Results

Footprint analysis

In the TMEV-infected mice, footprint analysis revealed a shortened, irregular, sometimes sliding gait, and a progressively declining stride length. At 196 dpi the stride length was reduced to 62.3% +/- 7.5% (standard error of mean, SEM) of the baseline measurement at 0 dpi (Fig. 4-1). Mock-infected mice showed no changes in the stride length over the study period. The two-way repeated measures ANOVA revealed a significant reduction in the stride length in TMEV-infected animals compared to the mock-infected mice at 147 and 196 dpi (time: p < 0.001, group: p = 0.008, time x group: p < 0.001, Fig. 4-1).

Figure 4-1: Footprint analysis. The measurement of hindlimb stride length revealed a chronic progredient reduction in Theiler`s murine encephalomyelitis virus-infected compared to mock-infected mice. Data are presented as the mean +/- standard error of mean of the percental change compared to the preinjection baseline measurement on day 0. A significant difference between the groups as detected by two-way repeated measures ANOVA with post-hoc independent t-tests for the different timepoints is marked as follows: *, p ≤ 0.05.

Light microscopy of semithin sections

At 14 dpi, minimal changes characterized by focal dilated myelin sheaths, infiltrated with microglia/macrophages, interpreted as early signs of demyelination were observed (Fig. 4-2a).

In addition, mild multifocal leptomeningeal lymphohistiocytic infiltrates were detected. At 42 dpi, multiple demyelinated foci, mainly in the ventral and lateral funiculi, displaying a crescent shape with its base at the meninges were present. Within these foci moderate to severe leptomeningeal, perivascular, and parenchymal lymphohistiocytic infiltrates were found. Microglia/macrophages contained intracytoplasmic dark dense onion skin-like inclusions, presumably representing phagocytosed myelin debris (Fig. 4-2b). In the vicinity of these infiltrates, small clusters of demyelinated axons were present. At 98 dpi, the further enlarged lesioned areas displayed a severe infiltration with densely packed microglia/macrophages displaying frequently Gitter cell morphology. (Fig. 4-2c). These lesions showed a severe myelin loss. At 147 dpi, lesions appeared similar to 98 dpi; however, additional changes were found in the dorsal funiculi. Interestingly, few remyelinated axons were present. At 196 dpi, many lesions showed a subsiding lymphohistiocytic infiltration, mild to moderate remyelination and a severe astrogliosis (Fig. 4-2d). Microglia/macrophages and Gitter cells were frequently enclosed by a rim of hypertrophic astrocytic processes. The remyelinated areas displayed a similar extent of OG- and Schwann cell-type of remyelination.

It appeared that the submeningeal compartment of the lesions showed Schwann cell-type remyelination, whereas OG-type related new myelin formation was present more frequently in the cental areas. In addition, multifocal areas with moderate meningeal, perivascular and parenchymal inflammation and demyelination without remyelination were still present within the same animals. Semiquantitative evaluation of the degree of demyelination revealed a progressively increasing significant myelin loss between 14 and 196 dpi in TMEV-infected mice (Fig. 4-3a). In contrast, demyelination was not observed in mock-infected mice.

Semiquantitative analysis of remyelination revealed a delayed, however, continuously increasing process of new myelin formation with significantly higher amounts of remyelinated axons at 147 and 196 dpi (Fig. 4-3b).

Figure 4-2: Light microscopic findings after Theiler’s murine encephalomyelitis virus (TMEV) infection. (A) A single vacuolated myelin sheath surrounding an intact axon that is infiltrated by a microglia/macrophage containing phagocytosed myelin debris (arrow) at 14 days post infection (dpi). (B) Focal infiltrates of macrophages/microglia containing phagocytosed myelin (arrows) in the vicinity of demyelinated axons at 42 dpi. (C) A severely demyelinated lesion with infiltrates of macrophages/microglia with Gitter cell morphology containing phagocytosed myelin and tissue debris (arrows) at 98 dpi. (D) A mild- to moderately remyelinated lesion showing clusters of remyelinated axons (arrows) at 196 dpi. Toluidine blue-stained semithin sections of epon embedded thoracic spinal cord of TMEV-infected mice. Scale bars = 20 µm.

Figure 4-3: Semiquantitative assessment of de- and remyelination after Theiler’s murine encephalomyelitis virus (TMEV)-infection. The area of de- and remyelinated white matter was evaluated with 0 (no changes), 1 (<

1/4 of the white matter affected), 2 (1/4 - 1/2 of the white matter affected), or 3 (> 1/2 of the white matter affected) points in toluidine blue-stained semithin sections of the thoracic spinal cord of TMEV-infected and mock-infected mice. Box and whiskerplots show the median and quartiles of the semiquantitative scores.

Extreme values are shown as circles. (A) The area of demyelination progressively increased from 14 until 196 dpi in the TMEV-infected mice. (B) Significant remyelination was first detected at 147 dpi and increased until 196 dpi in TMEV-infected mice. A significant difference between the groups as detected by the Mann-Whitney U test is marked as follows: *, p ≤ 0.05.

Electron microscopy

At 42 dpi many intralesional macrophages/microglia contained phagocytosed myelin debris, often still retaining its lamellar pattern, in membrane bounded cytoplasmic vesicles (Fig. 4-4a). Some myelin sheaths showed a separation of their lamellae at the outer mesaxon with interposed cellular processes, most likely of macrophage/microglial origin. Myelin sheaths without direct monocytic contact appeared unremarkable. Lymphohistiocytic perivascular infiltrates were present at this time point and persisted until the end of the observation period.

At 196 dpi, dense aggregates of intralesional macrophages/microglia and Gitter cells with abundant amorphous debris in membrane bound cytoplasmic vesicles represented the most prominent finding in many lesion centers (Fig. 4-4b). Only few intact preexisting axons were present within these regions. In other areas, completely demyelinated axons surrounded by reactive macroglial processes (Fig. 4-4b), or intermingled demyelinated and remyelinated axons were found (Fig. 4-4c, d). OG-type remyelination was characterized by the occurrence of a relatively thin myelin sheath (Fig. 4-4c). Schwann cell-type remyelination consisted of axons enwrapped in a 1:1 ratio of axons and cells (Fig. 4-4d). No viral inclusions or paracrystalline arrays were observed.

Immunohistochemistry

In the thoracic spinal cord white matter of all evaluated mock-infected mice (N=47), the NG2-positive cells represented a randomly distributed population with a density of 81.7 +/- 3.6 (SEM) cells/mm2. NG2-positive cells displayed a round to oval nucleus and an elongated bi- or oligopolar-shaped soma with long processes roughly oriented in a radial direction, interpreted as OPCs (Fig. 4-5A). Furthermore, a small fraction of stellate-shaped NG2-positive cells, interpreted as pre-OGs was detected [33]. Additionally, NG2-immunoreactivty was frequently identified in perivascular and leptomeningeal cells.

A moderately increased number of intralesional NG2-positive cells with a pronounced immunoreactivity was found 28, 42 and 98 dpi (Fig. 4-5B). Most of these cells displayed a moderately enlarged bi- or oligopolar phenotype resembling OPCs (Fig. 4-5B). At 98 and 196 dpi, an increased fraction of the NG2-positive cells in the lesion centers displayed a stellate-shaped phenotype, interpreted as pre-OGs (Fig. 4-5C). In contrast, enlarged bi- or oligopolar NG2-positive cells resembling OPCs were still present at the lesion border at 196 dpi (Fig. 4-5C). Statistical analysis employing two-way ANOVA revealed that the density of intralesional NG2-positive cells was significantly ~3.0-fold elevated at 28, 42 and 98 dpi

Figure 4-4: Electron microscopic findings after Theiler’s murine encephalomyelitis virus (TMEV)-infection.

(A) Infiltrating microglia/macrophages contained phagocytosed myelin debris, often still retaining its lamellar pattern in membrane bounded cytoplasmic vesicles (arrows) in the vicinity of demyelinated axons (arrowhead) at 42 days post infection (dpi). (B) The cytoplasm of intralesional Gitter cells contained abundant amorphous debris in membrane bound cytoplasmic vesicles (arrows) next to areas of completely demyelinated axons (arrowheads) at 196 dpi. (C) An area displaying multiple remyelinated axons with relatively thin myelin sheaths characteristic for oligodendrocyte-type remyelination (arrows) next to demyelinated axons (arrowhead) at 196 dpi. (D) An area with Schwann cell-type remyelination (arrow) next to demyelinated axons (arrowhead) at 196 dpi. Uranyl acetate and lead citrate stained ultrathin sections of epon embedded thoracic spinal cord. Electron microscopy. a = axon; gc = nucleus of a Gitter cell; m = nucleus of a macrophage/microglia; sc = nucleus of a Schwann cell. Scale bars = 4 µm.

Figure 4-5: Immunohistological findings after Theiler`s murine encephalomyelitis virus (TMEV)-infection.

Serial sections from the thoracic spinal cord of a mock-infected mouse from 196 days post infection (dpi, A, D, G, J) and two TMEV-infected mice from 28 dpi (B, E, H, K) and 196 dpi (C, F, I, L) showing immunoreactivity for nerve/glial antigen 2 (NG2, oligodendroglial progenitor cells), ABC method (A, B, C), 2´,3´- cyclic nucleotide 3´-phosphodiesterase (CNPase, oligodendrocytes), Dako Ark animal research kit (D, E, F), glial fibrillary acidic protein (GFAP, astrocytes), ABC method (G, H, I), and nonphosphorylated neurofilament (non-pNF, injured axons), Dako ark animal research kit (J, K, L). (A) Small bi- or oligopolar NG2-positive cells (arrow) were randomly distributed in the normal white matter. (B) An increased number of intralesional NG2-positive cells showing an enlarged bi- or oligopolar phenotype (arrow) and fewer NG2-NG2-positive cells with a stellate-shaped phenotype (arrowhead) were found at 28 dpi. (C) At 196 dpi, enlarged bi- or oligopolar NG2-positive cells were still detected at the lesion border (arrow). (D) The normal white matter showed a homogenous distribution of CNPase immunoreactivity. (E) At 28 dpi, foci of vacuolation and reduced CNPase immunoreactivity (star) were detected in the white matter. (F) The CNPase-negative demyelinated area (star) was prominently increased at 196 dpi. (G) GFAP-positive cells in the normal white matter showed a fibrillary phenotype. (H) An unchanged amount of GFAP-positive cells (arrow) showing a reactive phenotype with thickened processes was found in lesioned white matter areas at 28 dpi. (I) At 196 dpi, the demyelinated lesions showed an increased number of GFAP-positive cells (arrows) and a dense meshwork of GFAP-positive processes. (J) The white matter of mock-infected mice lacked positive axons. (K) At 28 dpi

non-pNF-positive axons were detected within the lesions (arrow) as well as in the normal appearing white matter (arrowheads). (L) At 196 dpi non-pNF-positive axons displaying a swollen morphology were still detectable especially in the periphery of the lesions (arrows) and in the normal appearing white matter. Immunohistology, slightly counterstained with Mayer’s hemalaun. Nomarski differential interference contrast. Scale bars = 20 μm.

compared to the white matter of mock-infected animals (Fig. 4-6, group p < 0.001, time p = 0.003, group x time p = 0.006). Additionally, a significantly increased density of intralesional NG2-positive cells was detected at 42 dpi compared to the normal appearing white matter of TMEV-infected mice, employing two-way repeated measures ANOVA (group p = 0.003, time p = 0.064, group x time p = 0.029).

The normal appearing white matter showed a homogenous distribution of CNPase-positive cells and myelin sheaths (Fig. 4-5 D). At 28 dpi, small foci with vacuolated myelin sheaths and a mildly reduced density of CNPase-positive cells were detected (Fig. 4-5E). At the following time points the density of intralesional CNPase-positive cells continuously declined. At 196 dpi, large areas in the lesion centers lacked CNPase immunoreactivity (Fig.

4-5F). In the periphery of the lesions, few CNPase-positive cells and circular structures, presumably resembling remyelinated axons, were found (Fig. 4-5F). Demyelination, defined as CNPase-negative white matter area, was significantly increased in TMEV-infected mice at 28, 98 and 196 dpi compared to mock-infected mice, and reached its maximal extension at 196 dpi with 21.6% +/- 7.8% (SEM; two-way ANOVA; group p = 0.001, time p < 0.001, group x time p < 0.001). The density of intralesional CNPase-positive cells was significantly

~0.6-fold reduced from 56 to 196 dpi compared to the white matter of mock-infected mice (Fig. 4-6, two-way ANOVA; group p < 0.001, time p = 0.001, group x time p = 0.886).

Additionally, a significantly decreased density of intralesional CNPase-positive cells was detected from 28 to 196 dpi compared to the normal appearing white matter of TMEV-infected mice (two-way repeated measures ANOVA; group < 0.001, time p = 0.087, group x time p = 0.005).

Figure 4-6: Quantitative evaluation of macroglial cells in the demyelinating lesions in Theiler`s murine encephalomyelitis (TMEV). The bars show the mean percental change +/- standard error of the mean of the density of intralesional immunoreactive cells/mm2 for nerve/glial antigen 2 (NG2, oligodendroglial progenitor cells), 2´,3´- cyclic nucleotide 3´-phosphodiesterase (CNPase, oligodendrocytes) and glial fibrillary acidic protein (GFAP, astrocytes) in TMEV-infected mice compared to the normal appearing white matter in mock-infected animals. The number of intralesional NG2-positive cells was significantly elevated at 28, 42 and 98 days post infection (dpi). The density of CNPase-positive cells was significantly declined 56, 98 and 196 dpi.

Intralesional GFAP-positive cells were increased at 98 and 196 dpi. § = no demyelinating lesions were detected by immunohistochemistry before 28 dpi in the TMEV-infected mice and at no time point in the mock-infected animals. Significant differences between the groups as revealed by two-way ANOVA with post-hoc independent t-tests for the different timepoints are marked as follows: *, p ≤ 0.05.

Most of the GFAP-positive cells in the normal appearing white matter resembled fibrous astrocytes (Fig. 4-5G). Although the number of intralesional GFAP-positive cells remained unchanged at 28 dpi, astrocytes appeared hypertrophic and exhibited thickened processes (Fig. 4-5H). At later time points, both the amount of GFAP-positive processes and the number of intralesional GFAP-positive cells progressively increased (Fig. 4-5I). The density of intralesional GFAP-positive cells was significantly ~5.0 fold elevated at 98 and 196 dpi compared to the white matter of mock-infected animals (Fig. 4-6, two-way ANOVA; group p

< 0.001, time p < 0.001, group x time p < 0.001). Similarly, a significant increase in intralesional GFAP-positive cells was detected at 98 and 196 dpi compared to the normal appearing white matter of TMEV-infected mice (two-way repeated measures ANOVA; group p < 0.001, time p = 0.002, group x time p < 0.001).

In the spinal cords of TMEV and mock-infected mice neurons and dendrites in the gray matter were positive for non-pNF, whereas no positive reaction was detected in the white matter of mock-infected animals (Fig. 4-5J). In contrast, few non-pNF-positive axons were detectable in TMEV-infected mice at 14 dpi. From 28 dpi towards 98 dpi a markedly increased number of non-pNF-positive axons was detected within the demyelinating lesions as well as the normal appearing white matter (Fig. 4-5K). Frequently, the non-pNF positive axons displayed an enlarged, swollen shape, interpreted as axonal degeneration. At 196 dpi non-pNF-positive axons were still detected at the lesion borders as well as in the normal appearing white matter (Fig. 4-5L). In contrast, the amount of non-pNF-positive axons appeared to be moderately reduced whitin lesion centers displaying severe astrogliosis.

Immunofluorescence and confocal laser scanning microscopy

Confocal laser scanning microscopy of double-stained frozen sections was used to evaluate the phenotype of NG2-positive cells and their relationship to CNPase and GFAP-positive cells, respectively. Small populations of both bi- or oligopolar NG2-positive cells resembling OPCs (Fig. 4-7A), and less frequently stellate-shaped NG2-positive cells resembling pre-OGs (Fig. 4-8A) were found in the normal appearing white matter of mock-infected mice.

Moreover, a strong NG2-immunoreactivity was associated with pericytes of the blood vessel walls, meninges and peripheral nerve roots. At 28 and 98 dpi, an increased number of mostly elongated, bi- or oligopolar NG2-positive OPCs was found within the lesions (Fig. 4-7D).

Notably, some lesions displayed an increased percentage of stellate-shaped NG2-positive pre-OGs at 98 dpi (Fig. 4-8D). At 196 dpi, a rim of bi- or oligopolar NG2-positive OPCs was observed at the lesion-borders (Fig. 4-7G), whereas in the lesion center a normal to reduced amount of stellate-shaped NG2-positive pre-OGs was detected. At 98 dpi, a loss of CNPase-expression was monitored within the lesions (Fig. 4-7E). Similarly, at 196 dpi most lesions lacked CNPase immunofluorescence. In contrast, some intralesional areas exhibited an especially intense and bright CNPase-positive immunofluorescent signal, interpreted as foci of remyelination (Fig. 4-7H). At 98 dpi, intralesional GFAP-positive cells showed prominent hypertrophic and thickened processes (Fig. 4-8E). At 196 dpi, the lesions displayed an irregularly arranged meshwork of densely packed hypertrophic GFAP-positive cellular processes (Fig. 4-8H).

Figure 4-7: Immunofluorescence was used to evaluate the phenotype of nerve/glial antigen 2 (NG2)-positive cells and their relationship to 2´,3´- cyclic nucleotide 3´-phosphodiesterase (CNPase)-positive cells (oligodendrocytes) after Theiler`s murine encephalomyelitis virus (TMEV) infection. Frozen sections of the spinal cord from a mock-infected mice from 28 dpi (A-C) and TMEV-infected mice from 98 dpi (D-F), and 196 dpi (G-I) were immunolabeled for NG2 in red (A, D, G) and CNPase in green (B, E, H). In the merged images (C, F, I) colocalization is shown in yellow / orange and nuclei were stained with bisbenzimide (blue). (A-C) In the white matter of mock-infected mice bi- or oligopolar NG2-positive cells (arrow) interpreted as oligodendroglial precursor cells (OPCs) were detected within the CNPase-positive white matter. (D-F), In the TMEV-infected mice, at 98 dpi an increased amount of bi- or oligopolar NG2-positive cells, interpreted as OPCs (arrow), was detected within the lesions. Furthermore, a prominent loss of CNPase-positive myelin sheaths was obvious (star). (G-I) At 196 dpi, a rim of bi- or oligopolar NG2-positive OPCs was observed at the lesion borders (arrow), whereas in the lesion centers (star) fewer NG2-positive cells were detected. An especially intense amount of CNPase immunofluorescence was found within some of the lesions (arrowhead). In these areas, a mildly increased amount of NG2-positive processes displaying a patchy colocalization with CNPase was observed (arrowhead). Immunofluorescence for NG2 with a Cy3-coupled secondary antibody, CNPase with Alexa-488-labeled Fab-fragments, and nuclei with bisbenzimide, confocal laser scanning microscopy. (A-C) Scale bars = 7 µm; (D-I) Scale bars = 12 µm.

Figure 4-8: Immunofluorescence was used to evaluate the phenotype of nerve/glial antigen 2 (NG2)-positive cells and their relationship to glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) after Theiler`s murine encephalomyelitis virus (TMEV) infection. Frozen sections of the spinal cord from a mock-infected mouse from 98 dpi (A-C) and TMEV-infected mice from 98 dpi (D-F), and 196 dpi (G-I) were immunolabeled for NG2 (A, D, G) and GFAP (B, E, H). In the merged images (C, F, I) NG2 is shown in red, GFAP in green, and colocalization in yellow / orange. The nuclei were stained with bisbenzimide (blue). (A-C) In the normal white matter of mock-infected mice bi- or oligopolar as well as stellate-shaped NG2-positive cells (arrow), interpreted as pre-oligodendrocytes, were detected next to GFAP-positive cellular processes. (D-F) In the TMEV-infected mice, at 98 dpi an increased amount of stellate-shaped NG2-positive cells (arrow) was detected in many lesions, next to perivascular inflammatory infiltrates (stars). (G-I) At 196 dpi, a prominent irregular meshwork of GFAP-positive cells and processes was found within the lesions. In these areas a mildly increased amount of colocalization of NG2 and GFAP was detected within single cells (arrow) and cellular processes (arrowhead). Immunofluorescence for NG2 with a Cy3-coupled secondary antibody, GFAP with Alexa-488-labeled Fab-fragments, and nuclei with bisbenzimide, confocal laser scanning microscopy. (A-C) Scale bars = 10 µm; (D-F) Scale bars = 12 µm; (G-I) Scale bars = 7 µm.

Colocalization studies revealed that the NG2-positive cells never displayed strong intrasomal colocalization with either CNPase or GFAP, either in the mock-infected or in the TMEV-infected mice (Figs. 7C, 7F, 7I, 8C, 8F, 8I). In the white matter of mock-TMEV-infected mice, only very rarely was a patchy colocalization of NG2 and CNPase found in cellular processes within the neuroparenchyma and some perivascular cells. Furthermore, streaks of NG2 and GFAP colocalization were infrequently found in the white matter of mock-infected mice. The latter seemed to represent locations where NG2-positive cellular processes were attached to or crossed GFAP-positive processes (Fig. 4-8C). In the TMEV-infected mice, intralesional colocalization of NG2 with either CNPase or GFAP was mildly increased at 196 dpi. NG2-positive processes displayed a patchy colocalization with CNPase and were especially prominent in those intralesional areas that exhibited an intense CNPase immunofluorescence, interpreted as remyelination (Fig. 4-7I). Similarly, an increased NG2 and GFAP colocalization was detected predominantly in areas with densely packed GFAP-positive hypertrophic cells and processes (Fig. 4-8I).

RT-qPCR

A low to moderate amount of TMEV RNA was found at 1 hpi and 7 dpi in spinal cord specimen of TMEV-infected mice (Fig. 4-9). This was followed by a marked ~240-fold increase until 28 dpi, and virus persistence at a high level was noticed until the end of the observation period. TMEV-specific RNA was never detected in mock-infected animals. The relative mRNA expression of both OPC marker-proteins, NG2 and PDGFα-R, was temporarily increased in TMEV-infected mice (Fig. 4-10). Statistical analysis employing two-way ANOVA revealed that NG2 mRNA was ~2.0 fold upregulated in the spinal cord of TMEV-infected mice at 14, 56 and 98 dpi (group p < 0.001, time p = 0.794, group x time p = 0.178). PDGFα-R mRNA was ~1.8 fold upregulated at 7, 14 and 56 dpi (two-way ANOVA;

group p < 0.001, time p = 0.698, group x time p = 0.095). In contrast, the mRNA transcripts of mature myelin proteins such as CNPase and MBP remained either unchanged or exhibited a decrease in the TMEV-infected mice (Fig.10). No significant difference in CNPase copy numbers was observed between the groups (two-way ANOVA; group p = 0.658, time p = 0.015, group x time p = 0.161). Total MBP mRNA was ~0.7-fold reduced at 14 and 98 dpi (two-way ANOVA; group p < 0.001, time p < 0.001, group x time p = 0.154).

Simultaneously, the expression of the exon 2 containing variants of MBP mRNA was reduced at 7, 56, 98 and 196 dpi (two-way ANOVA; group p < 0.001, time p < 0.001, group x time p

= 0.589).

Figure 4-9: Detection of Theiler murine encephalomyelitis virus (TMEV) RNA in the spinal cord by RT-qPCR.

The bars show the mean +/- standard error of the mean of the log10-transformed normalized copies / 10 ng RNA. In infected mice, TMEV RNA was detectable from 1 hour pi (0 dpi) on, and persisted until the end of the observation period (196 dpi), A marked increase of the amount of TMEV RNA occurred between 7 and 28 dpi.

TMEV RNA was never detected in mock-infected animals. Significant differences between the groups as revealed by two-way ANOVA with post-hoc independent t-tests for the different timepoints are marked as follows: *, p ≤ 0.05.

Interestingly, the fraction of the MBP exon 2 containing mRNA splice variants compared to

Interestingly, the fraction of the MBP exon 2 containing mRNA splice variants compared to