Dynamic expression of L-selectin in cell-to-cell interactions between neutrophils and endothelial cells in vitro.

Neutrophil-endothelial cell interactions are regulated by cell adhesion molecules and their cognate ligands. It has been proposed that L-selectin and Mac-1 (CD11b/CD18), two neutrophil adhesion receptors, have sequential roles in neutrophil extravasation during inflammation. In this model, L-selectin mediates rolling and initial adherence of neutrophils to endothelial cells, while Mac-1 strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. L-selectin and Mac-1 expression are known to be inversely regulated. Here an in vitro culture system has been developed to investigate in situ expression of L-selectin during cell-to-cell interactions between neutrophils and endothelial cell monolayers by confocal immunofluorescence analysis. Neutrophils underwent profound cell shape change from round to polarized cell morphology with pseudopod formation after 5 to 15 min coculture with IL-1-stimulated human endothelial cells. L-selectin was redistributed to the pseudopod of the polarized neutrophils in correlation with such cellular changes. During initial cell attachment, neutrophils bound to IL-1-stimulated endothelial cells expressed a high level of L-selectin in a polarized pattern. L-selectin expression decreased over time during neutrophil-endothelial cell interactions.

Mechanisms of inflammatory cell attachment in chronic relapsing experimental allergic encephalomyelitis: a scanning and high-voltage electron microscopic study of the injured mouse blood-brain barrier.

Brain and spinal cord blood vessels from mice subjected to chronic relapsing experimental allergic encephalomyelitis were examined by scanning (SEM) and high-voltage electron microscopy (HVEM). SEM analysis of veins and venules from affected tissue regions demonstrated inflammatory cells (ICs), primarily lymphocytes or monocytes, attached to the luminal endothelial cell (EC) surface adjacent to the junctional complexes. In transverse section these cells were shown by HVEM to extend and to insert filopodia (lymphocytes) or flap-like lamellapodia (monocytes) into the luminal EC surfaces. Affected ECs often expressed increased microvillar projection as well as parajunctional crater-like structures on their luminal surfaces. Based on scanning and high-voltage electron microscopy, we present morphological evidence that some populations of sensitized ICs do not penetrate the EC junctions initially during EC attachment but instead insert pseudopodial projections into specialized openings in the ECs that are formed in response to chronic inflammation.

Combined conventional transmission, scanning, and high-voltage electron microscopy of the same blood vessel for the study of targeted inflammatory cells in blood-brain barrier inflammation.

The microvasculature of brains and spinal cords from mice subjected to chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied using three different electron microscopic techniques. Blood vessels were initially examined by scanning electron microscopy. This allowed for the investigation of topographical changes of the luminal aspects of endothelial cells (ECs) and identification of targeted inflammatory cells (ICs) attached to the ECs. The same blood vessel areas with attached ICs examined by scanning electron microscopy were subsequently trimmed, processed for routine conventional transmission electron microscopy, and plastic embedded. Thin (80 nm) sections were cut and evaluated. Semithick (0.5-0.75 microns) serial sections of this material were examined by high-voltage electron microscopy. Data presented here described a useful technique for combining several ultrastructural techniques that permits simultaneous topographic and cross-sectional examination of selected regions of individual blood vessels or specifically targeted ICs.

Microanalysis of Alzheimer disease NFT and plaques.

Electron probe energy dispersive microanalysis of isolated andin situ neurofibrillary tangles (NFT) and neuritic (senile) plaque cores have been done to investigate the levels of Al, Si, Ca and Fe in the leading lesions of Alzheimer disease neuropathology. Varying levels of Si and Al, and to a lesser extent Ca, have been co-localized in about one half of the NFT and plaques examined using X-ray mapping. The variability of detection and the low levels of Al present indicates that aluminum is not required for the formation of the NFT and that aluminosilicates are not involved in the formation of the plaque core.

Aluminum neurotoxicity in mammals.

Although aluminum comprises a large percentage of the Earth's crust, it is excluded from body tissues, and especially from the central nervous system. When aluminum is experimentally introduced to the central nervous system, several neurotoxic effects are observed:i.e. neurofibrillary changes, behavioral and cognitive deficits and enzymatic and neurotransmitter changes, as well as certain types of epileptic seizures.The localization of relatively high levels of aluminum in Alzheimer disease, Guamanian amyotrophic lateral sclerosis and Parkinsonism-dementia has led to the implication of aluminum as a pathogenic factor in these diseases. Recent studies have shown that microtubule-associated proteins are part of the paired helical filaments which make up the intraneuronal neurofibrillary tangle. Other studies have identified the protein making the vascular and neuritic (senile) plaque amyloid and located the gene responsible for this protein to chromosome 21.Our electron microprobe analysis studies have not found the levels of aluminum or silicon in either the neurofibrillary tangles or amyloid cores reported elsewhere, nor have the levels of aluminum been elevated in approximately one half of the tangles and plaque cores examined to date.

Sites of egress of inflammatory cells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis.

Results are reported of experiments designed to focus at attachment sites of inflammatory cells (ICs) on the luminal surface of brain endothelial cells (ECs) and on the mechanisms of horseradish peroxidase (HRP) transport across the altered blood-brain barrier (BBB) in a murine model of chronic relapsing experimental allergic encephalomyelitis. Cationized ferritin (CF) served as a marker for evaluating the electrostatic nature of brain microblood vessels (MBVs) on the plasma membranes of ICs or normal mouse peripheral white blood cells and erythrocytes. SJL/J mice demonstrating clinical illness were given HRP or CF, in vivo or in situ, respectively. Light microscopy and conventional transmission electron microscopy of cerebellum or thoracic and lumbar spinal cord regions demonstrated HRP leakage most pronounced in MBVs with perivascular infiltrates. HRP traversed across the ECs via numerous vesicles and tubular profiles located mostly in the parajunctional regions, while EC junctions appeared closed. Scanning electron microscopy demonstrated that IC attachment was primarily at parajunctional sites on the EC surface. We also observed increased microvillar projections extending from the EC surface into the lumen. CF demonstrated a patchy decoration on both the luminal EC surface and IC membranes but did not label uncoated invaginating membrane pits or tubular structures. Our data indicate that the points of attachment of the ICs on the EC surface may reflect specific receptor sites where the ICs eventually gain entrance into CNS across the BBB during brain inflammation.

Genetic control of scrapie: incubation period and plaque formation in I mice.

The host component of control of scrapie incubation period in the mouse is manifested largely through the action of the Sinc gene. Only one mouse strain (VM) has been found that is p7p7 (prolonged incubation for ME7 agent) and two other strains have been derived from VM. All other strains, designated s7s7, have a short incubation for ME7. In the present study, the I strain was shown to fulfil the criteria that are characteristic of mouse strains with the p7 allele of Sinc: a comparatively long incubation period for ME7 and a short incubation period for 22A, the incubation period for F1 hybrid mice (s7s7 X p7p7) either fell between the incubation periods for the parental strains (with ME7) or were longer than either parent (with 139A and 22A), amyloid plaques occurred following injection of ME7 and 87V but not after 22A or 139A, lesion profiles for four scrapie strains were similar in I mice and p7p7 mouse strains, and injection of 87V led to disease in less than 300 days. Finally, allelism tests using F1 hybrid mice (I X a p7p7 mouse strain) and progeny of backcrosses between these F1 mice and I mice failed to reveal the segregation of additional major genes affecting scrapie incubation period.

Ultrastructural observations of spinal cord lesions and blood-brain barrier changes in scrapie-infected mice.

Spinal cord samples from IM or VM mice injected intracerebrally with the 87V scrapie agent were examined ultrastructurally at the clinical stage of disease for changes in blood vessel permeability and for pathological alterations. In several animals, (3 of 16), massive changes were noted in the cervical spinal cords in the subependymal area of the cortical gray matter immediately surrounding the central canal including ependymal cell changes, the presence of amyloid plaque in close association with microglial cells, extensive neuropil vacuolation, the appearance of reactive astrocytes, degenerating neurites and vacuolated neurons. In those regions showing structural damage, localized increased permeability to horseradish peroxidase across the blood-brain barrier was noticed along with the appearance of numerous vesiculo-canalicular profiles in micro-blood vessel endothelial cells with extravasation of the tracer to the neuropil. Some damaged neurons appeared flooded with this tracer. These changes were not observed in either the thoracic or lumbar spinal cord regions. The occurrence of pathological changes in the spinal cords of a small percentage of intracerebrally injected mice was probably due to a high concentration of the scrapie agent which localized in the cervical spinal cord, presumably after entering the spinal fluid via the lateral ventricle at the time of injection.

Feline maternal taurine deficiency: effects on retina and tapetum of the offspring.

The retina and tapetum of kittens born to taurine-deficient and taurine-supplemented mothers were compared. Retinal taurine concentrations typically reach adult levels 6 weeks postnatally. When measured at weaning at 8 postnatal weeks, the taurine concentrations in retina and tapetum of taurine-deficient kittens were 40% of normal levels. An ultrastructural correlate found in the retinas of taurine-deficient kittens was the presence of photoreceptor outer segments that were reduced in length and altered from the typical columnar configuration. Tapetal cells of taurine-deficient kittens were distinguished by accumulations of electron-dense droplets, the presence of tapetal rods with dilated limiting membranes and the presence of amorphous vesicles.

Postnatal taurine deficiency in the kitten results in a persistence of the cerebellar external granule cell layer: correction by taurine feeding.

Dietary deprivation of taurine in pregnant cats from approximately 1 week prior to giving birth is sufficient to reduce substantially the taurine concentration in feline milk but does not result in any abnormalities in kittens at birth. Kittens nursing on this low taurine milk have a lower growth rate than normal, have lower tissue taurine concentrations, and 8 weeks after birth have a persistence of cells in the cerebellar external granule cell layer. Mitotic figures are present also, indicating that cell division is occurring still, an event which normally is completed 3-4 weeks after birth. Daily oral supplementation with 40 mumoles taurine increases the growth rate almost to the level of normally nurtured kittens and results in normal tissue taurine concentrations and apparently normal migration of cells in the cerebellum. These findings indicate that nutritional taurine supplied in the milk is involved in the normal ontogeny of the cerebellum and that a taurine deficiency at this stage of development results in a maturational delay.

Taurine deficiency in the developing cat: persistence of the cerebellar external granule cell layer.

Dietary taurine deprivation adversely affects feline pregnancy and is associated with the frequent occurrence of fetal resorption, abortion, stillbirth, and low birthweight of live kittens at term. Taurine-deprived, live-born kittens have a poor postnatal survival rate and grow less well than kittens from taurine-supplemented queens. The postnatal dietary taurine intake of such kittens is reduced if they are nursed by their biologic mothers; the concentration of taurine in milk of taurine-deprived mothers is less than 10% of that in milk from taurine-supplemented queens. Surviving kittens from taurine-deprived mothers exhibit a constellation of neurological abnormalities (abnormal hind leg development, a peculiar gait characterized by excessive abduction and paresis, and thoracic kyphosis readily visible by X-ray). These findings suggest the presence of a developmental cerebellar deficit. Histological examination of the pre- and postnatally taurine-deprived kitten's cerebellum reveals a persistence of the external granule cell layer, which was confirmed by electron-microscopic examination. Numerous mitotic figures are present in the cells in the external granule cell layer of the cerebellum of kittens born from the nursed by taurine-deprived queens, but not in those from taurine-supplemented mothers. These findings suggest a maturational delay.

Preclinical changes in weight of scrapie-infected mice as a function of scrapie agent-mouse strain combination.

Several inbred strains of mice were injected with different scrapie agents and their total body weight was monitored throughout the incubation period. As a control, mice were injected with normal mouse brain homogenate. For most combinations of scrapie agent and mouse strain, weights during the preclinical phase were similar to or lower than the average weight of controls. For some combinations there was a significant increase in weight (compared to controls) during the latter part of the preclinical phase of disease. The effect was dependent on both agent and mouse strain, i.e., in some cases a mouse strain showed the increase with one scrapie agent but not another and some scrapie agents caused the increase in one inbred strain of mouse but not in another strain. The increase in weight was due to accumulations of fat rather than a generalized increase in weight of various organs. With one mouse strain (SJL), there was increased vacuolation seen in the hypothalamus of mice injected with scrapie agents that showed the increase in weight compared to the lesion intensity with an agent which did not cause the weight increase.

Increased blood-brain barrier permeability in scrapie-infected mice.

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of "slow" viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).

Retinal damage in scrapie mice.

Retinal damages in mice infected with scrapie are reported. The effects ranged from no histopathological changes, through partial loss of the outer nuclear layer (ONL) to the most severe changes with complete loss of ONL cells and photoreceptors. For the most part, cells of inner nuclear and ganglion layers were spared. These changes were found in 23% of C57BL/6J mice injected with the ME7 strain of scrapie and in 28.5% of VM mice injected with the 87 V strain, while no changes were found in 13 IM mice injected with the 87 V strain of scrapie. The possible relation of these changes to scrapie infection and to light induced retinopathy is discussed.

Evidence for induction of localized amyloid deposits and neuritic plaques by an infectious agent.

Mice inoculated intracerebrally with mouse brain homogenate infected with a particular strain of scrapie agent developed amyloid and neuritic plaques and amyloid tubercles along the injection track, solid wall-like infiltrations at the injection site, or both. Our observations implicate an infections agent in the localized formation of the amyloid and the neuritic plaque--the leading pathological change found in normal elderly people and animals and in patients with senile dementia of the Alzheimer type. Further, these studies indicate that microglia appear to play a central role in the pathogenesis of amyloid formation and could be involved in the processing, production, and assembly of the fibers.

A simple screening procedure for evaluating central nervous system tissue sections showing structural and cytochemical alterations of the blood-brain barrier.

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 micrometers and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.