Minocycline or iNOS inhibition block 3-nitrotyrosine increases and blood–brain barrier leakiness in amyloid beta-peptide-injected rat hippocampus

This work has examined levels of 3-nitrotyrosine (3-NT, a marker for peroxynitrite formation) and intactness of blood–brain barrier (BBB) in amyloid beta-peptide (Aβ_1–42)-injected rat hippocampus. Immunohistochemical analysis demonstrated 3-NT immunoreactivity in microglia/macrophages and astrocytes were significantly increased at 7 days post-Aβ_1–42 injection. Administration of the broad spectrum anti-inflammatory agent minocycline or the selective iNOS inhibitor 1400W markedly reduced 3-NT levels. Double immunofluorescence staining showed that 3-NT was prominently expressed in microglia/macrophages and astrocytes located in proximity to blood vessels. Additionally, Aβ_1–42 injection caused a marked increase in permeability of the BBB to immunoglobulin G (IgG); both minocycline and 1400W were highly effective in decreasing the leakiness of the BBB. Our results suggest the involvement of glial-derived reactive nitrogen species in mediating increased BBB permeability in Aβ_1–42 injected rat hippocampus.     

SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain

Although extracellular matrix (ECM) glycoproteins play important roles in neural development, their levels are generally believed to decrease in the adult brain. Immunohistochemical analysis indicates that the anti-adhesive ECM glycoprotein SPARC/osteonectin, which contains a follistatin ‘module’, is expressed in the adult rabbit nervous system. In the cerebellum, SPARC is present in Bergmann glia, with a strong signal along their radial fibres. SPARC, while enriched in membrane fractions, is not a transmembrane protein. In the hippocampus, colocalization of SPARC is observed in cells which express the astrocytic marker GFAP. The expression of SPARC by a subset of astrocytes, particularly in synaptic enriched areas, suggests a continuing role for the ECM in the adult brain.     

The influence of cytokines on the integrity of the blood-brain barrier in vitro

The effects of the cytokines tumour necrosis factor-α (TNF-α), interleukin (IL) -1 β and IL-6 on the permeability of monolayers of rat cerebral endothelial cells (RCEC) were investigated to assess potential changes in the integrity of the blood-brain barrier (BBB). RCEC were cultured to tight monolayers with a trans endothelial electrical resistance (TEER) of 100–150 ω- cm² on polycarbonate filters. Exposure of the RCEC to TNF-α, IL-1β and IL-6 induced a decline in the TEER, which could be completely abolished by 1 μM of indomethacin, a cyclooxygenase inhibitor. In addition, the effect of IL-1β on TEER across monolayers of RCEC could be completely inhibited by IL-1 receptor antagonist. In conclusion, cytokines induce a disruption of the BBB in vitro. In this process, cyclooxygenase activation within the endothelial cells seems to play a key role.     

The effect of interferon β-1b on lymphocyte-endothelial cell adhesion

Interferon β-1b (IFNβ-1b) (Betaseron®) has been recently approved forfor treatment of multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). The mechanism of action of IFNβ-1b is not understood, but its effect in reducing gadolinium enhanced MRI lesions suggest an effect at the blood brain barrier (BBB). Thus the objective of this study is to examine the effect of IFNβ-1b treatment of endothelial cells (EC) on lymphocyte-EC adhesion, and on the expression of the adhesion molecules (AM) ICAM-1, VCAM and E-selectin induced by IFN-γ, TNF-α, or IL-1β. Primary cultures of human umbilical vein EC (HUVEC) were used which under basal conditions expressed low levels of ICAM-1 but not VCAM or E-selectin. IFNβ-1b (1–1000 IU/ml) had minimal effect on basal expression of AM on HUVEC, but AM could be substantially upregulated by IFN-γ, IL-1β or TNF-α which was associated with a parallel increase in lymphocyte-EC adhesion. The effect of IFNβ-1b on AM expression induced by IFN-γ, IL-1β or TNF-α was slightly additive, and was associated with a modest increase in lymphocyte-EC adhesion. In contrast TGF-β, shown previously to downregulate lymphocyte-EC adhesion, inhibited this adhesion in our experiments. It is concluded that IFN-β does not downregulate the inducible expression of ICAM-1, VCAM or E-selectin on HUVEC and does not inhibit the adhesion of lymphocytes to HUVEC. These findings have implications on the mechanism of action of IFNβ-1b in MS.     

Glial pathology in development of cerebellar dysplasia in the hereditary cerebellar vermis defect (CVD) rat

The cerebellar vermis defect (CVD) rat is a new neurological mutant characterized by a cerebellar vermis defect and dysplasia in the cerebellum, especially at the cerebellopontine junctions. In this study, the cytokinetics of glia in terms of the development of cerebellar dysplasia in the CVD rat was investigated using glial fibrillary acidic protein (GFAP) and vimentin immunohistochemistry. In the cerebellar hemispheres, dislocation of the Bergmann glia was observed from postnatal day 5 (P5) in lesions with abnormally aggregated external granule cells (EGCs). Rearranging Bergmann glia were often seen around the EGCs penetrating into the white matter. In the cerebellopontine junctional areas, Bergmann glia were induced after penetration of the Purkinje cells, identified with calbindin immunohistochemistry, and EGCs into the pons from P10. Bergmann fibers were frequently arranged perivascularly. In the clusters of Purkinje cells without EGC settlement in the pons, a small number of Bergmann fibers were observed and their alignment was completely disturbed. These findings suggest that morphological changes in the Bergmann glia depend on their contact with Purkinje cells, but that the orientation of their processes may be influenced by EGC settlement. These glial fibers in the CVD rat may play an important role in the aberrant migration of EGCs, resulting in the development of cerebellar dysplasia. Received: 13 April 1999 / Revised, accepted: 20 July 1999     

Astrocyte-Targeted Expression of Interleukin-3 and Interferon-α Causes Region-Specific Changes in Metallothionein Expression in the Brain

Transgenic mice expressing IL-3 and IFN-α under the regulatory control of the GFAP gene promoter (GFAP-IL3 and GFAP-IFNα mice) exhibit a cytokine-specific, late-onset chronic-progressive neurological disorder which resemble many of the features of human diseases such as multiple sclerosis, Aicardi-Goutières syndrome, and some viral encephalopathies including HIV leukoencephalopathy. In this report we show that the metallothionein-I+II (MT-I+II) isoforms were upregulated in the brain of both GFAP-IL3 and GFAP-IFNα mice in accordance with the site and amount of expression of the cytokines. In the GFAP-IL3 mice, in situ hybridization analysis for MT-I RNA and radioimmunoassay results for MT-I+II protein revealed that a significant upregulation was observed in the cerebellum and medulla plus pons at the two ages studied, 1–3 and 6–10 months. Increased MT-I RNA levels occurred in the Purkinje and granular layers of the cerebellum, as well as in its white matter tracts. In contrast to the cerebellum and brain stem, MT-I+II were downregulated by IL-3 in the hippocampus and the remaining brain in the older mice. In situ hybridization for MT-III RNA revealed a modest increase in the cerebellum, which was confirmed by immunohistochemistry. MT-III immunoreactivity was present in cells that were mainly round or amoeboid monocytes/macrophages and in astrocytes. MT-I+II induction was more generalized in the GFAP-IFNα (GIFN12 and GIFN39 lines) mice, with significant increases in the cerebellum, thalamus, hippocampus, and cortex. In the high expressor line GIFN39, MT-III RNA levels were significantly increased in the cerebellum (Purkinje, granular, and molecular layers), thalamus, and hippocampus (CA2/CA3 and especially lacunosum moleculare layers). Reactive astrocytes, activated rod-like microglia, and macrophages, but not the perivenular infiltrating cells, were identified as the cellular sources of the MT-I+II and MT-III proteins. The pattern of expression of the different MT isoforms in these transgenic mice differed substantially, demonstrating unique effects associated with the expression of each cytokine. The results indicate that the MT expression in the CNS is significantly affected by the cytokine-induced inflammatory response and support a major role of these proteins during CNS injury.     

Mechanisms involved in the blood-brain barrier increased permeability induced by Phoneutria nigriventer spider venom in rats

We have recently demonstrated by electron microscopy, using lanthanum nitrate as an extracellular tracer, that the intravenous injection of Phoneutria nigriventer spider venom (PNV) induces blood-brain barrier (BBB) breakdown in rat hippocampus. One and nine days after PNV injection, tracer was found in pinocytic vesicles crossing the endothelium and in the interendothelial cleft, suggesting that BBB breakdown had occurred through enhanced transendothelial transport and/or tight-junction opening. In the present work, we investigated the mechanisms by which PNV (850 microg/kg, i.v.) increased the hippocampal microvascular permeability in rats 24 h after the endovenous administration. The expression and phosphorylation of some tight- and adherens junctions-associated proteins in hippocampal homogenate and hippocampal microvessel homogenate were assessed by Western blotting and immunoprecipitation. The microtubule-dependent transcellular transport was also evaluated by quantitative ultrastructural methods in pretreated rats with colchicine (0.5 mg/kg, i.p.), prior to PNV injection. Western blots showed no significant increase in the expression of the tight junction-associated proteins ZO-1 and occludin or in the adherens junction-associated beta-catenin after 24 h of PNV administration. Morphological study showed no alterations of the immunolabeling for occludin and ZO-1 in rat brain cryosection following PNV. In addition, no changes were observed in phosphotyrosine content of occludin and beta-catenin in PNV-treated rats compared with control animals. However, the disruption of microtubule-dependent transcellular transport by colchicine completely prevented (p<0.001) PNV-induced leakage of the BBB tracer. These findings indicate that the increased BBB permeability evoked by PNV in rats probably resulted from enhanced microtubule-dependent transendothelial vesicular transport, with no substantial involvement of the paracellular barrier in the time interval studied.     

Effects of diabetes mellitus on astrocyte GFAP and glutamate transporters in the CNS

Diabetes mellitus increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment. The cellular mechanisms responsible for the increased risk of these disorders are incompletely understood. Astrocytes are proving critical for normal CNS function, and alterations in their activity could contribute to diabetes-related disturbances in the brain. We examined the effects of streptozotocin (STZ)-induced diabetes in rats on the level of the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), number of astrocytes, and levels of the astrocyte glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate/aspartate transporter (GLAST), in the cerebral cortex, hippocampus, and cerebellum by Western blotting (WB) and immunohistochemistry (IH). Studies were carried out at 4 and 8 weeks of diabetes duration. Diabetes resulted in a significant decrease in GFAP protein levels (WB) in the hippocampus and cerebellum at 4 weeks and in the cerebral cortex, hippocampus and cerebellum by 8 weeks. Attenuated GFAP immunoreactivity (IH) was evident in the hippocampus, cerebellum and white matter regions such as the corpus callosum and external capsule at both 4 and 8 weeks of diabetes. Astrocyte cell counts of adjacent sections immunoreactive for S-100B were not different between control and diabetic animals. No significant differences were noted in astrocyte glutamate transporter levels in the cerebral cortex, hippocampus, or cerebellum at either time period (WB, IH). With the expanding list of astrocyte functions in the CNS, the role of astrocytes in diabetes-induced CNS disorders clearly warrants further investigation.     

Interleukin-1β System (Ligand, Receptor Type I, Receptor Accessory Protein and Receptor Antagonist), TNF-α, TGF-β1 and Neuropeptide Y mRNAs in Specific Brain Regions During Bacterial LPS-Induced Anorexia

Bacterial lipopolysaccharide (LPS) or endotoxin induces neurological manifestations including anorexia. It is proposed that LPS-induced cytokine production is involved in the generation of neurological manifestations and in neuroinflammatory/immunological responses during Gram-negative infections. For example, LPS-induced effects can be blocked or ameliorated by the interleukin-1 receptor antagonist (IL-1Ra). Here, sensitive and specific RNase protection assays were used to investigate the effects of the intracerebroventricular (i.c.v.) administration of LPS on mRNA levels of interleukin-1β (IL-1β) system components, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and neuropeptide Y (NPY) in the cerebellum, hippocampus, and hypothalamus. The same brain region sample was analyzed with all of the antisense probes. The data show simultaneous local induction of multiple cytokine components messenger ribonucleic acids (mRNAs) within specific brain regions in anorectic rats responding to i.c.v. administered LPS (500 ng/rat). Interleukin-1β and IL-1Ra had a similar mRNA induction profile (hypothalamus > cerebellum > hippocampus). Interleukin-1 receptor type I (IL-1RI) mRNA also increased in all three brain regions examined, and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA was induced in the hypothalamus. Tumor necrosis factor-α mRNA levels increased in the hypothalamus > hippocampus > cerebellum. Levels of membrane bound IL-1R AcP, TGF-β1, and NPY mRNAs did not change significantly in any brain region. The results suggest that: (1) endogenous up-regulation of IL-1β and TNF-α in the hypothalamus contribute to LPS-induced anorexia; and (2) the ratio IL-1Ra/IL-1β, and IL-1β ↔ TNF-α interactions may have implications for Gram-negative infections associated with high levels of LPS in the brain-cerebrospinal fluid.