Rehana K. Leak

Microglia/Macrophage Polarization Dynamics Reveal Novel Mechanism of Injury Expansion After Focal Cerebral Ischemia

Background and Purpose— Mononuclear phagocytes are highly plastic cells that assume diverse phenotypes in response to microenvironmental signals. The phenotype-specific roles of microglia/macrophages in ischemic brain injury are poorly understood. A comprehensive characterization of microglia/macrophage polarization after ischemia may advance our knowledge of poststroke damage/recovery. Methods— Focal transient cerebral ischemia was induced in mice for 60 minutes; animals were euthanized at 1 to 14 days of reperfusion. Reverse-transcriptase polymerase chain reaction and immunohistochemical staining for M1 and M2 markers were performed to characterize phenotypic changes in brain cells, including microglia and infiltrating macrophages. In vitro experiments using a transwell system, a conditioned medium transfer system, or a coculture system allowing cell-to-cell contacts were used to further elucidate the effect of neuronal ischemia on microglia/macrophage polarization and, conversely, the effect of microglia/macrophage phenotype on the fate of ischemic neurons. Results— Local microglia and newly recruited macrophages assume the M2 phenotype at early stages of ischemic stroke but gradually transformed into the M1 phenotype in peri-infarct regions. In vitro experiments revealed that ischemic neurons prime microglial polarization toward M1 phenotype. M1-polarized microglia or M1-conditioned media exacerbated oxygen glucose deprivation–induced neuronal death. In contrast, maintaining the M2 phenotype of microglia protected neurons against oxygen glucose deprivation. Conclusions— Our results suggest that microglia/macrophages respond dynamically to ischemic injury, experiencing an early “healthy” M2 phenotype, followed by a transition to a “sick” M1 phenotype. These dual and opposing roles of microglia/macrophages suggest that stroke therapies should be shifted from simply suppressing microglia/macrophage toward adjusting the balance between beneficial and detrimental microglia/macrophage responses.

Suprachiasmatic nucleus organization

Abstract. The suprachiasmatic nucleus (SCN) of the hypothalamus is a dominant circadian pacemaker in the mammalian brain controlling the rest-activity cycle and a series of physiological and endocrine functions to provide a foundation for the successful elaboration of adaptive sleep and waking behavior. The SCN is anatomically and functionally organized into two subdivisions: (1) a core that lies adjacent to the optic chiasm, comprises predominantly neurons producing vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP) colocalized with GABA and receives dense visual and midbrain raphe afferents, and (2) a shell that surrounds the core, contains a large population of arginine vasopressin (AVP)-producing neurons in its dorsomedial portion, and a smaller population of calretinin (CAR)-producing neurons dorsally and laterally, colocalized with GABA, and receives input from non-visual cortical and subcortical regions. In this paper, we present a detailed quantitative analysis of the organization of the SCN core and shell in the rat and place this in the context of the functional significance of the subdivisions in the circadian control of regulatory systems.

Microglial and macrophage polarization—new prospects for brain repair

The traditional view of the adult brain as a static organ has changed in the past three decades, with the emergence of evidence that it remains plastic and has some regenerative capacity after injury. In the injured brain, microglia and macrophages clear cellular debris and orchestrate neuronal restorative processes. However, activation of these cells can also hinder CNS repair and expand tissue damage. Polarization of macrophage populations toward different phenotypes at different stages of injury might account for this dual role. This Perspectives article highlights the specific roles of polarized microglial and macrophage populations in CNS repair after acute injury, and argues that therapeutic approaches targeting cerebral inflammation should shift from broad suppression of microglia and macrophages towards subtle adjustment of the balance between their phenotypes. Breakthroughs in the identification of regulatory molecules that control these phenotypic shifts could ultimately accelerate research towards curing brain disorders.

Topographic organization of suprachiasmatic nucleus projection neurons

The mammalian circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), has two subdivisions. The core is located above the optic chiasm, receives primary and secondary visual afferents, and contains neurons producing vasoactive intestinal polypeptide and gastrin‐releasing peptide. The shell largely surrounds the core, receives input from nonvisual sources and contains neurons producing arginine vasopressin and calretinin. In this study, we tested the hypothesis that SCN efferent projections are topographically organized with respect to the subdivision of origin. Injections of retrograde tracers were placed in major sites of efferent termination, described from prior studies that used anterograde tracers (Watts and Swanson, [1987] J. Comp. Neurol. 258:230–252; Watts et al. [1987] J. Comp. Neurol. 258:204–229). After retrograde tracer injections in the medial preoptic area, dorsomedial and paraventricular hypothalamic nuclei, bed nucleus of stria terminalis, paraventricular thalamic nucleus, zona incerta, and medial subparaventricular zone, retrogradely labeled SCN cells are clustered in the shell with few labeled neurons in the core. After injections centered in the lateral subparaventricular zone, peri‐suprachiasmatic region, lateral septum, or ventral tuberal area, the majority of neuronal label is in the core with moderate to sparse neuronal label in the shell. Both subdivisions are labeled after injections in the paratenial thalamic nucleus. The same pattern of retrograde labeling is found with four tracers, cholera toxin‐β subunit, Fluoro‐Gold, the Bartha strain of pseudorabies virus, and biotinylated dextran amine. These data extend our understanding of the significance of the division of the SCN into shell and core by demonstrating that the subdivisions differ in the pattern of projections. Together with prior observations that the subdivisions differ with respect to afferents, local connections, and neuroactive substances, the present study provides an anatomic basis for discrete control of circadian function by the SCN core and shell. In this novel view, the nature of the signal conveyed to areas receiving core or shell projections varies as a function of the subdivision from which innervation is derived. J. Comp. Neurol. 433:312–334, 2001.

Emerging Roles of Nrf2 and Phase II Antioxidant Enzymes in Neuroprotection

Phase II metabolic enzymes are a battery of critical proteins that detoxify xenobiotics by increasing their hydrophilicity and enhancing their disposal. These enzymes have long been studied for their preventative and protective effects against mutagens and carcinogens and for their regulation via the Keap1 (Kelch-like ECH associated protein 1)/Nrf2 (Nuclear factor erythroid 2 related factor 2)/ARE (antioxidant response elements) pathway. Recently, a series of studies have reported the altered expression of phase II genes in postmortem tissue of patients with various neurological diseases. These observations hint at a role for phase II enzymes in the evolution of such conditions. Furthermore, promising findings reveal that overexpression of phase II genes, either by genetic or chemical approaches, confers neuroprotection in vitro and in vivo. Therefore, there is a need to summarize the current literature on phase II genes in the central nervous system (CNS). This should help guide future studies on phase II genes as therapeutic targets in neurological diseases. In this review, we first briefly introduce the concept of phase I, II and III enzymes, with a special focus on phase II enzymes. We then discuss their expression regulation, their inducers and executors. Following this background, we expand our discussion to the neuroprotective effects of phase II enzymes and the potential application of Nrf2 inducers to the treatment of neurological diseases.

Microglia/macrophage polarization dynamics in white matter after traumatic brain injury

Mononuclear phagocytes are a population of multi-phenotypic cells and have dual roles in brain destruction/reconstruction. The phenotype-specific roles of microglia/macrophages in traumatic brain injury (TBI) are, however, poorly characterized. In the present study, TBI was induced in mice by a controlled cortical impact (CCI) and animals were killed at 1 to 14 days post injury. Real-time polymerase chain reaction (RT–PCR) and immunofluorescence staining for M1 and M2 markers were performed to characterize phenotypic changes of microglia/macrophages in both gray and white matter. We found that the number of M1-like phagocytes increased in cortex, striatum and corpus callosum (CC) during the first week and remained elevated until at least 14 days after TBI. In contrast, M2-like microglia/macrophages peaked at 5 days, but decreased rapidly thereafter. Notably, the severity of white matter injury (WMI), manifested by immunohistochemical staining for neurofilament SMI-32, was strongly correlated with the number of M1-like phagocytes. In vitro experiments using a conditioned medium transfer system confirmed that M1 microglia-conditioned media exacerbated oxygen glucose deprivation–induced oligodendrocyte death. Our results indicate that microglia/macrophages respond dynamically to TBI, experiencing a transient M2 phenotype followed by a shift to the M1 phenotype. The M1 phenotypic shift may propel WMI progression and represents a rational target for TBI treatment.

Suprachiasmatic pacemaker organization analyzed by viral transynaptic transport

The suprachiasmatic nucleus (SCN) of the hypothalamus, the principal circadian pacemaker, is a paired structure with two subdivisions, a ventral core receiving photic input and a dorsal shell receiving non-photic input. Rhythmicity is thought to be generated by individual SCN neurons which are coupled to achieve synchrony [D.K. Welsh, D.E. Logothetis, M. Meister, S.M. Reppert, Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing patterns, Neuron, 14 (1995) 697-706]. Normally, the core and shell, and the nuclei on each side, act in unison to transmit rhythmicity to effector systems. It is not known how coupling between neurons in the two subdivisions, and between the two SCNs, takes place. In the present study, we analyze the intrinsic, commissural, and efferent projections of the SCN using the swine herpesvirus (pseudorabies virus, PRV) as a tool for transynaptic analysis of circuits and small iontophoretic injections of the conventional tracer horseradish peroxidase (HRP) conjugated to fluorescein. We find that the core and shell each project through commissural efferents to homologous contralateral areas. The core projects densely to shell but we find little reciprocal innervation. The two subdivisions project to different hypothalamic areas, with the core projecting to the lateral subparaventricular zone and shell to the dorsomedial hypothalamic nucleus and medial subparaventricular zone. These data are the first demonstration that connections within the SCN, and from the SCN to effector regions, are topographically organized and lend insight into the flow of information through and out of the pacemaker.

Calbindin-D28K cells in the hamster SCN express light-induced Fos

ALTHOUGH the suprachiasmatic nuclei (SCN) have been intensively analyzed, they contain a population of cells that has not yet been characterized. In this study, we examined the distribution of cells immunoreactive (ir) for calbindin-D28K (CaBP), calretinin (CR), parvalbumin, vasopressin-associated neurophysin (NP), substance P (SP), vasoactive intestinal peptide (VIP), and light-induced Fos-like protein. Previously unidentified cells in the core of the hamster SCN contained CaBP. Photic stimulation during the night induced Fos expression in about 75% of the CaBP-positive SCN cells, and about 50% of the Fos-positive cells in the core region expressed CaBP. These findings provide new information in the search for the cellular localization of pacemaker cells in the SCN, as photic input entrains the circadian system, and cells that receive photic input must be either part of the clock itself, or an upstream component of the clock.

Organization of suprachiasmatic nucleus projections in Syrian hamsters (Mesocricetus auratus): an anterograde and retrograde analysis.

Circadian rhythms in physiology and behavior are controlled by pacemaker cells located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The mammalian SCN can be classified into two subdivisions (core and shell) based on the organization of neuroactive substances, inputs, and outputs. Recent studies in our laboratory indicate that these subdivisions are associated with functional specialization in Syrian hamsters. The core region, marked by calbindin‐D28K (CalB)‐containing cells, expresses light‐induced, but not rhythmic, clock genes. In the shell compartment, marked by vasopressinergic cells and fibers, clock gene expression is rhythmic. Given these findings, an important question is how photic and rhythmic information are integrated and communicated from each of these regions to effector areas. The present study used localized, intra‐SCN iontophoretic injections of the anterograde tracer biotinylated dextran amine (BDA) to investigate intra‐SCN connectivity and the neural pathways by which information is communicated from SCN subregions to targets. Intra‐SCN connections project from the core to the shell compartment of the SCN, but not from the shell to the CalB region of the SCN. Retrograde tracing experiments were performed using cholera toxin‐β (CTB) to determine more specifically whether SCN efferents originated in the core or shell using neurochemical markers for the rhythmic (vasopressin) and light‐induced (CalB) SCN subregions. The combined results from anterograde and retrograde experiments suggest that all SCN targets receive information from both the light‐induced and rhythmic regions of the SCN (albeit to varying degrees) and indicate that light and rhythmic information may be integrated both within the SCN and at target effector areas. J. Comp. Neurol. 468:361–379, 2004.

HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis

Significance Moderate or severe traumatic brain injury (TBI) damages white matter, thereby contributing to long-term neurological deficits. Currently, there are no satisfactory therapies to mitigate this white matter injury (WMI). Here we show that inhibition of histone deacetylases (HDACs) exerts robust structural and functional protection of white matter in a murine model of TBI/WMI by polarizing microglia/macrophages toward the beneficial M2 phenotype. HDAC inhibition shifted microglia/macrophage phenotype by up-regulating glycogen synthase kinase 3 beta (GSK3β), which inactivated phosphatase and tensin homologue (PTEN) through phosphorylation, thereby promoting PI3K/Akt signaling. The GSK3β-dependent M2 phenotype exerted potent anti-inflammatory effects that protected myelin-forming oligodendrocytes and diminished WMI. These results reveal a previously unexplored role for GSK3β/PTEN/PI3K signaling in the regulation of microglia/macrophages and demonstrate the promise of HDAC inhibition in the treatment of TBI/WMI. Severe traumatic brain injury (TBI) elicits destruction of both gray and white matter, which is exacerbated by secondary proinflammatory responses. Although white matter injury (WMI) is strongly correlated with poor neurological status, the maintenance of white matter integrity is poorly understood, and no current therapies protect both gray and white matter. One candidate approach that may fulfill this role is inhibition of class I/II histone deacetylases (HDACs). Here we demonstrate that the HDAC inhibitor Scriptaid protects white matter up to 35 d after TBI, as shown by reductions in abnormally dephosphorylated neurofilament protein, increases in myelin basic protein, anatomic preservation of myelinated axons, and improved nerve conduction. Furthermore, Scriptaid shifted microglia/macrophage polarization toward the protective M2 phenotype and mitigated inflammation. In primary cocultures of microglia and oligodendrocytes, Scriptaid increased expression of microglial glycogen synthase kinase 3 beta (GSK3β), which phosphorylated and inactivated phosphatase and tensin homologue (PTEN), thereby enhancing phosphatidylinositide 3-kinases (PI3K)/Akt signaling and polarizing microglia toward M2. The increase in GSK3β in microglia and their phenotypic switch to M2 was associated with increased preservation of neighboring oligodendrocytes. These findings are consistent with recent findings that microglial phenotypic switching modulates white matter repair and axonal remyelination and highlight a previously unexplored role for HDAC activity in this process. Furthermore, the functions of GSK3β may be more subtle than previously thought, in that GSK3β can modulate microglial functions via the PTEN/PI3K/Akt signaling pathway and preserve white matter homeostasis. Thus, inhibition of HDACs in microglia is a potential future therapy in TBI and other neurological conditions with white matter destruction.