Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Summary

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Abstract

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Introduction

Astrocytes are one of the most important key players in the central nervous system (CNS). Growing body of evidence indicates that the roles of astrocytes are more than providing neuronal support. In fact, the roles of astrocytes under physiological conditions can be very complex, such as guiding the migration of the developing axons¹, regulating CNS blood flow², maintaining the pH homeostasis of the synaptic interstitial fluid³, and participating in the blood brain barrier⁴ and synaptic transmission⁵. Under pathological conditions, astrocytes respond to injury with a process called reactive astrogliosis in wh....

Protocol

NOTE: This study was conducted in accordance with the recommendation of the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The animal protocol was approved by the University of Wisconsin-Madison, Institutional Animal Care and Use Committee. Primary Astrocyte Culture protocol that is presented here is adopted from the protocols presented by Zhang Y¹⁹ et al. and Cengiz P²⁰ et al. with some modifications.

1. Hippocampal Dissection and Astrocyte Culture

1. Prepare all necessary reagents and materials including surgical scissors, smooth fine forceps, flat tip forceps, paper towel....

Results

Understanding the roles of sexed astrocyte functions under physiologic or pathophysiological conditions have been immensely elucidated by culturing these cells under in vitro conditions. The important aspect of performing sexed culturing is to determine the sex of the mouse pup prior to its use. We determined the sex of the mouse genetically by PCR and by visual assessment (Figure 2)¹⁶. The methodology of sex determination using PCR was adopted with mo.......

Discussion

In order to study the sex differences in the properties and function of astrocytes under physiological and pathological conditions, preparation of sexed primary astrocytes in cell culture is an important tool to utilize. In the present study we report a highly efficient and a reproducible method to culture a highly enriched homogeneous population of sexed hippocampal astrocytes from newborn (P0-P2) C57Bl/6 (wild type) or K19F (GFAP null) mouse pups in-vitro. Establishing this methodology helps the investigators .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Astrocyte culture media
DMEM, high glucose	cellgro	10-013-CV
Horse Serum	Gibco	26050-070	Final Concentration: 10%
Penicillin-Streptomycin	Cellgro	30-002-CI	Final Concentration: 1%
L-Leucine methyl ester hydrochloride	Aldrich	L1002-25G	Final Concentration: 5 mM
Solution for brain tissue digestion
HBSS	Life Technologies	14170-088
Trypsin	cellgro	25-050-CI	Final Concentration: 0.25%
Other
70% (vol/vol) ethanol	Roth	9065.2
Poly-D-Lysine (PDL) 12 mm round coverslips	Corning	354087
Water	Sigma	W3500	Cell-culture grade
PBS	cellgro	21-040-CV	Cell-culture grade
0.05% Trypsin-EDTA	Life Technologies	25300-062
70 μm Sterile cell strainer	Fisher scientific	22363548
3.5 cm Petri dish	BD Falcon	353001
15 mL Falcon tube	BD Falcon	352096
50 mL Falcon tube	BD Falcon	352070
Forceps, fine	Dumont	2-1032; 2-1033	# 3c; # 5
Forceps, flat tip	KLS Martin	12-120-11
13 cm surgical scissors	Aesculap	BC-140-R
Confocal Microscope	Nikon	A1RSi
Centrifuge	Eppendorf	5805000.017	5804R
Orbital Shaker	Thermo Scientific	SHKE 4450-1CE	MaxQ 4450
Anti-IBA1	Wako	019-19741	Rabbit monoclonal
Anti-MAP2	Sigma	M2320	Mouse monoclonal
Anti-HIF1alpha	abcam	ab179483	Rabbit monoclonal
Anti-S100B	Sigma	HPA015768	Rabbit polyclonal
Anti-GFAP (cocktail)	Biolegend	837602
VECTASTAIN Elite ABC Kit (Rabbit IgG)	Vector Labs	PK-6101	Contains 4 Reagents
Goat Anti Rabbit Alexa-Fluor 488	Invitrogen	A11070
Goat Anti Mouse Alexa-Fluor 568	Invitrogen	A11004