Helge Andersen

HISTOCHEMISTRY AND DEVELOPMENT OF THE HUMAN EYELIDS

The development of the eyelids involves numerous complicated biological and embryological problems, which have received little attention in the past in particular problems concerning the development of human eyelids. In this connection the processes associated with the growth of the eyelids and their subsequent junction should be emphasized, as also the importance of this junction for the differentiation of the structures situated behind the eyelids. Furthermore, the disjunction of the eyelids at later stage of embryonic development should be mentioned, together with the formation of the cilia and associated glands and the role which they play in the disjunction process as well as their possible influence on the keratinization of the margins of the eyelids. Also the development of the lipid-producing Meibomian glands should be mentioned. The purpose of the present study was to clarify, if possible, these problems and a t the same time to draw up a biological time-table for the development of the different structures of the eyelids.

On the localization of a prostaglandin-dehydrogenase activity in the kidney

SummaryUsing the kidneys from white rats a method for the localization of a prostaglandin-dehydrogenase activity is presented.The activity demonstrated showed NAD dependence and displayed a high substrate specificity.The most pronounced activity was observed in the thick ascending limb of the loop of Henle and in the distale tubule. Lesser activity was found in the collecting tubules of the inner medulla, in the interstitial cells of the medulla, in the epithelial cells of the pelvis, in the tunica media of the cortical arteries and arterioles and in the visceral epithelium of the renal corpuscles.The pitfalls of the cytochemistry of the co-enzyme linked dehydrogenases are discussed and correlated to the present study. Similarly the observations noticed is discussed in relation to the metabolism and biological activity of the prostaglandins.

Simplified control experiments in the histochemical study of coenzyme-linked dehydrogenases

SummaryDifferent experiments concerning some of the most critical steps in the histochemical procedure for coenzyme-linked dehydrogenases (fixation procedure, cryoprotection, osmolar protection, substantivity of formazans, “nothing dehydrogenase” reaction, diffusion of enzyme, rediffusion of reduced coenzyme and/or reduced phenazine methosulphate) were carried out in order to improve or introduce simplified control methods by which the in situ localization of enzyme activity can be achieved without the need of expensive equipments.As a test-object glucose-6-phosphate dehydrogenase was used.Brief (5 min) prefixation of tissue (liver) at 0–4° C with 1% buffered (pH=7.2) methanolfree formaldehyde (from paraformaldehyde) gave excellent preservation of morphology during the procedures of freezing, cutting and incubation and caused no inhibition of G6PDH. With the named fixation no improvement was obtained by the simultaneous use of cryoprotection (DMSO) or osmolar protection (sucrose). Finally, the fixation caused an enhancement of Nitro BT penetration into the tissue as well as of formazan substantivity. On the other hand, the brief fixation did not abolish the diffusion of enzyme (proved by different methods) and of reduced coenzyme or reduced phenazine methosulphate.In a conventional aqueous incubation medium as well as in a gel incubation medium (PVA, grade Bo5/140) the rate of diffusion of reduced coenzyme and/or reduced phenazine methosulphate was investigated by using a special double-section incubation method. The concentrations of Nitro BT, NADP and PMS were balanced against each other and it was concluded that by using a gel medium containing 0.5 mg/ml Nitro BT, 0.1 mg/ml NADP and 0.003 mg/ml PMS, the in situ localization of G6PDH activity could be achieved at the cellular level with an incubation time not exceeding 10 min. With the incubation time mentioned, the “nothing dehydrogenase” reaction was out of the question. The sensibility of the double-section incubation method is discussed and provided that the dehydrogenase in question contains sulphydryl groups in the active enzyme centre, the method seems to exhibit a sufficient high level of sensitivity in the control of the diffusion of the different components operative in the histochemical dehydrogenase procedure.The recording of the incubation time needed for the appearance of the two formazans (red and blue) is recommended in order to follow the enzymatic reaction rate and the effect of different procedures (fixation, solvents added etc.) as well as the rates of “nothing dehydrogenase” reaction, diffusion of reduced coenzyme and/or reduced PMS.

The histiocyte in human foetal tissues: its morphology, cytochemistry, origin, function, and fate.

SummaryBased upon a material comprising human foetuses cytochemical studies of a widespread type of cell were carried out. Apart from amoeboid mobility the cell is characterized by pinocytotic and phagocytotic activity.In early development stages these cells are seen intravascularly and penetrating the vessels. Later they are seen in connection with the formation of the vascular epiphyseal cartilaginous canals and in the vitreous body, the synovial joints and dental anlage. Furthermore these cells are seen at the removal of the epithelial cells of the dental lamina and the junctional epithelium in the two palatine processes. The cells concerned are seen also in the deep periosteal layer at the centre of the diaphysis synchronously with the vaseularization of the periosteum and prior to the periosteal invasion. Based upon morphology and cytochemistry the theory is advanced that these cells form the chondroclasts and the multinucleated osteoclasts. By contrast, the diaphysial osteoblasts are derived from invading pre-osteoblasts from the cambium-layer of the periosteum.These cells are also seen along the basal surface of the neural apparatus and invading the brain vesicles.On the basis of morphology and cytochemistry the cell type is designated a histiocyte and its origin is traced back to primitive leucocytes.

The early development of the pars distalis of human foetal pituitary gland.

SummaryA histochemical and ultrastructural investigation of the early development of the adenohypophysis was carried out on a human material.Special attention was paid to an accumulation of hyaluronic acid and chondroitin-4-and/or-6-sulfate in the mesenchyma; its role in morphogenesis of the Rathkes pouch is discussed.The role of the vessels as a “critical factor” in the budding of the parenchyma and in the differentiation of secretory cells is discussed.Canalicular extensions from the original lumen of the pouch into the core of parenchymal buds, which migrate into the mesenchyma, is a new observation. The participation of canaliculi in formation of follicular structures of pars distalis is described and discussed. The primitive cell type lining the pouch is also found in the wall of canaliculi and follicular structures. The cell type is described and its role as the real progenitor cell of the adenohypophysis is discussed.Three types of colloid are noticed in pars distalis: 1. In the follicular structures, 2. in the slits or clefts caused by partial occlusion of the lumen of the pouch, and 3. mesenchymal extravasal colloid presumably representing material squeezed out from the aforementioned clefts or slits. It is concluded that the colloid contains material secreted from different types of granulated cells as well as material from the surface coat of the luminal cells.

Histochemistry and development of the human eyelids. II. A cytochemical and electron microscopical study.

In a previous study, the development of the human eyelids was investigated in a material comprising human foetuses of crown-rump lengths varying from 8 to 220 mm (Andersen, Ehlers and Matthiessen, 1965). The development passes through three stages: ( 1 ) The stage of growth: The period from the initial ectoderm proliferation till the junction of the eyelids (crown-rump length from about 12 mm to about 40 mm), (2) The stage of differentiation: From junction to the beginning of disjunction (crown-rump length from about 40 mm to about 150 mm), and (3) The stage of maturation: The remaining part of the development of the eyelids, characterized by continued growth and maturation of the various structures of the eyelids. The study dealt mainly with the first two stages, and time-tables were drawn up for the appearance and development of the different structures, and the development was discussed from a functional point of view. However, certain problems remained and will be dealt with in the present paper: ( 1 ) Does the junction of the eyelids give rise to an epithelial adhesion only between the two eyelids, or are desmosomes, or another epithelial contact, formed at the site of the junction? (2) The more intimate relationship between the epithelium of the junction

Platelet-derived growth factor stimulates chemotaxis and nucleic acid synthesis in the protozoan Tetrahymena

Platelet-derived growth factor (PDGF) is in concentrations of a few nanograms per ml a very active chemoattractant for the free-living ciliated protozoan Tetrahymena; at the same time it induces a rapid increase in incorporation of radioactive nucleic acid precursors into RNA and DNA. We find it remarkable that this lower eukaryote responds to platelet-derived growth factor in very much the same way as fibroblastic cells.

Studies in succinate dehydrogenase histochemistry

SummaryThe activity of succinate tetrazolium reductase was investigated in liver and kidney from the rat and mouse. The results obtained were related to the cellular level of succinate dehydrogenase (SDH) as well as to the level of CoQ.It was concluded that the low activity in centrolobular areas of the liver lobules compared with the perilobular areas, exclusively is due to a naturally deprivation of CoQ.The level of SDH as well as of CoQ was very high in renal cortical tubules rich in mitochondria (proximal and distal convoluted tubules, the ascending thick limb of Henle). This was indicated by the facts that the initial reaction rate was high and no enhancement was obtained by the addition of CoQ10.In all experiments the activity of fresh frozen sections were compared with the activity of sections from briefly prefixed tissue. The influence of different fixatives, variation in Nitro BT concentration, cryoprotection (dimethyl sulfoxide, DMSO) and osmolar protection (sucrose) was investigated and discussed. Further, the substrate-carrying effect of DMSO was investigated and discussed.Brief (5 min) fixation at 0–4° C—especially with 1% buffered (pH=7.2) methanol-free formaldehyde (from paraformaldehyde) gave excellent preservation of morphology and caused no inhibition of SDH activity. Furthermore, the fixation caused an enhancement of Nitro BT penetration into the tissue and an enhancement of formazan substantivity.The incubation time needed for the appearance of both the red and blue formazan was recorded in order to follow the initial reaction rate. This procedure proved to be a sensitive indicator, when the influence of components added (CoQ10, DMSO, sucrose etc.) was studied.

Specificity in steroid histochemistry, with special reference to the use of steroid solvents. distribution of 11 β-hydroxysteroiddehydrogenase in kidney and thymus from the mouse

SummarySeveral factors influencing the steroiddehydrogenase histochemistry were investigated: diffusion of enzyme; inactivation of enzyme; effects of the steroid solvents commonly used; the validity in localization of the enzyme activity; “nothing dehydrogenase” reaction. 1. The importance in controlling the diffusion of each enzyme system to be studied is emphasized. Provided that the presence of SH-groups in the active centre of the dehydrogenase can be proved, a control experiment using a double-section incubation method should be carried out. 2. A comparison between the use of unfixed and briefly prefixed sections is recommended in order to avoid a possible distortion of the tissue during the incubation. The influence of prefixation on diffusion of enzymes or reaction products as well as on inactivation of enzymes must be studied. 3. The steroid solvents—especially dimethyl formamide caused a morphological distortion, and an inactivation and/or extraction of reaction products (the red monoformazan) in fresh frozen sections, these solvents should therefore be handled with caution. A special mixture of dimethyl formamide and propylene glycol is recommended. 4. The steroid should be completely soluble in the incubation medium in order to secure zero order kinetics. 5. Avoidance of sulphydryl “nothing dehydrogenase” reaction, since the reaction predominantly manifests itself as a red formazan obscuring sites with low dehydrogenase activity. 6. The localization of the NAD(P)H oxidase systems must be controlled, in order to ensure that they should not be a limiting factor in the detection of the dehydrogenase activity. Secondly, this investigation may act as a control on diffusion of dehydrogenase and/or reduced coenzyme. 7. That the investigation of the incubation time needed for initial visual reaction allows a certain quantitative estimation of the concentration of enzyme localized at different sites in the same section. The investigation should also include the red formazan, since it has recently been proved to be an intermediary step in the enzymic reduction of Nitro BT, and as such may reflect sites with low enzyme concentration.Further, some of the functional aspects of the activity of 11β-hydroxysteroiddehydrogenaseNAD(P)H oxidase systems in the thymus were discussed, and lastly the localization of these systems in the kidney was revised.

Histochemistry of 3β-hydroxysteroid dehydrogenase in rat ovary

SummaryBy recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3β-HSD was investigated:1.Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced.2.Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% w/v for 5 min) thereby enhancing the Nothing dehydrogenase reaction.3.Preextraction of lipids with acetone for 20 min at −30° C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells).4.No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20° C.5.By using the double-section incubation method no diffusion of 3β-HSD or rediffusion of NADH or PMSH could be noticed within 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0 mg/ml).6.The utility of different inhibitors of alkaline phosphomonoesterase was tested and discussed.7.By inhibiting alkaline phosphomonoesterase with 0.1 mM ofl-p-bromotetramisole or 16 mM ofβ-glycerophosphate, 3β-HSD was shown to be exclusively NAD-linked.8.Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3β-HSD, but not of NADPH-tetrazolium reductase.9.3β-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited byn-ethyl maleimide.10.Whether alcohol dehydrogenases may use steroids as substrate is discussed. It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed.11.Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase.12.Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF).13.The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone.4.If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.