ILAR 43 (3)

Introduction. ILAR 43 (3): 121.
[Reviewer's Note - Any parts of this introductory article which recapped specific articles published in this ILAR issue were not reviewed as other LABSG members will be summarizing these articles for the LABSG list.] Rats and mice comprise more than 80% of the animals used in biomedical research, teaching, and testing. Use of rodents in biomedical research will continue to increase with the use of knockout technology, advances in genomics, and advances in surgical and anesthetic techniques, coupled with technological advances in electronics. Due to these technologies and advances, the mouse and perhaps the rat are re-emerging as the most versatile and valuable tools for scientists over the next few decades. Scientists will need relevant animal models and in vivo systems to validate scientific advances in molecular biology, genomics, and stem cell research. Ironically, rodent use will increase until new scientific disciplines and targets are sufficiently validated to allow us to eventually reduce and replace research animals. Continual refinement of rodent models allows researchers to gain more meaningful information from even just 1 mouse in real time. Conventional animal models in species such as dog and nonhuman primate can now be refined or replaced through miniaturization of these models using rats and mice. This strategy facilitates acceleration of the translation of new scientific knowledge into breakthroughs in medicine/surgery for both humans and animals. Miniaturization allows other animal alternatives through reduction and refinement.
1. Name 2 stocks of mice commonly used as pseudopregnant recipient females in the production of transgenic mice.
2. Name a mouse strain popular for both pronuclear microinjection and recipient blastocysts for embryonic stem (ES) cells.
3. Which method can be used to produce gene targeted "knock-in" or "knock-out (KO)" mice?
a. Pronuclear injection of DNA into a 1-cell embryo
b. Injection of modified ES cells into blastocysts
c. Retrovirus-mediated gene transfer
d. Any of the above
4. Describe 1 method for selection of ES cells that have successfully undergone homologous recombination.
5. True/False. Homologous recombination of a transgene construct is a common event.
1) ICR, CD-1;
2) C57BL/6 (FVB and SWR also used extensively for pronuclear microinjection);
3) B [A is random integration];
4) Most common technique is a "positive-negative" selection for neomycin resistance and HSV-TK (Herpes Simplex Virus-thymidine kinase) genes. The transgene construct contains both of these markers but is designed such that if successful homologous recombination occurs, the positive marker (neomycin resistance) is incorporated into the ES cell and the negative marker (HSV-TK gene) is lost. Exposure of ES cell cultures to neomycin kills cells that did not integrate the neo gene. By adding a negative selectable marker (e.g. HSV-TK) that is lost during homologous recombination, you can expose the cells to gancylovir and eliminate all cells that integrated the transgene in a non-homologous fashion.
5) False. It's a relatively rare event (1:10,000 1:25,000). This is why you need a screening system (i.e. PCR, survival selection techniques with positive-negative markers) to detect which cells had homologous recombination.

Understanding the human condition: experimental strategies in mammalian genetics. ILAR 43 (3): 123.
Genetically defined and engineered mice have become extremely valuable in biomedical research for many reasons. Mice are relatively inexpensive to maintain and have a high reproductive rate. Many technologies have been developed to analyze and manipulate the mouse genome. As a result, numerous inbred and/or mutant strains have already been produced. The mouse's biology and genome has been found to be very similar to that of humans allowing us to relate the normal function and pathologies of the mouse's immune, endocrine, nervous, cardiovascular, skeletal, and other complex physiological systems to that of humans. Manipulating the mouse genome and environment can often induce conditions of interest that do not occur naturally in mice. In addition, mice can serve as hosts for both normal and diseased human tissues.

Two different yet complementary approaches have been used as genetic research strategies in the mouse: phenotype-driven and genotype-driven.

Phenotype-driven approaches examine phenotypes and attempts to identify the genes responsible for them. This approach examines spontaneous single gene mutations, induced mutations, and natural polymorphisms.

Single gene, point mutations were studied extensively early in the field of genetics. However, examination of spontaneous single gene mutations can be complicated by the action of modifier genes that may significantly affect the expressed phenotype. The importance of modifier genes became evident through examination of "obese, ob, and diabetes, db mutations, which originally arose in the C57BL/6J (B/6) and C57BLKS/J (BKS) mouse strains, respectively….[T]hese mutations originally caused distinct phenotypes, ob, causing obesity, and db, causing both noninsulin-dependent diabetes and obesity. However, this difference in phenotype actually reflected differences in the genetic background of the inbred strains and not the mutations themselves." Therefore, researchers now place mutations on different genetic backgrounds to search for modifiers.

Mutational analyses of physiological pathways can be substantially accelerated through the use of mutagens (ex. ethynitrosourea) which increase the natural mutation rates by a thousand-fold or more. However, mutagenesis projects designed to search for specific phenotypes can be hindered by the efficiency of cloning the induced mutations. The use of bacterial artificial chromosomes and the availability of the sequence of the human and mouse genome have aided in this laborious process.

Natural Polymorphisms
Naturally occurring polymorphisms (NPs) describe both natural variation and the genetic interactions important in most serious human diseases. They can identify critical control steps in complex physiological processes. Single nucleotide polymorphisms are being developed as genetic marker systems in mice. If successfully developed, they may substantially reduce the cost of genotyping. One drawback with NPs in contrast to singe gene mutations is the increased difficulty in positionally cloning NPs.

Evidence suggests that the same genes may be involved in the polymorphisms affecting disease susceptibility in both mice and humans. These genes likely code for proteins that act at critical steps in phenotype _expression. These proteins are attractive targets for therapeutic or preventative intervention. In addition, cross-species concordance for major disease QTL has very large economic implications as identifying the chromosomal location of specific DNA segments in mice can be much cheaper than in humans. Therefore, QTL are frequently located in mice and then their presence confirmed in humans.

_Expression Profiling
_Expression profiling provides a quantitative description of the _expression of thousands of genes at one time. Two _expression profiling systems are available including Affymetrix chips and deposited arrays. In the Affymetrix system, the DNA sequences of genes are deposited on chips by a process similar to photolithography. In the deposited array system, DNA sequences are deposited on glass slides. In both systems, fluorescence-labeled cDNA is hybridized to the arrays and the fluorescence is quantitated. Deposited arrays are cheaper and more easily reconfigured. _Expression arrays have been used to identify the cell types from which tumors originated. This is significant as prognosis and response to chemotherapy correlate with the molecular classification of tumors.

Genotype-driven approaches examine the affect of genetically altering specific genes and then examining the resultant phenotype. This approach often utilizes DNA transgenesis or knockout technology.

Transgenic animals are created by injecting a gene into the pronucleus of a fertilized egg where it can incorporate randomly into chromosomal DNA. Transgenic technology is used primarily to determine the functional anatomy of a gene. Since the location and copy number of the transgene is different in every recipient, the level of gene _expression is not uniform across different lines.

Knockouts techniques use homologous gene replacement to replace an endogenous gene with an altered copy of the same gene. This provides a uniform insertion point and copy number and preserves the adjacent DNA regulatory sequences. Initially, this technique was used to produce null mutants in whom the function of a gene was lost in all cells. However, it has now been shown that a functional redundancy exists in which alternative mechanisms can compensate for the loss of the gene. In addition, timed, tissue-specific deletions have been developed which allow investigators to examine the effects of a gene in a specific tissue or at a specific period in the animal's life.

Phenotyping as a limiting factor
While numerous advances have been made in the study and manipulation of genes, technical problems exist with phenotyping mice, especially at the physiological level. Due to their small size, phenotyping methods have to be modified or specifically designed for use in mice. Noninvasive imaging techniques are often used since they allow serial examination of the same animal, cause little to no trauma, and can be fast and simple.

Mouse behaviors can be especially difficult to phenotype because they are highly sensitive to environmental factors. As a consequence, seemingly identical behavioral studies performed by multiple laboratories can have significantly variable results.

Partitioning the Phenotype
Instead of attempting to examine a large, complex phenotype such as cancer, atherosclerosis, or diabetes, it may be easier to partition the phenotype and examine only one portion of the process.

Humanized Mice
In order to alleviate some of the difficulties and ethical constraints involved in human experimentation, one can introduce human genes, proteins, and tissues into animal hosts. This is possible due to the evolutionary conservation of genetic regulatory mechanisms.

The Future

"We can confidently expect rapid increases in the speed and efficiency of every research strategy that is sequence dependent, especially positional cloning and genetic engineering." Due to the large amount of information that can be gathered from genetic studies, some trends will likely develop. One trend is a move toward very large-scale experimental programs such as with multi-institutional consortia examining one specific area of interest. The second trend involves the huge increase in the demand for bioinformatics and computational biology needed to deal with data storage, retrieval and analysis. Lastly, as we increase our knowledge in multiple components of biological processes, researchers will move away from studying very narrow topics and toward a systems analysis approach.

1. What is the name of the major histocompatibility complex in mice? In humans?
2. What are the two complementary approaches used in genetic research in the mouse?
3. T/F Modifier genes can have significant impact on an expressed phenotype.
4. Describe the phenotype of the ob and db mutations in the mouse strains in which they were originally derived.
1. H2;HLA
2. Phenotype-driven and genotype-driven approaches
3. True
4. ob - obesity; db - noninsulin-dependent diabetes and obesity

Miniaturization: an overview of biotechnologies for monitoring the physiology and pathophysiology of rodent animal models. ILAR 43 (3): 136.
Recent advances in bioengineering technologies have made it possible to collect high quality reproducible physiological data quantitatively in a wide range of laboratory animal species, including rodents. Several of these technologies are incorporated into a plan called miniaturization, which aims to design, develop, and maintain rodent animal models to study the pathophysiology and therapy of human diseases. The focus of this article is to cover technologies that include laser doppler flowmetry (LDF), digital sonomicrometry, bioelectrical impedance, and microdialysis. These methods are some of the most widely used miniaturization methods because they cause minimal pain and distress, reduce the number of animals used in biomedical research, allow chronic, non-terminal assessment of physiological parameters in rodents and are cost-effective. An overview of each of these technologies and their major applications in rodents used for biomedical research is provided below.

LDF- Laser doppler flowmetry is a method for determining blood flow through tissues, capillaries, arterioles and venules. It is commonly used to monitor the effect of environmental conditions, physical manipulations and drug treatments on tissue perfusion. The basic operating principles of LDF is that a laser beam is directed to an area of tissue. Upon contact with the red blood cells in the target tissue, light waves are reflected and scattered, and the resulting in broadening of the light wave frequency is detected and received by a photodetector. The photodetector converts the light waves into an electrical signal that is processed to provide an estimation of blood flow called flux.

Two types of laser doppler instruments used to measure blood flow are single point fiber optic methods and laser doppler images. The single point fiber optic monitor is the instrumentation most frequently used in rodents. Noninvasive continuous measures can be made using skin surface probes and needle probes have been designed to penetrate tissues. Endoscopic probes are less invasive than needle probes and measure flow on mucosal surfaces. The laser doppler imager is a noninvasive system that measures flux in tissues without the use of fiber optic probes. In experiments where repetitive analysis of blood flow is required on the same animal, the region of interest should be marked to allow appropriate realignment and to ensure the same region is scanned. LDF requires the measurement tissue to remain stationary in relation to the laser beam; therefore anesthesia of the animal is often required. Careful selection of anesthesia agents is a must to ensure it will not adversely alter blood flow in the vascular bed to be examined. The measurement of regional cerebral blood flow in the rat is the most widely reported experimental use of LDF. The second most common application is in the area of angiogenesis. Some of the most common models that use this technique are the hamster cheek pouch, disc angiogenesis, matrigel, and mouse and rat limb ischemia. Of all the angiogenesis models described, the mouse and rat limb ischemia models are the most commonly studied.

Digital Sonomicrometry Digital Sonomicrometry is an ultrasonic measurement system that uses piezoelectric crystals to measure multiple distances within an aqueous medium or soft tissue. It has been used extensively for non-terminal assessment of cardiovascular function, as well as to generate pulmonary, gastrointestinal, urogenital and musculoskeletal measures. The three major components in a digital sonomicrometry system are the piezoelectric crystals, the sonomicrometer, and the computer acquisition system. The basic operating principle is a system of two or more crystals are implanted onto a tissue of interest. Electrical impulses are directed to a crystal resulting in oscillation and production of an ultrasonic signal. The signal is transmitted to additional crystals in the nehtwork. The crystals alternate between transmitting and receiving modes. Data acquired from system grow exponentially with the number of crystals implanted. The system has been used in animals as small as the mouse.

Bioelectrical impedance Bioelectrical impedance analysis is a rapid noninvasive technique for estimating fluid distribution and body composition. The application of this technique in biomedical research has been limited to rats, cats, dogs, sheep and the non-human primate. Biometrical impedance assumes that the geometry of the living body can be approximated as a simple cylindrical conductor in which low-voltage electrical currents are delivered to a single fixed frequency or multiple frequencies. It assumes that resistance or impedance to these currents is inversely proportional to the amount of total body water and lean body mass. Aqueous tissues such as lean muscle and fluids are considered excellent conductors while fat and bone have poor conductance properties. In determining fluid distribution and body composition, bioelectrical impedance assumes that intracellular fluid (ICF) and extracellular fluid (ECF) behave as conductors and that cell membranes and tissue interfaces act as imperfect capacitators. Total body impedance is related to the amount of water in the body. Multifrequency bioelectric impedance analysis (MFBIA) is the most frequently used instrument. Measures are made on an anesthetized animal placed on a nonconductive surface with electrodes placed subcutaneously in a tetrapolar arrangement. MFBIA is an excellent tool for estimating changes in the ICF and ECF.

Microdialysis Microdialysis is an analytical technique used to evaluate changes in the chemical composition of tissues. Microdialysis does not require the removal of body fluids, therefore, exogenous and endogenous compounds in the ECF can be evaluated without altering the fluid balance or disturbing blood homeostasis. Microdialysis sampling is performed by surgically implanting a probe into an organ or biological fluid of interest. Probes consist of a semi-permeable membrane in the formation of a hollow fiber member. Tubing is affixed to the inlet and outlet port of the probe and a perfusate similar to the ionic composition and pH of the medium sampled is infused through the interior of the probe membrane. During sampling, animals are placed into a tethering system so the animal can move freely. The solution that diffuses out of the probe is representative of the concentration of the ECF and is termed the dialysate. The dialysate is collected into a fraction collector or can be directly transported to an analytical system. Analytes that are typically measured with microdialysis include drugs and their metabolites, neurotransmitters, and amino acids. Several factors must be considered when performing microdialysis procedures. These factors include probe type and composition, perfusate flow rate and temperature, surgical recovery time, and length and composition of tubing. Intracerebral microdiaylsis is the most frequently reported microdialysis technique described in literature. However the technique has been quickly expanded to include applications in pharmacokinetic studies and toxicology studies where tissues like skin, kidney, liver and tumors are evaluated. Because homeostasis is not affected by the application of microdialysis, numerous samples can be taken from the same animal, decreasing the number of animals that must be used in studies. Microdiaylsis is the most affordable technique to monitor ECF spaces in unanesthetized animals.
1. What does LDF stand for and what is the most common use of the technique?
2. Which LDF instrument is most frequently used with rodents?
3. What is microdialysis?
4. What is bioelectrical impedance and how is it used?
1. LDF- Laser doppler flowmetry is a method for determining blood flow through tissues, capillaries, arterioles and venules.
2. The single point fiber optic monitor.
3. Microdialysis is an analytical technique used to evaluate changes in the chemical composition of tissues. Microdialysis does not require the removal of body fluids, therefore, exogenous and endogenous compounds in the ECF can be evaluated without altering the fluid balance or disturbing blood homeostasis. Bioelectrical impedance analysis is a rapid noninvasive technique for estimating fluid distribution and body composition. Measures are made on an anesthetized animal placed on a nonconductive surface with electrodes placed subcutaneously in a tetrapolar arrangement. Low-voltage electrical currents are passed through the animal and delivered to a single fixed frequency or multiple frequency receiver allowing resistance or impedance to these currents to be measured and used to estimate the amount of total body water and lean body mass.

Noninvasive cardiovascular phenotyping in mice. ILAR 43 (3): 147.
In this article the authors summarize various diagnostic technologies that they have adapted from human medicine for use in the phenotyping of
transgenic mice. As more genetically engineered animals become available, the need for fast and reliable means of phenotyping these animals
increases. The need to perform serial assessments on these animals as they develop and age has prompted the development of noninvasive
methods for assessing cardiovascular function.
The need for anesthesia in the manipulation of mice is the first obstacle that researchers in this field faced as all anesthetic agents affect
cardiovascular function in some way. The authors have used a variety of agents including isoflurane, etomidate and sodium pentobarbital which
they found maintained heart rates in mice at physiological levels. They also used an acepromazine/ketamine/xylazine combination that decreased
heart rate significantly but maintained blood pressure.
As many phenotypes are subclinical, experimental manipulation of the animal (e.g., induction of stress) is often required to observe the
phenotype. Some examples of manipulations that are used to expose the phenotypes of these mice include the following: the administration of
chronotropic, iontropic or vasoactive compounds; exercise and surgical manipulations (coronary artery occlusion, aortic constriction). The
ability to perform noninvasive diagnostic testing in transgenic mice enables researches to monitor the response to these interventions over time.
Imaging Methods
The imaging methods used for the monitoring of cardiovascular function in mice include MRI, x-ray angiography, ultrasound (2-D, M-mode and
Doppler) and nuclear angiography. The small size and rapid heart rate of mice have presented the biggest challenges in adapting these
technologies for use in mice. Currently M-mode ultrasound is the most commonly used noninvasive method for evaluating cardiac function in
mice With the highest temporal resolution at 1 msec, it has been used to calculate the left ventricular diameter throughout the cardiac cycle. One
of the largest drawbacks to all of these techniques is that they do not allow for the estimation of loading conditions or pressures which require
more invasive techniques.
Invasive Physiological Measurements
Most cardiovascular physiology studies are conducted using pressure, flow and dimension sensors plasced into vessels and organs of larger
animals. While there are manufacturers producing miniature pressure, flow, volume and telemetry sensors and instrumentation designed for use in mice, the use of these devices is currently limited by size, resolution, fidelity, accuracy and calibration concerns. The procedures involving the
use of these devices often involve complicated surgeries and they often can not be repeated in the same animal also limiting their usefulness.
Noninvasive Physiological Methods
ECG - The first ECG recordings were done in mice over 70 years ago. Since that time 6 and 12 lead ECGs have been done on both wild type
and transgenic mice to determine various time intervals and conduction velocities. The authors emphasize that maintaining the body temperature
of mice is critical but they also note that heating lamps and pads typically result in noise interference in the 60 Hz range. To avoid this, they have
developed a device that incorporates a low voltage heating unit into an ECG/restraint board. A picture of this device is included in the article â€"
refer to Figure 1.
Doppler - These instruments measure blood velocity by detecting the change in frequency between an emitted burst of ultrasound (10 - 20
mHz) and the returning echoes from moving blood. Systems used in mice have been developed by mounting 1 mm diameter 10 or 20 MHz
ultrasonic crystals at the end of 2 mm x 10 cm long stainless steel tubes. Currently a murine pulsed doppler system allows for a velocity resolution of 5 mm/sec at 20 MHZ, a mazimum measurable velocity of 9.3 m/sec at 10 MHz and a temporal resolution of 0.1 msec.
Nuclear Angiography - This technique uses 178Ta to assess right and left ventricular ejection fractions in mice using a human scale nuclear
camera with a pin-hole lens to obtain 2 mm resolution with frame rates of 160/sec. While the resolution is marginal, this technique has been used
to calculate ejection fractions in mice. This technique requires placement of a catheter for administration of the radionuclide but can be repeated in
30 minute intervals or longer allowing for serial calculations to be obtained.
Examples and Applications
The authors provide several examples of the experimental uses of the noninvasive technology discussed in the article. These include the following:
1) Use of ECG to monitor S-T segment changes before, during and after coronary artery occlusion to confirm ischemia during the procedure. For additional information refer to Figure 2 in the article.
2) Use of Doppler measurements to assess peripheral vascular impedance and monitor progression of atherosclerosis in ApoE-KO mice. With the use of doppler, these mice have also been shown to have elevated aortic and mitral velocities thought to be due to decreased cross-sectional areas or increased cardiac output combined with decreased total peripheral resistance.
3) Use of peripheral doppler measurements of blood velocity to determine arterial pulse wave velocity (PWV) as an indicator of arterial stiffness. As vessels become more stiff, they propagate pressure and velocity waves faster than more compliant vessels. Using this technology, the authors have shown increased PWV in ApoE KO mice (atherosclerosis model) and in matrix GL protein KO mice (calcified arteries). Studies in alpha smooth muscle actin KO mice show the PWV at rest is only slightly lower than that of wild type mice at rest; however, when administered a phenylephrine bolus, the KO mice show markedly reduced response compared to the WT mice.
The authors also note that they are developing improved tail-cuff blood pressure monitoring devices to better measure and account for the effect of loading conditions. Currently, the noninvasive studies they conduct take approximately 15 to 30 minutes per mouse with analysis taking another 15 - 30 minutes thus allowing for fairly rapid phenotypic screening.
1) Which of the following agents or combination of agents when used in mice maintained heart rates in the physiological range:
a) sodium pentobarbital
b) isoflurane
c) ketamine/acepromazine/xylazine
d) a and b
e) b and c
2) True or False: Doppler ultrasound measures blood velocity by detecting the change in amplitude between an emitted burst of ultrasound and the returning echoes from moving blood.
3) Currently, the best temporal resolution acheived with the murine pulsed doppler ultrasound system is:
a) 1 msec
b) 0.1 msec
c) 0.01 msec
d) 0.001 msec
4) True or False: ApoE KO mice are used as models for the study of atherosclerosis
1) d, The authors found that sodium pentobarbital, isoflurane and etomidate maintained heart rates in the physiological range. The ketamine/acepromazine/xylazine combination depressed heart rates but maintained blood pressure.
2) False, Doppler ultrasound measures blood velocity by detecting the change in frequency between an emitted burst of ultrasound and the returning echoes from moving blood.
3) b, 0.1 msec is the best temporal resolution available for doppler ultrasound at this time.
4) True

Mechanical ventilation for imaging the small animal lung. ILAR 43 (3): 159.
Abstract This review emphasizes the challenges and benefits of in vivo imaging of the small animal lung. Mechanical ventilation is the key to high quality, high resolution images. The article focuses on problems of ventilation support, control of breathing motion and lung volume, and imaging during different phases of the breathing cycle. Solutions for these problems are discussed in relation to MRI using proton imaging and hyperpolarized helium imaging. Examples of applications are given in the rat and guinea pig. Pulmonary imaging is a valuable source of information about both the normal and diseased lung.

Summary The small animal is an important model of pulmonary disease. In vivo imaging is an important tool for many of these noninvasive, nondestructive studies. The lung is a difficult organ to image in vivo due to constant motion of the heart and lungs and low tissue density providing little substance for x-ray attenuation or proton signal for MRI. There are 2 types of MR studies: conventional proton imaging and the newer hyperpolarized helium imaging for pulmonary gas spaces. Mechanical ventilation is important for in vivo studies in small animals for many reasons. It maintains proper gas exchange, administers gaseous anesthetics, and provides a way to synchronize imaging to the breathing cycle. Imaging data can be captured from the lung during specific phases of the breathing cycle. The review focuses on the use of mechanical ventilation for motion control in the lung, synchronizing image data acquisition to selected phases of the breathing cycle and examples of applications of the use of MRI of pulmonary models in small animals. Much of this information is presented in the form of photographs. It would be valuable for those interested in the use of these techniques to read the actual article and to take advantage of these visual aids. It would be valuable for those studying for boards to look at the images and be able to identify them as MR images. Breathing motion causes blurring of images. Severity of the effect is based on rate and amplitude of the breathing motion. These effects can be minimized by breath holding in human patients and variations of breath holding produced by mechanical ventilation in animals. There are several ways to synchronize imaging to the breathing cycle. The ventilator can be set to deliver a pulse on each breath which triggers the imaging device. This is useful for studies of spontaneously breathing animals. Another method is to lessen the amplitude of the motion using very small tidal volumes with high frequency jet ventilation. This method utilizes tidal volumes that are a fraction of the conventional volumes delivered at high rates. This minimizes the partial volume blurring while normal gas exchange is maintained. There is still a sufficient degree of artifact present to make this method undesirable it some studies. However this method is sufficient for studies which do not rely on high spatial resolution such as studies involving the descending aorta, azygous vein, pulmonary vessels, vena cava and chest wall. Scan synchronous ventilation using a custom built, MR compatible ventilator was the method of choice of the authors. Commercially available systems were not adequate due to the long tubing required to get the ventilator a safe distance from the MR. This long tubing created too much dead space in the system. A key component of the custom made ventilator is a pneumatically controlled plastic breathing valve that attaches directly to the endotracheal tube thus eliminating the problem of large gas volumes (dead space) in ventilator hoses between inspiratory and expiratory valves. The ventilator is an open system without any rebreathing of gasses. This design allowed the authors to generate a wide variety breathing patterns that can be accurately synchronized to image acquisition. This design could be useful for any application that requires mechanical ventilation but for which access to the animal is restricted by distance or space requirements. The authors then describes specific breathing patterns they were able to generate using the ventilator. These included imaging during end expiration, long inspiration, short breath hold, longer breath holds and an extended breath hold. The breath patterns were computer generated. The change in breath patterns allows for imaging of the lungs for different purposes. The authors go on to describe the physiological monitoring and support they provide during these procedures. They are able to maintain adequate anesthesia and normal body temperature. The anesthesia protocol is described in detail. ECG, airway pressure, exhaled CO2, body temperature, and heart rate are monitored. There is a feedback loop in the MR system to provide warm air to maintain normal body temperature. The authors stress the importance of maintaining a stable physiologic state of the animal during the study. The factors they consider most important are anesthesia level, heart rate and body temperature. Examples cited by the authors include a rat and a guinea pig model and are best viewed by the reader as there are many photographic illustrations. The examples include, combined cardiac gated and scan synchronous ventilation and hyperpolarized He gas imaging of pulmonary airways. The final example compares hyperpolarized He imaging to proton imaging.

Conclusions The authors conclude that by combining various techniques is possible to acquire high resolution in vivo images of the pulmonary system of small animals using MR techniques. These techniques are useful in studying both normal and diseased lungs.
1. Why is it difficult do in vivo imaging of the pulmonary system of a small animal?
2. What two techniques can aid the reduction of artifacts when using MR techniques to image the lung?
3. What two imaging techniques are described to visualize the pulmonary system and how do they differ?
1. The area is in constant motion from heart activity and breathing and lung tissue is very low density.
2. Cardiac gated imaging and scan synchronous ventilation. Proton imaging and hyperpolarized helium gas imaging.
3. Proton imaging relies on tissue density so is better for denser tissues such as blood vessels. Hyperpolarized helium does not require dense tissues so is better for imaging actual airways.

Methods in vascular infusion biotechnology in research with rodents. ILAR 43 (3): 175.
The following review article was intended to summarize the advances made in vascular infusion biotechnology and to see how close we've come to miniaturizing vascular infusion and sample collection systems which are in place and being used by researchers today. By using those technological advancements already available, it has greatly enabled scientists to address animal welfare issues, as well as to assert 2 of the 3 Rs--reduction and refinement of animal models.

In addition to discussing the new and varied types of infusion systems, this article briefly touches on those items which should be included in the pre-approval and planning stages of a protocol: aseptic technique, post surgical care and catheter maintenance. In addition, it reviews pertinent technical issues (vehicles for infusion; infusion volume and rate; catheter type, coatings, sizes, and placement) to assist the scientist in ensuring and obtaining appropriate data.

The challenge/goal is now to miniaturize these devices for more efficient use in the small transgenic rodent model.
1. Complications associated with vascular infusion techniques in rodents have included all of the following, except
a. cellulitis and phlebitis
b. ocular vascular lesions
c. amyloid deposits
d. thrombosis
e. sepsis
2. Which of the following methods is best used for dosing, especially in rodents ?
a. body mass
b. body area
c. body volume
d. body weight
3. Which of the following catheter coatings was shown in human studies to improve resistance to thrombin formation, reduce the incidence of infection at the catheter site, and in rats revealed a significant increase in catheter patency ?
a. Hydrocoat
b. TDMAC heparin
c. CBAS heparin
4. What is the name given to the special needle used to penetrate the silicone septum on the vascular access port ? (See diagram, Figures 2 and 3, p. 178)
5. Which of the following tethered infusion devices is least desirable due to the potential for surgical complications ?
a. button tether
b. head block
c. jacket
d. tail cuff
e. harness
6. Which of the following curves best depicts the true accuracy of a given infusion pump comparing the flow accuracy of the pump over a period of time ?
a. bell-shaped curve
b. trumpet curve
c. regression curve
d. survival curve
1. e; 2. a; 3. c; 4. Huber needle; 5. d; 6. b