MARG is a histological technique that provides an image of the distribution of radiolabeled compounds in tissues and organs at the cellular level. This technique, which relies on direct exposure of the radioactive entity to a photographic emulsion, is prone to artifacts and cannot provide quantitative data. However, its strength lies in its ability to examine the spatial distribution of radioactivity at the tissue and cellular layer, and it serves as an important informational link from the whole-body, organ, and tissue levels to the cellular level. A further description of this technique and it applications and limitations will also be presented later in this manuscript.
Quantitative Whole-Body Autoradiography
A brief description of the QWBA technique (1
) begins with the administration of a radioactive test substance (beta particle emitters, such as 14
H, and 125
I) to a lab animal followed by euthanasia and snap freezing of the carcass (typically in a dry ice–hexane bath). Frozen carcasses are then freeze embedded in a carboxymethylcellulose block, cryosectioned at 30–50 µm, and sections are collected onto adhesive tape. The sections on tape are dehydrated and then apposed to phosphor-imaging plates, or X-ray film, along with a set of radioactive calibration standards. Exposure times vary from a few days to a few weeks for phosphor-imaging plates, or for weeks to months for X-ray film. Digital images are obtained to enable tissue quantification of radioactivity concentrations by image analysis.
WBA was a crude method in the beginning. In 1867, Niepse de Saint Victor first described the phenomenon of autoradiography, which he described as the “persistent activity due to an unknown chemical radiation” (3
). This observation lead London to performing an experiment in which an autoradiographic image of a frog treated with radium was first produced in 1904 (4
). To that end, ARG is truly the first molecular imaging technique used for the localization of radioactivity in biological specimens. Fifty years later, Dziewaitkovski used beta radiation to investigate the localization of compounds in biological samples (5
) followed by the development of the whole-body autoradiography technique by Ullberg in 1954 (6
) who pioneered the technique by administering 35
S-penicillin to mice followed by freeze embedding them in water-soaked cotton using dry ice. He then sectioned their entire body bodies using a large microtome in a walk-in freezer. The whole-body sections he produced were then opposed to X-ray film which produced the autoradiographs of tissue distribution. Methods for sectioning the whole bodies of lab animals were also developed and methods included the use of dry ice-cooled microtomes (7
); exposing the surface of ground down frozen carcasses (8
); abrasion of resin-embedded carcasses (9
); thick sectioning using a circular saw (10
); and finally, the development of a large microtome held inside a chest freezer (11
). Leica Microsystems, Inc. (Nussloch, Germany) began manufacturing commercially available large format cryomicrotomes, and they are the past and current leading provider of large cryomicrotomes used for WBA today.
Quantitation of autoradiographs was the next challenge for pioneering macroautoradiographers and Berlin and Ullberg (12
), and Kutzim (13
) made the first attempts to quantify tissue concentrations in autoradiographs using early image analysis techniques. Unfortunately, the results achieved were only semiquantitative, but in the following years (1974–1987), several investigators (14
) researched methods to better determine quantitative data from autoradiographs with limited success owing to the inherent nonlinearity of film. Also during this time, Schweitzer (20
) developed an image calibration method using 14
C-spiked blood standards at concentrations bracketing the sensitivity of phosphor-imaging plates. This robust technique continues to be used by many investigators today. Luckey revolutionized WBA in 1975 by developing and patenting phosphor-imaging technology or radioluminography (2
), which provided images capable of providing digital images from whole-body sections within days. Most importantly, these images enabled the direct determination of quantitative tissue concentrations over four to five orders of magnitude, and validation efforts began. A technical validation of the phosphor-imaging instrument, and QWBA methods, which more completely described the principals, specifications, and limitations of the instrumentation and QWBA, was published in 2000 in a special edition of the Journal of Regulatory Toxicology and Pharmacology
). In 1994, a group of autoradiographers in the pharmaceutical industry formed the Society for Whole-Body Autoradiography (SWBA) whose mission was to promote the use of QWBA over traditional organ dissection homogenate methods to determine tissue distribution of new drugs. Further work parameterized quantitative aspect to meet stringent bioanalytical expectations (22
), and in 1990, a Japanese collaboration of >20 companies proposed that QWBA should replace the use of traditional dissection and liquid scintillation counting (LSC) assay to determine true tissue distribution during the drug development (28
). Dr. Yasuo Ohno of the National Institute of Health Science (Tokyo, Japan) concluded his presentation at the 1997 meeting of the Society for Whole-Body Autoradiography by stating that the Japanese Ministry of Health and Welfare would accept QWBA data in lieu of traditional organ dissection distribution studies for the approval of new drugs as long as the procedures were appropriately validated. Today, pharmaceutical companies have almost entirely eliminated the use of dissection studies to determine the distribution of new pharmaceuticals, and anecdotal information to the author has suggested that the Food and Drug Administration is now requesting that “QWBA” studies be provided especially to answer certain questions that arise during drug development.
In addition to autoradiography and autoradioluminography, direct nuclear imaging technologies were also developed. These instruments utilize ionization chambers and different imaging technologies (e.g., scintillating sheet, charge-coupled device camera) and were developed by Jeavons in 1983 (29
). Today's instrument consists of a parallel plaque avalanche chamber, which is based on the invention by Charpak in 1989 (30
). These instruments, which are currently sold by Biospace Lab (Paris, France), also image radioactivity in whole-body and smaller tissue sections, and have the ability to acquire quantitative images in real time. These instruments have proved most useful in drug discovery because they are capable of dual-isotope analysis, and they provide data quickly. One limitation for these systems is that only a limited number of sections can be analyzed at one time, and the instruments require regular and careful maintenance.
The key strength of the QWBA technique is that it shows true tissue distribution of radiolabeled test articles in a relatively unadulterated, in situ
sample. Phosphor imaging has also been shown to be a very robust yet sensitive technology for the quantification of radioactivity in whole-body sections. Its wide linear range and sensitivity that can reliably quantify ~45 dpms distributed over an area of one half square centimeters far exceeds that achieved by LSC for similar sized samples. Phosphor imaging is also able to image the relatively weak energy of 14
C and 3
H, which fortunately are also long-lived isotopes so that drugs/metabolites with very long half-lives can be tracked in the body of animals over years, which is not possible using the relatively short-lived isotopes used for in vivo
positron emission tomography (PET) and single-photon emission computed tomography imaging. In general, QWBA provides a much higher quality data set than that provided by organ dissection and homogenate assay by providing concentration data on all tissues; not just those chosen for typical dissection studies, which eliminates the possibility of missing organs that may have high concentrations. The images can also be reviewed and analyzed at any time so that if an issue arises after the tissue distribution has been completed, then investigators can go back and review and quantify the nonroutine tissue(s) of interest. QWBA also eliminates the variables of cross-contamination of organs and the variable effects of organ exsanguination that inevitably occurs during organ removal. The whole-body-freezing procedure, which takes approximately 5–15 min, which depends on body weight, to halt biological and metabolic processes, also decreases the interanimal variability of the data, and this has been routinely experienced by whole-body autoradiographers. This observation supports the use of a study design where one animal/time point and more time points are used, which in turn provides more reliable pharmacokinetic (PK) parameters for a more accurate description of tissue compartment PK. This approach also satisfies and supports the clinical use of lab animals. The techniques used for QWBA can vary across different laboratories, however, the combined methods have been thoroughly developed, tested, and reviewed, and are very robust (31
). In short, QWBA provides a higher quality, more detailed, and more useful data, while using less animals, than is required for organ homogenate assay studies.
Of course, QWBA has several limitations that need to be considered. The first, which is common to all radiolabeled studies, is that the technique provides data on the concentration of radioactivity only. That is to say, investigators do not know the actual molecular identity of the radioactivity they are tracking and/or quantifying. Tissue concentrations determined by QWBA (or LSC) can include parent drug, plus its metabolites, and/or degradation products. This is most problematic when using 125
I-labeled peptides and proteins, and to a lesser extent, when using 3
H, because the in vivo
stability of these two radiolabels is often not too good, and the radiolabel can be lost as free 125
I or 3
O. When the radiolabel dissociates from the parent molecule, then investigators are faced with the responsibility of characterizing the extent to which the radiolabel has been lost and then adjusting their tissue concentration appropriately and/or stating the fact that the results are semiquantitative. Additionally, QWBA sections are typically dehydrated to facilitate further processing, and so, volatile metabolites are lost, although this situation probably does not occur that often and may be able to be predicted when the chemistry is well understood. This is where the strengths of MALDI-MSI can be realized, especially because the same sections used for QWBA can be used for analysis by MALDI-MSI as will be presented in the next section of this article. Another limitation of QWBA is that it is difficult to evaluate short-lived isotopes, like those used for radiopharmaceuticals, due to the processing time required, although it is possible to alter the processes to obtain data more quickly, but it requires special attention. For example, when using 90
C, or 18
F as the radiolabel, which have half-lives of 2.67 days, 21 min, and 60 min, respectively, as the radiolabel, which have a half-life of 2.67 days, respectively, the frozen “wet” sections may need to be exposed to imaging plates immediately and while under freezer conditions, otherwise it could take too long to dehydrate the sections, and half of the radioactivity could be lost thus decreasing the sensitivity of the technique. Despite these technical drawbacks, Kaim et al
) were able to demonstrate that the uptake of 18
F-FET in nonneoplastic inflammatory cells in an experimental soft tissue infection model was lower than that of 18
F-FDG, allowing thus to predict a higher specificity for the detection of tumor cells using 18
Another limitation is cost. The instrumentation for QWBA is relatively expensive, and highly trained staffs are needed to perform the processing and analysis, which can be a big limitation for some labs. QWBA is also limited in that it cannot adequately provide data at the microscopic level due to the freezing technique. Despite the “snap-freezing” techniques used, the freezing of all tissues is too slow to prevent cellular damage due to ice crystal formation. This makes receptor localization/staining difficult and cellular identification much more difficult, so that radiolabeled test articles cannot be easily colocalized to specific molecular targets. Other limitations, that also affect dissection studies, include the influence of the experimental conditions, such as radiochemical purity of the test compound, nominal and radioactive doses given to the animals, anesthesia, and methods of euthanasia. Investigators need to ensure that they are working with radiolabeled material that has the proper specific activity and radiopurity so that the proper dose of radioactivity can be given and to make sure that the radioactivity being quantified is actually compound related and not a radioimpurity to ensure quantifiable results related to the test compound. The use of anesthesia and method of euthanasia can also affect the tissue distribution of any test article and should be adjusted in cases where the investigator(s) think it may affect distribution of their test article. For example, it is commonly thought that CO2 alters the permeability of the blood–brain barrier (acidosis) and therefore, alters normal brain penetration. Thus, euthanasia by CO2 inhalation may not be a good technique to use if the compound is targeted for the brain. Likewise, euthanasia by exsanguination, which must be used for tissue dissection studies, will undoubtedly have an effect on tissue distribution due to the massive fluid changes that take place in the body during that process. This is another benefit of QWBA over organ dissection because most often animals for QWBA are not exsanguinated and remain intact.
There are also limitations that are related to the instrumentation and methods. Difficulties can arise during cryomicrotomy, where there are limitations on section thickness, sample size, and sample characteristics. The thickness of whole-body sections, which is determined by the cryomicrotome settings, needs to be thick enough to be collected on tape, and also so, there is enough tissue and radioactivity present to produce an image for quantitation. The section thickness and isotope of the calibration standards must also match the test sample to ensure proper imaging calibration and determination of tissue concentrations of drug-derived radioactivity. Additionally, the cryomicrotome has its own functional limitations, such as how thin it can section, vertical blade movement (for thicker samples), blade sharpness, cryochamber size and ability to dehydrate sections, and overall sample size that can fit in the microtome. These are just a few of the several functional limitations that must be considered when using the microtome. The phosphor imager used to obtain the images for analysis also has its limitations, such as image resolution (pixel size), lower limits of detection (typically ~2,220 dpm/g/0.5 mm2 image region from sections that are ~40-µm-thick section). Typical exposure times for QWBA range from 3 to 4 days depending on the isotope and radioactive dose administered to the animal. However, despite the current limitations, QWBA still provides the best way to determine tissue concentrations in situ.
Quantitative Whole-Body Autoradiography Applications
QWBA offers many applications for researchers in drug discovery and development and can best answer questions related to the biodistribution of new or old drugs. Schweitzer et al
) investigated the distribution of [14
C] Diclofenac sodium (Voltaren®), which is a drug that has been marketed for years, after a single oral administration in rats. These investigators showed that Diclofenac preferentially distributed into the inflamed tissues and achieved exposures that were 26- and 53-fold higher in the inflamed neck and inflamed paws of treated animals than in control rats. All other tissues in treated animals and control animals showed similar distribution and exposure. Most often, QWBA is used during the drug development stages and to support regulatory submissions. The data are used to show tissue pharmacokinetics and to predict human radiation dosimetry that might occur during human-radiolabeled absorption, distribution, metabolism, and excretion (ADME) studies. A routine tissue distribution performed using QWBA may routinely evaluate 35–40 tissues over various study periods that often go as long as 35 days postdose, and by having ten or more time points, which provides more reliable pharmacokinetic parameters for all tissues that can be measured. However, QWBA data is also used in drug discovery to support the selection of new drug candidates, based on tissue distribution/uptake/retention, and also to help evaluate and identify potential toxicological issues (35
). Foster et al
) demonstrated that the immunomodulatory drug FTY720 and its phosphorylation product were specifically localized to the central nervous system white matter, and preferentially along myelin sheaths, following single and multiple oral doses. Weiss et al
) showed that the bisphosphonate zoledronic acid was initially rapidly eliminated from plasma and noncalcified tissue but only slowly from bone, whereas the terminal half-lives of elimination from these tissues were similar, which suggest redistribution of drug from the bone rather than prolonged retention in the latter. Potchioba and Nocerini (38
) have described how tritiated compounds can be used to determine tissue distribution characteristics early on during the drug discovery phase to identify lead compounds. The key observation and rational for the use of 3
H was that the tritium-labeled material could be obtained much quicker than the more stably labeled 14
C compound (2 weeks versus
2–3 months), however, they did not discuss how they assured in vivo
radiostability of the 3
H, which is known to be exchanged with water in vivo
, and which can result in a loss of the 3
H, thus affecting tissue quantitation. This is something that needs to be addressed whenever 3
H will be used for quantitative tissue distribution by QWBA. The paper also failed to mention that the cost of using tritiated compounds for QWBA can be very expensive because the phosphor-imaging plates used for 3
H can be used only once. Furthermore, these imaging plates are relatively small and can fit only about six whole-body sections on them, and so, two or more plates are required for each rat. These authors also reported that a 17-day exposure time was used, which is much longer than that used for 14
C (typically approximately a 1–4-day exposure). The use of tritiated compounds to support drug discovery is not new though and was being routinely used by pharmaceutical companies (E. Solon, presented at the European Society for Autoradiography meeting in Heidelberg, 1998).
QWBA is a very good technique to study placental and milk transfer of xenobiotics in rodents, owing to the high resolution offered so that small tissues of the fetus can be positively identified and quantified (see Fig. ). Bruin et al
) showed that following oral dosing of 14
C-labeled deferasinox (Exjade, ICL670) to rats, that placental transfer was limited, but that approximately 3% of the dose was transferred into the breast milk. Phosphor imaging can also be used on sections of individual organs, such as kidney, brain, and eyes, where high resolving power may be needed to screen drug penetration into the finer structures of organs. Figure presents an example of an in vitro
competitive binding assay in brain sections that was imaged and quantified using phosphor imaging of brain sections incubated in different solution of test drug and known receptor antagonists. Figure shows the utility of using cryosectioning and phosphor imaging of dog eyes that have been exposed to various test articles to evaluate ocular distribution. This level of detail can also be obtained in rat eyes (Fig. ), and it can be very important when the data will be used to determine human dosimetry exposure and there is an association of the test article with melanin. QWBA is the probably the best technique currently available to enable the evaluation of test article association to melanin. This is particularly important because while tissue dissection may not detect concentrations of drug-derived radioactivity in the pigmented uveal tracts of lab rats (due to the small sample size and relative low sensitivity), QWBA can image these small pigmented tissues and can provide the concentration data necessary to enable a more precise prediction of a possible health risk to human volunteers participating in a human radiolabeled ADME study.
Fig. 1 An example of an image obtained during a placental transfer study conducted using QWBA. Whole-body autoradioluminographs of a pregnant rat (day 17; left) and a 17-day-old rat fetus (right) showing differential distribution of 14C-AZT-derived radioactivity (more ...)
Fig. 2 An example of the type of results that can be obtained after an in vitro rat brain section competitive binding assay screen using quantitative phosphor imaging. Control rat brain sections (~40 µm thick) can be incubated in solutions (more ...)
Fig. 3 Dog and rat eyes shown by autoradioluminography. a Dog eye individually cryosectioned at 40 µm thick and imaged using phosphor storage technology. Adjustments to the gray scale appearance facilitate tissue identification for quantification. (more ...) 125
I-labeled proteins and peptides are being used more often these days in QWBA analyses to study tissue distribution and pharmacokinetics in the Biotech arena. Although these studies are not often required for regulatory submission, Biotech companies are finding that the data gleaned from these studies have helped them to the better understand and select their compounds in development. Although the in vivo
stability of 125
I on most large molecule xenobiotics is not 100%, the image data can still be useful, especially when target tissue/organ distribution data are needed, and semiquantitative data can be obtained with confidence when the in vivo
stability and amount of free 125
I circulating in the body is estimated. However, it is important for investigators using 125
I to consider that the following tissues/organs either contain iodine symporters, and/or organify free iodine, thus these tissue concentrations must be interpreted with caution: thyroid gland, stomach, kidneys, mammary gland, salivary gland, thymus, epidermis, and choroid plexus (40
QWBA studies are also routinely used to evaluate tissue distribution of xenobiotics in large lab animals such as rabbits, dogs, and monkeys (see Fig. ). In these cases, the animal's carcass must be able to fit into a freezing frame block of approximately 40
15 cm for sectioning. However, if the frozen carcass is larger than those dimensions, then it can be subdivided so that all parts of the carcass can be sectioned and analyzed. In this way, even larger samples, such as the distribution into pigs, can be analyzed (41
Monkey QWBA. The image on the left shows a monkey carcass being cryosectioned for QWBA, and a sample phosphor image of a monkey is on the right
QWBA has also been used to answer very specific questions related to issues that can arise during drug discovery. For example, target organ penetration (tumor, brain), tracking the distribution of oligonucleotides and nanoparticles, and drug–drug and drug–food interactions (40
QWBA is a versatile tool which can provide pharmaceutical scientists with quantifiable high-resolution images of the distribution of xenobiotics in practically any biological sample, large or small, and when the proper study design is applied, a wealth of knowledge can be obtained from a single study. The benefits of QWBA and updated study designs over the outdated tissue dissection technique are numerous and substantial. To that end, regulatory authorities should encourage drug developers use QWBA instead of organ dissection and homogenization to conduct tissue distribution studies whenever possible. This is because QWBA provides the highest quality quantitative data and a more complete analysis of true tissue distribution, which is especially important when trying to predict human exposure to radiation during human-radiolabeled studies.
MARG provides pharmaceutical scientists with a high-resolution tool to investigate spatial localization of radiolabeled drugs at a tissue and cellular level. MARG is especially good at providing insight regarding in vivo receptor binding in various cell types and has predictive value for specific drug targeting. In this respect, it has been used widely in academic settings where it can provide important information on cellular mechanisms. MARG has applications in all areas of science, but this report will discuss examples in drug metabolism, pharmacology, toxicology, and molecular biology.
The methods used by the author which are described below are based on the methods of Appleton (42
) and Stumpf (43
), but it is important to realize that there are many variations of the method presented in the literature. To begin, an animal is dosed with a radiolabeled substance (typically 3
S, or 125
I), the animal is exsanguinated, and tissues are dissected and snap frozen in isopentane that is chilled in liquid nitrogen. The tissue is then cryosectioned at −20°C (or the optimal cutting temperature for a given tissue/organ) to obtain 4−5-µm-thick sections. Then, under darkroom conditions, sections are thaw mounted onto dry glass microscope slides that have been precoated with nuclear photographic emulsion. The slides are placed into a light-tight box with desiccant and allowed to expose for an appropriate amount of time. The collection of sections onto dry slides is a key step developed by Appleton (42
), and it eliminates the possibility of diffusion of soluble compounds, which can happen during slide and section dipping into an aqueous emulsion. In contrast, the original Stumpf and Roth method (44
) involved collection of the section into vials for freeze drying, which required very careful section handling and was very time consuming and prone to sample destruction. Following exposure, the slides are developed in a manner similar to developing photographic film before being stained using conventional histological staining protocols. This may include immunostaining techniques that can provide positive colocalization of drug-derived radioactivity to known cell types, receptors, and/or other structures/markers for which antibody staining protocols exist (43
The first microautoradiographic data were produced by Lacassagne in 1924 (45
), which lead to further work by Bélanger and Leblond (46
), who poured liquid photographic emulsion onto histological sections to reveal the location of radioactive substances in the tissues. Joftes and Warren in 1955 (47
) revised that technique and dipped slides into photographic emulsion, which gained wide use due to its ease of manipulation. This technique has survived the years and is sometimes used today. However, if diffusible radiolabeled compound are imaged, the results can be useless owing to the relocation of the radiolabeled substance, which produces telltale artifacts that can invalidate experiments and discourage investigators. During the 1940s, methods utilizing strips of dried emulsion were developed where the histological sample that contained a radioactive substance was placed in direct contact with the dried emulsion strip and allowed to expose it over time. This method proved cumbersome though because the technician would need to maintain the precise position of the strip on the samples during exposure, development, and staining, and often the alignment could not be maintained and the entire process would end in failure. However, in 1964, Appleton (48
) first developed the technique of collecting cryosections onto slides covered with strips of dried emulsion using a thaw-mounting technique. This required sectioning and collection of sections in a darkroom and under safelight conditions, which requires a dedicated staff who can master and maintain their skills. The use of cryopreservation and cryosectioning remains critical to the study of diffusible substances because it maintains the spatial locale of the radiolabeled substance in the matrix whereas liquid tissue fixation steps most often solubilize and relocate the diffusible test article. However, when substances are tightly bound to cellular structures (e.g., receptor proteins), positive results may still be obtained from samples processed using conventional histology techniques. MARG procedures are also still used reliably by molecular biologists to detect RNA molecules in situ
and to study the localization of genes and DNA sequences in histological preparations owing to their relatively stable positions in cells. Further refinement of MARG techniques occurred during the 1960s by Caro (49
), and shortly after that, Stumpf and Roth (44
) made additional improvements to establish receptor autoradiography as a more reliable technique. This established the basis for the current MARG techniques. Numerous elaborations on the techniques have been presented since then by different investigators (50
), but the basic principals have remained unchanged for >30 years. Today, as in the past, the MARG technique is very difficult to master, which continues to hamper its use. Researchers must use caution when reviewing the literature and relying on articles that used the emulsion-dipping technique and claim quantitative data. The conclusions may be questionable and may be disputed in some cases. Validation of the technique is lacking in most labs, and results can be very subjective. Claims of truly quantifiable results have never been clearly shown by any investigator, and they are at best semiquantitative. This is due to the lack of thickness uniformity of both the tissue sections and the emulsion detection media used. Additionally, there are few, if any, instances where calibration and/or quality control standards have been coexposed within the samples, which is the only way to clearly prove quantitation of samples.
Nevertheless, MARG has made important contributions to drug discovery and development over the years and has provided insight into the localization of pharmaceuticals to support proof of concept studies, mechanisms of toxicity, efficacy, physiology of hormone action, and cell regulation. For example, MARG is useful to study skin penetration of various compounds and is routinely used in the cosmetic industry, physiology research, pharmacology, and safety studies. Unilever is a company that makes consumer skin care products and as such is responsible to prove safety of substances that are applied to skin. They have developed MARG techniques to examine skin penetration in different in vitro
test models in rats, pigs, and human skin samples. They have also coupled MARG with confocal microscopy and other techniques to extend the usefulness of their studies (H. Minter presented at the 2007 Meeting of the Society for Whole-Body Autoradiography Meeting). Linoleic acid (LA) is commonly used in cosmetics, but its in vivo
human skin penetration characteristics were not very well demonstrated. However, in 2006, Rauvast and Mavon (51
) used a unique, in situ
, “virtual” microautoradiographic slide to examine transfollicular delivery of LA in human scalp. They combined their MARG data with an in vitro
permeation experiment and compartmental analysis to show that most of the LA was localized to the hair sheath, but that none was present in the dermal compartment and that 10% of the total LA recovered was found in the stratum corneum and dermis after 6 h. This supported their notion that the diffusion of LA occurred by a transfollicular route. It also demonstrated the value of high-resolution MARG for providing detailed cellular localization of the molecule. Skin receptor MARG techniques have also been used to study the absorption, penetration, and cellular localization of 3
H-Maxacalcitol, which is a vitamin D analog used for the treatment of psoriasis. Hayakawa et al
. treated the dorsal skin of rats with a 3
H-Maxacalcitol ointment and examined skin exposed for periods of 0.5, 2, 8, 24, 48, or 168 h (52
). They discovered two routes of skin penetration; one via epidermal cell layers and the other via hair follicles. They were also able to distinguish very fine regions of cellular localization, which supported theories on the mechanism of efficacy. Figure shows an example of a 14
C-labeled test article localized to the sebaceous gland in the skin of a rat after a dermal application (unpublished data from author). This example demonstrates the fine detail of localization that can be obtained with MARG.
MARG showing localization of a 14C-labeled test article in the sebacious glands of rat skin after a topical application. Arrows point to localizations of radioactivity
Both renal function and localization of various substances in the kidney have been studied by MARG. Young et al
) used 14
C-iodoantipyrine as a tracer to study intrarenal blood flow in nephrectomized rats, and they used their microautoradiographs and standards to determine blood flow rates. They noted how MARG was helpful in defining the morphological location of blood flow, and they made several conclusions regarding changes in regional renal blood flow; “the interaction between vasoactive mediators and the autonomic nervous system”; and “that medullary blood flow was dependent on local prostaglandin production and is also influenced by sympathetic nervous supply.”
Another laboratory used in vitro
MARG to show localization and density of atrial natriuretic peptide (ANP) in nephrectomy biopsy samples obtained from patients with renal disease (54
). These investigators used [125
I]-alpha-human (1–28) ANP, and they found localization in the glomerulus and tubular regions in the human biopsy specimens. They also observed that density of ANP binding generally decreased in patients with renal dysfunction and hypertension. Overall though, their study established an in vitro
MARG method to assess ANP binding in human biopsy specimens.
More recent work by Yamamoto et al
) used MARG in combination with immunohistochemistry, macroautoradiography, and positron emission topography to examine intestinal ulceration and healing in the rat. They used 18
F-FDG to examine ulcerations in the small intestine of rats, which were induced using indomethacin. MARG combined with immunohistochemistry showed an accumulation of 18
F-FDG in inflammatory cells and in granular tissue-forming cells, forming granulation tissue, and around ulcers. 18
F-FDG was also found to be present in proliferating intestinal crypt cells and in intact intestinal tissue taken from indomethacin-treated and control animals. They concluded ulceration could be visualized early by the prominent uptake of 18
F-FDG by inflammatory cells, and by the formation of granulation tissue by cells in, and around ulcers. This work also demonstrated the value of combining both in vivo
and ex vivo
imaging techniques, which provided robust data sets for analysis.
Several limitations have impeded the progress and wider use of MARG in drug discovery and development. These include: the processing time required to obtain results; the inability of the technique to provide quantitative results, which includes the inability to assure and prove uniformity of tissue and emulsion thicknesses, and lack of internal calibration and/or quality control standards; the high rate and ease of artifact production; and difficulties in collection tissue sections under darkroom conditions. The processing time to obtain results by MARG is a difficult thing to gauge as each tissue must be treated and evaluated differently depending on how much radioactivity is present. The exposure time can take anywhere from days to weeks and even months to obtain optimal results. This often discourages the drug discovery scientist who often works under much shorter timelines, and it becomes an overwhelming amount of work for the scientists in the development area, who need strong study designs, which require many more samples be included in the evaluation, and who may be challenged by regulators to assure high quality results, through the use of validated procedures, and quality control and calibration standards for each sample. Technology may help to solve some of these problems if the detection media (e.g., emulsions) can be more uniformly produced and made to have inherently linear quantitation. Technology may also help to develop easier methods of collecting uniformly thick tissue sections that can be automatically mounted onto slides for processing. Although this would be quite a challenge due to the varying matrices to be sectioned (e.g., hard bone, adipose, and eyes). Dependable microsized calibration and quality control standards that can be coexposed with every section would also need to be developed to assure reproducibility of quantitation. Finally, the new methods would need to enable a significant reduction in the amount and types of artifacts that are produced. Currently, the following types of artifacts (43
) must be controlled: (1) effects on emulsion by slight variations in light, humidity, temperature, tissue characteristics, fixation, freezing, chemicals, pH, developer, fixer, and miscellaneous debris in developer solutions; (2) tissue condition (e.g., freezing technique, fixation, autolysis, sectioning temperature, improper section mounting); (3) light leaks; (4) latent image fading; (5) reticulation of emulsion; (6) positive chemography; (7) negative chemography; (8) deviations of pH in processing fluids; (9) pressure artifacts; (10) ice crystals on knife; (11) crystalline deposits from developing process. Some of these are more easily controlled than others, but together they require a high level of skill by the analyst to overcome. Until methods and/or technologies can be developed that can better control tissue section and emulsion uniformity and also reduce the sources of and occurrence of artifacts, the current technique will remain strictly qualitative and will prove to be too daunting for routine use in pharmaceutical discovery and development. The lack of new developments in MARG methods has continued to make MARG an underutilized technique in drug discovery and development, but when performed correctly, the results can be of utmost value in promoting a drug candidate and in answering some pivotal questions for pharmaceutical investigators.