Stokes Lab Research
“If the human brain were so simple that we could understand it, we would be so simple that we couldn’t.”
-George Edgin Pugh, 1977
Why I love research and working with students in the lab:
My interest in research began as an undergraduate, at The University of Texas at Austin, where I was very fortunate to have a graduate student mentor who was driven, patient, and sincerely interested in allowing new students ask questions, fail, and try any technique they wanted. This supportive environment introduced to me the complexities of neural signaling in the brain and I was mystified by something that I knew we may never fully understand, but I wanted to try. In the hands of passionate and talented mentors, I was guided through a project investigating the effects of alcohol consumption on inflammation in the brain. I will never forget the first time I saw a mouse brain section light up in three different beautifully fluorescent colors: WOW! I was hooked. Right then I realized that I now had the knowledge and tools to color a brain, and I wanted to do this every single day. Fast forward to a PhD in Biomedical Sciences and Postdoctoral Research in the neural control of breathing, and I am still captivated by the colorful brain images that I get to create every day.
Figure 1: One of my first confocal images as a graduate student. Microglia in the mouse spinal cord.
What we are working on right now (the short version):
Exposure to sustained hypoxic (low oxygen) conditions, arising from a life-threatening injury, illness, surgery, or a trip to the mountains, evokes a rapid increase in ventilation (breathing) that may last from minutes to months, depending on the extent of the hypoxic event. While the central nervous system (brain and spinal cord) is integral to this process, the molecular and physiological mechanisms of ventilatory control are not entirely understood. Understanding how the brain interprets and responds to hypoxic respiratory signals has relevance to many disease states (e.g. COPD and sleep apnea) as well as helping us better understand the mechanisms behind ventilatory acclimatization to hypoxia (VAH), or your body’s continued enhanced ventilation even after the hypoxic signal is gone.
Currently, we are focusing on imaging respiratory control regions of the brain to better understand how changes in ventilation are centrally regulated by both neuronal and non-neuronal cells.
Cellular communication in the brain is not limited to neurons. Astrocytes and microglia are present in respiratory regions of the brainstem and surround neural synapses and neighboring blood vessels, modulating communication of the neural pathways. Astrocytes are a heterogeneous population with cell types that regulate neurotransmitter availability in the synapse and others that are chemosensitive, meaning that they can respond to changes in blood pH. Microglia are thought to be upstream of astrocytes in that upon activation by neurotransmitters released by neurons, microglia release their own gliotransmitters, activating neighboring astrocytes which then amplify neuronal signaling.
In respiratory centers of the brainstem, both microglia and astrocytes are activated as shown by immunofluorescent labeling, and my lab is interested in uncovering the role of these glial cells in respiratory control.
Figure 3: Microglia and astrocytes in the NTS during normoixa and after 24 hours of hypoxia.
Astrocyte activation can be quantitatively studied by immunofluorescent labeling of these cells using a specific antibody, GFAP. When astrocytes are activated they produce more GFAP protein, which translates to a “brighter” more intense signal in an image. Thus, we can calculate the average intensity over baseline (normoxic animals) to get an output of astrocyte “activation”. On the other hand, microglia can be visualized with the Iba-1 antibody and assessed for a morphology shift to the larger “active state”, with more amoeboid-shaped cell bodies. This also requires immunofluorescent labeling of these cells with Iba-1 and imaging them on a confocal microscope. Then I take those images and calculate cell body size and branch filament length.
Figure 4: Microglia activation profile in the NTS based on morphological changes from the ramified to amoeboid-shape.
Current projects in the lab include:
Optimizing astrocyte activation quantification methods
Confirming the astrocyte activation profile in previously published respiratory control regions
Assess astrocyte activation in different respiratory control regions of the brain
What you will learn working in my research lab:
How to form a research question, hypothesis, and experimental design based on current published literature
How to critically evaluate research methods and data
Both your own, and others'
Research techniques, including tissue staining and microscopy
This includes the principles behind immunofluorescent tissue labeling and fluorescence microscopy
How to quantitatively analyze microscope images, specific to cell type
How to communicate your research to anyone and everyone!
Patience! Research takes time and often requires multiple attempts with protocol modifications along the way
That research is fun and a great way to build both problem solving and time management skills
Still interested? Follow Toprosaur to the lab!
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