Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

National Institute of Neurological Disorders and Stroke

Complex odontoma from a female Sprague-Dawley rat is described histopathologically. Necropsy revealed a hard (bony), white mass (3.0 x 3.0 x 2.1 cm) on the left mandible. Microscopically, the mass consisted of islands or nests of epithelial and mesenchymal elements that formed abortive tooth structures. In other areas, tooth formation consisted of a pulp cavity lined by layers of odontoblasts, dentin, enamel, and ameloblasts. Concerning all features of normal tooth formation which was differentiated and mineralized yet completely disorganized, the diagnosis of complex odontoma was recommended.

Spontaneous complex odontoma in a Sprague-Dawley rat.

A total of 604 Escherichia coli strains isolated from weaned pigs with diarrhea or edema disease on 653 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of 5 fimbriae (F4, F5, F6, F18, and F41), 3 heat-stable (STa, STb, and EAST1) and 1 heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e) genes. Forty-five (7.5%) of the 604 E. coli isolates carried the gene for AIDA. Of these 45 isolates, 5 (11.1%) carried EAST1 genes only, 1 (2.2%) carried genes for at least one of the fimbrial adhesins, 12 (26.7%) carried genes for at least one of the toxins, and 27 (60%) carried genes for at least one of the fimbrial adhesins and toxins. Fifty-one percent of strains that carried AIDA genes carried Stx2e genes, and 40% of strains that carried AIDA genes carried F18ab. The isolation rate of enterotoxigenic E. coli strain carrying genes for AIDA was 87%, and the isolation rate of Shiga toxin-producing E. coil strain carrying genes for AIDA was 49%. AIDA may represent an important virulence determinant in pigs with postweaning diarrhea or edema disease.

Prevalence of a gene encoding adhesin involved in diffuse adherence among Escherichia coli isolates in pigs with postweaning diarrhea or edema disease.

Swine hepatitis E virus (HEV) was detected in formalin-fixed, paraffin-embedded tissues from naturally infected pigs by nested reverse transcription-polymerase chain reaction (RT-PCR). The results for seminested RT-PCR were compared with those determined by in situ hybridization. The results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested RT-PCR is a reliable detection method. Swine HEV nucleic acid was detected in formalin-fixed, paraffin-embedded hepatic tissues from 40 pigs. Distinct positive signals for swine HEV were obtained in the same hepatic tissues by in situ hybridization. Swine HEV nucleic acid was localized to the cytoplasm of hepatocytes and had a granular staining pattern. The rate of agreement between nested RT-PCR and in situ hybridization for the detection of swine HEV in formalin-fixed, paraffin-embedded hepatic tissues was 100%.

Development of nested RT-PCR for the detection of swine hepatitis E virus in formalin-fixed, paraffin-embedded tissues and comparison with in situ hybridization.

In this study the subtype of eae gene was determined by polymerase chain reaction for a total of 59 attaching and effacing Escherichia coli isolated from preweaned (38 isolates) and postweaned (21 isolates) pigs. The eae(beta) gene detected in 19 E. coli from preweaned pigs and 10 E. coli from postweaned pigs was found to be the most common subtype, followed by eae(gamma), eae(epsilon), and eae(zeta) genes. Subtypes were not determined for 7 E. coli isolates. No other subtype of the eae gene was detected in eae+ E. coli evaluated in this study.

Polymerase chain reaction analysis of eae gene subtypes present in attaching and effacing Escherichia coli isolated from pigs with diarrhea.

Obesity is a condition of abnormal adipose tissue storage and recently it has been recognized as a major factor in metabolic syndrome. High-fat diet-induced obesity in the C57BL/6 mouse is an important animal model because of its similarities with human obesity. The aim of the present study was to estimate obesity, liver injury and steatohepatitis, and the distribution of inducible nitric oxide synthase (iNOS) in mice with high-fat diet induced obesity. Three-week-old male C57BL/6J mice were fed either a high-fat diet (D-60: 60 kcal% fat, or D-45: 45 kcal% fat) or a normal diet (D-10: 10 kcal% fat) for 15 weeks. Oral glucose tolerance tests and intraperitoneal glucose tolerance tests showed that the D-60 mice had severely impaired glucose tolerance. In serum chemistry values and histopathological lesions, the D-60 group showed severe steatohepatitis. A distinct positive signal for iNOS was detected by immunohistochemistry in the cytoplasm of hepatocytes around the central vein in the D-45 and D-60 groups. Serum insulin levels and insulin immunohistochemistry in the pancreas showed pancreatic injury and insulin resistance in the D-60 group. We observed the presence of more iNOS in the high-fat diet-induced obese mouse, which has characteristics of non-alcoholic steatohepatitis (NASH) and diabetes, and expect that these background pathological data will be useful in research on obesity, diabetes mellitus, and non-alcoholic fatty liver disease.

Inducible nitric oxide distribution in the fatty liver of a mouse with high fat diet-induced obesity.

Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs) were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.

Altered cell cycle gene expression and apoptosis in post-implantation dog parthenotes.

Graft-versus-host disease (GVHD) remains a significant complication of allogeneic transplantation. We previously reported that the adenosine A(2A) receptor (A(2A)R) specific agonist, ATL146e, decreases the incidence and severity of GVHD in a mouse transplant model. There is increasing interest in treatments that increase CD4(+)CD25(high)Foxp3(+) regulatory T cells (Tregs) to suppress GVHD. Our current study found in vitro that A(2A)R selective agonists enhanced TGF-β-induced generation of mouse Tregs 2.3- to 3-fold. We demonstrated in vivo suppression of GVHD with specific A(2A)R agonists in two different murine GVHD transplant models associated with profound increases in both circulating and target tissue Tregs of donor origin. Three different A(2A)R agonists of differing potency, ATL146e, ATL370, and ATL1223, all significantly inhibited GVHD-associated weight loss and mortality. At the same time, Tregs shown to be of donor origin increased 5.1- to 7.4-fold in spleen, 2.7- to 4.6-fold in peripheral blood, 2.3- to 4.7-fold in colon, and 3.8- to 4.6-fold in skin. We conclude that specific activation of A(2A)R inhibits acute GVHD through an increase of donor-derived Tregs. Furthermore, the increased presence of Tregs in target tissues (colon and skin) of A(2A)R-specific agonist-treated mice is likely the mechanistic basis for the anti-inflammatory effect preventing acute GVHD.

Adenosine A₂A receptor agonist-mediated increase in donor-derived regulatory T cells suppresses development of graft-versus-host disease.

Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.

Intrapancreatic ectopic splenic tissue found in a cloned miniature pig.

Alcoholic fatty liver diseases (AFLD) and non-alcoholic fatty liver diseases (NAFLD) are two pathological conditions that are spreading worldwide. Both conditions are remarkably similar with regards to the pathophysiological mechanism and progression despite different causes. Oxidative stress-induced mitochondrial dysfunction through post-translational protein modifications and/or mitochondrial DNA damage has been a major risk factor in both AFLD and NAFLD development and progression. Cytochrome P450-2E1 (CYP2E1), a known important inducer of oxidative radicals in the cells, has been reported to remarkably increase in both AFLD and NAFLD. Interestingly, CYP2E1 isoforms expressed in both endoplasmic reticulum (ER) and mitochondria, likely lead to the deleterious consequences in response to alcohol or in conditions of NAFLD after exposure to high fat diet (HFD) and in obesity and diabetes. Whether CYP2E1 in both ER and mitochondria work simultaneously or sequentially in various conditions and whether mitochondrial CYP2E1 may exert more pronounced effects on mitochondrial dysfunction in AFLD and NAFLD are unclear. The aims of this review are to briefly describe the role of CYP2E1 and resultant oxidative stress in promoting mitochondrial dysfunction and the development or progression of AFLD and NAFLD, to shed a light on the function of the mitochondrial CYP2E1 as compared with the ER-associated CYP2E1. We finally discuss translational research opportunities related to this field.

Role of CYP2E1 in mitochondrial dysfunction and hepatic tissue injury in alcoholic and non-alcoholic diseases.

The role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in promoting aging-dependent hepatic disease is unknown and thus was investigated in this study. Young (7 weeks) and aged female (16 months old) wild-type (WT) and Cyp2e1-null mice were used in this study to evaluate age-dependent changes in liver histology, steatosis, apoptosis, fibrosis and many nitroxidative stress parameters. Liver histology showed that aged WT mice exhibited markedly elevated hepatocyte vacuolation, ballooning degeneration, and inflammatory cell infiltration compared to all other groups. These changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT mice although serum ALT and insulin resistance were not significantly altered. Aged WT mice showed the highest rates of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of mitochondrial nitroxidative stress and alteration of mitochondrial complex III and IV proteins in aged WT mice, although hepatic ATP levels seems to be unchanged. In contrast, the aging-related nitroxidative changes were very low in aged Cyp2e1-null mice. These results suggest that CYP2E1 is important in causing aging-dependent hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 could be a potential target for translational research in preventing aging-related liver disease.

Seung-Kwon Ha

Translational Neuroradiology Section

National Institute of Neurological Disorders and Stroke

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Seung-Kwon Ha

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Translational Neuroradiology Section

National Institute of Neurological Disorders and Stroke

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Translational Neuroradiology Section