Esther Leung

Myelin water imaging in multiple sclerosis: quantitative correlations with histopathology

Various magnetic resonance (MR) techniques are used to study the pathological evolution of demyelinating diseases, such as multiple sclerosis (MS). However, few studies have validated MR derived measurements with histopathology. Here, we determine the correlation of myelin water imaging, an MR measure of myelin content, with quantitative histopathologic measures of myelin density. The multi-component T2 distribution of water was determined from 25 formalin-fixed MS brain samples using a multi-echo T2 relaxation MR experiment. The myelin water fraction (MWF), defined as T2 signal below 30 milliseconds divided by the total signal, was determined for various regions of interest and compared to Luxol fast blue (myelin stain) mean optical density (OD) for each sample. MWF had a strong correlation with myelin stain [mean (range) R2-/0.67 (0.45+ 0.92)], validating MWF as a measure of myelin density. This quantitative technique has many practical applications for the in vivo monitoring of demyelination and remyelination in a variety of disorders of myelin.

Myelin water imaging of multiple sclerosis at 7 T: Correlations with histopathology

Myelin water imaging (MWI) promises to be invaluable in understanding neurological diseases like MS. However, a limitation of MWI is signal to noise ratio. Recently, a number of investigators have performed MWI at field strengths higher than 1.5 T. Our goal was to determine if myelin water imaging at increased SNR, arising from the use of a small bore 7 T MR system with optimized coil geometry, enables the production of superior myelin water maps with increased spatial detail and enables better correlations with histology. Ten formalin-fixed MS brain samples underwent a 32-echo T(2) relaxation experiment which measured myelin water fraction (MWF) on a 7-T animal MRI scanner. MWF correlated strongly qualitatively and quantitatively with luxol fast blue staining for myelin [mean (range): R(2)=0.78 (0.56-0.95), p<0.0001]. The quality and detail of 7 T myelin water maps were far superior to that previously seen at 1.5 T, allowing for visualization of fine structures such as the normal prominent myelination of the deeper cortical layers, the alveus of the hippocampus and rings of preserved myelin in a concentric Balos lesion. 7 T imaging will allow detailed assessment of myelin pathology to a degree not possible with lower field strengths.

A pathology-MRI study of the short-T2 component in formalin-fixed multiple sclerosis brain

Objective: To determine the pathologic basis of areas not exhibiting signal of the short-T2 component of the T2 relaxation distribution in MS, as studied in formalin-fixed brain. Background: A myelin-specific MRI signal would be of great importance in assessing demyelination in patients with MS. Evidence indicates that the short-T2 (10 to 50 millisecond) component of the T2 relaxation distribution originates from water in myelin sheaths. The authors present two cases of MS in which the anatomic distribution of the short-T2 component was correlated with the pathologic findings in postmortem formalin-fixed brain. Method:— One half of the formalin-fixed brain was suspended in a gelatin-albumin mixture cross-linked with glutaraldehyde, and scanned with a 32-echo MRI sequence. The brain was then cut along the center of the 5-mm slices scanned, photographed, dehydrated, and embedded in paraffin. Paraffin sections, stained with Luxol fast blue and immunocytochemically for 2′,3′-cyclic nucleotide 3′-phosphohydrolase for myelin and by the Bielschowsky technique for axons, were compared with the distribution of the amplitude of the short-T2 component of the comparable image slices. Results: The anatomic distribution of the short-T2 component signal corresponded to the myelin distribution. Chronic, silent MS plaques with myelin loss correlated with areas of absence of short-T2 signal. The numbers of axons within lesions were reduced, but many surviving axons were also seen in these areas of complete loss of myelin. Conclusion: In formalin-fixed MS brains the short-T2 component of the T2 relaxation distribution corresponds to the anatomic distribution of myelin. Chronic, silent demyelinated MS plaques show absence of the short-T2 component signal. These results support the hypothesis that the short-T2 component originates from water related to myelin.–1510

Dirty-appearing white matter in multiple sclerosis: preliminary observations of myelin phospholipid and axonal loss.

Abstract“Dirty-appearing white matter” (DAWM) in multiple sclerosis (MS) is defined as a region(s) with ill-defined borders of intermediate signal intensity between that of normal-appearing white matter (NAWM) and that of plaque on T2-weighted and proton density imaging. To delineate the histopathology of DAWM, four formalin-fixed cerebral hemisphere slices of three MS patients with DAWM were scanned with T2- weighted and proton density sequences. The myelin water fraction (MWF) was obtained by expressing the short T2 component as a fraction of the total T2 distribution. Hemispheric sections were then stained with Luxol fast blue (LFB) for myelin phospholipids, for myelin basic protein (MBP) and 2’,3’-cyclic nucleotide 3’-phosphohydrolase (CNP) for myelin; Bielschowsky silver impregnation for axons; and for glial fibrillary acidic protein (GFAP) for astrocytes. Compared to NAWM, DAWM showed reduction in MWF, corresponding to a reduction of LFB staining. DAWM also showed reduced Bielschowsky staining. Quantitatively, the change in MWF in DAWM most consistently correlated with the change in LFB staining. The findings of this preliminary study suggest that DAWM is characterized by loss of myelin phospholipids, detected by the short T2 component, and axonal reduction.

Immunohistochemical localization of peptidylarginine deiminase in the rat brain

The enzyme peptidylarginine deiminase is responsible for the post-translational modification of certain proteins by catalysing the deimination of arginine residues to citrullines. Recently, peptidylarginine deiminase has been purified from rat skeletal muscle and its primary structure determined by molecular cloning. We have used antibodies raised against this enzyme to examine its distribution in the rat central nervous system. A few discrete neuronal cell groups in the telencephalon were selectively stained. These were the septofimbrial nucleus, the anterior commissural nucleus of the hypothalamus, and the anterodorsal thalamic nucleus. The subcommissural organ was intensely stained; but other circumventricular organs were not. In addition, peptidylarginine deiminase-immunoreactivity was found in a large population of small cells throughout the grey and white matter of the rat brain. Some of these cells contained glial fibrillary acidic protein and could thus be identified as astrocytes. However, in many regions these peptidylarginine deiminase-positive cells appeared distinct from the glial fibrillary acidic protein-positive astrocytes. Light and electron microscopic examination indicated that these cells had the morphology of microglia, however they were not stained by a lectin marker for microglia. These cells often surrounded cerebral capillaries, and sent ramifying processes into the neuropil. Peptidylarginine deiminase has been suggested to be involved in the synthesis of the endothelium-derived relaxing factor nitric oxide from arginine-containing molecules. Thus the distinct peptidylarginine deiminase-positive glial cells surrounding cerebral blood vessels may be in a position to regulate local blood flow in response to neuronal activity.

Pathological basis of diffusely abnormal white matter: insights from magnetic resonance imaging and histology.

Background: The pathological basis of diffusely abnormal white matter (DAWM) in multiple sclerosis (MS) has not been elucidated in detail, but may be an important element in disability and clinical progression. Methods: Fifty-three subjects with MS were examined with T1, multi-echo T2 and magnetization transfer (MT). Twenty-three samples of formalin-fixed MS brain tissue were examined with multi-echo T2 and subsequently stained for myelin phospholipids using luxol fast blue, for axons using Bielschowsky, immunohistochemically for the myelin proteins myelin basic protein (MBP) and 2′,3′-cyclic nucleotide 3′ phosphohydrolase (CNP) and for astrocytes using glial fibrillary acidic protein (GFAP). Regions of interest in DAWM were compared with normal appearing white matter. Results: Fourteen of 53 subjects with MS in the in vivo study showed the presence of DAWM. Subjects with DAWM were found to have a significantly lower Expanded Disability Status Scale (EDSS) and shorter disease duration (DD) when compared with subjects without DAWM (EDSS: 1.5 versus 3.0, p = 0.031; DD: 5.4 versus 10.3 years, p = 0.045). DAWM in vivo had reduced myelin water and MT ratio, and increased T2 and water content. Histological analysis suggests DAWM, which shows a reduction of the myelin water fraction, is characterized by selective reduction of myelin phospholipids, but with a relative preservation of myelin proteins and axons. Conclusions: These findings suggest that the primary abnormality in DAWM is a reduction or perturbation of myelin phospholipids that correlates with a reduction of the myelin water fraction.

Diffusely abnormal white matter in multiple sclerosis: further histologic studies provide evidence for a primary lipid abnormality with neurodegeneration.

Abstract Although multiple sclerosis (MS) lesions have been studied extensively using histology and magnetic resonance imaging (MRI), little is known about diffusely abnormal white matter (DAWM). Diffusely abnormal white matter, regions with reduced mild MRI hyperintensity and ill-defined boundaries, show reduced myelin water fraction, and decreased Luxol fast blue staining of myelin phospholipids, with relative preservation of myelin basic protein and 2′,3′-cyclic-nucleotide 3′-phosphohydrolase. Because DAWM may be important in MS disability and progression, further histologic characterization is warranted. The MRI data were collected on 14 formalin-fixed MS brain samples that were then stained for myelin phospholipids, myelin proteins, astrocytes and axons. Diffusely abnormal white matter showed reduced myelin water fraction (−30%, p < 0.05 for 13 samples). Myelin phospholipids showed the most dramatic and consistent histologic reductions in staining optical density (−29% Luxol fast blue and −24% Weil’s, p < 0.05 for 13 and 14 samples,respectively) with lesser myelin protein involvement (−11% myelin-associated glycoprotein, −10% myelin basic protein, −8% myelin-oligodendrocyte glycoprotein, −7% proteolipid protein, −5% 2′,3′-cyclic-nucleotide 3′-phosphohydrolase, p < 0.05 for 3, 3, 1, 2, and 3 samples, respectively). Axonal involvement was intermediate. Diffusely abnormal white matter lipid and protein reductions occurred independently. These findings suggest a primary lipid abnormality in DAWM that exceeds protein loss and is accompanied by axonal degeneration. These phenomena may be important in MS pathogenesis and disease progression, which is prominent in individuals with DAWM.

Ultrastructure of cholinergic neurons in the laterodorsal tegmental nucleus of the rat: Interaction with catecholamine fibers

The ultrastructure of choline acetyltransferase (ChAT)-immunoreactive neurons in the laterodorsal tegmental nucleus (TLD) of the rat was investigated by immunohistochemical techniques. The immunoreactive neurons were medium to large in size, with a few elongated dendrites, contained well-developed cytoplasm, and a nucleus with deep infoldings. They received many nonimmunoreactive, mostly asymmetric synaptic inputs on their soma and dendrites. ChAT-immunoreactive, usually myelinated, axons were occasionally seen in TLD. Only one immunoreactive axon terminal was observed within TLD, and it made synaptic contact with a nonimmunoreactive neuronal perikaryon. The synaptic interactions between ChAT-immunoreactive neurons and tyrosine hydroxylase (TH)-immunoreactive fibers in the TLD were investigated with a double immunohistochemical staining method. ChAT-immunoreactivity detected with a beta-galactosidase method was light blue-green in the light microscope and formed dot-like electron dense particles at the electron microscopic level. TH-immunoreactivity, visualized with a nickel-enhanced immunoperoxidase method, was dark blue-black in the light microscope and diffusely opaque in the electron microscope. Therefore, the difference between these two kinds of immunoreactivity could be quite easily distinguished at both light and electron microscopic levels. In the light microscope, TH-positive fibers were often closely apposed to ChAT-immunoreactive cell bodies and dendrites in TLD. In the electron microscope, the cell soma and proximal dendrites of ChAT-immunoreactive neurons received synaptic contacts from TH-immunoreactive axon terminals. These results provide a morphological basis for catecholaminergic regulation of the cholinergic reticular system.

Thalamic stimulation in multiple sclerosis: evidence for a ‘demyelinative thalamotomy’:

The mechanism of action of deep brain stimulation (DBS) in the alleviation of tremor in multiple sclerosis (MS) and other neurological disorders is unknown. Moreover, whether the trauma accompanying this surgery is responsible for the induction of new MS plaques is controversial. Here we report the first description of the post-mortem imaging and pathologic findings in the brain of a MS patient who underwent thalamic DBS for the treatment of MS-induced tremor. MR imaging of formalin-fixed brain slices was carried out at 1.5, 3 and 7 Tesla and correlated with the histopathology. There were numerous demyelinative plaques in the white mater, cortex and deep gray matter. There were no plaques along the DBS tract within the sections that sampled the deep hemispheric white matter. However, deep within the thalamus focal demyelination approximated the tract, particularly in the region corresponding to the electrical field. The findings in this single case raise the possibility that focal demyelination may be induced by the electrical field and this may be responsible for long-lasting alleviation of tremor in the absence of continued electrostimulation.

Complement and Humoral Adaptive Immunity in the Human Choroid Plexus: Roles for Stromal Concretions, Basement Membranes, and Epithelium

The choroid plexus (CP) provides a barrier to entry of toxic molecules from the blood into the brain and transports vital molecules into the cerebrospinal fluid. While a great deal is known about CP physiology, relatively little is known about its immunology. Here, we show immunohistochemical data that help define the role of the CP in innate and adaptive humoral immunity. The results show that complement, in the form of C1q, C3d, C9, or C9neo, is preferentially deposited in stromal concretions. In contrast, immunoglobulin (Ig) G (IgG) and IgA are more often found in CP epithelial cells, and IgM is found in either locale. C4d, IgD, and IgE are rarely, if ever, seen in the CP. In multiple sclerosis CP, basement membrane C9 or stromal IgA patterns were common but were not specific for the disease. These findings indicate that the CP may orchestrate the clearance of complement, particularly by deposition in its concretions, IgA and IgG preferentially via its epithelium, and IgM by either mechanism.