Morphological analysis of selective destruction of pancreatic beta-cells by cytotoxic T lymphocytes in NOD mice.

Interactions of pancreatic islets and islet-associated mononuclear cells (IAMCs) from the nonobese diabetic (NOD) mouse were morphologically investigated. To obtain IAMCs, pancreatic islets isolated from adult NOD mice were cultured for 7 days with interleukin 2. Noted by light microscopy, interactions between IAMCs and freshly isolated islets from young NOD mice began 30 min after the initiation of the coculture, and 6 h later, normal cellular array of the islets was lost. By electron microscopy, most IAMCs had low nucleus-cytoplasm ratio, the nucleus was notched and exhibited condensed chromatin along the nuclear membrane, and well-developed Golgi complexes and several mitochondria were distributed in the cytoplasm. These IAMCs adhered to beta-cells, but not to alpha- or delta-cells, with their pseudopods and caused cytolysis of beta-cells. Immunohistochemical study with antibodies specific for pancreatic hormones demonstrated that only cells reacting with anti-insulin antibody were selectively lost as the incubation time proceeded. Electron immunohistochemistry by immunogold technique showed that effector cells in IAMCs reacted with anti-CD8 (Lyt-2) antibody, but not anti-CD4 (L3T4) or anti-asialogangliosideM1 antibody. In addition, the concentration of pancreatic hormones in the culture medium, used as a marker of cytolysis, also demonstrated that insulin was significantly increased after 6 h of culture, whereas glucagon and somatostatin were not. These results suggest that CD8+ cytotoxic T lymphocytes are involved in the selective destruction of pancreatic beta-cells in the NOD mouse.

Histochemical study of the differentiation of microglial cells in the developing human cerebral hemispheres.

Applying nucleoside diphosphatase (NDPase) histochemistry, the appearance and differentiation of microglial cells in the developing human cerebral hemispheres were investigated by light and electron microscopy. In the pallium of the 38 days old human embryo, a few round NDPase-positive cells (round cells) were observed in the expanding zone. Although distinct blood vessels had not yet formed within the wall of the pallium, some cellular elements resembling haemopoietic cells were noticed in the expanding zone. In the 51 days old fetus, blood vessels displaying NDPase activity were seen in the mantle and marginal layers, and some invaded the matrix. Several round NDPase-positive cells were distributed, mainly around the vascular sprouts (primitive blood vessels) in the matrix. In the marginal layer, NDPase-positive cells exhibiting short cytoplasmic processes were encountered (poorly ramifying cells). In the 58, 66 and 82 days old fetuses, the round NDPase-positive cells were seen mainly in the matrix or subcortical layer where vascular sprouts were conspicuous and the poorly ramifying cells were in the subcortical and marginal layers. In the two latter fetuses, NDPase-positive cells showing long highly ramifying cytoplasmic processes (highly ramifying cells) were noted mainly in the marginal layer and sometimes in the subcortical layer. In the 5 months old fetuses, numerous NDPase-positive cells were distributed in the mantle, subcortical and marginal layers, and most of them appeared to belong to the populations of the poorly or highly ramifying cells. On the basis of the ultrastructural features, the round cells and highly ramifying cells were regarded as amoeboid cells and microglial cells, respectively. These findings suggest that at least some amoeboid cells are transformed into microglial cells via the stages of poorly ramifying microglial cells, and also that, in the human cerebral hemispheres, appearance of the microglial elements is closely related with vascularisation, especially in the early developmental stages.

Inflammatory cell changes in haversian canals. A possible cause of osteoporosis in rheumatoid arthritis.

We studied the morphology of the haversian canals in the osteopenic cortical bone of the medial femoral neck from patients with rheumatoid arthritis and compared the findings with those in patients with osteoarthritis and with uncomplicated coxa valga. In the rheumatoid bone, the diameters of the canals were larger and many more contained osteoclasts. Fewer haversian canals showed only lining cells than in the osteoarthritic or coxa valga patients. In bone from rheumatoid patients, especially in canals with osteoclasts, small blood vessels were frequently lined by tall endothelial cells with an infiltration of mononuclear cells. These morphological differences are discussed with reference to the possible mechanisms of loss of cortical bone in rheumatoid arthritis and other conditions.

Localization and subcellular distribution of cellular ras gene products in rat brain.

Localization and subcellular distribution of the cellular ras gene products (c-ras p21s) in rat brain were studied by immunofluorescence and immunoblotting using a monoclonal antibody recognizing all of Ki-, Ha- and N-ras p21s. In immunohistochemical analysis, strong immunoreactivity for ras p21s was observed in the neuropile of cerebral and cerebellar cortex. On the other hand, the immunoreactivity of the neuronal perikarya and that of white matter were weak and that of non-neuronal cells was undetectable. In subcellular fractionation analysis of cerebrum, c-ras p21s were found mostly in the particulate fractions and almost half of the particulate-bound c-ras p21s were recovered in the P2 fraction containing myelin, synaptosomes and mitochondria, approximately one-third were in the P3 fraction containing microsomes, and the rest were in the P1 fraction containing nuclei and cell debris. In further fractionation of the P2 fraction, most of c-ras p21s were associated with synaptosomal fraction. In the synaptosomal fraction, c-ras p21s were highly concentrated in the fractions rich in synaptic plasma membranes and were poorly present in the other fractions rich in synaptic vesicles, intrasynaptosomal mitochondria or postsynaptic densities. The content of c-ras p21s of the original homogenate was calculated to be 0.05% of the total protein and c-ras p21s were distributed in the fractions rich in synaptic plasma membranes with approximately 4-fold enrichment over the original homogenate. These results indicate that c-ras p21s are mainly localized in the synaptic plasma membranes and microsomes and suggest that they may participate in some specific neuronal functions at these sites.

A fifteen-somite human embryo.

The morphological features of a well-preserved human embryo having 15 pairs of somites are described and illustrated with a complete set of photomicrographs. This embryo was found during a forensic dissection of a Japanese woman, fixed in 10% formalin for 5 days, and embedded in an epoxy resin mixture according to routine procedures. Serial sections about 0.75 microns thick were made and stained with toluidine blue. The most important features were as follows: 1. The embryo measured 4.1 mm in greatest length and was quite symmetrical viewed dorsally. 2. Closure of the neural tube took place from the level of the first branchial groove, corresponding to the level of the anterior one-third of the rhombencephalon, to that of about 0.4 mm posterior to the 15th somite, coming across the anterior end of the cloacal membrane. The neural plate and the wall of the neural tube consisted exclusively of a pseudostratified columnar epithelium, and neither the mantle nor marginal layers were identified anywhere. 3. At the anterior end of the embryo there was a conspicuous optic evagination, and at the dorsal end of the second branchial groove a round otic placode. 4. Of the three segments of the entodermal tract the midgut was the largest, constituting about half the entire length, and opened ventrally into the large yolk sac, whose surface was covered by a meshwork of highly developed blood vessels. In the foregut two branchial pouches were seen, corresponding to the branchial grooves. Primordia of the thyroid gland and of the liver were also recognized. Among the entodermal epithelial cells lining the ventral half of the hindgut anterior to the origin of the allantois were found numerous primordial germ cells. 5. Among the 15 pairs of somites, the first two were small and consisted of dermomyotome of epithelial cell arrangement and of sclerotome mingled into mesenchyme. The next three were large, triangular on transverse section, and consisted of dorsolateral dermomyotome and ventromedial sclerotome representing a densely packed mesenchymal cell aggregation. The sixth to the tenth somites showed a well-defined triangular contour, each containing a lumen, the myocoele. The last five somites were progressively smaller and were quadrangular in shape. 6. The intermediate mesoderm was encountered at levels from the 7th to the 15th somite, but no indications of pronephric differentiation were detected. 7. The heart tube was relatively large, took an S-shaped tortuous course, and occupied almost the entire pericardiac cavity. It consisted of the bulbus cordis, the ventriculus, and the atrium, each of which consisted of a thick myocardial tube and a thin endocardial tube.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of oxygen concentration on embryonic development in rats: a light and electron microscopic study using whole-embryo culture techniques.

By using a whole-embryo culture technique (New 1978), the effects of oxygen concentration (5%, 20% and 95% oxygen) on embryonic development in the rat were investigated by light and electron microscopy. The best embryonic development occurred when the 9.5-day-old embryos were cultured for 24 h with 5% oxygen, and the 10.5-day-old embryos with 20% oxygen (optimum oxygen concentration). When the 9.5- and 10.5-day-old embryos were cultured for 24 h with too little or too much oxygen, retardation of the embryonic growth and abnormal development was observed. Using light microscopy, numerous degenerating cells, exhibiting granular deposits in the cytoplasm, were seen, but the distribution of the degenerating cells was quite different between the two groups. With electron microscopy, the most striking feature of the degenerating cells in the embryos cultured with too little oxygen, was the extreme swelling of the mitochondria without any morphological alterations of the nucleus or the other cell organelles. On the other hand, the characteristic feature of the degenerating cells in the embryos exposed to too much oxygen, was the formation of phagolysosomes in the cytoplasm. Morphological alterations of the nucleus or mitochondria were not evident. In the present study, the possible teratogenic mechanism of too much or too little oxygen in the whole-embryo culture of the rat embryo is discussed.

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos.

Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5-12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinct regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in parallel with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical studies of the differentiation of microglial cells in the cerebral hemispheres of chick embryos and chicks.

Using histochemical procedures to reveal the presence of nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase), we investigated the appearance, distribution and ultrastructure of amoeboid and microglial cells in the cerebral hemispheres of chick embryos and young chicks, in order to elucidate the relationship between these two cell populations. On day 6 of incubation, a few round cells exhibiting NDPase, TPPase and AcPase activity were first detected in the thin mantle layer of the cerebral hemisphere. In the corpus striatum, these round cells increased rapidly in abundance until day 13 of incubation, after which their numbers gradually decreased, so that, on day 19 of incubation, they had entirely disappeared. Between day 10 and day 17 or 18 of incubation, round cells were located mainly in the zone of the mantle layer closest to the lumen. On day 10 of incubation, NDPase-, TPPase- and AcPase-positive cells that had a few short cytoplasmic processes (poorly ramified cells) were detected in the intermediate and basal zones of mantle layer. They increased in abundance until day 17 or 18 of incubation and thereafter rapidly decreased in number. Round and poorly ramified cells exhibited NDPase activity on their plasma membranes and in their cytoplasmic vacuoles, with TPPase and AcPase activity being localized within their vacuoles. On day 19 of incubation, NDPase- and TPPase-positive cells with long, well-ramified cytoplasmic processes (well-ramified cells) were observed in the corpus striatum, these being mainly localized in the basal zone. After hatching, these cells increased rapidly in abundance and were distributed throughout the corpus striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase activity in neural crest cells of the early chick embryo.

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.

Scanning electron microscopic studies on stigmas in rat ovaries.

Ultrastructural observations on stigmas in rat ovarian follicles have been performed by scanning electron microscopy. Stigmas were classified into two types under a dissecting microscope; extensively bulging vesicles (bleb-type stigmas) and small, flat avascular areas (flat-type stigmas). The bleb-type stigma had lost its surface epithelial cells extensively, and the flattened and densely arranged fibroblasts without fibrous structures were exposed. These fibroblasts had short, serrate, cytoplasmic projections where multivesicular structure-like granules were seen. By contrast, on the flat-type stigma a small region of exposed stroma was covered with fibrous structures. Removal of the fibrous structures by the HCl-collagenase method revealed that the exposed stroma consisted of stellate fibroblasts surrounding a small opening through which a few granulosa cells were about to discharge. When the cumulus mass was protruding, it was surrounded by the fibrous structures. These findings indicate that both the stellate fibroblast formation and the presence of fibrous structures are needed for the release of ova through the stigma.

Acetylcholinesterase activity in the myotome of the early chick embryo.

The myotome of early chick embryos was investigated histochemically by means of the acetylcholinesterase (AChE) reaction. Light-microscopically, at the cervical level, the myotome was first recognized and AChE activity demonstrated at stage 13 (2-day-old embryo). Subsequently, the myotome elongated ventro-laterally along the inner surface of the dermomyotome and reached the ventro-lateral end of the dermomyotome at stage 17 to 18 (3 day-old embryo). AChE activity in the myotome showed subsequent increase in intensity during the course of development. The myotome consisted mainly of AChE-positive cells displaying enzymatic activity along the nuclear membrane and within the cytoplasm. In contrast, almost all cells of the dermomyotome and the interstitial cells were AChE-negative. Electron-microscopically, the myotome cells of the 2 day-old embryo and the cells in the dorso-medial portion of the myotome of the 3 day-old embryo were morphologically undifferentiated; AChE activity was detected in the nuclear envelope and in single short profiles of the endoplasmic reticulum (ER). On the other hand, in the 3 day-old embryo the cells in the ventro-lateral portion of the myotome showed AChE activity in the nuclear envelope, numerous profiles of the ER and some Golgi complexes. These AChE-positive cells were regarded as developing myogenic cells based on their morphological characteristics. The present findings indicate (i) that the appearance of AChE activity in the cytoplasm is the first sign of the differentiation of myogenic cells, and (ii) that in these myogenic cells the increase in AChE activity is based on the development of the ER.