Grey matter demyelination and neurodegeneration in Multiple Sclerosis: a new animal model for studying disease mechanisms
Author(s)
Gardner, Christopher James
Type
Thesis
Abstract
Multiple sclerosis is the commonest neurological disease affecting young adults. Whilst the
initial relapsing-remitting disease phase is associated with inflammatory demyelination and is
treatable with immunomodulatory drugs, the secondary progressive phase (SP-MS) is
associated with ongoing axonal loss and cortical atrophy and is currently untreatable. Studies
of SP-MS have revealed the presence of extensive subpial demyelinated lesions within the
cerebral cortex. This pathology is associated with a high level of meningeal inflammation, a
gradient of cell loss from the cortical surface and high levels of microglia activation. To test
the hypothesis that pro-inflammatory cytokines diffusing from the cerebral meninges could be
responsible, we have established an animal model mimicking cortical grey matter pathology.
Female DA rats were immunised with 5μg recombinant myelin oligodendrocyte glycoprotein
(rmMOG) in incomplete Freunds adjuvant (IFA). This dose was insufficient to initiate
encephalomyelitis, but did initiate an anti-MOG humoral immune response in the periphery.
Twenty days post-immunisation animals received an injection of tumour necrosis factor (TNF)
and interferon gamma (IFNγ) into the subarachnoid space at the sagittal sulcus.
Immunohistochemistry revealed areas of subpial demyelination extending through cortical
layers I–III. Lesions were maximal after 7 days and had resolved by remyelination at 14 days.
A gradient of microglia/macrophage activation was present from the cortical surface. The
extent of demyelination correlated with activation of microglia in the cortex and macrophages
within the meninges. Activated microglia were observed contacting myelin, oligodendrocytes
and neurons. In the demyelinated cortex, expression of the TNF receptors TNFR1A and
TNFR1B was upregulated on oligodendrocytes and perivascular macrophages respectively.
CD8+ T cells were observed in the meninges, corpus callosum and scattered throughout the
grey matter, whereas CD4+ T cells and CD79a+ B cells were restricted to the meninges.
Oligodendrocyte numbers were reduced in the upper cortical layers prior to demyelination
(days 1 and 3 post-injection), but were still present in demyelinated lesions at day 7. Numbers
of neurons and astrocytes were not changed. Control animals immunised with IFA and
injected with cytokines had increased presence of inflammatory cells within the meninges but
no demyelination. Animals immunised with rmMOG and injected with PBS had no
demyelination or immune response within the meninges or cortex.
Thus, acute subpial demyelination was dependent on a pre-existing immune response
against myelin protein, coupled with generalised pro-inflammatory signalling within the
meninges. These findings support our hypothesis of a role for meningeal inflammation in the
cortical pathology of MS and describe for the first time an animal model that can be used to
study the molecular mechanisms involved. Future research will aim to maintain meningeal
inflammation and produce a model of chronic demyelination.
initial relapsing-remitting disease phase is associated with inflammatory demyelination and is
treatable with immunomodulatory drugs, the secondary progressive phase (SP-MS) is
associated with ongoing axonal loss and cortical atrophy and is currently untreatable. Studies
of SP-MS have revealed the presence of extensive subpial demyelinated lesions within the
cerebral cortex. This pathology is associated with a high level of meningeal inflammation, a
gradient of cell loss from the cortical surface and high levels of microglia activation. To test
the hypothesis that pro-inflammatory cytokines diffusing from the cerebral meninges could be
responsible, we have established an animal model mimicking cortical grey matter pathology.
Female DA rats were immunised with 5μg recombinant myelin oligodendrocyte glycoprotein
(rmMOG) in incomplete Freunds adjuvant (IFA). This dose was insufficient to initiate
encephalomyelitis, but did initiate an anti-MOG humoral immune response in the periphery.
Twenty days post-immunisation animals received an injection of tumour necrosis factor (TNF)
and interferon gamma (IFNγ) into the subarachnoid space at the sagittal sulcus.
Immunohistochemistry revealed areas of subpial demyelination extending through cortical
layers I–III. Lesions were maximal after 7 days and had resolved by remyelination at 14 days.
A gradient of microglia/macrophage activation was present from the cortical surface. The
extent of demyelination correlated with activation of microglia in the cortex and macrophages
within the meninges. Activated microglia were observed contacting myelin, oligodendrocytes
and neurons. In the demyelinated cortex, expression of the TNF receptors TNFR1A and
TNFR1B was upregulated on oligodendrocytes and perivascular macrophages respectively.
CD8+ T cells were observed in the meninges, corpus callosum and scattered throughout the
grey matter, whereas CD4+ T cells and CD79a+ B cells were restricted to the meninges.
Oligodendrocyte numbers were reduced in the upper cortical layers prior to demyelination
(days 1 and 3 post-injection), but were still present in demyelinated lesions at day 7. Numbers
of neurons and astrocytes were not changed. Control animals immunised with IFA and
injected with cytokines had increased presence of inflammatory cells within the meninges but
no demyelination. Animals immunised with rmMOG and injected with PBS had no
demyelination or immune response within the meninges or cortex.
Thus, acute subpial demyelination was dependent on a pre-existing immune response
against myelin protein, coupled with generalised pro-inflammatory signalling within the
meninges. These findings support our hypothesis of a role for meningeal inflammation in the
cortical pathology of MS and describe for the first time an animal model that can be used to
study the molecular mechanisms involved. Future research will aim to maintain meningeal
inflammation and produce a model of chronic demyelination.
Date Issued
2011-05
Date Awarded
2012-02
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Reynolds, Richard
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)