Emmprin (CD147), a cell surface regulator of matrix metalloproteinase production and function.
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Long-term intrathecal catheterization in the rat.
We report an intrathecal (i.t.) catheter system that permits repeated administration of volumes of 10 microl or more in the awake rat over many months. A small skin incision is made and a 32 ga polyurethane catheter is inserted in the sacral subarachnoid space using a modified 22 ga needle. The other end of the catheter is tunneled subcutaneously to the flank and exteriorized through a titanium port. The device is well tolerated, does not cause sensory or motor deficits, and does not interfere with behavioral testing. Rats equipped with this device can be housed with other rats. Over the 9 month observation period the function of the catheter was verified by repeated injection of 15 microl of 2% lidocaine that caused temporary paraplegia. Out of 12 implanted rats, the number of fully functional catheters was 10 at 3 months, seven at 6 months, and six at 9 months. At 3 months, i.t. injection of anti-dopamine-beta-hydroxylase antibodies conjugated to saporin (DBH-SAP, 5 microg/10 microl) resulted in noradrenergic denervation of the spinal cord in all rats (n=10). We propose that intrathecal catheterization is well suited for long term behavioral and pharmacological studies.
Animals↗
Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population.
Cells derived from the central nervous system (CNS) are usually characterised by manual counting on slides after specific immunolabelling. In this study, we investigated the possibility of using flow cytometry to determine the proportion of neurons, astrocytes or microglial cells in primary cultures. We show that parameters other than physical features are necessary to discriminate between these different cell types because of some overlap in their size and granulosity. We then used specific antibodies against intracellular markers such as Tuj-1 or GFAP to discriminate neurons from astrocytes by flow cytometry. The labelling was specific and reliable, allowing quantitative studies. Indeed, we did not find any significant difference in the number of Tuj-1 and GFAP-positive cells in primary cultures of neuronal and glial cells as determined by manual counting on slides or flow cytometry. More importantly, similar data were obtained in mixed populations, indicating that flow cytometry can be used for quantitative studies of heterogeneous cultures. The flow cytometry therefore appears to be a reliable method for the phenotypic characterisation of CNS-derived cells. This technique which enables a rapid analysis of numerous samples, might be particularly interesting for the study of neural stem cell differentiation.
Animals↗
Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein.
DNA immunization, in theory, is of great interest as a source of specific antibodies against different antigens. In an attempt to produce polyclonal and monoclonal antibodies against cell surface molecules by using the DNA immunization strategy, intramuscular and intrasplenic routes of DNA injection were compared. Two to five, but not a single, intramuscular DNA immunizations induced anti-CD54 and anti-CD147 antibody production. In contrast, a single intrasplenic immunization of CD54-encoding DNA could induce anti-CD54 antibody production. To produce monoclonal antibody (mAb), spleen cells obtained from an intrasplenic CD54-encoding DNA immunized mouse were fused with myeloma cells using the standard hybridoma technique. A hybridoma secreting specific mAb to CD54 was established. The generated mAb reacted to CD54 protein expressed on transfected COS cells and various cell types, the same as using standard CD54 mAb MEM-111. Our results demonstrated that direct immunization of antigen-encoding DNA into spleen is an effective route for production of both polyclonal and monoclonal antibodies to cell surface molecules. This finding is very useful for the production of antibodies to cell surface molecules where the protein antigen is not available or difficult to prepare, but cDNA encoding the corresponding protein is available.
Animals↗
Maturation of the blood-retina barrier in the developing pecten oculi of the chicken.
The major interest in the development of the blood-brain barrier and its underlying induction mechanisms is given by the crucial role they play in the maturation of the central nervous system in general. Whilst it is believed that it is the microenvironment in the brain that destines the endothelial cells to become committed to barrier properties, the analysis of the multitude of factors probably responsible for this commitment is extremely difficult. Therefore, in a previous study, we inaugurated the pecten oculi of the avian eye as a relatively simple in vivo model of the blood-brain barrier [Gerhardt, S. et al, Cell Tissue Res., 285 (1996) 91-100]. In the present study, we demonstrate data on the development of the pecten which allow us to understand better the commitment of barrier properties in endothelial cells in an environment which is considerably less complex than that realized in the brain. The pecten is built up by mainly two cell types, the pigmented glial cells and the endothelial cells. The pigmented cells, which are believed to originate from the retinal pigment epithelium, lose their tight junctions in the microenvironment of the vitreous body, whereas the endothelial cells, which originate from the permeable choroidal vessels, gain tight junctions and other barrier properties in the microenvironment of the vitreous body. On embryonic day 7 (E7), tight and gap junctions between epithelial-like glial cells line the vitreal border of the developing pecten. By E16, these junctions disappear, and the endothelial cells gradually acquire barrier characteristics (continuous and P-face associated tight junctions, no extravasation of lanthanum nitrate, and the exclusive expression of the glucose transporter isoform 1 and the barrier specific antigen HT7 in their luminal and abluminal membranes). The results are discussed considering the switch from an epithelial (glial) to an endothelial barrier.
Animals↗
Interferon beta-1a prevents the effects of lipopolysaccharide on embryonic brain microvessels.
By means of light and electron microscopy we have studied the effect of interferon beta-1a (IFNbeta-1a) in the optic tecta of 20-day-old chick embryos under normal conditions and after exposure to lipopolysaccharide (LPS) which mimics the blood-brain barrier (BBB) disruption in meningoencephalitis. Optic tecta were examined for: (i) ultrastructure by means of transmission electron microscopy; (ii) the immunohistochemical localization of HT7 antigen, a specific marker of differentiation of the brain microvessels; (iii) the brain microvessel permeability, by means of horseradish peroxidase (HRP) tracer; (iv) the expression of microvessel glycoconjugates, by means of lectin histochemistry, using Ricinus communis agglutinin-I (RCA-I), specific for beta-D-galactosyl moieties and Wheat Germ agglutinin (WGA) specific for sialyl and N-acetylglucosaminyl moieties. A morphometric evaluation of brain microvessel permeability and of glycoconjugate expression was also performed. In control- and in IFNbeta-1a-treated embryos, HRP was confined to the vessel lumina which were sealed by the interendothelial tight junctions. RCA-I binding sites were recognizable both in the basal membranes and in the tight junctions, while WGA sites were present on the luminal side of the endothelial cells. HRP was blocked in the vessels lumina by the interendothelial tight junctions. After LPS treatment, HRP showed an extravascular localization and the labeling of microvessels by anti-HT7 antibodies disappeared. RCA-I binding was only found ultrastructurally and appeared as irregularly clustered gold particles, in the cleft of damaged tight junctions, but were no longer detectable in the endothelial basement membranes. After pretreatment of LPS-treated embryos with IFNbeta-1a, the vessel permeability to HRP strongly decreased and the vessels showed the normal pattern of HT7 protein and of the RCA-I binding sites. These results indicate that the changes induced by LPS in the endothelial cells are prevented by IFNbeta-1a.
Animals↗
Calpain expression and infiltration of activated T cells in experimental allergic encephalomyelitis over time: increased calpain activity begins with onset of disease.
Calpain activity and expression at the protein level were examined in inflammatory cells, activated microglia, and astrocytes prior to or at onset of symptomatic experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). EAE was induced in Lewis rats by injection of guinea pig spinal cord homogenate and myelin basic protein (MBP) emulsified with Complete Freund's Adjuvant (CFA). Calpain translational expression, determined by Western blot and immunocytochemistry, was correlated with calpain activity, infiltration of inflammatory cells, and myelin loss at 2-11 days following challenge with antigen. Controls (CFA only) did not show any changes over time in these parameters and very few changes (CD11+ microglia/mononuclear phagocytes) were seen in either group from days 2 to 8 post-induction. In contrast, from days 9 to 11, the animals that developed the disease (at least grade 1) demonstrated extensive cellular infiltration (CD4+, CD25+, and CD11+ as well as increased calpain expression (content) and activity. This study demonstrates that cell infiltration and increased calpain activity do not begin in the CNS until the onset of clinical signs.
Animals↗
Characterization of an in vitro rhesus macaque blood-brain barrier.
The blood-brain barrier (BBB) has been modeled in vitro in a number of species, including rat, cow and human. Coculture of multiple cell types is required for the correct expression of tight junction proteins by microvascular brain endothelial cells (MBEC). Markers of inflammation, especially MHC-II, and cell adhesion molecules, such as VCAM-1, are not expressed on the luminal surface of the barrier under resting conditions. The rhesus macaque model has been used to study early events of HIV-neuropathogenesis in vivo, but a suitable in vitro model has not been available for detailed mechanistic studies. Here we describe an in vitro rhesus macaque blood-brain barrier that utilizes autologous MBEC and astrocytes. We believe that this model is highly relevant for examining immunological events at the blood-brain barrier and demonstrate its potential usefulness for examining early events in AIDS neuropathogenesis.
Acquired Immunodeficiency Syndrome↗
Mechanisms of recovery from experimental allergic encephalomyelitis induced with myelin basic protein peptide 68-86 in Lewis rats: a role for dendritic cells in inducing apoptosis of CD4+ T cells.
Spontaneous remission of experimental allergic encephalomyelitis (EAE) is usually associated with prominent apoptosis. The mechanisms behind apoptosis are unknown. We examined the functions of dendritic cells (DC) from Lewis rats with EAE induced by immunization with myelin basic protein peptide 68-86 (MBP68 - - 86). Recovery from EAE was associated with three major functional changes of freshly prepared DC: (1) elevated proliferation, (2) increased nitric oxide (NO) production, and (3) augmented IFN-gamma secretion. In Freund's complete adjuvant (FCA)-immunized control rats, no increase of proliferation, NO production or IFN-gamma secretion was observed on day 21 post-immunization (p.i.), i.e., recovery from EAE. In vitro effects of IFN-gamma, TNF-alpha, TGF-beta1, IL-4 and IL-10 on DC were examined. IFN-gamma enhanced proliferation and NO production by DC, while TNF-alpha and IL-4 induced only slight DC proliferation. DC from recovering EAE rats (day 21 p.i.) suppressed MBP68 - - 86-induced T cell proliferation compared to DC obtained at other time points in EAE and FCA-immunized rats. DC-derived NO induced apoptosis of CD4+ T cells, thereby inhibiting autoreactive T cell responses. Besides IFN-gamma stimulation, NO production by DC was mainly induced in an antigen-dependent manner when DC were co-cultured with T cells. The results suggest that spontaneous recovery from EAE is associated with augmented DC functions. Overproduction of NO by DC results in apoptosis of autoreactive CD4+ T cells, thereby decreasing autoreactive T cell reactivities. The existence of such a NO negative feedback loop may contribute to remission of EAE.
Animals↗
Delayed neuronal damage related to microglia proliferation after mild spinal cord compression injury.
In order to investigate the mechanism of delayed progressive or secondary neuronal damage after the spinal cord injury, we developed a mild-compression injury model in the rat thoracic spinal cord. Our compression device consists of a soft silicone point of contact to the dura, in order to prevent violent injury that may cause axonal tears or hemorrhages in the spinal cord. Since rats often assume a 'standing' posture, i.e. raising head with lifting their fore-limbs, damage to the thoracic spinal cord was evaluated by measuring the frequency of 'standing', which effectively indicates hind limb function. Twenty-four hours after compression by a 20 g weight for 10 or 20 min, the standing frequency of the injured rat was almost the same as that of sham animals that underwent laminectomy without compression. However, the standing frequency decreased with time; the frequency of standing at 72 h was approximately 30-50% that of sham animals. In the compressed spinal cord tissue, microglial cells, detected by lectin staining, proliferated with time. An enormous amount of microglia was observed at 48 and 72 h after compression, although only a small amount of cells were positive to lectin staining at 24 h after the compression. These results suggest that our mild-compression spinal cord injury model showed late-onset or delayed neuronal damage that may be related to pathological microglia proliferation.
Animals↗
Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats.
Recent studies have implicated chronic elevated exposures to environmental agents, such as metals (e.g. manganese, Mn) and pesticides, as contributors to neurological disease. Eighteen-month-old rats received intraperitoneal injections of manganese chloride (6 mg Mn/kg/day) or equal volume of saline for 30 days in order to study the effect of manganese on the dopamine- and GABA-neurons. The structures studied were substantia nigra, striatum, ventral tegmental area, nucleus accumbens and globus pallidus. First, we studied the enzymatic activity of mitochondrial complex II succinate dehydrogenase (SDH). We found an overall decrease of SDH in the different brain areas analyzed. We then studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra and ventral tegmental area following Mn treatment. Correspondingly, TH immunoreactivity was increased in substantia nigra and ventral tegmental area. Manganese treatment significantly decreased GAD mRNA levels in individual GABAergic neurons in globus pallidus but not in striatum. We also quantified the density of glial fibrillary acidic protein (GFAP)-labeled astrocytes and OX-42 positive cells. Reactive gliosis in response to Mn treatment occurred only in striatum and substantia nigra and the morphology of the astrocytes was different than in control animals. These results suggest that the nigrostriatal system could be specifically damaged by manganese toxicity. Thus, changes produced by manganese treatment on 18-month-old rats could play a role in the etiology of Parkinson's disease.
Aging↗
Engagement of CD147 molecule-induced cell aggregation through the activation of protein kinases and reorganization of the cytoskeleton.
CD147 is a broadly expressed cell surface molecule of the immunoglobulin superfamily whose expression is up-regulated upon T cell activation. Engagement of CD147 by CD147 monoclonal antibodies (mAbs) has been shown to induce homotypic aggregation of U937 cells. To study intracellular signal transduction induced by the engagement of CD147 molecules, protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitors were used to inhibit cell aggregation. The results indicated that a PKC inhibitor, sphingosine, and a PTK inhibitor, herbimycin A, inhibited CD147 mAb-induced cell aggregation in a dose-dependent manner. In contrast to herbimycin A, a PTK inhibitor, genistein, enhanced cell aggregation. This discrepancy may be due to the differential effect of herbimycin A and genistein on the target cells. Effect of actin filament polymerization blocking agent, cytochalasin B, was also studied and it was found that cytochalasin B completely inhibited CD147 mAb-induced cell aggregation. This result implied that U937 cell aggregation induced by CD147 mAbs is associated with cytoskeleton reorganization. Thus, our observations suggest that cell aggregation induced by the engagement of CD147 with specific mAbs depend upon the activation of protein kinases and a functional cytoskeleton.
Antibodies, Monoclonal↗
Microglial proliferation in the spinal cord of aged rats with a sciatic nerve injury.
Nerve injury may lead to chronic neuropathic pain syndromes. We determined whether the extent of central nervous system microglial activation that accompanies nerve injury is age dependent and correlated with behavioral manifestations of pain. We used the Bennett and Xie sciatic nerve chronic constriction injury model (Bennett, G.J., Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1998) 87-107) to induce neuropathic pain in three age cohorts of Fischer 344 FBNF1 hybrid rats (4-6, 14-16, and 24-26 months). Rats were assessed for thermal sensitivity (hyperalgesia) of their hind paws pre-injury (day 0) and up to 35 days post injury. On various days post injury, the L4-L5 levels of their spinal cords were reacted for localization of an antibody to OX-42, a marker for microlgia. OX-42 immunoreactivity (ir) was quantified by use of a Bioquant density analysis system. OX-42 ir was heavy in areas of sciatic nerve primary afferent terminations and in the motor columns of its neurons. Aging increases OX-42 ir in the absence of injury. After injury, OX-42 ir increased further, but the increases over control levels decreased with age. Ligation-induced analgesia and hyperalgesia were both correlated with the increases in OX-42 ir, regardless of age.
Aging↗
In vitro characterization of Mato's FGP cells isolated from rat cerebrum.
Mato's FGP cells surrounding cerebral arterioles play a significant role in the maintenance of a homeostatic microenvironment in the brain. In this study, the perivascular cells were isolated from rat cerebral microvessels and cultured in vitro to characterize their phenotype. Autofluorescence of the intracellular granules in cultured cells and the uptake of HRP and DiI-Ac-LDL by these cells were observed. The cells reacted positively to an anti-scavenger receptor A antibody. Positive immunoreactions of cultured cells to ED1 and ED2 antibodies were observed, whereas they were weak or negative to ED3 and OX42 antibodies. Acid phosphatase activity was detected in the granules of cultured cells. In conclusion, the cells cultivated under the present conditions revealed very similar characteristics to Mato's FGP cells in situ and therefore are useful for studies on FGP cells.
Acid Phosphatase↗
Upregulation of adrenocorticotrophic hormone in the corticotrophs and downregulation of surface receptors and antigens on the macrophages in the adenohypophysis following an exposure to high altitude.
Altitude exposures lead to the development of hypobaric hypoxia because of low oxygen tension in the ambient air. This study has shown the vigorous upregulation of adrenocorticotrophic hormone (ACTH) expression in corticotrophs of the pars distalis (adenohypophysis) of rats 1-7 days after an altitude exposure. Concomitant to this was the increase in number and hypertrophy of the immunoreactive corticotrophs. It was suggested that this had resulted in an upsurge of ACTH production which may have suppressed the immuno-expression of complement type 3 receptors and major histocompatibility complex class II antigens constitutively expressed by the parenchymal macrophages through paracrine action. Along with ACTH, altered levels of other hormones following such exposures may also contribute to suppression of antigen presenting function and phagocytic activity of macrophages. The effects of altitude (hypobaric hypoxia) exposure, however, were reversible as the above immunohistochemical changes returned to normal 21-28 days after the hypobaric hypoxic insult.
Adrenocorticotropic Hormone↗
Granulocyte macrophage-colony stimulating factor activates microglia in rat cortex organotypic brain slices.
Cytokines play an important role in the regulation of proliferation and migration in the central nervous system. The aim of this study was to determine if granulocyte macrophage-colony stimulating factor (GM-CSF) activates cells in the cortex of organotypic brain slice cultures. Our data show that murine GM-CSF markedly stimulated the proliferation and migration of small round microglia from a cortex slice. These round cells were strongly positive for integrin CD11b (OX-42), isolectin B4-lectin-binding, the monocytic marker ED1 and partly expressed major histocompatibility complex (MHC) class II antigen (OX-6). Only some differentiated microglia were visible which expressed the integrin CD11c and MHCII. GM-CSF enhanced the proliferation as analyzed by bromodeoxyuridine incorporation. The number of migrated cells decreased during culturing and enhanced terminal dUTP nick-end labelling positive nuclei were found. Taken together, our data conclude that GM-CSF is an important cytokine, which regulates the proliferation and migration of cortical microglia.
Animals↗
Selective microglial activation in the rat rotenone model of Parkinson's disease.
Chronic rotenone exposure reproduces features of Parkinson's disease (PD) (Nat. Neurosci. 3 (2000) 1301; Exp. Neurol. 179 (2003) 9). We investigated the role of glial activation in rotenone toxicity in vivo. Male Lewis rats received 2-3 mg/kg rotenone per day for up to 4 weeks. In 50% of surviving rotenone-treated animals, there was nigrostriatal dopaminergic degeneration, marked by reduced tyrosine hydroxylase immunoreactivity). Extensive microglial activation, determined by OX-42-ir, occurred in striatum and nigra of rotenone-treated animals, and was prominent before anatomical evidence of dopaminergic lesions. Microglia enlarged and developed short, stubby processes in rotenone-treated animals. Rotenone-induced microglial activation was less pronounced in cortex. Reactive astrocytosis was minimal and limited to a thin rim around the lesion. Marked microglial activation with minimal astrocytosis is another pathological feature of PD reproduced by rotenone treatment.
Animals↗
Microglial activation and tyrosine hydroxylase immunoreactivity in the substantia nigral region following transient focal ischemia in rats.
The temporal profiles of the changes of dopaminergic cells and microglial activation induced by transient cerebral ischemia were investigated in the substantia nigra pars compacta (SNc) located outside ischemic areas of rat brain. Transient cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion was continued for 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days. Dopaminergic cells immunostained with tyrosine hydroxylase (TH)-antibody in the ipsilateral SNc were significantly decreased at 7 days post-ischemia compared with those in the contralateral side (P<0.05). However, at 60 and 120 days, there were no significant differences between ipsilateral and contralateral side of the SNc. Unlike the TH immunoreactivity, activated microglial cells immunostained with OX-42 antibody were significantly increased at 2 and 3 days and then decreased gradually until 10 days post-ischemia. Activated microglial cells were increased at 2 weeks post-ischemia, and this pattern remained until 60 days. These results suggest that the transient changes of TH-immunoreactive cells in the SNc caused by transient focal ischemia are correlated with a biphasic microglial cell activation response.
Animals↗