Protection of rat primary hippocampal cultures from A

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Protection of rat primary hippocampal cultures
from Aß cytotoxicity by pro-inflammatory
molecules is mediated by astrocytesNeurobiology
of disease, Vol 19 (2005) 243-254presentation
byAshim Malhotra, Brian Scharf, Sushil Pai
Ann-Marie Matei
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Introduction

• Central nervous system (CNS) consists of the
  brain and the spinal cord
• In the brain there are two principle cell types,
  the neurons and the glia cells
• Part of the glia are the astrocytes which are
  most commonly thought to provide a host of
  essential support functions for neurons

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Protein misfolding disorders

• Alzheimers disease
• Huntingtons-Chorea
• Parkinsons disease

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Introduction contd

• Alzheimers disease (AD)
• - Multifactorial disease
• - The rate of synapse loss and neuronal cell
  death determines the onset and/or the progression
  of dementia
• - Impairment of mental function is preceded
  by the development of two lesions
• ? deposition of fibrillar ß-amyloid peptide
  (Aß) as insoluble extracellular aggregates
  forming the core of senile plaques
• ? appearance of intracellular neurofibrillary
  tangles

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Introduction contd

• AD contd
• - ß-amyloid plaques are characteristic
  hallmarks of AD
• - Aß is derived from the cleavage of the
  amyloid precursor protein and varies in length
  from 39 to 42 amino acids
• - Aß42 occurs more frequently and forms
  fibrillar aggregates more readily

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Introduction contd

• ß-amyloid
• - Can activate inflammatory pathways by
  enhancing microglial secretion of inflammatory
  cytokines
• - It can trigger production of reactive oxygen
  species (ROS), nitrogen intermediates and TNF-a
  from microglia cells

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Introduction contd

• Microglial cells
• - Phagocytic cells
• - Major immunocompetent component of CNS
• - Serve as scavenger cells in the event of
  infection, inflammation, trauma and
  neurodegeneration
• - Produce several cytokines responsible for
  autocrine regulation and communication with
  neurons, astrocytes and leukocyte infiltrates

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Introduction contd

• Microglial cells contd
• - Gradual activation safeguards CNS
  homeostasis, tissue defense and immune reactivity
• - The AD brain is characterized by the
  presence of senile plaques surrounded by abundant
  activated microglia

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Introduction contd

• Astrocytes
• - The most abundant cell type in the brain
• - These cells fill the spaces between neurons
• - Contribute to brain homeostasis in several
  ways
• ? buffering of extracellular K
• ? regulating neurotransmitter release
• ? forming the blood-brain barrier (BBB)
• ? releasing growth factors
• ? regulating the brain immune response
• - Play a role in a variety of diseases

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Introduction contd

• Astrocytes contd
• - Considered to be the structural and trophic
  support of the CNS
• - Antioxidant defense mechanism because they
  contain superoxide dismutase (SOD), glutathione
  peroxidase, glutathione
• - When stimulated by pro-inflammatory
  molecules they secrete IL-1ß and nerve growth
  factor (NGF) potentially increasing the viability
  of damaged neurons

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Introduction contd

• Astrocytes contd
• - TGFß1 mRNA increases in astrocyte cultures
  exposed to lipopolysaccaride from gram-negative
  bacteria (LPS), interferon gamma (IFN-?) or tumor
  necrosis factor alpha (TNF-a)
• - TGF-ß1 increases upon administration of
  IL-1ß elicits a synergism with NGF
• - There are however, contradictory reports
  regarding the protective role of astrocytes

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Introduction contd

• In this paper the effect of pro-inflammatory
  molecules LPS IFN-? was evaluated on the
  activation of glial cells and neurotoxicity
  induced by Aß
• LPS and IFN-? have been widely used in different
  in vitro in vivo experimental approaches for
  the study of Alzheimers other
  neurodegenerative diseases

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Hippocampus Anatomy
CA2
CA1
Corpus Callosum
Dentate gyrus
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Materials Methods

• Techniques
• Cell culture
• Hippocampal
• Glial
• Immunocytochemistry
• Apoptosis assay TUNEL
• Cell Viability assay MTT

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Cell culture Hippocampi
18 day SD rat hippocampi
HBSS, pH 7.4 trypsin
10 MEM stopped digestion
After 24h 2uMAraC
Anti- (? tubulin III GFAP)
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Principle of TUNEL assay
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3
DNA fragment w/ free 3 -OH
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TdT
BrDU
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Ab against BrDU
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MTT Assay
MOM
mitochondrion
SDH
Formazan
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