Accepted Manuscript Title: Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve Author: Mona G. Amer Nehad F. Mazen Nader M. Mohamed PII: DOI: Reference:

S0040-8166(14)00078-0 http://dx.doi.org/doi:10.1016/j.tice.2014.09.002 YTICE 891

To appear in:

Tissue and Cell

Received date: Revised date: Accepted date:

5-6-2014 20-8-2014 8-9-2014

Please cite this article as: Amer, M.G., Mazen, N.F., Mohamed, N.M.,Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve, Tissue and Cell (2014), http://dx.doi.org/10.1016/j.tice.2014.09.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve Mona G. Amer1,2, Nehad F. Mazen1, Nader M. Mohamed3 Histology & Cell Biology Department, Faculty of Medicine, Zagazig University, Egypt. Histology department, Faculty of Medicine, Taif University, Saudi Arabia

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Pediatrics department, Faculty of Medicine, Taif University, Saudi Arabia

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Email: [email protected]

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1- Dr. Mona Gomah Amer. Assistant professor of Histology and Cell Biology, Faculty of Medicine, Zagazig University, Egypt

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2- Dr. Nehad Mazen. Lecturer of Histology and Cell Biology, Faculty of Medicine, Zagazig University. Email: [email protected]

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3- Dr. Nader Mohamed Mohamed. Assistant professor of pediatrics, Faculty of Medicine, Taif University, Saudi Arabia.

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Email: [email protected]

Highlights: -

We investigate the effect of ageing on peripheral nerves

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The underlined oxidative stress in peripheral nerves with aging

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histopathological changes in aged peripheral nerves -

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Calorie

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protect the peripheral nerves against aging changes

Abstract:

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Background: Aging is associated with structural, functional and biochemical alterations in the nervous system. Calorie restriction (CR) was found to retard most physiological indices of aging. Objectives: This work aimed to investigate the effect of CR on age-related changes in sciatic nerves. Materials and Methods: Thirty male albino rats aged 1 month were equally divided into three groups; Group I [control adult-ad libitum AL]: fed a regular diet and sacrificed at the age of 6 months, group II (aged-AL group): fed a regular diet AL and sacrificed at the age of 18 months, and group III (aged CR) fed a 40% calorie restricted diet and sacrificed at the age of 18 months. Rats were anesthetized and sciatic nerves were processed for light, electron microscope and morphometric studies. Oxidative stress in sciatic nerves was investigated by estimation of lipid perioxidation by product malondialdehyde (MDA) tissue level and antioxidant enzyme; superoxide dismutase activity (SOD). Results: The aged (AL) sciatic nerves appeared disorganized, with thick perineurium and increased collagen fibers associated with decreased g- ratio. Abnormal myelin forms were seen as outfolded myelin loops, thin denuded myelin, splitting of myelin into myelin figures and interlamellar vacuoles. Schwann cells revealed vacuolated cytoplasm. There was also significant increase in MDA level and a significant decrease in SOD activity in comparison to control adult (AL). Apparent structural and histomorphological improvement were noticed after CR in aged rats. Conclusion: Ageing caused structural and biochemical alterations in sciatic nerves with alleviating effect of calorie restriction on such effects. Key words: sciatic nerve, ageing, calorie restriction, ultrastructure, oxidative stress

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Introduction:

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Aging is associated with structural, functional and biochemical alterations in the nervous system. Neuromuscular system function declines with age and manifests as dramatic decreases in muscle strength and size, often referred to as sarcopenia [Vandervoort, 2002]. Skeletal muscle atrophy and weakness lead to the loss of functional mobility and independence for many older adults (Roubenoff, 2001). Agerelated changes in the central nervous system (CNS) are well documented and include neuronal loss, demyelination, and deficits in cognitive function; however, little has been reported concerning age-related changes in the peripheral nervous system beyond a decline in nerve conduction velocities (Peters, 2002). Neurons with long processes are particularly vulnerable to degeneration which makes peripheral nerves (PN) susceptible to age-related modifications (Mattson and Magnus, 2006). Signal propagation along axons is facilitated by myelin, a lipid-rich membranous structure formed by Schwann cells (SC). Distinct domains within the myelin and the axonal plasma membrane are maintained by complex signaling events between neurons and glia (Garbay et al., 2000). Therefore, degenerative changes in either cell type have global influences on overall nerve structure and function. A clear understanding of the mechanisms underlying age-related changes in the peripheral nervous system is necessary to fully understand and prevent the decline in neuromuscular function that often accompanies aging (Sims-Robinson et al., 2013).

The health benefits of calorie restriction (CR) have been known to the scientists for decades. In the recent literature, CR or dietary restriction has been generally defined as consumption of nutritious diet that is 40% less in calories compared to ad libitum diet. (Wolf, 2006). Reduced calorie intake without malnutrition extends lifespan in rodents and delays the onset of multiple age-associated diseases (Colman et al., 2014). Although there have been extensive studies on the ability of CR to reduce agerelated changes in the central nervous system, peripheral organs and lifespan 3 Page 3 of 25

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(Mattson et al., 2001; Jolly, 2004), little is known about the effects of such approach on peripheral nerves. Previous studies (Rangaraju et al., 2009 &Opalach et al., 2010) showed that dietary restriction is an efficient means of defying age-related oxidative damage in peripheral nerves associated with reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier. However, no available data on the effect of such mechanisms on the age associated histopathological changes in peripheral nerves.

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We hypothesized that the mechanism of aging is mainly attributed to oxidative damage. Therefore, the present study examined the effect of a 40% caloric restriction (CR) versus ad libitum (AL) feeding on the structural, ultrastructural changes and oxidative parameters associated with aging in sciatic nerves

Materials and Methods:

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All experimental procedures were carried out in accordance with the research protocols following the recommendations of the Institute Review Board Instruction of Care and Use of Laboratory Animals, Faculty of medicine, Zagazig University.

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Animals:

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This study was carried out on 30 male albino rats, aged 1 month, weighing 50-75 g. They were obtained from the Animal House, Faculty of Medicine, Zagazig University, Egypt. Rats were housed in stainless steel cages with a 12-h light/dark cycle and allowed tap water ad libitum. They were allowed to acclimatize for 2 weeks before starting the experiment and observed for general health and suitability for induction in the experiment and included in the study. 2. Experimental design:

Rats were equally divided into three groups as follows:

(1) Group 1 [adult-ad libitum (AL) group]: Animals were fed a regular diet AL (Table A) and sacrificed when they reached the age of 6 months [Bhattacharyya and Thomas, 2004]. They served as the control group. (2) Group 2 (aged-AL group): Animals were fed a regular diet AL and sacrificed at the age of 18 months (3) Group 3 [aged-calorie restricted (CR) group] [Obin et al., 2000]: Animals were fed a 60% calorie diet (Table 1) started at 6 weeks age until the age of 18 months, when they were sacrificed.

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The 40% calorie restriction diet was formulated so that the animals received 40% fewer calories than the ad-libitium-fed animals.

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At the time of sacrifice, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (55 mg/kg bw). A longitudinal skin incision was performed at the upper hind limb level with blunt separation of gluteal muscles to expose the sciatic nerve and 10 mm long nerve fragments were collected and processed for histological and biochemical analysis. 3. Histological study:

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For light microscopy, specimens were fixed in 10 % formalin solution and processed to prepare 5µm thick paraffin sections that were stained with haematoxylin and eosin (Bancroft and Gamble, 2008).

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Parts of sciatic nerves of all groups were processed using simple pre-embedding protocol for staining myelin sheaths, described by Di Scipio et al., 2008. The method involved immersing the specimens in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections were then counterstained using Masson's trichrome counterstain, which permitted the imaging of connective structures in nerves.

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Specimens for electron microscope examination were cut into 1 mm3 pieces and stored in the same fixative overnight at 4º C (2.5% glutraldehyde buffered with 0.1mol/L phosphate buffer at pH 7.4 for 2h). They were postfixed in 1% osmium tetroxide for 1 h, dehydrated through graded alcohol series, and embedded in epoxy resin. Ultrathin sections (50 nm thick) were collected on copper grids and stained with uranyl acetate and lead citrate (Hayat, 2000). 4.Quantitative morphometric analysis:

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The measurements were obtained using computer-based image analysis software (Leica Qwin 500; Imaging Systems, Cambridge, UK). Sections from all groups were analyzed for certain parameters [Inserra et al., 2001]: The nerve cross-sectional area (in micrometer square) was determined by manually outlining the nerve image at ×10 magnification using H&E-stained sections. Total number of nerve axons, number of regularly organized myelinated axons, number of irregular and degenerated myelinated axons and area percentage of blue colored collagen fibers was determined using osmic acid and MT-stained sections at X40..

Fiber and axon diameters were measured using osmic acid and MT-stained sections at X1000 magnification in five different fields for each specimen (rat), following which they were stored and summarized for statistical analysis. The g- ratio (The ratio of axon diameter to fiber diameter) was calculated (Arbuthnott et al., 1980) .All results were statistically analyzed 5. Biochemical analysis: Sciatic nerves were rinsed in ice- cold saline solution and frozen in liquid nitrogen after removal of the adherent tissue. On the day of the homogenate preparation, sciatic 5 Page 5 of 25

nerve segments were measured, weighed and rinsed in ice-cold saline solution. Sciatic nerves were cut into small pieces and then homogenized at 4°C in 2 mL of ice-cold saline (11 mmol L-1 Tris buffer, pH 7.4) with glass homogenizer. The resulting homogenate was passed through a cellulose filter to remove impurities and divided into aliquots for biochemical analysis. All sciatic nerve samples were analyzed for determination of the following parameters:

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a. Determination of lipid perioxidation: Lipid peroxide was estimated by measurement of malondialdehyde (MDA) levels spectrophotometrically in sciatic tissue homogenate according to method done by Buege and Aust, (1978). MDA in the supernatant can react with freshly prepared thiobarbituric acid (TBA) to form a colored complex which has maximum absorbance at 535 nm. The nmol MDA/g wet tissue was calculated from the plotted standard curve prepared from 1,1,3,3tetraethoxypropane.

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b. Superoxide dismutase activity (SOD): sciatic tissue SOD activity was determined by colorimetric assay using the nitroblue tetrazolium method described by Sun et al. (1988) based on the ability of SOD to form H2O2 from superoxide radicals generated by an exogenous reaction involving xanthine and xanthine oxidase which converts nitrobluetetrazolium (NBT) to NBT-diformazan. The NBT-diformazan absorbs light at 550 nm. The extent of reduction in the appearance of NBT-diformazan is a measure of SOD activity.

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6. Statistical analysis

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Data were presented as Mean ± SD. Statistical differences between groups were assessed by one-way Analysis of Variance (ANOVA) followed by Post Hoc multiple comparison tests (least significant different test; LSD) from the SPSS statistical package (version 17; Chicago, IL, USA) to compare changes among individual groups.

Relationships between the morphometric parameters were done by pearson correlation (r) analysis and the correlation coefficients (r) were tested. P < 0.05 was considered statistically significant. Results:

.1 Histological results:

Examination of H&E stained sections of adult (AL) group (fig. 1a) showed sciatic nerve fascicles containing a mixture of large and small myelinated fibers, bounded by connective tissue perineurium. The perineurium appeared as thin layer of connective tissue. The axon contained within each myelin sheath appeared pale with no structure details. The aged (AL) sciatic nerves (fig. 1b,c) appeared disorganized, with thickened perineurium and prominent endoneureal nuclei. The degenerated axons were replaced by hyaline material. In aged (CR) sciatic nerves (fig.1d), most axons were apparently normal.

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In Osmic acid and MT-stained sections of control adult (AL) sciatic nerves (fig. 2a,b), myelin sheaths appeared as well-preserved darkly stained structures, often rounded or elliptical, surrounding pale axons, with minimal blue stained endoneurial collagen fibers. In aged (AL) sciatic nerves (fig. 2c,d), many myelinated nerve fibers appeared degenerated with disrupted axoplasm, and contained dark central cores in place of central axons. Increased blue stained perineurial and endoneurial collagen fibers were also observed. Most myelinated axons of aged (CR) group were apparently normal with minimal blue stained collagen fibers inbetween (fig. 2e,f).

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Ultrathin sections of control adult (AL) group (fig. 3a, b) revealed regular arrangement of the myelinated and unmyelinated nerve fibers. Myelin sheathes were compact and lineated. The axoplasms of both types of nerve fibers contained mitochondria, neurotubules and neurofibrils. Schwann cells with their euchromatic nuclei were seen surrounding the myelin sheath. The endoneurium could be seen between the nerve fibers. The aged (AL) sciatic nerves (fig. 3c, d, e, f) showed different forms of myelin degeneration as out folded myelin loops, thin denuded myelin and splitting of myelin into myelin figures. Mast cells appeared inbetween degenerated myelinated fibers (fig. 3c,d). Myelin sheathes also displayed inter lamellar vacuoles with shrinkage of axoplasm. Schwann cells revealed vacuolated cytoplasm (fig. 3e). Endoneureal blood capillaries were seen with obliterated lumen, heterochromatic irregular endothelial cell nuclei and perivascular collagen fiber infiltration (fig. 3f). In aged (CR) nerve fibers, most myelinated and unmyelinated nerve axons and Schwann cells were apparently normal (fig. 3g,h). 2. Quantitative morphometric results (table: 2 & fig. 4):

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The mean cross sectional area of sciatic nerves was significantly increased in both aged (AL) and (CR) groups compared to control adult (AL) group, with significant decrease in aged (CR) compared to aged (AL) groups(P

Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve.

Aging is associated with structural, functional and biochemical alterations in the nervous system. Calorie restriction (CR) was found to retard most p...
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