Introduction ~10 pages (double spaced)
Background on research topic
E.g. Astrocytes, oxidative stress, glutathione, brain disease where oxidative stress plays a role, VRAC. LRRC8 molecular structure, subunits and importance of subunits, physiological role of the channel, early papers on the discovery of the molecular identity of channel (journals “science” and “nature”), evolution of LRRC8 (how is this channel different across species), role of LRRC8 in disease, particularly in brain (stroke, hepatic encephalopathy)
Related studies from the literature that led to your hypothesis.
Be sure to build the case for your study (the rationale that leads to your hypothesis)
Goal: see how LRRC8 is the same and different across species, what does this tell us about the evolution of LRRC8 and importance as a housekeeping protein
Clearly state your hypothesis
1 – 2 sentences that describe your overall experimental strategy for testing your hypothesis
details Abstract
Neurodegenerative disease (e.g. Alzheimer and Parkinson Diseases) and acute brain disorders (e.g. stroke and traumatic brain injury) are characterized by neuronal death which follows a complex series of events involving oxidative stress and loss of supportive cell function.
Astrocytes, a type of glial cell in the brain, often convert from a beneficial phenotype to a destructive phenotype in brain disease or disorders.
The terms astrogliosis or reactive astrocytes describes the detrimental phenotype of astrocytes. In this reactive state, astrocytes adopt a hypertrophic morphology and release harmful substances such as reactive oxygen species and cytokines.
Converting astrocytes back to a beneficial phenotype is a promising strategy for preventing neuronal death in brain disease.
Astrocyte culture conditions in the Layton lab mimics astrogliosis, observed as enhanced neuronal death of neurons in response to oxidative stress in the presence of astrocyte-conditioned media.
We aim to determine the mode of astrogliosis in our chick embryonic primary astrocyte cultures.
We hypothesize that our astrocytes have increased activation of the proinflammatory transcription factor NF-kappaB (Nuclear factor kappa-light-chain-enhancer of activated B cells).
To test this hypothesis, we plan to monitor the release of proinflammatory cytokines, released in response to NF-kappaB activation in our astrocyte cultures. We will also inhibit NF-kappaB activation to determine if the reactive phenotype is ameliorated, as measured as enhanced neuronal survival in response to hydrogen peroxide-induced death. we will block cytokine release from astrocytes with salbutamol.Salbutamol is a medication commonly used to treat asthma by relaxing the muscles in the airway making it easier to breathe. We expect to see a decrease in neuronal death Reveals if the release of cytokines is the cause of neuronal death
will block cytokine release from astrocytes with salbutamol.
Salbutamol is a medication commonly used to treat asthma by relaxing the muscles in the airway making it easier to breathe.
We expect to see a decrease in neuronal death
Reveals if the release of cytokines is the cause of neuronal death
methods to measure cell viability.
Fluorescence: Calcein-AM to measure living cells (green) and ethidium homodimer to measure cell death (red)
MTT Assay: measures mitochondrial activity, indicating cell viability
Cell Dissection
The optic tectum brain region was extracted from chicken embryos at two different developmental stages: embryonic day 6 and embryonic day 8.
At embryonic day 6, we focus on neurogenesis to generate neuronal cultures,
while at embryonic day 8, we focus on gliogenesis to generate astrocyte cultures.
Cell Dissociation
First we use an enzyme called trypsin to dissociate the brain cells
Then, we add another enzyme called DNase to further break down any remaining DNA in the cell suspension. This helps to ensure that the cells are completely separated from each other and are in a single-cell suspension.
Next, the cell suspension is centrifuged
Finally, astrocytes and neurons are seeded onto plates.
