Danielle L. Brown

Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague-Dawley rats were administered either vehicle, Scl-Ab (3 or 50mg/kg/week subcutaneously), or human PTH (1-34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume. Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.

Congenital hepatic fibrosis in 5 dogs.

Congenital hepatic fibrosis is a disorder of biliary system development histologically characterized by diffuse periportal to bridging fibrosis with numerous small often-irregular bile ducts and reduction in the number of portal vein branches. The condition results from abnormal development of the ductal plate, the embryonic precursor to the interlobular bile ducts. It has rarely been reported in veterinary species, and it has never been reported in dogs. This article describes 5 cases of a ductal plate malformation in dogs consistent with congenital hepatic fibrosis. On light microscopy, all 5 livers had severe bridging fibrosis with a marked increase in the number of small bile ducts, which often had irregular, dilated profiles reminiscent of the developing ductal plate. In addition, 80% (4 of 5) of cases lacked typical portal vein profiles. Cytokeratin 7 and proliferating cell nuclear antigen immunohistochemistry was performed on the 3 cases for which paraffin-embedded tissue was available. The bile duct profiles were strongly positive for cytokeratin 7 in all 3 cases, and they were negative for proliferating cell nuclear antigen or only had rare positive cells. All 5 dogs presented with clinical signs of portal hypertension. Congenital hepatic fibrosis should be considered in the differential diagnosis in young dogs that present with portal hypertension and lesions that may have been interpreted as bridging biliary hyperplasia or extrahepatic biliary obstruction.

Mesenchymal Hamartoma of the Liver in a Late-Term Equine Fetus

Mesenchymal hamartoma of the liver is a rare congenital disorder of biliary tract development. During the necropsy of a late-term equine fetus, a markedly enlarged liver of more than two times normal weight was found. Light microscopic review revealed that the normal hepatic parenchyma had been obliterated, replaced, and expanded by abnormal bile ducts surrounded by abundant, myxoid stroma. The lesion was diagnosed as a mesenchymal hamartoma. Small portions of the liver had bridging septa of fibrosis and proliferations of small-caliber abnormal bile ducts, resembling another congenital biliary abnormality termed congenital hepatic fibrosis.

Stereological Principles and Sampling Procedures for Toxicologic Pathologists

Abstract This chapter outlines and discusses the principles and the practical tools of the scientific discipline of stereology with a focus on several established and novel (unpublished) sampling procedures. These sampling strategies are applied to whole three-dimensional organs, well-defined fragments of organs, and ultimately to two-dimensional sections interrogated using a range of stereological probes including points, lines, curves, frames, and optical and physical disectors. The chapter provides the toxicologic pathologist with a versatile toolbox of 9 sampling designs at the organ level and 38 distinct estimators of individual total structural characteristics. The designs and estimators are tailor-made for addressing frequently encountered quantitative problems in toxicologic pathology. For the sampling of tissue at the organ level, sampling designs are described in sufficient detail to allow their implementation. Numerous practical examples are illustrated and a few examples are explained in detail with accompanying computations in the appendices. Statistical error (coefficient of error, CE), the variability associated with the stereological procedure, is provided for most described estimators (with or without the use of the proportionator).

Bias in image analysis and its solution: unbiased stereology

Although the human eye is excellent for pattern recognition, it often lacks the sensitivity to detect subtle changes in particle density. Because of this, quantitative evaluation may be required in some studies. A common type of quantitative assessment used for routine toxicology studies is two-dimensional histomorphometry. Although this technique can provide additional information about the tissue section being examined, it does not give information about the tissue as a whole. Furthermore, it produces biased (inaccurate) data that does not take into account the size, shape, or orientation of particles. In contrast, stereology is a technique that utilizes stringent sampling methods to obtain three-dimensional information about the entire tissue that is unbiased. The purpose of this review is to illuminate the sources of bias with two-dimensional morphometry, how it can affect the data, and how that bias is minimized with stereology.

Aflatoxin B1 and/or Hepatitis B Virus Induced Tumor Spectrum in a Genetically Engineered Hepatitis B Virus Expression and Trp53 Haploinsufficient Mouse Model System for Hepatocarcinogenesis

The authors investigated the spectrum of tumors and Trp53 mutations in genetically engineered models using the FVB/N mouse that expressed the hepatitis B virus genome and/or carried a Trp53 null and wildtype allele and/or were exposed to aflatoxin B1. Liver tumor incidence was increased when all three risk factors were present. Without aflatoxin B1 exposure, neither Trp53 haploinsufficiency nor HBV expression affected liver tumor development. Liver tumor prevalence increased with aflatoxin B1 exposure (p < .001), as thirteen of fourteen mice with liver tumors were initiated with aflatoxin B1. Liver tumors were more frequent in males (12/190) than females (2/170). Seventy-three mice developed sarcomas. Trp53 haploinsufficiency was associated with increased sarcoma incidence in males and females (p < .001). In Trp53 haploinsufficient mice, the HBV transgene increased the risk of sarcoma in males and females (p < .001). Lymphoma was significantly increased in Trp53 haploinsufficient FVB/N mice. There was no loss of heterozygosity at the wildtype Trp53 locus in twenty-five sarcomas or four hepatocellular tumors examined. No mutations were identified in the mRNA (exons 2–11) of Trp53 in six liver neoplasms or twenty-four sarcomas. In this model system, HBV expression affected only hepatocellular neoplasia in association with both aflatoxin B1 initiation and p53 haploinsufficiency.

Decreased osteoprogenitor proliferation precedes attenuation of cancellous bone formation in ovariectomized rats treated with sclerostin antibody

Sclerostin antibody (Scl-Ab) stimulates bone formation, which with long-term treatment, attenuates over time. The cellular and molecular mechanisms responsible for the attenuation of bone formation are not well understood, but in aged ovariectomized (OVX) rats, the reduction in vertebral cancellous bone formation is preceded by a reduction in osteoprogenitor (OP) number and significant induction of signaling pathways known to suppress mitogenesis and cell cycle progression in the osteocyte (OCy) (Taylor et al., 2016). To determine if the reduction in OP number is associated with a decrease in proliferation, aged OVX rats were administered vehicle or Scl-Ab for 9 or 29 days and implanted with continuous-delivery 5-bromo-2′-deoxyuridine (BrdU) mini-osmotic pumps 5 days prior to necropsy. The total number of BrdU-labeled osteoblasts (OB) was quantified in vertebral cancellous bone to indirectly assess the effects of Scl-Ab treatment on OP proliferation at the time of activation of modeling-based bone formation at day 9 and at the time of maximal mineralizing surface, initial decrease in OP number, and transcriptional changes in the OCy at day 29. Compared with vehicle, Scl-Ab resulted in an increase in the total number of BrdU-positive OB (+260%) at day 9 that decreased with continued treatment (+50%) at day 29. These differences in proliferation occurred at time points when the increase in total OB number was significant and similar in magnitude. These findings suggest that reduced OP proliferation contributes to the decrease in OP numbers, an effect that would limit the OB pool and contribute to the attenuation of bone formation that occurs with long-term Scl-Ab treatment.

Meeting Report: Tissue-based Image Analysis

Quantitative image analysis (IA) is a rapidly evolving area of digital pathology. Although not a new concept, the quantification of histological features on photomicrographs used to be cumbersome, resource-intensive, and limited to specialists and specialized laboratories. Recent technological advances like highly efficient automated whole slide digitizer (scanner) systems, innovative IA platforms, and the emergence of pathologist-friendly image annotation and analysis systems mean that quantification of features on histological digital images will become increasingly prominent in pathologists’ daily professional lives. The added value of quantitative IA in pathology includes confirmation of equivocal findings noted by a pathologist, increasing the sensitivity of feature detection, quantification of signal intensity, and improving efficiency. There is no denying that quantitative IA is part of the future of pathology; however, there are also several potential pitfalls when trying to estimate volumetric features from limited 2-dimensional sections. This continuing education session on quantitative IA offered a broad overview of the field; a hands-on toxicologic pathologist experience with IA principles, tools, and workflows; a discussion on how to apply basic stereology principles in order to minimize bias in IA; and finally, a reflection on the future of IA in the toxicologic pathology field.

Immunopathology of the Hepatobiliary System

The liver is the largest internal organ in the body and contains many cell types that are involved in local and systemic immunity, including those of both the innate and adaptive immune systems. Approximately 10 % of the cells of the liver belong to the immune system, including the largest supply of tissue-specific macrophages and natural killer cells in the body. One of the main roles of immune cells in the liver is to establish an immunotolerant microenvironment, as the liver is the first tissue to receive portal blood flow from the gastrointestinal tract, which includes endotoxin, self-antigens, and ingested antigens. This prevents unwanted systemic immune responses towards these everyday encountered antigens. Kupffer cells and sinusoidal endothelial cells in the liver play important roles in removing pathogens and other possible antigens from the portal blood prior to them reaching the systemic circulation, also preventing unwanted reactions. Autoimmune disease can result if this immunotolerant microenvironment is disrupted. The liver also plays a role in local and systemic inflammation through the production of acute phase proteins, cytokines, and other pro-inflammatory mediators. Immune cells play an important role in many liver diseases of humans and animals, including drug-induced liver injury, viral infection, and neoplasia. In addition, the liver plays an important role in systemic disorders, particularly septicemia. This chapter outlines the normal immunobiology of the liver, including the cell types involved in the immune response, and their roles in various diseases of the liver as well as systemic disorders. Animal models of liver diseases involving the immune system are also discussed.