A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

State University of New York Upstate Medical University

Recent studies have shown that the role of the transcription factor interferon regulatory factor-1 (IRF-1) in the expression of major histocompatibility complex (MHC) class I molecules is tissue-specific. Our previous studies indicated a role for IRF-1 in expression of MHC class I genes in cultured astrocytes in response to interferon-gamma (IFN-gamma). However, the requirement for IRF-1 in MHC class I expression has not been directly analyzed in neural tissue. To further ascertain the importance of IRF-1 in the induction of MHC class I genes in astrocytes in response to IFN-gamma, we analyzed astrocytes from mice with a targeted disruption of the IRF-1 gene (IRF-1(-/-) mice). As expected, astrocytes from wild type (IRF-1(+/+)) mice showed a coordinate increase in both IRF-1 and MHC class I gene expression in response to IFN-gamma. To the contrary, astrocytes from IRF-1(-/-) mice had greatly reduced MHC class I mRNA expression. MHC class I gene promoter activity in astrocytes was controlled entirely through a single enhancer, the MHC-IRF-E, to which IRF-1 bound in response to IFN-gamma in wild type but not in IRF-1(-/-) mouse astrocytes. In vivo, astrocytes in brains of wild type mice readily responded to IFN-gamma to express MHC class I molecules. This correlated with increased MHC class I mRNA in the brain. In contrast, brains of IRF-1(-/-) mice showed no MHC class I gene induction following exposure to IFN-gamma indicating that all cells in the central nervous system (CNS) including astrocytes with the potential to express MHC class I molecules were dependent on IRF-1. These studies conclusively demonstrate a major role for IRF-1/MHC-IRF-E interactions in controlling MHC class I gene expression in astrocytes in response to IFN-gamma.

Interferon regulatory factor-1 is required for interferon-gamma-induced MHC class I genes in astrocytes.

We previously characterized the expression and function of the protein tyrosine phosphatase SHP-1 in the glia of the central nervous system (CNS). In the present study, we describe the role of SHP-1 in virus infection of glia and virus-induced demyelination in the CNS. For in vivo studies, SHP-1-deficient mice and their normal littermates received an intracerebral inoculation of an attenuated strain of Theiler's murine encephalomyelitis virus (TMEV). At various times after infection, virus replication, TMEV antigen expression, and demyelination were monitored. It was found that the CNS of SHP-1-deficient mice uniquely displayed demyelination and contained substantially higher levels of virus than did that of normal littermate mice. Many infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of SHP-1-deficient but not normal littermate mice, showing that the virus replicated and spread at a much higher rate in the glia of SHP-1-deficient animals. To ascertain whether the lack of SHP-1 in the glia was primarily responsible for these differences, glial samples from these mice were cultured in vitro and infected with TMEV. As in vivo, infected astrocytes and oligodendrocytes of SHP-1-deficient mice were much more numerous and produced more virus than did those of normal littermate mice. These findings indicate that SHP-1 is a critical factor in controlling virus replication in the CNS glia and virus-induced demyelination.

Critical role for protein tyrosine phosphatase SHP-1 in controlling infection of central nervous system glia and demyelination by Theiler's murine encephalomyelitis virus.

We have shown previously that myelin-forming oligodendrocytes express the protein tyrosine phosphatase SHP-1 and that myelin formation was decreased in SHP-1-deficient motheaten mice compared to that in normal littermates. These studies suggested a potential importance for SHP-1 in oligodendrocyte and myelin development. To address further this possibility, we analyzed myelin formation by microscopy and myelin basic protein (MBP) gene expression in motheaten mice at ages when myelination occurs in the developing central nervous system (CNS). Furthermore, we correlate these findings with MBP gene expression in oligodendrocytes grown in vitro. We have found that CNS myelination was significantly reduced in SHP-1-deficient mice relative to their normal littermates at multiple times during the active period of myelination. Under electron microscopy, greater numbers of axons in spinal cords of motheaten mice were either unmyelinated or had thinner myelin sheathes compared to those in matched areas of normal littermates. Accordingly, MBP protein and mRNA levels were reduced in SHP-1-deficient mice compared to that in the CNS of normal littermates. In vitro, O1(+) oligodendrocytes from motheaten mice expressed much less MBP than O1(+) oligodendrocytes of normal littermates indicating an alteration in oligodendrocyte differentiation. The latter correlated with reduced MBP mRNA relative to cerebroside galactosyl transferase (CGT) gene mRNA in SHP-1-deficient oligodendrocytes in purified cultures. We propose that SHP-1 is a critical regulator of developmental signals leading to terminal differentiation and myelin sheath formation by oligodendrocytes.

Dysmyelination and reduced myelin basic protein gene expression by oligodendrocytes of SHP-1-deficient mice.

Nuclear factor kappaB (NFkappaB) is a dynamically modulated transcription factor with an extensive literature pertaining to widespread actions across species, cell types and developmental stages. Analysis of NFkappaB in a complex environment such as neural tissue suffers from a difficulty in simultaneously establishing both activity and location. Much of the available data indicate a profound recalcitrance of NFkappaB activation in neurons, as compared with most other cell types. Few studies to date have sought to distinguish between the various combinatorial dimers of NFkappaB family members. Recent research has illuminated the importance of these problems, as well as opportunities to move past them to the nuances manifest through variable activation pathways, subunit complexity and target sequence preferences.

NFkappaB in neurons? The uncertainty principle in neurobiology.

Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic viruses often spread to the CNS, causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus-induced myositis. Of particular importance, the key immune regulator src homology region 2 domain-containing phosphatase 1 (SHP-1), which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler's murine encephalomyelitis virus (TMEV) infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification, with loss of ambulation in wild-type (WT) mice. Surprisingly, although similar extensive myofiber infection and inflammation are observed in SHP-1(-/-) mice, these mice neither accumulate dead calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-1(-/-) muscle, and an increased infiltration of immature monocytes/macrophages correlated with an absence of clinical disease in SHP-1(-/-) mice, whereas mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that, following TMEV infection of muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differentiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus-induced myopathy. 

SHP-1-dependent macrophage differentiation exacerbates virus-induced myositis.

Glycolysis and mitochondrial respiration are essential for oligodendrocyte metabolism in both the developing and adult CNS. Based on recent reports on the effects of the proinflammatory cytokine IFN-γ on metabolism and on oligodendrocytes, we addressed whether IFN-γ may affect oligodendrocyte bioenergetics in ways relevant to CNS disease. Oligodendrocytes of mice treated with IFN-γ showed significant reductions in aerobic glycolysis and mitochondrial respiration. As expected, IFN-γ treatment led to the induction of STAT1 in oligodendrocytes indicating active signaling into these cells. To determine the direct effects of IFN-γ on oligodendrocyte metabolism, cultured oligodendrocytes were treated with IFN-γ in vitro, which resulted in suppression of glycolysis similar to oligodendrocytes of animals treated with IFN-γ in vivo. Mice lacking SHP-1, a key regulator of IFN-γ and STAT1 signaling in CNS glia, had high constitutive levels of STAT1 and decreased aerobic glycolysis and mitochondrial respiration rates relative to wild type mouse oligodendrocytes. Together, these data show that IFN-γ and SHP-1 control oligodendrocyte bioenergetics in ways that may relate to the role of this cytokine in CNS disease.

Paul T. Massa

Department of Neurology

State University of New York Upstate Medical University

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

Paul T. Massa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology

State University of New York Upstate Medical University

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

Department of Neurology