Секс Стратифицированная нейронов культур для изучения ишемических: Подготовка гибель клеток

Summary

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Abstract

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Introduction

Mammals are sexually dimorphic with males and females exhibiting different traits and characteristics. Sex differences extend even to the brain and are evident in neuronal susceptibility to injury and disease (reviewed in^1-5). For example, the male brain is more susceptible to ischemic neuronal injury from stroke or cardiac arrest followed by resuscitation (reviewed in⁶). Parkinson's disease (reviewed in⁷) and schizophrenia (reviewed in⁸) are more common in human males than in females, while the female human brain appears more susceptible to Alzheimer's disease (reviewed in⁹), and mood disorders such as depression (reviewed in¹⁰). Despite observable differences between sexes in susceptibility to neuronal injury and disease, most current treatment paradigms fail to consider sex and are similar in both men and women. Greater success might be achieved if sex differences were accounted for and understood.

In vivo animal models of neuronal injury and disease demonstrate sexual dimorphism similar to humans and are critical to enhancing our understanding of sex differences and how those differences contribute to neuronal survival and function. Evidence has demonstrated, for example, that male rodents are more susceptible than females to ischemic neuronal injury^11-15. This difference is thought to be at least partially dependent upon the molecular signaling of testosterone, the primary male sex steroid hormone, and estrogen, the primary female sex steroid hormone. Studies with gonectomized male and female rodents reveals that testosterone and the testosterone metabolite, dihydrotestosterone (DHT) exacerbate and estrogen decreases neuronal injury following cerebral ischemia^12-14,16-18. However, sex differences in cerebral ischemia are present in both neonate and adult animals^19,20. Neonates experience surges in estrogen or testosterone in utero during development, but have relatively low levels of hormone after birth until puberty (reviewed in²¹). Thus, the presence or absence of primary sex hormone is not the only determinant of sex-influenced neuronal susceptibility to injury. Indeed, it is likely that sexual dimorphisms are established during development that result in differences in cell signaling and response to ischemia even when hormone levels are low.

Regardless of whether sex differences are dependent on the presence of sex steroid hormone or whether they are due to dimorphisms established during development, it is becoming clear with further study that there are differences in cell signaling between male and females in the brain. In the Herson Lab at the University of Colorado School of Medicine, we have identified one such difference. We have found that the calcium, sodium, and potassium permeable ion channel, TRPM2 (reviewed in^22,23), induces neuronal cell death following ischemic insult in vivo in the male but not the female mouse²⁴. We demonstrate that pharmacological inhibition or genetic knockdown of TRPM2 is protective in the male but not the female mouse in our in vivo middle carotid arterial occlusion (MCAO) model of stroke²⁴.

Despite their usefulness, in vivo animal models of ischemic neuronal injury may, at times, be limited for molecular studies of neuronal death, due to the difficulty or inability to specifically target neurons for investigation. For this reason, our lab uses primary disassociated cortical or hippocampal neurons in vitro to complement our in vivo work. Primary disassociated neuron cultures present a useful tool to investigate the cell signaling pathways involved in neuronal injury and disease. Neurons in culture can be maintained in a tightly controlled environment, with or without glial input. In the absence of glia, any responses observed in culture are neuronal. Pharmacological agents can be administered to culture without concerns about absorption across the blood brain barrier, allowing for the inhibition or activation of specific components of a given signal transduction pathway. Molecular biology can be used to overexpress or knockdown various proteins of interest, and electrophysiological measurements can be conducted on individual cells, something that would not be possible in an in vivo model system.

The Herson Lab expands the usefulness of primary disassociated neuronal cultures by first sexing the embryos used to prepare the cultures. Male and female sex-stratified neuron cultures demonstrate clearly that male neurons are more susceptible than female neurons to neuronal cell death following oxygen and glucose deprivation²⁵. Oxygen and glucose deprivation is an accepted in vitro model of neuronal ischemia as ischemia reduces oxygen and glucose availability to the brain. Furthermore, we have demonstrated with electrophysiological experiments in disassociated, sex-stratified neurons that TRPM2 is activated in male but not female neurons^24,26. We are currently investigating the molecular sex-specific regulators of TRPM2 activity^24,26. Here, we share and discuss our protocol for establishing sex-stratified primary embryonic disassociated hippocampal neuron cultures and present representative data using this method that suggests that the DNA repair enzyme, PARP-1, contributes to ischemic cell death in a sex specific manner similar to TRPM2. PARP-1 is activated by DNA breakage due to oxidative stress (reviewed in^27,28). When DNA damage is minimal, PARP-1 activity enhances cell survival, but when damage is excessive, increased PARP-1 activity exacerbates cell death. PARP-1 produces poly-(ADP)ribose, a known activator of the TRPM2 channel^28-31, and some evidence suggests that PARP-1 is activated preferentially in the male but not female brain in response to oxidative stress^32,33. Thus, we hypothesize that PARP-1 activates TRPM2 in the male but not female, inducing neuronal cell death in response to oxygen and glucose deprivation.

Protocol

All procedures in this protocol were conducted according to the National Institute of Health guidelines for the ethical treatment and care of animals, and according to an approved IACUC animal use protocol. 1. Preparation of Tissue Culture Dishes, Culture Media, and Dissection Instruments to be Performed at Least One Day Before Dissection Prepare 1 L borate buffer: 3.1 g/L boric acid and 4.75 g/L Borax in double distilled H2O. Adjust pH to 8.4. Filter-sterilize and store…

Representative Results

Proper sexing of embryonic mice is a critical step in the preparation of sex-stratified primary neuron cultures. To ensure the male and female hippocampi were cultured separately from correctly sexed pups, embryos were separated by sex and their cortex and hippocampus were harvested. Genomic DNA was isolated from the harvested tissue of each embryo according to standard procedures, being careful to keep sex separate. Polymerase Chain Reaction (PCR) was performed using primers for the male-specific gene, SRY and the unive…

Discussion

Sex stratified primary disassociated hippocampal neuronal cultures in which male and female hippocampi are dissected, disassociated, and cultured separately present a useful tool for investigating sex-specific cell signaling in neuron cell death. While neuroscientists commonly use primary neuronal cultures, protocols vary between labs depending on their specific experimental aims and interests. Many researchers choose to coculture neurons with glia, either in mixed culture or by plating the neurons on a glass cover slip …

Materials

B27 Supplement	Invitrogen	17504-044
Borax	Sigma	71997
Boric Acid	Sigma	B6768
Bovine Serum Albumin	Sigma	A4503
Fetal Bovine Serum	Invitrogen	26140-079
GlutaMAX	Invitrogen	35050-061
Glutamine	Invitrogen	25030-081
HBSS, no calcium, no magnesium, no phenol red	Invitrogen	14175-095
MEM, no glutamine, no phenol red	Invitrogen	51200-038
Neurobasal, no glutamine, no phenol red	Invitrogen	12348-017
Penicillin/Streptomycin	Invitrogen	15070-063
Poly-D-lysine, MW 70,000-150,000	Sigma	P0899
Trypan Blue 0.4%	Invitrogen	15250-061
EQUIPMENT
Name of the Equipment	Company	Catalogue Number
Dumont #5/45 Angled Forceps	Fine Science Tools	11251-35
Dumont #7 – Fine Curved Forceps	Fine Science Tools	11274-20
Serrated (London) Forceps	Fine Science Tools	11080-02
Extra Fine Graefe Forceps 1 x 2 teeth	Fine Science Tools	11153-10
Moria Spring Scissors	Fine Science Tools	15396-00
Medium Operating Scissors	Roboz Surgical	RS-6812
Extra Fine Microdissecting Scissors	Roboz Surgical	RS-5880
24-well Tissue Culture Plates	Fisher	87721
Cell Strainer, 40 µM Nylon Mesh	BD Biosciences	352340