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Effects of D-Galactose on the Structure of Nerve Fibers in Cerebellar White Matter.

Byline: Qingfeng Zhu Xueping Ren and Changzheng Zhang

Abstract Chronic administration of D-galactose (D-gal) has been widely reported to mimic brain aging however the potential mechanism underlying this phenomenon remains largely unclear. Here we investigated whether and how the nerve fibers are altered by long-term D-gal exposure. After mice were treated with D-gal (100 mg/kg/day s.c.) for 8 weeks the cerebellar white matter was processed for transmission electron microscopy. The mean diameter of the myelinated fibers and axons as well as the mean thickness of the myelin sheaths was measured. The fiber diameter was 6.80% higher in the D-gal group compared with the controls (P less than 0.01) with the peak diameter distribution shifting from1.01.2 m in the control to 1.21.4 m in the D-gal-treated group. Such an increase was principally due to the increase in the myelin sheath (19.43%) and less in the axon (6.68%).

Myelin sheath thickening in D-gal-treated fibers may result from intra-myelin degeneration such as loosening or splitting of the myelin lamellae which ultimately alters the ratio of the sheath thickness to the fiber diameter. This is likely to impair impulse conduction velocity and play an important role in neural dysfunction in D-gal-treated animals.

Key words: Nerve fiber myelin sheath axon cerebellar white matter; D-galactose INTRODUCTION

It has been demonstrated that chronic administration of D-galactose (D-gal) is capable of causing morphological and functional impairments in the brain resembling symptoms of normal aging insults and thus it is considered an effective paradigm for establishing aging brain models (Cui et al. 2006; Chen et al. 2010; Park and Choi 2012; Gu et al. 2013). The neural impairments underlying the D-gal exposure are presumed to result from increased oxidative stress and disrupted neurotransmitter balance in nervous tissues (Cui et al. 2006; Marosi et al. 2012; Gu et al. 2013). Intriguingly a recent study reports that the number of neurons remains consistent in the cerebral cortex of animals with D-gal-induced aging (Gu et al. 2013) and therefore it is worthy of investigating whether the neuronal configurations undergo D-gal- dependent degeneration.

The nerve fibers are an important base for neural functioning. It is clear that myelin sheaths in nerve fibers in the normal aging brain exhibit marked degenerative alterations such as forming splits and balloons (Bowley et al. 2010; Peters and Kemper 2012) which may correlate with the decline in nerve conduction velocity and disruption in neuronal circuit timing (Lu et al. 2013; Kemp et al. 2014). The present study was designed to identify changes in the diameters of the myelinated fibers and axons as well as the thickness of the myelin sheaths in the cerebellar white matter of control and chronic D-gal-treated mice. We sought to understand the influence of D-gal on the structures of nerve fibers attempting to provide evidence for further investigations on mechanisms of chronic D-gal administration mimicking brain aging.

MATERIALS AND METHODS

Animals

Male Kunming mice (6 weeks of age weighing 202 g) were randomly assigned into two groups: saline-treated group (0.9% saline 1 ml/day s.c.; n = 4) and D-gal-induced aging group (D-gal Sigma St Louis MO USA dissolved in 0.9% saline 100 mg/ml/kg each day s.c.; n = 4); the treatment lasted for 6 consecutive weeks according to a well-established protocol (Prakash and Kumar 2013; Hao et al. 2014). Mice were individually raised in a temperature-controlled (2224C) and 12 h light/dark cycle with food and water ad libitum Drug injection was performed at 16:00-17:00 h daily. All mice were monitored daily and weighed weekly in order to evaluate their health status. Animal housing and the experimental procedures were in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 80-23 revised 1996).

Tissue preparation

Mice were anesthetized with sodium pentobarbital (Sigma; 40 mg/kg i.p.) and transcardially perfused with a solution of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The anterior cerebellum were collected and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer overnight at 4C. Four lobes containing the subcortical white matter were cut into pieces of around 1A-1A-1mm prior to fixation in 1% OsO4 in 0.1 M sodium cacodylate buffer for 2 h at room temperature and dehydration in an ascending acetone series. The osmicated tissue blocks were embedded in Epon-812 (Electron Microscopy Science Hatfield PA USA) and trimmed under a light microscope. Semithick sections were first taken and stained with toluidine blue. The subcortical white matter was identified; then the blocks were turned to obtain horizontal sections of the subcortical white matter where the vertically oriented nerve fibers were arranged.

Ultrathin sections (5070 nm) were subsequently cut with a diamond knife on an LKB-11800 ultramicrotome (LKB Stockholm Sweden) and collected on 300- mesh copper grids. The ultrathin sections were stained with uranyl acetate and lead citrate and observed under an electron microscope (JEOL1400 Tokyo Japan) and electron micrographs were captured at the same time.

Quantification of the fiber diameter axon diameter and myelin sheath thickness

Electron micrographs were taken at a primary magnification of 10000100000 at which the myelin lamellae were distinct. For each block the fiber diameter axonal diameter and myelin sheath thickness were determined by at least 50 nerve fibers. The mean diameters of the fiber and axon were estimated according to our previously used formula (Zhang et al. 2011a; Zhu et al. 2011): d = (aA-b)1/2 (where a and b are the longitudinal and transversal diameters from the section respectively). Each measurement was made three times and the mean values were taken as the values for a single fiber. All the measurements were made blind to the animal identity in order to avoid investigator bias.

The data were then arranged according to the fiber diameter of less than 0.4 0.40.6 0.60.8 0.81.0 1.01.2 1.21.4 1.41.6 and greater than 1.6 m in both the control and D-gal-treated groups. It should be noted that as some fibers were sectioned obliquely our quantitative measurements might be biased to an unknown degree due to the simply visual examination thus the measurements in this study were rough estimates. However the results should not have been affected since the same criteria were used for both groups.

Quantitative analysis

The data are expressed as the mean SD. Student's t test and one-way analysis of variance followed by Fisher's Least Significant Difference post hoc test were used for statistical analysis. P values less than 0.05 were considered to be significant.

RESULTS

Compared with the control group D-gal- treated mice developed mild dyskinesia and signs of slow reaction to various stimuli which were consistent with other observations (Cui et al. 2006; Gu et al. 2013) indicating a successful D-gal- induced aging mouse model. The body weight showed a slight tendency to be elevated in the D- gal-treated mice when compared with these in the controls but showed no statistical difference (data not shown).

Nerve fiber structure in D-gal-treated mice Myelinated fibers from the control mice appeared compact and homogeneous with integral sheaths; whereas the fibers from the D-gal-treated mice showed dilatation with irregular contours in which some sheaths displayed obvious splitting and vacancy in some places (Fig. 1). Some sheaths appeared too loose for the enclosed axons leaving a lot of space between the axon and myelin (Fig. 1). The fiber diameter ranged from 0.28 to 2.18 m however the average fiber diameter in the D- gal-treated group was 1.10 m which was 6.80% thicker than that in the control group (1.03 m). Such thickening might have been mainly due to the sheath thickness (19.43% increase) and less in the axon (only 6.68% increase) (P less than 0.01; Fig. 2) indicating that the myelin sheath underwent more severe alterations due to D-gal. Fiber diameter myelin sheath and axon diameter To map the effects of D-gal on the structures of the specific fibers we had a range of fiber diameters of less than 0.4 0.40.6 0.60.8 0.81.0 1.01.2 1.21.4 1.41.6 and less than 1.6 m (Fig. 3). Within-group analysis revealed that the fiber diameter was highly dependent on these segments [F(7984) = 4995.341 P less than 0.01 in the control group; F(71051) = 4476.551 P less than 0.01 in the D-gal-treated group]. The peak fiber diameter distribution was 1.01.2 m in the control group and shifted to 1.21.4 m in the D-gal-treated mice.

Compared with the controls fiber diameters in the D-gal-treated mice showed no significant difference in the less than 0.4 and 0.40.6 m segments (P greater than 0.05) whereas they were increased by 4.38% 6.54% 5.60% 6.36% 5.28% and 13.02% in 0.6 0.8 0.81.0 1.01.2 1.21.4 1.41.6 and greater than 1.6 m segments respectively (P less than 0.05 or 0.01; Fig. 4). We analyzed alterations in the sheath thickness between the control and D-gal-treated mice based on the fiber diameter. Within-group analysis revealed that the sheath thickness was highly dependent on the diameter segments in both groups [F(7984) = 205.444 P less than 0.01 in the control group; F(71051) = 206.235 P less than 0.01 in the D-gal- treated group]. Compared to the controls a mean thickness of less than 0.4 and 0.40.6 m showed no significant difference in the D-gal-treated mice (P less than 0.05) whereas the thickness significantly increased by 10.39% 13.93% 14.54% 8.71% 15.62% and 12.81% in 0.60.8 0.81.0 1.01.2 1.21.4 1.41.6 and less than 1.6 m segments respectively (P less than 0.05 or 0.01; Fig. 5).

Finally we investigated how D-gal affected the axonal diameter. In both the control and D-gal- treated mice axonal diameter also showed a significant difference dependent on the fiber diameter [F(7984) = 117.262 P less than 0.01 in the control group; F(71051) = 228.244 P less than 0.01 in the D-gal-treated group]. Compared to the control group the mean thickness markedly increased only in the 1.41.6 and greater than 1.6 m segments (7.88% and 17.84% respectively; P less than 0.01; Fig. 6) and showed no significant difference in other diameter segments in the D-gal-treated mice (P less than 0.05) indicating a weak increase in the axon due to D-gal exposure.

DISCUSSION

Several lines of evidence have demonstrated that chronic systemic exposure to D-gal can induce acceleration of brain aging and this paradigm has now been widely applied for establishing brain aging models (Cui et al. 2006; Chen et al. 2010; Park and Choi 2012; Banji et al. 2013; Gu et al. 2013). It is reported that long-term treatment with D-gal induces many kinds of neurodegenerative symptoms such as cognitive decline (Yu et al. 2013; Zhou et al. 2013; Stefanova et al. 2014; Zhu et al. 2014) deficits in learning and memory (Li et al. 2012; Prisila-Dulcy et al. 2012) and impaired locomotion (Banji et al. 2013; Gu et al. 2013). Potential contributing mechanisms may include induction of oxidative stress (Cui et al. 2006; Banji et al. 2013; Hao et al. 2014; Zhu et al. 2014) mitochondrial dysfunction (Prakash and Kumar 2013) retrogression of neuronal organelles (Lei et al. 2013)

Disruption in neurotransmitter balance (Gu et al. 2013) and accumulation of AY- amyloidprotein (Stefanova et al. 2014). The results from our present study indicate that significant alterations also occur in the structures of nerve fibers.

From our observations the myelinated nerve fibers from the D-gal-treated mice exhibited dilatation with irregular morphology and the myelin lamellae were loosened with obvious splitting. These phenomena indicate chronic administration of D-gal caused marked degeneration of nerve fiber structures which is in line with many reports in normal-aging fibers (Peters et al. 2001; Peters 2002) and correlates to our previous reported degenerations in cortical Purkinje cells (Zhang et al. 2011b). We found that the dimensions of the fibers appeared to increase in D-gal-treated mice compared with the controls and this mainly resulted from an increase in the myelin sheath thickness. The increase in the myelin sheaths in D-gal-treated fibers may have been due to splitting and loosening of the myelin. In addition as the demyelinated axons can be remyelinated (Fancy et al. 2011; Yang et al. 2013; Xie et al. 2014)

We speculate that the mechanism underlying the myelin alterations in D- gal-treated animals is complicated due to the concomitance of myelin breakdown and myelin re- formation.

In summary our present study found that the fiber dimensions increased in D-gal-treated mice which may have been mainly due to the increase in myelin sheath thickness. Such alterations may result in deficits in impulse conduction velocity and damage in neural circuits which might be a potential mechanism for behavioral deficits produced by chronic D-gal administration.

ACKNOWLEDGEMENTS

This work was supported by grants from the Natural Science Foundation of Anhui Province (No. 1308085MH127) Natural Science Foundation of Anhui Provincial Education Bureau (No. KJ2013B124; KJ2013A179) and the Scientific Foundations of Anqing Normal University.

Conflict of interest The authors have no conflict of interest.

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Publication:Pakistan Journal of Zoology
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Date:Feb 28, 2015
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