Cytoplasmic Vacuolation and Tapetal Changes Induced by a Novel Analgesic Agent in Beagle Dogs

Drug-induced unique cytoplasmic vacuolation was found in the subchronic oral toxicity study of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), a potential therapeutic agent for neuropathic pain, in beagle dogs. In the first study, DMIP was administered at a dose of 250, 500, or 1,000 mg/kg/day once daily for 14 days. Discoloration of tapetum lucidum accompanied by tapetal swelling was observed at ≥250 mg/kg/day. The tapetal swelling was correlated to the light microscopic observation of cytoplasmic vacuolation in tapetal cells, and similar vacuolation was observed in several other tissues, including the coronary artery and aortal arch, in a dose-dependent manner. Immunohistochemistry for lysosomal-associated membrane protein 2 indicated that the vacuoles were enlarged lysosomes. However, the nature of these vacuoles was different from that of phospholipidosis because no lamellar bodies were observed. In the second study, DMIP was administered at a dose of 10, 50, or 250 mg/kg/day once daily for 14 days followed by a 14-day recovery period. Tapetal changes and systemic vacuolation were not observed at ≤50 mg/kg/day, and vacuolation observed at 250 mg/kg/day was reversible. A few reports have described the enlargement of lysosomes not attributable to phospholipid accumulation. Our findings provide further information about the toxicological implications of drug-induced lysosomal swelling.

Hyaloperonospora camelinae on Camelina sativa in Washington State: Detection, Seed Transmission, and Chemical Control

Camelina (Camelina sativa) plants with symptoms of downy mildew were obtained from three different locations in Washington State. Based on polymerase chain reaction (PCR) and sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, the causal pathogen was identified as Hyaloperonospora camelinae. The PCR primers consistently amplified 699-bp bands from the infected plants but not from the asymptomatic plants. A comparison of the sequences with those in GenBank revealed 100% sequence similarity to H. camelinae. Growth and development of the H. camelinae was observed in different tissues using light microscopy and scanning electron microscopy (SEM). Light microscopic observation revealed the presence of oospores in the infected leaves and SEM revealed the presence of conidia and conidiophores on the seed surface. To determine whether H. camelinae is a seed-transmitted pathogen, seed collected from infected plants were planted in Sunshine professional growing mix maintained in a growth chamber. Disease symptoms were observed in 96% of the seedlings compared with 3% of the seedlings grown from seed from asymptomatic plants, which indicates that H. camelinae is a seed-transmitted pathogen. Seed treated with mefenoxam, a fungicide specific for Oomycetes, significantly reduced the incidence of the disease.

A Simple but Effective Demineralizing Method for Osteogenic Allograft Preparation

Bone regeneration with demineralized bone preparations has demonstrated its potential in grafting procedures in surgical disciplines of both medicine and dentistry. To improve the effectiveness in preparation and osteogenic regeneration, we developed a simple and rapid demineralizing method for osteogenic allograft preparation and evaluated its osteogenic effect using an ectopic bone formation assay. The rat diaphyseal cortical bones were demineralized in heated 0.6N HCl at 60° C for 10 to 30 mins using a controlled-heat ultrasonic cleaner, washed with sterilized distill water, and dehydrated with graded alcohol after 30% H2O2 treatment. The prepared grafts were implanted in rat dorsal subcutaneous pouches for 1-3 weeks and then the harvested tissue samples were prepared for routine light microscopic observation. The allografts demineralized with 60°C HCl for more than 20 mins in an ultrasonic condition were completely demineralized and effectively induced ectopic formation without specific pathologic findings. These findings suggest that demineralization with 60°C HCl for around 20 mins using a controlled-heat ultrasonic cleaner and dehydration with graded alcohol after short treatment with 30% H2O2 is a very simple but effective osteogenic allograft preparation method with minimal antigenicity and sterilizing effects.

In Vitro Osteogenic Activity of Rat Mesenchymal Cells Cultured on Transparent β-Tricalcium Phosphate Ceramics

We have cultured mesenchymal cells (MSC) on various types of ceramic disks and used these tissue-engineered ceramics for hard tissue regeneration. In this approach, observation of cultured cell morphology is important even if culture substrata are calcium phosphate ceramics, which usually show bioactive nature. However, due to the opaque nature of the ceramics, cells observation is very difficult. Here, we demonstrate light microscopic observation of rat MSC cultured on transparent β-tricalcium phosphate ceramics (β-TCP). The culture was performed in osteogenic medium. Thus, the cell differentiated into bone-forming osteoblasts, which fabricated a mineralized matrix on the ceramic disks. Microscopic observation revealed that the cascade of osteogenic differentiation after attachment/proliferation of MSC on the ceramic disks was similar to that on a culture grade polystyrene dish. These results confirmed the excellent property of β-TCP for MSC culture leading to hard tissue regeneration.