Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman microspectroscopy

Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm^?1, and 2,945 cm^?1, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.     

Lipid Droplets in Schwann Cells During Tellurium Neuropathy Are Derived from Newly Synthesized Lipid

Exposure of weanling rats to a diet containing elemental tellurium results in a peripheral neuropathy characterized by segmental demyelination and minimal axonal degeneration. One of the earliest ultrastructural abnormalities in tellurium neuropathy is an increased number of cytoplasmic lipid droplets in myelinating Schwann cells. The pathogenesis of these lipid droplets was investigated using light and electron microscopic autoradiography. Nerve lipids were either "prelabeled" with [3H]acetate via in vivo intraneural injection 3 days before a 2-day exposure to tellurium, or "postlabeled" via in vivo intraneural injection or in vitro incubation with [3H]acetate following a 2-day exposure to tellurium. In the prelabeled nerves, myelin became heavily labeled, but the tellurium-induced cytoplasmic lipid droplets were rarely labeled. In the postlabeled nerves, the tellurium-induced cytoplasmic lipid droplets were the most heavily labeled structures within the nerve. These data indicate that the tellurium-induced lipid droplets in Schwann cells are derived from newly synthesized lipid rather than from the early breakdown and internalization of myelin lipids. The earliest biochemical abnormality observed in tellurium neuropathy is an inhibition of cholesterol synthesis at the squalene epoxidase step. This leads to an accumulation of squalene within the nerve. We conclude that the cytoplasmic lipid droplets in Schwann cells contain this accumulated lipid.     

Investigations of thermotropic phase changes in peripheral nerve of frog and rat. A spin label study

The temperature-dependent fluidity of myelin of frog and rat peripheral nerve (Nervus ischiadicus) was studied using the spin label technique. In frog nerve a phase change was detected at 38 °C. In rat nerve no sharp phase change could be established, and the lipid-depleted frog and rat nerve also showed no transition. From the spectral data, it was concluded that in frog and rat nerve the lipid-protein interactions are different, i.e. species dependent. Ca²⁺-depletion of frog nerve caused a loss of transition, while rat nerve remained unaffected. Thus it was indicated that, in frog nerve, Ca²⁺ is involved in the phase change. In the total lipid extract of frog nerve a phase change centered at 32 °C occurred, while the total lipid extract of rat nerve again showed no transition. It is suggested that a connexion exists between our results and investigations on the temperature dependence of an axonal conduction block of nerve.     

Exchange rates at the lipid-protein interface of the myelin proteolipid protein determined by saturation transfer electron spin resonance and continuous wave saturation studies.

The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.     

A comparison of sciatic nerve neuropathy in diabetic and aged rats

We compared the development of sciatic nerve neuropathy in young diabetic rats with that in non-diabetic aged rats. Diabetes was induced in six-month old rats by injection with alloxan and was moderately controlled by single daily injections of insulin. Blood insulin levels in diabetic rats were significantly reduced compared to the aged animals, and glucose was significantly higher in diabetic rats. Sciatic nerve conduction velocities were measured monthly. Both motor and sensory conduction velocities decreased in the diabetic rats to a level that was similar to those in 36-month old rats. The decreases in conduction velocities in the diabetic rats were most dramatic during months 6 through 12 of diabetes. After 6 and 12 months of diabetes, sciatic nerves were examined by electron microscopy and compared to nerves from 24- and 36-month old rats respectively. Ultrastructural changes in the sciatic nerves of diabetic rats at 6 months included disruptions of myelin and dense axoplasm. In comparison, the 24-month old rats only had distorted contours of the nerve fibres. After 12 months of diabetes, the axoplasm had large spaces and the myelin was thickened and deformed. The axoplasm of 36-month old rats was normal in appearance; however the myelin sheath was thickened and split into layers. The Schwann cells were vacuolated and irregular in shape. These observations indicate that diabetes results in the early onset of age-like changes in the sciatic nerve. It suggests that the control of hyperglycemia in humans may preserve sciatic nerve structure and function.     

Altered metabolic incorporation of fucose and leucine into PNS myelin of 25-week-old diabetic (C57BL/Ks〈db/db〉) mice: Effects of untreated diabetes on nerve metabolism

Sciatic nerves of 25-week-old genetically diabetic (C57BL/Ks 〈db/db〉) mice and their litter-mate controls were removed, and their metabolic incorporation of [³H]fucose and [¹⁴C]leucine into myelin was studied in vitro. Untreated diabetic animals showed significant increases (p<0.05) in the fucose/leucine incorporation into myelin when compared to values found for their litter-mates. These results correlated well with previous experiments performed on alloxan or streptozotocindiabetic rats and thus show the in vitro incubation procedure to be a good indicator of altered metabolic conditions in peripheral nerves due to diabetes mellitus. The resulting ratio increases seen in diabetic animals is at variance with the decrease in ratios found in animals undergoing typical Wallerian degeneration. These results suggest that different metabolic processes operate in untreated diabetics than in normals or in those undergoing other degenerative nerve processes.     

A comparison of composition and fluidity of multiple sclerosis and normal myelin

Myelin was isolated from normal-appearing white matter from the brains of 11 multiple sclerosis (MS) patients, 11 normal individuals, and 1 patient with subacute sclerosing panencephalitis (SSPE). Ratios of protein, cholesterol, and phospholipid content, total fatty acid content, and total amino acid content were determined. The lipid bilayer fluidity of the myelin was measured using electron spin resonance and fluorescence polarization spectroscopy. The protein-to-phospholipid ratio was higher than normal, the cholesterol-to-protein ratio was lower than normal while the cholesterol-to-phospholipid ratio was normal in MS myelin. This suggested a relative increase of protein to lipid content in MS myelin. Of the fatty acid content of a total lipid extract, MS myelin had relatively more 16:0, less 18:3, and less 22:3, 24:1 than normal. The total amino acid content of MS myelin was altered in a way which suggested a decreased percentage of basic protein and an increased percentage of proteolipids. SSPE myelin had similar changes in amino acid content but not in protein-to-cholesterol or-phospholipid ratios or fatty acid content as MS myelin. There was no significant difference in myelin fluidity, however, between MS and normal myelin using fatty acid spin labels or fluorescent probes. A correlation was found between the fluidity and the cholesterol-to-phospholipid ratio but the MS and normal samples varied over a similar range. Although these alterations in lipid and protein composition had no effect on fluidity, they may nevertheless have serious consequences for myelin structure.     

Alterations in mRNA Expression of Myelin Proteins in the Sciatic Nerves and Brains of Streptozotocin-induced Diabetic Rats

Diabetic neuropathy is the most common complication of diabetes. We examined the levels and the mRNA expression of myelin proteins in the sciatic nerves and the brains of streptozotocin-induced diabetic rats. The diabetic rats exhibited a decrease in body weight, elevation of the blood glucose level and a decrease in motor nerve conduction velocity at 2 weeks after streptozotocin injection. In the sciatic nerves of diabetic rats, the level of P0 protein and its mRNA expression were markedly reduced at 20 weeks after the injection. In the brains, the levels of proteolipid protein and myelin-associated glycoprotein and their mRNA expression were selectively decreased at 20 weeks after the injection. This affected expression of myelin proteins was found even when no histological abnormalities were detectable. Considering the functional significance of myelin proteins, this impairment of protein expression is possibly involved in the pathogenesis of diabetic neuropathy, including that in brain disorders.     

ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions.

Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.     

Correction of abnormal lipid fluidity and composition of rat ileal microvillus membranes in chronic streptozotocin-induced diabetes by insulin therapy

Diabetes was induced in rats by administration of streptozotocin. After 90-120 days, one group of chronic diabetic animals was treated with insulin for chronic diabetic animals was treated with insulin for 10 days. The lipid fluidity and composition of microvillus membranes prepared from ileal enterocytes of control, diabetic, and insulin-treated diabetic animals were determined. Lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the probes 1,6-diphenyl-1,3,5-hexatriene, DL-2-(9-anthroyl)stearic acid and DL-12-(9-anthroyl)stearic acid, was decreased in membranes of diabetic animals compared to membranes of control and insulin-treated diabetic membranes. The differences in fluidity resulted from an increased cholesterol content and cholesterol/phospholipid molar ratio in membranes of diabetic animals. The activities of sucrase and alkaline phosphatase were also found to be higher in membranes of diabetic animals. Insulin treatment, however, failed to significantly influence the enzymatic activities of these membranes. These studies, therefore, demonstrate that alterations in the lipid fluidity, lipid composition, and certain enzymatic activities exist in microvillus membranes of enterocytes prepared from chronic streptozotocin-induced diabetic rats. Administration of insulin for 10 days to these animals restored membrane fluidity and lipid composition but not enzymatic activities to control membrane levels.     

Fluorescence and ESR spectroscopy studies on the interaction of isoflavone genistein with biological and model membranes

Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein.     

Morphometric studies of peripheral nerves and spinal ganglion cells in streptozotocin diabetic rats

Morphometric studies of sural nerves and dorsal root ganglia cells L5 were performed in 8 diabetic rats 35 or 44d after the administration of streptozotocin and in 8 controls. Morphometry of photographed semithin sections was realized after whole body glutaraldehyde perfusion with semiautomatic MOP AM 02 and MOP Videoplan. The peripheral nerve showed no decrease of the total nerve area, number of nerve fibres or of myelinated area. Parameters area of fibres decreased in diabetic animals caused by reduction of myelin sheath thickness. There were no changes of mean neuron volume and maximal cell diameter in the dorsal root ganglia. Whereas in experimental short-term diabetes the peripheral nerves demonstrate morphologic changes correlating to biochemical abnormalities, no such correlations can be observed in the perikarya. It is suggested, that primary SCHWANN cell lesion is responsible for the observed myelin thickness reduction.     

An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy

Changes in membrane and macromolecular fluidity which may accompany the differentiation processes of sporulation and germination in Bacillus megaterium K.M. are examined by electron spin and nuclear magnetic resonance spectroscopy. No change in membrane lipid fluidity is observed in isolated forespores up to stage VI. Between stage VI and release of mature spores, the ESR spectrum of doxylstearic acid spin labels becomes polycrystalline. This change in spectral fluidity is completely reversed during germination and is paralleled by the rapid release of Ca2+ from the spore. NMR studies also show that the mature spore has reduced macromolecular mobility and an increased nonexchangeable water pool compared with vegetative cells.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis.

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

ESR研究急性运动及恢复期大鼠RBCM脂流动性的改变

本文以大鼠递增负荷力竭性运动为模型,利用电子自旋共振（ＥＳＲ）技术测定急性运动中及不同时程恢复期红细胞膜（ＲＢＣＭ）脂流动性的改变。ＲＢＣＭ脂尾部流动性在短时间中等强度运动（三级负荷末）即有明显下降（Ｐ＜０．０５）,在运动后恢复期进行性加重,并于运动后１２ｈ达极显著改变（Ｐ＜０．０１）：此时膜脂头部流动性也明显下降（Ｐ＜０．０５）。之后二者均有恢复趋势,提示递增负荷力竭性运动对ＲＢＣＭ脂流动性有重要影响,并且ＲＢＣＭ脂流动性与膜功能相互作用,互相影响     

The effects of aerobic exercise training on the age-related lipid peroxidation, Schwann cell apoptosis and ultrastructural changes in the sciatic nerve of rats

The potential role of exercise in preventing the age-related spontaneous peripheral neuropathy has not been studied. We examined the effects of long-term aerobic exercise training on lipid peroxidation, Schwann cell (SC) apoptosis and ultrastructural changes in the sciatic nerve of rats during aging. Three groups of 12-week old Wistar rats ran on a treadmill for 6, 9 and 12 months (exercise trained (ET) group, n=10 each) according to an exercise training program targeted at a speed of 22 m/min (at 7 degrees incline), 60 min/day, 6 days/week. Three corresponding groups of untrained rats were used as the controls (sedentary (SED) group). At the end of each period, sciatic nerve biopsies were performed, and processed for biochemical, immunohistochemical and ultrastructural analyses. The results showed that aging was associated with an increased level of nerve malondialdehyde (MDA, marker of lipid peroxidation) and a higher number of SC apoptosis in SED group. The SED group showed irregular nerve fibers with thin myelin sheaths and areas of myelin-axon detachment. However, the ET group had significantly diminished nerve lipid peroxidation and SC apoptosis. In the ET group, nerve fibers had a thick myelin sheath with frequent folding. These findings suggest that aerobic exercise training protects peripheral nerves by attenuating oxidative reactions, and preserving SCs and myelin sheath from pathologic changes, which occur during normal aging.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

A role for Nogo receptor in macrophage clearance from injured peripheral nerve

We report a role for Nogo receptors (NgRs) in macrophage efflux from sites of inflammation in peripheral nerve. Increasing numbers of macrophages in crushed rat sciatic nerves express NgR1 and NgR2 on the cell surface in the first week after injury. These macrophages show reduced binding to myelin and MAG in vitro, which is reversed by NgR siRNA knockdown and by inhibiting Rho-associated kinase. Fourteen days after sciatic nerve crush, regenerating nerves with newly synthesized myelin have fewer macrophages than cut/ligated nerves that lack axons and myelin. Almost all macrophages in the cut/ligated nerves lie within the Schwann cell basal lamina, while in the crushed regenerating nerves the majority migrate out. Furthermore, crush-injured nerves of NgR1- and MAG-deficient mice and Y-27632-treated rats show impaired macrophage efflux from Schwann cell basal lamina containing myelinated axons. These data have implications for the resolution of inflammation in peripheral nerve and CNS pathologies.     

An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy

Changes in membrane and macromolecular fluidity which may accompany the differentiation processes of sporulation and germination in Bacillus megaterium K.M. are examined by electron spin and nuclear magnetic resonance spectroscopy. No change in membrane lipid fluidity is observed in isolated forespores up to stage VI. Between stage VI and release of mature spores, the ESR spectrum of doxylstearic acid spin labels becomes polycrystalline. This change in spectral fluidity is completely reversed during germination and is paralleled by the rapid release of Ca²⁺ from the spore. NMR studies also show that the mature spore has reduced macromolecular mobility and an increased non-exchangeable water pool compared with vegetative cells.