The angiotensin II type 2 (AT2) receptor promotes axonal regeneration in the optic nerve of adult rats.

The renin-angiotensin system (RAS) has been traditionally linked to blood pressure and volume regulation mediated through the angiotensin II (ANG II) type 1 (AT1) receptor. Here we report that ANG II via its ANG II type 2 (AT2) receptor promotes the axonal elongation of postnatal rat retinal explants (postnatal day 11) and dorsal root ganglia neurons in vitro, and, moreover, axonal regeneration of retinal ganglion cells after optic nerve crush in vivo. In retinal explants, ANG II (10(-7)-10(-5) M) induced neurite elongation via its AT2 receptor, since the effects were mimicked by the AT2 receptor agonist CGP 42112 (10(-5) M) and were entirely abolished by costimulation with the AT2 receptor antagonist PD 123177 (10(-5) M), but not by the AT1 receptor antagonist losartan (10(-5) M). To investigate whether ANG II is able to promote axonal regeneration in vivo, we performed optic nerve crush experiments in the adult rats. After ANG II treatment (0.6 nmol), an increased number of growth-associated protein (GAP)-43-positive fibers was detected and the regenerating fibers regularly crossed the lesion site (1.6 mm). Cotreatment with the AT2 receptor antagonist PD 123177 (6 nmol), but not with the AT1 receptor antagonist losartan (6 nmol), completely abolished the ANG II-induced axonal regeneration, providing for the first time direct evidence for receptor-specific neurotrophic action of ANG II in the central nervous system of adult mammals and revealing a hitherto unknown function of the RAS.

The C-Band All-Sky Survey (C-BASS): Simulated parametric fitting in single pixels in total intensity and polarization.

The cosmic microwave background (CMB) B-mode signal is potentially weaker than the diffuse Galactic foregrounds over most of the sky at any frequency. A common method of separating the CMB from these foregrounds is via pixel-based parametric-model fitting. There are not currently enough all-sky maps to fit anything more than the most simple models of the sky. By simulating the emission in seven representative pixels, we demonstrate that the inclusion of a 5 GHz data point allows for more complex models of low-frequency foregrounds to be fitted than at present. It is shown that the inclusion of the C-BASS data will significantly reduce the uncertainties in a number of key parameters in the modelling of both the galactic foregrounds and the CMB. The extra data allow estimates of the synchrotron spectral index to be constrained much more strongly than is presently possible, with corresponding improvements in the accuracy of the recovery of the CMB amplitude. However, we show that to place good limits on models of the synchrotron spectral curvature will require additional low-frequency data.

On the redshift distribution and physical properties of ACT-selected DSFGs.

We present multi-wavelength detections of nine candidate gravitationally-lensed dusty star-forming galaxies (DSFGs) selected at 218GHz (1.4mm) from the ACT equatorial survey. Among the brightest ACT sources, these represent the subset of the total ACT sample lying in Herschel SPIRE fields, and all nine of the 218GHz detections were found to have bright Herschel counterparts. By fitting their spectral energy distributions (SEDs) with a modified blackbody model with power-law temperature distribution, we find the sample has a median redshift of z = 4.1 - 1.0 + 1.1 (68 per cent confidence interval), as expected for 218GHz selection, and an apparent total infrared luminosity of log 10 ( µ L IR / L ⊙ ) = 13.86 - 0.30 + 0.33 , which suggests that they are either strongly lensed sources or unresolved collections of unlensed DSFGs. The effective apparent diameter of the sample is µ d = 4.2 - 1.0 + 1.7 kpc , further evidence of strong lensing or multiplicity, since the typical diameter of dusty star-forming galaxies is 1.0-2.5 kpc. We emphasize that the effective apparent diameter derives from SED modelling without the assumption of optically thin dust (as opposed to image morphology). We find that the sources have substantial optical depth. ( τ = 4.2 - 1.9 + 3.7 ) to dust around the peak in the modified blackbody spectrum (λ obs ⩽ 500µm), a result that is robust to model choice.

Validation of risk adjustment models for in-hospital percutaneous transluminal coronary angioplasty mortality on an independent data set.

OBJECTIVES: We sought to validate recently proposed risk adjustment models for in-hospital percutaneous transluminal coronary angioplasty (PTCA) mortality on an independent data set of high risk patients undergoing PTCA. BACKGROUND: Risk adjustment models for PTCA mortality have recently been reported, but external validation on independent data sets and on high risk patient groups is lacking. METHODS: Between July 1, 1994 and June 1, 1996, 1,476 consecutive procedures were performed on a high risk patient group characterized by a high incidence of cardiogenic shock (3.3%) and acute myocardial infarction (14.3%). Predictors of in-hospital mortality were identified using multivariate logistic regression analysis. Two external models of in-hospital mortality, one developed by the Northern New England Cardiovascular Disease Study Group (model NNE) and the other by the Cleveland Clinic (model CC), were compared using receiver operating characteristic (ROC) curve analysis. RESULTS: In this patient group, an overall in-hospital mortality rate of 3.4% was observed. Multivariate regression analysis identified risk factors for death in the hospital that were similar to the risk factors identified by the two external models. When fitted to the data set, both external models had an area under the ROC curve >0.85, indicating overall excellent model discrimination, and both models were accurate in predicting mortality in different patient subgroups. There was a trend toward a greater ability to predict mortality for model NNE as compared with model CC, but the difference was not significant. CONCLUSIONS: Predictive models for PTCA mortality yield comparable results when applied to patient groups other than the one on which the original model was developed. The accuracy of the two models tested in adjusting for the relatively high mortality rate observed in this patient group supports their application in quality assessment or quality improvement efforts.

Validation in a community hospital setting of a clinical rule to predict preserved left ventricular ejection fraction in patients after myocardial infarction.

BACKGROUND: A previous study showed that patients with previous myocardial infarction (MI) who meet 4 simple clinical and/or electrocardiographic criteria have a left ventricular ejection fraction (LVEF) of 40% or greater, with a positive predictive value of 98%. The objective of this study was to validate this clinical rule in the community hospital setting. METHODS: Retrospective chart review in a 330-bed community hospital. Two hundred thirteen consecutive patients with MI were identified between June 1, 1993, and March 31, 1995. Left ventricular ejection fraction was predicted in a blinded fashion by means of the clinical rule before the actual LVEF test was reviewed. RESULTS: We identified 213 patients admitted with the primary discharge diagnosis of acute MI. All patients met standard clinical and enzymatic definitions for acute MI and had at least 1 measure of LVEF, such as echocardiography, ventricular angiography, or gated blood pool scan. The clinical rule predicted that 83 patients (39.0%) would have an LVEF of 40% or greater. Of these 83 patients, 71 had an ejection fraction of 40% or greater, for a positive predictive value of 86%. Of the 12 patients who were incorrectly predicted to have a preserved LVEF, 6 (50%) had an index non-Q-wave anterior MI (P<.001). Reanalyzing the patient population with a fifth variable (anterior non-Q-wave MI) added to the original 4 variables increased the positive predictive value to 91%. CONCLUSION: This simple clinical prediction rule has a positive predictive value of 86% when applied in the community hospital setting. Patients with anterior non-Q-wave MI may be 1 group in whom the rule is inaccurate, and expanding the clinical rule to 5 variables may increase the positive predictive value. When a technology-based assessment of left ventricular function is considered in patients after an MI, this prediction rule may allow for a more cost-effective patient selection, and as many as 40% of patients who have had acute MIs may require no testing at all.

Riboflavin-mediated axonal degeneration of postnatal retinal ganglion cells in vitro is related to the formation of free radicals.

It is well known that glial cells produce several neurotrophic factors. We detected a neurogedegenerative/neurite growth inhibiting activity in serum-free astrocyte-conditioned medium (ACM). After high performance liquid chromatography (HPLC)-purification, spectral analysis and test of biologic activity in tissue cultures of postnatal retinal explants we isolated a fraction containing a riboflavin-(vitamin B2)-like compound which caused the neuronal degeneration. We therefore investigated the influence of pure riboflavin on axonal regeneration in vitro. Riboflavin is a normal compound of Dulbecco's modified Eagle medium (DMEM) and other tissue culture media in various concentrations. The removal of riboflavin from ACM by reversed phase chromatography abolished the neurite growth inhibiting effect and enhanced the regenerative response of axonal outgrowth from postnatal rat retinal explants. However, doubling of the normal medium concentration (1 microM) of riboflavin lead to strong degenerative alteration of the outgrowing axons in a dose-dependent manner, even under maximal growth stimulation by cultivating the explants in astrocyte-conditioned medium. To check the possibility that riboflavin-mediated cytotoxicity is related to the production of free radicals through photoabsorption from daylight, we irradiated culture medium with UV light, and induced radical stress by incubating the explants with Fe2+/3+. In an other set of experiments, we proofed, if antioxidants/free radical scavengers like pyruvate or vitamin C and E are able to overcome the neurite growth inhibiting influence of riboflavin or the radical stress. Our findings suggest an involvement of riboflavin-mediated formation of free radicals/reactive oxygen species and subsequent neurite degeneration in in vitro-assays of neuronal regeneration or neuronal cell cultures. How far the riboflavin/free radical-induced axonal degeneration could be an explanation for neurological degenerative disorders has to be elucidated.

Cerebellar external granule cells are attached to the basal lamina from the onset of migration up to the end of their proliferative activity.

The development of cerebellar external granule cells in rats was studied from the time of demarcation of the cerebellar anlage on embryonal day 12 up to the time of their disappearance on postnatal day 20. Two types of cells were found. The first was orientated tangentially to the cerebellar surface and was characterized by a persistent contact to the basal lamina via an external process, with a lamellopodial tip and a cytoskeleton characteristic for migratory cells, and a retracting internal process featuring a single cilium. This cell type was the first to appear on embryonal day 14 in the caudolateral angle of the cerebellar anlage and, later, spread over the whole cerebellar surface. It disappeared after the external granular layer was completely expanded over the cerebellum. The second cell type appeared for the first time on embryonal day 16 in the caudal part of the cerebellar anlage and disappeared on postnatal day 20. It was orientated radially and also had contact with the basal lamina either with its cell body or with one or two short, radial processes, whose morphology differed from that of the external process of tangential cells by the absence of a lamellopodium and a prominent cytoskeleton. After postnatal day 17 contacts of external granule cells with the basal lamina decreased rapidly in length and number and were absent on postnatal day 20. We interpret these findings to indicate that tangential external granule cells are migrating before taking on a radial orientation characteristic for the mitotic cycle of proliferating external granule cells. In the light of increasing evidence implicating extracellular matrix in various developmental events of the nervous system we propose that the basal lamina of the cerebellum may be used as substrate and guidance structure by migrating external granule cells, and, furthermore, that the persistent contact with the basal lamina may mediate stimuli maintaining external granule cells in a proliferative state.

Fetal brain grafts rescue adult retinal ganglion cells from axotomy-induced cell death.

After intraorbital transection of the optic nerve of adult rats, 90% of the retinal ganglion cells die within 30 days. Since fetal brain extracts and cocultured fetal target regions support the survival of retinal ganglion cells in vitro (Nurcombe and Bennett: Exp. Brain Res. 44: 249-258, '81; McCaffery et al.: Exp. Brain Res. 48: 377-386, '82; Armson and Bennett: Neurosci. Lett. 38: 181-186, '83) we investigated whether cell death in the adult retina could be prevented by transplanting fetal (E16) thalamus and tectum to the proximal stump of the optic nerve of adult rats that was completely transected 2-3 mm behind the optic disc. Unoperated eyes contained 119,973 (+/- 939, SEM) retinal ganglion cells, estimated from axon counts of the intact optic nerve. Of these, 11,601 (+/- 1,857) remained in control operated eyes at 30 days postoperation while in the eyes of grafted rats, 35,086 (+/- 2,278) retinal ganglion cells were counted. Thus, 23,485 (= 22% of those normally dying after transection of the optic nerve) ganglion cells were rescued by the fetal grafts from cell death normally following axotomy. These results indicate that fetal target regions of retinal ganglion cells contain and/or produce neurotrophic molecules that promote the survival of adult axotomized retinal ganglion cells.

Regeneration of axons into the trochlear rootlet after anterior medullary lesions in the rat is specific for ipsilateral IVth nerve motoneurones.

The fibre projection from the IVth nerve nucleus to the superior oblique muscle was determined quantitatively in the normal rat by defining fibre numbers in transverse sections of the IVth nerve, and neurone numbers after retrograde labelling by horseradish peroxidase (HRP) injection into the muscle. There were 183 +/- 27 (S.E.) labelled neurones in the nucleus contralateral to the injected muscle and only 2 +/- 1 ipsilateral. The ipsilateral fibre number was 234 +/- 7 and the cell/axon ratio 0.8 +/- 0.1. Extensive analysis of all HRP retrogradely labelled material revealed no central fibre contribution to the IVth nerve other than from neurones resident in the trochlear nucleus. The central portion of the trochlear nerve tract was severed at its point of decussation in the anterior medullary velum. Ninety days after lesion, 10 +/- 4 (6% of control) neurones were labelled in the ipsilateral trochlear nucleus; none were labelled in the contralateral nucleus or in any other part of the midbrain, pons, medulla, or cerebellum. The number of myelinated fibres in the IVth nerve had decreased to 21 +/- 5 (9% of control) so that the cell/axon ratio was 0.4 +/- 0.2, thus suggesting that a single motoneurone has more fibres after lesion. In electron micrographs of the IVth nerve, larger than normal numbers of unmyelinated fibres were seen. Many myelinated fibres displayed signs of abnormal myelination. After regeneration, the projection was exclusively ipsilateral and not crossed as in the normal. These findings establish that there is a high degree of specificity after regeneration since no myelinated central nervous system axons other than trochlear fibres select the IVth nerve root as a trajectory over which to regenerate.

6-OHDA-induced ectopia of external granule cells in the subarachnoid space covering the cerebellum. III. Morphology and synaptic organization of ectopic cerebellar neurons: a scanning and transmission electron microscopic study.

The present report describes the ultrastructure, surface morphology, and synaptic connectivity of ectopically placed cerebellar neurons after treatment of newborn rats with 100 micrograms 6-hydroxydopamine (6-OHDA), administered intracisternally. In addition to granule cells which form the majority of ectopic neurons, neurons exhibiting the ultrastructure of basket/stellate cells are found in the subarachnoid space over the cerebellum. The ectopic neurons present an almost complete spectrum of homologous efferent and afferent connections. Parallel fiber synapses are found on thorns of spiny branchlets of Purkinje cell dendrites which also have grown out into the subarachnoid space, and on the somata and dendrites of basket/stellate cells. Many ectopic parallel fibers are seen to pass into the molecular layer of the underlying cerebellar cortex through defects in the pial surface, presumably connecting with intracortical postsynaptic partners. Synapses between ectopic Purkinje cell dendrites and basket/stellate cell axons are also observed; however, the source of these axons remains uncertain. Granule cell dendrites are engaged in glomeruli with mossy fibers. Moreover, ectopic granule cell colonies are densely innervated by noradrenergic fibers. Our results show that the technique of generating ectopia of external granule cells provides an additional model for investigating influences of epigenetic factors on the development of nerve cells.

6-Hydroxydopamine induced ectopia of external granule cells in the subarachnoid space covering the cerebellum. II. Differentiation of granule cells: a scanning and transmission electron microscopic study.

The present report describes the morphological differentiation of ectopic granule cells from external granule cells that have been induced to escape from the cerebellar cortex into the subarachnoid space by injecting neonatal rats with 100 microgram 6-hydroxydopamine (6-OHDA) into the cisterna magna. The following cell types were observed in the period between 5 and 25 days postinjection (dpi): (1) unipolar cells with one process bearing a growth cone at its tip; (2) bipolar cells with two thin beaded processes originating from opposite cell poles, bearing growth cones at their tips; (3) bipolar cells with a T-like process at one pole and a short process lacking a terminal growth cone at the opposite pole; (4) multipolar cells with one thin beaded process and two or more short processes bearing growth cones of a different morphology at their tips; (5) intermediate stages. In the late second week p.i., cell aggregates were observed that continually increased in size up to 30 dpi. On the basis of our light, transmission, and scanning electron microscopic findings, we interpret these cell types to be equivalent to the individual stages of granule cell differentiation that characterize axon formation, migration, and aggregation. In the period between 30 and 365 dpi, granule cells were almost exclusively organized into cell colonies of different sizes, but small cell clusters and single granule cells exhibited the scanning electron microscopic features of adult granule cells, i.e., a small spherical cell body, a single axon with parent axonal stem, T-junction, and parallel fiber, and dendrites engaged in synaptic glomeruli. The parallel fibers ran in fasciculi of different sizes, often parallel to each other, but without preferential orientation over the cerebellar surface. During migration and aggregation, the granule cells and their processes were associated with a substrate of glial sheets that in turn were connected to intracortical Bergmann glia fibers. Our findings indicate that (1) granule cells differentiate normally in an ectopic environment in the presence of glia, (2) ectopic Bergmann glia contain no directional information to guide aberrant migratory granule cells to their correct destination, (3) granule cells can survive outside the brain parenchyma for periods up to one year (the longest postinjection interval studied).

Regeneration of axons from the adult rat optic nerve: influence of fetal brain grafts, laminin, and artificial basement membrane.

After transection of the optic nerve of adult rats, most of the axons in the proximal stump die and the surviving ones are unable to regenerate into the distal optic nerve. Since the fetal brain has an inherent capacity to regenerate axons, we investigated whether fetal (E16) target regions of optic axons (thalamus and tectum) transplanted to the completely transected optic nerve of adult rats would promote axon regeneration. In control operated rats, axon growth beyond the site of transection was restricted to a few fibers that grew irregularly within the connective tissue scar. By contrast, in grafted animals directed outgrowth of optic axons toward the transplant started at 6 days postoperation (p.o.) and reached its maximum 15 days p.o. and later, when numerous single optic fibers and small axon fascicles had grown toward and into the graft, where they formed arborizations and terminal varicosities. Regenerating optic axons were further advanced than GFAP-positive strands of astroglia that emanated from the proximal optic nerve stump. Laminin immunoreactivity appeared at 6 days p.o. in the zone of reactive astroglia in the terminal part of the optic nerve stump. Later it showed a distribution complementary to the pattern of GFAP immunoreactivity, which it seemd to circumscribe. There was no unequivocal codistribution of laminin immunoreactivity with regenerating axons. In further experiments, target regions from different ontogenetic stages (E14 to neonate and adult) and nontarget regions (E16, cerebral cortex or spinal cord) were grafted to the optic nerve stump. With the exception of the adult grafts, all transplants had effects on axon regeneration comparable to those of E16 target regions. In order to test the effects of extracellular matrix molecules on axon regeneration, a basement membrane gel reconstituted from individual components of the Engelbreth-Holm-Sarcoma (EHS) sarcoma was implanted between proximal and distal optic nerve stumps. No axons were induced to regenerate by this matrix. Likewise, laminin adsorbed to nitrocellulose paper and implanted at the lesion site did not stimulate axon growth from the proximal optic nerve stump. These results indicate that fetal brain is able to induce and direct regrowth of axons from the optic nerve toward the graft across a substrate that is not composed of astroglia or basement membrane components like laminin. The directed growth of axons in the absence of a preformed substrate implies a chemotactic growth response along a concentration gradient mediated by neurotropic molecules released from the graft.

Derivation of cerebellar Golgi neurons from the external granular layer: evidence from explantation of external granule cells in vivo.

The present report provides evidence to challenge the traditional view that cerebellar Golgi cells are derived from the ventricular neuroepithelium, postulating instead that they originate from external granule cells. Supporting evidence for this assertion comes from three sources: 1) Typical Golgi cells are found in ectopic granule cell colonies, both outside the cerebellum (in the subarachnoid space) and also within the cerebellar cortex between fused folia. Because ectopic granule cell colonies are derived from external granule cells, which become displaced after treatment with 6-hydroxydopamine (6-OHDA), it was assumed that the ectopic Golgi cells also stem from such displaced external granule cells. 2) In order to demonstrate that Golgi cell precursors migrate from the external granular layer into the Purkinje cell plate, the development of the cerebellar cortex was studied over the period of Golgi cell genesis. On E19 the external granular layer in the rat is subdivided into an outer proliferative and an inner subproliferative zone. At the inner margin of the external granular layer, and in the marginal zone, radially oriented, darkly staining cells are present that exhibit all the characteristics of migrating neurons possessing a leading process oriented toward the Purkinje cell plate, a somatic cilium, and a close association with radial glia fibers. In later stages, these cells are also found deep to the Purkinje cell plate. Because Golgi cells arise during the period between E19 and postnatal day 2 in the rat (Altman and Bayer, '77, '78) and as the basket cells, the first neurons of proven origin from the external granular layer, are not produced before the second postnatal day (Altman, '72), the earlier migrating neurons are presumed to be Golgi cells. 3) Available data from cell kinetic 3H-thymidine studies show that there is no unequivocal evidence for Golgi cell genesis from the ventricular neuroepithelium, because, at the time of Golgi cell birth, ventricular and external granular stem cell populations are proliferating, and with the present methods it is not possible to decide which of these are the precursors of Golgi cells. Thus, taken together, the findings of this study show that Golgi cells are more likely to arise from the external granular layer than from the ventricular neuroepithelium. This concept would unify cerebellar histogenesis by proposing that projection neurons arise from the ventricular neuroepithelium, whereas all interneurons of the cerebellar cortex are descendants of the external granular layer.

Three morphologically distinct types of interface develop between adult host and fetal brain transplants: implications for scar formation in the adult central nervous system.

The development of the host/graft interface of cerebellar and cerebral transplants was studied 1-60 days after operation. Grafts from fetal Wistar rats were transplanted to a cavity over the superior colliculus of adult rats by removing parts of the overlying cortex and hippocampus according to the Björklund/Stenevi technique. In sham-operated control rats, in which a cavity was made in the brain but no graft was implanted, the parenchyma bordering the entire cavity developed a complete glial-meningeal scar within 2 weeks after operation consisting of multilayered glial processes, a basal lamina, and fibroblasts (meningeal cells). A similar interface also developed between graft and host in the most superficial parts of the transplantation cavity. In the basal parts of the transplantation cavity, the host/graft interface consisted either of an incomplete sheet of astrocyte processes aligned in parallel to each other but without a covering basal lamina or of completely fused neuropil without any morphological signs of separation between host and transplant. It is concluded that these three zones of host/graft interface are established by differential interaction between the growing transplant and the host cicatrix. At the basal host/graft parenchymatous interface the fetal transplant interferes with the normal adult cicatrization process of the host, possibly by either releasing inhibitory factors or by preventing contact between the astroglia of the host and fibroblasts (meningeal cells). In white matter regions of the transplantation cavity, voluminous cysts developed, both in sham-operated controls and in graft recipients, which were invaded by transplanted neurons.

Development of astroglial cells in the proliferative matrices, the granule cell layer, and the hippocampal fissure of the hamster dentate gyrus.

The histogenesis of the hamster dentate gyrus was studied with light and electron microscopy and antisera against the astrocyte-associated antigens vimentin and GFAP, in order to follow the differentiation of radial glial cells and astrocytes. The formation of the stratum granulosum is preceded by the establishment of successive dentate matrices, which are formed by cells that leave the ventricular neuroepithelium and occupy positions above the fimbria (suprafimbrial), below the pial surface (subpial), and within the dentate hilus (hilar dentate matrix). The subpial dentate matrix invades the marginal zone of that region of the cerebral wall, where the stratum granulosum will later develop. From the beginning of its existence on embryonal day 13 (E13) up to its disappearance about postnatal day 7 (P7), it is characterized by a high content of GFAP-positive cells and mitoses. This indicates early gliogenesis in the dentate anlage, long before the appearance of the stratum granulosum. Many of the bipolar GFAP-positive cells are oriented parallel to the pial surface and have focal contacts to the pial basement membrane. The establishment of the subpial dentate matrix splits the primordial radial glial scaffold of the hippocampal/dentate anlage into two bundles: 1) the suprafimbrial bundle that retains its original radial position between ventricle and pial surface; and 2) the dorsal glial bundle that traverses the ventral tip of the pyramidal cell layer of future CA3. The latter is pushed dorsolaterally, away from the pial surface, by the enlargement of the subpial dentate matrix and, later, by the suprapyramidal blade. The latter emerges around birth as small radial columns of granule cells located between the bent basal parts of the ventralmost fibers of the dorsal glial bundle and the subpial dentate matrix. From the beginning of its existence it is traversed by unipolar "secondary" radial glial fibers that appear to originate from the subpial dentate matrix. Both the supra- and the infrapyramidal blades seem to elongate by the addition of postmitotic granule cells and "secondary" radial glial cells from the subpial dentate matrix to the growing end of the primordial stratum granulosum. The hilar dentate matrix that is localized in the prospective hilar region, inside the growing stratum granulosum, also contains glial cells that seem to be incorporated into the stratum granulosum. The dentate gyrus is demarcated from the CA1 region of the hippocampus proper by GFAP-positive cells that populate the hippocampal fissure, and that also originate from the subpial dentate matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Destruction of meningeal cells over the medial cerebral hemisphere of newborn hamsters prevents the formation of the infrapyramidal blade of the dentate gyrus.

Meningeal cells participate in the development of the cerebellum both by stabilizing the extracellular matrix of the pial surface and by organizing the radial glial scaffold and the lamination of the cerebellar cortex. In the present study we investigated possible influences of meningeal cells on the development of the dentate gyrus, whose ontogenesis has many similarities to that of the cerebellum. Meningeal cells were selectively destroyed by injecting newborn hamsters with 25 micrograms 6-hydroxydopamine (6-OHDA) into the interhemispheric fissure. Twenty-four hours postinjection (p.i.) the meningeal cells over the medial cerebral hemispheres were completely destroyed. Thirty days p.i. the infrapyramidal blade of the dentate gyrus was almost completely missing, while the suprapyramidal blade was hypertrophied, extending with its medial tip almost up to the medial surface of the cortex. In order to ascertain that this maldevelopment was caused by the destruction of meningeal cells, another group of hamsters was pretreated with normetanephrine (NMN) which inhibits the extraneuronal uptake of 6-OHDA into meningeal cells. In this group the meningeal cells were unaffected by the treatment, and the morphology of the dentate gyrus was normal 30 days p.i. of 6-OHDA plus NMN. When the meningeal cells were destroyed in later stages of development (postnatal days 1-5), alterations of the dentate gyrus could be induced only up to the fourth postnatal day; thereafter, 6-OHDA treatment left it unchanged. This indicates a critical period of meningeal cell influence that coincides with the period of existence of the subpial dentate matrix. Analysis of the time course of the defective development revealed that in the first 5 days p.i. 1) meningeal cells over the medial cerebral hemisphere were destroyed and removed, 2) the pial basement membrane over both the dentate anlage and the diencephalon thinned and ruptured, and the adjacent brain parts fused focally, 3) many cells of the subpial dentate matrix disappeared from their subsurface position, 4) the number of "immature" cells increased in the hilus and the subgranular zone of the suprapyramidal blade, 5) the suprapyramidal blade elongated and thickened considerably, while the infrapyramidal blade did not form. Beyond 5 days p.i. those parts of the pial surface of the dentate anlage that had not fused with the diencephalon were repopulated with meningeal cells. This reappearance of meningeal cells was accompanied by 1) the restitution of the normal morphology of the basement membrane, 2) the reappearance of neuronal and glial cells below the pial surface, and 3) the formation of fragments of the infrapyramidal blade which later developed a normal appearing lamination.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental studies on cerebellar foliation. I. A qualitative morphological analysis of cerebellar fissuration defects after neonatal treatment with 6-OHDA in the rat.

The present report describes the natural history of defective cerebellar fissuration in the rat after neonatal treatment with 6-hydroxydopamine (6-OHDA). Within 24 hours after an intracisternal (IC) injection of 100 micrograms 6-OHDA cerebellar pial fibroblasts degenerated almost completely and were phagocytosed b macrophages within 2-5 days postinjection (dpi) leaving the cerebellar surface denuded of pia. Bergmann glia end feet at first exhibited morphological signs of gliosis and later formed new sprouts that penetrated the basal lamina and grew into the subarachnoid space covering regenerating pial fibroblasts and also invading ectopic colonies of external granular layer (EGL) cells. Breaches in the basal lamina appeared after the pial fibroblast had been destroyed and were confined to areas where Bergmann glia end feet were absent and where EGL cells were opposed to the basal lamina. EGL cells escaped through these fractures into the subarachnoid space in the fissures, where they proliferated to form large colonies of granule and stellate cells. In those fissures in which EGL ectopia featured, opposing folia fused and fissures were lost. These findings suggest that pial fibroblasts and the basal lamina have an important role in maintaining lobular partition during development of the cerebellum, in establishing cerebellar fissures, and in consolidating Bergmann glia-EGL cell relationships as a prerequisite for orderly migration of EGL cells.

In vitro-staining specificity of the antibody 5-D-4 for microglia but not for monocytes and macrophages indicates that microglia are a unique subgroup of the myelomonocytic lineage.

We have previously shown that (i) the ramified phenotype and (ii) the microglia-specific pattern of membrane currents are induced not only in microglia, but also in monocytes and macrophages if they are cultured in the presence of astrocytes. These findings indicated that microglia are not a separate type of cell of the myelomonocytic lineage, but are induced to take on their unique characteristics by astrocytes. Recently, it was discovered that the antibody 5-D-4 selectively stains ramified microglia in situ. We therefore studied the influence of astrocytes and other epithelial cells on the expression of the keratan sulfate epitope recognized by 5-D-4 in microglia and other myelomonocytic cells. Our findings show that this antigen is exclusively expressed in microglia only if they are induced to ramify by coculture with either astrocytes or epithelial cells. By contrast monocytes and macrophages, even if induced to take on the ramified phenotype do not stain positive with 5-D-4. These findings indicate (i) that 5-D-4 is a specific marker for ramified microglia in vitro, and (ii) that microglia are a separate class of myelomonocytic cells, distinct from monocytes and macrophages.

Intrathecal synthesis of monocyte chemoattractant protein-1 (MCP-1) in amyotrophic lateral sclerosis: further evidence for microglial activation in neurodegeneration.

Autopsy studies and animal experiments suggest that microglial inflammation contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS). Monocyte-chemoattractant protein (MCP-1) might play an important role in microglial recruitment. We studied MCP-1 levels in sera and cerebrospinal fluid of 29 ALS patients and compared the results with 11 control patients with tension headache. The MCP-1 level was determined using enzyme-linked immunosorbent assays (ELISA). A significant increase in cerebrospinal fluid MCP-1 level but not serum level was seen in the patients with ALS compared to the control subjects. These results suggest that cerebrospinal fluid MCP-1 activity may be a sensitive marker for neuroinflammation in ALS useful for monitoring treatment trials in ALS.

ATP and adenosine induce ramification of microglia in vitro.

Microglial cells in the healthy adult brain possess a characteristic ramified morphology with multiple branched processes, small somata and down-regulated inflammatory properties. In contrast, microglial cells isolated from new-born rat brain inevitably show a non-ramified amoeboid phenotype, which is observed in vivo after pathologic activation or during development. To identify factors that control microglial morphology we investigated the effects of purines alone or in combination with astrocyte-conditioned medium (ACM). Under optimized culture conditions postnatal rat microglial cells developed an amoeboid to ovoid phenotype. Addition of 0.6-1 mM ATP or adenosine induced the outgrowth of numerous processes after 2-3 days that could be observed also in the presence of ACM as previously reported. Culture in ACM plus ATP or adenosine yielded an optimized ramified phenotype. ATP or adenosine, but not ACM alone, also prevented the formation of a flat, amoeboid morphology induced by lipopolysaccharide (LPS); however, at 0.6-1 mM they did not reduce the initial LPS-induced activation of the transcription factor NF-kappaB. By using specific agonists or antagonists the morphological transformations could not be confined to a distinct purinoreceptor subtype, but appeared to be mediated by long-term presence of adenosine in the medium to which phosphorylated purines were rapidly hydrolyzed by microglial cells. Since ACM did not contain sufficient concentrations of ATP or adenosine, purines are not the only ramification-inducing factors present in ACM; however, they are a valuable tool to induce microglial ramification in vitro.