This document describes a study using spectral-domain optical coherence tomography (SD-OCT) to examine long-term cerebrovascular changes in response to voluntary exercise in rats. 14 rats were assigned to either an exercise or control group. SD-OCT scanning was performed under anesthesia to image the motor cortex at high resolution before and after inducing hypoxia, allowing non-invasive longitudinal assessment. Histology will also be performed and compared to SD-OCT. The results will help explore exercise-induced cerebrovascular plasticity and validate SD-OCT for mapping vascular changes over time.

1. Methods
Pre-Imaging
14 rats were pseudo-randomly assigned to either an inactive control or voluntarily
exercise group. Animals are housed individually, on a 12-hour light/dark schedule, with no
environmental stimuli that could influence brain plasticity. Exercising animals have access
to a running wheel with a revolution counter.
Surgery
In preparation for SD-OCT analysis, animals are anesthetized using 1.5 - 4% v/v
isoflurane in a gas mixture of 80% air and 20% oxygen; temperature is monitored, and
maintained by a heating blanket. Following anaesthetization, the head of each rat is fixed
in a stereotaxic apparatus. Next the head is shaved, and iodine applied as antiseptic.
Then the scalp of the animal is surgically retracted. A craniotomy is performed over a
small, defined area of motor cortex, and the objective lens of the SD-OCT scanner is
aligned over the region of interest.
Scanning
The scanning procedure lasts from 10-12 minutes. The scanner does not touch the tissue;
rather, it scans the field of view with 3mW of near-infrared light with a wavelength of
~1300nm, consistent with previous published manuscripts. Cross sections within a 2mm x
2mm scanning field are imaged 10 times before moving to the next cross-sectional
position. The recorded images are then stacked to create 2 and 3-dimensional
angiograms via algorithms on offsite computers. Upon completing the first imaging
sequence, a hypoxic condition (10%) is supplied, and the imaging procedures are
repeated to assess vascular response to hypoxia.
Histology
In order to compare the efficacy of SD-OCT to traditional methods, immunohistochemistry
will be performed on pre-scanned brains, using CD31/34 antibody. A two-way analysis of
variance (ANOVA) will be conducted to analyze changes in blood vessel density relative
to the two methods of data collection. Furthermore, standard t-tests will be conducted to
analyze differences in the mean percent change of blood vessel dilation for data collected
using SD-OCT. Additional analyses will include correlations between running behavior and
measures of capillary density and flexibility.
Introduction
Vascular pathologies are the leading cause of fatality worldwide, killing 17.5 million
people in 2012 and accounting for 3 in every 10 deaths. Recent evidence has
demonstrated that exercise is a powerful activator of compensatory mechanisms for
the lack of oxygen and glucose supply inherent in many neurovascular disorders (such as
stroke, Alzheimer’s, and ALS). Traditional methods of histological analysis have enabled
researchers to successfully examine changes in cerebrovascular architecture in response
to exercise; however, these techniques typically require the sacrifice of the animal, which
inhibits longitudinal data collection and prevents translation to human medicine. More
advanced techniques, such as fMRI, have been useful in investigating long-term
changes in brain anatomy and function, but are prohibitive in terms of cost and image
resolution.
The present study is an ongoing collaboration between the Swain Neurobiology Lab and
Dr. Ramin Pashaie’s BIST lab (Bio-Inspired Sciences and Technologies). Here, we
introduce spectral-domain optical coherence tomography (SD-OCT) as a means to fill the
respective gaps in both traditional analyses and established brain imaging techniques.
SD-OCT produces high-resolution, 2-D and 3-D angiograms, and allows for non-
invasive in vivo imaging within the same animal at multiple time points. This enables us to
map long-term cerebrovascular growth, in addition to real-time changes in blood vessel
dilation and blood velocity.
The primary goal of our current study is to examine long-term cerebrovascular
changes in the adult rat in response to voluntary exercise, as the effect of exercise
training on cerebrovascular structure and function has not been fully explored. Using
SD-OCT to analyze blood vessel density between exercised and non-exercised animals
has allowed us to investigate the cerebrovascular system and the effects of exercise with
micrometer precision. Utilizing SD-OCT to examine changes in blood vessel diameter
and dilation between exercised and non-exercised animals will allow us to more
precisely explore exercise-induced cerebrovascular plasticity, while using statistical
methods to compare the accuracy of this new platform to more traditional histological
techniques.
Results from Pilot
Fig. 1.
1A – SD-OCT axial scans, arranged to represent stacking of images. Images show sufficient
axial resolution with minimal noise. Displayed in LabView GUI environment. BIST lab.
1B – 2-D maximum intensity projection of the 3D angiogram generated through computational
synthesis of SD-OCT scans. Vasculature is clear, in focus, and displays high resolution. BIST
lab.
1C-D – Confirmation via light microscopy revealed the craniotomies of animals TC01 and
TC02 were localized correctly over the intended region of M1 (motor cortex). Associated page
from Paxino’s Rat Brain in Stereotaxic Coordinates (4th ed.) shown for comparison. Analysis
revealed high accuracy at the surgical site, and absence of significant damage to cortex from
the procedure. Swain lab.
Cerebrovascular Plasticity
Assessed by Spectral-Domain Optical Coherence Tomography
UWM Neuroscience Department
UNDERGRADUATE: Alexander T. Wickstrom
MENTOR: Dr. Rodney Swain
Data from Previous Study
Acknowledgements
The SD-OCT scanner was built and provided by the
BIST Lab. I am grateful for all of the hard work and
brain power of Farid Atry, and indebted to Ramin
Pashaie for allowing the use of the scanner. Also to Erin
Adams (UWM) for providing surgical insight.
Brief Overview of SD-OCT
OCT was invented in the early 1990s, and has recently seen an explosion of development
and applications in the fields of biomedical engineering and medicine. It is analogous to
ultrasonography in that its axial resolution is derived from the arrival time of “echoes,” yet the
detection in OCT is based on the principle of interferometry, and thus utilizes a Michelson
interferometer. The use of light waves compared to sound results in an approximately 10- to
100- fold increase in resolution. In organs as subtle and complex as brains, this level of
precision is indispensible in both the laboratory and the clinic..
SD-OCT Schematic Interferogram Transform
Conclusion
The pilot results gained from our current study, combined with the previous data published
by the BIST lab, allow us to confidently proceed with the final stages of our project. The
high-resolution, reproducibility, and efficiency of SD-OCT makes it an ideal research tool
for non-invasive imaging of living tissue. By finishing our current experiments, we aim to
promote and further validate the use of SD-OCT as an attractive tool in both laboratory
and clinical settings, well-suited to provide new insights into neurological diseases
involving the cerebrovascular system.
1A 1B
1C 1D
Fig. 2.
Dilation of cerebral blood vessels during a previous experiment (Atry, et al., 2015).
Top Left: (a) Bright field image taken from the charge-coupled device camera utilized
in our present study. Coordinates labeled for reference. (b) Maximum-intensity
angiogram generated via SD-OCT. The red color indicates areas of increased dilation
following optogenetic stimulation, with intensity of color proportional to degree of
change. A similar trend has been observed in our pilot study. (c) and (d) show a closer
view of the region marked by the yellow square in (b), before and after stimulation.
Top Right: Hemodynamic response of transgenic mice (ChR2) compared with
controls. (b) shows fractional change in flow and velocity before and after stimulation.
(c) displays a quantitative comparison of fractional change in the vessel diameter after
stimulation. Bottom: Fluctuation in blood flow and blood velocity following optical
stimulation in (a) transgenic and (b) control animals.
References