Ongoing Oxidative Stress Causes Subclinical Neuronal Dysfunction in the Recovery Phase of EAE

Abstract

Most multiple sclerosis (MS) patients develop over time a secondary progressive disease course, characterized histologically by axonal loss and atrophy. In early phases of the disease, focal inflammatory demyelination leads to functional impairment, but the mechanism of chronic progression in MS is still under debate. Reactive oxygen species generated by invading and resident central nervous system (CNS) macrophages have been implicated in mediating demyelination and axonal damage, but demyelination and neurodegeneration proceed even in the absence of obvious immune cell infiltration, during clinical recovery in chronic MS. Here, we employ intravital NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX1-4, DUOX1, 2) and, thus, to identify the cellular source of oxidative stress in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) in the remission phase of the disease. This directly affects neuronal function in vivo, as monitored by cellular calcium levels using intravital FRET-FLIM, providing a possible mechanism of disease progression in MS.

Keywords: EAE/MS; FLIM–FRET; NOX; calcium; intravital imaging.

Figure 1

Peripheral immune infiltration of the CNS has largely resolved in the remission phase of EAE. (A) FACS analysis of CNS cells after recovery of EAE shows a low immune infiltrate with few monocytes/macrophages (CD45^highCD11b⁺; MP) and CD3⁺ cells. Most of the CD45 expressing cells are CD45^lowCD11b⁺ (microglia; MG). CD45^highCD11b⁺ frequencies are comparable to healthy untreated mice (n = 5; ±SD). We applied an unpaired t-test to statistically evaluate the results. In the CD45^highCD11b⁺ fraction (MP), only few cells express CX3CR1 but most of the cells in the CD45^lowCD11b⁺ fraction (MG). The overlap of CX3CR1 and tdRFP was comparable in both cell types around 3% (n = 3; ±SD, clinical information listed in Table 1) (B) Exemplarily gating strategy of the FACS analysis of whole CNS in mice after recovery of EAE. (C) Projection of 3D intravital fluorescence image in the brain stem of a CX3CR1^+/− EGFP mouse in health and during the remission phase of EAE. The astrocytes (and blood vessels) are labeled by i.v. injected SR101 (red), while the microglia are expressing EGFP (green). Scale bar = 50 μm. The colocalization of the EGFP and SR101 signals, i.e., overlap of the microglial and astrocytic markers, respectively, amounts to 3.6 ± 1.8%.

Figure 2

Intravital imaging reveals that remission in EAE correlates with lack of overt immune infiltration, with persisting disruptions of the microglial and astrocytic networks. (A) 3D intravital fluorescence images in the brain stem of CerTN L15 × LysM tdRFP mice in health (n = 5), at peak EAE (n = 6) and in the remission phase (n = 2). λ_exc = 850 + 1110 nm, λ_em = 525 ± 25 nm (Thy1-Citrine in neurons depicted in green), λ_em = 593 ± 20 nm (LysM tdRFP in phagocytes depicted in red), scale bar = 50 μm. (B) 3D intravital fluorescence images in the brain stem of CX3CR1^+/− EGFP mice in health (n = 3), at peak EAE (n = 4) and in the remission phase (n = 5). λ_exc = 935 nm, λ_em = 525 ± 25 nm (CX3CR1^+/− EGFP in microglia/macrophages depicted in green), scale bar = 50 μm. (C) Using higher-order Fourier coefficients, we describe the complex shape of microglia. The first Fourier coefficient describes the position of the cells, the second coefficient the sphericity of the cell body and starting from the third Fourier coefficient, the ramification of all cell processes is reproduced: the higher the values of high-order Fourier coefficients with respect to the second Fourier coefficient, the higher the degree of ramification and length of cellular processes of microglia. (D) The different shapes of the microglia, shown in (B), were classified in health (71 cells) at peak EAE (57 cells) and in its remission phase (63 cells). The difference between the values of the third, fourth, and fifth Fourier coefficients is significant between healthy controls and remission, but not significant between peak of EAE and remission of EAE. Statistical significance was determined by ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). (E) Projection of 3D intravital fluorescence images in the brain stem of C57/B6 mice i.v. injected with sulforhodamine 101 (SR101) in health (n = 2), at peak EAE (n = 4) and in the remission phase (n = 3). λ_exc = 880 nm, λ_em = 593 ± 20 nm (SR101 in astrocytes depicted in red), scale bar = 50 μm.

Figure 3

NOX enzymes activation correlates with neuronal dysfunction in the remission phase of EAE. (A) τ₂ (enzyme-bound) NAD(P)H–FLIM images acquired in the brain stem of healthy mice (n = 5) and of mice affected by EAE at peak of the disease (n = 6) and in the remission phase (n = 7). The results encompass four independent EAE experiments. Scale bar = 30 μm. (B) Intravital fluorescence intensity image, NAD(P)H–FLIM image and FRET–FLIM neuronal calcium image acquired in the brain stem of a CerTN L15 × LysM tdRFP mouse in the remission phase of EAE. Scale bar = 30 μm. (C) Quantification of the mean NOX activation area of individual mice at peak EAE (n = 6) and in the remission phase (n = 7), four independent EAE experiments. While the mean are of NOX activation is strongly increased in the remission phase of EAE as compared to healthy controls, we could observe only a slight decrease of the NOX activation area as compared to peak of EAE (at lesion sites). (D) Quantification of the neuronal dysfunction area characterized by a neuronal calcium concentration larger than 1 μM at peak EAE (n = 4) and in the remission phase (n = 2), two independent EAE experiments. The area of elevated neuronal calcium (area of neuronal dysfunction) is slightly reduced in the remission phase of EAE as compared to the peak of the disease. However, since in healthy controls there is no elevated neuronal calcium, in both phases of EAE, the elevated calcium indicates massive neuronal dysfunction. (E) Direct correlation between NOX enzymes over-activation area and neuronal dysfunction area in the remission phase of EAE, within the brain stem (n = 2 mice). All images are acquired at 30–150 μm depth within the brain stem (z-step = 2 μm). (F) Intravital 3D images of the brain stem of a CerTN L15 × tdRFP mouse affected by EAE, at peak of the disease, at an arbitrary time point t = 0 and 120 min later. The upper panels show intensity images of axons (cyan, Thy1) and of immune cells (red), whereas the lower images show the corresponding FRET ratio images (Calcium images) of the axons. At the contact sites between axons and immune cells, we observe strongly increased neuronal calcium. After 2 h, at exactly these sites, we observed dramatic morphological changes of the axons, i.e., appearance of ovoid bodies and axonal disruption. The axonal disruption and ovoid bodies formation along the axon is indicated by white arrows in the lower panels of (F). Statistical evaluation in (C,D) was determined by ANOVA tests (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4

Cellular origin of oxidative stress in the remission phase of EAE. (A) Intravital fluorescence images and enzyme-bound NAD(P)H fluorescence lifetime images of all cells and of specific cell subtypes acquired in the brain stem of transgenic mice affected by EAE, in the remission phase. We evaluated CerTN L15 × LysM tdRFP mice, in which neurons Thy1 express the calcium indicator TN L15 and LysM⁺ phagocytes express tdRFP, and CX3CR1^+/− EGFP mice, in which microglia/macrophages express EGFP. Astrocytes were intravitally labeled with sulforhodamine 101 by i.v. injection at least 2 h before imaging. The white lines in the fluorescence and fluorescence lifetime images at the bottom of (A) demarcate the outline of a blood vessel. Hence, it becomes obvious that most of LysM⁺ cells reside within the blood vessels, and only few can be found at the border to the CNS parenchyma. Scale bars = 50 μm. (B) Contribution of the individual cell subsets to the over-activation of the NOX enzymes in the CNS of mice during the remission phase of EAE (n = 2 CerTN L15 × LysM tdRFP mice – neurons/Thy1 and LysM; n = 5 CX3CR1^+/− EGFP mice – microglia/CX3CR1 cells; n = 3 mice labeled with sulforhodamine 101 – astrocytes). The results encompass four independent EAE experiments.