Rapid appearance of β-amyloid precursor protein immunoreactivity in glial cells follwing excitotoxic brain injury

Clinical and experimental data have indicated an up-regulation of amyloid precursor protein (APP) after various types of CNS injury. In the present study the cellular source of lesion-induced APP has been investigated an a neurotoxic CNS model. Quinolinic acid injection into the striatum results in neuronal degeneration, while glial cells survive. APP immunoreactivity was detected in glial cells starting at postoperative day 3 and persisted until day 21, the last time point studied. Double immunocytochemistry identified the majority of APP-immunoreactive cells as glial fibrillary acidic protein-immunoreactive astrocytes. There was no evidence of amyloid fibril deposition during this time. It is concluded that following excitotoxic neuronal degneration APP is mainly produced by reactive astrocytes in the lesioned area.     

Selective localization of gelatinase A,an enzyme degrading β-amyloid protein,in white matter microglia and in Schwann cells

Gelatinase A is an enzyme capable of cleaving soluble -amyloid protein (AP), and may function as an -secretase to produce secretory forms of amyloid precursor protein. We examined gelatinase A immunoreactivity in the brains and posterior roots of neurologically normal, lacunar stroke, Alzheimer disease (AD), amyotrophic lateral sclerosis, progressive supranuclear palsy and myasthenia gravis cases. The gelatinase A antibody stained only microglial cells in the white matter in all the brain tissues. In AD brain, the reactive microglia located in the center of classical senile plaques, as well as in other microglial cells in the gray matter, showed no immunoreactivity. Gelatinase A in white matter microglial cells may play a role in preventing local deposition of AP. In the posterior root, Schwann cells had positive immunoreactivity. As with other metalloproteases, gelatinase A in Schwann cells may play an antiproliferative role.     

Fluorescence automatic cell sorter and immunohistochemical investigation of CD68-positive cells in meningioma

Infiltration of brain neoplasms by mononuclear cells including monocytes/macrophages has attracted little attention since they have marked morphological heterogeneity. Twenty-seven meningiomas were studied by anti-CD68 antibody-gated flow cytometry and by immunohistochemical analysis using the anti-CD68 antibodies. Flow cytometric analysis divided cells contained within tumor tissues into CD68-positive and -negative cells. In addition, eight gliomas, eight metastatic brain tumor, and 12 pituitary adenomas were investigated in the same way to compare meningiomas. The mean contents of CD68-positive cells were 24.0±3.7% in meningiomas, 4.4±1.4% in gliomas, 9.5±3.9% in metastatic brain tumors, and 4.5±1.8% in pituitary adenomas. Immunohistochemically, CD68-positive cells showed significant heterogeneity and were detected as round, rod-shaped, ameboid and ramified cells in meningiomas. Although the infiltrated mononuclear cells in gliomas have been investigated to some degree and showed that they express cytokines and/or growth factors, these infiltrated cells in meningioma have barely been studied. The CD68-positive cells detected in this study are likely to be monocytes, macrophages and microglias, and are presumed to be in various functional stages and to play important roles in growth regulation in meningioma.     

Increased senile plaques without microglia in Alzheimer's disease

Summary To clarify the association of microglia with senile plaques, the brains from 13 patients with Alzheimer's disease (AD) and 23 nondemented aged controls were investigated immunohistochemically by a double-labeling method using anti--protein antiserum and anti-ferritin antibody, which is a recently reported microglia marker. In addition, a quantitative analysis was performed. The senile plaques which appeared initially in the nondemented aged controls consisted of a diffuse type without any amyloid cores and these were found in the group aged 50–59 years. The great majority of them were found to contain no ferritin-positive microglia. The number and proportion (percentage) of microglia-containing diffuse plaques increased with age. Classical and compact plaques began to appear in the brains of the group aged 70 years and over, and practically all of them contained microglia. These results suggest that microglia are not associated with initial plaque formation, but correlate with amyloid core formation. In AD, the most prominent feature was that the diffuse plaques, which contained either no or only a few ferritin-positive microglia, increased markedly.Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Extracellular acidification decreases the basal motility of cultured mouse microglia via the rearrangement of the actin cytoskeleton

The present study was undertaken to examine the effect of extracellular pH (pH(0)) on the locomotor function of murine microglial cells in vitro. We have found that basal motility of microglia, as measured by a computer-assisted video assay, decreased in an acidic, but not in an alkaline environment. Extracellular acidification affected the architecture of F-actin cytoskeleton, inducing bundling of actin and the formation of stress fibers. The change in intracellular pH (pH(i)) resulting from the change in pH(0) seems to be a prerequisite for the motility decrease since other means to decrease pH(i), namely Na(+)-free solution (in the absence of HCO(-)(3)) and nigericin-containing solution, mimicked the extracellular acidification. In contrast to its pronounced effect on basal motility of microglial cells, the motility increase, as induced by the chemoattractant complement 5a (C5a), was not affected by the acidic environment. The relationship of pH(0) to the locomotor function was also studied in a long-term microchemotaxis assay where microglia migrated within a pH gradient. Intracellular acidification induced by lowering pH(0) to 6.0 or removal of Na(+) from the assay medium decreased basal microglial cell migration. The C5a-induced chemotactic migration was moderately decreased by the acidic environment. In conclusion, our results suggest that acidification of the microglial extracellular milieu leads to a decrease in pH(i) and thereby reduces the basal microglial motility and C5a-induced chemotaxis via a rearrangement of the cytoskeleton. We would therefore like to speculate that changes in pH(i) constitute an important control mechanism in regulating the locomotor function of microglia in culture and probably also in the intact tissue.     

Activation of mouse microglial cells affects P2 receptor signaling

Microglial cells are the immunocompetent cells of the CNS, which are known to exist in several activation states. Here we investigated the impact of microglial activation on the P2 receptor-mediated intracellular calcium ([Ca(2+)](i)) signaling by means of fluo-3 based Ca(2+)-imaging. Cultured mouse microglial cells were treated with either astrocyte-conditioned medium to induce a ramified morphology or LPS to shift the cells toward the fully activated stage. The extracellular application of ATP (100 microM) induced a [Ca(2+)](i) elevation in 85% of both untreated and ramified microglial cells, whereas only 50% of the LPS-activated cells responded to the stimulus. To characterise the pharmacological profile of microglial P2 receptors we investigated the effects of various P2 agonists on [Ca(2+)](i) in cultured microglial cells. Untreated and ramified microglial cells demonstrated a very similar sensitivity to the different P2 agonists. In contrast, in LPS-activated microglia, a sharp decrease of responses to P2 agonist stimulation was seen. This indicates that microglial activation influences the capability of microglial cells to generate [Ca(2+)](i) signals upon P2 receptor activation.     

Mononuclear cell proliferation and hyperplasia during Wallerian degeneration in the visual system of the goldfish in the presence or absence of regenerating optic axons

Patterns of proliferation and changes in non-neuronal cell number in the visual system of the goldfish have been quantitatively examined during optic axon regeneration after an optic nerve crush (ONC). In addition, in order to examine the effect of the regenerating axons on cellular responses in the visual pathways, we did a similar analysis of animals with the right eye removed (ER). Finally, we used double labeling protocols to demonstrate that the proliferating cells that we were counting were mostly phagocytic cells of the mononuclear lineage. In animals with an ONC, we observed an early burst of proliferation that peaked between 7 and 14 days after surgery in all parts of the visual system. In the optic tract, there was also a secondary rise that peaked at 21 days. Levels of proliferation returned to normal by 32 days postoperative in the tract and tectum, while they remained somewhat elevated in the optic nerve for at least 93 days. The total number of non-neuronal cells in the visual paths also rose to peak values between 7 and 14 days after ONC surgery. In the optic tract and tectum, the values fell rapidly after this time, while in the optic nerve, there was a secondary peak at 32 days after which values remained elevated for the duration of the experiment. As compared to animals with an ONC, enucleation resulted in elevated proliferation and hyperplasia at early postoperative intervals. However, because these differences occurred when axons had not yet regenerated into the affected structures, these data do not provide strong evidence for a direct effect of regenerating optic axons on the early cellular responses during Wallerian degeneration in the goldfish. In addition, in the tectum, there was an early increment in cell number that was not associated with elevated levels of proliferation. We believe that this increment represents immigration of resident microglia from other regions of the brain.     

A novel effect of an opioid receptor antagonist, naloxone, on the production of reactive oxygen species by microglia: a study by electron paramagnetic resonance spectroscopy

Microglia as the first line of defensive cells in the brain produce free radicals including superoxide and nitric oxide (NO), contributing to neurodegeneration. An opioid receptor antagonist, naloxone, has been considered pharmacologically beneficial to endotoxin shock, experimental cerebral ischemia, and spinal cord injury. However, the mechanisms underlying these beneficial effects of naloxone are still not clear. This study explores the effects of naloxone on the production of superoxide and NO by the murine microglial cell line, BV2, stimulated with lipopolysaccharide (LPS) as measured by electron paramagnetic resonance (EPR). The production of superoxide triggered by phobol-12-myristate-13-acetate (PMA) resulted in superoxide dismutase (SOD)-inhibitable, catalase-uninhibitable 5,5-dimethyl-1-pyrroline N-oxide (DMPO) hydroxyl radical adduct formation. LPS enhanced the production of superoxide and triggered the formation of non-heme iron-nitrosyl complex. Cells pre-treated with naloxone showed significant reduction of superoxide production by 35%. However, it could not significantly reduce the formation of non-heme iron-nitrosyl complex and nitrite. Taken together, the results expand our understanding of the neuroprotective effects of naloxone as it decreases superoxide production by microglia.     

Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat

HSP70 and heme oxygenase-1 (HO-1) are thought to be markers of cell injury and oxidative stress, respectively. We have immunolocalized these proteins in the spinal cord at 1-14 days after contusion. HSP70 and HO-1 were co-induced in glia and macrophages within the injured segment at all time points. This co-induction may reflect complementary functions that serve to protect these cells as they respond to the postcontusional environment.     

Microglial dysfunction in brain aging and Alzheimer's disease

Microglia, the immune cells of the central nervous system, have long been a subject of study in the Alzheimer's disease (AD) field due to their dramatic responses to the pathophysiology of the disease. With several large-scale genetic studies in the past year implicating microglial molecules in AD, the potential significance of these cells has become more prominent than ever before. As a disease that is tightly linked to aging, it is perhaps not entirely surprising that microglia of the AD brain share some phenotypes with aging microglia. Yet the relative impacts of both conditions on microglia are less frequently considered in concert. Furthermore, microglial “activation” and “neuroinflammation” are commonly analyzed in studies of neurodegeneration but are somewhat ill-defined concepts that in fact encompass multiple cellular processes. In this review, we have enumerated six distinct functions of microglia and discuss the specific effects of both aging and AD. By calling attention to the commonalities of these two states, we hope to inspire new approaches for dissecting microglial mechanisms.