ILAR J
Volume 49, Number 1, 2008
Noninvasive Bioimaging of Laboratory Animals

Davis.  Introduction: Bioimaging of Laboratory Animals: The Visual Translation of Molecular Insights, pp. 1-3

Task 3 – Provide Research Support, Information, and Services

SUMMARY: This article is the introduction to a collection of reviews focused on laboratory animal imaging. The articles describe the methods, limitations and most common applications of small animal imaging.

Imaging modalities used with laboratory animals provide ways to explore the molecular mechanism of several diseases, minimize the limitations of static tissue-based techniques (e.g., histopathology), and may decrease the numbers of animals required for a study.

The first manuscript describing the MRI of a rat was published almost 25 years ago and the technology has evolved to include dynamic contrast-enhanced MRI (DCE-MRI). One recent publication describes the use of DCE-MRI to detect vascular permeability and study the inflammatory response and vascular remodeling associated with chronic airway inflammation in rat models.

A promising aspect of functional MRI (fMRI) is that it can provide maps of brain activities with millimeter spatial resolution.

Some diagnostic imaging techniques developed for human use that are available for lab animal research include computed tomography (CT or microCT), single photon emission computed tomography (SPECT), positron emission tomography (PET), intravascular ultrasound, optical coherence tomography, near-infrared fluorescence and spectroscopic imaging, and bioluminescence imaging.

Multispectral imaging allows the separation of five or more fluorophores, with each signal quantitated and visualized separately. The advantages to this technique include sensitivity, quantitation, and multiplexing. The use of multiple and varied fluorophores allows the examination of three different or interactive processes simultaneously. Microscopy-based multispectral techniques are an excellent complement to other in vivo imaging techniques.

Multiphoton microscopy is a cell-based, functional complement to anatomic imaging. Using labeled fluorophores, dynamic events can be studied with subcellular resolution in the functioning organ.

The discovery of bioluminescence has been applied for use as a biotechnology via luciferase systems.

QUESTIONS:
1. 	Define the acronym DCE-MRI.
2. 	Define the acronym fMRI.
3. 	Define the acronym CT.
4. 	Define the acronym SPECT.
5. 	Define the acronym PET.
6. 	What is a fluorophore?

ANSWERS:
1.	Dynamic contrast-enhanced magnetic resonance imaging. 
2.	Functional magnetic resonance imaging. 
3.	Computed tomography. 
4.	Single photon emission computed tomography. 
5.	Positron emission tomography. 
6.	From Wikipedia: A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. This technology has particular importance in the field of biochemistry and protein studies, e.g. in immunofluorescence and immunohistochemistry. 

Klaunberg and Davis.  Considerations for Laboratory Animal Imaging Center Design and Setup, pp. 4-16

Task 3:  Provide Research Support, Information, and Services
 
Imaging facilities have grown in use, due to the ability to study disease or response to therapy via a noninvasive means.  Designing an in vivo imaging facility for laboratory animals is a major endeavor.  An initial large capital investment is imperative, so it is most efficient to design a shared imaging facility.  The following items are important considerations when designing such facilities:  

1.  	Specific building requirements for particular types of imaging modalities, such as MRI.
2.  	Animal housing and imaging support areas.
3.  	Personnel workspaces and comfort.
4.  	Biological and radiation safety.
5.  	Animal anesthesia and monitoring equipment to maximize animal safety.
 
MRI (magnetic resonance imaging) has 3-dimensional imaging that uses the nuclear property of spin in certain isotopes of elements to form anatomical images.  MRI can be used to study blood flow, perfusion, brain activity, etc.  It is the most expensive imaging modality and requires specific building design considerations, as well as poses unique health and safety risks.  People with metal implanted devices (e.g. pace makers, cochlear implants, etc.) should not work in close proximity to the magnet.   Animal motion can cause image artifacts and blurring, so "gating" captures images at time points triggered by specific events.  This will eliminate the motion artifact.
 
X-ray CT (computed tomography) uses a series of radiographic images, obtained at different angles around the animal to construct a 3-dimensional image.  This modality of imaging is great for visualization of bone structure, fat tissue, and air spaces, due to the high contrast between these tissues.
 
PET (positron emission tomograph) constructs an image based on radioisotope decay of a tracer agent given, just prior to the imaging session.  The most commonly used PET agent is 2-FDG (fluoro-2-deoy-glucose).
 
Other sources of imaging are include:  ultrasound, optical imaging, laser Doppler imaging, fluorescence imaging, and bioluminescence imaging.  
 
Ultimately, the imaging devices in the planned / designed facility need to reflect the needs of the users.  Current and future experimental study goals need to be considered.  All (e.g. scientists, designers, builders, animal personnel, management, etc.) must work together to ensure successful completion and use of the animal imaging facility.

QUESTIONS:
1.  	Which is the FDA-recommended limit of a magnetic field for people with pacemakers or other internal devices?
a.  	10 Gauss 
b.  	1 Gauss
c.  	5 Gauss 
d.  	3 Gauss
e.  	None of the above
2.  	Which image modality is great for visualizing bone structures, fat tissue, and air spaces?
a.  	MRI 
b.  	CT
c.  	PET 
d.  	Ultrasound
e.  	BLI
3.  	Which term describes the process that enables the capture of images a time points triggered by specific events, thus eliminating motion artifacts?
a.  	Doppler
b.  	Tracking
c.  	Time trigger
d.  	Gating
e.  	None of the above
4.  	What is the most advantageous aspect of in vivo imaging?

ANSWERS:
1.  	c
2.  	b
3.  	d
4.  	The ability to follow an individual animal over time

Hildebrandt et al.  Anesthesia and Other Considerations for in Vivo Imaging of Small Animals, pp. 17-26

Task 2 - Prevent, Alleviate, and Minimize Pain And Distress
Task 3 - Provide Research Support, Information, and Services
Task 6 - Design and Operate Laboratory Animal Facilities
Task 9 - Collaborate on the Selection and Development of Animal Models
Task 10 - Design and Conduct Research

SUMMARY: This article discusses several issues of preparing animals for and using them in several imaging techniques. Imaging has become increasingly common for small animals, as it permits a research study to follow individual animals longitudinally and noninvasively. This article discusses the use of positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging. The advantages to using imaging techniques include the ability to follow a single animal throughout the disease course and the ability to localize altered biochemical and physiological processes.

PET and SPECT rely on detection of photons emitted from radiolabeled probes in the body. The imaging probe is injected either i.v. or i.p. PET has a high sensitivity and small amounts of a probe are needed. CT uses an x-ray source and detector to produce 3-D images. This technique differentiates between bone and soft tissues, but cannot separate soft tissues very well. CT is less sensitive than PET and requires higher concentration of contrast agents, which may have pharmacological effects on the animals. MRI uses strong magnetic fields to detect emissions from the hydrogen nuclei in tissues. MRI can be used for both anatomic identification as well as physiological parameters. MRI is better that CT for identification of soft tissues. Optical imaging is the detection of either bioluminescence or fluorescence. Bioluminescence requires the injection of a substrate into the animal. Fluorescence is usually produced by the cells or genes previously introduced into the animal, making it unnecessary to manipulate the animal again before imaging.

Factors that may influence imaging data include animal strain and sex, as parameters such as pharmacokinetics, metabolism, and stress are different, all of which affect the imaging. Also fasting and diet, and time of day can alter the final image in small animals, as circadian rhythms and food intake alter the metabolism of animals and thus alter the image quality.

Anesthetic drugs are routinely used to image rodents, as the process requires the animal to be still. It is important for anesthetized animals to be kept warm, as they easily become hypothermic while anesthetized. Barbiturates, like pentobarbital, ketamine, and inhalation anesthesia are the most commonly used agents for rodents. Length of sleep time varies with species, strain, age, and sex. These different drugs alter physiological parameters, such as cardiac function. It has been documented that glucose uptake is significantly altered depending on the method of anesthesia, as well as if the animal has been fasted or kept warm during the procedure. The authors recommend that all methods are well described in any reporting of the study. Monitoring of anesthetized animals is also difficult, as respiration and heart rates are very rapid. Monitoring equipment must be positioned out of the imaging area. One technique used to minimize the motion artifact of respiration is called “gating.” This technique attempts to synchronize the heart and respiratory data with the imaging data. One method has been developed to image an awake, conscious rat. This is called “RatCAP” and involved fitting a device around the animal’s head. This allows to images of the rat brain, but permits the animal to be awake and moving during the procedure.

One advantage to imaging methods is the possibility of repeated images of the same animal. There are concerns, however, about the repeated administration of agents to animals. Volume of a substance is often a limiting factor. Those imaging modalities in which radioactive substances are used must be aware that repeated doses are harmful. The added handling and injections also increases the stress to each animal. Further research must be done to optimize each trial so the most humane methods are used.

QUESTIONS:
1. 	How long a fasting time does it take to clear a rodent stomach of food?
2. 	T or F Ketamine has been demonstrated to depress cardiac output and respiration.
3. 	T or F Cardiac function is better maintained with isoflurane than with injectable anesthetics.
4. 	What tissues are exposed to greater radiation dose when radiolabels are administered for imaging studies?

ANSWERS:
1. 	About 6 hours, but since most of the eating is done during dark hours, food removal during the dark hours has a greater impact than removal during light hours.
2. 	False
3. 	True
4. 	Tumors, brain, heart, kidney, bladder

Johnson.  Introduction to Rodent Cardiac Imaging, pp. 27-34

SUMMARY: Ultrasound (U/S), microcomputed tomography (MicroCT), and magnetic resonance (MR) imaging can be used to examine the structure and function of the rodent heart.

U/S provides real-time assessment of cardiac function while MR and microCT provide 3D and 4D views (4D imaging is based on 3D images that change over time, for example a 3D image of a beating heart).

All three modalities can be used to assess cardiac function including:
1	Fractional  shortening - % change in left ventricle dimension from  end-diastole to end-systole
2	Ejection fraction – fraction of end-diastolic volume that is ejected  each beat; measures contractility
3	Stroke volume – difference between end-diastolic and end-systolic  volumes
4	Cardiac output – amount of blood pumped by the left ventricle into  the aorta per minute; *cardiac output = stroke volume X heart rate

The normal heart rate of a mouse is approximately 500 bpm; a rat, approximately 300 bpm.

Cardiac function can be directly (or indirectly) affected by any drugs administered and anything that can affect the animal’s physiologic parameters.  Therefore these must be considered prior to any imaging studies.

Animal Preparation
Ideal anesthesia for imaging = easy to administer, reproducible, rapid in onset and recovery, causes minimal heart rate and cardiac depression, and has low toxicity.  Common forms of anesthesia include ketamine/xylazine injection and inhalation anesthesia.

Ketamine/xylazine can cause bradycardia and hypotension in mice and can cause significant cardiac depression.  Ketamine may also stimulate hypersalivation.

Inhalation anesthesia is recommended for procedures longer than 30-40 minutes as it is easier to control and provides more stable and physiologically relevant heart rates than do injectable anesthetics.   However, attempts should be made to limit the use of gas anesthesia to 3 hours since longer anesthetic episodes are associated with increased negative effects.

The choice of anesthetic can significantly influence cardiac function. Differences have been found between injectable and inhalation anesthesia in heart rate, cardiac output, and fractional shortening.  Transgenic and wild-type animals may have differing sensitivities to anesthetics.

Electrocardiograms (ECG) and body temperature should be monitored during imaging procedures.  ECGs can be affected by an animal’s position, lead attachment(s), and number of leads, noise suppression, and amplifier bandwidth. Body temperature influences heart rate and cardiac function.  Use of heating devices is often indicated, however, they can interfere with ECG signals.

All equipment near the magnet of an MRI must be nonmagnetic.

Animals must be properly restrained to reduce motion artifacts.  In addition to the use of anesthesia, animals may be restrained with bite bars, immobilizing splints, cradles, and medical tape.

Gating 
Normal motion associated with breathing and cardiac function can produce image artifacts.  “Gating” reduces the motion artifacts by synchronizing image acquisition with the cardiac cycle or respiratory pattern.  To facilitate respiratory gating, animals may either 1) be intubated and a pressure transducer placed in the breathing system or 2) an external pressure sensor be applied to the abdomen to detect respiratory movement.   The external pressure sensor is difficult to use if respiratory rate is unstable.

In ‘retrospective gating,’ image data collection occurs continuously with the corresponding physiological data and the projection data are retrospectively sorted based on where the images fell in the cardiac cycle.  It has the advantage over prospective gating of enabling imaging of all time points in a cardiac cycle during one scan.   However, it has the disadvantage of less motion control than prospective gating.

Ultrasound
The basic components of a U/S system are a transducer, a computer, and a monitor.  The acoustic impedance is the product of transmission velocity and tissue density.  Sound waves are reflected at the boundary between differing tissue densities.   U/S modes include
1	b-mode – 2D cross-section view of target in real time
2	m-mode – the image generated by a single ultrasound beam is displayed over time
3	Doppler – used to determine the speed and direction of blood flow.

For U/S, animals must be prepared by
1	Removing hair
2	Application of coupling gel (usually)
3	Positioning and securing the animal’s position

It is possible to image unanesthetized mice.  Acclimating the animal to restraint and imaging may be necessary.  The influence of induced stress must be considered.

U/S also has the advantage of usually requiring short scan times however, it involves a certain level of subjective interpretation by someone experienced with the modality.

MicroCT
“A projection image is produced from radiation, transmitted from an X- ray source, that is attenuated by the tissue as it passes through the  body and then picked up by a detector that converts the signal to an  image.  A CT scanner produces 3D images by taking x-ray projection  views, acquired at hundreds of equally spaced angular positions  around the animal, and reconstructing them by means of a back- projection algorithm.”  The X-ray attenuation is proportional to the  density of the tissue.  Therefore different tissue types are assigned  differing grayscale values.

MicroCT cannot easily differentiate different soft tissue types  therefore, use of a contrast agent may be necessary.  The animal’s  exposure to ionizing radiation inherent to microCT must be considered  and is a limitation of the modality.

Magnetic Resonance
MR does not involve the use of ionizing radiation, but the generation  of magnetic resonance from spinning protons in the animal’s body.  MR  signal indicates two different relaxation times, T1 and T2.
1	T1 (recovery) = due to the nuclei giving up energy to the  surrounding environment
2	T2 (decay) = due to the nuclei exchanging energy with each other.

Advantages of MR =
1	High tissue contrast
2	Ability to acquire 3D images
3	Capacity for functional imaging.
4	Potential for multiple mouse imaging

Disadvantages of MR=
1	Cost of equipment
2	Limited availability of systems for use with rodents
3	Cost and resources needed to maintain a facility
4	Depending on the study, need for long scan times.

NOTE:  Table 1 in the article provides a succinct comparison of the advantages and disadvantages of U/S, microCT, and MR use with rodents.

QUESTIONS:
1.  	List 4 factors related to animal preparation that can influence  cardiac imaging.
2.  Gating can be used to reduce motion artifacts associated with  what two biologic functions?
3.  	You need to purchase some equipment that will be used very close  to the bore of an MRI.  What is one thing related to the equipment’s  composition that you must consider?
4.  	With which modality (U/S, microCT, MRI) is the animal subject  exposed to ionizing radiation?
5.  	T/F.  Ultrasound evaluation of mice requires that mice be  anesthetized at the time of the scan?

ANSWERS:
1.	Choice of anesthesia regime (injectable or inhaled), intubated or  free-breathing animals, physiological monitoring (ECG, respiration,  and temperature), and animal restraint.
2.  	Respirations and cardiac function
3.  	The presence or absence of any ferromagnetic metal that would be  drawn toward the  MRI’s magnetic
4.  	MicroCT
5.  	False, however image artifacts may be induced with any animal  movement and the effects of any stress induced by restraint must be  considered.

Driehuys et al.  Small Animal Imaging with Magnetic Resonance Microscopy, pp. 35-53

Introduction: Small animal magnetic resonance microscopy (MRM) is a valuable tool in noninvasive biomedical investigations and provides functional information about living animals and  high-definition anatomical images, with a resolution of 100 micrometers in all dimensions.  The extension of clinical MRI to MRM had required the development of specific technology as magnets, imaging coils, imaging acquisition sequences and biological support for small animals in high magnetic fields. Challenges in MRM are no direct access of the animal to the investigator, remote monitoring and delivering of agents, restrictions in the use of certain types of equipment and distortion of electrical recordings. However this technique provides valuable information as high tissue contrast and spatial resolution.

Technical basis: MRM signal source originates from the nuclear magnetic moments of the constituent atoms and molecules. The most common detected nucleus is the proton H, because of its abundance in tissues. The MR scanner  detects and spatially localizes these nuclei. A powerful static magnetic field is used to align these nuclei and determine their detection frequency. A radiofrequency coil surrounding the sample elicits and receives the nuclear magnetic signal. Gradient coils encode the spatial distribution of the nuclei. Finally, a computer synchronizes the application of radiofrequency pulses, switching of gradients, reading and digitization of signal, image reconstruction and image display.

MRM can be used in fixed specimens, increasing the achievable anatomic image resolution from 100 to 20 micrometers. It requires specialized specimen preparation techniques  to achieve maximum resolution, tissue/organ contrast and structural definition. MRM achieves fixation and staining with a single solution (Bouińs-Gadolinium or Formalin-Gadolinium). However, imaging the live small animal is one of the MRḾs most valuable advantage. Biological motion introduces a significant obstacle to microscopic resolution, so it is essential to control not only the gross body movement but also motion from cardiac and breathing activity, to reduce longer image acquisition times and image blurring. The animal preparation includes anesthesia (either injectable or inhalational), body temperature support and physiologic monitoring (body temperature, heart rate and breathing rate), and sometimes additional support such as mechanical ventilation. at the completion of the imaging, the animal’s recovery will be hastened by maintaining normal body temperature with heat lamps or heating pads, monitoring body temperature, aspiration of any excess fluid in the oral cavity and subcutaneous delivery of fluids.

QUESTIONS:
1.	True or false? MRM is an imaging technique that has been extended from magnetic resonance imaging that requires a tenfold increase in image resolution in all three dimensions and specific technology (magnets, imaging coils, imaging acquisition sequences and biological support for small animals in high magnetic fields).
2. 	The most commonly detected nuclei are:
a. 	1H, 3He, 13C, 17O, 19F, 23Na, 31P, 129Xe.
b. 	1H, 5O, 15C
c. 	In MRM only 1H is used.
d. 	In MRM, we only use non-native nuclei that have to be administered.
3. 	The first step of an MR scan is:
a.	To sacrifice the animal and fix the sample
b.	To place the sample in a magnetic field which provides an axis for the nuclei to align along or against.
c.	To anesthetize the animal with injectable anesthetics.
4. 	True or false? The use of perfusion-fixation methods employing contrast media increases the achievable anatomic image resolution of MRM from 1000 to 200 micrometers.
5. 	Fixed-specimen preparation for MRM....
a.	Requires specialized preparation techniques to achieve maximum resolution, tissue/organ contrast and structural definition.
b.	Only requires a PBS 1X embbebing of the sample.
c.	MRM can only be achieved with in vivo samples, because it required biological activity to obtain images.
6.	Animal preparation for MRM involves:
a.	Anesthesia, body temperature support and physiologic monitoring.
b.	Simple preparation, because it could be done with awake animals.
c.	Only anesthesia, because this technique needs a low metabolic activity of the animal maintaining body temperature below 35ºC.

ANSWERS:
1.	True
2.	a
3.	b
4.	False
5.	a
6.	a

Hutchins et al.  Small Animal PET Imaging, pp. 54-65

Positron Emission Tomography (PET) measures the in vivo distribution of positron-emitting radionuclides that have been used to label imaging agents.  The principle goal of PET is to characterize biological processes in tissues using these radioactive tracer agents/biomarkers.

Physics of PET Imaging (production of annihilation photons)
1.	Proton-rich radionuclides undergo positron decay (emission of a positron and conversion of a proton to a neutron)
2.	The positron combines with a nearby electron to form a positronium
3.	The positronium rapidly produces 2 annihilation photons (gamma rays) that travel in nearly 180 degrees opposition
4.	The gamma rays are detected by radiation detectors (inorganic scintillators) to generate a line of response to pinpoint the source of the radionuclide

PET scanner performance is determined by:
1	Efficiency of the inorganic scintillation detectors at interacting with annihilation photons
2	Spatial resolution of adjacent annihilation photons
3	Ability of the detector to determine the time of photon arrival (resolving ability)
4	Background noise - ability to differentiate annihilation photons from other lower energy photons

Ideal qualities of imaging agents:
5	Administered at low concentrations to prevent perturbation of the biological processes under study
6	Tracer interaction with the protein target reflects biology (saturability, specificity, stereoselectivity, affinity, retention, accumulation)
7	Biodistribution allows delineation of target tissue from surrounding tissues
8	Resistant to metabolism to prevent confounding the study through the presence of multiple metabolites
9	Low toxicity
10	Interact at rate limiting step in biological process

QUESTIONS:
1.	Match the PET tracer with the most commonly used study objective.
a.	[F-18] fluoro-2-deoxy-D-glucose		      i.   Bone Studies
b.	[F-18] NaF					      ii.   Blood pool imaging
c.	[C-11] carbon monoxide			      iii.  Brain and myocardial studies
2.	Explain what pathway FDG is associated with.

ANSWERS:
1.	a-iii, b-i, c-ii
2.	Glycolytic pathway - phosphorylation by hexokinase results in entrapment within the cell

Dunn and Sutton.  Functional Studies in Living Animals Using Multiphoton Microscopy, pp. 66-77

RDD- Tasks 1, 3, 9, 10
All species

SUMMARY: Although in vivo imaging techniques such as MRI, PET and CT are being used for laboratory animals to address characterization of physiological processes and tissue distribution of drugs and their targets, these techniques will not resolve critical events that are happening at the cellular or sub-cellular level. In vivo fluorescence microscopy is an effective way to do this. Another advantage to this technique is that with the use of multiple fluorophores multiple parameters can be tested at the same time.

In vivo microscopy has been performed for 200 years by the recent development of the multiphoton microscopy is capable of collecting high resolution fluorescent images hundreds of microns into tissues. It is also significantly less toxic to mammalian cells.

Applications of in Vivo Multiphoton # microscopy

Brain Function: Investigators can label 2/3 of pyramidal neurons with a calcium-sensitive fluorescent probe, and conduct multiphoton microscopy through a cover glass attached to the brain of anesthetized rats, with a craniotomy. This technique has also been effective in identifying calcium dynamics in astroglia and neuronal networks. Injecting bulk fluorescent tracers into the vasculature really makes monitoring blood flow and subsequent brain function easier. Studies can be performed on the same animals over a period of months allowing for studying of aging changes in the brain, and neural remodeling. This technique is also very useful for studying induced type of brain injuries.

This microscopy technique has been very useful in the study of Alzheimer’s disease to evaluate amyloid deposition in the brain.

Skin Structure and Function: As skin is easily accessible it is a logical target for imaging. However, in practice most published studies are using ex vivo skin preparations.

Autofluorescence from endogenous compounds in the skin such as NADPH and elastin coupled with secondary harmonic generation from collagen fibers allows examination of the skin without any exogenously administered fluorescent probes. A possible drawback is that absorption of single-photon infrared light by melanin granules present at the dermal-epidermal junction can cause cavitation and damage to skin  In 2004 refinements were made to the delivery of laser pulses and multiphoton ( MP) microscopy is well suite to provide a non-invasive “optical biopsy” of the skin .

Skeletal Muscle and Function: Skeletal muscle can also be easily accessed by a simple incision in the skin, making it another good target for the use of multiphoton microscopy.

Several studies have use the anterior tibialis muscle in mice. Many studies involving mitochondrial function, uptake of Calcium during contraction have been performed.

Tumor Biology: Tumors that have been implanted in lab animals in subcutaneous locations are also easy to image using MP microscopy. Studies have been done on the behavior of cancer cells during metastasis. In vivo MP microscopy has also provided crucial insights into the relationship between immune cells and tumor cells.

Immune Cell Biology MP: microscopy has been able to provide analyses of immune cell trafficking and interactions deep within lymph nodes. Studies have been done both in explanted lymph nodes as well as in living animals with lymph nodes exposed.

Visceral Organ Structure: The organ which has been studied the most with multiphoton microscopy is the kidney. Motion in the heart and lungs make these organs more difficult to image.

Limitations to in Vivo Multiphoton Microscopy: The first challenge is to generate contrast through fluorescence. This usually requires fluorescent labeling of a specific cell or compartment. One solution to this is to use transgenic animals that express fluorescent proteins in specific types of cells. Viral infection and in vivo transfection are also means for expressing fluorescent proteins in experimental animals.

Depth of Imaging by Multiphoton microscopy: The working image of the microscope objective is the limiting factor. It has been measured at 150 microns in the kidney and no more than 1000 microns in the brain.

Speed of Imaging: Due to technical difficulties at the present time scanning these images results in a capture rate of one to tow frames per second.

Future prospects for in vivo Multiphoton Microscopy: Presently this powerful technique is being driven by a few labs. The technology has developed to the point where commercial MP systems are as simple to use as confocal microscopes. It is reasonable to expect that in the next 10 years there will be a proliferation of in vivo MP microscopy as new methods of fluorescent labeling of cells in animals are developed.

QUESTIONS:
1.	What capabilities does MP microscopy have that differentiates it from MRI, PET and CT? 
a.	Can image to a deeper level in the body 
b.	Can resolve critical events that occur at cellular and subcellular level 
c.	Requires no anesthesia of surgery on live animal 
d.	Completely non-toxic to animal used 
2.	This paper states that a preponderance of the MP microscopy done in brains has been to support research involving which human disease? 
a.	HIV 
b.	Parkinson’s Disease 
c.	Alzheimer’s Disease 
d.	Lou Gehrig’s Disease 
3.	A disadvantage to MP microscopy compared to a PET scan is that…. 
a.	While they both require anesthesia of the animal MP may also require a surgical incision 
b.	It cannot be used to assess the mechanism of toxicity in the kidney of a specific drug 
c.	They both require anesthesia and use of radioactive compounds 
d.	It cannot characterize the variations in vascular permeability that occurs at the single cell level? 

ANSWERS: 
1. 	B. 
2 	C 
3. 	A

Levenson et al.  Muliplexing with Multispectral Imagining: From Mice to Microscopy, pp. 78-88

RDD Task 3: Provide Research Support, Information and Services
Species: Mouse and Rat
 
SUMMARY: In vivo imaging modalities can be categorized as traditional anatomic imaging (e.g. ultrasound, radiography, MRI etc.) and functional imaging, which can examine dynamic physiologic and pathologic processes. Optical functional imaging includes bioluminescence, in which an enzymatic reaction is produced (light producing construct inserted and enzymatic substrate administered) and detected, and fluorescent imaging, in which an external light source is used to stimulate emission from a fluorescent probe. Photoacoustic imaging, in which sound waves caused by rapid light induced expansion of absorbers are detected, is also in development. Bioluminiscent imaging offers the advantage of low background signals although the absolute light levels generated are low. The benefits of fluorescent imaging include permission of a wider range of probes (500-950 nms) and multiplexing (or the simultaneous imaging of multiple processes flagged by different probes). Major challenges to optical imaging include limited image depth and fluorescent imaging has the added disadvantage of inciting autofluorescence, which can make image analysis difficult. 
 
The Maestro in vivo imaging system includes a cooled megapixel CCD camera, a Xenon light sources with spectral filters, a heated stage and anesthesia manifold and a light-tight enclosure. Sophisticated software allows the separation (or uncoupling) of multiple fluorescent signals.  Data is semi-quantitative in that sophisticated post-imaging multispectral analysis can yield quantitative information. Problems of autofluorescence of hair, skin and food can be helped by animal preparation (clipping and cleaning area to be imaged) and the feeding on non-fluorescent diets. A similar system is available for microscopic imaging and both systems can be used both in vivo and in vitro. 
 
QUESTIONS:
1. 	Advantages of fluorescent imaging include all of the following except: 
a.	A wide range of probes can be imaged
b.	Multiplexing is possible
c.	Fluorescent probes in deep tissues can be easily visualized
d.	Fluorescent probes can be attached to cancer cells, antibodies, etc.
e.	All of the above are advantages of fluorescent imaging
2. 	True or False? Feeding non-plant based rodent chow prior to imaging is an effective way to reduce complicating autofluorescence in fluorescent imaging.
3. 	Bioluminscent imaging is _____ sensitive then fluorescent imaging.
4. 	Disadvantages of bioluminescent imaging include which of the following?
a.	Substrates must be given at a specific time prior to imaging studies
b.	In vitro imaging is not possible due to the need for an enzymatic reaction in an in tact organism.
c.	Low levels of background noise
d.	Relatively complicated manipulations are required to insert light producing constructs
e.	All of the above are disadvantages of bioluminescent imaging
5. 	True or False? It is not necessary to clip animal subjects before optical imaging.
 
ANSWERS:
1. 	c; light signals are attenuated in deep tissues in fluorescent (and bioluminescent) imaging
2.	True; chlorophyll fluoresces and feeding non-plant based diets can reduce background noise in fluorescent imaging.
3. 	More; bioluminescent imaging can detect small numbers of light producing cells at early time points
4. 	a, b, and d; relatively low levels of background autofluorescence is an advantage of bioluminescent over fluorescent imaging
5. 	False; hair removal by clipping or depilatories is recommended prior to bioluminescent or fluorescent imaging b/c hair is autofluorescent.

Nader and Czoty.  Brain Imaging in Nonhuman Primates: Insights into Drug Addiction, pp. 89-102

Task 9 - Collaborate on the selection and development of animal models

Primary species - macaques (secondary species - squirrel monkeys is also mentioned in one reference [Czoty PW, et al. J Pharmacol Exp Ther 2002;300:831-837]).

SUMMARY: The purpose of the review article was to describe in vivo brain imaging techniques used in nonhuman primate (NHP) models of drug addiction and provide basic background information on in vivo imaging techniques. 

Introduction
Advantages to use of in vivo brain imaging in NHPs include the ability to image experimentally naïve animals before drug exposure to assess trait variables (whether a finding is a pre-existing characteristic predictive of an outcome) and to repeatedly image the same NHP throughout a study to determine whether a finding was changed by an independent variable (whether the independent variable is a state variable). Compared with rodents, NHPs are more similar to humans in the pharmacokinetics and metabolism of the mu opiate receptor antagonist naltrexone, the serotonin-releasing drug fenfluramine, and 3,4-methylenedioxymethamphetamine. Cocaine-induced changes in brain metabolism in NHPs are different from that in rodents. Nonhuman primate societies have complex social behaviors similar to human social behaviors that enable investigation of the influence of social variables on sensitivity to abuse-related effects associated with drug use.

Positron emission tomography
Positron emission tomography (PET) permits in vivo examination of brain function. Injection of a radioactive PET ligand is followed by decay of the ligand, which produces positrons that randomly travel in space until collision with an electron; this collision results in formation of 2 gamma-ray photons that project at 180 degrees. Detectors in PET cameras are arranged in a ring around an animal. Stimulation of 2 detectors at 180 degrees results in a 3-dimensional location of the collision and thus the radioactive molecule or tracer, which produces a 2-dimensional image of a slice through the brain. Frequently used radiotracers include 15^O (half-life, 2 minutes [15^O water is used to assess blood flow]), 11^C (half-life, 20 minutes), and 18^F (half-life, 110 minutes).

Analyses of PET images require use of magnetic resonance imaging (MRI) to identify regions of interest (ROIs). The PET images are superimposed on magnetic resonance images and ROIs are used to measure tissue content of a radioactive molecule. For receptor ligand studies, analysis of PET data follows a 3-compartment model: radioactivity in blood, in the extracellular space of the brain, and bound to a receptor. Rates of movement of a tracer between compartments are analyzed to generate a distribution volume (DV) in an ROI, which is a measure of the percentage of injected dose of radioactive substance across time. To quantify and compare PET data, the DV for ROI is considered in relation to a control region containing few receptors bound by ligand; the ratio of these DVs is the distribution volume ratio (DVR), which is the primary dependent variable in most PET studies. A related dependent variable is the binding potential (BP), defined as the DVR-1. These dependent variables are unit less numbers that represent the ratio of receptor density to affinity for a receptor. Changes in DVR may result from changes in receptor density or receptor affinity; this is considered an advantage of PET because it provides a measure of receptor binding in a living brain (i.e., receptor binding is assessed in the presence of neurotransmitters that can compete with the radioligand). 

Single photon emission computed tomography: Single photon emission computed tomography (SPECT) is an imaging technique similar to PET; however, SPECT studies use radioactive tracers with long decay times. Additionally, these tracers emit single photons instead of pairs of photons; therefore, SPECT is less sensitive than PET and SPECT images are less detailed. Compared with PET, SPECT is less expensive and easier to implement.

Conscious versus unconscious animals: In imaging studies, main issues of concern include whether an NHP should be anesthetized or remain conscious during the procedure and whether the anesthetic agent used may influence the dependent variable. The authors recommend performing within-subjects, repeated-testing experiments to determine the best anesthetic to use. The authors also caution that changes in blood flow during PET studies in conscious and unconscious animals have not been examined.

Correlating neurochemistry with behavior: Positron emission tomography can be use to examine neurochemical mechanisms underlying changes in behavior and in the PET signal via 2 methods: administration of nonradiolabelled compounds can indirectly affect concentrations of neurotransmitters to provide information concerning interactions of a PET tracer with extracellular neurotransmitters and PET data can be correlated with behavioral measures known to be sensitive to neurotransmitter concentrations, thus providing a better understanding of mechanisms mediating behavior. Several examples for each method are provided in the article.

Other imaging modalities
Techniques based on MRI offer advantages of increased spatial (1 mm) and temporal (seconds) resolution, compared with PET and SPECT. Additionally, because MRI techniques do not require use of radioactivity, it is safer and the number of times an NHP can be studied is unlimited, compared with PET and SPECT.

Perfusion techniques: Two MRI-based techniques used for examining cerebral blood flow include dynamic susceptibility contrast (DSC) MRI and arterial spin labeling (ASL). With DSC MRI, contrast is provided by injection of an exogenous paramagnetic agent before imaging. In ASL, labeling protons in arterial blood upstream of a brain ROI provides contrast. Both techniques may be useful in longitudinal studies of the effects of cocaine exposure on cerebral perfusion or within-subject studies of pre-existing traits in individuals vulnerable to abuse-related effects of cocaine or consequences of long-term cocaine use.

Functional and pharmacologic MRI: Functional MRI (fMRI) provides high spatial resolution for mapping brain function. Functional MRI is based on neurovascular coupling, the local increase in blood flow that occurs providing energy to brain regions in which activity has increased in response to a stimulus. Increased perfusion results in an increase in the ratio of oxygenated to deoxygenated hemoglobin. Deoxygenated hemoglobin distorts the magnetic field, which can be detected by use of appropriate parameters resulting in a type of contrast that is dependent on the homogeneity of the magnetic field in each voxel. The intensity of signal in each voxel is modulated by blood flow (called blood oxygen level-dependent [BOLD] contrast). The BOLD signal is superimposed on an anatomic map to localize the response with excellent spatial resolution; pixel intensity represents the strength of correlation between the BOLD signal and presentation of a stimulus. Various colors correspond to positive and negative correlations. Functional MRI has been used in studies investigating the visual system in NHPs; indicating that use of fMRI in conscious NHPs is feasible.

Use of BOLD contrast to study effects of drugs that interact with brain dopamine, serotonin, opioid, glutamate, GABA, and cannabinoid systems in rodents is referred to as pharmacologic MRI (phMRI). In NHPs, phMRI has been use to characterize alterations in function of brain dopamine systems in association with aging and models of Parkinson's disease. 

Limitations of fMRI and phMRI include an incomplete understanding of the association between neuronal activity and blood flow changes, interpretation of data is limited by relying on relative rather than quantitative measures, the possibility that drugs can influence vascular function independent of neural activity, age-related changes in perfusion are not completely understood, a low signal-to-noise ratio, sensitivity to subject movement during imaging, and uncertainty concerning interactions between the drug of interest and anesthesia in anesthetized subjects.

Diffusion tensor imaging: Diffusion tensor imaging (DTI) is used to examine the integrity of axonal projections by measuring the direction of water diffusion in the brain. In gray matter, water tends to diffuse randomly (isotropic diffusion). In white matter, axonal membranes and myelin sheaths restrict diffusion of water. White matter is tightly packed into directionally oriented fiber bundles; therefore, water tends to diffuse along a vector parallel to a fiber tract (anisotropic diffusion). By use of appropriate MRI parameters, the extent to which water flows along or perpendicular to a fiber tract can be described. The tendency of water to flow along a fiber tract rather than perpendicular to it is determined by the amount of resistance to flow provided by an axonal membrane and myelin sheath. Variations in fractional anisotropy (FA) describe reductions in anisotropy that may be associated with underlying pathologic lesions. 

Compared with ex vivo tract-tracing methods, DTI is less time consuming and multiple experiments can be performed in an NHP, making longitudinal studies possible. Limitations include a lack of standard methods of voxel placement for statistical analysis, an incomplete understanding of confounding variables, differences in topography of fiber tracts in individual NHPs, sensitivity to movement, a low signal-to-noise ratio, and disturbances in magnetic field in areas where brain and bone interface. 

Magnetic resonance spectroscopy: Magnetic resonance spectroscopy (MRS) provides information about concentrations of chemicals in discrete brain regions by taking advantage of differential sensitivity of atomic nuclei in various molecular environments to an applied magnetic field to provide relative or quantitative measures of concentrations of specific metabolites. Most MRS studies use the abundant hydrogen nucleus. Magnetic resonance spectroscopy has been used to examine pharmacologic effects of abused drugs and characterize the effects of long-term drug use on brain function. The most relevant neurotransmitters that can be measured with MRS are GABA and glycine. Other components that can be measured include N-acetyl-aspartate (a neuron-specific component of myelin synthesis), choline (concentrations of which include several phospholipids involved in synthesis of cell membranes), and creatine. Decreases in concentrations of these components may represent cell loss or decreases in metabolism or demyelination, or both.

Electroencephalography and magnetoencephalography: Electroencephalography (EEG) and magnetoencephalography (MEG) are used to directly measure electromagnetic signals resulting from neuronal activity and provide temporal resolution in the range of milliseconds. Electroencephalography provides the most direct measure of neuronal activity possible with noninvasive techniques; however, these signals are distorted as they pass through brain tissue, skull, and skin, which complicate the point of signal origin (poor spatial resolution [cm]).

Magnetoencephalography can be use to detect small magnetic fields generated by electrical currents produced by neuronal activity without distortion; therefore, localization of the origin of activity is easier and more accurate with MEG than with EEG, and MEG provides excellent temporal resolution.

Conclusion 
The greatest advantages of in vivo brain imaging techniques in NHPs include use of within-subjects study designs and the ability to perform longitudinal studies. In vivo imaging of animal models of human disease will provide a basis for understanding genetic predispositions and environmental modulation of many human diseases and may be use to evaluate treatment strategies.

QUESTIONS:
1. 	Define PET and briefly describe how an image is produced with this technique.
2. 	Why is anesthesia a concern when performing imaging studies in NHPs?
3. 	Other than PET, name 3 methods of in vivo brain imaging that has been used in NHPs.
4. Why is the ability to perform longitudinal studies and noninvasive techniques important?

ANSWERS:
1. 	Positron emission tomography: Injection of a radioactive ligand is followed by decay of the ligand, which produces positrons that randomly travel in space until collision with an electron resulting in formation of 2 gamma-ray photons that project at 180 degrees. Stimulation of 2 PET camera detectors at 180 degrees results in a 3-dimensional location of the collision and thus the radioactive molecule or tracer producing a 2-dimensional image of a slice through the brain.  
2. 	Because the anesthetic agent used may influence the outcome of the study.
3. 	Any 3 of the following: single photon emission computed tomography (SPECT), dynamic susceptibility contrast (DSC) MRI, arterial spin labeling (ASL), functional MRI (fMRI), pharmacologic MRI (phMRI), Diffusion tensor imaging (DTI), magnetic resonance spectroscopy (MRS), electroencephalography (EEG), and magnetoencephalography (MEG).
4. 	The ability to perform longitudinal studies (i.e., one animal can be used for multiple studies) and noninvasive techniques represent reduction and refinement of an animal model.

Zinn et al.  Noninvasive Bioluminescence Imaging in Small Animals, pp. 103-115

SUMMARY: Molecular imaging using bioluminescence requires the use of a reporter construct encoding either firefly luciferase or Renilla luciferase.  Firefly luciferase uses D-luciferin as a substrate delivered via IV or IP injection or via osmotic pump or by addition to the drinking water to provide 30-60 minutes of stable light emission.  Renilla luciferase uses coelenterazine analogs for substrate and can be used together with firefly luciferase with minimal interference.  Light emission can be measured via relative units (counts of photons per unit time) or absolute units of radiance (photons/second/square centimeter/steradian).  The absolute number is independent of animal position or camera settings and allows comparison between systems and experiments.

Bioluminescence has been used in the following research applications:
1	Cancer biology:
o	Tumor size is correlated with light emission 
o	Luciferase positive cells can be detected shortly after implantation – prior to detection by palpation 
o	Metastasis of cells can be determined by tracking foci of light intensity 
2	Immunology and Infectious disease: 
o	Tissue distribution of viral vectors or pathogens 
o	Bacterial amounts (peak infection levels and clearance of the pathogen) 
o	Tracking of naïve T-cell populations 
3	Tissue specific gene expression 
o	Detection of apoptosis: Activation of luciferase by caspase 3  (active only during apoptosis) 
o	In vivo signaling: using a luciferase reporter of signal transduction and activation to identify signaling pathways 

Sensitivity can be influenced by the following:
1	Instrumentation and imaging parameters 
o	Sensitivity is increased as the animal is placed closer to the CCD camera, during longer acquisition times, and higher binning 
2	Presence of hair 
3	Tissue depth of luciferase expressing cells 
4	Level of luciferase expression from cells 
5	Amount of substrate administered 
6	Diet induced background phosphorescence 
7	Animal body temperature 
o	Anesthesia induced hypothermia will decrease light emission 

QUESTIONS:
1.	What enzyme and substrate are required for bioluminescence? 
a.	Beta-galactosidase and Xgal 
b.	Beta-galactosidase and luciferase 
c.	Luciferase and luciferin 
d.	Luciferase and oxyluciferin 
e.	Luciferase and Xgal 
2.	What other molecules are required in addition to the substrate for light transmission? 
f.	ATP and calcium 
g.	ATP and magnesium 
h.	Calcium and magnesium 
i.	Oxygen and calcium 
3.	True or False.  Bioluminescence tends to have a lower background signal than fluorescence. 
4.	What dietary material should be eliminated from mouse chow to reduce spontaneous phosphorescence? 

ANSWERS:
1. 	c
2. 	b
3. 	T
4.	Plant material (replaced by casein)

Roe and Chen.  High-Resolution fMRI Maps of Cortical Activation in Nonhuman Primates: Correlation with Intrinsic Signal Optical Images, pp. 116-123

Species: Squirrel monkey, secondary species

SUMMARY: BOLD fMRI is blood oxygen-dependent functional magnetic resonance imaging and is the most widely used functional brain mapping tool.  Its ability to map with high spatial resolution is untested however.  Other methods of studying the brain include single- or multiple-neuron electrophysiology which have high spatial resolution but are limited in spatial scale.  Brain imaging modalities such as PET, fMRI, etc. can image large areas of brain simultaneously but lack spatial resolution.  BOLD fMRI allows neuroscientists to monitor activity in many brain regions and study how these areas participate in sensation, motor activity and perception.  Neurons that are active increase their oxygen consumption which leads to changes in blood oxygenation.  BOLD fMRI detects this as differences between the magnetization of oxy- and deoxyhemoglobin.

Millimeter and submillimeter scale brain imaging currently requires optical imaging of intrinsic signals (OIS) that is based on activity-dependent changes in reflectance of cortical tissue.  This requires a physical “window” on the brain and is limited to surface activations.  This method has very high spatial resolution (~100um).  Voltage-sensitive dyes are used the same way but may be associated with phototoxicity to living tissues.  

This study investigated whether BOLD fMRI could resolve millimeter sized activations and if it correlates to results obtained by optical imaging techniques.  They conducted somatosensory mapping experiments in anesthetized squirrel monkeys.  BOLD fMRI showed consistent activation size and topography with previous studies with proven techniques.  Images from the BOLD fMRI and OIS of the same animal showed a high degree of alignment.  They concluded that fMRI methods can achieve high spatial resolution.  They also showed that the method could differentiate spatial events of 0.5mm proving that fMRI does have submillimeter spatial resolution.

QUESTIONS:
1.	What is one strength of the following imaging modalities: a) Single-electrode recording? b) fMRI? c) optical imaging?
2.  	What neuroanatomical feature of squirrel monkeys makes them good animal models for neuroscience research?
3.  	List the three factors the authors' feel contributed to their success.

ANSWERS:
1. a) High spatial and temporal resolution (limited to superficial cortical structures, invasive technique); b) can monitor any region of the brain including deep structures, non-invasively (limited spatial resolution); c) high spatial resolution (limited to superficial surface structures, invasive technique).
2.  The have relatively flat cortices making them ideal for surface imaging techniques such as optical imaging as compared to species with numerous sulci and gyri.  
3. 1) The subtle nature of the physical stimulus used that resulted in very little contamination of the signal; 2) High magnetic field used; 3) attention to fine-tuned anesthesia.