key: cord-0007826-sxst1cxp
authors: Ohno, Masaki; Higashi, Yasuto; Suzuki, Kinuko
title: Microglial cell response to neuronal degeneration in the brain of brindled mouse
date: 1992-05-22
journal: Brain Res Dev Brain Res
DOI: 10.1016/0165-3806(92)90023-p
sha: a3077f03ce8e40acf26956b043c89074409d9b57
doc_id: 7826
cord_uid: sxst1cxp

Reactive changes of microglia in response to neuronal degeneration were investigated in the brains of brindled mottled mice with immunocytochemical technique. This mutant has a genetic defect in copper metabolism and spontaneous neuronal degeneration develops around postnatal day 10, in particular in the parasagittal regions of the cerebral cortex and thalamus. The antibodies to macrophage specific antigen, F4/80 and to type-three complement receptor, Mac-1 were used for the study. Reactive morphological changes of microglia, which are immuno-reactive to the antibodies to F4/80 and/or Mac-1, were demonstrated in areas corresponding to those of neuronal degeneration, coincident with the emergence of cells expressing major histocompatibility complex class II, Ia, antigen. Some of the Ia expressing cells had morphological features of ramified microglia, while others were rod shaped with few processes and were mostly located in the perivascular regions. The focal nature of such cellular changes suggests that signal(s) from the degenerating neurons may be responsible for microglial activation and cellular expression of the Ia antigen in the brain of the brindled mouse.

Microglia, first described by del Rio-Hortega 6'7 has been well recognized in human neurodegenerative lesions. For many years, there had been controversies as to the origin of microglia but by now it is generally accepted that microglia are cells of the monocyte lineage. More recently, microglia and brain macrophages have drawn the special attention of many investigators because of their presumed active roles in the Acquired Immune Deficiency Syndrome (AIDS), Alzheimer's disease and multiple sclerosis s'14.t~'2°'27'2s'3°. Reactions of microglia to neuronal damage have been investigated experimentally in 6-hydroxydopamine-induced lesions in the substantia nigra t and in the facial nerve nuclei induced either by axotomy ts or by an intra-axonal injection of toxic ricin 43. However, the reactions of microglia to these experimentally induced lesions are temporal events and do not reflect the spontaneous and gradual neuronal degeneration seen in neurodegenerative diseases.

Spontaneous neuronal degeneration in the well defined areas of the cerebral cortex and thalamus is a neuropathological feature in the hemizygous brindled mottled mouse, an x-linked recessive mutant with a genetic defect in copper metabolism 5°'51. In this murine mutant, neuronal degeneration gradually develops around the postnatal day (P)10, and death usually occurs before reaching P15 with extensive neuronal degeneration. Chronological events of neuronal degeneration in this mutant have been studied in detail s°. Therefore, we investigated temporo-spatial microglial reactions to neuronal degeneration, caused by a metabolic perturbation using this mutant. We also investigated the effects of neonatal malnutrition (or starvation) on microglial reactions, since these mutant mice became markedly emaciated at the terminal stage. The results indicate that morphological alterations of microglia occurred regionally, coincident with neuronal degeneration. Also, as has been noted in several neurodegenerative diseases in humans 27 as well as in an experimentally induced neuronal injury 42, major histocompatibility complex (MHC) class II antigen, Ia, expressing cells appeared in or in close vicinity to the site of neuronal degeneration. Some of these Ia expressing cells had distinct morphological features of ramified microglia but others were rod shaped with few processes resembling perivascular microglia 24.

The mice used in the experiments were offsprings of a brindled heterozygous female (C3H/HeJ-MObr'J/+) and a C57BL/6J male, originally from the Jackson Laboratory (Bar Harbor, Maine, USA). The colony of brindled mice was maintained by interbreeding of the offspring. The mice were housed in constant temperature and under a 12 h light/dark cycle. A total of 23 hemizygous males and 21 littermate male controls was used for the study. They were killed at postnatal days (P)3, 5, 7, 10, 12 and 14.

A total of 7 normal littermate mice were used to examine the effects of malnutrition. Following the methods applied previously s°, normal pups were allowed to be fed only 6 h per day from P5 and killed at P8, 10 or 15. The body weights of these mice were comparable to those of hemizygous mice.

The mice were anesthetized with ethylether and were perfused briefly via the left cardiac ventricle with physiological saline, followed by a periodate-lysine-paraformaldehyde (PLP) fixative 29 for 10 rain. The brains were removed immediately and immersed in the same fixative for 2 h and rinsed for 24-48 h in 0.1 M phosphate buffered saline (PBS), pH 7.4 containing 15% sucrose at 4°C. Coronal sections of the cerebrum, 30-50 pm thick were cut 3erially with a vibratome and processed for immunocytochemistry. The immunocytochemical procedures were essentially the same as reported previously 19. In brief, the sections were incubated with primary antibodies overnight at 4°C and then sequentially incubated with biotinylated rabbit anti-rat IgG (1:100 in PBS) for 2 h at room temperature, and in 1% ABC-Elite reagent for 1 h. Primary antibodies were diluted with PBS (1:300 for !:4/80; 1:500 for MAb-Mac-l: 1:300 for MAb-la). An antibody for GFAP was diluted at 1:20,000 with PBS containing 0,3% triton-X-100. After the first and second incubations, the sections were washed 3 times for 10 rain each in the PBS and then developed with 0.05% diaminobenzidine (DAB) (Sigma, St Louis, Me) or DAB-nickel It in 0,t M Tris buffer containing 0.01-0.005% hydrogen peroxide for 5 rain. DAB reaction was intensified by incubating sections with a solu. tion of 1% osmium tetroxide for 30 rain, After being placed on the silane coated glass slides, the sections were dehydrated and mounted with Permount (Fisher Scientific, Pittsburgh, PA) Control sections were incubated without primary antibody.

After immunocytochemical visualization with MAb-Mac-1 or MAb-Ia as stated above, the sections were dehydrated in graded concentrations of ethanol and embedded in poly/Bed 812 (Polyscience Inc, Warrington, PA). One #m thick sections were stained with Toluidine blue and examined with a light microscope. Immune-reactive cells were identified on the section and the block trimmed for thin sectioning. Ultrathin sections were lightly stained with lead citrate and uranyl acetate and examined with a Zeiss 10A electron microscope.

Normal control mice. In agreement with the previous report by Perry et ai. 34, there were many 1:4/80 positive cells in the cerebrum of the mice younger than P7. Mac-1 positive cells were also identified but were very faintly stained in the brains of these young mice. On P3, many F4/80 positive cells had round cell bodies with a few processes (primitive microglia 32) or without processes (ameboid microglia) and were concentrated in the corpus callosum, paraventricular white matter and internal capsule. A few F4/80 positive ramified microglia were also recognized mostly in the cerebral cortex ( Brindled hemizygous mice. On P3 and 5, most of the F4/80 positive cells in the cerebrum were primitive microglia ( Fig. 1B) but gradually ramified microglia increased in number. On P7, the morphology of both F4/80 positive and Mac-1 positive microglia in the cerebral cortex in hemizygous mice were similar to that of controls. However, after P10, Mac-1 positive cells with various morphological features (reactive microglia/macrophages) appeared in the cerebrum, in addition to the ramified microglia (Fig. 2) . Some of these reactive microglia/macrophages were rod-shaped or round-shaped with only a few processes, while others had numerous convoluted thick processes suggestive of the transitional form from the ramified microglia. These reactive micro- gliaJmacrophages were most numerous in the cingulate cortex (Fig. 3A) , cortex around the sulcus rhinalis, piriform cortex (Fig. 3B) , hippocampus (Fig. 3C) , lateral thalamus and the internal capsule (Fig. 3D ). In the frontal and parietal cortices where a laminar pattern of neuronal degeneration was observed (Fig. 4A) , these reactive microgliaJmacrophages were distributed in a well defined laminar pattern (Fig. 4B) . Reactive microglia/ macrophages were more numerous with intense immunoreactivity on P14 when neuronal degeneration was more advanced. At ultrastructural level, Mac-1 positive ramified microglia had slender processes and scant perikaryal cytoplasm and were often observed as perineuronal cells (Fig. 5A) . Some degenerating neurons were surrounded by the processes of Mac-I immune-reactive cells (Fig.  5B) , which sometimes contained electron-dense granular tissue debris.

Malnourished mice. There appeared to be some delay in differentiation of microglia in these mice and even on PI0, primitive microglia were still well recognized in the cerebral cortex (data not shown). However, reactive mi-crogliaJmacrophages were never seen in the cerebrum of these mice.

Normal control mice. Ia positive cells could be detected in the leptomeninges and choroid plexuses but rarely in the brains of control mice at any age. When they were found in the brail~, they tended to be located adjacent to blood vessels.

Brindled hemizygous mice. Coincident with the emergence of reactive microglia/macrophages in the cerebrum, a considerable number of la positive cells were identified in the cingulate and piriform cortices and lateral thalamus where neuronal degeneration was most extensive. A few Ia positive cells were also noted in the hippocampus and basal ganglia (Fig. 4C) . Some Ia positive cells had morphological features of ramified micro-gila with delicate processes (Fig. 6) , while others were rod-shaped (Fig. 7A) . The former was predominantly found in the piriform cortex. The latter was located in the cingulate as well as in the piriform cortices. These rod-shaped Ia positive cells were often located in the perivascular region (Fig. 7B ) although ultrastructurai features of the nuclear chromatin pattern and perikarya of these cells were closely similar to those of Mac-1 positive perineuronal microglia (Fig. 5A) . Ia positive cells were scattered in the leptomeninges over the cingulate and piriform cortices where some Ia positive cells were found in the subpial regions (Fig. 8) .

Malnourished mice. Similar to control mice, only rare Ia positive cells were recognized in the perivascular regions in the brain.

Astrocytes immune-reactive with anti-GFAP antibody were recognized in the brains of both control and hemizygous mice. In the latter at P12, intensely immune-re. active astrocytes tended to be present in laminar distribution corresponding to the site of neuronal degeneration (Fig. 4A) . On P14, however, intensely immune-reactive astrocytes were seen throughout all layers of the cerebral cortex. The immune-reactivity was also enhanced on astrocyte~ in hippocampus, piriform cortex and internal caF~ule (data not shown) of the hemizygous mouse. '

Our results show that the developmental patterns of microglial maturation and differentiation in the brindled hemizygous mice were closely similar, if not identical, to those of littermate control mice. In agreement with an earlier report by Perry et al. 34 , we noted that immunoreactivity of immature microglia to MAb-Mac-1 was very weak before P10, With age, immature microglia de- creased and Mac-1 positive ramified microglia increased. After P10, the majority of microglia in the brains of control mice were Mac-1 positive ramified microglia. In the brindled hemizygous mice, there appeared to be more immature microglia on P5 suggesting some delay in maturation but such a difference was no longer ap-preciated on P7 when the majority of microglia were ramified in both hemizygous and control brains. After P10, with progression of neuronal degeneration, various shaped Mac-1 positive cells (reactive microglia/macrophages) emerged in the brains of brindled hemizygous mice. Morphological features of reactive microglia/macrophages described here were similar to those of cells described as activated, phagocytic or reactive microglia in other pathological conditions 4'6'7'35'4°.

Our previous neuropathological study of brindled hemizygous mice showed that in the cingulate cortex early neuronal changes, as manifested by enlargement of mitochondria, became noticeable as early as 1'4 but neuronal degeneration was observed after Pl0 s°'s~. Mac-1 positive reactive microglia/macrophages appeared on Pl0 predominantly in the regions where neuronal degeneration was noted, suggesting that degenerating neurons release some signals or factors causing such morphological transformation of microglia. Our observation is in agreement with that of Streit and Kreutzberg who noted reactive changes of microglia in ricin injected facial nuclei only after irreversible neuronal damage had occurred 43.

The Mac-1 antigen is intimately associated with the type three complement receptor (CR3) on macrophages 2'39 and mediates the attachment and phagocytosis of particles coated with C3bi by granulocytes and mac- rophages 49. It also appears tO play an important role in adhesion dependent functions such as granulocyte chemotaxis, adherence to surface and aggregation 3g. Thus, the pronounced immuno-reactivity of these reactive microglia/macrophages with MAb-Mac-1 may indicate elevated cellular functions that include phagocytic, secretory or adhesive capacities s'ts '39,49. Although mechanisms and/or regulation of microglial transformations in vivo are not well understood, several factors such as gamma interferon, the interleukins and granulocytes/macrophage colony stimulating factor ((3/ M-CSF) are known to induce microglial transformations in vitro ~°'~2'46. More recently Giulian et al. found two microglial growth factors in the cerebral cortex of rat between embryonic day E-16 and postnatal day PN-1 and in the injured brain tissue ~s. These authors found that one of them, MM~, stimulated proliferation of mononuclear phagocytes from bone marrow but not the other, MM2. Interestingly, when the activities of these growth factors were monitored using astrocytes in vitro, they found that astrocytes did not secrete MMI. Thus, their result suggests that the cells, other than astrocytes, may be responsible for the stimulation of mononuclear phagocytes.

Shafit-Zagardo et al. reported intensely GFAP immuno-reactive astrocytes throughout the cortex in the hemizygous brindled mouse on P1537. When we studied GFAP immuno-reactivity of astrocytes on P12, preferential distribution of strongly immuno-reactive astrocyte~ was recognized in the areas corresponding to neuronal degeneration. Th;s result also suggests that astrocytes may be regionally activated by degenerating neurons oi by activated microglia.

In the areas of neuronal degeneration in the brain of the hemizygous mouse, major histocompatibility complex class II antigen, Ia, expressing cells were observed. Many of them were rod shaped but some were morphologically identical to ramified microglia. Some rod shaped Ia positive cells were located in the subarachnoid space and also in the subpial region of cingulate cortex where neuronal degeneration was most extensive, suggesting penetration of Ia positive cells into the brain parencb.yma through the pia-arachinoid in response to signals derived from degenerating neurons. Some Ia immunoreactive cells had morphological features of ramified microglia. Whether rod shaped Ia immuno-reactive cells transformed to ramified microglia or Ia expression was induced in ramified microglia is yet to be studied.

Ia expressing cells have been considered to play an important role in the initiation of cellular immune response 3'4s. In the central nervous system, Ia expression was reported in varieties of cell types including microglia ls'26'3s'44'47 and was thought to be related to immunomediated disease processes such as multiple sclerosis, ex-perimental allergic encephalomyelitis (EAE), experimental allergic neuritis (EAN), etc. However, more recently, Ia expression has been reported in microglia in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease 27 and in animals following axotomy 4t'42 or Wallerian degeneration 23. We have seen many la expressing cells in the brain and peripheral nerves of the twitcher mouse, an authentic murine model of the metabolic demyelinating disorder Krabbe's disease in humans ~9.

These recent observations of Ia expression in apparently non-immune mediated diseases suggest some additional role of la molecule in cellular degeneration. Alternatively, it is possible that some immunological mechanisms are involved in cellular degeneration in these pathological conditions in humans as well as in experimental animals. In the twitcher mouse with major pathological changes in the white matter, Ia expressing cells were more numerous in the white matter ~9, while in the brindled mouse with pronounced neuronal degeneration, Ia expressing cells were found strictly in the gray matter. These observations suggest that Ia expression is reg-ulated by local pathological processes. Ia antigen expression could be induced by gamma interferon in vivo 31 and in vitro 36 '45 , and systemic treatment with interferon gamma alone or in combination with tumor necrosis factor alpha causes microglial cell processes to express class I and class II MHC antigens ~7. Gamma-interferon-like immunoreactivity has been shown to be present in certain neurons of the central and peripheral nervous system in normal animals 9'22'25. More recently, transient expression of gamma-interferon-like immunoreactivity has been demonstrated in the axotomized rat motor neurons 33. Therefore, it is possible that gamma-interferonlike immunoreactivity may be expressed in the degenerating neurons in the brindled mouse, although the expression of such immuno-reactivity by neurons may not be necessarily related to the la induction in nonneuronal cells 21.

Microglial response to 6-hy. droxydopamine-induced substantia nigra lesions

Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor

Role of MHC gene production in immune regulation

Anti-macrophage CR3 antibody blocks myelin phagocytosis by macrophages in vitro

Production of superoxide anions by a CNS macrophage, the microglia

tercer elemento' de los centros nerviosos. I. La microglia en estado normal. II. Intervention de la microglia en los processos pathologicos, lIl. Naturaleza probable de la microglia

Cytology and Celhdar Pathology of the Nervous System

Biology of disease. Microglia in human disease, with an emphasis on acquired immune deficiency syndrome

lnterferon-~,.like.immunoreactivity in developing rat spinal ganglia neurons in vivo and in vitro

Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages

Postnatal develop. ment of serotonin nerve fibers in the somatosensory cortex of mice studied by immunocytochemistry

Colony-stimulating factors as promotors of ameboid microglia

Microglial mitogens are produced in the developing and injured mammalian brain

Microglia: immune network in the CNS

Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells

Dual label immunocytochemistry of the active multiple sclerosis lesionsl major histocompatibility complex and activation antigens

T-lymphocyte entry and antigen recognition in the central nervous system

Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat

The twitcher mouse: immunocytochemical st,dy of Ia expression in macrophages

Relationships of microglia and astmcytes to amyloid deposits of Alzheimer's disease

Gamma interferon-like immunoreactive material in rat neurons: evidence against a close relationship to gamma interferon

Gamma interferon-like immuno-reactivity in the rat nervous system

Wallerian degeneration induces la-antigen expression in the rat brain

Microglial cells are a component of the perivascular gila limitans

Interferon-gamma-like immuno-reactivity in certain neurons of the central and peripheral nervous system

Antigen presentation in the brain: MHC induction on brain endothelium and astrocytes compared

Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains

Expression of the histocompatibility glycoprotein HLA-DR in neurological disease

Periodate-lysine-paraformaldehyde fixative, a new fixative for immunoelectron microscopy

Microglia in the giant cell encephalitis of acquired immune deficiency syndrome: proliferation, infection and fusion

Differential expression of Ia and la-associated invariant chain in mouse tissue after in vivo treatment with IFN-?

Morphological studies on neuroglia. VI. Postnatal development of microglial cells

Gamma-interferon-like immunoreactivity in axotomized rat motor neurons

Immunohistochem. ical localization of macrophages and microglia in the adult and developing mouse brain

Microglial cells: origins and reactions

Comparison and quantitation of Ia antigen expression on cultured macroglia and ameboid microglia from Lewis rat cerebral cortex: analyses and implications

Rapid increases in glial fibrillary acidic protein mRNA and protein lev

els in the copper-deficient brindled mouse

The immunopathology of experimental allergic encephalomyelitis. II, Endothelial cell Ia increases prior to inflammatory cell infiltration

Leukocyte complement receptors and adhesion proteins in the inflammatory response: insights from an experiment of nature

Functional plasticity of microglia: a review

Expression of Ia antigen on perivascular and microglial cells after sublethal and lethal motor neuron injury

Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system

Response of endogenous glial cells to motor neuron degeneration induced by toxic ricin

Coronavirus infection induces H-2 antigen expression on oligodeadrocytes and astrocytes

MHC antigen expression on bulk isolated macrophagemicroglia from newborn mouse brain: induction of Ia antigen expression by gamma-interferon

Effects of colony stimulating factors on isolated microglia in vitro

Expression and synthesis of routine immune response-associated (la) antigen by brain cells

Antigen-presenting function of the macrophages

Identification of C3bi receptor of human monocytes and macrophages by using monoclonal antibodies

Neuronal degeneration in the brain of the brindled mouse. A light microscope study

Neuronal degeneration in the brain of the brindled mouse. An ultrastructural study of the cerebral cortical neurons

Acknowledgements. This work was supported in part by U.S.EH.S. grants NS-24453, HD-03110 and ES-01104. The authors thank Ms. Clarita Langaman for tissue preparation for electron microscopy, Ms. Deborah Langford for photographic work and Ms. Deborah Sears for manuscript preparation.