Tempol ameliorates murine viral encephalomyelitis by preserving the blood-brain barrier, reducing viral load, and lessening inflammation

Abstract

Multiple sclerosis (MS) is a progressive inflammatory and/or demyelinating disease of the human central nervous system (CNS). Most of the knowledge about the pathogenesis of MS has been derived from murine models, such as experimental autoimmune encephalomyelitis and viral encephalomyelitis. Here, we infected female C57BL/6 mice with a neurotropic strain of the mouse hepatitis virus (MHV-59A) to evaluate whether treatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) affects the ensuing encephalomyelitis. In untreated animals, neurological symptoms developed quickly: 90% of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits. Treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mM tempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptoms were attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. Not surprisingly, tempol substantially preserved the integrity of the CNS, including the blood-brain barrier. Furthermore, treatment with tempol decreased CNS viral titers, macrophage and T lymphocyte infiltration, and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription of tumor necrosis factor-alpha and interferon-gamma, and protein nitration. The results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated CNS inflammatory response. Overall, our study supports the development of therapeutic strategies based on nitroxides to manage neuroinflammatory diseases, including MS.

Fig. 1

(A) Evolution of clinical symptoms and (B) histology of brain tissues 7 days postinfection of female C57BL/6 mice infected with MHV-A59 (500 pfu; ic). Infection and analysis were performed as described under Experimental procedures. (A) Neurological symptoms of the mice (n = 12) were scored as described in the text and under Experimental procedures. In summary, scores 0, 1, 2, 3, 4, and 5 correspond to normal, mildly sick, moderately sick, severely sick, moribund, and dead mice, respectively. (B) Representative hematoxylin/eosin- and Weil-stained (bottom right) sections from brains of MHV-A59-infected mice 7 days postinfection. The regions are labeled as Co (cortical area), Amyg (amygdaloid nucleus), Hip (hippocampus), and CC (corpus callosum). Massive inflammatory cellular infiltration and loci of myelin disruption are marked by single and encircling arrows, respectively. Scale bars, 10 μm.

Fig. 2

Representative EPR spectra of brain homogenates of sham and MHV-59A-inoculated female C57BL/6 mice 1 day postinfection sacrificed at different times after tempol administration (24 mg/kg; ip). The spectrum of each homogenate was scanned before and after addition of 1 mM ferricyanide, as indicated. Instrument conditions: microwave power, 10 mW; modulation amplitude, 0.1 mT; time constant, 327 ms; scan rate, 0.029 mT/s.

Fig. 3

(A) Evolution of clinical symptoms and (B) histology of brain tissues 7 days postinfection of female C57BL/6 mice infected with MHV-A59 (500 pfu; ic) and treated with tempol. Infection, treatment, and analysis were performed as described under Experimental procedures. (A) Neurological symptoms of the mice (n = 12) were scored as described for Fig. 1. (B) Representative hematoxylin/eosin- and Weil-stained (bottom right) sections from brains of MHV-A59-infected mice treated with tempol 7 days postinfection. The regions were labeled as Co (cortical area), Amyg (amygdaloid nucleus), Hip (hippocampus), and CC (corpus callosum). In contrast to the brains of untreated mice (Fig. 1B), massive inflammatory cellular infiltration and loci of myelin disruption are largely absent.

Fig. 4

Survival of female C57BL/6 mice inoculated with MHV-A59 and treated with tempol (▪) or not treated (▴). Inoculation of MHV-A59 (500 pfu; ic) and treatment were performed as described under Experimental procedures. Control animals (●) were those that received PBS intracranially instead of the virus and were treated with tempol. The values shown correspond to the means ± standard error of five groups of animals (n = 5 or 6 each).

Fig. 5

Permeability of the BBB 7 days postinfection in female C57BL/6 mice inoculated with MHV-A59 and treated or not with tempol. Inoculation of MHV-A59 (500 pfu; ic), treatment, and measurement of BBB permeability were performed as described under Experimental procedures. Fluorescein uptake is expressed as (μg fluorescence spinal cord tissue/mg protein)/(μg fluorescence sera/μl blood). The values shown correspond to the means ± standard error of eight (control), seven (tempol-treated), and four (sham) mice; ⁎p < 0.002, ⁎⁎p < 0.0002, unpaired t test. Representative photographs of brains are also shown for qualitative assessment of BBB permeability.

Fig. 6

Representative photomicrographs of brain sections 7 days postinfection of female C57BL/6 mice inoculated with MHV-A59 and treated or not with tempol. Sections were immunohistochemically stained using Mac-2, iNOS, and nitrotyrosine antibodies as specified. Sections were counterstained with Harris's hematoxylin. The procedures employed are described under Experimental procedures. Slides were examined with a 40× lens, photographed, and printed under the same conditions. Scale bars, 50 μm.

Fig. 7

Relative quantification of markers of inflammation in the CNS 7 days postinfection of female C57BL/6 mice inoculated with MHV-A59 and treated or not with tempol. Inoculation of MHV-A59 (500 pfu; ic), treatment, and analysis were performed as described under Experimental procedures. (A) Semiquantitative microdensitometric/morphometric image analysis of immunoreactive areas of Mac-2, iNOS, and 3-nitrotyrosine in the brain of the animals as specified. The data obtained in all analyzed regions were normalized in a sampled field of 8.96 × 104 μm² and pooled together to be expressed. The values shown correspond to the means ± standard error (n = 3); ⁎p < 0.0002, ⁎⁎p < 0.02, ⁎⁎⁎p < 0.004, unpaired t test. (B) Relative quantification of 3-nitrotyrosine in the spinal cord of the specified animals by immune slot blot. The values correspond to the means ± standard error (n = 3); p < 0.03, unpaired t test (not shown for clarity). The inset shows representative blots. (C) Relative quantification of TNF-α and IFN-γ transcripts in the spinal cords of the specified animals. The values correspond to the means of two independent experiments. The inset shows the results of one of the experiments.

Fig. 8

Relative quantification of CD4 and CD8 mRNA in the spleen and spinal cord 1 and 7 days postinfection of female C57BL/6 mice inoculated with MHV-A59 and treated or not with tempol. Inoculation of MHV-A59 (500 pfu; ic), treatment, and analysis were performed as described under Experimental procedures. (A) Representative results obtained from three infected mice each, untreated or treated with tempol as specified. (B) Representative results obtained from naive mice. (C) Relative quantification of CD4 and CD8 transcripts in the spinal cords by densitometry of the corresponding bands shown in (A). The values correspond to the means ± standard error; ⁎p < 0.01, ⁎⁎p < 0.02, unpaired t test.