TRAUMATIC BRAIN INJURY
An analysis of the presence of preferential damage that occurs after brain injury
Over 2.8 million people sustain a traumatic brain injury annually.
Traumatic brain injury, or TBI, is a severe injury that usually is caused by a violent blow to the head. TBI can disrupt the normal function of the brain and can cause subsequent medical issues, leading to millions of dollars being spent on healthcare and recovery. The most common ways for TBI to occur include motor-vehicle accidents, falls, and from violence or physical abuse. Typically, TBI occurs in the form of a concussion; and repeated concussions can be particularly dangerous, especially as they are prevalent in sports such as football and boxing. Furthermore, multiple brain injuries can increase the susceptibility for additional TBI’s and can engender a cascade of pathophysiological mechanisms that can be exacerbated by the presence of multiple injuries. Thus, in order to characterize the pathology of TBI, it is important to study the damage to the brain’s vasculature as a result of injury.
TBI typically occurs as a result of car accidents, falls, and physical abuse.
Brain or Jello with Fruit?
And the vasculature of the brain falls within the cerebrovascular network of the brain, or the system in the brain that is responsible for the blood flow. The energy demands of the brain are massive: it accounts for at least 20% of the body’s energy consumption. Thus, there has to be an incredibly intricate circulatory system to help deliver oxygen and nutrients to the brain so that you can think normally. The cerebrovascular network of the brain primarily consists of blood vessels and surrounding parenchyma tissue, and these two components of the brain have varying densities. Consequently, if a head is moved quickly, different parts of the brain may be impacted differently as you would imagine that the brain would be altered most at the junctions where densities are varied quickly.
A jello block with fruit tears at the junctions between the fruit and jello after a fall, similar to a brain and its junctions between blood vessels and brain tissue after TBI
To help you picture it out a little, imagine a block of jello with fruit chunks inside. The jello would be the parenchyma tissue and the fruit chunks would be the blood vessels. If you were to drop this block of jello with fruit, you would imagine that the jello would split apart primarily where the fruit and jello come together. Similarly, we hypothesize that much of the damage that occurs after TBI occurs near the area where the blood vessels and parenchyma tissue come together; and we define this area immediately surrounding the blood vessel as the perivascular domain.
Damaging the Brain
To measure the amount of damage that the brain suffered, we observed a process called cell permeabilization, which occurs within the neuronal cells of the brain.
A STRONG BLANKET
In a normal functioning cell, each of the internal organelles are working properly. All of the chemicals and free radicals surrounding the cell are harmless in the extracellular matrix and remain there because of the protective layer that covers the cell called the neural membrane.
Penetration
After a violent blow has been suffered to the head, the membrane of the neuron can tear open. As a result, the chemicals and free radicals that were once harmless outside of the cell can flow into the cell and disrupt the normal balance and activity of the internal organelles.
permanent damage
After the chemicals and free radicals have entered the neuron, they can permanently damage the neuron, affecting its long-term performance. Even if the neuron survives the injury and the neural membrane closes back up, it will not be able to function properly.
With all of this in mind, we want to know if neurons that are in the perivascular domain are especially vulnerable to this cell permeability because of the “jello effect” caused by varying densities of the parenchyma tissue and blood vessels. We hypothesized that neurons that are physically closer to multiple mid-sized blood vessels are more likely to undergo damage following repetitive TBI as compared to isolated neurons.
Subjects and Injury
Pigs were rotated on the sagittal plane to induce TBI
To conduct our analysis, we performed tests utilizing a porcine model. We used 6 female yorkshire swine, weighing about 20-25 kilograms each. The traumatic brain injury was induced in these pigs using a closed-head rotational acceleration model, where the heads were rotated on the sagittal plane at a peak angular velocity of 90-110 radians per second to replicate the inertial loading that occurs in TBI for humans.
Visualization of Permeabilized Cell Distribution
bilateral intracerebral ventricular (ICV) infusion of lucifer yellow (LY)
In order to visualize which particular cells were permeabilized, we used a special dye called lucifer yellow (LY). Prior to TBI, we infused the lucifer yellow into the lateral ventricles, where it diffused throughout the interstitial space and was exposed to the neurons in the pig brain.
disruption
When neurons undergo permeabilization, the cell membrane tears open. As a result, the lucifer yellow can penetrate the cell.
A strong blanket, revisited
After the lucifer yellow is diffused throughout the interstitial space of the pig midbrain, it approaches neurons. On the cellular level, lucifer yellow is impermeable because of the protective neural membrane.
Permanent Visualization
Once the neuron recovers and the neural membrane is able to close back up, the lucifer yellow becomes trapped inside the cell. We can then utilize imaging techniques to visualize the cells and see which cells became permeabilized. Cells that are permeabilized are classified as LY+.
Experimental Timeline
The experimental timeline denoting the timing of the administration of injuries and ly within the two repetitive injury groups
Image Analysis
To analyze our images, we programmed a macro with java and utilized it in a software called imageJ to help count our permeabilized cells and collect our data.
All data analysis was focused to the midbrain of the pig, highlighted in the yellow box.
The midbrain data was visualized via coronal section images, example shown above.
Zoomed in photos of the midbrain images, displaying blood vessels (dark circles in A) and LY+ cells (neon ellipses in B)
blood vessel identification
With our coded macro, mid-sized blood vessels (defined as blood vessels with a diameter between 25 and 115 microns in length) were identified as regions of interest and highlighted.
blood vessels with a diameter between 25-115 microns in left were outlined (left), picture of midbrain with blood vessels selected (right)
Drawing the perivascular domain
After the blood vessels were identified, we extended the macro to draw a perimeter zone around each blood vessel that had a radius of 200 microns, which we defined as the perivascular domain. 200 microns of length was chosen as we wanted the perivascular domain to be approximately double the average length of a blood vessel’s radius. The updated macro then drew this perivascular domain on the images on imageJ.
(Left) A schematic of the perivascular domain (dashed green circle), perivascular domain is 200 microns in radius. (Middle) A picture of the midbrain with perivascular domains drawn. (Right) A zoomed in picture of individual perivascular domains
Data Collection
To best determine if perivascular domains (and more specifically, cells that are closer to multiple blood vessels/in multiple perivascular domains) are more preferential to cell permeabilization, we needed a metric in which we were to analyze to establish our answer. We determined that conducting a cell density analysis was the proper statistic to compare.
1. Apply the extended macro to the midbrain pictures and draw the perivascular domain regions for every blood vessel
2. Identify all LY+ cells and categorize them based on the number of perivascular domains the fall within (black circles for two perivascular domains and yellow circles for one)
3. Measure the area of the entire midbrain of the pig, including blood vessels and perivascular domains
4. Measure the area in which there is only one perivascular domain
5. Measure the area in which there are 2 intersecting perivascular domains or 3+ intersecting perivascular domains
6. Use data from steps 1-5 to conduct density analysis with excel
To see the raw excel data sheet, click here.
Results
GRAPHS DEPICTING THE CELL COUNT (a), ZONE AREAS (B), AND LY+ DENSITY (C) OF THE PIGS. *** denotes p < 0.001, **** denotes p < 0.0001
After all of the data was collected, we performed the density analysis for the permeabilized cells. For uniformity, we pooled all of the animals together, regardless of their TBI administration timing groups. We observed that as the number of vessels in proximity (measured by the number of perivascular domains the neuron fell within) increased (A), the number of LY+ cells decreased. However, this was matched by a decrease in the zone area as well (B). As a result, there was not an obvious trend in the LY+ density as the vessels in proximity increase. Furthermore, despite the 2 vessels and 3+ vessels groups showing a slight increase in LY+ cell density, none of the vessels in proximity groups displayed a significant difference. Thus, the number of vessels in proximity did not appear to have a significant effect on the LY+ density.
GRAPH THAT DEPICTS LY+ DENSITY OF THE PIGS, SEPARATED BY TBI ADMINISTRATION TIMING GROUPS
Additionally, we separated the data into their respective TBI administration groups. We did not find any significant trends that occurred within vessels in proximity group or the timing groups. Thus, the timing of the TBI administration did not cause for any preferential damage after TBI.
Conclusions/Implications for Future Research
As seen in the first graph, the number of vessels in the proximity of a neuron did not appear to have an effect on the density of the LY+ cells. The groups where there were 2 vessels or 3+ vessels in proximity displayed a small increase, but it was not significant.
As seen in the second graph, the time passed in between TBI administration did not play a significant role in the density of the LY+ cells either as no trends were present in the data.
Because only 6 test subjects were used, the variability of the data was very large. As a result, the error bars spanned a large area and caused for overlaps which led to insignificance. With more test subjects and data, the results would be more precise and significance would have been easier to achieve. Future testing can increase the number of subjects.
With a greater number of subjects and more definitive results, then structural continuities at vascular-brain tissue interfaces could create biomechanical stress concentrations. Therefore, therapies can be tailored to treat neurons near perivascular domains where damage is more likely to occur.
If time taken in between TBI administration does have an effect on neuron permeabilization, then information about timings of repetitive TBI can be utilized to identify the severity of injury for patients.
More anatomical justification in the definition of the perivascular domain should be utilized for future experiments.
Future endeavors can implement pressure-sensitive nanocrystals and biomarkers for secondary injury in order to further investigate the damage that occurs after TBI.
Want More Information?
You can view below for the official poster.
