Morphological study of cultured cardiac ganglionic neurons from different postnatal stages of rats.

This study sought to establish a culture model of cardiac ganglia (CG) neurons of the Sprague-Dawley (SD) rat which could by used to study the distinct characteristics of CG neurons. After culturing, the morphology and immunocytochemistry of CG neurons obtained on different days after birth were compared. Samples of CG neurons were taken from the posterior atrial wall of rats aged 7, 14, 21 and 40 postnatal days (designated as P7, P14, P21 and P40, respectively). During 3-6 days of culture, the morphological changes of the cultured neurons were monitored using a light microscope. Immunocytochemical staining of the neurofilaments (NF-L, -M and -H) was performed to identify the CG neurons and the changes in morphology. The differences in size of the CG soma of each culture were compared by morphometry. Frozen sections of CG neurons were used as the in vivo control of the above experiments. The results showed that the rate of growth in size of the CG soma was highest in the P7 group, and was slower after weaning (21 days after birth). Cultured neurons were categorized into unipolar-like (Type I), multipolar-like (Type II), and bipolar-like (Type III) based on their morphological characteristics. In NF immuocytochemical staining, there were strong responses to NF-H and NF-M in all cultures, but not to NF-L. More specifically, responses to NF-H were mainly observed in perikaryons and neurites, whereas the responses to NF-M were mainly in perikaryons. The present study has established a culture system for cardiac ganglia neurons of SD rats. Our results show that the intracardiac neurons were still developing in their somata and the processes and that various responses to different antibodies of NF for CG neurons occurred in different postnatal stages in rats.

Growth characteristics of normal and dystrophic myoblasts in primary myoblast cultures.

This study employed immunocytochemistry and toluidine blue counterstaining to compare different procedures utilized in primary myoblast cultures, from which an optimal culture model for normal myoblasts could be derived. The growth characteristics of normal and dystrophic myoblasts were also investigated by means of this model. Results indicate that the requirements for an ideal myoblast culture should include a combined enzyme of 0.25% trypsin and 0.2% collagenase (type IV) (1:1), a preplating time of approximately 15-20 minutes, and a seeding density of 1 x 10(5) cells/ml. Furthermore, the mouse samples should be newborn mice. A better proliferative capacity of myoblasts was noted in an incubator with 10% CO2 coupled with Dulbecco's MEM plus 15% fetal calf serum. With regard to the growth characteristics of normal and dystrophic myoblasts, the doubling time of normal myoblasts was shorter than that of dystrophic myoblasts. In terms of the fusion percentage of myoblasts, dystrophic myoblasts tended to fuse earlier than normal ones, especially after 5 days in culture. The findings of this study are valuable in understanding the myogenesis of myoblasts under different culture conditions. The establishment of requirements for good growth of myoblast cultures will facilitate myoblast transfer therapy. Finally, the growth characteristics of normal and dystrophic myoblasts as well as variances in the proliferation and differentiation of these two types of cells are clarified.

Primary culture of mouse myoblasts.

The literature has revealed variations in the protocols for myoblast cultures, and little information is available on myoblast and fibroblast proliferation. Therefore, the purposes of this study were to establish a prudent protocol for myoblast cultures by comparing a variety of culturing procedures used in previous research and to quantitate myoblast proliferation and fusion under different culture conditions. In addition, the growth status of myoblasts and fibroblasts was investigated. Results indicate that the requirements for an ideal myoblast culture should include a combined enzyme of 0.25% trypsin and 0.2% collagenase type IV (1:1), a preplating time of approximately 15-20 minutes, and a seeding density at 1 x 10(5) cells/ml. Furthermore, the mouse sample should be those of newborns. A better proliferative capacity of myoblasts was noted in an incubator of 10% CO2, coupled with Dulbecco's MEM plus 15% fetal calf serum. The doubling times of myoblasts were shorter than those of fibroblasts, and myoblast number reached its highest at 4 and 5 days. The findings of this study are valuable in understanding the growth status of myoblasts and fibroblasts in primary cultures. Moreover, the establishment of requirements for a good growth of myoblast cultures will facilitate myoblast transfer therapy.

Seasonal variation in cell cycle during early development of the mouse embryo.

Studies of the cell cycle of mouse embryos before implantation were conducted using Giemsa and DAPI stains. The time of embryo recovery did not affect the success rate of cultures during the winter, but embryos cultured during the summer showed the 'two-cell block' phenomenon at the early two-cell stage, 30-37 h after the injection of human chorionic gonadotrophin. There was no significant difference in the number of embryos collected per mouse between summer and winter, but cleavage from the two-cell to the four-cell stage occurred later in the summer than in the winter. Cell cycle of mouse embryos may therefore show seasonal variation.

Effect of unilateral vagotomy on capsaicin-induced neurogenic inflammation in the trachea of rats.

This study reports the effect of unilateral vagotomy on neurogenic inflammation in the trachea. Neurogenic inflammation was produced in the trachea of vagotomized rats by a venous injection of capsaicin, the pungent ingredient of red pepper. Monastral blue was used as tracer dye to label the affected blood vessels. Vagus nerves and tracheal tissues were processed for light and electron microscopy. The damaged right vagus nerve was found to have degenerated. However, unilateral vagotomy did not completely block neurogenic inflammation in the trachea ipsilaterally and contralaterally, as shown in the whole mount preparations. Microscopic observations of tracheal tissue sections showed that neurogenic inflammation did not evenly occur on both sides of the trachea. The possible reason for this is discussed.