2. ii
ABSTRACT
Hoxb8 microglia are a particular breed of scavenger cells in the brain which have
been related to obsessive compulsive disorder (OCD)-like behavior among other
behavioral disorders. These cells have been found to reach full maturity during a period of
neurological development known as the critical period. However, it has yet to be
determined whether a correlation between the critical period and Hoxb8 microglial cell
growth exists or if they are merely occurring at the same time due to unknown external
factors. To better understand the extent to which the critical period plays a role in Hoxb8
microglial cell growth in mice brains, whisker trimming was conducted over four different
periods of time. Whisker trimming was used as a source of manual sensory deprivation in
order to lengthen the critical period in the tested mice brains. Given the direct connection
between mice whiskers and the barrel field region of the brain, any resulting effects of
manipulating the critical period on the density of Hoxb8 microglial cells would arise
independently of outside factors in that particular region. Upon analysis of the barrel field
regions of the brain for each mouse, it was found that whisker trimming from the eighth
day after birth to the thirty-ninth day (P8-P39) produced a mean Hoxb8 microglial density
456% greater than was found in the control. Though this experiment serves as a preliminary
study, the results provide insight into the connection between Hoxb8 microglia and the
critical period. Furthermore, they simultaneously suggest the possibility of future
applications in the use of manual sensory deprivation to control Hoxb8 microglial cell
growth. Paired with further testing, the possibility of induced Hoxb8 microglial cell growth
presents a possible treatment method for behavioral orders such as obsessive compulsive
disorder through controlled sensory deprivation.
4. 1
INTRODUCTION
Microglia are scavenger cells in the brain believed to be related to synaptic pruning,
a process in which microglia engulf and eliminate excess synapses in the brain to balance
neural development (1) (2). A balanced number of strongly connected nerve-nerve
synapses in the brain allows human beings to process sensory input in a healthy manner.
Improper pruning during early neural development can result in multiple mental and
behavioral diseases, such as obsessive compulsive disorder (OCD), clinical depression, and
epilepsy (3). Furthermore, a particular type of microglia, called Hoxb8 microglia, has been
tied to behavioral abnormalities in mice. Disruption of Hoxb8 microglia and their growth
in the brain have been found to produce OCD-like behavior (1). Given the impact of Hoxb8
microglia on behavioral disorders, there is an urgent need to understand their controlling
factors.
One particular area of Hoxb8 microglia research is the time period in which they
reach their peak density and complexity in the brain. This time, known as the critical period,
is the portion of the animal’s life in which neural connections are shaped and sensory
functions reach full maturity (4). For a mouse, the critical period lasts from the eighth day
after birth to the sixteenth (P8-P16), with Hoxb8 microglia generally reaching their peak
density between P4 and P16 (1). However, it is unclear whether or not the critical period
actually plays a role in Hoxb8 microglial proliferation or if it is merely a coincidence that
Hoxb8 microglia reach their peak density during the critical period. As a result, the effect
of changes in the critical period on the proliferation of Hoxb8 microglia needs to be
determined.
5. 2
To determine the impact of the critical period on the proliferation of Hoxb8
microglia, the critical period of mice was manipulated and the resulting effects on the
microglial density was analyzed. The critical period was manipulated via whisker
trimming, as it’s been found in previous studies that the resulting sensory deprivation is
capable of prolonging the critical period (4). Furthermore, there is an established pathway
between the whiskers of mice and the barrel field, the region in the brain that is thought to
be critical for the development of neural functions [5]. Therefore, for the critical period of
be linked to Hoxb8 microglial cell growth, trimming the whiskers of mice should produce
a correlated response in the barrel field of the brain that can be calculated and found to be
statistically significant.
Given that the whisker-barrel field networks operate in a contralateral manner, any
change in Hoxb8 microglial proliferation as evidenced by the density of Hoxb8 microglia
should occur in the hemisphere’s barrel field, opposite to the side of whiskers that were
trimmed. Controlled whisker trimming can provide a quantifiable comparison in microglial
density between the barrel fields linked to the trimmed and untrimmed sides of the face of
each tested mouse. In turn, these findings are used to promote the study of both synaptic
pruning and the treatment of the behavioral disorders with direct ties to Hoxb8 microglia.
6. 3
METHODOLOGY
A. Animals
Sixteen C57BL/6 male mice weighing between 10-15 grams and containing the gene
Cx3CR1 GFP/+: Hoxb8-Icre/+ Rosa TdT Tomato were used for this experiment. Cx3CR1
GFP/+: Hoxb8-Icre/+ Rosa TdT Tomato served to bind TdT Tomato, a red fluorescent
protein, to Hoxb8 microglia in mice. Four mice were designated for each trimming period,
providing the four testing conditions with individual sample sizes of n=4 and an overall
sample size of n=16. The first trimming period was conducted between the fourth day after
birth and the sixteenth day after birth (P4-P16); The second between the fourth and
seventeenth days after birth (P4-P17);The third between the eight and thirty-ninth days after
birth (P8-P39); and the fourth for the first 32 days following birth (P0-PP32). Animals were
sacrificed by rapid decapitation, after anesthetization using a single injection of 5 ml of
Avertin to produce complete loss of feeling. To ensure complete loss of sensory activity,
the pad of the mouse’s foot was pinched using forceps over a period of 5 seconds, with no
observed reaction from the mouse being deemed acceptable. The research and method of
sacrificing the mouse met the guidelines of IACUC and Capecchi lab practice.
B. Preparation of Mice
Mice were bred together so that all offspring would contain the Cx3CR1 GFP/+: Hoxb8-
Icre/+ Rosa TdT Tomato gene. Upon birth, this was tested and confirmed by removing the
tip of the tail from each mouse pup and sending it off to the Capecchi Laboratory to be
genotyped and placed in the records. All mice found not to contain the acceptable gene
were either sacrificed using IACUC guidelines or set aside for future experiments. To
7. 4
ensure the acquisition of data from every trimming period, breeding and genotyping were
conducted until sixteen mice containing the Cx3CR1 GFP/+: Hoxb8-Icre/+ Rosa TdT
Tomato gene were born and whisker trimming had been completed on every mouse.
C. Study Design
To study whether manipulation of the critical period caused changes in microglial
proliferation, we studied four sets of four mice. Every set of mice was kept in a ventilated
12”x7.7”x5.25” cages bought from Allentown and provided a continuous source of food
and water. Trimming was conducted on whiskers on the right side of the face for each set
of mice using a Phillips Norelco trimmer. Trimming was conducted on every mouse once
every 24 hours over the duration of their respective trimming periods. The first set of mice
were trimmed between the fourth and sixteenth days after birth; the second set between the
fourth and seventeenth days; the third set between the eighth and thirty-ninth day after
birth; and the final set from birth until the thirty second day after birth. Upon completion
of the trimming period, the mice were anesthetized using 0.5 ml of the anesthetic Avertin
as specified in the Animals subsection and sacrificed via rapid decapitation. At death, the
mice heads were perfused for 30 min before the brain was extracted using a medical spatula
and placed in 25 mL of 1 M phosphate buffered saline solution. Each brain was kept at
10˚C for 24 hours to ensure that no blood remained in the brain prior to slicing. Each brain
was then sliced into 100 µm slices using a Leica Vibratome and placed onto a glass slide.
Each slice underwent treatment with 105 µL of a primary antibody composed of anti-RFP
and a second treatment with 105 µL of an Alexa555-labeled secondary antibody. Before
and between each treatment, 100 µL of 1M Phosphate Buffered Saline (PBS) was used
8. 5
three times to rinse the slices in five minute intervals. Once treated, the slides were again
kept at 10˚C for 24 hours and imaged using a Leica TCS SP5 X confocal microscope. The
resulting image files were then analyzed using the number Imaris 7.2.3 software (Bitplane).
D. Methods of Measurement
The number of Hoxb8 microglia in the barrel field and their density relative to the size of
the barrel field were measured using Imaris 7.2.3 Data Visualization and Processing
software. Each image image was processed so that only the Hoxb8 microglia were visible.
The barrel field was then specified and manually selected for every brain slice that was
imaged using a combination of the default Imaris software and the Atlas Brain Map (Atlas).
Upon selection, Imaris was set to find every point above a specific color and light intensity
threshold, as the confocal microscope would make the Hoxb8 microglia appear brighter
than the surrounding tissue. By adjusting the color and intensity threshold for each brain
slice, all microglia present in each brain slice were found. Each slice was then manually
checked to ensure that no microglia had been missed. Upon completion, the area of each
barrel field and the number of Hoxb8 microglia present were collected and extracted into
a Microsoft Excel file for analysis. For every brain slice, data was collected from the
control (untrimmed) barrel field first followed by the experimental (trimmed) barrel field.
E. Quantification and Statistics
The Hoxb8 microglial density was calculated for each brain slice so as to determine the
mean densities for the experimental (trimmed) and control (untrimmed) sections for each
9. 6
trimming period. The mean Hoxb8 microglial density was calculated using Equation 1 as
seen below:
𝑑̅ =
∑ 𝑑 𝑛
𝑛
𝑖=1
𝑛
Equation 1. Mean Hoxb8 microglial Density
Where 𝑑̅ represents the mean, d represents the Hoxb8 microglial densities in each
mouseforanindividual trimming period,and n representsthe numberofmice in each
trimming period. Given that every trimming period contained four mice, n may be
replaced in the calculations with 4, as seen in Equation 2.
𝑑̅ =
∑ 𝑑 𝑛
𝑛
𝑖=1
4
Equation 2. Adjusted Mean Hoxb8 microglial Density
Upon calculation of the mean for each set of data, the standard deviation was calculated
using Equation 3 as seen below:
𝜎 = √
∑(𝑑 − 𝑑̅)2
𝑛
Equation 3. Standard Error of the Mean
Where 𝜎 represents the standard error of the mean, and d, 𝑑̅, and n represent the values
as specified previously. For every trimming period, data is presented as mean ± standard
error of the mean. Statistical significance of the results was determined by using the sample
10. 7
size and standard deviation of each trimming period to conduct a t-test. Results were
deemed statistically significant and thus accepted at p ˂0.05.
11. 8
RESULTS
A. Mean Hoxb8 microglial density
Fig. 1: Mean Hoxb8 microglial densities of multiple trimming periods; Data for the mean microglial
densities are in terms of the number of Hoxb8 microglia per cubic millimeter. Trial 1 represents the P4-P16
trimming period; Trial 2 represents the P4-P17 trimming period; Trial 3 represents the P8-P39 trimming
period; Trial 4 represents the P0-P32 trimming period;
All sixteen mice designated and prepared for trimming met the preparation
conditions as specified in the Methodology section. Figure 1 shows the mean Hoxb8
microglial densities for each of the trimming periods used as trials in this experiment. As
seen in Figure 1, mean Hoxb8 microglial densities ranged from 2.59 x 10-6 to 1.83 x 10-5
microglia per cubic millimeter for the untrimmed side of the face of the tested mice. For
the trimmed side of the face, mean Hoxb8 microglial densities ranged from 4.21 x 10-6
and 1.18 x 10-5 microglia per cubic millimeter. For all but the first trial (P4-P16), there was
a larger mean Hoxb8 microglial density in the trimmed barrel fields than the untrimmed
fields. However, no linear trends were visible for the tested trimming periods. The largest
1 2 3 4
Untrimmed 1.83E-05 6.21E-06 2.59E-06 2.17E-06
Trimmed 1.32E-05 9.39E-06 1.18E-05 4.21E-06
-5.00E-06
0.00E+00
5.00E-06
1.00E-05
1.50E-05
2.00E-05
2.50E-05
MeanMicroglialDensity(microglia/mm^3)
Trimming Period
12. 9
density for both the trimmed and untrimmed Hoxb8 microglial densities occurred in trial
1, where the untrimmed density was found to be 1.83 x 10-5 and the trimmed density was
found to be 1.32 x 10-5 microglia per cubic millimeter.
B. Differences in mean Hoxb8 microglial density
Fig. 2. Difference in mean trimmed and untrimmed Hoxb8 microglial densities for multipletrimming
periods;Data for the difference in means are interms of microgliaper cubic millimeter. Trial 1
represents the P4-P16 trimmingperiod; Trial 2 represents the P4-P17 trimmingperiod; Trial 3 represents
the P8-P39 trimmingperiod; Trial 4 represents the P0-P32 trimmingperiod.
All differences in mean Hoxb8 microglial density were calculated from the results
gathered in Figure 1, as shown in the results subsection Mean Hoxb8 Microglial Density.
The differences in the mean trimmed and untrimmed Hoxb8 microglial densities are
shown in Figure 2, with the data plotted in terms of microglia per cubic millimeter. The
largest difference between the trimmed and untrimmed mean densities was found in the
trial 3 (P8-P39), with a difference of 1.15 x 10-5 microglia per cubic millimeter. The
1 2 3 4
Difference -6.10E-06 3.18E-06 1.15E-05 2.04E-06
-8.00E-06
-6.00E-06
-4.00E-06
-2.00E-06
0.00E+00
2.00E-06
4.00E-06
6.00E-06
8.00E-06
1.00E-05
1.20E-05
1.40E-05
Difference(microglia/mm^3)
13. 10
smallest difference was found in trial 4 (P0-P32), with a difference of 2.04 x 10-6 microglia
per cubic millimeter. For every trial but trial 1 (P4-P16), the difference in mean Hoxb8
microglial density was found to be positive. No linear trends were found between trials.
C. Statistical Significance
Table 3. T-test and P-test values for each trimming period.
Trial Mean Untrimmed
Density
(mm-3)
Mean Trimmed
Density (mm-3)
t Value P Value
1
(P4-P16)
1.83 * 10-5 1.32* 10-5 2.0276 2.447
2
(P4-P17)
6.21 * 10-6 9.39 * 10-6 .7103 2.447
3
(P8-P39)
2.59 * 10-6 1.18 * 10-5 6.4097 2.447
4
(P0-P32)
2.17 * 10-6 4.21 * 10-6 1.4149 2.447
All t-test and P-test values were calculated from the results gathered in Figure 1,
as shown in the results subsection Mean Hoxb8 Microglial Density. Therefore, all sixteen
mice were raised and prepared to the specifications as set forth in the Methodology
section. The results of the t-tests and P-tests are shown in Table 3, with a data set being
deemed statistically significant if the t-test value was greater than that of the P-test. For
every testing period, given the same number of test subjects per testing period, the P
value was found to be 2.447. Of the calculated t-values, only that of trial 3 was larger
than the associated P-value, with a t-value of 6.4097. Trial 1 was the only other trial
14. 11
found to have a t-value greater than 2. As such, the only conclusions that may be drawn
from the experiment must be drawn from the P8-P39 trial in which there was a 1.15 x10-5
increase in Hoxb8 microglial density as a result of the controlled whisker trimming.
15. 12
DISCUSSION
The purpose of this experiment was to determine the impact of the critical period on the
proliferation of Hoxb8 microglia. To determine this effect, the mean Hoxb8 microglial
density of each trimming period was calculated and shown in the results subsections
Mean Hoxb8 Microglial Density and Difference in Mean Hoxb8 Microglial Density. The
results show that in trials 2-4, there were greater microglial densities in the barrel fields
of the trimmed whiskers as opposed to those of the untrimmed whiskers. The largest
difference, 1.15 x 10-5, was found in Trial 3 which suggests that a trimming period of P8-
P39 could possibly induce the largest increase in Hoxb8 microglial proliferation. The
only negative difference, -6.10 x 10-6, was found in Trial 1 which suggests that a
trimming period of P4-P16 could possibly induce the largest reduction in Hoxb8
microglial proliferation.
In subsection C of the results, Statistical Significance, the differences in the mean
Hoxb8 microglial density were tested for statistical significance. Trial 3 was found to
have a t-test value greater than that of the critical t value, and deemed statistically
significant. As such, any conclusions drawn in regard to the effects of whisker trimming
on Hoxb8 microglial proliferation must be drawn solely from that trial as of this point.
The increase in Hoxb8 microglial density as a result of controlled whisker trimming
would indicate that the rate of microglial proliferation increased as a result of the sensory
deprivation provided by whisker trimming. This is possibly due to the sensory
deprivation extending the critical period, thereby increasing the amount of time over
which the Hoxb8 microglial cells could proliferate and grow (4). As a result, this would
suggest that there is a direct correlation between the critical period and the growth of
16. 13
HOxb8 microglial cells in the brain. Used in conjunction with the other research being
conducted in the Capecchi Lab, we are able to help provide a better understanding of the
factors that affect Hoxb8 microglia. Through future experiments, this study may be
applied in the continuation of synaptic pruning research and that of the mental and
behavioral disorders linked to Hoxb8 microglia. By understanding how Hoxb8 microglial
cell growth is affected by external stimuli, we can then predict how external sources will
affect synaptic pruning and the development of related behavior. This can then be used to
develop methods of detecting behavioral disorders early and treating them before they
become overwhelming.
Furthermore, the results found from trial 3 might indicate the possibility of using
sensory deprivation as a method of manually extending the critical period and increasing
Hoxb8 microglial cell growth. Doing so would serve as method of treatment for
behavioral disorders stemming from imbalanced synaptic pruning, such as epilepsy and
OCD. Being able to increase microglial proliferation through controlled sensory
deprivation would possibly allow for the rate of synaptic pruning to be increased or
decreased, thereby allowing for manual manipulation of a patient’s brain to achieve a
healthy balance of synapses. Increasing Hoxb8 microglial proliferation would result in
more Hoxb8 microglial cells, possibly leading to more synapses being consumed in
pruning and leaving the patient with a balanced number of synapses in the brain. Since
imbalanced synaptic pruning is a primary source of multiple mental and behavioral
disorders, using sensory deprivation to control Hoxb8 microglial proliferation and
through it synaptic pruning could possibly allow for the treatment of such disorders [3].
17. 14
However, further testing both independently and in conjunction with other
members of the Capecchi Lab is required before more conclusions can be drawn in
regards to the effects of whisker trimming on Hoxb8 microglial proliferation. Animals
with Hoxb8 mutations have naturally fewer microglia on average than normal mice,
making it difficult to conduct statistical analysis and determine significance [8]. Paired
with the small size of each testing period(N=4), it is unclear whether the lack of statistical
significance in trials 1, 2, and 4 was a reflection of the results being valid or some
external factors interfering with the experiment. Since past studies have found the effects
of sensory deprivation to be evident only when the manipulation of the sensory input is
made during the critical period, the specific trimming periods used in the experiment may
have possibly been ineffective in manipulating the microglial density [4]. Furthermore,
trimming for trial 3 commenced on the first day of the critical period, which could
explain why trial 3 was found to have statistically significant results. Another possible
factor that might have affected the results is that the mice were kept only with those in
the same trimming period. Given that social dynamics play a large role in the
development of the brain, the mice in each testing period may have inadvertently affected
the brain growth of each other in a manner different to those in the other periods. (9)
The results may also have been affected by factors which could not be controlled
within the confines of the experiment, such as the physiology of the test subjects. In past
studies, it was speculated that Hoxb8 may also be produced in the bone marrow of mice
(6). Therefore, sensory deprivation would be unable to entirely control the effect of
Hoxb8 microglial proliferation, as some would be naturally arising from the bone marrow
and be transported into the brain. Though not controllable within the confines of the
18. 15
experiment and something to be noted, marrow transplant and sensory deprivation
experiments have been found to be related yet separable in regards to microglial
proliferation (7). Therefore, the results of this study may be deemed valid when held
within the confines of a sensory deprivation experiment and when the mice are bred in a
matter so as to minimize differences in bone size and density.
In conclusion, the results of this experiment serve as a valid preliminary study upon
which to base and develop further experiments. This may and most likely will focus on
investigating the effects of critical period and external stimulus manipulation on Hoxb8
microglial proliferation. From those experiments, clinical treatments for mental and
behavioral disorders such as OCD, ADHD, and autism could be developed. Therefore,
further experiments with larger sample sizes would be needed for the next step moving
forward. Furthermore, experiments conducting whisker trimming solely during the
critical period would allow for alignment of this initial experiment with the discoveries
made by Dr. Berardi [cite his/her work]. And in the distant future, upon completion of
further testing and the acquisition of FDA approval, clinical testing may be possible to
treat OCD-like behavior in mice and humans.
19. 16
CONCLUSION
Our primary aim is to determine whether a correlation exists between the critical period
and the proliferation of Hoxb8 microglial cells. Our data indicates that controlled whisker
trimming is able to alter the Hoxb8 microglial densities in mice brains by manually
providing a source of sensory deprivation, thereby lengthening the critical period. When
the critical period is lengthened, there’s more time in which Hoxb8 microglial cells may
grow, thereby increasing the number of Hoxb8 microglial cells in the affected region of the
brain.
These results confirm the connection between Hoxb8 microglial proliferation and the
critical period. Furthermore, they provide insights into future methods of manipulating
Hoxb8 microglial cell growth so that they may be used in the treatment of behavioral
disorders such as OCD.
20. 17
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22. 19
Name of Candidate: Brian Flach
Birth date: February 1, 1993
Birth place: Minneapolis, Minnesota
Address: 19 Valley Road
Madison, New Jersey, 07940
