Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

A porcine model of neurofibromatosis type 1 that mimics the human disease.

Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.

Keratoacanthoma Pathobiology in Mouse Models.

Recently we described skin tumors driven by skin-specific expression of Zmiz1 and here we define keratoacanthoma pathobiology in this mouse model. Similar to human keratoacanthoma development, we were able to segregate murine keratoacanthomas into three developmental phases: growth, maturation, and regression. These tumors had areas with cellular atypia, high mitotic rate, and minor local invasion in the growth phase, but with development they transitioned to maturation and regression phases with evidence of resolution. The early aggressive appearance could easily be misdiagnosed as a malignant change if the natural pathobiology was not well-defined in the model. To corroborate these findings in the Zmiz1 model, we examined squamous skin tumors from another tumor study in aging mice, and these tumors followed a similar biological progression. Lastly, we were able to evaluate the utility of the model to assess immune cell infiltration (F4/80, B220 Granzyme B, CD3 cells, arginase-1) in the regression phase; however, because inflammation was present at all phases of development, a more comprehensive approach will be needed in future investigations. Our study of keratoacanthomas in selected murine models suggests that these squamous tumors can appear histologically aggressive during early development, but with time will enter a regression phase indicating a benign biology. Importantly, studies of squamous skin tumor models should be cautious in tumor diagnosis as the early growth distinction between malignant versus benign based solely on histopathology may not be easily discerned without longitudinal studies to confirm the tumor pathobiology.

Genetically modified species in research: Opportunities and challenges for the histology core laboratory.

Translational research using animal models has traditionally involved genetically modified rodents; however there is increasing use of other novel genetically engineered species. As histology laboratories interface with researchers studying these novel species there will be many situations in which protocols will need to be adapted to the species, model and research goals. This paper gives examples of protocol adaptations to meet research needs and addresses common considerations that should be addressed for all research tissues submitted to the histotechnology laboratory. Positioning the histotechnologist, as well as the investigator, to meet the challenges associated with novel research models will help maximize research efficacy and quality.

Extended sequence preferences for oligodeoxyribonucleotide activity.

Synthetic type B phosphorothioate oligodeoxyribonucleotides (ODN) activate mouse B cells via Toll-like receptor 9 (TLR9). Starting with closely related 15-mer prototype ODN, the sequence requirements for stimulatory (ST-) and inhibitory (IN-) activity were contrasted, by measuring apoptosis protection, G(1) entry and interleukin-6 secretion. ST-ODN and IN-ODN differ in that (1) ST-ODN require a 5' T, (2) the central CG is obligatory, (3) CG must be flanked 3' specifically by TT at the position where IN-ODN have GG, and (4) IN-ODN tolerate truncation of the 3' end better than ST-ODN. Features shared by ST-ODN and IN-ODN include (1) requiring CC adjacent to the 5' end, and (2) avoiding CC immediately 5' to the CG. This pattern is used to create a model of how ST-ODN binding might function to aggregate TLR9 so as to initiate the signal, and how the 5' ends of ST-ODN and IN-ODN compete for binding. Further justification for considering TLR9 to be the ODN receptor was provided by a demonstration that in HEK293 cells transfected with TLR9, the potency of a panel of ODN for activating NF-kappaB roughly parallels that seen in the biological assays in mouse B cells.

Innate immune responses in lupus-prone Palmerston North mice: differential responses to LPS and bacterial DNA/CpG oligonucleotides.

Inadequate immune response to infectious danger may contribute to the pathogenesis of systemic autoimmune diseases, e.g., systemic lupus erythematosus. To test this hypothesis, we studied innate responses of prediseased lupus-prone Palmerston North (PN) mice to lipopolysaccharide (LPS), bacterial DNA, and synthetic CpG oligonucleotides. LPS and bacterial DNA/CpG oligodeoxyribonucleotides (ODNs) drove PN splenocytes into the cell cycle and protected B cells against spontaneous apoptosis, as in control lupus-free DBA-1 mice. LPS induced significantly higher IL-6 production in PN than in control splenocytes. In contrast, in PN splenocytes bacterial DNA and CpG ODNs induced approximately four- to sixfold lower IL-12p40 and approximately twofold lower IL-6 secretion than controls. This reduction in cytokine secretion in PN mice was not due to delayed kinetics but was related to significantly higher constitutive and CpG-inducible IL-10 secretion. Neutralizing anti-IL-10 antibodies almost completely restored PN IL-6 and IL-12p40 secretion to DBA-1 levels, whereas exogenous IL-10 inhibited in vitro IL-6 and IL-12p40 production in DBA-1 mice. Importantly, treatment with either IL-10 or anti-IL-10 antibody did not modulate CpG-induced cell cycle entry and apoptosis protection in either strain. In conclusion, lupus-prone PN mice show abnormal innate responses through their pattern-recognition TLR9 receptors, characterized by higher inducible IL-10 and lower IL-12p40 and IL-6 secretion, thus implying that response to infectious danger in PN mice is inappropriate and may be linked to lupus pathogenesis.