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Alden, Katrina

 
 
Wheaton Journal of  
Neurobiology	  Research	  	  
 
Issue 9, Spring 2017: 
  
"Modeling disease using primary neuronal tissue culture"  
 
R.L. Morris, Editor.  Wheaton College, Norton, Massachusetts. 
 
 
The effect of lipoplysaccharide on 
glial cell motility 
 
Katrina Alden 
 
BIO 324 / Neurobiology  
Final Research Paper 
3 May 2017 
 
 
 1 
 
 
The Effect of Lipoplysaccharide on Glial Cell 
Motility 
  
Katrina Alden 
Final Research Paper written for 
Wheaton Journal of Neurobiology Research 
BIO 324 / Neurobiology 
Wheaton College, Norton Massachusetts 
3 May 2017 
 
Introduction 
Amyotrophic lateral sclerosis (ALS), commonly referred to as Lou Gehrig’s Disease, is a 
fatal neurodegenerative disease that affects an individual at the neuromuscular level. The cause is 
largely unknown; 2% of cases are thought to come from a SOD1 gene mutation and the other 
98% is still being explored (Guadagno, Xu, karajgikar, Brown & Cregan, 2013). 
Nueroinflammation is one potential cause of Lou Gehrig’s that many scientists are researching. 
Inflammation of the nervous system is found in many neurodegenerative conditions such as 
Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (Guadagno, et al., 
2013).  
One factor that has been found to accelerate the onset of ALS is neuroinflammation 
(Boillee, Velde & Clevland, 2006). Research in ALS has indicated that over-activation of pro-
inflammatory glial cells may lead to the exacerbation of neurotoxic factors and cell death (Xiao, 
Zhao, Beers, Yen, Xie, et. al, 2007). ALS may be directly affected by the overproduction of pro-
inflammatory cytokines causing excess glutamate and oxidative reactions within neurons, which 
can cause cell death (Henkle, Beers, Zhao, Appel 2009). 
Glial cells regulate neuroinflammatory responses within the central nervous system 
(Whitney, Eidem, Peng, Haung  & Zheng, 2009). These cells release both pro- and anti-
inflammatory cytokines and chemokines. Acute exposure to inflammation has been known to 
stimulate neurogenesis yet in uncontrolled high concentrations pro-inflammatory cytokines 
promote cell death (Whitney, et al., 2009). In one study where Tumor Necrosis Factor (TNFα) , 
released by glial cells, was stimulated using the compound lipoplysaccharide (LPS), the amount 
of TNF α,  a pro-inflammatory cytokine, correlated with decreased survivability in hippocampus 
progenitors and neural precursor cells. TNFα at high levels triggered apoptosis in mouse neural 
precursor cells in vitro (Guadagno, Xu, karajgikar, Brown & Cregan, 2013). These findings 
suggest that in an isolated cell culture exposure to TNF α is likely to produce toxic and 
inhospitable environments for cells. This could help show correlations of pro-inflammatory 
cytokines and cell survivability rates.  
   In this experiment LPS was used to stimulate the production of TNFα in Gallus gallus 
sympathetic cells from a 10 day old chick embryos. The research on glial cells suggests that in 
acute doses LPS, which stimulates the release of TNFα, may promote cell health. In high doses 
LPS, which leads to high levels TNFα, cell death occurred. Due to the high resting motility of 
 2 
gila found in a healthy adult brain, it is thought that injury-induced neuroinflammation causes 
immediate activation/movement of microglia (Nimmerjhan, Kirchkoff & Helmchen, 2005). In 
this paper, it is predicted that glial cells will move more at the onset of LPS in small doses than 
the control. As LPS increases and as more TNFα is released, glial cells will be less motile due to 
the toxic environment. Therefore this experiment hypothesizes that glial cell motility will 
decrease with high doses of LPS and increase with small doses of LPS.  
 
Materials and Methods 
 
 Primary Culture 
 In this experiment, preparatory measures such as coverslip cleaning and flame 
constriction of pasture pipettes, were conducted using Preparation Day Steps in the Primary 
Culture of Chick Embryonic Peripheral Neurons 1:Dissection protocol (Morris, 2015a).  The 
addition of polylysine and laminin was ongoing during the dissection of Sympathetic nerve 
chains and Dorsal root ganglia from 10-day-old chick embryos. The dissection followed the 
guidelines of Preparation Day Steps in the Primary Culture of Chick Embryonic Peripheral 
Neurons 1:Dissection protocol (Morris, 2015a).  Hanks Balanced Salt Solution  (HBSS) was 
used for dissection medium and washing agent.  
 
Lipopolysaccharides Concentrations 
For this experiment three different doses of lipopolyscharide were prepared. LPS was 
made into a 1mg/ml solution by adding 1ml HBSS to 1mg of powdered LPS. HBSS and LPS 
were swirled gently to help mixture dissolve. Measurements used in this experiment were based 
from previous experiments performed by Liebermen et. al, 1989, which suggested a maximum 
dose of LPS at 10µg/ml (Liebermen et. al, 1989).  Using the maximum dose as a basis for the 
other two measurements, each sample differed by an increment of 5µg. The doses used 
were10µg/ml, 5µg/ml and 2.5µ,g/ml. Using the LPS stock solution of 1mg/ml, each dose was 
prepped as a 2x stock to allow for proper mixing of LPS in cultured Petri dishes. 1 ml of Growth 
Medium was added in 20µg/ml LPS, 10µg/ml LPS, and 5 µg/ml LPS and kept in chilled storage. 
  
Experimental and Control  
 After 48 hours of incubation, LPS in it’s three different 2x stock doses of 20µg/ml LPS, 
10µg/ml LPS, and 5 µg/ml LPS were applied with a sterile pipette to the three experimental 
dishes (each dish already had 1 ml growth medium for incubated cells, which mixed LPS to 100x 
stock ensuring proper mixture would occur) and 1ml Growth Medium without LPS was applied 
to the control dish. After 20 hours of incubation, a recommended time by Lieberman et. al 
(1989), cells were observed. 
 
Preparing Slides 
 Four chip chambers were created using the protocol of Primary Culture of Chick 
Embryonic Peripheral Neurons 2:Observation of live unlabeled cells (Morris, 2015b).  Growth 
medium was removed allowing for the removal of treated coverslips, which were placed on 
slides and sealed with VALAP (1:1:1 mix of Vaseline, Lanolin, Paraffin).  
 
 
 
 3 
Microscopy and Imaging  
 A Nikon Eclipse E200 microscope was used to view each slide labeled as control or LPS 
dosage. These slides were observed by a Sony digital input Camera (DFW−X700) with Nikon 
diagnostics at a 1. 0 C mount. BTV 6.0 b1 software was used to capture still frames. To ensure 
cell movement slides were kept at 37°C an electrical space heater was placed adjacent to the 
microscope with a digital thermometer taped to the microscope stage.  
All slides were first scanned at a 10x objective at phase one light looking for ganglia surrounded 
by glial cells. Glial cells were identified by their ruffled leeward edges, oblong shape and 
misshaped movement. Neurons were identified by static cell bodies with long extensions 
growing outward. When ganglion, a mass of cell bodies and glial, which surrounded the ganglia 
were found in the same frame, it was imaged at 40x objective with phase 2 light. The BTV 
software was used for time−lapse microscopy, capturing still frames every ten minutes for 30 
minutes, focusing on one ganglion.  
After all four slides were complete each still frame was uploaded into ImageJ 1.50i, on a 
Mac OS X 10.6.8 laptop.  Images were taken with collaborator Georgia Michalovic in the ICUC 
lab at Wheaton College, Ma. 
 
Image Analysis  
For purpose of quantification, the cell’s position was considered to be the center of the 
darkest nucleolus continuously visible for the entire image series. Cells that did not show 
movement of nucleolus or whose nucleus was not seen in each frame were excluded from 
quantification.  It should be noted that cells with dramatically dynamic nucleoli might produce 
different results than cells with static nuclei even if cells are similar. Each experimental group 
averaged the movement of two dark nucleoli. Using the multi−point tool, frame1 was marked at 
the previously defined, darkest visible nucleolus within the nucleus.  The XY coordinates, 
measured in pixels, were recorded for the starting nucleus position and tracked in each frame. 
The difference of pixels in XY coordinates was calculated from picture to picture in order to 
obtain net movement. XY coordinates measured movement of both forward backward and up 
and down motion.  Frame 1 of each experiment was marked with the multi−point tool from the 
Image J main tool bar, to show visible movement. To save each photo a screen shot was taken by 
holding command, shift 4. This process was repeated for all frames. XY coordinates from each 
cell were averaged per experimental group and compiled into two categories: average net 
movement and average glial motility/frame  
 
Results  
Strong patterns were found in the experimental doses, at 2.5 µg/ml dose of LPS glial cell 
motility spiked higher than the control, around 5 µg/ml the same trend was observed, there was 
higher glial motility than the control, but less movement than the first dose. At the highest dose 
of LPS 10 µg/ml motility was lower than the control. Patterns seen within average motility per 
frame showed that 30 minutes of imaging both control and experimental motility increased from 
0 to 30 minutes. 
 
 4 
A)    
                B)                                                   C) 
                              
 
Figure 1.  
Control cell image A, were viewed at a 40x magnification under transmitted light microscope. In 
image B, points 1−4 show net movement of control glial cell 1 recorded over 30 minutes. I 
image C, Points 5−8 show net movement of control glial cell 2 recorded in 30 minutes. The 
control net movement averaged was 34.5 pixels/frame.  A and B show a close up of data points 
recoded. Notice how the motility in the control images shows a forward and long distance 
trajectory.   
  
 
 
 
 
 
 
 
 5 
A) 
 
 
B)      C) 
   
 
Figure 2. 
Image A of 5µg/ml  LPS-treated cells imaged at a 40x magnification by transmitted light 
microscope. Two glial cells were marked at the center of the darkest nucleolus every 10 minutes 
for 30 minutes. In image B, points 1−4 represent net motility of cell 1. In image C, points 5−8 
represent net motility of cell 2. The averaged movement was recorded at 38.5 pixels in 30 
minutes. Note the back and forth movement along with long distance span represented by each 
point. 
 
 6 
 
Figure 3. 
Two glial cells from each experimental group were averaged. Averages were recorded in each 
group every 10 minutes measured in pixel/frame for 30 minutes. The control average and and all 
three experimental groups at 10 minutes showed  increase in motility at 30 minutes.   
 
 
 
Figure 4. 
Net movements of two glial cells per frame were averaged, this includes back/forth and up/down 
movements as recorded in XY coordinate pixels. Movement between beginning and end of trial 
were averaged between two glail cells to get a net total for each experimental group. Notice the 
highest dose of 10µg/ml LPS had the lowest net travel (n=22 pixels), while at a dose of 2.5µg/ml 
LPS had the highest net movement overall (n=47pixels). The 2.5µg/ml LPS dose traveled 12.5 
more pixels than the control and the 5µg/ml LPS traveled 4 pixels more. The 10µg/ml LPS 
traveled 12.5 pixels less than the control. 
 7 
 
Discussion & Conclusion  
These results support the proposed hypothesis that high levels of LPS treatment slow 
glail cell motility, whereas a smaller dose of LPS promoted cell motility. The slowed movement 
may be a result of overexposure to pro inflammatory TNFα. Glial cells promote neurogenesis, 
structural support and synaptic activity in neurons. When inflammation is present glia cells are 
immediately activated (Nimmerjhan et al., 2005).  The data presented supports Nimmerjhan’s 
study, as the control shows a high resting motility and the LPS activated glial cells which may 
have been producing TNFα showed greater activity through out a 30 minute trail (Nimmerjhan 
et al., 2005). The 2.5µg/ml LPS dose traveled a 1.362 fold change from the control, whereas the 
5µg/ml LPS dose traveled a 1.116 fold change from the control. The 10µg/ml LPS traveled a 
0.638 fold change from the control. These changes seen affirm the research from Whitney et. al 
and Guadagno et al., that suggest small amounts of TNFα produced from LPS may cause a 
neuroprotective response seen in high motility rate of glial cells, whereas  overexposure to TNFα 
produced from LPS may cause cell death or toxicity represented by decreased motility (Whitney, 
et al. 2009) (Guadagno, et al. 2013).  
However it is important to note that not all studies agree with this hypothesis. In one 
study in vitro microglia cells that were exposed to LPS for 72 hours were found to have more 
neuroprotective qualities than LPS treated glia with a 24-hour exposure.  TNFα was released at 
smaller quantities during long-term exposure causing higher cell mortality than acutely exposed 
cells (Cacci, Ajmone−Cat, Anelli, Biagoni, 2008).  This study adds a time component that was 
not considered in the experiment shown above. The glial cells tested were exposed to LPS for a 
20 hour period, this doesn’t meet the criteria for chronic or acute exposure according to Caccui 
et, al.  Using time of exposure to LPS as another factor to study the effects of neuroinflammation 
would be a logical follow up to this experiment. It would be beneficial to test motility at 
exposure times longer than 20 hours and to test effects of LPS after 20, 24, 42 and 84 hours. This 
type of experiment may lead to a different conclusion about what does and how much LPS 
exposure is needed to see neuroprotective properties in glial cells.  
 If this experiment were repeated it could be strengthened by a longer time-lapse period 
for imaging. The current study was also limited by a small sample size, if it were repeated, more 
than two glial cells should be measured to create a more representative average. Having a larger 
set of data/ multiple sets of data would show stronger correlations of dosage and glail cell 
motility to support the hypothesis. To ensure results of this experiment were reliable and not a 
result of experimental bias, the experiment should be repeated following exact methods to 
choose quantifiable glial cells. Exposure time to LPS should also be explored at different 
exposure periods to further explore results of Cuccie et, al.   
 Most neurodegenerative diseases show early signs of neuroinflammation (Guadagno, et 
al. 2013). Using different doses of LPS to find optimal exposure, would allow scientist to 
discover optimal levels of proinflammatory cytokines and lethal levels. This information could 
be essential to finding ways to prevent neurotoxicity and neural degeneration from occurring in 
ALS, Alzheimer’s and Parkinson’s patients. Identifying main factors of inflammation and 
neurotoxicity, such as glial cells would be an extraordinary step forward in finding a way to 
prevent these life consuming diseases. 
 
 
 8 
 
References 
 
Boillée, S., Vande Velde, C., & Cleveland, D. W. (2006). ALS: A Disease of Motor Neurons and 
Their Nonneuronal Neighbors. Neuron, 52(1), 39–59. 
http://doi.org/10.1016/j.neuron.2006.09.018 
 
Cacci, E., Ajmone-Cat, M. A., Anelli, T., Biagioni, S., & Minghetti, L. (2008). In vitro neuronal 
and glial differentiation from embryonic or adult neural precursor cells are differently 
affected by chronic or acute activation of microglia. Glia, 56(4), 412–425. 
http://doi.org/10.1002/glia.20616 
 
Guadagno, J., Xu, X., Karajgikar, M., Brown, A., & Cregan, S. P. (2013). Microglia-derived 
TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 
family member Puma. Cell Death and Disease, 4(3), e538. 
http://doi.org/10.1038/cddis.2013.59 
 
Henkel, J. S., Beers, D. R., Zhao, W., & Appel, S. H. (2009). Microglia in ALS: The Good, The 
Bad, and The Resting. Journal of Neuroimmune Pharmacology, 4(4), 389–398. 
http://doi.org/10.1007/s11481-009-9171-5 
 
Lieberman, A. P., Pithatt, P. M., Shint, H. S., & Shin, M. L. (1989). Production of tumor necrosis 
factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic 
virus (interleukin 1/ype I interferons/interleukin 6). Medical Sciences, 86, 6348–6352. 
 
Morris, R.L. (2015a) Neurobiology Bio324 primary culture of chick embryonic peripheral 
neurons 1: dissection.  Accessed <insert date you accessed article here>.  
Availableat: http://icuc.wheatoncollege.edu/bio324/2015/NBlabPrimaryTissCultureProc1Dis
sn2015.htm 
 
Morris, R.L. (2015b) Neurobiology Bio324 primary culture of chick embryonic peripheral 
neurons 2: observation of live unlabled cells.  Accessed 11/04/2015.  
Availableat: http://icuc.wheatoncollege.edu/bio324/2015/NBlabPrimaryTissCultureProc2Obs
ervnOfLliveUnlabeledCells2015.htm 
 
Nimmerjahn, A., Kirchhoff, F., & Helmchen, F. (2005). Resting Microglial Cells Are Highly 
Dynamic Surveillants of Brain Parenchyma in Vivo. Science, 308(5726). Retrieved from 
http://science.sciencemag.org/content/308/5726/1314.full 
 
Whitney, N. P., Eidem, T. M., Peng, H., Huang, Y., & Zheng, J. C. (2009). Inflammation 
mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and 
neurodegenerative disorders. Journal of Neurochemistry, 108(6), 1343–59. 
http://doi.org/10.1111/j.1471-4159.2009.05886.x 
 
 
 


The effect of lipoplysaccharide on glial cell motility.

Black, Sophia E.

 
 
 
  
Wheaton Journal of  
Neurobiology Research  
 
Issue 9, Spring 2017: 
  
"Modeling disease using primary neuronal tissue culture"  
 
R.L. Morris, Editor.  Wheaton College, Norton, Massachusetts. 
 
Effects of intracellular beta amyloid on 
mitochondria in triturated glial cells of Gallus 
gallus  
 
Sophia E. Black  
 
BIO 324 / Neurobiology  
Final Research Paper 
3 May 2017 
 
 1 
 
Effects of intracellular beta amyloid on 
mitochondria in triturated glial cells of Gallus 
gallus  
  
Sophia E. Black 
Final Research Paper written for 
Wheaton Journal of Neurobiology Research 
BIO 324 / Neurobiology 
Wheaton College, Norton Massachusetts 
3 May 2017 
 
Introduction 
  
Alzheimer’s disease is a neurodegenerative disease characterized by memory loss and 
loss of various cognitive abilities. Alzheimer’s was declared the sixth leading cause of death in 
the United States in 2017 (alz.org,2017). Alzheimer’s is believed to correlate with an abnormal 
accumulation of β-Amyloid peptide in the CNS, due to reduced clearance of the peptide 
(Mawvenyega et al., 2010). The Beta Amyloid peptide is a fragment of 40 amino acids from the 
integral membrane protein, beta-APP (beta- Amyloid precursor protein) (Haass,1993).  
Intracellular β-Amyloid specifically has been found to effect neurons by disrupting and 
inhibiting fast axonal transport, a disruption which has been linked to neurological disease and is 
associated with synaptic dysfunction (Wang, 2008). Mitochondria are organelles that are 
transported throughout the cell as sources of energy, the failure of mitochondria to be transported 
to the synapse is largely implicated in synaptic dysfunction and degeneration in AD cells 
(Verberg & Hollenbeck, 2008). The overproduction of β-Amyloid has also been found to effect 
the distribution and morphology of mitochondria in primary hippocampal neurons. A study in 
2008 found that cells exposed to β-Amyloid expressed mitochondrial fragmentation and an 
 2 
imbalance of mitochondrial fission/fusion (Wang, 2008). Fission and fusion of mitochondria are 
important for growth, redistribution, and maintenance of a healthy mitochondrial network. 
Decreased fusion and increased fission often occur when high levels of stress are present in the 
cell (van der Bliek, 2013). Mitochondria index reflects the total mass of mitochondria per unit 
length of a neurite, and can be indicative of the fusion fission events of the mitochondria. A 
study on mouse hippocampal neurons found that the length of mitochondria, mass of 
mitochondria, and percent of neurite occupied by mitochondria was decreased when treated with 
the active β Amyloid peptide 25-35. (Calkins, 2011). The percent of mitochondria per cell area was 
particularly interesting for our experiment as glial cells often internalize debris material, 
resulting in a range of detrimental effects on their cytoplasmic contents (Nagele, 2004). 
We chose specifically to study glial cells because they have been found to participate in 
β-Amyloid degradation and clearance in AD (Alzheimer’s Disease)  brains. Activated astrocytes 
(glial cells) in AD brains can accumulate substantial amounts of β-amyloid intracellularly, 
sometimes to the point that the peptide dominates the cytoplasm (Nagele, 2004). Based on the 
evidence of the effects of intracellular β-Amyloid on neurons, the evidence of AB25-35 on the 
index of mitochondria in hippocampal mouse neurons, and what is known about glial cells, we 
chose to test the effects of intracellular β-Amyloid on mitochondrial coverage in glial cells of 
Gallus gallus (domestic chicken). A concentration of 25µM was used to treat our experimental 
groups, as it was found in previous literature to have effects in CNS cells (Allaman et. al, 2010). 
The domestic chick, Gallus gallus was used as a model as their development is well known, 
allowing us to easily obtain the cells of the dorsal root because we know when the dorsal column 
has developed. We hypothesized that glial cells exposed to intracellular Aβ25-35 will have reduced 
 3 
mitochondrial coverage as measured by percent area of mitochondria staining by Rhodamine 123 
florescence, using ImageJ analysis.  
 
Materials and Methods 
Dissection 
The experiment was performed on harvested sympathetic nerve chain and dorsal root 
ganglion cells from 10 day old chick eggs.  The dissection followed the protocol of R.L.Morris, 
adapted from Peter J Hollenbeck, provided by R.L. Morris (Morris, 2015a).  Neurons and glia 
were grown in modified Leibovitz L-15, containing F-plus medium, as F-plus contains neuron 
growth factor. It is important to note that pen/strep was also added to the medium in order to 
prevent contamination.  
Trituration 
Before the neurons and glia were plated, we triturated the cells in order to rip the 
membranes and allow our reagents, FITC Dextran solution and Beta Amyloid, to enter into the 
cytoplasm. The trituration method used followed the protocol of Hollenbeck (Verberg & 
Hollenbeck, 2008). Control and Experimental cells were triturated with a 25mg/ml solution of 
FITC Dextran, of a volume equal to that of the cells and growth medium. In addition to being 
triturated with FITC Dextran, experimental cells were triturated along with a 25µM solution of 
Beta Amyloid25-35 one one-hundredth of the volume of cells and medium; 3.24µL. Both control 
and experimental cells were then plated and incubated at 37degrees Celsius for twenty four 
hours.  
Rhodamine 123 and Florescence Imaging 
 4 
After 24 hours, all experimental dishes and control were washed with HBSS and were 
administered a 1:11,200 dilution of Rhodamine-123 in HBSS, following Rhodamine dilution 
protocol (Sapiente, 2017). Two control dishes were exposed to RH-123, while two were not. The 
cells that received Rhodamine-123 and control dishes were incubated for ten minutes at 37 
degrees C and then washed again with HBSS. Each dish received new growth medium after the 
wash. Coverslips from each dish were then transferred to slides, using chip chamber protocol, for 
imaging (Morris, 2015a). Images were obtained using a fluorescent microscope and the SPOT 
software (Spot Insight Firewire 2 MegaPixel Camera). The microscope used was a Nikon Eclipse 
E400, using 40x mag, on phase 2. The exposure time for Rhodamine 123 used was 45,000-
50,000ms.  
Quantification 
Images were quantified using ImageJ Version 1.49, selecting the cell area in the 
florescence images using the free selection tool. We compared florescence images to their 
respective transmitted images and adjusted the threshold to 49-255 under the adjust menu, to see 
only the brightest staining within the selected cell. Each image was converted to an 8-bit image 
rather than an RGB image. The particles were then analyzed by ImageJ (command: analyze 
particles 1-Infinity) and the percent area of brightness within each cell was collected from the 
ImageJ summary, and copied into an Excel notebook. The threshold, 49-255, was held constant 
for all images analyzed. Only cells stained with Rh123 were used for data analysis in this 
experiment. 
Results 
After the triturated control with FITC Dextran solution, and experimental cells with FITC 
Dextran solution and β- Amyloid25-35, were incubated for 24 hours and stained with Rhodamine 
 5 
123, they were taken to the ICUC for imaging. In the current study, FITC Dextran was first 
successfully loaded into the cytoplasm of glial cells in culture using trituration loading (Vergerb 
&Hollenbeck, 2008)) (Figure 1). The trituration loading technique was then used to deliver Beta 
Amyloid 25-35 into the cytoplasm of experimental cells (Fig 2). The percent area of staining within 
the cell was used as a way to quantify the amount of space mitochondria took up in each cell. 
Three experimental images were adjusted to threshold and analyzed, while four control images 
were adjusted and analyzed. It was observed that the control cells appeared to have a greater area 
of the cell stained with Rhodamine-123 than the experimental cells, as the control cells had more 
densely stained areas, see Figure 2 and 3. The average percent area of red pixels within the cell 
area was slightly higher in the control images than the experimental images, see Figure 4. 
 
 
 
 
  
 
 
 
Figure 1: Transmitted image of glial cell (left) and florescence image displaying FITC Dextran 
loaded by trituration in cytoplasm  
 
 
 
 
 
 
 
 
 
 
Figure 2: Transmitted image and Fluorescent image, adjusted to threshold (49-255), of 
experimental glial cell treated with FitC Dextran, Rhodamine 123 and Beta Amyloid25-35. Notice 
the pattern of florescence coverage in the experimental image, representing mitochondria. 
 
 6 
 
Figure 3: Florescent Image adjusted to threshold (49-255) (left) and transmitted image (right) of 
glial cell treated with FitC Dextran and Rhodamine 123. Notice the pattern of florescence 
coverage in the control image (left), representing mitochondria. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4: The average percent area of brightness corresponding to stained mitochondria within 
selected cells from control and experimental groups. The error bars represent the standard 
deviation from the average of each group; 10.5 for experimental and 14.3 for control. 
(Experimental group n=3, Control group n=4) 
 
 
 
14.81	  
21.29	  
1	  
Av
er
ag
e	  
Pe
rc
en
t	  A
re
a	  
	  
Average	  Percent	  Area	  of	  RH-­‐123	  
staining	  Mitochondria	  in	  
Experimental	  and	  Control	  Groups	  	  
Experimental	  (25uM)	   Control	  	  
 7 
Discussion and Conclusion 
The data collected does not support the hypothesis that intracellular β Amyloid25-35 would 
decrease the percent area of the cell occupied by mitochondria. Although the average percent 
area of cell occupied by mitochondria was greater in the control group than in the experimental 
group, the standard deviation of the two group averages overlap (Figure 4), meaning that the 
difference in averages is not significant. The differences we found in our results can only be 
considered to be due to chance.  
If this experiment were to be replicated n values would need to be increased , providing a 
better opportunity for finding significant differences between the groups. A significant difference 
between groups would lead us to believe that β-Amyloid negatively impacted the health of the 
mitochondria network; fragmenting mitochondria and reducing the amount of cytoplasm 
occupied by the organelle, which may lead to reduced supply of ATP and contribute to cell 
dysfunction (Calkins, 2011). If results were found significant we may gain a better understanding 
of how intracellular β-Amyloid may have a detrimental effect on the fusion and fission of the 
mitochondria and the maintenance of a healthy network.  
Another area of improvement for a replication of this experiment may involve a slight 
alteration to the washing procedure. As a precautionary measure, we would wash the cells in 
HBSS once more, making it four washes rather than three. Rhodamine 123 is very effective at 
staining mitochondria due to its specificity, staining mitochondria directly, without passage 
through endocytic vesicles and lysosomes (Lincoln, 1979).  One source of error however could 
come from Rhodamine-123 not being fully removed from our HBSS washes, effecting the 
quality of our images, as there is the possibility of free floating stain.  
 8 
Future experiments should be done in order to get more conclusive results and to better 
understand the mechanism by which accumulation of β-Amyloid effects the occupancy of 
mitochondria in glial cells. One possible way to extend our results to future experiments would 
be to follow this procedure on neuronal synaptic terminals of Gallus gallus, rather than glial 
cells, as neuron terminals are also very dependent on mitochondria, having high demands of ATP 
(Verberg & Hollenbeck, 2008). Damage to neurons is also largely implicated as a pathology for 
Alzheimer’s disease (alz.org, 2107). The goal of future research is to find an early therapeutic 
target for the degenerative disease that effects so many people. 
 
 
 
References 
 
Allaman, I., Gavillet, M., Bélanger, M., Laroche, T., Viertl, D., Lashuel, H. A., & Magistretti, P. 
J. (2010). Amyloid-β aggregates cause alterations of astrocytic metabolic phenotype: 
impact on neuronal viability. Journal of Neuroscience, 30(9), 3326-3338. 
Calkins, M. J., & Reddy, P. H. (2011). Amyloid beta impairs mitochondrial anterograde 
transport and degenerates synapses in Alzheimer's disease neurons. Biochimica et 
Biophysica Acta (BBA)-Molecular Basis of Disease, 1812(4), 507-513. 
Haass, C., Hung, A. Y., Schlossmacher, M. G., Teplow, D. B., & Selkoe, D. J. (1993). beta-
Amyloid peptide and a 3-kDa fragment are derived by distinct cellular 
mechanisms. Journal of Biological Chemistry, 268(5), 3021-3024. 
Mawuenyega, K. G., Sigurdson, W., Ovod, V., Munsell, L., Kasten, T., Morris, J. C., ... & 
Bateman, R. J. (2010). Decreased clearance of CNS β-amyloid in Alzheimer’s disease. 
Science, 330(6012), 1774-1774. 
Mercedes, Cynthia, (2017) β Amyloid Chemical Compound Sheet 
Morris, R. L., (2015b) Neurobiology Bio324 primary culture of chick embryonic peripheral 
neurons 2:observation of live unlabeled cells. Accessed 11/04/2015. Available at 
http://icuc.wheatoncollege.edu/bio324/2015/NBlabPrimaryTissCultureProc2ObservnOfL
iveUnlabeledCells2015.htm 
Nagele, R. G., Wegiel, J., Venkataraman, V., Imaki, H., Wang, K. C., & Wegiel, J. (2004). 
Contribution of glial cells to the development of amyloid plaques in Alzheimer’s disease. 
Neurobiology of aging, 25(5), 663-674. 
Reddy, P. H. (2009). Amyloid beta, mitochondrial structural and functional dynamics in 
Alzheimer's disease. Experimental neurology, 218(2), 286-292. 
Sapiente, Joe, (2017) Rhodamine 123 Chemical Compound Sheet 
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Pigino, G., Morfini, G., Atagi, Y., Deshpande, A., Yu, C., Jungbauer, L., ... & Brady, S. (2009). 
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van der Bliek, A. M., Shen, Q., & Kawajiri, S. (2013). Mechanisms of mitochondrial fission and 
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with Local NGF or Semaphorin Signaling. The Journal of Neuroscience  : The Official 
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Collaborators: Jake Newberry, Chris Lafen, Claudine Humure 
 
 
I have abided by the Wheaton Honor Code in this work. 
Sophia Black 


Effects of intracellular beta amyloid on mitochondria in triturated glial cells of Gallus gallus.

ii,  81 leaves.Includes bibliographical references: leaves 79-81

Lowell, Rowan

Fortresses, fields and fens.

Fisher

Stokes, Delaney

Whittles, Lindsay

Oral History: Kersti Yllo

Danga, Kristina

Evidence That Cellular Stress Increases Autophagy In Neuronal Gallus gallus Cells As Measured By Lysosomal Activity.

Smith, Rebecca A.

3-nitropropionic acid influenced mitochondrial abundance in axons in a chick neuron model of Huntington's disease.

77 leavesIncludes bibliographical references: leaves 75-77

Dubois, Audrey

Meter? I barely even know'er ! Encoded information in poetry.

Shao, Jintan, TT, 1995-

Preliminary study of effects of Ginkgo biloba extract on axonal growth of Gallus gallus embryo neurons.

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