Immunocytochemical Investigations Research Articles

Immunocytochemical investigation of the in vivo endocytosis by renal tubular epithelial cells.

The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho-cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8. It was introduced into the circulation of normal mice for 5, 10, and 30 minutes and the distribution of the labeling was determined by protein A-gold immunocytochemistry, using specific antiDNP antibodies on tissue sections of routinely aldehyde-fixed, osmiumpostfixed, and Epon-embedded kidneys. Cationic BSA-DNP was detected at the endothelial and epithelial sides of the glomerular basement membrane, and over capillary and tubular basement membranes. In the proximal tubular epithelial cell, labeling was present over microvilli as well as over endosomal and lysosomal compartments, with labeling intensities varying from one compartment to the other. Morphometric evaluations of the labeling demonstrated a progressive incorporation of the probe from microvilli and endocytic compartments at 5 minutes to endocytic and lysosomal compartments at 10 and then 30 minutes. When considering labeling densities, no significant differences were found on microvilli and basolateral membranes between times of circulation; however, the labeling density over endosomal and lysosomal compartments was very intense at 10 minutes compared with 5 minutes, decreasing at 30 minutes. Results from this study validate the cationic albumin tagged with DNP as a tool in the study of the quantitative aspects of protein endocytosis at the ultrastructural level, in the kidney tubular epithelium.

The immunocytochemical localisation of potential candidate vaccine antigens from the salmon louse Lepeophtheirus salmonis (Kroyer 1837)

Vaccination is potentially one of the most valuable methods of control against the caligid copepod Lepeophtheirus salmonis, the salmon louse, a major pathogen of farmed salmonids in the Northern hemisphere. A search for suitable potential antigens from this complex metazoan parasite forms the basis of this study. Salmon louse homogenate was separated by column chromatography into specific fractions which were assayed for a range of enzyme activities. Fractions found to be associated with enzymic activity or peaks of UV absorbance were used to raise antisera in 44 Wistar rats. An immunocytochemical investigation of the midgut of the adult louse using these specific antisera revealed immunostaining of the lumenal and basal surfaces of the louse gut. It is suggested that the proteins in the chromatographic fractions may represent digestive enzymes and/or structural or functional components of the salmon louse gut.

Ultrastructural aspects of platelet adhesion on subendothelial structures.

Platelet adhesion to ligands within the subendothelium induces platelet activation. Ultrastructural and immunocytochemical investigations were carried out to obtain information on the microtopography of the adhesive interactions. Rabbit platelets which react in vivo with endothelial lesions (V. jugularis), and human platelets which react in vitro with collagen were observed. It was shown that the reaction of adhering platelets depends on the micro-topography of the adhesive surface 1. On plane surfaces the platelets spread 2. Collagen fibers induce the formation of focal contacts. They are initiated by binding of ligands to certain transmembrane receptors. The contact mediates the formation of the contractile gel, which acts as a constricting sphere internalizing surface bound ligands, i.e., retraction of collagen networks. Most of the experiments hitherto dealt with platelets that spread. The described findings show that the reaction mentioned above plays a more important role than previously imagined and may be the physiological response of platelets interacting with subendothelial components in vessel lesions.

Opioid growth factor modulates corneal epithelial outgrowth in tissue culture.

In addition to neuromodulation, endogenous opioid peptides serve as growth factors. To determine involvement of opioids in the homeostatic renewal and repair of the corneal epithelium, epithelial outgrowths from 3-mm explants of rabbit cornea were investigated. Blockade of opioid-receptor interaction by the potent opioid antagonist naltrexone (NTX) for 7 days significantly increased the extent of outgrowths and the number and labeling index (DNA synthesis) of epithelial cells, relative to control levels. Outgrowths exposed to the opioid growth factor (OGF) [Met5]enkephalin for 7 days were subnormal in extent and labeling index and displayed alterations in architectural pattern. The effects of OGF on epithelial outgrowth were blocked by concomitant exposure to the opioid antagonist naloxone; naloxone alone had no effect on growth at the concentration utilized. NTX and OGF were active in both serum-containing and serum-free cultures. Immunocytochemical investigations showed that both OGF and its opioid receptor zeta (zeta) were present in epithelial cells growing in control media. The results indicate that an endogenous opioid peptide and its receptor are present in mammalian corneal epithelium and serve to modulate cell proliferation, migration, and organization.

The expression of cytokeratins in human hepatocellular and cholangiocellular carcinomas

In order to determine the usefulness value of the antibodies to cytokeratins (CK) of “bile duct type” in the differential diagnosis between hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CC), we have made an immunocytochemical investigation, using the antibodies specifically recognizing CK19 and CK18, seperately, in liver, and laminin (LN) antibody. All the CC examined (10 cases) were found CK19-positive; interestingly, CK19-positive cancer cells were also observed in 38% of HCCs (14/37). Therefore, CK19 was not a reliable marker in differentiating HCC from CC, in our consideration. The CK19 expression in HCC was showed to be irrelevant to their differentiation degree, but related to the histologic subtypes which indicated the directions of their differentiation. CK19 expression was observed in all the HCC cell nests with glandular differentiation, and an uncontinuous LN-positive basement membrane-like structure was immunolocalized around these cells, which indicated that the glandular differentiation and CK19 expression in HCC were also related to the LN deposition, as in fetal liver and some chronic liver disorders.

Pathological changes of myocardial cytoskeleton in cardiomyopathic hamster.

Immunocytochemical investigation was performed on the cytoskeletal proteins in cardiac tissue of the cardiomyopathic hamster. Male cardiomyopathic UM-X7.1 hamsters at 180 days of age (n = 8) and age- and sex-matched normal BIO-RB hamsters (n = 8) were used in this study. Immunofluorescence microscopy using monoclonal antibodies against desmin, alpha-actinin, titin, and vincullin was employed. The heart weight to body weight ratio was significantly increased in the heart of cardiomyopathic hamster compared with that of normal hamster. In cardiomyopathic hamster, the left ventricular cavity was markedly dilated. Light microscopically, hypertrophy and atrophy of myocytes and myocardial fibrosis were prominently observed in cardiomyopathic myocardium. Immunocytochemically, desmin, alpha-actinin and titin showed the cross striations along the myofibers in normal myocardium. In contrast, in cardiomyopathic myocardium, desmin was irregularly distributed in myocytes and the amount of desmin was increased. Loss of cross striations of alpha-actinin and titin were frequently observed. Immunofluorescence against vinculin was not significantly altered. We conclude that the alterations of cytoskeletal proteins in myocardial cells may relate to decreased myocardial function in cardiomyopathic hamster failing heart.

EGF Receptor Expression in Mineralized Tissues: AnIn SituHybridization and Immunocytochemical Investigation in Rat and Human Mandibles

There is extensive evidence that growth factors play a central part in the autocrine/paracrine regulation of cell growth and differentiation in mineralized tissues. In order to investigate involvement of the EGFr receptor (EGFr) in forming mineralized tissues, its expression was studied by in situ hybridization and immunocytochemistry in mandibles of growing rats, as well as in human embryos. In Hertwig's epithelial root sheath of rat molar, EGFr mRNAs appeared strongly expressed, while dental pulp and dental follicle showed weak labeling. The lingual epithelium of rat incisor showed strong labeling, which decreased after epithelial dislocation. Cells of the adjoining lingual dental pulp and dental follicle, as compared to epithelium, contained a low level of EGFr mRNAs. In contrast, a significant signal with antisense RNA probe was observed in bone. Sense RNA probes provided a regular background or no labeling. Undifferentiated cells located in the periosteum and endosteal spaces were labeled. EGFr mRNAs were also present in osteoblasts and in lesser amounts in some osteocytes. In rat and in human bone, both osteoblasts and osteocytes were positive on immunostaining. Similarly in the Hertwig's root sheath, EGFr immunostaining and in situ hybridization labeling were closely related. These data show that different patterns of EGFr expression in forming mineralized tissues are tissue- and stage-specific. However, in all these cells, the present in situ investigation supports the assumption that EGFr is involved in the early stages of cellular proliferation and differentiation. This report also suggests that EGFr may play a role in differentiated and mature cells of mineralized tissues.

Catalytic and immunocytochemical detection of xenobiotic metabolizing enzymes in the olfactory organ of rainbow trout ( Oncorhynchus mykiss)

The fish olfactory organ, which is covered with a sensory epithelium, is in direct contact with aquatic pollutants. In mammals, presence of xenobiotic metabolizing enzymes has been reported in the olfactory epithelium, but few studies have been carried out on this topic in fish. In the present study, xenobiotic metabolizing enzymes were investigated in the rainbow trout olfactory organ. Data show that cytochrome P450 reductase, p-nitrophenol hydroxylase and glutathione S-transferase specific activities were in the same range as that in the liver, whereas 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase specific activities were 80 and 30 times lower, respectively, in the olfactory organ compared to the liver. Immunocytochemical investigation shows that cytochrome P4501A1 was located in both the epithelial non-sensory cells and the basal cells of the sensory epithelium, as well as in goblet and microvilli cells of the non-sensory epithelium. The presence of such enzyme activities in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.

Localization of neuropeptides in the nervous system of the brittle star Ophiura ophiura

Immunocytochemical investigations using antisera against the SALMFamide neuropeptide S1 and RFamide were carried out on whole mounts of the radial nerve cord and circumoral ring of the brittle star Ophiura ophiura. Both antisera show an abundant immunoreactivity in the ectoneural tissue and give similar patterns of distribution. They show that in the radial nerve cord there are discrete populations of neurons organized in an identical pattern in each segment. At the junction with the ring, however, the two proximal segments of the nerve cord are differentiated from the distal ones by increased numbers of immunoreactive neurons. This is probably due to the more complex integrative function in that area. In the ring, immunolabelling is simple and consists of fibre tracts and two local ganglia. In each segment both antisera label either one or two giant neurons the shape of which is similar to that of a class of fibres that have been described from intra-cellular dyefills. The S1- and RFamide-like distributions, although very similar, show some neurons which are only labelled by one antiserum. Since preabsorption controls indicate no cross-reactivity between antisera, there is evidence of two distinct neuropeptides in the nervous system of the brittle star.

Generation of a monoclonal antibody against the myelin protein CNP (2?,3?-cyclic nucleotide 3?-phosphodiesterase) suitable for biochemical and for immunohistochemical investigations of CNP

The functional role of CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase), a minor component of central and peripheral myelin is still unclear. Here we describe preparation of a monoclonal antibody directed against CNP. The antibody, of the immunoglobulin IgG1 type, raised with a basic 46 kDa membrane-associated protein solubilized from pig cerebellar membranes, can be used to detect immunoreactivity in solubilized brain homogenates from pig, mouse, rat, sheep, cow and man, in cerebrum and cerebellum, but not in other tissues such as liver, skeletal and heart muscle. The antibody recognizes the CNP doublet band and shows no cross-reactivity with any of the other brain proteins solubilized. In tissue sections from paraformaldehyde-fixed rat brain the antigen was localized in oligodendrocytes. In cultured glial cells from newborn mice the antibody stained cells which were identified as oligodendrocytes by co-localization of myelin basic protein. Even cells from a C6 rat glioma cell line, which contain very little of CNP, were labeled by the monoclonal antibody. Thus the monoclonal antibody recognizing CNP from several species is suitable for immunocytochemical investigations and also for biochemical studies of CNP, since the antibody has been employed for immunoprecipitation and immunopurification of CNP in crude brain homogenates.

Presence of Angiotensin II Immunoreactivity in the Ovary of the Rainbow Trout, Oncorhynchus mykiss

Renin-angiotensin system is present in mammalian ovarian follicular fluid and follicles. The demonstration of specific binding sites for angiotensin II in the follicular structures of rats suggests that angiotensin II may be related to the regulation of steroid biosynthesis in the mammalian ovary. Little is known about the presence and the action of angiotensin II in nonmammalian ovaries. An immunocytochemical investigation of angiotensin II in developing ovarian follicles of trout has been carried out. The specific antiserum was raised against (Val5)-angiotensin II of the trout. There was strong immunoreactivity in the ooplasm of endogenous vitellogenic follicles. Angiotensin II immunoreactive material was present in the follicular cells, but not within oocytes at the end of vitellogenesis. Immunocytochemically angiotensin II-like material in the ovary of rainbow trout fluctuates during the cycle of development.

An immunocytochemical investigation of glial morphology in the Pacific hagfish: radial and astrocyte-like glia have the same phylogenetic age

This study attempts to reconstruct the early phylogenetic history of macroglial cells among craniates. Since glia does not fossilize, such a reconstruction must be based on a cladistic comparison of glial characters in the Recent craniate taxa (hagfishes, lampreys, and gnathostomes); however, there are only few data on glial morphology and none on glial immunocytochemistry in hagfishes. Therefore, we investigated the presence and localization of various macroglia-specific epitopes in the brain and spinal cord of the Pacific hagfish, Eptatretus stouti (Myxinoidea) by means of immunocytochemistry. Antibodies directed against S100-protein and vimentin showed no cross reactivity. Antibodies directed against glial fibrillary acidic protein and glutamine synthetase labelled various glial structures. Glial fibrillary acidic protein-like immunoreactivity was observed in ependymal cells with radially oriented processes in some regions. However, throughout the entire CNS, labelling of non-ependymal cells and their processes prevailed. The processes of these cells made occasional vascular contacts and they also made contacts with neuronal perikarya. Glutamine synthetase-like immunoreactivity was also found in some processes with radial orientation and in ependymal cells; but the antibody stained mainly non-ependymal cells which gave rise to a felt-like meshwork of interdigitating fine and very fine processes penetrating the neuropil of the entire brain. Additionally, there was labelling in the walls of blood vessels and in processes enwrapping individual neurons. The occurrence of glial fibrillary acidic protein- and glutamine synthetase-like immunoreactivity in non-ependymal glial elements in the brain of hagfishes and the relative scarcity of labelling in radial glial elements necessitates a re-interpretation of the evolutionary history of glial cells. Non-ependymal macroglia with immunocytochemical and morphological characters resembling typical (mammalian) astrocytes appears to be as primitive as the various forms of radial ependymal glia.

Pigment-dispersing hormone-immunoreactive neurons in the cockroach Leucophaea maderae share properties with circadian pacemaker neurons

Neurons immunoreactive with antisera against the crustacean peptide beta-pigment dispersing hormone fulfill several anatomical criteria proposed for circadian pacemakers in the brain of the cockroach Leucophaea maderae. These include position of somata, projections to the lamina and midbrain and possible coupling pathways between the two pacemakers through commissural fibers. In behavioral experiments combined with lesion studies and immunocytochemical investigations we examined whether the presence of pigment-dispersing hormone-immunoreactive arborizations in the midbrain of the cockroach correlates with the presence of circadian locomotor activity. No rhythm was detected after severing both optic stalks in any animal for at least 12 days. Within the same time pigment-dispersing hormone-immunoreactive fibers in the midbrain disappeared. Two to seven weeks after the operation some of the cockroaches regained circadian locomotor activity, while others remained arrhythmic. In all cockroaches which regained rhythmic behavior pigment-dispersing hormone-immunoreactive fibers had regenerated and had largely found their original targets within the brain. In all arrhythmic cockroaches either none or very little regeneration had occurred. The period of the regained circadian activity inversely correlated with the number of regenerated immunoreactive commissural fibers. These data provide further evidence for the involvement of pigment-dispersing hormone-immunoreactive neurons in circadian clocks of orthopteroid insects.

Organization of the serotoninergic system in the brain of two amphibian species, Ambystoma mexicanum (Urodela) and Typhlonectes compressicauda (Gymnophiona)

An immunocytochemical investigation was made of the distribution of serotonin (5-HT) in the brain of larval and adult Ambystoma mexicanum and adult Typhlonectes compressicauda. Immunoreactive perikarya can be identified in the caudal diencephalon (paraventricular organ and infundibular nucleus), in the ventral mesencephalon (interpeduncular nucleus) and in the raphe of the rhombencephalon. Immunopositive fibers and terminal arborizations are widely distributed, extending from the whole telencephalon to the spinal lemniscus area. However, the retinorecipient structures of the thalamus and mesencephalon are either very weakly innervated (Ambystoma) or completely immunonegative (Typhlonectes). The habenular system also exhibits very few 5-HT-positive structures. The major serotoninergic neuron clusters, in both Urodela and Gymnophiona, tend to gather, from the paraventricular organ to the raphe, on both sides of the sagittal plane, showing no tendency to "lateralization". A new interpretation of the limited development of the serotoninergic system in amphibians is given.

Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: a combined in situ hybridization and immunocytochemical investigation.

Neurofibrillary tangle formation, a cardinal characteristic of Alzheimer's disease, is also a feature of several other neurodegenerative disorders, including subacute sclerosing panencephalitis (SSPE). In the present study the association of measles virus genome with neurofibrillary tangle formation has been studied in five cases of SSPE, using in situ hybridization (measles genome) and immunocytochemistry (tau, ubiquitin and beta/A4 amyloid). In two cases with duration of disease less than one year, neurofibrillary tangle formation was not observed. However, in those cases in which the disease was of several years duration, numerous tau- and ubiquitin-positive neurofibrillary tangles were demonstrated. In the two cases of longest duration, double-labelling techniques demonstrated the frequent association of neurofibrillary tangle formation with neuronal measles virus genome positivity. Immunocytochemistry for beta/A4 amyloid failed to demonstrate amyloid in any of the five cases. These findings support the hypothesis that neurofibrillary tangle formation can occur independently of amyloid formation and that this mechanism may operate in both Alzheimer's disease and in virally-induced disease.

GABAergic interneurons in the somatosensory thalamus of the guinea-pig: A light and ultrastructural immunocytochemical investigation

This work was performed to confirm previous data reporting the presence of GABAergic interneurons in the ventrobasal complex of guinea-pig, and to investigate the intrinsic organization of this nucleus compared to that of thalamic nuclei lacking interneurons. Immunocytochemical experiments were performed on the thalamus of adult guinea-pigs perfused with mixed aldehydes using an anti-GABA serum. At light microscopy, the immunoreaction on floating Vibratome sections showed that GABAergic neurons are present only in the reticular and lateral geniculate nuclei and in the ventrobasal complex. Quantitative evaluation of their number indicated that they are 20 and 15% of the total neuronal population in lateral geniculate nucleus and ventrobasal complex, respectively, while they are less than 1% in ventrolateral nucleus. At the ultrastructural level, the postembedding immunogold procedure showed the presence, in the ventrobasal complex, of GABA-labeled profiles involved in complex synaptic arrangements similar to those found in carnivores and primates. Conversely. GABA-labeled terminals in thalamic nuclei devoid of interneurons formed exclusively axo-dendritic or axo-somatic contacts, like in rats and mice. The present data suggest that GABAergic neurons in the ventrobasal complex of guinea-pigs give rise to functionally important rearrangements of its intrinsic synaptic organization and that they represent the morphological basis for an intrinsic modulatory mechanism that is absent in other thalamic nuclei lacking inhibitory interneurons. The phylogenetic implications of these findings are also discussed in comparison to other animal species.

Expression and functional roles of neural cell surface molecules and extracellular matrix components during development and regeneration of peripheral nerves

By combining both immunocytochemical and functional investigations, a hypothetical framework will be developed for the molecular mechanisms underlying neuron-glia interactions during development and regeneration of peripheral nerves. In particular, the immunoglobulin-like molecules L1, N-CAM, MAG and P0, the extracellular matrix molecules laminin and tenascin, and the carbohydrates PSA and L2/HNK-1 will be considered. During early stages of limb bud innervation in embryos, L1 and N-CAM are expressed on axons and Schwann cells and are involved in axonal fasciculation, whereas tenascin is thought to be involved in forming a scaffold around the nerve possibly preventing axons and/or Schwann cells from leaving the nerve. PSA has been shown to be involved in pathway selection at initial stages of limb bud innervation. Later on, when motor axons enter muscles, the carbohydrates determine the branching pattern of the nerves. During myelination, L1 appears to play a pivotal role during the formation of the first Schwann cell loops around the prospective myelin-containing axons. MAG and P0 appear also to be functionally involved at initial stages of myelin formation. Additionally, MAG may contribute to the formation and maintenance of non-compacted myelin and axon-Schwann cell apposition whereas P0 is involved in myelin compaction. Under regenerative conditions, L1, N-CAM, laminin, and tenascin are strongly up-regulated by denervated Schwann cells. In vitro observations strongly suggest that these molecules might foster axonal regeneration. The carbohydrate PSA is confined to regrowing axons and is also a candidate to support axonal regrowth. L2/HNK-1, which is found on motor axon-associated Schwann cells, may provide regenerating motor axons with a selective advantage over others resulting in appropriate reinnervation of motor pathways. Since many of the functional studies this review refers to have been performed in vitro, some of the conclusions drawn need reexamination in vivo. Gene manipulations, such as the generation of null mutants followed by a thorough morphological and immunocytochemical investigation may be a powerful tool to resolve this problem.

Textured Biomaterials as a Model for Studying Formation of Focal Contacts and Rearrangement of the Contractile Cytoskeleton in Platelets

Fibres of textured biomaterials (BM) enable platelets to adhere with formation of focal contacts. The contact structure and the reaction of the contact associated contractile cytoskeleton were studied using fibres of different flexibility/mobility: butyl-S-Sepharose (S), Polysulfone (PS) and Polyurethane (PU). Ultrastructural and immunocytochemical investigations were carried out to obtain information on the influence of tension on (1) the structure of the focal contacts; (2) the constricting cytoskeleton known to retract adherent collagen or fibrin fibres and (3) the cable-like bundles of actomyosin as observed in the clot. Fibre network from S spheres and 0.3 mm thick frozen sections of PS or PU were incubated with citrated PRP or with washed platelets at 37 C for 6 to 30 min while stirring for contact or activation with ADP or thrombin. Flexible fibres of the BM were found in deep invaginations of the plasmalemma associated with the constricting cytoskeleton. Focal contacts (mediated by fibrinogen as shown immunocytochemically) with fibres which were fixed in the texture or inflexible (PU) induce cable-like bundles of micofilaments containing myosin. These bundles pass across the cytoplasm and connect the contacts with the fibres or with other platelets, as demonstrated by computer-assisted 3-dimensional reconstruction. The model used indicates that retraction is possible as long as fibres are mobile and that cable-like bundles occur when the locomotion of platelets is blocked by immobile fibres. The interaction of platelets with textured BM reflects the situation during collagen or fibrin condensation. The findings may contribute to an understanding of platelet reactions on textured surfaces in grafts.

Cytological and Immunocytochemical Approaches to the Study of Corneal Endothelial Wound Repair

The vertebrate corneal endothelium represents a unique model system for investigating many cellular aspects of wound repair within an organized tissue in situ. The tissue exists as a cell monolayer that resides upon its own natural basement membrane that can be prepared as a flat mount to observe the entire cell population. Thus, it readily avails itself to many cytological and immunocytochemical methods at both the light microscopic and ultrastructural levels. In addition, the tissue is easily explanted into organ culture where further investigations can be carried out. These techniques have enabled investigators to use many approaches to explore function and changes in response to injury. In vivo, the endothelium acts as a transport tissue to actively pump Na+ and bicarbonate ions from the corneal stroma into the aqueous humor to control corneal transparency. Physiological findings indicate that fluid diffuses back into the stroma, across the endothelium, and thus hydration is said to be controlled by a pump-leak mechanism. Ultrastructural investigations, some employing horseradish peroxidase and lanthanum, have established the morphological basis for this mechanism as apical focal junctions that are not the classical tight junctions and do not constitute a complete zona occludens. Along with these apical focal junctions are gap junctions that appear identical to their counterparts in other cell types. Cytochemical studies localized both Na+K(+)-ATPase and carbonic anhydrase, the main pump enzymes associated with corneal hydration, to the lateral plasma membranes. Corneal endothelial cells of noninjured tissue do not traverse the cell cycle and are considered to be in the "Go" phase of the cell cycle as determined by microfluorometric analysis with DNA binding dyes such as auramin O and pararosaniline-Feulgen. However, injury can initiate cell cycle transverse and histochemical and cytological methods have been used to understand the tissue's response. Classical histochemical studies revealed that increased staining was observed for metabolic (NADase and NADPase) and lysosomal enzymes in cells bordering the wound area. The use of radiolabelled agents has further lead to an understanding of the endothelial wound response. Autoradiographic analyses of 3H-actinomycin D incorporation indicated that injury initiates changes in chromatin leading to increased binding levels of the drug in cells surrounding the wound. This change suggests that those cells undergo heightened macromolecular synthesis and this was confirmed by examining 3H-uridine and 3H-thymidine incorporation. The major mechanism involved in corneal endothelial repair is cell migration. Cytochemical and immunocytochemical investigations have allowed investigators an opportunity to gain some insight into changes that occur during this cellular process.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurofilament reorganisation and neurofilament antigen redistribution in spinal motoneurones following retrograde axonal transport of diphtheria toxin.

Single unilateral injections of diphtheria toxin (DTX) into the external anal sphincter muscle or internal intercostal nerve of cat induced characteristic ultrastructural lesions in corresponding ipsilateral spinal motoneurones 6-8 days later. The chief neuronal lesion was a progressive disruption of Nissl body composition and organisation, which between days 8-19 post injection was accompanied by a progressive accumulation of neurofilaments in motoneuronal perikarya and dendrites. Some axons in the ipsilateral ventral horn became hypertrophied due to neurofilamentous accumulation. Related immunocytochemical investigations 6-35 days after injection of DTX revealed abnormal immunoreactivity intoxicated motoneurones for 200-kDa and 160-kDa phosphorylated neurofilament proteins, but not in contralateral motoneurones. By day 35 abnormal neurofilament immunostaining also occurred in ipsilateral and some contralateral interneurones but not contralateral motoneurones. Abnormalities of Nissl body endoplasmic reticulum, neurofilament organisation, and neurofilament protein immunostaining were identical after either intraneural and intramuscular injections of DTX, indicating abnormalities were attributable to toxicity and not injection-related axonal damage. Since DTX acts specifically in the soma to inhibit protein synthesis, neurofilament abnormalities are secondary to cytotoxicity and probably result from deficits in transference of existing partially phosphorylated neurofilaments to the axonal transport system, or axonal transport per se.