Bovine White Matter Research Articles

Modeling T1 and T2 relaxation in bovine white matter

The fundamental basis of T1 and T2 contrast in brain MRI is not well understood; recent literature contains conflicting views on the nature of relaxation in white matter (WM). We investigated the effects of inversion pulse bandwidth on measurements of T1 and T2 in WM. Hybrid inversion-recovery/Carr–Purcell–Meiboom–Gill experiments with broad or narrow bandwidth inversion pulses were applied to bovine WM in vitro. Data were analysed with the commonly used 1D-non-negative least squares (NNLS) algorithm, a 2D-NNLS algorithm, and a four-pool model which was based upon microscopically distinguishable WM compartments (myelin non-aqueous protons, myelin water, non-myelin non-aqueous protons and intra/extracellular water) and incorporated magnetization exchange between adjacent compartments. 1D-NNLS showed that different T2 components had different T1 behaviours and yielded dissimilar results for the two inversion conditions. 2D-NNLS revealed significantly more complicated T1/T2 distributions for narrow bandwidth than for broad bandwidth inversion pulses. The four-pool model fits allow physical interpretation of the parameters, fit better than the NNLS techniques, and fits results from both inversion conditions using the same parameters. The results demonstrate that exchange cannot be neglected when analysing experimental inversion recovery data from WM, in part because it can introduce exponential components having negative amplitude coefficients that cannot be correctly modeled with nonnegative fitting techniques. While assignment of an individual T1 to one particular pool is not possible, the results suggest that under carefully controlled experimental conditions the amplitude of an apparent short T1 component might be used to quantify myelin water.

Inelastic Behavior in Repeated Shearing of Bovine White Matter

Understanding the brain's response to multiple loadings requires knowledge of how straining changes the mechanical response of brain tissue. We studied the inelastic behavior of bovine white matter and found that when this tissue is stretched beyond a critical strain threshold, its reloading stiffness drops. An upper bound for this strain threshold was characterized, and was found to be strain rate dependent at low strain rates and strain rate independent at higher strain rates. Results suggest that permanent changes to tissue mechanics can occur at strains below those believed to cause physiological disruption or rupture of axons. Such behavior is characteristic of disentanglement in fibrous-networked solids, in which strain-induced mechanical changes may result from fiber realignment rather than fiber breakage.

Characterization of the NMR behavior of white matter in bovine brain

In vitro experiments on 15 white matter samples from five bovine brains were performed on a 1H-NMR spectrometer at 24 degrees C and 37 degrees C. The average myelin water fractions (MWFs) were 10.9% and 11.8% for samples at 24 degrees C and 37 degrees C, respectively. The T1 relaxation time at 37 degrees C was found to be 830 ms, exhibiting monoexponential behavior. A four-pool model including intra/extracellular (IE) water, myelin water, nonmyelin tissue, and myelin tissue was proposed to simulate the NMR behavior of bovine white matter. A cross-relaxation correction was introduced to compensate for shifting of the measured data points and T2 times over the duration of the Carr-Purcell-Meiboom-Gill (CPMG) measurement due to cross relaxation. This correction was found to be slight, providing evidence that MWFs measured using a multiecho technique are near physical values. At 24 degrees C the cross-relaxation times between myelin tissue and myelin water, myelin water and IE water, and IE water and nonmyelin tissue were found to be approximately 227, 2064, and 402 ms, respectively. At 37 degrees C these same cross-relaxation times were 158, 1021, and 170 ms, respectively. The exchange rate between myelin water and myelin was found to be 11.8 s-1 at 37 degrees C, while the exchange rate between IE water and nonmyelin tissue was found to be 6.8 s-1. These exchange rates are of similar magnitude, which indicates that the interaction between IE water and nonmyelin tissue cannot be ignored.

Solubility of Volatile Anesthetics in Bovine White Matter, Cortical Gray Matter, Thalamus, Hippocampus, and Hypothalamic Area

Although known for whole brain, values are lacking for solubilities of modern volatile anesthetics in specific brain regions. Some regions should differ from others (e.g., gray matter versus white matter) because they differ in lipid content and because potent inhaled anesthetics are lipophilic. In the present report, we examined this issue in bovine brain, finding that white matter/gas partition coefficients are 1.6 (desflurane) to 2.4 (halothane) times larger than gray matter/gas partition coefficients, with values for isoflurane and sevoflurane lying between these at 1.9. Values for thalamus/gas, hypothalamic area/gas, and hippocampal/gas partition coefficients lie between those for gray and white matter. These data may be useful in defining the parts of the brain involved with return to consciousness during recovery from anesthesia.

A Neutralizing Anti-Nogo66 Receptor Monoclonal Antibody Reverses Inhibition of Neurite Outgrowth by Central Nervous System Myelin

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

Acid hydrolase activity of cultured bovine oligodendrocytes.

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8-11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.

Analysis of changes in MR properties of tissues after heat treatment.

To characterize changes in the MR parameters of tissues due to thermal coagulation, a series of T(1), T(2), diffusion, and magnetization transfer measurements were performed on a variety of ex vivo tissues: murine slow twitch skeletal muscle, murine cardiac muscle, murine cerebral hemisphere, bovine white matter, murine liver tissue, bovine retroperitoneal adipose tissue, hen egg white, human prostate and human blood. Standardized heat treatments were performed for each tissue type, over the temperature range from 37 degrees C to 90 degrees C. For all tissues, changes in each MR measurement resulting from thermal coagulation were observed above a threshold temperature of approximately 60 degrees C. These changes are explained based on biophysical knowledge of thermal damage mechanisms and the MR properties of normal tissues, and are particularly relevant for interpreting the changes in image contrast that are observed when MRI is used to guide and monitor thermal coagulation therapy procedures. Magn Reson Med 42:1061-1071, 1999.

AMPA-selective glutamate receptor subunits in glial cells of the adult bovine white matter

We have investigated the presence and distribution of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) preferring glutamate receptor subunits GluR1–4 in glial cells of the adult bovine corpus callosum, optic nerve and fornix. To this end, reverse transcription and polymerase chain reaction (RT-PCR) analysis and immunohistochemical experiments were carried out using specific primers and antibodies for each subunit. In the three areas studied, we observed that the main subunits expressed were GluR1–3 and that they were present in the majority of astrocytes. These subunits were located throughout the cell body and processes of fibrous astrocytes and were particularly rich in endfeet and in the glial adventice surrounding the capillaries. In addition, we also observed by immunohistochemistry that the GluR4 subunit was present in a small subpopulation of cells which, based on their morphological and antigenic features, may correspond to immature cells of the oligodendroglial lineage. These results demonstrate a differential expression of AMPA-selective glutamate receptor subunits with respect to glial cell type and raise the possibility that the expression of particular subunits may be associated with specific functions in adult white matter glial cells.

Study of the interaction of the myelin monomeric GTP-binding proteins with other brain proteins.

Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.

Expression of kainate-selective glutamate receptor subunits in glial cells of the adult bovine white matter.

It is now well established that glial cells, like neurons, express a large variety of non-NMDA glutamate receptors. However, knowledge about the subunits forming the receptors and the types of glial cells which express them is incomplete. The aim of this study was to determine the presence and distribution of kainate-preferring glutamate receptor subunits in glial cells in the white matter of adult cattle. To this end, reverse transcription and polymerase chain reaction (RT-PCR) analysis of mRNA extracted from the corpus callosum, fornix and optic nerve of brains of adult cattle was carried out using specific primers that allowed selective amplification of the kainate transcripts for all five subunits were detected. Restriction digestion of the amplified products confirmed the specificity of the PCR amplifications and showed that the extent of Q/R editing in the GluR6 subunit was lower than that described in the rat whole brain. Immunohistochemical experiments revealed that GluR5-7 and KA2 subunits appeared in approximately 50% of astrocytes and 40% of oligodendrocytes. In astrocytes the receptor subunits were located throughout the cell body and processes, whereas in oligodendrocytes they were mostly restricted to the soma. Double-labelling studies demonstrated that in the white matter areas studied GluR5-7 and KA2 subunits are coexpressed in the majority of macroglial cell endowed with kainate receptors. In addition, astrocytes, but not oligodendrocytes, expressing kainate-selective receptors were found to possess the GluR1 subunit, an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit which has previously been found in this macroglial cell type. Taken together, these results provide evidence for the presence and molecular diversity of kainate-selective receptor subunits in glial cells of the adult white matter and for their coexpression with AMPA receptor subunits. The widespread distribution of kainate receptors in glial cells of the white matter suggests that these receptors may be important in axon-glia and/or glia-to-glia signalling.

Proteolipid Protein from the Peripheral Nervous System Also Contains Covalently Bound Fatty Acids

Proteolipid protein, the major protein component of central nervous system (CNS) myelin, is known to contain approximately 2% by weight of covalently bound fatty acids. Recently, this protein was also found, though at a lower concentration, in the peripheral nervous system (PNS). The present study was undertaken to determine whether PNS proteolipid protein, like its CNS counterpart, was fatty acylated. Myeliri PLP and DM-20, the two proteins that constitute the proteolipid protein fraction, were isolated from bovine sciatic nerves by chromatography on a methylated Sephadex G-100 column. The identity of the isolated proteins was determined by (a) SDS-PAGE, (b) Western blot analysis, and (c) amino acid analysis, and fractions containing only PLP and DM-20 were subjected to acid-methanolysis. Gas-liquid chromatography of the released methyl esters revealed the presence of fatty acids (2.9% w/w) distributed as approximately 47% palmitic, 22% stearic, and 19% oleic acid. Similar results were obtained for bovine white matter proteolipid protein when isolated and analyzed in parallel with the peripheral nerve protein. These data unequivocally demonstrate that both PNS and CNS proteolipid protein are equally acylated.

Kinesin heavy chain from bovine brain and Drosophila appear to be highly homologous molecules

A microtubule-enriched fraction was prepared from bovine white matter, and kinesin and other microtubule-associated proteins were extracted from taxol-stabilized microtubules by homogenization and ultracentrifugation in the presence of nucleotides (guanosine triphosphate and adenosine triphosphate). The kinesin-enriched fractions were subjected to preparative SDS-PAGE, and the band representing the kinesin heavy chain was excised, homogenized, and subjected to partial enzymatic digestion with Staphylococcus aureus V8 protease. Four peptides were selected for sequence analysis and compared to the previously published sequence for the Drosophila kinesin heavy chain (Yang JT, Laymon RA, Goldstein LSB, Cell 56:879-889, 1989). All four peptides matched closely with portions of the Drosophila sequence corresponding to the central, alpha-helical domain. Total amino acid composition analysis of bovine kinesin heavy chain also reveals a high degree of homology to the Drosophila sequence.

Lipid-bound, native-like, myelin basic protein

Batch purification of the myelin basic protein (MBP) in the lipid-bound form was obtained from bovine brain white matter by using the slightly polydisperse nonionic detergent, n-octyl-pentaoxyethylene (octyl-POE) and hydroxyapatite. This large-scale procedure can also be carried out in laboratories without chromatographic equipment, and is applicable to small amounts of myelin. More interestingly, removal and inhibition of the proteolytic activity associated with myelin allowed us to obtain more stable and intact forms of the protein when compared with MBP isolated in the lipid-bound form by our previous method. Since it retains binding to all myelin lipids, this purified MBP may be considered as being in a native-like form. In this article, we suggest why this more intact form of MBP could be used to advantage as an alternative to lipid-free, water-soluble MBP in the study, detection, and treatment of myelin damage in pathology.

The regional and subcellular distribution of calcium activated neutral proteinase (CANP) in the bovine central nervous system

Calcium-activated neutral proteinase (CANP) activity was determined in subcellular fractions and in different regions of bovine brain. The CANP specific activity in spinal cord and corpus callosum, areas rich in myelin, were almost six-fold greater than cerebral cortex and cerebellum. Treatment of whole homogenate and myelin with 0.1% Triton X-100 increased the CANP activity by tenfold. Subcellular fractions were prepared from bovine brain gray and white matter. Most of the CANP activity (70%) was in the primary particulate fractions P1 (nuclear), P2 (mitochondrial) and P3 (microsomal). On subfractionation of each particulate fraction, the majority of the activity (greater than 50%) was recovered in the myelin-enriched fractions (P1A, P2A, P3A) which separate at the interphase of 0.32 M- and 0.85 M-sucrose. The distribution of activity was P2A greater than P1A greater than P3A. Further purification of myelin (of P2A) increased the specific activity over homogenate by more than three-fold. The same myelin fractions contained the highest proportion (60%) and specific activity (five-fold increase) of CNPase. The enzyme activity in different regions of brain and in subcellular fractions was increased by 20-39% after the inhibitor was removed. Electron microscopic study confirmed that the myelin fractions were highly purified. The cytosolic fraction contained 20-30% of the total homogenate CANP activity. Other fractions contained low enzyme activity. CANP was identified in the purified myelin fraction by electroimmublot-technique. It is concluded that the bulk of CANP in CNS is tightly bound to the membrane, may be masked or hidden and is intimately associated with the myelin sheath.

Isolation and characterization of bovine brain myelin distribution of 5'-nucleotidase.

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2',3'-cyclic nucleotide 3'-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5'-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH:cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5'-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.

Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter.

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN3, 5 mM beta-mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000 g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72-76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 micrograms). It required 0.8 mM Ca2+ for half-maximal activation and 5 mM Ca2+ for optimal activation. Mg2+ (5 mM) was ineffective while Zn2+ and Hg2+ were inhibitory. The pH optimum was ranged from 7.5-8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purified enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.

Myelin‐specific domain on the plasmalemma of oligodendroglia: Differential expression in the rat and hypomyelinating mouse mutants jimpy and quaking

Monoclonal antibody, 1A9, prepared against bovine white matter, recognizes a proteinaceous, myelin-specific domain in the CNS that is restricted to the surface of oligodendroglia in primary dissociated cell cultures. The antigen is not detected in the PNS or non-neural tissues. Antibody binding is abolished by heating, exposure to SDS and delipidation, indicating that a conformationally sensitive epitope is recognized. The antigen is present in tracts of developing white matter in rat cerebellum beginning at 5 days postnatally. In developing cultures of fetal rat brain the period of rapid onset for the phenotypic expression of 1A9 antigen is similar to that of galactocerebroside, corresponding to 2-4 postnatal days of age. The 1A9 antigen is not observed in white matter or cultured oligodendroglia of the hypomyelinating jimpy mutant mouse, but its expression is qualitatively normal in the quaking mutant. The possibility is raised that 1A9 may be the primary target of the jimpy mutation.

Immunoreactivity and ouabain-dependent phosphorylation of (Na+ + K+)-adenosinetriphosphatase catalytic subunit doublets.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 6% polyacrylamide was used to resolve the 100-kDa catalytic (alpha subunit) polypeptide of (Na+ + K+)-adenosinetriphosphatase from various tissues. The catalytic subunit was identified on immunoblots with antisera against mouse brain catalytic subunit and lamb kidney holoenzyme. Immunoblots and Coomassie Blue-stained companion gels showed double species of the 100-kDa subunit in sucrose gradient fractions of mouse brain and kidney, bovine grey and white matter, purified lamb kidney and duck salt gland holoenzyme, electroplax microsomes, and NaI-extracted microsomes of goldfish and rat brain. The apparent molecular mass differences between the two species in each tissue all ranged between 5 and 8 kDa. Both forms in rat brain and lamb kidney enzyme contain common epitopes reactive with antibodies immunoaffinity-purified on either species from mouse brain. In addition, ouabain-dependent acid-stable inorganic phosphate incorporation was tested with mouse brain, lamb kidney, and electroplax enzyme. Ouabain-dependent phosphorylation was demonstrated in both species in lamb kidney and electroplax and in the larger of the two forms in mouse brain. These results suggest that double species of the phosphorylatable subunit are present generally in epithelial as well as excitable tissues and in fish and avian as well as mammalian species. Work is needed to elucidate their qualitative and quantitative characteristics in different tissues.

Experimental allergic encephalomyelitis induced by proteolipid apoprotein in Lewis rats

Experimental allergic encephalomyelitis (EAE) was induced in inbred Lewis rats by sensitization with bovine white matter proteolipid apoprotein (PLP). 18–61 days after a single injection of 100 μg of PLP, 12 of 31 rats (39%) developed clinical EAE and 18 of 23 (78%) showed pathologic EAE with significant demyelination. Lymphocyte proliferative responses and antibodies to PLP were elevated but did not correlate with the clinical or pathologic state. This is the first demonstration of PLP-induced EAE with significant demyelination in rats and will contribute to the study of autoimmune demyelination.

Complement potentiates the degradation of myelin proteins by plasmin: Implications for a mechanism of inflammatory demyelination

A previous finding, that the basic protein in lyophilized bovine myelin was degraded by macrophage-conditioned media in the presence of plasminogen, suggested that the macrophage-secreted plasminogen activator, along with plasminogen, might have a role in destruction of myelin during inflammatory demyelination. To approximate more closely the conditions expected in vivo, plasmin, or macrophage supernatants plus plasminogen, were incubated with freshly homogenized bovine white matter or freshly isolated myelin, as distinguished from lyophilized myelin. Under these conditions basic protein was not degraded. Phospholipase or lysolecithin potentiated the degradation of basic protein in fresh bovine myelin by plasmin; however, the cultured macrophages did not secrete significant amounts of phospholipase and plasminogen activator simultaneously into the cultured media after activation with any of several different agents. Recently myelin was shown to activate complement. After preincubation of fresh myelin with guinea pig serum, as a source of complement, the basic and proteolipid proteins were vulnerable to plasmin or to macrophage-conditioned media plus plasminogen. C 3-depleted and C 4-deficient sera were not effective, suggesting that these complement components were required for the serum effect. Hypothetically, then, degradation of myelin proteins in the CNS could be initiated by plasminogen activator, secreted by infiltrating macrophages, plus complement and plasminogen, which could enter the CNS through lesions in the blood-brain barrier.