This document describes an experimental study that used image reconstruction software to compare the ventricular systems of normal rabbits and rabbits with induced hydrocephalus. MRI and digital subtraction angiography were used to obtain images of the rabbits' brains and cerebrospinal fluid spaces. The images were then processed using the software applications ImageJ and MIMICS. ImageJ was used to subtract angiography images before and after contrast injection to highlight the subarachnoid spaces. MIMICS was used to generate 3D reconstructions of the subarachnoid spaces from the images. The goal was to quantify volume changes in the ventricular system between normal and hydrocephalic models to help analyze hydrocephalus. The study found challenges in acquiring high

1. Image Reconstruction of Hydrocephalic Rabbit Ventricular System
Final 2013 RET Report
Prepared by
Zenaida Almodovar
William Penn Nixon Elementary School
Chicago, Illinois
August 14, 2013
Laboratory for Product for Process Design
Department of Bioengineering, University of Illinois at Chicago
Director: Professor Andreas Linninger
National Science Foundation - Grant EEC # 1132694
_____________________________ ___________________________
Zenaida Almodovar Dr. Andreas A. Linninger
RET Fellow RET Director

2. 2
Abstract
Hydrocephalus is the accumulation of cerebrospinal fluid (CSF) in the ventricles of the brain. The
cerebrospinal fluid is generated in four central cavities at the base of the brain. It allows the
relatively heavy brain to float within the skull, cushion the brain to prevent injury, regulates the
composition of the fluid bathing the neurons and glial cells of the central nervous system (CNS)
and provides a route through which certain chemical messengers can be widely distributed in the
nervous system. It flows through the ventricles by way of interconnecting channels and eventually
flows into subarachnoid spaces (SAS) bathing the brain and spinal cord. The shape and volume of
the ventricles can be connected with clinical hydrocephalus illness. The aim of this study is to
compare the cerebrospinal fluid filled spaces of the normal rabbit and hydrocephalus model to
compare the volume changes using image reconstruction software applications. This study used
four (4) healthy 4 month old New Zealand white rabbits, weighing 3 kg. Sterile kaolin (aluminum
silicate) suspension (0.03 ml) was injected into the cistern magna using a 25 gauge butterfly needle
to induce hydrocephalus. Several angiogram x-ray images were taken using iohexol contrast agent
(0.2 - 0.5 ml) to resolve the SAS. The animals with cisternal kaolin injection did not develop any
degree of hydrocephalus. This study employed both manual and automated segmentation methods,
in vivo and ex vivo to perform 3-D reconstruction of the ventricular system. Two diagnostic
imaging modalities (angiography and MRI) provide medical image data for the reconstruction of
the ventricular structures. The goal of the study is to reveal the normal and hydrocephalus SAS
using these software applications and use these findings to improve the study and treatment of
hydrocephalus. In this study, I have described the experimental design on how to perform MRI on
a rabbit model, how to process the image reconstruction and the general methods on how to acquire
quality images. Imaging modalities, 3-D angiography and MRI, in conjunction with image
reconstruction contribute to the analysis of hydrocephalus study. There are challenges still to be
tackled for the tissue preparation and image acquisition of small animal ex-vivo MRI. ImageJ and
MIMICS are powerful image processing tools which can be used to analyze and understanding
how the brain responds to a hydrocephalic state.

3. 3
Table of Contents
Abstract............................................................................................................................................2
Introduction......................................................................................................................................4
Purpose.............................................................................................................................................4
Ventricles and Cerebrospinal Fluid..................................................................................................5
Disruption of CSF Circulation Can Cause Hydrocephalus ...............................................................6
Measurement of Ventricular Volume................................................................................................7
Image J.............................................................................................................................................7
Digital Subtraction Angiography......................................................................................................8
Magnetic Resonance Imaging (MRI) ................................................................................................9
Image Reconstruction.....................................................................................................................11
Materials & Methods......................................................................................................................13
Hydrocephalus Administration.......................................................................................................13
Data Acquisition.............................................................................................................................14
Ex-Vivo Preparation........................................................................................................................14
Ex-vivo MRI...................................................................................................................................17
Tissue FixationMethod...................................................................................................................17
Results............................................................................................................................................17
MIMICS on Rat’s SAS...................................................................................................................17
Conclusion......................................................................................................................................21
Discussion and Future Works.........................................................................................................22
Acknowledgements .........................................................................................................................22
References.......................................................................................................................................23
Appendix I: Synopsis of Author’s Notes..........................................................................................25

4. 4
Introduction
Purpose
The purpose of this study is to compare angiography images from a healthy rabbit ventricular
system and the experimental kaolin model for hydrocephalus in order to assess ventricular
enlargement. My research goal is to use two software applications to conduct the volume analysis
for a normal rabbit and a hydrocephalus model. The research project’s primary aim is to use the
volume measurement from 3D image reconstruction for quantification of a hydrocephalus
symptom.
My aim is to subtract two rabbit images that are obtained before and after contrast media is
administered using image processing software, ImageJ. Digital Subtraction Angiography is a way
of taking images of the cerebrospinal fluid of the central nervous system of a rabbit using complex
computerized x-ray equipment. It requires an injection of a contrast agent to highlight the
SAS; a contrast agent is radioopaque. Background image is determined from a digitized image
taken a few moments before injection of the contrast material. The resulting angiogram is a high-
contrast image of the SAS.
The second software application, MIMICS, is used to generate an image reconstruction of the rat
model subarachnoid spaces (SAS). Three dimensional (3-D) tissue reconstruction from the digital
images of serial sections has significant potential to improve the understanding of the growth
patterns in the ventricular system and the spatial arrangement of CSF, enhance the study of
biomechanical behavior of the tissue structures towards better treatments (e.g. tissue-engineering
applications). A rat model of obstructive hydrocephalus was created by injecting kaolin
(2.5 mg/mouse) into the cisterna magna in previous work done by Basati (CITATION).
This review will provide a critical evaluation of the available research evidence and various
theories and methods about effective practices in 3-D reconstructions of the rat and rabbit
ventricles, MRI for small animals and identify some key factors for effective modeling of the
ventricular system. This 3-D model helps to understand the complex anatomical structure.
This paper is organized as follows: In section I, the brief introduction on hydrocephalus. Then after
an overview of the software application systems (both hardware and software), I describe in detail
the designing and implementation of different mask analysis modules of the software system. I
present the experimental methods and materials on the image reconstruction in section II from a
rabbit imaging procedures. Finally, in section III, some important results and implications of this
system and the possibility for future works using this system for different computational analysis
tasks are discussed.

5. 5
Ventricles and Cerebrospinal Fluid
There are four ventricles: the two (right and left) lateral ventricles, the third ventricle, and
the fourth ventricle. The ventricles are connected by narrow aqueducts.
Cerebrospinal fluid (CSF) is a watery liquid similar in composition to blood plasma, low in cells
and proteins. The prime purpose of the CSF is to support and cushion the brain and help nourish
it. Figure 1 illustrates the flow of CSF through the central nervous system.
CSF is formed within the lateral ventricles flows through them to the subarachnoid space (SAS)
via apertures in the fourth ventricle where it is returned to the dural venous sinuses by the arachnoid
villi (Kurtcuoglu, et al. 2007). It passes through the interventricular foramina into the third
ventricle, from there through the cerebral aqueduct into the fourth ventricle, and then through the
median and lateral apertures into cistern magna and the pontine cisterna (Figure 2) (Basati &
Linninger, 2011; Nolte, 2008). Some CSF flows through the central canal of the spinal cord.
Figure 1: Flow of Cerebrospinal fluid.
http://www.baileybio.com/plogger/?level=picture&id=411
Figure 2: Ventricles are CSF filled cavities (blue) positioned deep inside the brain. The
two lateral ventricles have anterior, posterior and inferior horns. The interventricular
foramen connects the lateral ventricles and the third ventricle. The cerebral aque-
duct connects the third and fourth ventricles together. http://antranik.org/central-
nervous-system-intro-to-brain-and-ventricles-medulla-oblongata-pons-mid-brain-and-
cerebellum/.

6. 6
CSF is believed to be primarily produced within the ventricles by delicate clusters of specialized
tissue called the choroid plexus (Nolte, 2008; Owen-Lynch, et. al 2003).
The rate of formation of new CSF, an average of about 350 µl/minute-half a liter/day-in humans,
is relatively constant and little affected by systemic blood pressure or intraventricular pressure.
<<State the total volume of CSF >> This means that the total volume of CSF is renewed about
three times per day (Nolte, 2008).
Disruption of CSF Circulation Can Cause Hydrocephalus
Because the rate of production of CSF is relatively independent of blood pressure and
intraventricular pressure, the fluid will continue to be produced even if the path of its circulation
is blocked or is otherwise abnormal. When this happens, CSF pressure rises and ultimately the
ventricles expand at the expense of surrounding brain tissue, creating a condition known as
hydrocephalus (Del Bigio & Bruni, 1991; Olopade, et. al, 2012; Basati et. al, 2012; Linninger et.
al, 2007) (Figure 3).
In principle, hydrocephalus could result from excess production of CSF, from blockage of CSF
circulation, or from a deficiency in CSF reabsorption. Hydrocephalus is caused by an imbalance
between how much CSF is produced and how much is absorbed into the bloodstream: partial
obstruction--either from one ventricle to another or from the ventricles to other spaces around the
brain, poor absorption--a problem with the mechanisms that enable the blood vessels to absorb
CSF and, overproduction-- create more than normal and more quickly than it can be absorbed.
To alleviate the problem of the accumulation of cerebrospinal fluid in the ventricles of the brain,
a shunt system can be surgically inserted in order to drain the excess fluid (Basati, 2012; Linninger,
2007; Del Bigio & Bruni, 1991). According to an Information Sheet published by the
Hydrocephalus Association, it outlines three major components within a shunt system which
consists of a long, flexible tube with a valve that keeps fluid from the brain flowing in the right
direction and at the proper rate. One end of the tubing is placed in one of the brain’s ventricles.
The tubing is then tunneled under the skin to another part of the body where the excessive buildup
of CSF can be more easily absorbed in the abdomen cavity (Basati, et. al 2011).
Figure 3: FatimaKhatu, 25, kissesthe head ofher 18-month-old daughter RoonaBegum, who suffersfrom hydrocephalus.
Read more: http://www.news.com.au/lifestyle/health-fitness/indian-girl-with-swollen
-head-needs-miracle-parents/story-fneuzlbd-1226619955744#ixzz2YUeo8X2z

7. 7
Shunt system therapy has been around for over 60 years. Hydrocephalus is believed to occur in
about two out of 1,000 births in United States; the incidence of acquired hydrocephalus is not
known exactly due to the variety of disorders that may cause it. Incidence of acquired
hydrocephalus is unknown. About 150,000 shunts are implanted each year in the developed
countries, but little information is available for other countries
(http://thehealthscience.com/showthread.php?168552-Hydrocephalus).
Shunt system treatment has remained relatively unchanging since its inception and has a high
failure rate. It can encounter several problems such as mechanical malfunctions, infections,
material degradation and blockages (Basati, et. al 2011, Hydrocephalus Association). Shunt
dependence occurs in 75% of all cases of treated hydrocephalus and in 50% of children with
communicating hydrocephalus. The attraction of giving patients the chance of escaping the
numerous inherent complications of CSF shunts has led to a rapid expansion of enthusiastic
neurosurgeons and bioengineers offering alternative approaches for the treatment of
hydrocephalus.
Measurement of Ventricular Volume
In this paper the volumes of the CSF filled spaces of rabbits will be analyzed for the diagnosis of
hydrocephalus, which will serve as reference values for future studies. We present an automatic
method to estimate those volumes from a 3-D whole brain magnetic resonance imaging (MRI)
sequence. This enables us to statistically analyze the fluid volumes, and to characterize a range for
the ratio of SAS volume to ventricular volume in the healthy rabbit and hydrocephalus model. This
ratio will be used as an index to indicate a robust distinction between pathological and healthy
cases . The ventricular volume was measured using image reconstruction techniques on
computerized angiography scans and magnetic resonance images obtained in a rabbit with
hydrocephalus and control.
In 2011, Dr. Sukhraaj Basati performed n acute experiment which he had designed that consisted
of sequential shunting and infusing CSF from the ventricles of a hydrocephalic rat. Fifteen 3-
weekold Sprague-Dawley rats were injected with 20 μl of 25% w/v sterile kaolin suspension into
the cisterna magna using a 26-gauge needle. The objective was to measure dynamic volume
changes. Radiography with contrast administration was performed in 1 hydrocephalic and 1 non-
hydrocephalic animal to assess the location of the implant, as well as to discern ventricle integrity
during implantation (Basati, et. al, 2011)
Image J
ImageJ is a public domain image processing program implemented in Java and developed by
Wayne Rasband since 1997 (National Institutes of Health [NIH]; (Collins, 2007; Maurizi, et. al,
2009; Abramoff, et. al, 2004). It is a powerful software package which includes a series of tools
for 2D image analysis, image acquisition, image sequences and final production of figures.
There are now a number of efforts going forward to extend the architecture and functionality of
ImageJ using modern software programming techniques.
(http://rsbweb.nih.gov/ij/docs/menus/process.html).
The applications to which ImageJ have been applied are astounding. It is being used in imaging
applications ranging from astronomy, skin analysis, to neuroscience (Abrámoff, et.al, 2004).

8. 8
Digital Subtraction Angiography
Digital subtraction angiography is a way of taking images of the CSF of the central nervous system
of the body of a rabbit using complex computerized x-ray equipment. This usually requires an
injection of a contrast agent to highlight the SAS. The contrast agent is a clear liquid which shows
on x-rays due to its high density. This 'dye' is harmless and will pass out of the rabbit’s body in
the urine over the hours following the test. First, the background image is determined from a digital
image taken a few moments before injection of the contrast material. After contrast is injected and
the second image taken, the background is subtracted; the process for subtraction is shown in
Figure 4. The resulting angiogram is a high-contrast image of the SAS. My aim is to subtract two
rabbit angiography images that are obtained before and after contrast media is administered.
Figure 4: Digital Subtraction Angiography
http://ric.uthscsa.edu/personalpages/lancaster/DI-II_Chapters/DI_chap10.pdf
ImageJ offers various tools and functions for analyzing digital images.
Figure 5: ImageJ Menu Toolbar in Windows operating system which lists 8 commands.
Various steps were used from the tool-box to subtract two images. The following steps were
utilized to generate the subtraction image.
Enhance contrast: The enhance contrast tool that can be found under Process>Enhance Contrast,
has three different functions. When neither Normalize nor Equalize is selected, the display is
adjusted in a way that the given number of pixels becomes saturated. In this case the intensity
values in the image are not changed. When Normalize is selected, a contrast stretching or
normalization is done. http://rsbweb.nih.gov/ij/docs/guide/146-29.html#toc-Subsection-29.5 Use
Process>Enhance Contrast>
Image Calculator:It performs arithmetic and logical operations between two images selected from
popup menus. It allows images to be combined using one of several mathematical functions,

9. 9
including add, subtract, multiply, and divide as well as AND, OR, XOR, min, max, and average.
I will perform the following arithmetic operation: [img1 = img1-img2] Use Process>Image
Calculator>select operation>create new window. http://rsbweb.nih.gov/ij/docs/guide/146-
29.html#toc-Subsection-29.13
Saturated pixels: It determines the number of pixels in the image that are allowed to become
saturated. Increasing this value increases contrast. This value should be greater than zero to prevent
a few outlying pixels from causing the histogram stretch to not work as intended.
These were the steps that I utilized to perform arithmetic operations between two images.
Image A-In-vivo Image 1: Slice 50
Image B-In-vivo Image 2: Slice 52
Use Process>Image Calculator>Enhance Contract.
Magnetic Resonance Imaging (MRI)
Hydrocephalus can be detected with magnetic resonance imaging (MRI). To create an MRI image
of the brain, a strong magnetic field is rotated about the head. Exposure to this energy field induces
Image 1
Image 2
Figure 6: Image A: Rabbit induced withkaolin injection into cistern magmawithabutterfly needle-2/22/11. Image B: An “iohexol” contrast
agent wasinjected to assessthe location for the kaolin administration and measure ventricular volume. Image C: Image subtraction
manually using ImageJ -484 x 459 and each pixel wascoded 8 bit; thissingle image occupied 217 K. Image D: Automatic subtraction.
Image A Image B
Image C Image D

10. 10
the brain’s many hydrogen atoms to resonate, or leap to a higher energy state. As the field passes
through, some of the hydrogen returns to a lower energy state. Sensors detect these up and down
jumps in energy, and computers coordinate the data gathered. MRI scans can produce strikingly
detailed images of the brain. It requires tenfold increase in image resolution in all three dimensions,
resulting in signal reductions of at least a factor of 1000 (Driehuys et. al, 2009).
There are several challenges in trying to achieve high 3D spatial resolution with small animal
imaging. The smaller field of view requirements for small animal imaging indirectly result in
improved resolution over clinical scanners designed for human imaging. It requires the
development of technology specific to MRM-magnets, imaging coils, image acquisition sequences
and biological support for small animals in high magnetic fields. Basic procedures must be
followed for the MRI scanning of the rabbit ventricular system because in order to retain the same
relative anatomical definition as the human image; it must be acquired with a voxel volume
approximately 3,000 times smaller than of a human and the accompanying signal loss must be
“won back.” Additional challenges are low signal-to-noise ratios, biological support for small
animals in high magnetic field, and demanding data processing requirements.
High resolution MRI of the rabbit brains were done to estimate changes in the neuroanatomy to
understand both normal developmental as well disease processes and mechanisms of
hydrocephalus. Experimental studies have been done that explained the differences between these
two imaging techniques. “In-vivo imaging allows for the longitudinal analysis of structural
change.” (Graham, 2012; Lerch, et. al, 2012). The duration of in-vivo imaging sessions is,
however, limited by the approximately 3 hrs anesthesia tolerance of small animals, resulting in
isotropic voxel sizes of around 100 µm.
Ex-vivo fixed-brain imaging has greater resolution and sensitivity due to the lack of constraints of
imaging time, the use of tighter fitting radiofrequency coils, high concentration contrast agents
such as gadolinium Gd chelates (Dazai, et. al, 2011) and a lack of movement artifacts (Graham,
2012: Lerch, el. al 2012; .
Quantitative MRI requires accurate, reliable and efficient brain segmentation techniques. Manual
segmentation allows full user control. However, it is user-dependent, tedious and time-consuming.
Figure 7: Dr. Basati induced hydrocephalus with a butterflyneedle with kaolin solution (7/24/2013).
Figure 8: A photograph image of the angiography set up was takenby Nicholas Giovanni. (7/24/2013)

11. 11
Our aim is to use 3-D rotational angiography for reconstruction which provides volume-rendered
three-dimensional images (J. Moret, et. al, 1998). This technique produces excellent resolution
and can be rotated in any directions to show the ventricular structures from any required angle.
Acquisition takes few seconds with an average of several images per run.
It will provide all the anatomical information required for the entire experimental study. The
Philips Integris 3D Rotational Angiography (3D-RA) must be calibrated correctly in order to take
a series of images while the C-arm performs a continuous rotation around the region of interest.
The region of interest is centered on the rotational axis to ensure complete acquisition during the
entire rotation. Then the images are acquired in the rotational angiography mode over an angle of
180˚. The C-arm is positioned manually at the starting and ending positions and confirmed on the
acquisition console sequentially. Once these positions are confirmed, images are acquired by
depressing the acquisition switch. After the acquisition, 120 contrast images are transferred to a
computer workstation. Around the isocenter of rotation, a predefined default volume is
automatically reconstructed as a 3-D volume.
Image Reconstruction
To define image reconstruction, Dr. Andreas A. Linninger (2007) states that “image reconstruction
tools facilitate the collection of patient-specific geometry data such as the exact dimensions of the
ventricular and subarachnoidal spaces (SAS) as well as the computer-aided reconstruction of the
CSF-filled spaces”. The author elucidates that the “image reconstruction also involves the
physiologically accurate interpretation of the different substructures of the brain in his study”
(Linninger, et. al, 2007). In his investigation, the authors used MIMICS to import one-hundred-
twenty MRI slices and reconstructed the complex pathways of the ventricles and the cerebral. In
addition, 3-D geometry was reconstructed of the patient with hydrocephalus (Linninger, et. al,
2007). Dr. Linninger and co-researchers in the Laboratory for Product and Process Design,
Figure 9: The image plane produced
by an MRI scan. http://www.my-
ms.org/mri_plane_math.htm
g
depicts a virtual “section”
of the brain along the plane
of the scan.)

12. 12
Department of Bioengineering at UIC have used it to reconstruct the ventricles, the SAS and the
parenchyma.
Ying Hsu and co-researchers at the Department of Bioengineering at University of Illinois at
Chicago use MIMICS imaging software to reconstruct a patient-specific computational model for
the CSF-filled SAS (Hsu, et. al, 2013).
The reconstruction has been performed by using both MIMICS v. 1.5 and 3-Matic software
applications. First, a sequence of 2-D cross-sectional medical x-ray data slices is generated by a
medical scanner. They are downloaded onto the image reconstruction tools using this program to
create a segmentation mask. The MIMICS image reconstruction software was used to further
segment the 3-D volume into a finite number of small triangular or tetrahedral elements (Linninger,
et. al, 2007). Analysis of medical image data usually proceeds by identifying the objects to be
measured and then actually measuring their properties. Identification of objects is called
segmentation. Figure 10 illustrates an image reconstruction flow chart.
Figure 10: Image Reconstruction Flow Chart
This computer program also can create a simulated three-dimensional rendition of the data by
stacking a series of images together. It needs to be able to sort through the data to match the slices
appropriately and must overlay them accurately to create images of internal structures.
Medical Images
Segmentation
Masks
3 D Object
Surface MeshVolume Mesh
Computational
Dynamic Flow
Simulation

13. 13
The general principle of 3-D reconstruction is composed of the following steps:
1. A sequence of 2-D cross-sectional medical x-ray data slices is generated by a medical scanner.
Then they are downloaded onto the image reconstruction tools: MIMICS program. Then they
were imported into MIMICS.
2. Thresholding is the first action performed to create a segmentation mask. One can select a
region of interest by defining a range of grey values. The boundaries of that range are the lower
and upper threshold value. All pixels with a grey value in that range will be highlighted in a
mask. The usage of the magnifying glass tool from the tool-box and click multiple times on
the image to get the maximum zoom so I can see the individual pixels.
3. Then it will convert the selected mask region into a 3-D model mask.
4. The 3-D model can then be converted into STL format.
5. Then export 3-D models to 3-matics to optimize the surface and volume meshes for CFD. It
segments the 3-D volumes into a finite number of small triangular or tetrahedral elements
(Linninger, 2007).
6. One can modify these models and their internal structures using 3-matic. Boolean operations
allow one to make different combinations of two segmentation masks (Subtraction, Union, and
intersection).
7. Compute data into Finite Element simulation-Computation dynamic flow simulation
We will attempt to compare the cerebrospinal spaces of the normal rabbit and hydrocephalus model
and to compare the volume changes using image reconstruction software applications.
Materials & Methods
Hydrocephalus Administration
All experiments described were approved by University of Illinois Chicago’s Animal Care
Committee. The objective is to induce hydrocephalus into rabbits. Each rabbit were weighed prior
to induction of hydrocephalus. The animals were obtained from the animal holding facility of the
Biologic Resources Laboratory (BRL) at University of Illinois Chicago.
This study used four (4) healthy averagely 4 month New Zealand white rabbits, weighing 3 kg.
Fur from the head is removed using a shaver to provide a better view of needle insertion site. Pulse
and temperature monitoring devices were utilized by BRL staff. Sterile kaolin (aluminum silicate)
suspension (0.03 ml) was inserted into the cistern magna using a 25 G2 butterfly needle to induce
hydrocephalus. The rabbits were anesthetized with isoflurane and monitored by BRL personnel.
Several angiogram x-rays images were taken using iohexol contrast agent (0.2 - 0.5 ml) to resolve
the SAS to give us a clear resolution of SAS. Sample photographs were taken of the control and
hydrocephalic rabbits.
According to observations, the hydrocephalic rabbit exhibited a general reduction in activity.

14. 14
Data Acquisition
Ex-Vivo Preparation
The rabbit are sacrificed; the brain was removed, fixed and stored in formalin at 4 0C. The brains
were embedded into an agarose gel. Then the brain was sliced into the coronal plane. During the
gross examination, inflammation of the meninges was found.
Figure 11: Photograph taken of an ex-vivo Rabbit Brain on 7/24/2013.
Figure 12: Photograph taken of an ex-vivo on 7/18/2013. A coronal section of fixed brains
from kaolin-injected (right) on rabbit after 5 days. Scale bar=2 mm.
Rabbit Model result of hydrocephalus by kaolin injection into the CSF
Rabbit #13-029920 Rabbit #13-033592 Rabbit #13-031134 Rabbit brain 4
06/26/13 07/10/13 07/18/13 7/26/2013
No kaolin injection,
2 contrast injection
procedures
No prior procedures Contrast injection,
kaolin injection
Figure
11
Figure
12

15. 15

16. 16
Reference photo showing rabbit 13-033592 on left and 13-029920 on right, with ruler, to show
size and perspective:
Reference photo with rabbit 13-031134 on the left and rabbit #4 on the right with a ruler to
show size and perspective.

17. 17
Ex-vivo MRI
Tissue Fixation Method
The brains can be fixed using one of two brain tissue fixation methods: aldehyde solution or
focused beam microwave irradiation (FBMI) (Liu, et. al, 2013) Based on their results, aldehyde
fixation maintained in vivo manganese enhancement for ex vivo MEMRI.
Results
Image reconstruction was done with the rat’s SAS since our rabbit ex-vivo has not developed
hydrocephalus among the four (4) rabbit model.
Figure 13: The parameters used for obtaining the MRI images on a 9.4T animal MR scanner
(Agilent) were as indicated: Tr =2800ms, Te =16.38ms, averages =2, matrix =256x256, slices
=15, Thickness =0.195mm, gap =0mm, orientation: axial
Figure 14: MRI angiography image of the rat’s CNS.
MIMICS on Rat’s SAS
The image reconstruction has been performed by using both ‘Mimics’ and ‘3-matic’ softwares on
the rat’s SAS. Figure 12 is a MRI image showing a slice of the rat CNS. 3-D domain is divided
which we can apply a mask. The region of interest (ROI) can be highlighted manually or
automatically by threshold settings. The specific structures of interest were “colored” with the help
of the layer tool on slice. Figure 13 is a MRI angiography image of the rat’s CNS.
The procedure utilizes medical image data on which regions of interest are selected. In this case
we have used sequential axial images of the rat CNS. We have made 2 masks:
Figure 13 Figure 14

18. 18
Mask I for the required CSF space (as indicated by the region in Figure 12 in the pink region of
interest)
Mask II for the spinal cord and brain tissue (as indicated by the blue region in Figure 13)
Once the medical image data were loaded into MIMICS projects, the next stage is to convert the
raw data into 3-D objects. A 3-D object was generated from the segmentation mask of 2-D slices.
Figure 15 illustrates three orthogonal slices of the MRI data with the cerebrospinal fluid in the
brain and spinal cord indicated in blue. The three dimensionally reconstructed the brain and spinal
are shown for a better space orientation. We made two masks. A segmentation mask of CSF
cerebrospinal fluid space and the central nervous space tissue regions were generated. Then, I
remesh for automatic upload to the 3-Matic software and then I saved it as a STL file.
Figure 15: Screenshot ofthe 3D ofthe CSF space ofthe rat-mid spine to the end ofthe spinal canal
region. Figure 16: MRI image showing an axial slice ofthe rat CNS with the segmentation masks
corresponding to CSF space (pink) and CNS tissue regions (blue)- Upper brain to mid spine region.
Next, I employed the 3-Matic software. It is used for improving the surface mesh and generating
a volume mesh. Then, the two masks are then converted into two 3-D surface meshes as shown
in Figure 15 and 16. The green object represents the surface mesh for the CNS tissue. I will do
the same procedures for the pink region of CSF.
Figure 15
Figure 16

19. 19
Figure 17 and 18- Surface Meshes
“Diagnostics” from the tool bar were used to check the mesh integrity. Then I create a volume
mesh from the completed surface mesh to export to Fluent. For example, I used the Easy
automatic remeshing which the software searches for all triangles whose quality is below the
indicated level and transform them to triangles with a better shape. We can apply the Manual
remeshing. It will help to eliminate those triangles with a shape inferior to the required quality
level which may remain after automatic remeshing. The Remesher offers a unique toolbox to
reshape those triangles manually.

20. 20
Figure 19: Volume Mesh
The volume mesh can then be used for performing computations using computational fluid
dynamics (CFD) software such as ANSYS Fluent. Figure 19 is the blue volume mesh. It has to be
done for the red surface mesh as well.
Next I applied the Boolean subtraction application which allows you to make different
combinations of two segmentation masks (Subtraction, Union and Intersection).
Figure 20: Subtracted surface mesh obtained by Boolean subtraction of meshes corresponding to CNS space
and CNS tissue.

21. 21
Conclusion
In this study, fully automatic commercially available software for the assessment of SAS image
reconstruction was evaluated and found to be very fast and user-friendly. Both imaging modalities
provided invaluable information. Hydrocephalus was not produced in our rabbits by the infusion
of kaolin injection into the subarachnoid spaces in the cistern magna. During the gross
examination, inflammation was found in the meninges. Meninges is the three membranes (dura
mater, arachnoid, and pia mater) that line the skull and vertebral canal and enclose the brain and
spinal cord.
The image reconstruction has been performed by using both MIMICS and 3-Matic software.
MIMICS software was used for creating masks and surface meshes. 3-Matics software was
utilized to improve the surface mesh and generate the volume mesh.
Rotational angiography run was done on the rabbit during the kaolin injection administration into
the subarachnoid spaces in the cistern magma. The 3D rotational angiography did not yielded
clear images.
Thus, the obtained 3D volume can be rotated and viewed in any direction. Cut planes can be made
at any position in the volume, and measurements can be made.
However, beyond our limitations on the usage of MIMICS licensing requirement, the last step was
not completed.
is subtracted from the equals to

22. 22
Discussion and Future Works
Small animals such as rodents and rabbits have been utilized and the kaolin model allows the study
of acute and chronic hydrocephalus (Kondziella et. al, 2009; Basati et. al, 2012; Olopade et. al,
2012). According to Del Bio and Bruni, (1991), the authors used silicone oil-induced model of
hydrocephalus because they stated that it avoids the complicating inflammatory changes associated
with kaolin (Wisniewski, et. al, 1969). Further improvement is needed in the infusion process. One
technique to be considered for future research is to utilize the infusion of viscous 100,000 c.s.
silicone oil instead of kaolin injection which would be ideal as it tends to eliminate the
complications of mechanical damage and inflammation of the meninges (Wisniewski, et. al, 1969).
Another improvement is the calibration of the rotational angiography run in order to produce image
reconstruction of the ventricular system of a hydrocephalus model.
Based on these data, we were not able to compute the ventricular volume measurements for the
rabbits and to reconstruct the ventricular system using rotational angiography. There are challenges
still to be tackled for the tissue preparation and image acquisition of small animal ex-vivo. The
rate of formation of CSF was not calculated.
Our data further suggest that imaging modalities, 3-D angiography and MRI, in conjunction with
image reconstruction contribute to the analysis of hydrocephalus study. ImageJ and MIMICS are
powerful image processing tools which can be used to analyze and understanding how the brain
responds to a hydrocephalic state.
Acknowledgements
RET Research Program was possible by the RET 2013 National Science Foundation Grant #
I would like to thank the following individuals who have assisted me on completing this study:
 The Teacher Research Program is based upon the UIC Bioengineering Department and
Laboratory for Product and Process Design works and grant supported by National Science
Foundation - EEC # 1132694.
 Professor Dr. Andreas Linninger, Program Director of the Laboratory for Product and
Process Design who provided the motivation to become a great scientist student and
encouraged us to share our knowledge to my students.
 This paper could not have been written without the invaluable assistance of Mr. Kevin
Tangen who had the patience and encouragement to supply invaluable information and
training sessions on digital subtraction angiography, small animals experiments, imaging
process programs and the hydrocephalus experiment.
 Laboratory for Product and Process staff members
 Dr. Sukhraaj Basati who had taught me about the experimental procedures and set up for
small animals.

23. 23
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Appendix I: Synopsis of Author’s Notes
Cerebrospinal fluid volume measurement in hydrocephalic rats
This paper validates a method to measure dynamic changes in ventricular volume using
impedance sensors. Fifteen-three week old Sprague-Dawley rats were injected with 20 µl
of 25% w/v/ sterile kaolin suspension into the cisterna magna using a 26 gauge needle.
Two phenomena were observed: CSF volume can be controlled acutely from the ventricles
of a hydrocephalic rate and local dynamic volume and pressure changes can be recorded.
Hydrocephalus was induced in weanling rats by kaolin injection into the cisterna magna.
At 28 days after induction, the sensor was implanted into the lateral ventricles. One
implication is that future experiments will use models with larger animals with a chronic,
wireless volume-pressure monitoring system. These measurements, coupled with high-
resolution MRI, may allow for long-term studies of brain tissue. Future experiments
consist of performing 3D reconstruction of the ventricles before and after shunting to
improve the calibration curve using high-solution MRI.
Effect of spinal micro-anatomy of CSF flow patterns-Comparative analysis of in-vivo data
and computations (Tangen, Hsu, Nguyen, Kaewken, Zhu, Linninger)
This paper investigates the effect of spinal nerves and arachnoid trabeculate on pulsatile
CSF flow. The aim is to study the effect of the micro-anatomy on CSF flow; all 31 pairs
of spinal nerves were reconstructed. The researcher constructed a patient-specific model
to predict the spatio-temporal CSF flow fields and validate them against in-vivo flow
measurement. CINE MRI measurement of CSF pulsatile flow was taken in a specific
patient. Data were collected for the spinal canal of a 29 year old healthy male on a 3T
Signa HD x MR scanner.
Fact Sheet on Shunt Systems for the Management of Hydrocephalus (2012)
(www.hydroassoc.org)
This fact sheet explains how CSF shunts are commonly used to treat hydrocephalus. It
explains on how shunt can come in a variety of form, types of valves mechanism
The Fixation Protocol alters Brain morphology in ex-vivo MRI mouse phenotyping (de
Guzman, et. al,)
The purpose of this study was to characterize the effect that increased fixation by chemical
cross-linking has on brain structure volume detectable with MRI. 8 V57BL/6J mice
underwent transcardiac perfusion through the left ventricle. Prohance contrast agent was
used. It concluded that the rate at which structure volume changes with fixation time varies
depending on the location of the brain. It states that further investigation will be performed
to determine if long term storage of the samples in PBS may affect structure volume further.
The Frequency and Magnitude of Cerebrospinal fluid Pulsations influence intrathecal drug
distribution (Hsu, Hettiarachchi, Zhu, Linninger, 2012)
This paper propose a method, medical image-based computational fluid dynamics
(miCFD) for investigating IT drug delivery. It is used to construct a patient-specific model
to quantify drug transport as a function of a spectrum of physiological CSF pulsations.

26. 26
CSF velocities at C4, T6, and L4 in the spine were measured. Magnetic resonance imaging
(MRI) and CINE (MRI) to capture central nervous system anatomy and CSF pulsatile flow
velocities. A miCFD model was reconstructed from these MRIs and the patient’s CSF
flow velocities were computed.
In vivo vs. ex vivo magnetic resonance imaging in mice (Mackenzie-Graham, 2012)
A commentary: Wanted dead or alive? The tradeoff between in vivo vs. ex vivo MR brain
imaging in the mouse (Lerch, et. al, 2012) The journalist compares the differences between
in vivo and ex-vivo.
Image Processing with ImageJ (Abrámoff et. al, 2004)
This paper explains the ImageJ’s origin, image processing and analysis.
ImageJ for microscopy (Collins, 2007)
This paper explains how ImageJ incorporates a number of useful tools for image processing such
as intensity processing and analysis, three-dimensional (3-D) reconstruction routines.
MIMICS
Case study: Aneurysms are balloon-like swellings of arterial walls. They occur most
frequently at or near the place where an artery bifurcates (divides). An aneurysm can cause
neurological deficits in two ways. As it grows, it can push against and compress brain
structures, much as a growing tumor would.
Silicone oil-induced hydrocephalus in the rabbit
This paper investigates the pathological changes resulting from hydrocephalus, to correlate
these changes with intracranial pressure (ICP) and ventricular enlargement; to determine
the potential for reversal of these changes by shunting.
Materials and Methods: Male New Zealand white rabbits (2.5-3.0 kg, 3-4 months of age)
were used in these investigations. Hydrocephalus was induced by injection of 0.5 ml/kg
silicone oil
An Optimized Java Basedsoftware Package for Biomedical images and Volumes Processing
(Maurizi, Franchi, Placidi
This paper explains an efficient, Java based software package founded on a kernel that
implements functions and tools common to various biomedical imaging techniques and a
series of modules implementing functions for specific applications.
Definition of kernel: is a set of basic tools to visualize and process images and volumes
which is common to various biomedical applications.
Multiple-mouse Neuroanatomical Magnetic Resonance Imaging (Dazai, et.al, 2011)

27. 27
This video explains the experimental design, data acquisition and processes on both in vivo
and ex-vivo brain imaging of multiple mice.
Ventricle wall movements and cerebrospinal fluid flow in hydrocephalus (Penn, Basati,
Sweetman, Guo, Linninger, 2011)
This paper provide additional clinical data obtained in patients with normal pressure
hydrocephalus and supplement these data with computer simulations to better understand
the CSF flow and ventricular wall displacement and emphasize its clinical implications.
Three NPH patients and 1 patient with aqueductal stenosis underwent cine phase-contrast
MR imaging (CINE MR imaging) for measurement of CSF flow and ventricle wall
movement during the cardiac cycle. In healthy volunteers, net CSF aqueductal flow was
1.2 ml/minute in the craniocaudal direction. In patients with NPH, the net CSF flow as in
the opposite direction. Shunting has a major effect on CSF dynamics and in particular on
the pressure volume relationship. After shunting, the magnitude of the abnormal fluid flow
decreased or reversed.
Wanted dead or alive? The tradeoff between in-vivo versus ex-vivo MR brain imaging in the
mouse (Lerch, et. al, 2012)
This paper explains the tradeoffs between in-vivo and ex-vivo mouse imaging. The author
investigated these trade-offs by determining the source of variability in anatomy measurements
derived from high field mouse MRI and then simulating an experiment designed to recover a subtle
change in hippocampal volume.