John A. Olschowka

Neuroinflammation and M2 microglia: the good, the bad, and the inflamed.

The concept of multiple macrophage activation states is not new. However, extending this idea to resident tissue macrophages, like microglia, has gained increased interest in recent years. Unfortunately, the research on peripheral macrophage polarization does not necessarily translate accurately to their central nervous system (CNS) counterparts. Even though pro- and anti-inflammatory cytokines can polarize microglia to distinct activation states, the specific functions of these states is still an area of intense debate. This review examines the multiple possible activation states microglia can be polarized to. This is followed by a detailed description of microglial polarization and the functional relevance of this process in both acute and chronic CNS disease models described in the literature. Particular attention is given to utilizing M2 microglial polarization as a potential therapeutic option in treating diseases.

The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain

Using the indirect immunofluorescent technique, corticotropin releasing factor (CRF)-like immunoreactive nerve fibers and cell bodies were observed to be widely distributed in rat brain. A detailed stereotaxic atlas of CRF-like immunoreactive neurons was prepared. Large numbers of CRF-containing perikarya were observed in the nucleus paraventricularis, with scattered cells in the following nuclei: accumbens, interstitialis stria terminalis, preopticus medialis, supraopticus, periventricularis hypothalami, amygdaloideus centralis, dorsomedialis, substantia grisea centralis, parabrachialis dorsalis and ventralis, tegmenti dorsalis lateralis, vestibularis medialis, tractus solitarius and reticularis lateralis. The most intense staining of CRF-containing fibers was observed in the external lamina of the median eminence. Moderate numbers of CRF-like fibers were observed in the following nuclei: lateralis and medialis septi, tractus diagonalis, interstitialis stria terminalis, preopticus medialis, supraopticus, periventricularis thalami and hypothalami, paraventricularis, anterior ventralis and medialis thalami, rhomboideus, amygdaloideus centralis, habenulae lateralis, dorsomedialis, ventromedialis, substantia grisea centralis, cuneiformis, parabrachialis dorsalis and ventralis, tegmenti dorsalis lateralis, cerebellum, vestibularis medialis, reticularis lateralis, substantia gelatinosa trigemini and lamina I and II of the dorsal horn of the spinal cord. The present findings suggest that a CRF-like peptide may be involved in a neurotransmitter or neuromodulator role, as well as a hypophysiotropic role.

Sustained hippocampal IL-1β overexpression mediates chronic neuroinflammation and ameliorates Alzheimer plaque pathology

Neuroinflammation is a conspicuous feature of Alzheimer disease (AD) pathology and is thought to contribute to the ultimate neurodegeneration that ensues. IL-1 beta has emerged as a prime candidate underlying this response. Here we describe a transgenic mouse model of sustained IL-1 beta overexpression that was capable of driving robust neuroinflammation lasting months after transgene activation. This response was characterized by astrocytic and microglial activation in addition to induction of proinflammatory cytokines. Surprisingly, when triggered in the hippocampus of the APPswe/PS1dE9 mouse model of AD, 4 weeks of IL-1 beta overexpression led to a reduction in amyloid pathology. Congophilic plaque area fraction and frequency as well as insoluble amyloid beta 40 (A beta 40) and A beta 42 decreased significantly. These results demonstrate a possible adaptive role for IL-1 beta-driven neuroinflammation in AD and may help explain recent failures of antiinflammatory therapeutics for this disease.

Inflammatory Responses to Amyloidosis in a Transgenic Mouse Model of Alzheimer’s Disease

Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimers disease (AD). Mice carrying both mutant genes (PS/APP) develop AD-like deposits composed of beta-amyloid (Abeta) at an early age. In this study, we have examined how Abeta deposition is associated with immune responses. Both fibrillar and nonfibrillar Abeta (diffuse) deposits were visible in the frontal cortex by 3 months, and the amyloid load increased dramatically with age. The number of fibrillar Abeta deposits increased up to the oldest age studied (2.5 years old), whereas there were less marked changes in the number of diffuse deposits in mice over 1 year old. Activated microglia and astrocytes increased synchronously with amyloid burden and were, in general, closely associated with deposits. Cyclooxygenase-2, an inflammatory response molecule involved in the prostaglandin pathway, was up-regulated in astrocytes associated with some fibrillar deposits. Complement component 1q, an immune response component, strongly colocalized with fibrillar Abeta, but was also up-regulated in some plaque-associated microglia. These results show: i) an increasing proportion of amyloid is composed of fibrillar Abeta in the aging PS/APP mouse brain; ii) microglia and astrocytes are activated by both fibrillar and diffuse Abeta; and iii) cyclooxygenase-2 and complement component 1q levels increase in response to the formation of fibrillar Abeta in PS/APP mice.

Hypothalamic and Extrahypothalamic Distribution of CRF-Like Immunoreactive Neurons in the Rat Brain

Using an antiserum to synthetic ovine corticotropin-releasing factor (CRF), CRF-like immunoreactive neurons were observed in the central nervous system of normal and colchicine-treated rats. CRF immunoreactivity was observed in the hypothalamus, thalamus, amygdala, cerebral cortex, midbrain, pons medulla, cerebellum and spinal cord. The significance of the extrahypothalamic distribution of CRF-like immunoreactivity is unknown.

Chronic Interleukin-1β Expression in Mouse Brain Leads to Leukocyte Infiltration and Neutrophil-Independent Blood–Brain Barrier Permeability without Overt Neurodegeneration

The proinflammatory cytokine interleukin-1β (IL-1β) plays a significant role in leukocyte recruitment to the CNS. Although acute effects of IL-1β signaling in the mouse brain have been well described, studies elucidating the downstream effects of sustained upregulation have been lacking. Using the recently described IL-1βXAT transgenic mouse model, we triggered sustained unilateral hippocampal overexpression of IL-1β. Transgene induction led to blood–brain barrier leakage, induction of MCP-1 (monocyte chemoattractant protein 1) (CCL2), ICAM-1 (intercellular adhesion molecule 1), and dramatic infiltration of CD45-positive leukocytes comprised of neutrophils, T-cells, macrophages, and dendritic cells. Despite prolonged cellular infiltration of the hippocampus, there was no evidence of neuronal degeneration. Surprisingly, neutrophils were observed in the hippocampal parenchyma as late as 1 year after transgene induction. Their presence was coincident with upregulation of the potent neutrophil chemotactic chemokines KC (keratinocyte-derived chemokine) (CXCL1) and MIP-2 (macrophage inflammatory protein 2) (CXCL2). Knock-out of their sole receptor CXCR2 abrogated neutrophil infiltration but failed to reduce leakage of the blood–brain barrier.

Ultrastructural demonstration of noradrenergic synapses in the rat central nervous system by dopamine-beta-hydroxylase immunocytochemistry.

Noradrenergic (NA) cell bodies and axonal processes were identified in the electron microscope by the immunocytochemical localization of the norepinephrine-synthesizing enzyme, dopamine-beta-hydroxylase (DBH). DBH immunoreactivity, visualized by the peroxidase-antiperoxidase method, was observed in the somata and proximal processes of locus coeruleus neurons and in the distal axons of several NA terminal fields. DBH immunoreactivity is distributed throughout the cytoplasm of the NA neuron, but demonstrates a selective association with endoplasmic reticulum, Golgi apparatus, mitochondrial outer membranes, large granular vesicles, and small, round synaptic vesicles. DBH-positive axonal profiles, typically interspersed between unlabeled dendrites, form two distinct populations: a) thin, unmyelinated intervaricose segments (ca. 0.28 micron) and b) spherical varicosities (ca. 1.00 micron). No DBH-positive varicosities were observed in contact with intracerebral capillaries. In order to determine whether or not NA axons typically form synaptic contacts, a quantitative analysis of selected areas of the diencephalon, cerebellum, and limbic cortex was carried out. More than half (58%) of all DBH-positive varicosities form axodendritic synapses characterized by specialized junctional appositions. The results suggest that NA neurons typically exert their influence on other neurons through highly restricted synaptic contacts.

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalamus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immuno-reactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase of interleukin-1β mRNA and protein in the spinal cord following experimental traumatic injury in the rat

Interleukin-1 beta (IL-1beta) is a major mediator of inflammation and a growth promoter for many cell types that could play an important role in the consequences of traumatic spinal cord injury (SCI). In the present study, the expression of IL-1beta and its mRNA was determined in the rat spinal cord following a standardized contusion injury. IL-1beta mRNA, measured with quantitative RT-PCR, was significantly increased in the lesion site by 1 h after SCI (35.2 +/- 5.9 vs. 9.1 +/- 2.1 pg/mg RNA, n = 3, P < 0.05) and remained significantly higher than in the normal spinal cord for at least 72 h post-injury (p.i.). IL-1beta mRNA levels in tissue immediately caudal to the lesion site did not change after the injury. IL-1beta protein levels, measured by an ELISA, were determined at the lesion site and in cerebrospinal fluid (CSF) and serum samples. IL-1beta levels in the CSF and serum were much lower than in the spinal cord. At the lesion site, IL-1beta was increased significantly by 1 h p.i., peaked at 8 h (32.3 +/- 0.1 vs. 7.6 +/- 1.9, ng/g tissue, n = 5, P < 0.05) and remained significantly higher than normal through at least 7 days p.i. These results suggest that the increased IL-1beta mRNA and protein levels are an early and local response at the lesion site that could trigger other, later, responses to traumatic SCI.

Sustained Interleukin-1β Overexpression Exacerbates Tau Pathology Despite Reduced Amyloid Burden in an Alzheimer's Mouse Model

Neuroinflammation is an important component of Alzheimers disease (AD) pathogenesis and has been implicated in neurodegeneration. Interleukin-1 (IL-1), a potent inflammatory cytokine in the CNS, is chronically upregulated in human AD and believed to serve as part of a vicious inflammatory cycle that drives AD pathology. To further understand the role of IL-1β in AD pathogenesis, we used an inducible model of sustained IL-1β overexpression (IL-1βXAT) developed in our laboratory. The triple transgenic mouse model of AD, which develops plaques and tangles later in its life cycle, was bred with IL-1βXAT mice, and effects of IL-1β overexpression on AD pathology were assessed in F1 progeny. After 1 and 3 months of transgene expression, we found robust increases in tau phosphorylation despite an ∼70–80% reduction in amyloid load and fourfold to sixfold increase in plaque-associated microglia, as well as evidence of greater microglial activation at the site of inflammation. We also found evidence of increased p38 mitogen-activated protein kinase and glycogen synthase kinase-3β activity, which are believed to contribute to tau phosphorylation. Thus, neuroinflammation regulates amyloid and tau pathology in opposing ways, suggesting that it provides a link between amyloid accumulation and changes in tau and raising concerns about the use of immunomodulatory therapies in AD.