventricle (LLV) could influence the cardiovascular responses and mortality caused by TsTX central action. Urethane anesthetized wistar male rats were submitted to vessels femoral cannulation and to injections of PBS (Control group; n=4) or tempol (10 and 32µmol;2µL, respectively, n=5 and n=3), followed by TsTX injections (0.4µg;1μl) in LLV. Mean arterial pressure (MAP) and Heart Rate (HR) records were done before (10min for baseline; 20min for PBS or Tempol periods) and after TsTX injection (60min). Results expressed as mean±SEM; p<0.05. F ratio=mean square (MS) for the contrast/residual MS of the groups. Baseline MAP and HR were not different between groups.TsTX evoked hypertension and tachycardia (ΔMAP:35±5mmHg, ΔHR:121±22bpm, p<0.05) and 75% of death in control group. Tempol 32 µmol injection abolished the hypertension (for treatment; ΔMAP: F(2, 351) = 6, p<0.05) and the mortality (0%) arising from the TsTX central action. These results suggest that the pressor response and death could be mediated by ROS in CNS. Supported by: UFOP, CAPES and CNPq.
Relationship between hippocampal CA1 neuronal and sympathetic nerve activity during fear conditioning in rats
Authors: Yoshimoto Misa , Kanayama Misaki , Miki Kenju
Published: June 2021

The hippocampus has been implicated in the emotional responses of sympathetic nerve activity (SNA) to fear. However, there is a lack of direct evidence of the changes in hippocampal neuronal activity and SNA and on the functional relationships between these activities during the fear response. The aim of the present study was to measure the relationship between hippocampal neuronal activity and SNA during fear conditioning. Male Wistar rats were chronically implanted with multiple electrodes to monitor hippocampal CA1 neuronal activity, and bipolar electrodes to monitor renal SNA (RSNA) and lumbar SNA (LSNA). Electroencephalography was performed. Each rat was given a 5‐s tone terminating with a 1‐s, 5‐mA foot shock twice a day over 3 days. Hippocampal CA1 neuronal activity, RSNA, and LSNA were measured simultaneously during the quiet awake state in the rat's own cage. Heart rate decreased during the 3‐day fear conditioning. Hippocampal CA1 neuronal activity and RSNA increased, while LSNA decreased during the 3‐day fear conditioning. These data suggest that there is a positive neuronal coupling between hippocampal neural activity and RSNA during fear conditioning in rats. JSPS Grant.
Effects of Combined Oxytocin Receptor (OTR) with Vasopressin Receptor (V1aR or V1bR) Blockade within Paraventricular Nucleus (PVN) on Neurocardiovascular Responses
Authors: Rossi Noreen , Li Cailian
Published: June 2021

Vasopressin (VP) released from dendrites of magnocellular neurons within PVN may influence systemic hemodynamics and sympathetic nerve activity. PVN neurons possess all three receptor subtypes: V1aR, V1bR and OTR. We have shown that inhibition of any one of these receptors alone dose dependently blocks the rise in MAP and reflex changes in heart rate (HR) and renal sympathetic nerve activity (RSNA) with injection of VP into PVN. Here we test the hypothesis that a subthreshold dose of OTR antagonist combined with either subthreshold V1aR or V1bR blockers within PVN will result in additive inhibition of hemodynamic and RSNA changes induced by 100 ng VP into PVN. Male Sprague Dawley rats were instrumented and studies performed in awake unrestrained rats 24 ‐ 48 hr after recovery from anesthesia. Baseline parameters did not differ. VP increased MAP by 18.2 ± 3.1 mmHg (P < 0.001) while HR and RSNA decreased 33 ± 14 bpm and 13.6 ±10.6 %baseline, respectively (P <0.05). The response to combined subthreshold inhibitory doses of (d(CH₂)₅¹,Tyr(Me)²,Arg⁸)‐Vasopressin (V1aR, 20 ng), nelivaptan (V1bR, 0.5 ng) and (d(CH₂)₅¹,Tyr(Me)²,Thr⁴,Orn⁸,des‐Gly‐NH₂⁹)‐Vasotocin (OTR, 1 ng) attenuated the rise in MAP by 43% as well as the reflex changes in HR and RSNA. Neither V1aR+OTR blockade nor V1bR+ OTR blockade resulted in greater blockade of the responses (45% and 49%, respectively. Vehicle did not block any responses. These findings suggest that V1aR, V1bR and OTR activated pathways share intracellular and/or neural pathways that regulate system hemodynamics and sympathoexcitatory responses to VP within the PVN.
Tumor Necrosis Factor‐α Receptor 1 (TNFR1) Knockdown in Subfornical Organ (SFO) Reduces Sympathetic and Hemodynamic Responses to Blood‐Borne TNF‐α
Authors: Yu Yang , Wei Shunguang , Felder Robert
Published: June 2021

We recently reported that the SFO, a forebrain circumventricular organ that lacks a blood‐brain barrier, plays a major role in mediating sympathetic and hemodynamic responses to blood‐borne proinflammatory cytokines (PICs). Blood‐borne PICs upregulate inflammation and renin‐angiotensin system (RAS) activity in cardiovascular regions of the brain to activate the sympathetic nervous system, but the underlying mechanisms are not fully understood. We tested the contribution of TNFR1 in the SFO to the sympathetic and hemodynamic responses elicited by blood‐borne TNF‐α. Rats received SFO microinjections of a TNFR1 shRNA or scrambled shRNA lentiviral vector, or vehicle. Two weeks later, RT‐PCR showed that TNFR1 shRNA reduced TNFR1 mRNA by 43% (p<0.05) in the SFO but not in the paraventricular nucleus (PVN). Intracarotid artery injection of TNF‐α (200 ng) increased blood pressure, heart rate, and renal sympathetic nerve activity in rats treated with scrambled shRNA or vehicle. These excitatory responses to TNF‐α were attenuated (p<0.05) in rats treated with TNFR1 shRNA. Four hours after TNF‐α injection, mRNA for NF‐κB, cyclooxygenase‐2 and angiotensin II type 1 receptor had increased in both SFO and PVN in rats treated with scrambled shRNA or vehicle. In rats treated with SFO TNFR1 shRNA, the TNF‐α‐induced increases in these parameters were reduced (p<0.05) not only in SFO but also in PVN. These data suggest that blood‐borne TNF‐α acts upon TNFR1 in SFO to upregulate inflammation and RAS activity in SFO, with downstream effects on excitatory mediators in the PVN that contribute to sympathetic excitation. Supported by NIH Grants HL‐073986 and HL‐096671 and Department of Veterans Affairs.