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Publications (32)45.17 Total impact

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    ABSTRACT: Routinely, compounds are assessed by developmental and reproductive toxicology (DART) studies to evaluate the potential for drug-induced birth defects. High-throughput micro-CT images are being used to evaluate skeletal abnormalities due to its ability to provide high quality images of bone structures. Currently, these micro-CT images are visually inspected for skeletal abnormalities, which is a time and resource intensive process. To reduce the resources needed for skeletal evaluation, we developed image analysis strategies that allow for automatic segmentation of whole body CT images into individual bones and use structural variations of shape characteristics to classify bones as normal or abnormal. Extraction of various structures in the skull and torso were accomplished sequentially starting with skull bones and moving towards the neck, vertebrae, ribs, and limbs. A total of 17 skull bones/structures (supraoccipital, mandible, squamosals, zygomatics, etc.) and 20 torso structures (ribs, spine, humerus, femur, tibia, etc.) were identified and isolated using this algorithm. Next, we used geometrical (volume, length, width, etc.) and shape-based characteristics to identify bones lying outside the normal distribution of numbers, shapes and sizes to flag fetuses for potential abnormalities. We applied this tool to a test data set of 167 fetuses with verified skeletal abnormalities and received sensitivity of 0.959 and specificity of 0.805. This analysis platform allows for fully automated batch processing of images. Future work will include further development of the current platform to improve performance.
    No preview · Article · Jul 2015

  • No preview · Article · Sep 2014 · Reproductive Toxicology
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    ABSTRACT: Preclinical imaging technologies are increasingly being applied to developmental toxicology studies in drug development to determine potential compound toxicity. Although most of these studies are conducted in a non-regulatory setting, there is interest in performing these imaging studies under applicable regulations, for example Good Laboratory Practices (GLP), to support regulatory decisions concerning drug safety. This manuscript will describe regulations and processes to consider when bringing an imaging technology into GLP compliance.
    No preview · Article · May 2014 · Reproductive Toxicology
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    ABSTRACT: High-throughput micro-CT imaging has been used in our laboratory to evaluate fetal skeletal morphology in developmental toxicology studies. Currently, the volume-rendered skeletal images are visually inspected and observed abnormalities are reported for compounds in development. To improve the efficiency and reduce human error of the evaluation, we implemented a framework to automate the evaluation process. The framework starts by dividing the skull into regions of interest and then measuring various geometrical characteristics. Normal/abnormal classification on the bone segments is performed based on identifying statistical outliers. In pilot experiments using rabbit fetal skulls, the majority of the skeletal abnormalities can be detected successfully in this manner. However, there are shape-based abnormalities that are relatively subtle and thereby difficult to identify using the geometrical features. To address this problem, we introduced a model-based approach and applied this strategy on the squamosal bone. We will provide details on this active shape model (ASM) strategy for the identification of squamosal abnormalities and show that this method improved the sensitivity of detecting squamosal-related abnormalities from 0.48 to 0.92.
    No preview · Article · Feb 2014 · Proceedings of SPIE - The International Society for Optical Engineering
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    ABSTRACT: Preclinical imaging technologies are increasingly being applied to developmental toxicology studies in drug development to determine potential compound toxicity. Although most of these studies are conducted in a non-regulatory setting, there is interest in performing these imaging studies under applicable regulations, for example Good Laboratory Practices (GLP), to support regulatory decisions concerning drug safety. This manuscript will describe regulations and processes to consider when bringing an imaging technology into GLP compliance.
    No preview · Article · Jan 2014
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    ABSTRACT: Micro-computed tomography (micro-CT) is a high resolution imaging technique that has expanded and strengthened in use since it was last reviewed in this journal in 2004. The technology has expanded to include more detailed analysis of bone, as well as soft tissues, by use of various contrast agents. It is increasingly applied to questions in developmental biology and developmental toxicology. Relatively high-throughput protocols now provide a powerful and efficient means to evaluate embryos and fetuses subjected to genetic manipulations or chemical exposures. This review provides an overview of the technology, including scanning, reconstruction, visualization, segmentation, and analysis of micro-CT generated images. This is followed by a review of more recent applications of the technology in some common laboratory species that highlight the diverse issues that can be addressed. Birth Defects Research (Part C) 99:71-82, 2013. © 2013 Wiley Periodicals, Inc.
    No preview · Article · Jun 2013 · Birth Defects Research Part C Embryo Today Reviews
  • L David Wise
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    ABSTRACT: Relying on previous regulatory guidelines from multiple countries, the ICH S5(R2) guideline outlines the preclinical safety studies needed for registration of new medicinal products in the member countries (European Union, Japan, and the United States). The primary purpose of the guideline is to provide a testing strategy to detect and reveal toxicity to the reproductive system including development of the embryo. There are basically three study designs outlined by the guidance, assessment of fertility in adults, pre- and postnatal development of exposed offspring, and morphological evaluation following exposure during major organogenesis. This chapter discusses the major points addressed in the guidance for each study type, and points to additional references that discuss the practical details for conducting such studies.
    No preview · Article · Jan 2013 · Methods in molecular biology (Clifton, N.J.)
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    Full-text · Article · Nov 2012 · Reproductive Toxicology
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    ABSTRACT: Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, reduces de novo cholesterol biosynthesis primarily in the liver. Since cholesterol is a major component of brain myelin and peak periods of brain myelination occurs after birth, this study was designed to encompass this period in rats and evaluate the potential neurotoxic effects. The pharmacologically active, open-acid form of lovastatin was administered to groups of 50 Sprague-Dawley rats per sex subcutaneously once daily at dose levels of 0 (vehicle), 2.5, 5, or 10 mg/kg/day beginning on postnatal day 4 and continuing until termination on postnatal day 41 to 51. Physical signs and body weights were monitored during the study. Animals were assessed in a battery of behavioral tests, and at termination a set of animals were examined for gross and histological changes. There were no test article-related deaths, physical signs, or effects on preweaning and postweaning body weights during the study. In the behavior tests there were no test article-related effects in the passive avoidance, auditory startle habituation, open-field motor activity, or FOB. No test article-related postmortem findings were observed, including brain weights and histomorphology of brain, spinal cord, eye, optic nerve, or peripheral nerve. Based on these results, the no-effect level for general and neurobehavioral toxicity in neonatal rats was ≥10 mg/kg/day for open-acid lovastatin.
    No preview · Article · Aug 2011 · Birth Defects Research Part B Developmental and Reproductive Toxicology
  • L David Wise · Dahai Xue · Christopher T Winkelmann
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    ABSTRACT: Our laboratory has been conducting positive control studies to evaluate the utility of micro-computed tomography (micro-CT) for qualitative evaluation of fetal skeletal morphology. All-trans-retinoic acid (atRA) was used to produce a different spectrum of defects compared to our previous studies with boric acid and hydroxyurea. Groups of five mated Crl:CD(SD) female rats each were administered vehicle or atRA (2.5-50 mg/kg) on GD 10, and groups of four mated Dutch Belted rabbits each were dosed with vehicle or atRA (6.25-25 mg/kg) on GD 9. Cesarean sections were performed on GD 21 and 28, respectively. Following external examination the viscera were removed and fetuses scanned in a micro-CT imaging system. Fetuses were subsequently stained with alizarin red. Skeletal morphology was evaluated by each method without the knowledge of treatment group. Total bone mineral content (BMC) of each fetus was quantitated using the micro-CT images. In rats there were dose-related increases in the incidence of extra lumbar vertebra and non-dose-related increases in supernumerary ribs at all dose levels. There were decreases in mean number of ossified sacrocaudal vertebra at ≥ 5 mg/kg, and increases in skull bone malformations at ≥ 10 mg/kg. Rabbits were less sensitive on a mg/kg basis since skeletal malformations and a decrease in mean number of ossified sacrocaudal vertebra were observed only in the 25-mg/kg group. Micro-CT evaluation detected essentially the same incidence of skeletal abnormalities as seen in alizarin red-stained rat and rabbit fetuses. BMC analysis showed a trend toward slight decreases in atRA-treated rats, but no notable changes in rabbits. These results add support to our previous work that demonstrates that micro-CT imaging can effectively assess rat and rabbit fetal skeletal morphology.
    No preview · Article · Oct 2010 · Birth Defects Research Part B Developmental and Reproductive Toxicology
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    ABSTRACT: Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases in both men and women. A recently developed quadrivalent HPV vaccine, Gardasil, has been shown to be highly effective in the prevention of several HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on male fertility including reproductive performance, sperm evaluations, and histology of the testes. In addition, anti-HPV antibodies were measured during the study. Group 1 (30 male rats) received the full human dose of vaccine (0.5 mL, ∼200-fold excess based on body weight) by intramuscular injection at 6 weeks, 3 weeks, and 3 days prior to cohabitation. Group 2 males received only 1 dose at 3 days prior to cohabitation. Additional groups (20 male rats each) were administered PBS or Merck Aluminum Adjuvant similarly to Group 1. Ten males in the vaccine-treated groups were bled for immunogenicity assays after each dose. Twenty males per group were mated to untreated female rats. Cesarean sections were performed on Gestation Day 15 or 16. Cohabited males were necropsied and sperm count and motility were evaluated. There were no unscheduled deaths during the study and no evidence of toxicity in vaccine-treated male rats. The vaccine induced a specific antibody response to the 4 HPV types after each injection. There were no effects on the cesarean-section parameters of females or reproductive parameters of the cohabited male rats, including histomorphology of testes and epididymis, sperm count, and sperm motility. These results demonstrate that this quadrivalent HPV vaccine had no detectable adverse effects on routine measures of male fertility in rats.
    No preview · Article · Oct 2010 · Birth Defects Research Part B Developmental and Reproductive Toxicology
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    ABSTRACT: Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the ICH S5(R2) document. The studies that assess the hazard of both pre- and postnatal exposure are predominantly conducted in rodents (rat and mouse). Utilizing the collective experience of the authors, acceptable designs for both the range-finding and definitive studies are presented with detailed descriptions for the presentation of data. In addition, the suggested initiation and then total duration of these studies in relation to clinical studies are described. Optional parameters that may be included in the studies, as well as possible combination with other study designs are discussed. The details described herein will assist all laboratories performing these studies, individuals who need to plan for the studies, and regulatory agencies that ultimately review these studies.
    No preview · Article · Dec 2009 · Birth Defects Research Part B Developmental and Reproductive Toxicology
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    ABSTRACT: Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the International Conference on Harmonization (ICH) S5(R2) document (available at http://www.ich.org). The studies that assess developmental hazard are generally conducted in rodents and rabbits. Based on the authors' collective experience, adequate designs (including range-finding studies) and the presentation of data for these studies are described in detail. In addition, the suggested initiation and then total duration of these studies in relation to clinical studies that enroll women of childbearing potential are described. Optional parameters that may be included in the studies are discussed, as are study designs that combine assessments of fertility and developmental toxicity. New methods that may replace or enhance current procedures are outlined. The details described herein will assist all laboratories performing these studies, individuals who need to plan for the studies, and regulatory agencies that ultimately review these studies.
    No preview · Article · Dec 2009 · Birth Defects Research Part B Developmental and Reproductive Toxicology
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    ABSTRACT: Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the ICH S5(R2) document, "Detection of Toxicity to Reproduction for Medicinal Products and Toxicity to Male Fertility." Studies that assess a candidate drug's effect on fertility are generally conducted in rats. The evolution of, and ultimate harmonization of, fertility study designs are reviewed, and specific elements of an acceptable design, as well as the recommendations for presentation of data, are described in detail. Additionally, the timing of nonclinical fertility studies in relation to clinical studies that enroll men and women of reproductive potential is reviewed. Possible strategies for combining fertility assessment with other study designs are also presented. This article provides testing laboratories, sponsors, and regulatory agencies with a comparison of current methods and designs, with the aim of providing a common understanding of the critical design features.
    No preview · Article · Dec 2009 · Birth Defects Research Part B Developmental and Reproductive Toxicology
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    ABSTRACT: This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) incorporates improvements and enhancements to both content and organization of the terminology to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e. rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, 'malformation' or 'variation' remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis or interpretation. The skeletal terms have been augmented to accommodate cartilage findings.
    Full-text · Article · Sep 2009 · Congenital Anomalies
  • L David Wise · Christopher T Winkelmann
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    ABSTRACT: Assessment of developmental toxicity has historically included assessment of fetal skeletal morphology after alizarin red staining. X-ray micro-computed tomography (micro-CT) produces high-resolution images of skeletal structures and was investigated as an alternative method. Groups of 5 mated Crl:CD (SD) female rats each were administered vehicle or boric acid (40 to 500 mg/kg/day) from GD 6 through 11. On GD 21, all live fetuses were weighed, euthanized, and viscera removed. Each litter was placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 sec ( approximately 20 litters per hour) and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 min later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. Micro-CT evaluation of fetal rat skeletons detected essentially the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. The specific skeletal abnormalities that did not match exactly involved the smallest skeletal elements with minimal degrees of ossification (i.e., cervical ribs, hypoplastic 13(th) ribs, supernumerary ribs, the 5(th) sternebra, and numbers of caudal vertebrae), but the differences did not impact the overall conclusions. Additional measures such as femur length were easily measured by micro-CT. These results indicate that micro-CT imaging can effectively assess rat fetal skeletal structures, and for those laboratories with this resource, it may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.
    No preview · Article · Jun 2009 · Birth Defects Research Part B Developmental and Reproductive Toxicology
  • L David Wise · Christopher T Winkelmann
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    ABSTRACT: This laboratory has been investigating the utility of X-ray micro-computed tomography (micro-CT) to produce high-resolution, 3D images of skeletal structures in common laboratory species. The present investigation uses micro-CT evaluation of skeletons from rabbit fetuses exposed to the known teratogen, hydroxyurea. Groups of 4-6 mated Dutch Belted female rabbits each were administered vehicle or hydroxyurea (62.5 to 500 mg/kg) once on GD 12. On GD 28, all live fetuses were weighed, euthanized, and viscera removed. Up to 7 fetuses per litter were placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 seconds, and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 minutes later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. Except for a few isolated cases, micro-CT evaluation detected the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. Skeletal elements that are very small (e.g., caudal-most vertebrae, metacarpal no. 1) or those with a minimal degree of ossification were occasionally not observed with micro-CT. However, this difference did not impact the overall study conclusions. Femur length was easily measured by micro-CT. These results indicate that micro-CT imaging can effectively assess rabbit fetal skeletal structures, and for those laboratories with this resource, may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.
    No preview · Article · Jun 2009 · Birth Defects Research Part B Developmental and Reproductive Toxicology
  • Christopher T. Winkelmann · L. David Wise
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    ABSTRACT: Fetal skeletal assessments are routinely conducted as a part of preclinical safety studies to support the development of novel therapeutic agents. Alizarin red staining with visual inspection of fetal skeletons is the gold standard in evaluating skeletons for the presence of developmental abnormalities. X-ray based micro-computed tomography (micro-CT) imaging has been used to evaluate small skeletal structures, both in vivo and ex vivo. Recent technological advances have reduced micro-CT image acquisition time making this technology practical for routine fetal skeletal evaluations. Herein we report on the use of micro-CT imaging as a method to perform high-throughput assessment of fetal skeletons.
    No preview · Article · May 2009 · Journal of pharmacological and toxicological methods
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    ABSTRACT: To assess whether ovarian histopathological examination in repeated-dose rodent toxicity study could reliably anticipate toxic effects on female reproductive function and to assess whether ovarian change could be detected in a 2-week repeated-dose toxicity study, tamoxifen was administrated orally to female rats at 0.005, 0.03, or 0.2 mg/kg/day for 2 and 4 weeks in the repeated-dose toxicity studies, and for 2 weeks prior to cohabitation, during cohabitation, and through Gestation Day 7 in a female fertility study. The relationship between ovarian histopathological findings and fertility results was investigated. Findings at 0.03 and 0.2 mg/kg/day included decreases in body weight gains associated with decreases in food consumption, in 2- and 4-week repeated-dose toxicity studies and fertility study. The ovarian histopathological findings included increases in large atretic follicles, increases in interstitium cells and absence of newly-formed corpus lutea at 0.2 mg/kg/day in the 2-week study and at 0.03 and 0.2 mg/kg/day in the 4-week study. The treatment induced estrogenic and antiestrogenic reactions in the uterus, while mucinous degeneration was detected in the vagina. Effects on female fertility consisted primarily of disturbance of estrus cycle and decreases in numbers of pregnant rats which were considered to be related to ovarian histopathological changes. Based on these findings, ovarian histopathological evaluation in the repeated-dose toxicity study could anticipate the effects of tamoxifen on female fertility via ovarian dysfunction at slightly toxic doses, and 2-week treatment of tamoxifen at appropriate dose could be sufficient to detect ovarian toxicity by microscopic examination.
    No preview · Article · Feb 2009 · The Journal of Toxicological Sciences
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    ABSTRACT: Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases, with approximately half of the HPV-infected people being adolescents and young adults. A recently developed quadrivalent HPV vaccine, GARDASIL((R)), has been shown to be highly effective in the prevention of a number of HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on female fertility and F1 development, growth, behavior, and reproductive performance. In addition, anti-HPV antibodies in the F0 females and F1 offspring were measured during the study. Two groups of 65 virgin Sprague-Dawley rats were administered two or four intramuscular injections of the vaccine (full human dose of 0.5 mL at 5 and 2 weeks prior to mating, on Gestation Day [GD] 6, and Lactation Day [LD] 7; or GD 6 and LD 7 only). Additional groups of rats were administered phosphate-buffered saline or Merck Aluminum Adjuvant (MAA) at the same four times. All females were mated to males of the same stock. Cesarean sections were performed on 22/group on GD 21, 22/group were allowed to deliver, and remaining females used for blood collections or replacements. F0 female fertility parameters were evaluated. An extensive number of prenatal, perinatal, and postnatal parameters were evaluated in the F1 generation. There were no unscheduled deaths during the study. There was no evidence of toxicity in the F0 females given either MAA or vaccine. There were no effects on the fertility or reproductive performance of the F0 females. There was no evidence of developmental toxicity to the F1 generation, including fetal body weight and morphology, postnatal growth and development, behavior, and reproductive performance. The quadrivalent vaccine induced a specific antibody response to the four HPV types in the F0 female rats following one or multiple injections. Antibodies against all four HPV types were transferred to the F1 generation during gestation and/or lactation, likely via the placenta and milk, respectively. The passively transferred antibodies persisted up to Postnatal Day 77 when they were last measured. These results demonstrate that this quadrivalent HPV vaccine had no detectable adverse effects in either the treated F0 female rats or the F1 generation.
    No preview · Article · Dec 2008 · Birth Defects Research Part B Developmental and Reproductive Toxicology