Abstract We examined the expression of N‐myc, c‐myc, and c‐src in four embryonic carcinoma (EC) cell lines during different states of cell growth and following induction of in vitro differentiation. N‐myc mRNA was detected in undifferentiated cells of four EC cell lines (PCC7, PCC3, PCC4, F9) neither of which showed N‐myc gene amplification. No N‐myc transcripts could be detected in mRNA prepared from a murine neuroblastoma cell line and from a murine fibroblast line. The level of N‐myc mRNA decreased by 85% when PCC7 EC cells were induced by retinoic acid and cAMP treatment to form nerve‐like cells. Six days after induction, the PCC7 cells changed into aggregates of neurofilament positive cells with massive neurite outgrowths. At this stage DNA replication had been reduced by more than 95%. The decreased N‐myc expression in induced PCC7 cells was parallelled by 300–500% increase in c‐src expression. Slowing of cell multiplication by serum starvation, on the other hand, did not affect the level of N‐myc or c‐src mRNA levels in PCC7 cells. C‐myc was expressed in all EC lines except PCC7, which surprisingly did not express c‐myc even at an exponential rate of proliferation. Chemical induction of F9 EC cells to form visceral endoderm or parietal endoderm resulted in markedly reduced (85%) levels of N‐myc transcripts. A similar decline in c‐myc expression was found in differentiated F9 cells. No c‐src transcripts were detected in proliferating or differentiated F9 cells. These results suggest that N‐myc may be expressed not only in neural development, but also in very early, undetermined embryonic cells. The activation of c‐src expression when PCC7 EC cells differentiate into nerve‐like cells shows that the pattern of proto‐oncogene expression may change during a differentiation process, some proto‐oncogenes increasing, others decreasing their representation in the mRNA pool.
Using immunohistochemistry with antisera raised against the glial fibrillary acidic protein (GFA), we have studied the appearance and distribution of GFA-like immunoreactivity in whole mounts of rodent iris and in sectioned cat and cow iris. In the adult rat iris, a dense plexus of GFA-positive fibers was seen in both the dilator plate and the sphincter. The fluorescent fibers formed large meandering bundles and a dense irregular network of thinner fibers. In the sphincter, mainly thinner fibers were seen. Thin fibers were also seen winding around blood vessels in the dilator plate. In adult mouse iris, the GFA-positive fibers had a quite different distribution with a few radially oriented fiber bundles superimposed on a more regular network of thinner fibers. Adult guinea pig irides showed still another pattern of GFA-positive fibers with a low number of bundles and thinner fibers forming a sparse irregular network. In thicker fiber bundles of all three rodent species, as well as at branching sites of the thinner fibers, negative or weakly fluorescent swellings surrounded by GFA-like immunoreactivity were present. These structures probably represent the cellular origin of the GFA-positive fibers. Thick, strongly fluorescent fiber bundles, as well as numerous thinner fibers, were seen in sections of cat and cow iris. Prenatally, fibers were visualized at embryonic day 18 in the rat. In these irides as well as in irides from 21-day-old embryos and 1-day-old pups, most fibers were organized in a gradually increasing system of thin meandering fiber bundles that showed limited branching. At postnatal day 6, a more mature network of thinner fibers had developed between the now more numerous fiber bundles. No obvious increase or decrease in the amount of GFA-positive fibers was seen in irides grafted to the anterior eye chamber of adult rat recipients examined 1 and 6 days after grafting. However, in these irides, as well as in the host irides, strongly fluorescent spider-like cells with short branching processes and a negative nucleus were seen. These cells were more numerous and more strongly fluorescent in grafted irides as compared to recipient irides and in the 6-day iris grafts as compared to the 2-day grafts. In all probability, the GFA-positive fibers and cells forming a network in adult irides from different species and in embryonic and grafted rat irides represent Schwann cells and their processes. The cellular origin of the spider-like cells in the iris grafts is less clear.
The presence and morphology of GFA- and vimentin-positive astrocytes were studied immunohistochemically in rats using smear preparations of single intraocular grafts of the cortex cerebri anlage and of cortex pieces grafted to eyes containing a previously grafted piece of the locus coeruleus area. Similarly, astrocytes were studied in lesioned cortex cerebri in situ. A high number of GFA and vimentin-positive cells were found in smears of both types of cortex grafts as well as in smears of the lesioned cortex cerebri in situ. In contrast, only a limited number of GFA-positive astrocytes were seen in smears of normal cortex. Using computerized image analysis, the two-dimensional cell area and cell perimeter were found to be significantly increased in individual GFA-positive astrocytes in both types of intraocular cortex grafts as well as in the lesioned cortex when compared to GFA-positive astrocytes in normal cortex cerebri. GFA-positive cells in smears of cortex grafts from locus coeruleus-cortex combinations had significantly smaller cell area and cell perimeter values compared to similar cells from single cortex grafts. A similar, although less pronounced difference was observed between vimentin-positive cells from the same type of grafts. This suggests that the presence of the mature locus coeruleus graft in some as yet unknown way influences the development of the adjacent cortex graft towards a more normal astrocytic maturation. An additional finding was the large size difference between GFA- and vimentin-positive cells in the intraocular grafts. Since most evidence indicates that vimentin-positive cells are also GFA-positive, this may indicate that the two intermediate filament systems have a partially different distribution within individual astrocytes. It is concluded that computerized image analysis of smears processed for immunohistochemistry with antisera against GFA and vimentin is a useful technique for studies of astrocyte morphology in normal as well as experimentally perturbed CNS tissue. Cortex tissue that develops in contact with a locus coeruleus graft in the eye chamber show a significantly lesser degree of gliosis than cortex tissue developing in isolation in the eye.
Abstract Dependency of astrocytes on thyroid hormones during development was studied in intraocular cortex cerebri grafts, using antibodies to the glial fibrillary acidic protein (GFA). Fetal cortex cerebri was left in the anterior eye chamber of normal and thyroidectomized adult recipient rats 3 and 8 weeks to mature. All cortex grafts survived and became vascularized in the eye. The grafts were cryostat‐sectioned, and reacted with an antiserum specific for GFA. The density of GFA immunoreactivity in the cortex graft neuropil was subjectively estimated with fluorescence microscopy. After 3 weeks in oculo , the number of positive structures was significantly lower in the thyroidectomized group as compared to the euthyroid control group. This effect of thyroidectomy on the cortex grafts was entirely counteracted by daily injections of thyroxine (100 μg/kg s.c.) to similarly thyroidectomized recipients. The distribution of GFA‐positive structures was even within individual grafts of all three groups. Neither were any differences in morphology of fluorescent astroglial processes observed. The overall density of fluorescent GFA‐positive structures was considerably higher than that found in age‐matched normal in situ counterparts, even in the thyroidectomized group. After 8 weeks in oculo , the difference between the control and thyroidectomized groups in density of GFA‐positive structures in the neuropil had disappeared, and no general increase in density of immunoreactivity was noted in the control group. These results indicate that thyroid hormones might normally play a role for the development of GFA in the early postnatal brain, as thyroid hormone deficiency causes a transient delay in development of GFA immunoreactivity in intraocular cortex cerebri grafts.
The biocompatibility of heparin surface modified poly(methyl methacrylate) intraocular lenses (IOLs) was evaluated in two experiments following implantation in the anterior and posterior eye chambers of adult cynomolgus monkeys. Throughout the study, large inflammatory cells and prominent pigment deposits were seen on the unmodified lenses, whereas the heparin surface modified IOLs remained almost free of precipitates. Similarly, fewer posterior synechias were observed in eyes implanted with surface modified IOLs in the posterior chamber than in eyes implanted with control lenses. Histopathological examination of enucleated eyes confirmed the clinical findings. These experiments strongly support the idea that surface modification with heparin is a useful way to reduce clinical complications following cataract surgery with IOL implantation.
Abstract The influence of neonatal hypothyroidism on the development of immunoreactivity to glial fibrillary acidic protein (GFA) was studied in parietal cortex of rats treated from birth with the antithyroid agent propylthiouracil (PTU) for 3 or 8 weeks. Density of GFA immunoreactivity was evaluated in cryostate sections reacted with an antiserum specific for GFA. Three weeks postnatally, the density of GFA‐immunoreactive structures in the cortical layers II‐V was 70% lower in PTU‐treated animals than in controls injected with the solvent. This marked difference between the groups was, however, not seen in either the molecular layer, layer VI or white matter. The inhibited development of GFA immunoreactivity was not persistent in animals treated with PTU for 8 weeks continuously. Plasma from animals treated with PTU for 1,2,3 and 8 weeks was collected and the TSH level in each group compared with samples from age‐matched controls and newborn pups. The treatment with PTU resulted in a more than 10‐fold increase in TSH level after 1 week of injections. In longterm groups of 8 weeks, the TSH level decreased in the PTU‐treated animals, but stayed considerably higher than control values throughout the experiments. The results described in the present paper indicate a thyroid hormone dependent development of the GFA immunoreactivity in cortex cerebri astrocytes.
