Immunostaining and Fluorescence Imaging of Nanoparticles and Cells in the Brain

Jenna Nekl Author

08/04/2020 Added

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This study will examine what types of cells in the brain the nanoparticles interact with following a TBI using indirect immunostaining and fluorescence imaging. This will be done by targeting proteins of specific cells. This will help determine and design nanoparticles that best prevent ROS from damaging the brain after a TBI has occurred.

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[00:00:01.426]Hello my name is Jenna Nekl and my research
[00:00:03.765]project for summer UCARE 2020 was on
[00:00:07.264]immunostaining and fluorscence imaging of
[00:00:09.809]nanoparticles and cells in the brain under
[00:00:12.309]the direction of Dr. Forrest Kievit
[00:00:18.304]The project as a whole is about researching
[00:00:20.818]neuroprotective therapies that improve
[00:00:22.286]patient outcome after a traumatic brain
[00:00:24.133]injury occurs
[00:00:26.640]This is important because traumatic
[00:00:27.745]brain injury is the leading cause of
[00:00:29.441]injury-related death worldwide
[00:00:31.453]yet there are no approved
[00:00:33.105]neuroprotective therapies that improve
[00:00:35.340]neurological outcome post-injury
[00:00:38.157]Currently, TBI treatment entails stablizing
[00:00:40.667]the patient, reducing inflammation, and
[00:00:43.554]controlling the blood flow pressure to the
[00:00:45.532]brain. Those treatments focus on the patient's
[00:00:47.940]short-term injuries. However, they do not
[00:00:50.890]account for the long-term injuries that will
[00:00:52.229]occur in the patient's brain. Following a TBI,
[00:00:56.880]reactive oxygen species collect in the brain
[00:00:58.411]due to the blood-brain barrier breakdown
[00:01:00.976]and inflammation. ROS are naturally produced
[00:01:05.112]and discarded during physiological processes
[00:01:07.944]in the body. However, TBIs cause ROS production
[00:01:12.942]to increase to a rate at which discarding
[00:01:15.658]processes are not able to keep up.
[00:01:19.126]ROS can cause damage to cells and inhibit
[00:01:22.611]the brain from repairing itself by preventing
[00:01:25.431]neuron formation and axon development
[00:01:27.871]because they are highly reactive molecules.
[00:01:31.607]Therefore, ROS are partially responsible for
[00:01:35.214]further injury after the initial insult.
[00:01:38.229]The hippocampus, which is involved with
[00:01:40.592]learning and memory, is not impacted
[00:01:42.390]by the TBI until 48-72 hours after.
[00:01:48.252]This indicates that damage to the hippocampus
[00:01:50.797]is due to ROS traveling to healthy parts of the
[00:01:54.705]brain, which can cause cognitive impairments.
[00:01:59.593]Antioxidant nanoparticles have been shown
[00:02:01.956]to reduce neuron damage because the NPs
[00:02:04.520]are able to inactivate ROS to prevent spreading
[00:02:09.129]to the surrounding healthy brain tissue.
[00:02:12.724]NPs have the opportunity to be delivered into the
[00:02:15.542]brain because of the opening in the blood-
[00:02:17.531]brain barrier cause by a TBI. NPs are
[00:02:21.661]opitmal delivery systems because they can
[00:02:23.864]be manipulated to have certain characteristics
[00:02:26.295]such as size, what sites in the body to target
[00:02:28.820]diffusion rate, and permeability. NPs can be
[00:02:32.926]given a fluorscent tag and the cells that they
[00:02:35.612]interact with can be detected through indirect
[00:02:37.814]immunostaining and fluorescence imaging.
[00:02:41.395]Indirect immunostaining uses antibodies with
[00:02:43.435]fluorescent tag to target a specific antigen
[00:02:46.171]of a cell. The cells get stained with antibodies
[00:02:48.869]that bind to proteins that are specific to certain
[00:02:51.799]types of cells. Before using indirect immunostaining
[00:02:55.293]and fluorscence, the already dissected brains
[00:02:57.771]from the mice would be sectioned into slices
[00:02:59.959]and then imaged using a fluorescence microscope
[00:03:04.609]Through this, the exact type of cell that the
[00:03:06.526]NPs are interacting with will be able to
[00:03:08.946]be detected. With a better understanding
[00:03:11.531]of what type of cells different NPs interact
[00:03:14.593]with, more precise designs for future
[00:03:16.996]NPs can be engineered.
[00:03:21.906]My goals that I made when filling out the
[00:03:23.749]application for UCARE in January were to
[00:03:26.193]get trained on how to use the lab equipment
[00:03:28.389]so I could be more independent while
[00:03:29.567]working in the lab.
[00:03:30.908]I also wanted to learn how research is carried
[00:03:32.808]out in the lab because I had only read
[00:03:34.916]articles about it. Next, I wanted to find new
[00:03:37.162]information to help the research project
[00:03:39.085]grow and make connections with people
[00:03:41.080]in the lab and strengthen my communication
[00:03:43.517]skills along the way. I also wanted to learn
[00:03:46.448]more about what actually happens after a
[00:03:48.712]traumatic brain injury occurs and how
[00:03:50.834]nanoparticles work and how they are made
[00:03:53.418]and manipulated. My part in the project
[00:03:57.445]was to analyze the mice brain samples injected
[00:04:00.612]with nanoparticles through immunostaining
[00:04:02.919]and fluorescent imaging. By analyzing I would
[00:04:06.533]examine what types of cells in the brain the
[00:04:08.902]nanoparticles interact with following a
[00:04:11.211]TBI. The questions that I wanted to ask
[00:04:14.382]myself along the way were, what cells in the
[00:04:17.148]brain do different NPs interact with? and Are
[00:04:20.720]there specific characteristics nanoparticles
[00:04:23.027]have, such as size or surface properties, that
[00:04:25.759]promote their interaction with one certain
[00:04:27.762]type of cell over another?
[00:04:29.899]By asking myself those questions I will be able
[00:04:32.752]to tell what characteristics of the nanoparticles,
[00:04:35.175]if any, attract those NPs to certain cells
[00:04:37.945]of the brain and from that, the most efficient
[00:04:40.308]NP can be created and long-term injuries
[00:04:42.752]from a TBI can be minimized.
[00:04:48.475]Due to the COVID pandemic all UNL labs
[00:04:51.594]were closed to undergraduate students
[00:04:53.633]and so I was not able to do what I had planned
[00:04:56.459]to do in the labs and I was forced to do my
[00:04:58.867]research from home. While I was at home I
[00:05:01.553]talked with a graduate student I had been working
[00:05:03.636]with about the different projects he has planned
[00:05:06.940]for the fall. Most of my time this summer was
[00:05:12.404]spent reading articles to prepare myself
[00:05:14.804]for projects this fall about different imaging
[00:05:17.442]techniques, softwares, mass spectrometry, and
[00:05:20.385]nanoparticles.

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Immunostaining and Fluorescence Imaging of Nanoparticles and Cells in the Brain

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