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The possible protective role of ginseng on the sciatic nerve neuropathy induced experimentally by acrylamide in adult male albino rat: a histological and morphometric study

El-Derieny, Ezzat A.; Moustafa, Khaled A.; Soliman, Gehan M.

The Egyptian Journal of Histology: June 2014 - Volume 37 - Issue 2 - p 350–359
doi: 10.1097/01.EHX.0000446580.88948.42
Original articles

Introduction Ginseng’s active compounds exert beneficial effects on central and peripheral nervous system disorders.

Aim of the study The sciatic nerve was used as a model to study the possible protective effect of ginseng on peripheral neuropathy induced by acrylamide.

Materials and methods The study was carried out on 35 adult male albino rats. The animals were divided into three groups: group I (control), group II treated daily with acrylamide (30 mg/kg body weight) orally for 4 weeks, and group III (protective) treated with acrylamide at same dose, route, and duration as in group II concomitantly with ginseng (20 mg/kg body weight). After 4 weeks, rats were sacrificed. Samples from sciatic nerve were taken and processed for light and electron microscopic and morphometric studies.

Results Light and electron microscopic observations of group II revealed infoldings, splitting, and degeneration of myelin. Changes in axons included degeneration, compression, irregularity, and shrinkage with swollen mitochondria. Large vacuoles and swollen mitochondria were seen inside the Schwann cells. Changes in the myelin and axons in group III were much less frequent than those observed in group II. Only mild splitting and irregular thickening of the myelin with few swollen mitochondria were observed in some axons and Schwann cells. Morphometric study revealed a highly significant reduction (89.6%) in the mean g-ratio (axon/fiber ratio) and body weight in group II compared with the control and group III.

Conclusion Ginseng protected the sciatic nerve from the harmful effect of acrylamide to a great extent.

Department of Histology, Faculty of Medicine, Tanta University, Tanta, Egypt

Correspondence to Gehan M. Soliman, MD, Department of Histology, Faculty of Medicine, Tanta University, 3111, Tanta, Egypt Tel: +20 101 119 1096; fax: +20403339604; e-mail: gehan1969@yahoo.com

Received December 24, 2013

Accepted March 25, 2014

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Introduction

Ginseng refers to the root of several species in the plant genus Panax (C.A. Meyer Araliaceae). The species name ‘ginseng’ comes from the Chinese word ‘rénshēn’, which means ‘human’ as ginseng roots resemble the human body. It is a traditional medicine in Korea, China, and Japan that has been used as a general tonic to increase vitality, health, and longevity, especially in older persons, and it is also used as cancer-preventing agent 1. Ginseng helps the body to resist the adverse influences of harmful factors and to improve restoration of homeostasis. Some ginseng’s active compounds exert beneficial effects on central nervous system disorders and neurodegeneration 2,3.

Ginseng has a wide range of pharmacological activities including immunomodulatory effects, anti-inflammatory activity, and improvement of physical stamina, providing energy and strength in addition to stimulation of the appetite. It is also thought to have effects on learning, memory, and behavior. In addition, it increases the efficiency of the endocrine, metabolic, circulatory, and digestive systems, resulting in increased alertness and reflex actions. American species of ginseng are taken orally as adaptogens, nourishing stimulants, and in the treatment of type II diabetes as well as sexual dysfunction in men. The WHO reported no known contraindications for ginseng 4.

Acrylamide is a reactive, highly water-soluble vinyl monomer. It is a chemical intermediate used for production of polyacrylamide, which is utilized in the synthesis of dyes, contact lenses, waste water treatment, soil conditioning agents, and laboratory gels. Hence, exposure to acrylamide can occur in workplaces or in the environment through air, land, and during its production or use 5,6. Acrylamide is produced when certain foods particularly those rich in carbohydrates are cooked at high temperatures. Potato products, including chips and other potatoes cooked at high temperature, may comprise a large percentage of the total acrylamide intake from food 7,8. Acrylamide is a well-documented neurotoxicant in both human and laboratory animals. Subchronic, low-level occupational exposure of human to acrylamide produces neurotoxicity characterized by ataxia, skeletal muscle weakness, and numbness of the hands and feet 6. As acrylamide has an environmental persistence and widespread distribution and its neurotoxicity was clinically established 6–8, this research was conducted to study the histological changes in the sciatic nerve of adult male albino rat exposed experimentally to acrylamide and to evaluate the possible role of ginseng in alleviating these changes.

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Materials and methods

The present study was carried out on 35 adult healthy male albino rats weighing from 230 to 250 g each. The animals were divided into three groups: group I (the control and ginseng group) consisted of 15 rats and was subdivided into three equal subgroups, five animals each. In subgroup Ia each rat was kept without any treatment for 4 weeks. In subgroup Ib each rat was given distilled water orally by gastric tube at a dose of 0.75 ml daily for 4 weeks. In subgroup Ic each rat was given ginseng alone at a daily dose of 20 mg/kg body weight by a gastric tube for 4 weeks as in group III. Group II included 10 rats treated orally with acrylamide at a dose of 30 mg/kg body weight daily for 4 weeks 9; it is in the form of whitish, odorless water-soluble crystals with the purity above 99%. The solution was prepared by dissolving 1000 mg of acrylamide in 100 ml distilled water; hence, 1 ml solution contained 10 mg acrylamide. Group III (the protective group) included 10 rats treated with acrylamide at the same dissolution, dose, route, and duration as in group II concomitantly with ginseng orally at a daily dose of 20 mg/kg body weight 10 in the form of solution (each 100 ml of solution contains ginseng extract 0.933 g). Acrylamide was purchased from El-Gomhouria Company for Chemicals and Medical Trading (Cairo, Egypt). Pure ginseng was purchased from PHARCO Pharmaceutical Co. (Alexandria, Egypt).

The animals were weighed and examined daily for neurological signs. After 4 weeks, the rats were anesthetized with ether inhalation and perfused after insertion of intracardiac cannula with 500 ml saline followed by 500 ml of a mixture of 4% paraformaldehyde in 0.1 mol/l sodium phosphate buffer (pH 7.4) and 2.5% glutaraldehyde solution. After perfusion, both sciatic nerves were exposed near the greater sciatic foramen and a segment from each was taken (5 mm in length) and cut into specimens. The specimens were immediately fixed in 2.5% phosphate-buffered glutaraldehyde for 2 h at 4°C. After two to three rinses in the buffer, they were postfixed in freshly prepared 1% phosphate buffer osmium tetroxide at 4°C for 1 h. Then they were washed twice in phosphate buffer for 30 min and dehydrated in ascending grades of ethanol. To obtain cross-sections, the nerve samples were oriented longitudinally in a flat mould and embedded in Epon 11.

Semithin cross-sections (1-ìm-thick) were cut with the ultramicrotome, stained with toluidine blue, and examined with a light microscope. For ultrastructural observations, ultrathin sections (70–80 nm) were cut, double stained with uranyl acetate and lead citrate 11, then examined and photographed with a Jeol Electron Microscope (Jeol, Columbia, South Carolina. USA) connected to AMT CCD camera XR 40 (Advanced Microscopy Techniques), Corp 242 WEST Cummings Park, Woburn, MA, USA) using a software AMT Image Capture Engine–V600 for measuring the diameter of the myelinated fibers of the sciatic nerve and the diameter of their axons then estimating the axon/fiber ratio, which is known as g-ratio in some previous studies 12. EM Unit, Faculty of Medicine, Tanta University, Tanta, Egypt.

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Statistical study

Student’s t-test was used to compare the means of the weight of animals of different groups with the control group. Statistical presentation and analysis of morphometric data was conducted, using the mean, SD, percentage of change, and analysis of variance test (the Scheffe test) by SPSS (V.16; SPSS Inc., Chicago, Illinois, USA). Differences were considered significant when P value was 0.05 or less.

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Results

Animals treated with ginseng alone showed the normal behavior as control animals and did not exhibit any signs of neurological abnormality. In contrast, all animals treated with acrylamide developed variable degrees of neurological disturbances in the form of loss of equilibrium, reduction in activity, followed by abduction of hind limb, dragging of the feet along the floor, and finally hind limb paralysis (Photo 1). These signs started 14 days after the beginning of treatment and progressively increased in severity. However, these signs were less noted in animals treated with acrylamide concomitant with ginseng.

Photo 1

Photo 1

With respect to the death record, no animal mortality occurred in group I and gained normal weight during the period of treatment. In the acrylamide received group (group II), six animals died between day 10 and 20 of treatment (60% mortality). The living animals in this group showed a highly significant decrease in their weight as compared with group I. Only one rat in the group treated with acrylamide and ginseng (group III) died on day 25 (10% mortality), and there was an insignificant decrease in the weight of the living animals in this group as compared with group I (Table 1).

Table 1

Table 1

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Histological study of semithin and ultrathin sections

The histological findings were the same in all subgroups of group I. In sciatic nerve of group I, the toluidine blue-stained section showed normal nerve fibers with normal spaces and Schwann cells in-between (Fig. 1). Ultrathin sections of group I showed myelinated axons ensheathed with regular myelin having preserved compact lamellar structure. The outlines of the axons appeared regular and the axoplasm contained microtubules, neurofilaments, and mitochondria. Schwann cell cytoplasm surrounded both myelinated and unmyelinated axons and was surrounded with basal lamina (Fig. 2).

Figure 1

Figure 1

Figure 2

Figure 2

In the sciatic nerve of the acrylamide-treated group II, the toluidine blue-stained sections showed moderate and marked increase in the space between the nerve fibers, with infoldings and irregularity of the myelin and distorted axoplasm (Figs 3 and 4). In ultrathin sections of the same group, variable degrees of myelin abnormalities were observed; the most common and most frequent observation was disruption, splitting, and loss of compact lamellar structure of myelin sheath with excessive infoldings (Figs 3, 5–8, 10 and 12). Fragmentation, thinning out, and abnormal configuration were noticed in medium-sized and large myelinated fibers (Figs 8 and 10). Changes in the axons were more frequent than those observed in the myelin. The affected axons appeared shrunken and compressed by splitted or degenerated myelin or by vacuolated Schwann cell (Figs 5–9). The axoplasm of some nerve fibers contained vacuoles, destroyed swollen mitochondria, membrane-bounded vacuoles with clumped microtubules, and neurofilaments (Figs 7, 8, 11 and 12). Schwann cells showed vacuoles (some of them were autophagic vacuoles) and destroyed swollen mitochondria (Figs 7 and 9–11).

Figure 3

Figure 3

Figure 4

Figure 4

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

Figure 8

Figure 8

Figure 10

Figure 10

Figure 12

Figure 12

Figure 9

Figure 9

Figure 11

Figure 11

In semithin sections of the sciatic nerve of group III (received acrylamide concomitant with ginseng for 4 weeks), there was a mild increase in the space between the nerve fibers, which appeared more or less normal with mild focal lysis of the myelin (Fig. 13). In ultrathin sections of the same group, most of the nerve fibers appeared normal with normal mitochondria, microtubules, and myelin sheaths surrounded by normal Schwann cells (Figs 14 and 16). Changes in the myelin and axons in group III were much less frequent than those observed in group II. Only mild splitting and irregular thickening of the myelin with wide incisures and few swollen mitochondria were observed in the axons and Schwann cells (Figs 15 and 16).

Figure 13

Figure 13

Figure 14

Figure 14

Figure 16

Figure 16

Figure 15

Figure 15

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Morphometric and statistical studies

Table 2 and Histogram 1 show that the mean values of axon/fiber ratio (g-ratio) in group I, II, and III were 0.66±0.21, 0.29±0.06, and 0.55±0.16, respectively.

Table 2

Table 2

Histogram 1

Histogram 1

There was a significant difference between the three studied groups; group II had a significant decrease as compared with group I and group III (P<0.05), and there was a significant increase in group III as compared with group II (P<0.05).

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Discussion

The studies about toxicity of acrylamide have been developed for more than 30 years, and neurotoxicity was the only documented toxicity of acrylamide in human. As acrylamide was found in certain foods in recent years, its toxicity was concerned as a public subject. Furthermore, because of the cumulative nature of acrylamide-induced neurological defects, its neurotoxicity became the focus of research and debate 13.

Previous studies 14 suggested that glycidamide played a causal role in producing the neurological deficits and axonal degeneration induced by acrylamide intoxication of rats. In contrast, other studies have indicated that the parent compound acrylamide and not glycidamide is primarily responsible for induction of neurotoxicity 15,16.

Free radicals are continuously produced in vivo, and there are number of protective antioxidant enzymes for dealing with these toxic substances. Examples of these protective enzymes are superoxide dismutase, catalase, glutathione transferase, glutathione peroxidase, and antioxidant glutathione. The balance between the production and catabolism of oxidants is critical for maintenance of the biological function 17. It is established that the acrylamide is oxidized to glycidamide and conjugates with glutathione mainly in the liver. By depleting glutathione, acrylamide may decrease the antioxidant levels of the cells, leading to an overall increase in intracellular reactive oxygen species and cellular oxidative damage 18. Some investigators 19 stated that the effects of these free radicals are wide ranging, but three reactions are particularly relevant to cell injury. The first reaction is lipid peroxidation of membranes, which can result in extensive membrane, organelles, and cellular damage. The second reaction is oxidative modification of proteins, which can result in protein fragmentation. The third one is the reaction of these radicals with thymine in nuclear and mitochondrial DNA, which can result in single-stranded breaks in DNA.

In the present research, group II showed that the axons of nerve cells were shrunken, with clumping of cytoskeletal elements and splitting and discontinuity of myelin sheath with increase in the spaces between the nerve fibers (most probably due to edema). These findings are in accordance with a previous researcher 13 who reported similar results in his study. The formation of infolded myelin loops is a characteristic response of a large caliber myelin sheath to axonal atrophy, probably reflecting the presence of redundant myelin 20.

Discontinuity and demyelination of axons were attributed to the changes in myelin basic protein secondary to a toxic insult. The myelin membrane is highly vulnerable to damage from exposure to toxic substances, which can result in the loss of the myelin sheath (demyelination) or in alterations in the myelin sheath without producing actual demyelination (dysmyelination). Demyelination can occur following a direct perturbation to the myelinating cell or its myelin sheath or as a response to axonal degeneration. Dysmyelination, however, can include folding and edematous splitting at various levels of the myelin lamella 21. Oxidative stress may impair the axonal membrane, leading to demyelination in mammals 22.

The present study revealed cytoplasmic vacuolation in the axons and Schwann cells of the acrylamide-treated rats, which could be a result of lipid peroxidation, in addition to damage of the cell membrane as well as membranes of other cell organelles 19. This will lead to increased permeability of the membranes and disturbance of the ion concentration in the cytoplasm and cell organelles. Such damage is specifically followed by an increase in the plasma membrane permeability to sodium, which exceeds the capacity of pump to extrude the sodium. Accumulation of sodium leads to an increase in water content in the cell leading to its swelling. As a result, all organelles including mitochondria will also be affected in the form of swelling and destruction 23. The alterations and irregularity in shape that were found in the acrylamide-treated group could be coincided with the results of some previous studies 24.

In the present study, it was observed that daily administration of ginseng with acrylamide markedly reduced the behavioral signs of neuropathy (i.e. paralysis and ataxia) caused by chronic acrylamide administration. Moreover, examination of light and electron microscopic specimens revealed that ginseng showed reduction in the structural changes in sciatic nerve induced by acrylamide. Only mild focal splitting of myelin was observed in some nerve fibers, with few vacuoles and swollen mitochondria in some Schwann cells. These results are in agreement with the results of others 25 who found that ginseng had a protective effect against acrylamide toxicity. This effect may be either direct by inhibiting lipid peroxidation and scavenging free radicals 26 or indirect through the enhancement of the activity of glutathione peroxidase and superoxide dismutase, the enzymatic free radicals scavengers in the cells 10.

Neuroprotection by ginseng was suggested to be, in part, due to its effect on neuroglial cell populations. In this respect, it has been reported that ginseng total saponins prevented Schwann cell and astrocytic swelling induced by glutamate 27, and ginsenoside Rg1 inhibited microglial respiratory burst activity and decreased the accumulation of nitric oxide produced by activated microglia 28. In addition, inhibition of Na+ channels and improved energy metabolism by retarding ATP breakdown in cultured neurons are also involved 29.

A number of studies have shown that some ginsenosides can modulate neurotransmission in the brain. Ginsenosides Rb1 and Rg1, the most abundant ginsenosides in ginseng root, can modulate acetylcholine release and reuptake and the number of choline uptake sites, especially in the hippocampus. They also increase choline acetyl transferase levels in rodent brains. These results suggested that these compounds may improve central cholinergic function in humans 3.

Morphometric analysis of the sciatic nerve 4 weeks after receiving acrylamide (group II) revealed a significant reduction in the g-ratio (axon/fiber ratio), which normally ranges from 0.76 to 0.77 12. This reduction may be due to a decrease in the axon diameter or an increase in the fiber diameter. The increase in the fiber diameter was most probably due to splitting of myelin and presence of large incisures and intramyelinic edema 20. The decrease in the axon diameter might be due to shrinkage, irregularity, and compression by the abnormal myelin observed in this research. Hence, when the morphological changes improved with ginseng (in group III), the g-ratio increased again and showed significant increase compared with group II and insignificant decrease compared with group I.

It is concluded that ginseng reduces the damaging effect of acrylamide on the structure and subsequently the function of peripheral nerves. Hence, ginseng must be prescribed to the workers who may be exposed to acrylamide and can be added to potato products such as chips.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Table

Table

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Keywords:

acrylamide; ginseng; neuropathy; rat

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