Ultrastructural and immunohistochemical studies in callitrichid renal glomeruli

The “normal” mesangium of callitrichids exhibits certain features pointing to enhanced activity. Protrusions of the mesangial cellular cytoplasm (blebs) into the capillary lumen were observed very frequently as were electron-dense granules in mesangial matrix channels. Histochemical, α-actin expression was invariably observed in the mesangial cells of callitrichids.     

Ultrastructural immunohistochemical localization of adrenocorticotropin and beta-lipotropin in the rat brain.

In an attempt to identify the cells and organellel containing ACTH and beta-lipotropin in the rat brain, an immunocytochemical localization of these two peptides was performed at the electron microscopic level. Both ACTH and beta-lipotropin were localized in dense core vesicles of about 60-80 nm in diameter. Using serial sections, it has been possible to demonstrate that these peptides are contained not only in the same neuronal cell bodies, but also in the same dense core vesicles.     

Ultrastructural study of experimental muscle degeneration and regeneration in the adult rat

Summary Methyl-bupivacaine is a local anaesthetic with a selective myotoxic action. A single subcutaneous injection of the drug into the hind leg of adult rats produces a uniform, complete and irreversible destruction of superficial layers of fibres in the underlying extensor digitorum longus muscle. The degeneration of muscle fibres is followed by phagocytosis and a rapid and complete regeneration.The first stage in the regeneration process is the appearance of presumptive myoblasts within the original basement membrane of the sarcolemmal tube. On the second day after injury aggregates of myoblasts are present and fusion is observed between the cells. The myotubes thus formed increase in size by fusing with additional myoblasts. Myotubes are also observed to fuse with one another. On the fifth day after injury the regeneration process has proceeded to the stage of early muscle fibres with fully differentiated myofibrils with typical sarcomere structures. By ten days only mature muscle fibres of about normal size are present and regeneration appears complete.In previously denervated and methyl-bupivacaine treated muscles the stages of regeneration are similar to those observed in innervated muscles, the only apparent difference being a slowing of cell differentiation and incomplete maturation.An electrophysiological study shows that the motor nerve at the third day after injury forms synaptic contacts with regenerating muscle cells. At that stage of myogenesis the myotubes are highly sensitive to applied acetylcholine.1 (1-n-butyl-DL-piperidine-2-carboxylic acid-2,6-dimethyl-anilide-hydrochloride); Marcaine®, manufactured by AB Bofors, Nobel-Pharma, Mölndal, Sweden.The study was carried out under the auspicies of The Czechoslovak Academy of Sciences and the Royal Academy of Sciences in Sweden.     

Ultrastructural and immunohistochemical studies of microfibril-associated components in the posterior chamber of the eye

Connective tissue microfibrils were observed in tissues prepared with methods believed to minimize the loss of tissue components. The eyes of C57BL/6J mice were fixed with glutaraldehyde followed by either freeze substitution, or embedding in glycol methacrylate, a water-miscible embedding medium, after limited or no dehydration. In these preparations, microfibrils were present within sheet-like layers observed in the posterior chamber of the eye. The material enclosing the microfibrils that formed the layer was also preserved, at least partially, by fixation of the tissue with uranyl acetate or potassium permanganate (KMnO₄) as observed in the chick eye. This microfibril-associated material was found to be composed of heparan sulfate proteoglycan (HSPG) as shown by positive immunostaining for HSPG, as well as by identification of 4.5 nm-wide HSPG double tracks as its major constituent. When a considerable amount of this material was lost in KMnO₄-fixed tissues, the remaining portion was preserved in the form of clusters of about 50 nm in width which were periodically adhered along the length of microfibrils. At the center of each cluster, a minute dark particulate structure was present. It was composed of an approximately 10 nm-wide polygonal assembly of 3.5 nm-wide ring-like structures, and was, in unfixed chick eyes, positively immunostained for fibrillin. The periodicity of HSPG clusters, and of fibrillin, along the length of immunostained microfibrils was similar, ranging from 45 nm to 65 nm. These observations indicate that fibrillin is periodically associated at the surface of classical microfibrils, and it may mediate the association of large amounts of HSPG to microfibrils.     

Palisade Tissue Chloroplasts and Spongy Tissue Chloroplasts in Spinach: Biochemical and Ultrastructural Differences

Palisade tissue chloroplasts (P-Chlts) and spongy tissue chloroplasts(S-Chlts) were separately isolated from spinach leaves, andtheir photosynthetic properties were compared. The followingresults were obtained: (1) At saturating light, the activities of overall electrontransport and CO₂ fixation in P-Chlts were respectively 1.6–2.0and 2.5–3.0 times higher than those in S-Chlts on a Chlbasis. (2) The contents of PS I and PS II reaction centers (P700 and47 kDa polypeptide, respectively) were slightly higher in P-Chltsthan in S-Chlts, while the contents of plastoquinone, Cyt f,plastocyanin, ferredoxin, ferredoxin-NADP⁺ reductase, couplingfactor and ribulose-bisphosphate carboxylase were 1.6–2.2times higher in P-Chlts than in S-Chlts on a Chl basis. (3) Electron microscopic examination of chloroplast ultrastructureshowed that S-Chlts have highly stacked grana accompanied byhigher proportion of appressed thylakoids relative to non-appressedthylakoids, while P-Chlts have poorly stacked grana. The volumeratio of thylakoids to stroma was higher in S-Chlts than inP-Chlts. These results indicate that mesophyll chloroplasts adapt tothe light environment within a leaf in a similar way that thesun and shade plant chloroplasts adapt to the light environmentwithin a canopy. (Received July 19, 1984; Accepted October 13, 1984)     

An immunohistochemical and stereological analysis of PSI-induced nigral neuronal degeneration in the rat

Systemic administration of the proteasomal inhibitor I (PSI) to rats was reported to cause progressive nigral dopaminergic neuronal loss but this is disputed. A major controversy centres over the use of manual counting of tyrosine hydroxylase (TH) positive neurons at the level of third cranial nerve as opposed to employing systematic stereological analysis of cell loss in the entire substantia nigra (SN). To provide a method of marking SN neurones independent of protein expression, fluorogold^™ (FG) was stereotaxically injected bilaterally into the striatum of male Wistar rats to retrogradely label nigral dopaminergic neurons. After 1 week, animals were treated with six doses of PSI (8 mg/kg, s.c.) or its vehicle (dimethyl sulphoxide) on alternate days over a 2-week period. Five weeks after the last treatment, PSI-treated animals showed decreased spontaneous locomotor activity and reduced TH positive SN cell number at the level of the third cranial nerve compared to control rats. Manual cell counting showed loss of FG-labelled SN neurones at this level, with a subpopulation of surviving neurons displaying abnormal morphology. Manual counting of all FG-labelled cells in the entire SN also showed regional PSI-induced loss of neurones with both normal and compromised morphology. Stereological optical fractionator estimates of total FG-labelled cell number confirmed the manual cell counting data both at the level of the third cranial nerve and throughout the entire SN. These findings confirm that PSI does cause a persistent nigral dopaminergic neuronal loss. The reason for the lack of reproducibility between laboratories requires further investigation. We suggest that a failure to distinguish between TH-positive neurones with normal and abnormal morphology following PSI administration contributes to equivocal results.     

The connective tissue of the rat lung: electron immunohistochemical studies

The ultrastructural distribution of specific connective-tissue components in the normal rat lung was studied by electron immunohistochemistry. Three of these components were localized: type I collagen, fibronectin and laminin. Type I collagen was present not only in major airways and vascular structures, but also in alveolar septa. Laminin was found in all basement membranes, and only in basement membranes, demonstrating once more that this glycoprotein is an intrinsic component of the basement membrane. Fibronectin was found free in the interstitium and on the surfaces of collagen fibers. The basement membranes of bronchial, glandular and endothelial cells of large vessels lacked fibronectin; however, capillary endothelial and occasionally epithelial alveolar basement membranes contained some fibronectin in an irregular, spotty distribution. This localization suggests that in the lung, as in other tissues, fibronectin is not an intrinsic component of the basement membrane, but rather a stromal and plasma protein. Only basement membranes in the alveolar parenchyma contained "trapped" plasma fibronectin.     

Ultrastructural sequence of myelin breakdown during Wallerian degeneration in the rat optic nerve

Summary Adult albino rats were subjected to unilateral surgical removal of the eyeball. After survival times of 7–140 days, the numerical response of the neuroglial cells, and the progressive disintegration of the myelin sheaths in the optic nerves, were studied qualitatively and quantitatively in electron-microscopic montages. The distribution density of microglia and astroglia in degenerating optic nerve increased to peaks after 35 and 56 days respectively, whereas, the oligodendroglia gradually decreased. During the early stage of degeneration, microglial cells appeared and invaded the sheath at the intraperiod line, peeling off the outer lamellae, which were then engulfed by phagocytosis. Within the microglia, myelin sheath fragments were surrounded by a membrane curled to form a myelin ring. In the intermediate stage of degeneration, the paired electrondense lines of the ring, made up of myelin basic protein, decomposed and formed a homogenous or heterogenous osmiophilic layered structure, the myelin body, which, in the final stages, disintegrated and transformed into globoid lipid droplets and needle shaped cholesterol crystals.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

The spermatozoa of two closely related species of ophiuroids, Ophiocoma echinata and Ophiocoma wendti, were examined ultrastructurally. Morphologically, these spermatozoa resemble those of other non-echinoid echinoderms. The acrosomal complex, completely contained within an anterior fossa in the spherical nucleus, consists of a membrane-limited acrosomal vesicle and periacrosomal material. Events of the acrosomal reaction in O. echinata and O. wendti are presented. In both species, the reaction results in the establishment of an extracellular coat of acrosomal vesicle origin on the anterior surface of the spermatozoon. The possible role of this extracellular coat in the species-specific binding of sperm and ova is discusses. The origin of acrosomal tubule membrane is elucidated.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

Summary The general ultrastructural features of the hypothalamo-neurohypophysial system in rats with hereditary hypothalamic diabetes insipidus (DI-rats, Brattleboro strain) are described. There is no decisively distinguishing difference between the neurons of the supraoptic and paraventricular nuclei. The neurons of both nuclei show signs of active protein synthesis. The perikarya of the neurons are markedly hypertrophic, the nuclei are large and the nucleoli prominent. In the cytoplasm there are numerous ribosomes, abundant rough-surfaced endoplasmic reticulum and extensive Golgi complexes. However, very few neurosecretory granules are to be seen. The axons of the hypothalamo-neurohypophysial tract are likewise enlarged and the paucity of neurosecretory granules is a striking feature also in the area of the tract. The majority of nerve endings in the posterior pituitary of DI-rats are devoid of neurosecretory granules. Microvesicles are abundant in the nerve endings and there are findings which suggest that microvesicles are involved either in endoor exocytosis. The signs of active protein synthesis and the concomitant paucity of neurosecretory granules are interpreted to imply transportation of the secretory proteins in an extragranular phase. The possible mode of release of the secretory proteins from the nerve endings and the role of microvesicles therein are discussed.This study has been supported by grants from the Finnish Cultural Foundation and the Sigrid Jusélius Foundation. The collaboration of Professors Antti Arstila and Tapani Vanha-Perttula is gratefully acknowledged.The Brattleboro-rats were kindly provided by Dr. Heinz Valtin, to whom we express our thanks.     

Ultrastructural studies on the blood-testes barrier of the rat.

Typical pinocytic vesicles were visible in electron micrographs in both outer and inner cellular layers of rat boundary tissue. FF technique revealed that they formed characteristic ribbons of foveoli. Foveoles less numerous were present in outer lamina for the most part of the investigated material. So we believe that this phenomenon speaks in favor of myoblastic origin of that layer.     

Ultrastructural studies on the hypothalamic neurosecretory neurones of the rat

Summary The ultrastructural features of the paraventricular neurones of the non-treated rat are presented comparing them with those of the supraoptic neurones. No striking differences are seen between the general electron microscopic characteristics of the paraventricular and supraoptic neurones.The importance of adequate fixation to obtain good preservation of the neurones is emphasized, since inadequate fixation can cause e.g. artefactual appearance of dark neurones. The previously presented classification of the neurosecretory neurones into two categories (e.g. light and dark neurones) on the basis of the number of ribosomes is not considered justifiable, since their number can vary to a very great extent even within a single cell.The synthesis of neurosecretory products in the paraventricular neurones obviously follows the general mode of the synthesis of secretory proteins: ribosomes—RER—Golgi complex—secretory vesicles.On the basis of the localization of heavy metal deposits after osmium impregnation and demonstration of acid phosphatase the Golgi complex of the paraventricular neurones is found to be polarized. The direction of the polarity is discussed.The substructures of the dense cores of the neurosecretory granules and of the contents of the lysosomal dense bodies are nearly identical. Therefore it is considered impossible to determine positively the nature of the dark condensed material within the Golgi complex. The characteristics of the immature neurosecretory granules and the possibility of releasing neurosecretory products into the cytoplasm already within the perikarya are speculated.This study was supported by a grant from the Emil Aaltonen Foundation, Tampere. I express my best thanks to Docent Antti U. Arstila, Head of the Laboratory of Electron Microscopy, and Professor Urpo K. Rinne, Head of the Department of Neurology, for the guidance of this work.     

Secretin activates vagal primary afferent neurons in the rat: evidence from electrophysiological and immunohistochemical studies

In this study, we evaluated the vagal afferent response to secretin at physiological concentrations and localized the site of secretin's action on vagal afferent pathways in the rat. The discharge of sensory neurons supplying the gastrointestinal tract was recorded from nodose ganglia. Of 91 neurons activated by electrical vagal stimulation, 19 neurons showed an increase in firing rate in response to intestinal perfusion of 5-HT (from 1.5 +/- 0.2 to 25 +/- 4 impulses/20 s) but no response to intestinal distension. A close intra-arterial injection of secretin (2.5 and 5.0 pmol) elicited responses in 15 of these 19 neurons (from 1.5 +/- 0.2 impulses/20 s at basal to 21 +/- 4 and 43 +/- 5 impulses/20 s, respectively). Subdiaphragmatic vagotomy and perivagal application of capsaicin, but not supranodose vagotomy, completely abolished the secretin-elicited vagal nodose neuronal response. In a separate study, 9 tension receptor afferents among 91 neurons responded positively to intestinal distension but failed to respond to luminal 5-HT. These nine neurons also showed no response to administration of secretin. As expected, immunohistochemical studies showed that secretin administration significantly increased the number of Fos-positive neurons in vagal nodose ganglia. In conclusion, we demonstrated for the first time that vagal sensory neurons are activated by secretin at physiological concentrations. A subpopulation of secretin-sensitive vagal afferent fibers is located in the intestinal mucosa, many of which are responsive to luminal 5-HT.     

Morphometric and immunohistochemical studies of the suprachiasmatic nucleus in the hereditary microphthalmic rat]

Morphometric and immunohistochemical analyses of the suprachiasmatic nucleus (SCN) were performed on hereditary microphthalmic rats. In normal rats, the number of cells and the volume of the SCN were 11, 631 and 6.7 x 10(-2) mm3 (an average taken from 12 SCNs). However, the neuronal population and volume of the SCN in hereditary microphthalmic rats were 7,450 and 4.5 x 10(-2) mm3 (an average taken from 14 SCNs), respectively. There were no significant differences in the size of neurons between normal and microphthalmic SCN neurons. Immunohistochemical studies showed that a considerable number of antivasopressin positive neurons were present in microphthalmic rats, despite their lack of the optic nerve. However, further detailed studies revealed that the number of antivasopressin positive neurons present in microphthalmic rats was only 68% of those found in normal rats. These findings suggest that the complete development of the SCN and vasopressin neurons depends on the visual input.