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Shiraz University of Medical Sciences

11 ARTICLES PUBLISHED IN JoVE

Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Hassan Azari^1,2, Maryam Rahman², Sharareh Sharififar², Brent A. Reynolds²

¹ Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran , ²Department of Neurosurgery, University of Florida

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Neuroscience

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Hassan Azari^1,2, Sharareh Sharififar², Maryam Rahman², Saeed Ansari², Brent A. Reynolds²

¹Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran , ²Department of Neurosurgery, The University of Florida

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neuroscience

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

Hassan Azari^1,2, Sharon A. Louis³, Sharareh Sharififar¹, Vinata Vedam-Mai¹, Brent A. Reynolds¹

¹Department of Neurosurgery, University of Florida, ²Department of Anatomical Sciences, Shiraz University of Medical Sciences, ³STEMCELL Technologies, Inc.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Medicine

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Saeed Ansari^*1, Hassan Azari^*1,2, Douglas J. McConnell¹, Aqeela Afzal¹, J Mocco¹

¹Department of Neurosurgery, University of Florida, ²Department of Anatomical Sciences, Shiraz University of Medical Sciences

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Neuroscience

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Hassan Azari^1,2, Sebastien Millette¹, Saeed Ansari¹, Maryam Rahman¹, Loic P. Deleyrolle¹, Brent A. Reynolds¹

¹Department of Neurosurgery, University of Florida , ²Department of Anatomical Sciences, Shiraz University of Medical Sciences

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Neuroscience

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Hassan Azari^1,2, Sharareh Sharififar¹, Jeff M. Fortin¹, Brent A. Reynolds¹

¹Department of Neurosurgery, The University of Florida, ²Laboratory for Stem Cell Research, Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Loic P. Deleyrolle¹, Mark R. Rohaus¹, Jeff M. Fortin¹, Brent A. Reynolds¹, Hassan Azari^1,2

¹Department of Neurosurgery, The University of Florida, ²Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Medicine

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Saeed Ansari¹, Hassan Azari², Kenneth J. Caldwell¹, Robert W. Regenhardt³, Vishnumurthy S. Hedna⁴, Micheal F. Waters⁴, Brian L. Hoh¹, Adam P. Mecca³

¹Department of Neurosurgery, University of Florida , ²Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran , ³Department of Physiology and Functional Genomics, University of Florida , ⁴Department of Neurology, University of Florida

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Medicine

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Robert W. Regenhardt¹, Saeed Ansari², Hassan Azari³, Kenneth J. Caldwell², Adam P. Mecca¹

¹Department of Physiology and Functional Genomics, University of Florida , ²Department of Neurosurgery, McKnight Brain Institute, University of Florida , ³Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

JoVE Journal

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Javad Alizadeh^1,2, Shahla Shojaei^1,2,3, Simone da Silva Rosa¹, Adel Rezaei Moghadam^1,2, Amir A. Zeki⁴, Mohammad Hashemi⁵, Marek J. Los^6,7,8, Joseph W. Gordon^1,9, Saeid Ghavami^1,2,10

¹Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, ²Biology of Breathing Theme, Children Hospital Research Institute of Manitoba, University of Manitoba, ³Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, ⁴Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Center for Comparative Respiratory Biology and Medicine, ⁵Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, ⁶Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, ⁷Centre de biophysique moléculaire - UPR 4301, Centre national de la recherche scientifique (CNRS) CS80054, ⁸LinkoCare Life Sciences AB, ⁹College of Nursing and Children's Hospital Research Institute of Manitoba, Rady Faculty of Health Sciences, University of Manitoba, ¹⁰Health Policy Research Center, Institute of Health, Shiraz University of Medical Sciences

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

Biochemistry

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Sanaz Dastghaib^1,2, Zahra Shahsavar³, Zahra Karimian⁴, Pooneh Mokarram^1,5

¹Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, ²Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, ³English Language Department, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, ⁴Department of e-Learning in Medical Sciences, Virtual School and Center of Excellence in e-Learning, Shiraz University of Medical Sciences, ⁵Autophagy Research Center, School of Medicine, Shiraz University of Medical Sciences

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.