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December, 2010
It is with great pleasure that we present you this semester’s issue of The Oculus: Virginia
Journal of Undergraduate Research. As the culmination of eight weeks of peer reviews, this
Fall issue stands as an assortment of some of the most well-written and original pieces of
undergraduate research that can be found at our University. Representing several fields of
study, from history of religion to environmental policy to biomedical engineering, this edi-
tion is truly representative of the breadth and depth of research conducted at the University
of Virginia by students and their faculty mentors.
Sincerely,
Dear Readers:
Thomas Jefferson believed that learning and discovery lead naturally to human betterment.
He wrote, “I look to the diffusion of light and education as the resource to be relied on for
ameliorating the condition, promoting the virtue, and advancing the happiness of man.”
Our undergraduate students who are dedicated to research, discovery, and scholarship
continue the important work that began when Mr. Jefferson founded this University nearly
200 years ago.
Very truly yours,
Teresa A. Sullivan
President, University of Virginia
The Oculus
The Virginia Journal of Undergraduate Research
Volume 9 Issue 2
Contents
Coeditors-in-Chief
Tudor Cisu and Mitchell Leibowitz
Editoral Staff
Gunjan Amarnani, Upasana Bhattacharya, Ja Hyun Cho,
Michelle Choi, Li “Nelly” Fan, Marina Freckmann,
Jessleen Kanwal, Caroline Plowden, Sharon Rogart,
Catherine Schretter, Ko Eun “Janet” Shin, David Wu, Jason Ya
Layout Editor
David Wu
Photographer
Jessleen Kanwal
vectors, it is inconvenient and rarely used in early healthy contour. For myocarditis, the inflamed myo-
examinations. This limits the extent of the diagnostic cardium was expected to result in an abnormal QRS
use of VCG. complex and a slower propagation time. In the case
In view of this, the goal of this project was to of valvular heart disease (VHD), the diseased valves
develop a simple, user-friendly computational pro- were hypothesized to cause a non-directional con-
gram that utilized two sets of two simultaneous ECG tour orientation.
signals to generate an animated 3D QRS contour. By
comparing the resultant 3D contours of healthy and Materials and Methods
diseased hearts, this program allowed for instant di- Materials
agnosis at an earlier clinical examination. Currently, Materials needed for this experiment include
ECGs are used as a screening tool. However, after two ECG preamplifier circuits, two 3-patient cables,
successful implementation of this innovative and an oscilloscope, a multimeter, a DC power source,
cost-efficient software, VCG has the potential to be- OpenChoice, multiple resistors, capacitors, non-po-
come a new standard in diagnosing heart abnormali- larizable electrodes, alcohol wipes, and wires with
ties. crocodile clips.
Nivedha Panneer (middle) is a fourth-year pursuing a cardiac modeling laboratory involves modeling the intra-
Bachelor’s of Science in Biomedical Engineering. Her in- cellular pathways of cardiac myocytes through beta adren-
terests include infectious diseases and drug delivery. Her ergic receptors. After graduation in May 2011, he plans to
current capstone research involves the design of an assay matriculate in a biomedical engineering doctoral program,
and diagnostic device for the detection of Bordetella per- specializing in biomaterials with intentions to serve as a
tussis, commonly known as whooping cough, as well as faculty member at an engineering university.
the development of a computational model that studies the
spread and transmission of B. pertussis in a population. Hsuan Su (second from right) is a fourth year majoring
Upon graduation in May 2011, she plans to obtain a Mas- in Biomedical Engineering with a minor in Engineering
ter’s in Public Health with a focus on global health and Business. She has past experience working in cancer, bio-
health education in underdeveloped countries. materials, and chemical synthesis labs. For her current
capstone project, she aims to design a novel insole for run-
Wyatt Shields (right), a Virginia native, is a fourth-year ning shoes that mimics the properties of barefoot running
Biomedical Engineering major and a Materials Science and will conduct biomechanics testing in a gait and mo-
and Engineering minor. His interests include biomateri- tion laboratory. After graduation in May 2011, she hopes
als, tissue engineering, and ultrasound imaging. His cur- to pursue a lifelong career in the healthcare field anywhere
rent research and senior thesis project in Dr. Saucerman’s in the world.
which blocked low frequency noise whereas Stage 4 ECG and Vectorcardiography
and 5, low-pass filters, blocked high frequency noise. The circuits were used to acquire simultaneous
In order to reduce the baseline 60Hz noise in the ECG ECG measurements to represent the QRS complex
signals and to obtain a sharper pass band, a 1MΩ re- of a specific heartbeat. Frontal plane ECGs were re-
sistor was used in parallel with the capacitor in both corded using Lead I, where electrodes were on the
low-pass filters. Before the electrodes were attached, right and left arms, and Lead II, where electrodes
the patient’s skin was cleaned to remove oil and dead were on the right arm and left leg. The two circuits
skin cells that could disrupt the measured signal. recorded leads I and II simultaneously to produce
The finished ECG preamplifier system was then con- frontal plane ICVs of a specific heartbeat. Horizontal
nected with a 15V DC power supply to measure the plane ECGs were measured using precordial chest
analog ECG signals across the different leads of the
patient. Using Kirchhoff’s Laws, transfer functions
for each stage as well as the overall transfer function
for both circuits were generated and are shown in
Table 1.
From the theoretical transfer functions, the gains
for Stages 3, 4, and 5 were calculated by determin-
ing the respective magnitudes at a range of frequen-
cies, where s was equal to jω. The theoretical gains for
all five stages were multiplied together to obtain the
total gain of the circuit. The total gain was plotted
against time on a log-log scale to construct a Bode
plot. This process was repeated for both circuits. The
experimental gains of the two circuits were obtained
by applying a 100mV sinusoidal input and recording
the peak-to-peak output with an oscilloscope. These
experimental gains were calculated by the relation-
ship:
Figure 3. Frontal Plane ICV This figure illustrates how an
ICV, which is indicated by the red arrow, was constructed
in the frontal plane. The dashed green lines indicate the
perpendicular lines drawn from the lead axes.
leads V4 and V6. Using right arm, left arm, and right included BBB, anterior MI, myocarditis, and VHD.
leg as negative inputs, V4 and V6 were recorded si- Frontal leads I and II and horizontal leads V4 and V6
multaneously to produce horizontal plane ICVs of a were used from these data files to obtain 2D ICVs of
specific heartbeat. frontal and horizontal planes, respectively. From this,
Inverse VCG was performed by transforming the 3D ICVs were generated. Comparisons were then
analog signals from the frontal and horizontal planes made between the contours of the hearts.
into ICVs, while assuming the heart was stationary.
Since analog ECG signals are 1D, whereas ICVs are Results
2D, inverse VCG requires two analog signals to gen- Stages of the Circuit and Bode Plots
erate one ICV. Frontal plane and horizontal plane The overall theoretical and measured Bode plots
ICVs were computed using the amplitudes within for both circuits are shown in Figure 1. The theoreti-
the QRS complex for leads I and II and leads V4 cal corner frequencies for circuit-1 were 149.4 and
and V6, respectively. The planes were converted to 0.97 rad-s-1 and for circuit-2 were 147.4 and 1.24 rad-
Cartesian coordinates and the ICVs were plotted in s-1. The errors between the theoretical and measured
MATLAB; refer to the results for further discussion. gains for circuits-1 and 2 were 2.64 and 6.30%, respec-
Since only two circuits were constructed, the frontal tively. A 100mV peak-to-peak sinusoidal input was
leads were not recorded at the same time as the hori- applied to obtain gains since higher input voltages,
zontal leads. To account for this limitation, boundar- such as 1V, distorted the sinusoidal output.
ies were established to mark the beginning and end
of the QRS complex for ECG recordings in the two ECG and Vectorcardiography
planes. In order to create the 3D ICVs, the number of In order to create a visual representation of the
sampling points within this interval was required to electrical propagation within the heart, the following
be the same in both planes. steps were implemented in the software. First, the
data was filtered using a Butterworth filter to smooth
Abnormal ECG Data the ECG data, as can seen in Figure 2. To obtain one
Abnormal ECG data was obtained from the frontal plane ICV from two ECG signals, two perpen-
Physikalisch-Technische Bundesanstalt (PTB) Diag- dicular lines were constructed from the amplitudes of
nostic ECG Database. The different types of heart leads I and II, as seen in Figure 3. A resultant ICV was
conditions that were implemented into the software then constructed from the origin to the intersection
Fall 2010: Volume 9, Issue 2 7
Figure 5. Contour Plots 5A) 3D QRS contour plot. 5B) The contour for the frontal plane,
shown with respect to the x and z axes. 5C) The contour for the horizontal plane, shown with
respect to the x and y axes.
point of the two perpendicular lines. To accomplish ro-action potential and by comparing the shape of
this, the Cartesian coordinates of this intersection the experimental to the theoretical contour, as seen
point were determined. Since the Lead I axis cor- in Figure 6. The expected macro-action potential is
responded to the x-axis of the Cartesian coordinate directed down the heart and towards the patient’s
system, the x-coordinate of this intersection point back. This orientation is towards the thicker left ven-
was simply the amplitude of Lead I. Next, to deter- tricle wall that contains more cardiac myocytes. The
mine the z-coordinate of the intersection point, the experimental macro-action potential exhibited this
perpendicular line from Lead II was first extended to orientation, with slight deviations in the frontal and
the z-axis. The z-intercept of this line was obtained by horizontal contours. In the frontal plane, the only mi-
dividing the Lead II amplitude by the cosine of 30 de- nor deviation was at the end of the contour. Instead
grees. This is analogous to the y-intercept of the lin- of completing the loop, the end deviated from the ex-
ear slope-intercept equation. The slope of this equa- pected trajectory by curling in the opposite direction.
tion was the negative reciprocal of the slope of Lead These deviations could be due to oversampling the
II. Finally, the z-coordinate could be obtained by in- data in which parts of the P or T waves were sampled
serting the previously determined x-coordinate into instead of solely the QRS complex. In the horizontal
the aforementioned linear equation. The z-intercepts plane, the experimental contour had a smaller loop
were multiplied by negative one because a positive within the larger loop, as seen in Figure 5C. Also,
Lead II amplitude corresponds to a perpendicular the contour in the horizontal plane of the experi-
line with a negative z-intercept and a negative Lead mental data exhibited a more circular shape instead
II amplitude corresponds to a perpendicular line of a pear-shape in the theoretical horizontal plane
with a positive z-intercept, as seen in Figure 3. This contour. These deviations were likely due to minor
entire process was repeated for all data points within differences in the shape and orientation between the
the QRS complex to form a series of frontal ICVs; see analyzed hearts.
Figure 4B. Using V4 and V6 signals, the developed
software contained code using a similar process to Abnormal ECG Data
form a series of horizontal ICVs; see Figure 4C. Last, The ECG voltage values collected from the PTB
the ICVs from both planes were summed in 3D space database were about two orders of magnitude great-
to produce a series of 3D ICVs. A contour was formed er than the ECG voltage values collected experimen-
by connecting the ends of each subsequent ICV for tally, as seen in Figure 7. The most likely explanation
the frontal and horizontal planes, as well as in 3D for this discrepancy was that the database values
space; see Figure 5. A movie was generated at an were collected at a higher gain, therefore amplifying
increased time span for a better visualization of the the signal more than the experimental values. Along
contour formation. with the diseased states, a control from the database
The shape and orientation of the experimental was also analyzed using the software; see Figure 7B.
3D contour was used to validate the accuracy of the The direction of the largest ICV for the control fol-
software. This was done by comparing the direction lows the same trend as the experimental ICV, which
of the largest ICV, corresponding to the peak of the indicates that both healthy hearts follow the expected
QRS complex, to the expected direction of the mac- tendency for macro-action potential propagation.
BBB is typically marked by a wider QRS complex MI is an interruption of blood flow, usually from
because the travelling time of an impulse from cell to an occlusion of fatty thrombotic plaque in a coronary
cell in a BBB heart is longer than through normal car- artery, resulting in ischemia and necrosis of cardiac
diac myocytes. This explains the wide pear-shaped myocytes. Once the affected myocardium dies, scar
QRS contour of the patient; see Figure 7C. Although tissue forms and it can no longer conduct electric-
not specifically mentioned in the patient information, ity or contract properly.10 As seen in Figure 7D, the
it was further hypothesized that the patient was diag- macro-action potential did not fully propagate down
nosed with left BBB because the QRS contour appears the length of the MI heart, which was illustrated by
to begin in the right ventricle and travels towards the the z-to-y ratios of 0.3, 0.24, and 0.53 for the experi-
left atrium, which is the typical depolarization path mental, control, and MI hearts, respectively. Here,
for left BBB. This supported the hypothesis that the the macro-action potential in the MI heart traveled
direction of the macro-action potential was affected 43.4% and 54.7% less down the y-axis as compared
by BBB. The frontal plane of the left BBB also has a to experimental and control, respectively. This sup-
counter-clockwise rotation, which matches the pa- ports the expected propagation of the macro-action
tient’s contour formation.9 potential for a heart with an anterior MI.
Fall 2010: Volume 9, Issue 2 9
Myocarditis is an inflammation of cardiac muscle Motion artifacts were observed throughout the
due to infectious diseases. After the original onset of experiment, where very small movements led to
myocarditis, the response from the immune system increased vibrational effects in the ECG signal. One
prolongs the disease. It may cause heart failure, heart main source of motion artifacts was noise introduced
rhythm irregularities, or injuries to cardiac myocytes. by electrode shifting and skin deformation during
There is increased blood flow to the inflamed region ECG recording. This caused distortion in the signal
and an elongated QT interval in ECG readings that obtained from the ECG. To collect frontal and hori-
represents abnormal depolarization of the heart11. zontal plane data simultaneously, two additional
There may also be electrical conduction blockages ECG circuits would need to be constructed; the four
at the AV junction in myocarditis12. As a result, the resulting ECG circuits would then be compiled into
biopotentials take longer to propagate through the a master circuit. In theory, this master circuit would
QRS complex. As seen in Figure 7E, the predominant be attached to ECG monitors to output the 3D ICV
propagation occurs in the horizontal plane with little contour along with the real time ECG data. For this
change in the z-direction. This widened contour in result, the software must undergo minor alterations
the horizontal plane is likely due to longer propaga- to allow ECG data to be entered and plotted in real
tion time through the QRS complex, which was also time. This will reduce the error from analyzing a
seen in BBB. Since this patient suffers from both myo- single heartbeat. A final alteration would be to have
carditis and BBB, it is difficult to discern which dis- preset various diseased-states such as BBB, MI, myo-
ease could attribute to the wider contour. carditis, and VHD into the software. This would al-
In VHD, the valve is either stenosed and restricts low doctors to quickly locate and diagnose heart ab-
blood flow or is leaky and allows backflow. In both normalities in patients without the use of expensive
cases, the heart has to work harder to pump more equipment. Last, additional input from cardiologists,
blood to maintain normal output. When comparing physicians, and patients would help improve the de-
the contour of the patient with VHD to the experi- velopment and final software used to image the 3D
mental and control contours, the VHD contour ex- electrical configuration of the heart.
hibits a loop with a wider base. Since electrical ac- The primary goal of producing software that
tivity precedes mechanical contraction in the heart, plots a 3D contour of the heart from four ECG leads,
irregularity in heart contractions reflects abnormality using a series of calculated ICVs, was achieved. The
of electrical stimulation and propagation. As seen in software effectively isolated the QRS complex of the
Figure 7F, the contour does not have a downward ori- heartbeat and performed the necessary calculations
entation as that of the experimental or control, thus for graphic visualization of the biopotential propa-
indicating that the largest ICV was not in solely one gation. The final result would allow cardiologists to
direction. This irregularity could lead to arrhythmia. analyze the heart in an unprecedented way, through
electrical configuration, to diagnose a range of car-
Discussion diac diseases. With further development, this inno-
Several assumptions were made throughout the vative tool has the potential to impact the medical
investigation to compensate for limitations in the industry worldwide.
ECG data. These included that the frontal and hori-
zontal plane data sets were precisely simultaneous, References
the singular analyzed heartbeat was representative of 1. “Deaths and Mortality” Center for Disease Con-
all heartbeats for each patient, and that the spatial ori- trol and Prevention. http://www.cdc.gov/nchs/
entation for each heart was the same. From these gen- fastats/deaths.htm. Accessed 27 April 2010.
eralizations, improvements could be made to model 2. Cardiovascular Medicine: Diagnostics, Drugs,
the heart more precisely. For instance, electrogram and Devices (HLC041B). BCC Research. As re-
methods that digitally synthesize simultaneous ECG ported by http://www.redorbit.com/news/
signals from incomplete lead data are currently be- health/1145566/bcc_research_ market_data_in-
ing investigated.13, 14 This could account for the fron- dicates_cardiovascular_diagnostics_drug_and_
tal and horizontal planes being recorded at different devices/index.html. Accessed 27 April 2010
times. Research to account for the disparities in diag- 3. Yanowitz, F.G. The Standard 12 Lead ECG. Univ.
nosis caused by beat-to-beat variation may also help of Utah School of Medicine. library.med.utah.
to reduce error.15 Limitations of the software include edu/kw/ecg/ecg_outline/Lesson1/index.html
the inability to account for the variability in the size, 4. McDaniel, N. Electrophysiology Testing. Univ.
shape, and orientation among individual hearts. This of Virginia Health System. http://www.health-
reduced the effectiveness of comparing the contours system.virginia.edu/internet/childrens-heart/
of the diseased conditions from the database to the pted/pcrd003.cfm Accessed 24 April 2010.
healthy experimental contours. Therefore, a screen- 5. Dahlin, L.G. et al. Vectorcardiography is superior
ing early in life, where the heart would most likely be to conventional ECG for detection of myocardial
in a healthy state, would be beneficial to identify later injury after coronary surgery. Scandinavian Car-
potential diseased states using the software. diovascular Journal 2001. 35: 2:125-128.
Figure 8. Circuit Schematic Diagram of the original circuit with the five stages. Circuit-1 and circuit-2 were built using this
schematic. The values of resistors and capacitors were measured by a multimeter and are listed in Table 2 and Table 3 for
circuit-1 and circuit-2, respectively. RA, LL, and RL refer to the right arm, left leg, and right leg, respectively.
Table 2. Circuit-1 Resistor and Capacitors Values Table 3. Circuit-2 Resistor and Capacitors Values
The above table lists the resistor values, in kilo- The above table lists the resistor values, in kilo-
ohms (k-ohms), and capacitor values, in nano- ohms (k-ohms), and capacitor values, in nano-
Farads (nF) for circuit-1. Farads (nF) for circuit-2.
Introduction
hindlimb received no injections. Control samples, in MD), SMA+ vasculature was analyzed for diameter
which control PLAGA were applied, were adminis- measurements of large collateral vessels. For inves-
trated as described above. tigation of the angiogenic effects of the FTY-720,
specimens will be bisected about their midlines per-
Laser Doppler Perfusion Measurements5 pendicular to muscle fiber (MF) direction. BS-1 lec-
Animals anesthetized by IP injection animals were tin+ vessel profiles per MF, SMA+ vessel profiles per
placed on a surgical heating pad inside a small cham- MF, metrics that indicate the nature of the vascular
ber that eliminates ambient light. Feet were aligned remodeling response, were determined.
under the scan head of the Lisca PIM laser Doppler
imager in a single plane. The regions of interest were Quantification Metrics and Statistical Analysis8
scanned at a resolution of 256×256 pixels to produce To summarize the purposes of the protocols de-
a color perfusion image. The LDPI software was then scribed previously, there were three main metrics
used to quantify the mean voltage per region, which used to quantify the arteriogenic and angiogenic ef-
in turn was used to calculate relative perfusion. fects of FTY-720. The ischemic:contralateral perfusion
ratio was used to determine blood perfusion recov-
Muscle Preparation and Harvest5 ery, the diameter of the major collateral vessel im-
Gracilis muscles for excision and cross-sectioning aged from the harvested and excised gracilis muscle
were superfused with Ringer’s solution containing was used to examine vascular remodeling, and the
10-4 M adenosine for 30min. The abdominal aorta SMA+ and lectin+ vessel profiles per muscle fiber
was retrograde cannulated following euthanization. were used to investigate arteriogenesis and angio-
Following blood removal with a PBS (pH=7.4) per- genesis. The LDPI was analyzed using a two-way
fusion, muscles were perfusion-fixed with 4% para- variance analysis, while changes in arteriolar collat-
formaldehyde in PBS (4˚C) for 30min at 100 mmHg. eral diameter and SMA+ and lectin+ vessel profiles
Specimens for cryosectioning were excised. per muscle fiber were analyzed by two-way repeated
measures ANOVA with Holm-Sidak multiple pair-
Whole Muscle Processing and Analysis5 wise comparison. Statistical testing was performed in
Whole-mount gracilis muscles were treated with SigmaStat 2.0 using p<0.05 to evaluate significance.
type I collagenase (3 mg/ml, Sigma, St. Louis, MO)
in PBS for 30 minutes to digest the type I collagen Results
in the connective tissue between muscle fibers. After Characterization of FTY-720 Nanoparticles
being washed in PBS for 10 minutes, specimens were To characterize the size and structure of the PL-
placed in a 1:200 Cy3-conjugated monoclonal anti- AGA nanoparticles loaded with FTY-720, scanning
smooth muscle alpha-actin (anti-SMA) (clone 1A4, electron microscopy was performed (FIG. 1). Analy-
Sigma, St. Louis, MO) solution of 0.1% saponin and sis shows that the nanoparticles applied in this exper-
2% bovine serum albumin (BSA) in PBS at 4°C for iment were spherical in shape and structure. Using a
3 nights. The tissues were washed in 0.1% saponin submicron particle analyzer, the number-weight dis-
in PBS once for 60 minutes and in PBS twice for 30 tribution of average nanoparticle diameter was found
minutes on a shaker plate. Samples were stored in a to be 207nm. Separate analysis in ImageJ software
50/50 mixture of PBS and glycerol until mounted on where individual nanoparticles were measured, the
microscopic slides for confocal microscopy. Whole average diameter was 115nm. While variance exists
mounted muscles were observed with a ×4 objective between these values, they nonetheless characterize
on a Nikon TE-300 inverted microscope (Nikon, Mel- the spherical structure and order of magnitude of
ville, NY). Using ImageJ software (NIH, Bethesda, size of the FTY-720 nanoparticles.
made at the onset of the study and the significant the natural remodeling response. In ischemic tissues,
increase in perfusion at days 14 and 18 was not ex- there is an increase in the pressure gradient within the
pected. A potential explanation for this sudden in- vasculature as a result of an occlusion15. The resulting
crease could relate to the release of FTY-720 from the change in the blood flow profile and increased shear
PLAGA nanoparticles in vivo. It is known that the re- experienced at the fluid-vessel interface activates the
lease of a material, such as a synthetic material like growth-factor receptors in the ischemic tissue. The
FTY-720, is a function of its degradation and erosion subsequent increased interactions between growth
profiles as well as the hydrophilicity of the mate- factors and their upregulated receptors activate sig-
rial11. Furthermore, as PLAGA is characterized as a naling transduction pathways associated with cell
bulk-eroding copolymer, the release of a hydrophilic growth, proliferation, and differentiation16. Each of
material like FTY-720 would not be marked by a con- these cell-fate responses is characteristic of new ves-
stant release profile5,12. As such, the observed increase sel development and growth.
in perfusion at days 14 and 18 might be a result of a In summary, while the data obtained in this ex-
bulk-release profile of FTY-720 from the PLAGA co- periment did not successfully confirm the hypoth-
polymer13. Further repetitions of this experiment are eses regarding the therapeutic ability of FTY-720 via
required to verify at what time point this influx of vasculature remodeling, the desired trend increases
FTY-720 occurs. in the collected data were observed.
The observed average diameter of the collateral
main feeder vessels in ischemic hindlimbs increased Limitations
significantly relative to the non-ischemic controls The difficulty in acquiring significant data can be
with the FTY-720 treatment, a result consistent with attributed to limitations in the design of the experi-
the aforementioned conclusions from LDPI analy- ment and analysis techniques. Regarding the former,
sis. As perfusion measurements revolve about the the sample sizes of treatment groups was initially
ligated:contralateral average perfusion ratio, it is log- n=8. Four mice in the PLAGA and FTY-720 treatment
ical that the observed increase in blood flow recovery groups were euthanized at various times during the
might result from an increase in the diameter of this study as a result of poor recovery from the initial
vessel14. hindlimb ligation surgery. The resulting relatively
Characterization of the arteriogenesis and angio- small sample size (n=4) introduced a significant
genesis induced by FTY-720 resulted in increased av- amount of variance and deviation during statistical
erage diameter of the collateral main feeder vessels testing. Furthermore, the timeline for this experiment
as well as SMA+ and lectin+ vessels per MF. While was such that only one trial was completed to evalu-
these trend increases exist, it is difficult to validate ate the therapy of the FTY-720 treatment.
initial hypotheses regarding the efficacy or efficiency In regard to error in analysis techniques, it was
of FTY-720 as a therapeutic agent for vascular remod- difficult to measure significant increases in perfusion
eling as statistical analysis of the data showed there recovery due to significant noise in collected LDPI
was no significant change as a result of treatment. data. This also led to large values of variance and
These results imply that the vessel development in- standard deviation in statistical analysis testing. Ad-
duced by FTY-720 is not significantly greater than ditionally, it was difficult to compare the results from
the LDPI perfusion analysis and the characterizations Future Research & Refinements
of vasculature remodeling. LDPI data is such that In addition to further experimental trials, there
blood flow throughout the entire hindlimb is quan- are several components of this experiment that merit
tified17. Measurements of average collateral main further investigation. One of the most compelling of
feeder vessel diameter as well as SMA+ and lectin+ these is characterizing the release of FTY-720 from
vessel profiles per MF, however, are metrics localized the PLAGA copolymer. By quantifying the kinetics
to the targeted area of the gracilis muscle. As such, if of the drug delivery to the ischemic hindlimb, FTY-
further trials of this experiment yielded statistically 720 concentration, nanoparticle fabrication and load-
significant results, it would become challenging to ing, and methods of drug delivery become additional
draw consistent conclusions from these tests. variables for optimization in further experiments.
Future plans include developing a mathematical and
computational time-dependent model of the release
profile of FTY-720 to surrounding tissue.
Erin Dale
This study measured the effect of stomatal structure on gas-exchange processes in three differ-
ent ozone (O3) concentrations (20ppb, 80ppb, 160ppb) and compared the stomata morphologies
across five native and eight invasive tree species from the mid-Atlantic region. Two leaves from
103 trees were measured under 400x magnification after preparation and staining with safra-
nin O dye. Significant variations in stomatal complex size, guard cell width, number of contact
cells, and stomatal density were measured across species. The stomatal complex size, guard cell
width, guard cell length, and number of contact cells were all positively correlated to one anoth-
er. Novel correlations between stomatal type and guard cell width, number of contact cells, and
stomatal complex size were discovered. No significant morphological differences were found
between native and invasive species groups. Stomatal characteristics were found to correlate to
ozone uptake, cumulative ozone uptake, and net photosynthesis, but not stomatal conductance.
Cumulative ozone uptake at the medium ozone treatment departed from other trends and sug-
gests protective mechanisms in trees to avoid ozone uptake. At the high ozone treatment, the
traits that contributed to net photosynthesis changed from stomatal density to guard cell width
and stomatal complex size. This switch could be means for a new competitive advantage in
environments where ozone levels are growing. Individual tree species also exhibited variation
in physical stomatal characteristics and their strength of influence on gas-exchange processes.
Introduction
Another component of the atmosphere that af- (Patterson et al. 2000). This air polluting gas hinders
fects plants due to diffusion through the stomata plant maturation, decreases tree growth by ten per-
is ground-level O3 gas. While a beneficial layer of cent (Patterson et al. 2000), and lowers water-use ef-
stratospheric O3 protects the Earth from ultraviolet ficiency (Krupa et al. 2000). Through model predic-
rays from the Sun, the O3 in the troposphere acts a tions, O3 will also decrease net primary production by
pollutant and greenhouse gas. Ozone is derived from 16% at current ambient levels (Patterson et al. 2000).
nitrogen oxides and volatile hydrocarbons, both of However, most models ignore non-stomatal conduc-
which humans contribute to through automotive tances and recent data depicts non-stomatal conduc-
emissions (Krupa et al. 2000). In the Eastern part of tances should be incorporated into calculations be-
the United States, the ambient levels of O3 range from cause it can account for up to 63% of total O3 flux of a
30ppb to 50ppb, but in some areas reach over 100ppb given area (Hogg et al. 2007). Nevertheless, stomatal
the summers of 2008 and 2009. The leaves were ex- Statistical Analysis
posed to three O3 treatments (20ppb, 80ppb, 160ppb) All data collected were analyzed using SAS 9.1
for eight hours in cuvettes. Measurements on gas- (version 9.1.3; SAS Institute, Cary, NC, USA). Repeat-
exchange processes included O3 uptake per second, ed measure mixed model ANOVAs were run for the
cumulative O3 uptake over the course of one day dependent variables: the number of contact cells, size
(COU), net photosynthesis (An), and stomatal con- of the stomatal complex, guard cell length, guard cell
ductance (gs). width, and stomatal density. For all of these analyses,
tact cells, 15.255 µm stomatal complex size, and 2.971 morphologies, as A. altissima had a mean of 3.95 µm
µm guard cell width. All types had no significant dif- and C. occidentalis had a mean of 1.97 µm (X2=2.22,
ferences between guard cell length (Figs. 3, 4, 5, Table p=0.0066) (Fig. 8, Table 5).
6). When comparing native and invasive species,
The native species consisted of five laterocytic however, there was no significant variation between
types and three stephanocytic types. The invasive the two groups in regards to stomatal complex size
species included one anomocytic, one laterocytic, (F1, 11= 0.23, p= 0.6394), guard cell length (F1, 11=0.87,
and three stephanocytic stomatal types. p=0.3699), and guard cell width (F1, 11=1.38, p=0.2656)
(Fig. 11).
Stomata density
The stomatal density varied across species rang- Contact cells
ing from 36.38 stomata in one reference frame For the number of contact cells surrounding the
(.1662mm2) of P. calleryana to 103.33 stomata for Q. guard cells, there were significant differences across
alba (X2=2.31, p=0.0103) (Fig. 6, Table 5). In native and species between a range of 5.372 µm in C. glabra and
invasive species however, there was no significant 3.964 µm in L. tulipifera (X2=1.79, p=0.0366) (Fig. 10,
density difference, as invasive species had an aver- Table 5). There were no significant differences be-
age of 61.9 and native species had an average of 61.4 tween invasive and native species.
(F1, 12=0.32, p=0.5829) (Fig. 11).
Correlations between traits
Stomatal complex size, length, and width Stomatal complex size, length, width, and num-
Stomatal complex size also exhibited significant ber of contact cells were all significantly positively
differences across species. Ailanthus altissima had an correlated to each other (Table 2). Guard cell length
average stomatal complex size of 20.9 µm and C. occi- and width were most strongly correlated (r=0.76031,
dentalis of 11.8 µm (Fig. 7, Table 7) (X2=2.13, p=0.0088). p=<0.0001) while guard cell length and contact cell
Variation existed across tree species in regards to number were the weakest positively correlated traits
guard cell length with a range between A. altissima of (r=0.16162, p=0.006). Stomatal density exhibited nega-
11.3 µm and A. platanoides average of 6.4 µm, but was tive correlations to stomatal complex size (r=-0.29541,
not significant (X2=2.26, p=0.1020) (Fig. 9, Table 7). p=0.0427), guard cell width (r=-0.38874, p=<0.0001),
Guard cell width displayed distinctions in stomatal and guard cell length (r=-0.38652, p=<0.0001). All the
Figure 4. Relationship between stomatal type and stomatal complex size (F2,252=21.3, p=<0.0001) L=laterocytic
S=stephanocytic P= paracytic
individual tree species also followed these trends, Stomatal morphology and gas-exchange
except for L. tulipifera which demonstrated a nega- Net photosynthesis and stomatal morphology
tive correlation between contact cells and guard cell Of the traits measured, multiple morphologi-
length (r =-0.46649, p=0.0216). cal characteristics were correlated to gas-exchange
Traits in invasive species were also all positively processes including net photosynthesis (An). None
correlated. Native species were significantly positive- of the characteristics were positively correlated to
ly correlated with the exception of the number of con- An. Stomatal density negatively influenced the to-
tact cells and guard cell length (r=0.0732, p=0.3118). tal An (R2=0.2374, F= 27.4, p= <0.0001) (Tables 3, 4).
The traits that correlated to An varied with differ-
ent ozone treatments (Table 3). In the low treatment,
both the increasing percentage of radial cells (partial fects emerged (Table 3). The high and the medium
R2=0.1849) and density (partial R2=0.1058) resulted in treatments of ozone were not significantly correlated
a decreased An (p= 0.0115, F= 5.33). At the medium to stomatal traits. At the low ozone treatment, which
treatment, guard cell width strongly and positively most resembled a normal functioning leaf, the nega-
(partial R2= 0.6059) and stomatal density slightly tive correlation to specialized cells was still present
and negatively (partial R2= 0.0532) contributed to An and slightly stronger than the total ozone uptake per
(F= 27.06, p=<0.0001). At the high treatment, An was second (partial R2=0.1059, F= 6.43, p= 0.0174).
influenced positively by guard cell width (partial Ozone uptake of five species was directly impact-
R2=0.1213) and negatively by stomatal complex size ed by stomatal attributes including: A. rubrum (de-
(partial R2=0.1778) (F=5.76, p=0.0082). creasing stomatal complex size, partial R2=0.06988,
In one of the species, C. occidentalis, there was a F= 16.24, p= 0.005), C. occidentalis (increasing guard
significant negative correlation between stomatal cell length, partial R2=0.982; decreasing width, partial
complex size (partial R2=0.3097), density (partial R2=0.0162; decreasing radial cells, partial R2=0.0018;
R2=0.6385), and the presence of polar cells (partial F= 20482, p=0.005), P. serotina (decreasing stomatal
R2=0.0514) (F=1015.55, p= 0.023) (Table 7). Stomatal complex size, partial R2=0.5818; increasing guard cell
morphology of native and invasive species affected width, partial R2=0.0374; decreasing radial cells, par-
An through different traits (Table 8). Invasive species tial R2=0.1442; decreasing density, partial R2=0.2176;
were impacted positively by the increasing length F= 51.29 p =0.001), Q. acutissima (decreasing radial
of guard cells (partial R2=0.2414, F=9.23, p=0.005) cells, partial R2=0.4875; decreasing stomatal complex
and native species by radial cells (partial R2=0.3123) size, partial R2=0.1656; F= 5.65, p= 0.042), and R. pseu-
and stomatal density (partial R2=0.0627) (p=<0.0001, doacacia (decreasing radial cells, partial R2=0.1838;
F=16.8). decreasing number of contact cells, partial R2=0.5726;
F= 9.32, p= 0.015) (Table 7). There was no significant
Ozone uptake and stomatal morphology effect in either the native or invasive species groups
Ozone uptake was positively correlated to guard (Table 8).
cell width (partial R2=0.0235), but negatively related
to the presence of specialized cells (partial R2=0.0271) Stomatal conductance and stomatal morphology
and guard cell length (partial R2=0.226) (F=2.77, Overall, total stomatal conductance (gs) values
p=0.0465) (Tables 3, 4). However, when the ozone could not be explained by any stomatal attributes.
data was separated into the high, low, and medium Also, there were no significant contributions at the
treatments that were assigned to the trees, other ef- high, low, and medium ozone treatments.
Three species, however exhibited significant sto- The COU of A. rubrum, C. occidentalis, P. serotina,
matal trait correlation (Table 7). Guard cell length Q. acutissima, and R. pseudoacacia are affected by sto-
positively (partial R2=0.3841), width negatively (par- matal morphology (Table 7). A. rubrum was negative-
tial R2=0.1098), and radial cells positively (partial ly correlated to guard cell length (partial R2=0.7382,
R2=0.343) influenced A. rubrum (F= 8.55, p=0.021). F= 19.74, p=0.003). C. occidentalis was positively cor-
Stomatal complex size negatively (partial R2=0.0721), related to guard cell length (partial R2=0.9694) and
guard cell length negatively (partial R2=0.3797), ra- negatively to stomatal complex size (partial R2=0.025)
dial cells positively (partial R2=0.4305), contact cells (F= 177.03, p=0.006). P. serotina was negatively cor-
positively (partial R2=0.0937), and specialized cells related to specialized cells (partial R2=0.1179), radial
negatively (partial R2=0.0158) influenced P. serotina cells (partial R2=0.2483), density (partial R2=0.0239),
(F= 73.26, p=0.003). The number of contact cells nega- and stomatal complex size (partial R2=0.5378), and
tively (partial R2=0.5745) influenced Q. rubra signifi- positively correlated to guard cell length (partial
cantly (F= 6.75, p=0.048). There were no significant R2=0.056) (F= 36.39, p=0.0070). Q. acutissima was
effects on native and invasive species (Table 8). negatively correlated to stomatal complex size (par-
tial R2=0.5117, F= 7.33, p=0.0300). R. pseudoacacia
Cumulative ozone uptake and stomatal was negatively correlated to contact cells (partial
morphology R2=0.5622) and radial cells (partial R2=0.1910) (F=
The presence of specialized cells changes in- 9.15, p=0.0150).
versely to the COU across all three ozone treatments In native species, there was no significant correla-
collectively (F=4.46, p=0.0143), as well as in the low tion between COU and stomatal traits, but there was
and high treatments (Table 3). The medium treat- an inverse relationship between COU and stoma-
ment departs from this trend (Table 4). The medium tal complex size (partial R2=0.1218) and specialized
treatment regression has a negative slope suggesting cells (partial R2=0.1471) in invasive species (F= 5.15,
that at the medium ozone level, the stomata are not p=0.013) (Table 8).
uptaking an increasing amount of ozone as they do
at both the low and high treatments. This difference Discussion
could be driven by the negative correlation of the Stomatal morphology among tree species
number of contact cells (partial R2=0.0933) and posi- The results of this study identified and compared
tive correlation to the presence of polar cells (partial the variation of morphological stomatal characteris-
R2=0.0871) (F=3.08, p=0.0871). tics of 13 mid-Atlantic trees across species. By gath-
Figure 10. Comparison across species of number of contact cells (X2=1.79, p=0.0366)
49
Table 8. Stepwise multiple regression data: the influence of stomatal traits on native and invasive trees
COU= cumulative ozone uptake An= net photosynthesis gs= stomatal conductance
ering data on a large number of species, this study the larger the guard cells, the larger the stomatal
can categorize potential similarities and differences complex. This suggests that the correlation between
within a community to predict possible areas of com- guard cells and stomatal complex may be a morpho-
petitive advantage. logical response to the physical demand of stoma
size. This positively proportional trend was appar-
Stomatal type ent in all species, except L. tulipifera, between guard
As a common taxonomic differentiation (Ziegler cell length and number of contact cells (r= -.46649,
1987, Baranova 1983), the morphological stomatal p=0.0216). The number of subsidiary cells in Lirioden-
type was classified and retained within genus. For dron tulipifera oscillated often between two and four
example, A. rubrum and A. platanoides both had lat- contact cells. This trend implies that larger guard cell
erocytic stomata types, while all three Quercus spp. length correspond to two contact cells (paracytic sto-
possessed stephanocytic stomata types (Table 1). matal type), while smaller stoma lengths correspond
Genetic controls account for many of the stomatal at- to four subsidiary cells (laterocytic stomatal type).
tributes measured in this study. Therefore, including
closely related native and invasive species led to no Stomatal morphology and gas-exchange
significant difference between these species groups. This study also focused on the influence of physi-
Stomatal type is defined by the arrangement of cal stomatal traits on gas-exchange across species in
subsidiary cells around the guard cells. But, each order to help predict variation in gas-exchange. As
type also showed a significant correlation to other gas-exchange can differ across species (Novak et.al.
morphological traits, not included within classifica- 2005), in turn, the method in which species respond
tion criteria. The variation in mean number of contact to pollutants such as ozone also may vary. While the
cells, stomatal complex size, and guard cell width ex- effects of ozone (Dizengremel et al. 2008, Booker et
pressed significant relationships in these species. No al. 2009, Pfleeger et al. 2010), as well as the effects of
previous study has linked any stomatal trait other stomatal traits on gas-exchange (Russo 2010), have
than the number and orientation of subsidiary cells been studied, this is the first study to link the role
to stomatal type (Carpenter 2005, Baranova 1983). of morphological traits to the effect of ozone on gas-
exchange processes across multiple tree species.
Stomatal density
Native and invasive species did not differ in Net photosynthesis and stomatal morphology
stomatal density counts, but did display significant A measure of An and its possible correlation to
variation across species. Stomatal density was found stomatal traits were analyzed to help explain stoma-
to negatively correlate to guard cell length. This re- tal modulation of gas-exchange. The leaves that the
lationship has also been published in past studies stomatal measurements were measured from were
(Ohsumi et al. 2007, Kundu and Tigerstedt 1998), but different from the leaves in which the gas-exchange
the negative relationships between stomatal complex measurements were collected, although they were
size and guard cell width have not been previously from the same tree. A positive correlation between
studied. Larger stoma on a leaf correlate to lower An and stomatal density was observed in Azadi-
stomatal density, which was evident across species rachta indica (Kundu and Tigerstedt 1998), but very
such as A. altissima (stomatal density= 42.3, stomatal little research has been completed on direct corre-
complex size= 20.94 µm, guard cell width= 3.95 µm, lations between An and stomatal characteristics of
guard cell length= 11.34 µm). mid-Atlantic trees. This study did find a direct influ-
ence of stomatal density on An, but contradicts other
Stomatal complex size, length, and width studies on trees from other areas of the world. This
Other morphological traits including stomatal study found an inverse relationship between An and
complex size, guard cell length, and guard cell width stomatal density at overall, low, and medium ozone
exhibited significant correlations. The positive re- treatments (Table 4). This result indicates that the
lationship between all of these traits indicates that stomata on leaves with a lower stomatal density are
more efficiently assimilating CO2 than stomata on Also, guard cell length and width were the strongest
leaves with higher stomatal densities. Density in- positively correlated traits (Table 2). A smaller width
fluenced An at the low and medium treatments, but was hypothesized to most resemble a dumbbell
not at the high treatment. This may illustrate that at shaped guard cell, which is known to more efficiently
extremely high concentrations of ozone, normal sto- and quickly respond to light and increased opening
matal morphological influences no longer play a role (Hetherington and Woodward 2003). However, the
in net photosynthesis. This follows the hypothesis dumbbell shaped stomata seen in the Quercus spp.
that at ozone levels above 150ppb, stomatal regula- were closer to the average guard cell width across
tion is no longer under the control of the plant due to species. The departure of C. occidentalis and Quercus
injury (Elton). The switch from An being influenced spp. may be due to the fact that these traits have not
by stomatal density and the percentage of radial cells been well studied in past literature. Also, this study
at low treatments to stomatal density and guard cell does not address all possible influences on stomatal
width at medium treatments to stomatal complex regulation.
size and guard cell width could provide an avenue
for a change in competitive advantage. The An of trees Stomatal conductance and stomatal morphology
growing in higher ozone environments will be influ- Stomatal condutance, though a measure of water
enced by different stomatal characteristics than those flux, estimates the passage of gas through a leaf. Little
in low ozone environments. With different stomatal variation or influence of stomatal attributes appeared
contributors, different trees may succeed in these two across species. These findings support other studies
atmospheric environments on account of stomatal that have discovered little correlation to g and physi-
morphology. cal stomatal traits (Ohsumi 1998, Russo et.al. 2010).
The lack of correlation may be explained because
Ozone uptake and stomatal morphology other environmental factors such as light availability
It was also hypothesized that certain morphologi- and air humidity strongly influence stomatal conduc-
cal traits would be more highly correlated with O3 tance.
uptake and in combination these traits would explain
more of the variance in O3 uptake than they would Cumulative ozone uptake and stomatal morphology
alone (Patterson et al. 2000). This variation in number The presence of specialized cells strongly influ-
and type of traits was apparent across species (Table ences overall COU, but when separated by individu-
7). It was predicted that the stomata with the longest al tree speices, only Q. acutissima and P. serotina were
guard cell length and shortest width would have the influenced by specialized cells. A slightly significant
most O3 uptake. This was thought to be because the negative relationship appeared at the medium ozone
larger the aperture, the more stomatal conductance, treatment. At the medium ozone treatment, the
leading to an increase in O3 uptake (Patterson et al. leaves responded to ozone differently than at the oth-
2000; Sack 1987). The data presented in this study did er treatment levels. This departure from the overall
not corroborate this idea. Both A. altissima and P. cal- trend suggests that certain species possess ozone pro-
leryana possessed the highest mean guard cell lengths tective mechanisms and that these mechanisms are
(Fig. 9, Table 5), but ozone uptake was not influenced influenced by different traits than normally function-
by stomatal traits in either species (Table 7). Celtis oc- ing stomata at the high and low ozone treatments.
cidentalis possessed stomata with the shortest guard The stomata have been proposed to resist ozone by
cell,but, they were most prominently influenced by closing faster at these specific levels (Unsworth and
length in regards to ozone uptake (partial R2= 0.982). Black 1981; Krupa et.al. 2000).
Therefore, this species contradicts the expected trend.