This study is based on previous results from our group describing upregulation of mainly MMP-12 and -3 as well as TIMP-1 in association with a demyelinating meningoleukomyelitis in TME (ULRICH et al. 2006). These findings resulted in the hypothesis, that MMP-3 and MMP-12 are key-molecules in the pathogenesis of demyelinating diseases (Figure 4). The aims of this study were 1.) to determine the influence of TMEV-infection on astroglial and microglial MMP-synthesis in vitro, 2.) to elucidate whether injected MMPs contribute to demyelination by a direct destruction of myelin and/or oligodendrocytes or indirectly by facilitating infiltration of inflammatory cells or breakdown of the BBB, and 3.) to investigate the effect of Mmp3 and Mmp12 deficiency using knockout mice upon the clinical and morphological outcome during TME. Furthermore, the incidental finding of a spinal epidermoid cysts (ECs) without associated clinical signs in one control animal has been described.
Figure 4: Possible mechanisms how MMPs can contribute to demyelination in the CNS (adapted from YONG et al. 2001).
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6.1. Matrix metalloproteinase transcription in vitro
MMPs are associated with demyelination in TME (ULRICH et al. 2006). The in vitro experiment aimed to investigate the impact of TMEV-infection upon MMP transcription in astrocytes and microglia cells. Both cell types represent besides invading monocytes/macrophages an important source of MMP production in the CNS (NUTTALL et al. 2007). In vitro, a constitutive expression of Mmp2, -3, -9, -10, -12, -13, -14, -15, -24 and Timp2 to -4 in normal astrocytes and microglia cells (derived from brain cultures of SJL/J mice) was detected while Timp1 and Mmp11 were found in astrocytes only, and Mmp7 was absent in both cell populations. TMEV-infection of astrocytes induced a prominent upregulation of Mmp3, -9, -10, -12, and -13 as well as Timp1 transcripts in vitro. The observed upregulation of Mmp3 and -12 as well as Timp1 is in accordance with described in vivo observations during TME (ULRICH et al. 2006), while Mmp9 transcripts are not differentially expressed in vivo.
However, in contrast to observations in TME an upregulation of MMP-9 transcripts is reported in EAE and MS (ANTHONY et al. 1997, AVOLIO et al. 2003, COSSINS et al. 1997). This in vitro investigation revealed that TMEV-infection induces an increased MMP transcription in astrocytes, while TMEV-infection of microglia cells was associated with a down-regulation of MMP- and TIMP-transcription. However, Mmp12 expression was about 100-fold higher in microglia compared with astrocytes.
In addition, demyelination in TME is associated with a progressive intralesional astrogliosis as well as an increased number of microglia/macrophages (HAIST et al.
2012). Conclusively, MMPs produced by TMEV-infected astrocytes and inflammatory cells might contribute to the process of demyelination and BBB disruption.
6.2. Stereotaxic injection of matrix metalloproteinases
This experiment aimed to elucidate the mechanisms by which MMPs especially MMP-3 and -12 contribute to the process of demyelination. MMPs are known to cleave MBP, an essential constituent of the myelin sheaths in vitro (CHANDLER et al. 1995, SHIRYAEV et al. 2009). This raises the question whether these enzymes will be able to destroy myelin sheaths in vivo. MMP-9 was injected in addition to
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MMP-3 and -12 because this MMP is among others associated with demyelination in MS (AVOLIO et al. 2003, COSSINS et al. 1997). In addition, a loss of LFB-staining intensity is reported following stereotaxic injection of MMP-9 into the rat brain (ANTHONY et al. 1998). Stereotaxic injection of 440 ng recombinant, in vitro activated murine MMP-3, -9 and -12 into the caudal cerebellar peduncle (CCP) of adult SJL/J mice induced a severe demyelination before infiltration of inflammatory cells occurred (Figure 5). In addition to demyelination most prominent in MMP-3-injected animals a severe loss of oligodendrocytes was noticed. To confirm these histological findings transmission electron microscopy was performed.
Ultrastructurally, myelin sheath edema, fragmentation of myelin membranes and an extracellular edema were found. These observations demonstrate that MMP-3, -9 and -12 are able to induce demyelination in vivo. However, the mechanism whether MMPs induce demyelination by disintegration of the cell membrane possibly due to cleavage of MBP or its intrinsic oligodendrocyte-toxic-potential remains unknown. In addition, a leakage of the BBB, detected by extravasation of Evans blue and IgG, was observed following stereotaxic injection of MMP-3, -9 and -12. Opening of the BBB leading to extravasation of plasma proteins may be a second, important mechanism involved in the process of demyelination, complement activation and inflammatory cell extravasation.
Figure 5: Consequences of MMP-3, -9 and -12 following stereotaxic injection into the caudal cerebellar peduncle of adult SJL/J mice
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6.3. Infection of MMP-3 and -12 knock-out mice with TMEV
Microarray analysis of transcriptional changes during TME revealed a marked and progressive upregulation of Mmp12 (Figure 6). In addition, Mmp2, 3, 13, 14, 16 -19 and Timp1, -2 were upregulated while Mmp-9, -17, -24, -28 and Timp4 were downregulated at various time points (Figure 6). Furthermore, a variable expression pattern was observed for Mmp23.
Figure 6: Transcriptional changes associated with the ECM in the spinal cord of TMEV-infected mice (adapted from HANSMANN et al. 2012)
Thermometer-like icons display the fold-changes of significantly expressed genes in TMEV-infected mice compared with mock-TMEV-infected mice employing pairwise Mann-Whitney U-tests (p≤0.05) at four time points: (1) = 14 dpi, (2) = 42 dpi, (3) = 98 dpi and (4) = 198 dpi, respectively. Thermometer-like icons: red = upregulation, blue = downregulation;
green arrows = positive functional interaction; red arrows = negative functional interaction;
grey arrows = technical link; hollow green arrays = pathway start; B = binding; C = cleavage
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These observations are supported by previous RT-qPCR investigations of TMEV- and mock-infected mice (ULRICH et al. 2006). Stereotaxic injection of activated MMP-3 and -12 demonstrated the potential of MMPs to induce demyelination as well as a disruption of the BBB in vivo. Considering that MMP-3 and -12 are key-molecules in the process of demyelination the next question was whether disruption of Mmp3 or Mmp12 gene expression may abolish/reduce demyelination during TME.
To answer this question Mmp3-/- and Mmp12-/- mice (both on a SJL/J genetic background) as well as SJL/J wild-type mice were infected with TMEV. Clinical investigation employing a scoring system and rotarod-test revealed a significant difference between TMEV and mock-infected animals starting at 70 dpi (wild-type mice) and 91 dpi (Mmp3-/- mice) while significant differences between TMEV- and mock-infected Mmp12-/- mice were lacking. This leads to the conclusison that Mmp12 deficiency ameliorates clinical signs observed during TME.
These results may be explained by a significantly reduced degree of demyelination at 98 days post infection (dpi) in Mmp12-/- mice compared to wild-type and Mmp3 -/-mice. In accordance with a reduced demyelination a reduced leukomyelitis was detected at 98 dpi while the degree of meningitis and poliomyelitis was similar between the TMEV-infected groups. Immunohistochemistry revealed that inflammatory cells in the white matter were mainly composed of microglia/macrophages and lymphocytes. However, the density of microglia/macrophages was significantly reduced in Mmp12-/- mice compared to wild-type mice from 1 to 98 dpi. The observation that MMP-12 deficiency leads to a reduced degree of demyelination in the spinal cord in association with a reduced density of microglia/macrophages indicates that MMP-12 has a detrimental effect upon myelin preservation. Although the number of microglia/macrophages in Mmp12-/- mice was significantly reduced during the demyelinating phase of TME, the activation/infiltration of these cells was not completely abolished. This is an important finding because numerous studies show that several aspects of inflammation following CNS injury are beneficial (GIULIAN et al. 1990, GONCALVES DASILVA et al. 2009, MABON et al. 2000, POPOVICH et al. 1999, SKULJEC et al. 2011) and macrophages also facilitate recovery (BATCHELOR et al. 1999, GUTH et al. 1994,
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RAPALINO et al. 1998). These data indicated that an excessive infiltration of macrophages may contribute to a detrimental outcome. Furthermore, it has been shown that MMP-12 has a deleterious effect following spinal cord injury and intracerebral hemorrhage (WELLS et al. 2003). Macrophages and gitter cells contribute to demyelination and axonal damage in MS and TME by producing mediators of tissue damage such as MMPs, reactive oxygen species and pro-inflammatory cytokines (BATCHELOR et al. 1999). Macrophages represent an important source of MMPs with a broader range and often higher amounts of proteolytic enzymes compared to lymphocytes (BAR-OR 2008, BAR-OR et al. 2003, ULRICH et al. 2006).
6.4. Epidermoid cysts in the spinal canal of mice
The incidental finding of a spinal epidermoid cyst (EC) in one control animal leads to the first description of an EC in this respective mouse strain (HANSMANN et al.
2011). The cyst was attached to the meninges and compressed a part of the lumbar spinal cord from dorsolateral. In general the caudal part of the spinal cord represents a location where most of the described spinal ECs in other strains and species were located (JUNG et al. 1981, KULWICH 1994, STROOP 1984). ECs in mice have a small diameter and are not detected grossly. The occurrence of ECs in inbred mouse strains is variable, with C58/J, AKR/J, B6C3F1 and C57L/J strains lacking reported lesions, whereas albino swiss mice, Balb/c, quaking mice, C57BR/cdJ, Crl:CD® and C57Bl/6J strains have incidences of up to 6.3% (JUNG et al. 1981, KULWICH 1994, NOBEL et al. 1987, STROOP 1984). The incidences of ECs are variable among different mouse strains indicating a genetic predisposition for their development.
Most studies dealing with animal models of CNS diseases include clinical investigations like scoring systems behavioral tests or motor coordination tasks (e.g.
rotarod analysis). Clinical investigation is an important feature because most histopathological results as well as effects of applied substances were correlated with clinical outcome. The lack of clinical signs despite the spinal EC in the present case is consistent with previously described cases in mice. Additionally, performed rotarod
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tests confirmed the lack of motor coordination deficits. The lack of clinical signs in mice and rats is in contrast to findings in dogs and man where clinical signs including paraparesis, sensory loss and back and/or leg pain are reported (FERRARA et al.
2003, PLATT et al. 1999, STEINBERG et al. 2007). Dermoid cysts are an important differential diagnosis to ECs. These cyst formations are rare in mice but in contrast to ECs dermoid cysts can induce clinical signs (NGUYEN 1988). Dermoid cysts can be distinguished from EC by histopathology because dermoid cyst lack adnexal structures like hair follicles, sebaceous or sweat glands. Conclusively, the occurrence of spinal ECs in mice seems to be strain dependent and ECs are mainly located in the caudal part of the spinal cord. Furthermore, ECs in mice are neither detected grossly nor associated with clinical signs and therefore the detection of an EC within a mouse study should be interpreted as an incidental finding which will not influence the results of clinical investigations.
6.5. Concluding remark
In this thesis in vitro analysis of MMPs secreted by astrocytes and microglia, microarray analysis of transcriptional changes in the spinal cord of TMEV-and mock-infected mice, stereotaxic injection of activated MMP-3, -9 and -12 into the caudal cerebellar peduncle of mice and TMEV-infection of Mmp3- and Mmp12-knockout mice has been performed. In vitro Mmp3, -9, -10, -12 and -13 transcripts were upregulated in TMEV-infected astrocytes. Furthermore, in vivo Mmp3 and Mmp12 transcripts were upregulated in the demyelinating phase of TME. During TME Mmp12 knockout mice showed reduced demyelination, macrophage infiltration and motor deficits compared with wild-type mice. In addition, stereotaxic injection of MMP-12 into the CCP revealed demyelination and a reduced number of oligodendrocytes prior to the infiltration of leukocytes indicating a direct myelin and/or oligodendrocyte-toxic mode of action.
Conclusively, these data indicate that MMP-12 plays an essential role in the pathogenesis of demyelinating diseases and an inhibition of MMP-12 may be a suitable approach for preventing chronic progressive demyelination.
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