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Published online before print April 13, 2007
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From the Experimental Pathology Laboratory,* Pathology and Physiology Research Branch, and the Chronic Stress and Neurotoxicology Laboratory, Molecular Neurotoxicology Laboratory, and Receptor Biology Laboratory, Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia
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Abstract |
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We recently reported Nrf2 deletion induces a lupus-like autoimmune syndrome in female mice characterized by inflammatory lesions in liver and kidney, anti-DNA antibodies, intravascular deposition of IgG, and altered ratios of CD4+ and CD8+ T lymphocytes.9 Biochemical analysis revealed that mutant mice were lacking certain phase II detoxification enzymes in hepatic and lymphoid cells, rendering the mice unable to maintain peripheral lymphocyte homeostasis and autoimmune surveillance.9 Brains from these animals were collected and analyzed for the presence of immune-mediated disease and cause of death and morbidity. Although Nrf2-null mice did not demonstrate a substantive immune response in brain, and neurological disease did not cause death in these mice, vacuolar (spongiform) leukoencephalopathy was observed in several Nrf2-null mice. We were concerned that the absence of Nrf2 was promoting neural damage and/or leukodystrophy, a condition associated with degeneration of myelin, and initiated a systematic evaluation of the brains of Nrf2-null mice.
Our standard neurohistological evaluation includes stains that positively depict neuronal damage and glial activation, such as cupric-silver, Fluoro-Jade B, and glial fibrillary acidic protein (GFAP) immunohistochemistry. These stains reveal neuropathological alterations that are not readily observed with standard hematoxylin and eosin (H&E) stains.10 Fluoro-Jade B is a high-affinity and high-specificity fluorescent stain that facilitates detection of neuronal degeneration.11,12 In the brain, changes in other cell types can also reflect neuronal injury. In particular, astrocytes respond to neural injury through a hypertrophic process termed "reactive gliosis" that involves increased production of glial intermediate filaments, the major protein component of which is GFAP. Elevation in the GFAP level is a generic biomarker of underlying brain damage and is observed across a variety of central nervous system (CNS) injuries.10,13-18 To characterize further the morphological changes in the brains of Nrf2-null mice, we conducted necropsies using perfusion fixation of control and aged knockout mice before H&E light microscopy and transmission electron microscopy. Finally, we performed immunohistochemical staining to identify Iba-1, a calcium-binding protein that reveals microglial activation state, and to localize sites of oxidant injury using immunofluorescent double labeling for the lipid peroxidation product 4-hydroxy-2-nonenal and the myelin marker myelin basic protein. These morphological and neurohistochemical studies revealed the presence of a unique vacuolar leukoencephalopathy with myelin unwinding, intramyelinic cysts, and widespread astrogliosis in mice lacking Nrf2.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Nrf2 knockout mice with a 129/SvJ background were generously provided by Dr. Y.W. Kan (University of California, San Francisco, CA)19 and were re-derived at The Jackson Laboratory (Bar Harbor, ME) to ensure that the mice were free of specific pathogens. Nrf2 knockout and wild-type 129/SvJ mice were housed in the environmentally controlled National Institute for Occupational Safety and Health barrier facility, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The mice were provided with water and an irradiated diet (Harlan Teklad 7913; Harlan Teklad, Madison, WI) ad libitum. They were housed in microisolator cages on sterile Beta chip bedding (Northeastern Products, Warrensburg, NY) and received positive pressure HEPA-filtered air to each individual cage using a Maxi-Miser System (Thoren Caging Systems, Hazleton, PA).
For neuropathology analysis, Nrf2-null mice ages 1 year or older without clinical renal failure were used for necropsy. The Nrf2-null mice had a mean (±SE) age of 15.7 ± 1.5 months and a median age of 14 months (range, 12 to 25 months); six were female and five male. Control wild-type 129/SvJ mice had a mean (±SE) age of 12.3 ± 1.3 months and a median age of 14 months (range from 6 to 14 months); three were female and three male. Only one of the wild-type mice was less than 1 year of age.
For brain GFAP and Fluoro-Jade B staining, male Nrf2-null mice over 10 months of age without clinical renal failure were selected for necropsy. Three wild-type 129/SvJ and two wild-type C57BL/6J mice over 9 months of age were used as controls. For Iba-1 staining and for the immunofluorescence studies, an additional four Nrf2-null and four wild-type male mice over 15 months of age were used.
Neuropathology Necropsies
Surveillance necropsies were conducted as previously described.9 Mice were observed for signs of illness daily. Mouse breeding, morbidity, and mortality were recorded using the Colony software, version 3.0 (Locus Technology, Inc., Orland, ME). Brain tissues from surveillance necropsies were preserved by immersion in 10% neutral buffered formalin.
For systematic neuropathology examinations, brains were preserved for neuropathology as previously described.20 In brief, mice were deeply anesthetized by intraperitoneal injection of pentobarbital (Sleepaway; Fort Dodge Laboratories, Fort Dodge, IA), intravascularly perfused via the heart with 25 ml of calcium- and magnesium-free phosphate-buffered saline (PBS) containing heparin (10 units/ml) at 100 cc of pressure, followed by intravascular perfusion with room temperature Karnovsky’s fixative.21 For light microscopy, three cross sections of brain from 11 Nrf2-null and six control mice were collected as previously described22 and embedded in paraffin. For electron microscopy, specimens of the left cerebellar white tracts, pontine white tracts, and cerebellar vermis from six of the Nrf2-null and four of the control mice were postfixed in 1% osmium tetroxide and embedded in Epon. Ultrathin sections were cut at 70 nm, collected onto copper grids, and stained with uranyl acetate and lead citrate. Ultrastructural sections were viewed and photographed using a JEOL JEM-1220 analytical transmission electron microscope (Tokyo, Japan).
For histopathology evaluation, morphological alterations in the brains of Nrf2-null and wild-type mice were evaluated by a board-certified veterinary pathologist (A.F.H.). Light microscopic changes in H&E-stained sections were separately scored for severity and distribution on a scale of 0 to 5, and the two scores were added to produce a semiquantitative pathology score as previously described.23
Five Nrf2-null mice and five wild-type mice were used for GFAP immunohistochemistry and Fluoro-Jade B staining. An additional four null and four wild-type mice were used for Iba-1 immunohistochemistry and double-labeled immunofluorescent staining to simultaneously detect myelin basic protein and the lipid peroxidation product 4-hydroxy-2-nonenal. Mice were deeply anesthetized with pentobarbital (Sleepaway) and perfused transcardially with 100 ml of 0.9% saline followed by 150 ml of 4% paraformaldehyde. Brains were removed from the skulls and postfixed overnight in the perfusion solution. Following a rinse in Dulbecco’s phosphate-buffered saline (DPBS), brains were cryoprotected for 24 hours each in 10, 20, and 30% sucrose in DPBS. Frozen sections were cut at 35 microns on a Leica cryostat (model CM3000) and collected in DPBS + 0.8 g/L sodium azide.
Immunoperoxidase Staining
To visualize GFAP (astrocytic) and Iba-1 (microglial) immunoreactivity, free-floating sections were stained using the ABC method. Sections were rinsed in DPBS three times for 5 minutes each. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 + 10% methanol in DPBS for 15 minutes. Following three more rinses, sections were permeabilized (1.8 g of poly-L-lysine, 20 ml of 5% Triton X-100, 4 ml of equine serum, and 76 ml of DPBS) for 30 minutes, and incubated with GFAP (1:10,000 dilution, 4% equine serum; DAKO, Carpinteria, CA) or Iba-1 antisera (1:250 dilution, 4% equine serum; Wako, Richmond, VA) overnight at 4°C. The next day, sections were brought to room temperature, rinsed three times with DPBS, and incubated with species-appropriate secondary antisera [1:10,000 (GFAP), 1:1000 (Iba-1), 4% equine serum; Vector Laboratories, Burlingame, CA] for 2 hours at room temperature. Following three rinses with DPBS, sections were incubated with Avidin-D horseradish peroxidase (1:1000; Vector Laboratories, Burlington, CA) for 1 hour at room temperature. Sections were rinsed three times and incubated with a Chromagen solution [25 mg/50 ml of 3-3' diaminobenzidine (Sigma, St. Louis, MO) in DPBS + 50 µl of 30% H2O2] for 5 minutes. Following three rinses, sections were mounted onto microscope slides (Colorfrost Plus; Fisher Scientific, Pittsburgh, PA), dehydrated through an ethanol series, and coverslipped with Permount (Fisher Scientific).
Stained sections were viewed with an Olympus BX-50 microscope (Lake Success, NY) equipped with a Spot II digital camera (Diagnostic Images, Sterling Heights, MI) controlled by a Macintosh G4 computer (Apple Computer, Inc., Cupertino, CA). Images were captured with Spot 4.5 software and were assembled and labeled in Photoshop CS2 (Adobe Systems, Mountain View, CA) following image resizing only.
Fluoro-Jade B Staining
Selected sections were stained with Fluoro-Jade B, a fluorescent marker for the localization of degenerating neurons, which was obtained as a gift from Dr. Larry Schmued (National Center for Toxicological Research/Food and Drug Administration, Jefferson, AR).11 Unstained sections were mounted onto microscope slides (Colorfrost Plus) and were immersed in a solution of 1% NaOH in 80% ethanol for 5 minutes, followed by 70% ethanol for 2 minutes, and distilled water for 2 minutes. Background staining was suppressed by incubation in 0.06% potassium permanganate for 10 minutes with shaking. A rinse in distilled water for 2 minutes was followed by immersion in the staining solution for 20 minutes (0.01% stock solution, 4 ml of stock solution diluted in 96 ml of 0.1% acetic acid). After staining, slides were washed three times in distilled water for 1 minute each and were allowed to air dry overnight. Slides were placed on a warmer at 55°C for 5 minutes, cleared in xylene for 5 minutes, and coverslipped with DPX (Fluka, Buchs, Switzerland).
Immunofluorescent Double Labeling for Myelin Basic Protein and 4-Hydroxy-2-Nonenal
4-Hydroxy-2-nonenal is a common lipid peroxidation product resulting from oxidative stress. Because response to oxidative stress is controlled by Nrf2, we hypothesized that myelin degeneration in Nrf2-null mice could result from oxidant injury within the myelin sheath. Therefore, we used immunofluorescence to co-localize 4-hydroxynonenal and myelin basic protein.
Brain sections were mounted on Probe-On Plus slides (Fisher Scientific) and allowed to adhere for 1 day at room temperature. Slides were rehydrated in PBS for 5 minutes, blocked with 5% bovine serum albumin in PBS for 5 minutes, rinsed with PBS, and then blocked with 5% porcine serum (Biomeda, Foster City, CA) for 10 minutes at room temperature in a humidity chamber. Excess liquid was blotted away, and the slides were incubated overnight at 4°C in a humidity chamber with a 1:40 dilution of goat anti-human myelin basic protein (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-4-hydroxy-2-nonenal (AlphaDiagnostic, San Antonio, TX). The slides were rinsed three times in PBS and incubated for 2 hours at room temperature with a 1:40 dilution in PBS of Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 594 donkey anti-goat IgG (Molecular Probes/Invitrogen, Carlsbad, CA). For the negative control, rabbit serum (Biogenex, San Ramon, CA) was substituted for primary antibody.
Visualization of Immunofluorescent Double Labeling
Slides were visualized with an Olympus AX-70 photomicroscope equipped with a Quantix Digital camera (Photometrics, Tucson, AZ) and QED camera software (QED Imaging, Inc., Pittsburgh, PA). Fluorescence images were captured in monochrome using green (460 to 500 nm excitation) and red (532.5 to 587.5 nm excitation) cubes. The QED software adds green and red pseudocolor back into these monochrome images to reproduce the fluorescent colors of these cubes with images displayed as single-labeled as well as double-labeled images.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mice with targeted disruption of the Nrf2 gene exhibited certain disease manifestations and mortality. Both clinical and pathological examinations revealed that a majority of the diseased mice developed a lupus-like autoimmune syndrome characterized by multiorgan inflammatory lesions, intravascular deposition of immunoglobulin complexes, and premature death due to rapidly progressing membranoproliferative glomerular nephritis with a female predominance.9 A diagnostic examination of the brain specimens of the mice using standard light microscopy, formalin-fixed tissue, and H&E-stained sections, in which at least one section of the brain was examined, revealed marked vacuolar leukoencephalopathy characterized by formation of vacuoles in the white matter of the brain (see below). The prevalence of vacuolar leukoencephalopathy was 54% (7 of 13 mice). No apparent gender bias was observed for the brain lesion. The mean age of the mice with vacuolar leukoencephalopathy was 8.3 ± 2.6 months (mean ± SE), whereas the mean age of the mice without evidence of vacuolar leukoencephalopathy was 5.2 ± 1.7 months (mean ± SE). This prevalence rate of 54% in the diagnostic series represents a minimal prevalence rate for vacuolar leukoencephalopathy in the mice examined because of the use of formalin fixation and the absence of level III sections for some mice. Thus, a systematic evaluation of the brain for spontaneous neurodegenerative lesions associated with the absence of the Nrf2 transcription factor was performed.
To systematically evaluate leukoencephalopathy in Nrf2-null mice and to distinguish it from other lesions described previously,9
clinically healthy Nrf2-null mice of both genders were chosen for neuropathological evaluation at three standard brain sections.22
Nrf2-null mice at 
1 year and older were found to have vacuolar leukoencephalopathy with a prevalence of 100% (11 of 11). All levels of the brain showed vacuolar leukoencephalopathy, but the pathology scores reached statistical significance only in the cerebellar white tracts (P = 0.001, Mann-Whitney rank sum test, Figure 1
), where the prevalence was 100%. In level III pons (Figure 2)
, 6 of 11 Nrf2-null mice had multifocal to multifocal and coalescent, moderate to marked, vacuolar degeneration of the pontine white tracts. Light microscopic examination of H&E-stained sections revealed foci of vacuolar degeneration in six and three of the Nrf2-null mice in levels I and II, respectively. In the age- and gender-matched wild-type mice in this series, only one animal had a locally extensive area of mild vacuolar degenerative changes in the cerebellar white tracts. There were no other lesions in the brains of wild-type mice.
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. At higher magnifications, these foci of rarefaction represented vacuolar (spongiform) degeneration of the cerebellar and pontine white tracts (Figure 2B)
. The morphological alterations were characterized by multifocal and coalescing cyst-like spaces (Figure 2C)
that replaced much of the white matter normally seen in the cerebellum of the wild-type mice (Figure 2D)
. Foci of vacuolar degeneration in levels I and II were morphologically similar, although less extensive and less consistently observed than in level III. Neither gross examination of the brains nor gross measurements of the different brain levels detected any evidence of cerebellar atrophy.
In the Nrf2-null mice, multiloculated cystic dilation of myelin was a consistent ultrastructural finding (Figure 3)
. The lesion was observed in the pontine white tracts of all Nrf2-null mice examined, in the left cerebellar white tracts of three of the five Nrf2-null mice, and in the white tracts of the cerebellar vermis of one mouse. Typical lesions included large multiloculated cystic spaces and whorls of myelin within the myelin sheaths of axons (Figure 4A)
. Severe vacuolar degeneration with associated fibrous astrocytes was seen in the cerebellar vermis of an Nrf2-null mouse (Figure 4B)
. Oligodendrocytes, the cells that secrete and maintain myelin in the central nervous system, were morphologically normal even when adjacent to cystic spaces. Rare aggregates of glial cells were observed (Figure 3B)
. In the wild-type mice, ultrastructural alterations in the cerebellar vermis, left cerebellar white tracts, and pontine white tracts were limited to low levels of vacuolation within the neuropil, between axons (glial cell vacuolation) in the cerebellar vermis of one mouse, and low levels of myelin splitting in three of the four mice. These were considered to be within the normal limits of the mice (Figure 3A)
. Together, the results revealed that loss of Nrf2 function caused vacuolar leukoencephalopathy in multiple white tracts of mouse brain that is primarily manifested as cystic dilation of myelin sheaths in the absence of apparent exposure to toxicants, thus implicating Nrf2 in the physiological maintenance of myelin integrity and function. Likewise, the lack of Nrf2 function is related to the pathogenesis of spongiform myelin degeneration in the knockout model.
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The high prevalence of vacuolar leukoencephalopathy, which primarily affects the myelin sheath, prompted us to examine whether the loss of Nrf2 caused damage to neurons. Fluoro-Jade B, a specific stain for neuronal damage,11,12
revealed no significant neuronal degeneration or death in the brains of Nrf2-null or wild-type mice (Figure 5)
, despite the striking vacuolation in the white tracts. Immunostaining for Iba-1, a calcium-binding protein that reveals microglial activation state, showed no differences in distribution or morphology of microglia between Nrf2-null or wild-type mice, indicating microglial activation is not enhanced by myelinopathy observed in Nrf2-null mice (data not shown).
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compared with wild-type controls (Figure 6, A, C, and E)
. In striatum, immunoreactive astrocytes were observed throughout the striatal neuropil of Nrf2-null mice (Figure 6B)
and were virtually absent from control animals (Figure 6A)
. The corpus callosum was heavily laden with hypertrophic astroglia (Figure 6B)
compared with the wild-type control (Figure 6A)
. In the hippocampus of Nrf2-null animals, elevated immunoreactivity was observed throughout the neuropil and within the hilus (Figure 6D)
. Especially evident was the immunoreactivity of astrocytes in alveus and tapetum (Figure 6D)
, which was virtually absent from the controls (Figure 6C)
. The cerebellum of Nrf2-null mice contained many activated astrocytes dispersed throughout all white matter tracts (Figure 6F)
compared with wild-type control animals (Figure 6E)
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. Activated astrocytes were observed throughout the radiato corporis callosi (Figure 7A)
, dispersed throughout the entire striatal neuropil (Figure 7B)
, in all hippocampal subregions (Figure 7C)
, within the hilus and throughout the molecular layers of the dentate gyrus (Figure 7D)
, within the hippocampal alveus and tapetum (Figure 7E)
, and throughout the cerebellar white matter (Figure 7F)
. The coexisting myelin degeneration and astrocyte activation suggests an astrocytic response to myelinopathy and/or dysregulation of the GFAP gene associated with Nrf2 deletion.
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The lipid peroxidation product 4-hydroxy-2-nonenal was frequently expressed in white tracts of Nrf2-null mice where it co-localized with myelin basic protein to the sites of vacuolar degeneration (Figure 8, A–C)
. This indicated peroxidation of the lipid component of the myelin sheath. In Nrf2-null mice, and to a lesser extent in wild-type mice, 4-hydroxy-2-nonenal was occasionally expressed in the cytoplasm of cells that did not express myelin basic protein.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Myelin, the insulating layers of membrane wrapped around axons by oligodendrocytes, is essential for normal impulse conduction in the CNS.25 Aberrant structure and function of myelin can result from defects in the formation of myelin, or myelin degeneration, and result in various CNS disorders including death.25,26 The leukoencephalopathy and astrogliosis observed in Nrf2-null mice appears consistent with a new leukodystrophy because it is a condition characterized by progressive destruction of myelin due to a metabolic defect. Morphologically, the leukoencephalopathy of Nrf2-null mice is similar to leukoencephalopathy with vanishing white matter in humans, a disease caused by mutations in the subunits of the translation initiation factors, and is characterized by rarefaction and cystic vacuolar degeneration of white matter.27,28 However, leukoencephalopathy with vanishing white matter generally involves hypomyelination and oligodendrocyte abnormalities suggesting dysmyelination. Based on the cystic vacuolation of myelin and normal oligodendrocytes observed in the Nrf2-null mice, and the survival of the mice into adult life, the vacuolar leukoencephalopathy consistently associated with the absence of Nrf2 seems, instead, to be a result of myelin degeneration. The neuropathologic changes in Nrf2-null mice also have similarities to the gliosis and intramyelinic vacuolation noted in mice lacking mitochondrial superoxide dismutase29,30 ; however, in the latter case, vacuolation was also observed in the cortex and in brainstem nuclei, areas that were relatively spared in Nrf2-null mice. Thus, the unique pathological features of the leukoencephalopathy of Nrf2-null mice and the notion that Nrf2 is essential for up-regulation of antioxidant responses to oxidant injury suggest that the intramyelinic vacuolar leukoencephalopathy may represent a new type of leukodystrophy that is caused by impaired myelin homeostasis due to oxidative myelin damage and degeneration in the absence of Nrf2 activation of antioxidant responses.
Although neurodegeneration as assessed by Fluoro-Jade B staining was not evident throughout brain regions of the Nrf2-null mice, the widespread astrogliosis was a prominent feature and was especially evident in the white tracts where vacuolar degeneration was also apparent. Such a coexistence of astrogliosis and myelin degeneration has been observed in other natural and experimental disorders/diseases of CNS myelin,31-33 suggesting myelin degeneration can activate astrocytes. Indeed, demyelination has been shown to be a major stimulus for the subsequent induction of astrogliosis.14 Our analysis of microglial activation status revealed no apparent differences between wild-type and knockout mice; however, the wild-type animals were a median age of 18 months old and may have some age-related basal microglial activation, or the microglia may have been previously activated and returned to a ramified state while the astrocyte activation was persistent. Future studies will be required to fully determine the extent of age-dependent microglial activation and free radical-induced lipid peroxidation of myelin.
It is reasonable to hypothesize that myelin degeneration in Nrf2-null mice could result from oxidant injury within the myelin sheath in the absence of antioxidant responses. The localization of the lipid peroxidation product 4-hydroxy-2-nonenal to the dilated myelin of Nrf2-null mice is consistent with this hypothesis. Our findings demonstrate the importance of Nrf2 to normal myelin homeostasis and suggest that in its absence, oxidant injury leads to degeneration of the myelin sheath. Thus, the Nrf2-null mouse has a vacuolar leukoencephalopathy, which is a new member of the leukodystrophies, and seems to be associated with oxidant injury that accumulates within the myelin sheath in the absence of Nrf2.
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Acknowledgements |
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Footnotes |
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The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.
Accepted for publication March 13, 2007.
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