Optimization of decalcification techniques for histologic examination of the rat maxillary and mandibular incisors for toxicity studies.

The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.

Myostatin Inhibition Using ActRIIB-mFc Does Not Produce Weight Gain or Strength in the Nebulin Conditional KO Mouse.

Mutations in at least 12 genes are responsible for a group of congenital skeletal muscle diseases known as nemaline myopathies (NMs). NMs are associated with a range of clinical symptoms and pathological changes often including the presence of cytoplasmic rod-like structures (nemaline bodies) and myofiber hypotrophy. Our recent work has identified a variable degree of behavioral benefit when treating 2 NM mouse models due to mutations in Acta1 with myostatin inhibition. This study is focused on the effects of delivering ActRIIB-mFc (Acceleron; a myostatin inhibitor) to the nebulin conditional knockout KO (Neb cKO) mouse model of NM. Treatment of Neb cKO mice with ActRIIB-mFc did not produce increases in weight gain, strength, myofiber size, or hypertrophic pathway signaling. Overall, our studies demonstrate a lack of response in Neb cKO mice to myostatin inhibition, which differs from the response observed when treating other NM models.

Sdha+/- Rats Display Minimal Muscle Pathology Without Significant Behavioral or Biochemical Abnormalities.

Mitochondrial diseases (MDs) result from alteration of the mitochondrial respiratory chain (MRC) function. Despite the prevalence of MDs in the population, the paucity of animal models available limits the understanding of these disorders. Mutations in SDHA, a gene that codes for the alpha subunit of succinate dehydrogenase (SDH), can cause some forms of MD. SDHA is a crucial contributor to MRC function. In order to expand the range of MD animal models available, we attempted to generate a Sdha knockout rat. Since homozygous Sdha-/- rats could neither be identified in newborn litters, nor as early as embryonic day 14, we evaluated wild-type (WT) and heterozygous Sdha+/- genotypes. No differences in behavioral, biochemical, or molecular evaluations were observed between WT and Sdha+/- rats at 6 weeks or 6 months of age. However, 30% of Sdha+/- rats displayed mild muscle fiber atrophy with rare fibers negative for cytochrome oxidase and SDH on histochemical staining. Collectively, our data provide additional evidence that modeling SDH mutations in rodents may be challenging due to animal viability, and heterozygous rats are insufficiently symptomatic at a phenotypic and molecular level to be of significant use in the study of SDH deficiency.