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

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.

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Table of ContentsAbstract..............................................................................................................................................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)................................................................................................10

Image Reconstruction.......................................................................................................................11

Materials & Methods........................................................................................................................13

Hydrocephalus Administration.........................................................................................................13

Data Acquisition...............................................................................................................................14

Ex-Vivo Preparation..........................................................................................................................14

Ex-vivo MRI.....................................................................................................................................17

Tissue Fixation Method....................................................................................................................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

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Introduction

PurposeThe 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.

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Ventricles and Cerebrospinal FluidThere 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.

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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/.

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 HydrocephalusBecause 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.

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Figure 3: Fatima Khatu, 25, kisses the head of her 18-month-old daughter Roona Begum, who suffers from hydrocephalus.Read more: http://www.news.com.au/lifestyle/health-fitness/indian-girl-with-swollen

-head-needs-miracle-parents/story-fneuzlbd-1226619955744#ixzz2YUeo8X2z

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).

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 VolumeIn 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 JImageJ 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).

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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).

Digital Subtraction AngiographyDigital 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

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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, 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.

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Image 1Image 2

Figure 6: Image A: Rabbit induced with kaolin injection into cistern magma with a butterfly needle-2/22/11. Image B: An “iohexol” contrast agent was injected to assess the location for the kaolin administration and measure ventricular volume. Image C: Image subtraction manually

using ImageJ - 484 x 459 and each pixel was coded 8 bit; this single image occupied 217 K. Image D: Automatic subtraction.

Image A Image B

Image C Image D

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 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.

10Figure 7: Dr. Basati induced hydrocephalus with a butterfly needle with kaolin solution (7/24/2013). Figure 8: A photograph image of the angiography set up was taken by Nicholas Giovanni. (7/24/2013)

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 ReconstructionTo 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

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Figure 9: The image plane produced by an MRI scan. http://www.my-

ms.org/mri_plane_math.htm

Process Design, 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.

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Medical Images Segmentation Masks 3 D Object

Surface MeshVolume MeshComputational Dynamic Flow

Simulation

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 AdministrationAll 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.

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Data Acquisition

Ex-Vivo PreparationThe 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 406/26/13 07/10/13 07/18/13 7/26/2013

No kaolin injection,2 contrast injection

procedures

No prior procedures Contrast injection,kaolin injection

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Figure 11

Figure 12

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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.

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Ex-vivo MRI

Tissue Fixation MethodThe 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.

ResultsImage 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 SASThe 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:

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Figure 13 Figure 14

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 of the 3D of the CSF space of the rat-mid spine to the end of the spinal canal region. Figure 16: MRI image showing an axial slice of the 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.

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Figure 15Figure 16

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.

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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.

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ConclusionIn 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.

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is subtracted from the equals to

CNS CNStissue

SAS

Discussion and Future WorksSmall 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.

AcknowledgementsRET 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.

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Abstract ID: 9970 Title: Small Animal Magnetic Resonance Imaging: Current Trends, Challenges and Perspectives for Pathological Imaging.

Basati, S., B. Desai, A. Alaraj, F. Charbel and A.A. Linninger (2012). “Cerebrospinal fluid volume measurement in hydrocephalic rats.” J Neurosurg Pediatrics 10 ( ): 347-354

Basati, S., T.J. Harris, and A.A. Linninger (2011). “Dynamic Brain Phantom for Intracranial Volume Measurements.” IEEE Tran. Biomed. Eng., 54 (2): 291-302

Benveniste H. Blackband S. 2002. MR microscopy and high resolution small animal MRI: Applications in neuroscience research. Prog Neurobiol 67: 393-420.

Budinger, T.F. Lauterbur, P.C., 1984. Nuclear magnetic resonance technology for medical studies. Science 226, 288-298.

D’Arceuil H. Liu, C., Levitt, P. Thompson, B., Kosofsky, Crespigny, A. 2007. Three-dimensional high-resolution diffusion tensor imaging and tractography of the developing rabbit brain. Dev Neurosci 30, 262-275

Dazai, J., Spring, S., Cahill, L. S., Henkelman, R. M. Multiple-mouse Neuroanatomical Magnetic Resonance Imaging. J. Vis. Exp. (48), e2497, doi:10.3791/2497 (2011). http://www.jove.com/video/2497/multiple-mouse-neuroanatomical-magnetic-resonance-imaging

This website provides an overview on how to take magnetic resonance imaging of multiple mice.

Del Bigio, M.R., and J. Edward Bruni (1991). “Silicone oil-induced hydrocephalus in the rabbit.” Child’s Nervous System 7 ( ) 79-84

Driehuy B., N. J., B. A., Bucholz, E., Ghaghada K., Petiet A., and L. W. Hedlund L. W. (2008). Small animal imaging with magnetic resonance microscopy. Volume 49, 1. 35-53

Hsu, C.Y., I. Venugopal, Tangen, K., and A.A. Linninger (2013). “Image Reconstruction Using MIMICS.” LPPD Project Report. 1-30

Hsu, Y., H.D. M. Hettiarchchi, D.C. Zhu and A.A. Linninger (2012). “The Frequency and Magnitude of Cerebrospinal Fluid Pulsations Influence Intrathecal Drug Distribution: key factors for Interpatient Variability.” Anesthesia and Analgesia 115 (2): 386-394

http://hstalks.com/main/view_talk.php?t=1669&r=443&j=762&c=252

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http://mouseatlas.caltech.edu/13.5 pc/ -This website provide mouse brain atlases. http://rsbweb.nih.gov/ij/docs/index.html .- This website provides an overview on how to

utilize the ImageJ. http://www.loni.ucla.edu/MAP This website provides information on digital web based

mouse brain atlases that are based on MR microscopy data.Linninger, A.A., M. Xenos, D. C. Zhu, M. R. Somayaji, S. Kondaplli, and R.D. Penn, (2007). “Cerebrospinal fluid flow in the normal and hydrocephalic human brain.” IEEE Trans.

Magnetic Resonance Imaging (23 min) Video

Mayo Clinic Staff (2011). “Hydrocephalus.” www.mayoclinic.com/health/hydrocephalus/DS00393/METHOD=print&DSECTION=all

MIMICS Materialize. Available at: http://www.materialize.com/mimics. Accessed July 15, 2013.

NIH (2013). “Hydrocephalus Fact Sheet: National Institute of Neurological Disorders and Stroke (NINDS).” National Institutes of Health NIH Publication No. 08-385. www.ninds.nih.gov/disorders/hydrocephalus/detail_hydrocephalus.htm

Owen-Lynch, P. Jane, C.E. Draper, F. Mashayekhi, C. M. Bannister and J. A. Miyan (2003). “Defective cell cycle control underlies abnormal cortical development in the hydrocephalic Texas rat.” Brain 126: 623-63.

Ozdemir, M. B., I. Akdogan, E. Adiguzel and N. Yonguc (2005). “Three dimensional (3D) reconstruction of the rat ventricles.” Neuroanatomy 4: 49–51.

Penn, R.D., S. Basati, B. Sweetman, X. Guo and A. A. Linninger (2011). “Ventricle wall movements and cerebrospinal fluid flow in hydrocephalus.” J Neurosurg 115 (1): 159-164.

Prodanov, D., and Verstreken K. Automated Segmentation and Morphometry of Cell and Tissue Structures. Selected Algorithms in ImageJ. Bioelectronic systems group, BIONE, Imec, Belgium.

Wisniewski, H., Weller R. O., and R.D. Terry (1969). “ Experimental hydrocephalus produced by the subarachnoid infusion of silicone oil.” J. Neurosurg 31: 10-14.

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Appendix I: Synopsis of Author’s NotesCerebrospinal 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)

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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. 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 Based software 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.

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Multiple-mouse Neuroanatomical Magnetic Resonance Imaging (Dazai, et.al, 2011)

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.

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