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Effects of safranal, a constituent of saffron, and vitamin E on nerve functions and histopathology following crush injury of sciatic nerve in rats.

ABSTRACT

Safranal is one of the major components of saffron and has many biological effects such as antioxidant property. The present study investigated the effects of safranal on sciatic nerve function after induction of crush injury. We also used of vitamin E as a reference potent antioxidant agent.

In anesthetized rats, right sciatic nerve was crushed using a small haemostatic forceps. Functional recovery was assessed using sciatic functional index (SFI). Acetone spray and von Frey filament tests were used for neuropathic pain assay. Histopathological changes including severities of Wallerian degeneration of sciatic nerve and gastrocnemius muscle atrophy were investigated by light microscopy. Blood levels of malodialdehyde (MDA) were also measured.

The SFI values were accelerated, cold and mechanical allodynia were suppressed, the severities of Wallerian degeneration and muscular atrophy were improved, and the increased MDA level was reversed with 10 consecutive days intraperitoneal injections of 0.2 and 0.8mg/kg of safranal and 100mg/kg of vitamin E.

It is concluded that safranal and vitamin E produced same improving effects on crushed-injured sciatic nerve functions. Inhibition of oxidative stress pathway may be involved in improving effects of safranal and vitamin E on functions and histopathology of an injured peripheral nerve.

Keywords:

Safranal

Vitamin E

Sciatic nerve function

Oxidative stress

Rats

Introduction

Crocus sativus L., commonly known as saffron, is used in folk medicine for various purposes such as an antispasmodic, nerve sedative, expectorant, eupeptic, anticatarrhal, carminative, diaphoteric, stomachic, aphrodisiac and emmenagogue (Schmidt et al., 2007). Saffron contains carotenoid pigments called tricrocin, bicrocin and crocin, a bitter glycoside called picocrocin, and the volatile, aromatic substance safranal (Rios et al., 1996). Safranal as the most abundant chemical in saffron essential oil accounts for 60-70% of volatile fraction (Rezaee and Hosseinzadeh, 2013). Pharmacological studies have suggested antioxidant (Assimopolou et al., 2005), tissue protective (Kianbakht and Mozaffari, 2009), antinociceptive (Tamaddonfard et ah, 2013b), anti-inflammatory (Boskabady et ah, 2012), anti-cancer (Escribano et ah, 1996), antiepileptic (Panthan et ah, 2009), and memory enhancing (Hosseinzadeh and Ziaei, 2006) effects for safranal.

Peripheral nerves form an extensive network that links the brain and spinal cord to all other parts of the body. A nerve injury can interfere with communication between the brain and the muscles controlled by a nerve, and produced disturbances in sensory, motor and autonomic functions (Burnett and Zager, 2004). Inflammatory mediators such as prostaglandins, histamine, cytokines, chemokines, growth factors are involved in degeneration and regeneration processes and subsequent phenomena such as neuropathic pain (Moalem and Tracey, 2006; Camara-Lemarroy et ah, 2010; Dubovy, 2011). It is known that oxidative stress is one of the main causes of nerve damage after injury. Antioxidant therapy can cause fiber regeneration and oxidative stress reduction in injured nerves (Vanotti et ah, 2007). The production of ROS increases in the nociceptive system during neuropathic pain (Kallenborn-Gerhardt et al., 2013).

The effects of safranal on nerve function after peripheral nerve injury, to the best of our knowledge, have not been studied before. In the present study, we investigated the effects of safranal on nerve function and histological changes following sciatic nerve crush injury. In addition, to identify the mechanism that possibly mediates the effect of safranal on peripheral nerve function after sciatic nerve crush injury, the effect of safranal on plasma level of MDA was also investigated. We also compared the effects of safranal with vitamin E (as a potent antioxidant). Vitamin E (alpha-tocopherol), a fat soluble vitamin, is the major chain-breaking antioxidant in body tissues and is the first line of defense against lipid peroxidation, protecting cell membranes from free radical attack (Kamal-Eldin and Appelqvist, 1996).

Materials and methods

Animals

Healthy adult male Wistar rats, weighing 250-280 g were used in this study. Rats were maintained in groups of six animals per cage in a light-dark cycle (light on at 07:00 h) at a controlled ambient temperature (22 [+ or -] 0.5[degrees]C) with ad libitum food and water. All experiments were performed between 12:00hand 19:00 h. All research and animal care procedures were approved by the Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University and were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Chemicals

Chemical used in the present study included safranal (Kosher, purity of [greater than or equal to] 88%) and vitamin E were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. Safranal [(2,6,6-trimethyl-l, 3cyclohexadien- l-carboxaldehyde) or [Cl.sub.0][H.sub.14]O (Fig. 1) (Srivastava et al., 2010; Rezaee and Hosseinzadeh, 2013)] was dissolved in liquid paraffin (Tamaddonfard et al., 2013b). Vitamin E was dissolved in rice bran oil. Sodium dudecyl sulphate, acetic acid, thiobarbituric acid, n-butanol and pyridine were purchased from Merck Chemical Co., Darmstadt, Germany.

Treatment groups

The animals were divided into following 10 groups of six rats each:

Group 1: This group received no injection and no surgery, and served as intact group.

Group 2: In this group, the sciatic nerve was exposed but not crushed and served as sham surgery group.

Group 3: In this group intraperitoneal injection of paraffin was done for 10 consecutive days after surgically-induced crush injury in sciatic nerve and served as crush plus paraffin group.

Group 4-6: In these groups intraperitoneal injections of safranal at doses of 0.05, 0.2 and 0.8 mg/kg were performed after induction of a crush injury in sciatic nerve and served as safranal groups.

Group 7: In this group subcutaneous injection of rice bran oil was done for 10 consecutive days after surgically-induced crush injury in sciatic nerve and served as crush plus rice bran oil group.

Groups 8: In this group subcutaneous injection of vitamin E at a dose of 100 mg/kg was performed after induction of a crush injury in sciatic nerve and served as vitamin E groups.

Group 9: In this group subcutaneous injection of normal saline was done for 10 consecutive days after surgically-induced crush injury in sciatic nerve and served as crush plus normal saline group. This group was compared with paraffin and rice bran oil groups.

Intraperitoneal injections of safranal and paraffin were performed at a volume of 1 ml/kg. Subcutaneous injection volume for rice bran oil and vitamin E was 0.2 ml/rat. In the present study, the used doses of safranal and vitamin E were designed according to previous studies in which the used doses of safranal and vitamin E were 0.025-1 mg/kg and 30-100 mg/kg, respectively (Morani and Bodhankar, 2010; Vigueras-Villasenor et al., 2011; Boskabady et al., 2012; Amin and Hosseinzadeh, 2012).

Surgery

Rats were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). The area above the left lower thigh was shaved and sterilized with betadine. A 2 cm incision was made over the lateral aspect of the hind limb, and muscles are separated in order to expose the sciatic nerve. The nerve was crushed at 0.5 cm proximal to its trifurcation point using a small haemostatic forceps, the jaws of which were covered with teflon tubing to provide smooth surface. The nerve was crushed for 60 s with an estimated pressure of 0.5-1 kg/[mm.sup.2]. The crushed zone was approximately 2-3 [mm.sup.2] and uniformly transparent for several minutes thereafter (Tamaddonfard et al., 2013a). The muscle layers were re-approximated using 4/0 chromic gut sutures, and the skin was closed with 3/0 silk sutures.

Sciatic functional index

Evaluation of SFI was performed one day before and on days 7, 14 and 21 following surgery. Rats were held by the chest and their hind feet were pressed down onto a stamp pad soaked with water soluble blue ink. Rats were immediately allowed to walk along a confined walkway 7.5 cm wide by 60 cm long with a dark shelter at the end of the corridor leaving its foot prints on the paper that is cut to the appropriate dimensions and placed on the floor of the corridor. The following measurements were taken from the footprints: 1, distance from the heel to the third toe, the print length (PL); 2, distance from the first to fifth toe, the toe spread (TS); and 3, distance from the second to the fourth toe, the intermediary toe spread (ITS). All three measurements were taken from the experimental (E, undergoing sciatic nerve crush) and normal (N) limbs. Three factors that comprised the SFI were calculated as follows: 1, print length factor (PLF) = (EPL-NPL)/NPL; 2, toe spread factor (TSF) = (EST-NST)/NST; 3, intermediary toe spread factor (ITF) = (EIT - NIT)/NIT. Using these data, the SFI, which indicates the differences between the injured and the intact contralateral paw, was calculated by the following formula derived by Bain et al. (1989):

SFI = -38.3 [[EPL - NPL]/NPL] + 109.5 [[ETS - NTS]/NTS] + 13.3 [[EIT - NIT]/NIT] - 8.8

The SFI was analyzed as: an SFI equal to--100 indicates significant impairment, whereas an SFI oscillating around 0 is considered to reflect normal function.

Nociceptive tests

Cold allodynia was measured as the number of foot withdrawal responses after application of cold stimuli to the plantar surface of hind paw (Choi et al., 1994; Tamaddonfard et al., 2013b,c). One drop of 100% acetone was gently applied to the mid-plantar surface of the rat with a syringe connected to a thin polyethylene tube. A brisk foot withdrawal response after the spread of acetone over the plantar surface of the paw was considered as a sign of cold allodynia. The testing was repeated 10 times with an interval of approximately 3-5 min between each test. The response frequency to acetone was expressed as a paw withdrawal frequency (PWF: number of paw withdrawals/number of trails x 100).

Mechanical allodynia was assessed using an electronic von Frey Anaesthesimeter (IITC-Life Science Instruments, Woodland Hill, CA) as described by Chaplan et al. (1994) and Tamaddonfard et al. (2013b,c). Briefly, the rats were placed in individual plexiglass chambers (18 cm x 10 cm x 20 cm) with wire mesh floor, and allowed to explore and groom until they settled down. A set of von Frey filaments with bending force ranging from 1 to 60g (No. 5-15, respectively) were applied in an ascending order to the plantar surface of the right hind paw. Hind paw withdrawal was considered as positive response. The stimulation with one filament was repeated five times at 10-15 s intervals, when lack of a response, the next filament with greater bending force was applied. The lowest force required to elicit a paw withdrawal response was recorded as the paw withdrawal threshold (PWT) (g).

Biochemical assays

At day 21 after sciatic nerve lesion, the animals were sedated using diethyl ether and blood samples (0.5 ml) were collected in vials containing heparin by heart puncture. The plasma was separated and kept at -80[degrees]C until analysis of MDA. MDA, an index of free radical generation/lipid peroxidation, was determined as described by Ohkawa et al. (1979). Briefly, the reaction mixture consisted of 0.2 ml of 8.1% sodium dudecyl sulphate, 1.5 ml of 20% acetic acid (pH 3.5), 1.5 ml of 0.8% aqueous solution of thiobarbituric acid and 0.2 ml of blood plasma. The mixture was made up to 4 ml with distilled water and heated at 95[degrees]C for 60 min. After the cooling the contents under running tap water, 5 ml of n-butanol and pyridine (15:1, v/v) and 1 ml of distilled water was added. The contents were centrifuged and the organic layer was separated out and its absorbance was measured at 532 nm. Plasma MDA concentrations were expressed as nmol/ml.

Histopathological evaluation

At the day 21st following the blood collection, the sedated rats were euthanized and distal segment of sciatic nerve and gastrocnemius muscle were removed and fixed in 10% buffer formal saline. The formalin fixed nerves and muscles routinely processed for paraffin embedding, thin (4-5 [micro]m) transverse sections from nerves and muscles were cut and stained with hematoxylin and eosin (H&E) for light microscopic observations. The evaluation of the sections was based on the severity of pathological changes on a scale from normal (0) to sever (3) changes (Tamaddonfard et al., 2013a).

Statistical analysis

All data are presented as mean [+ or -] SEM. Significance of the SFI and cold allodynia, mechanical allodyni were assessed by factorial analysis of variance (ANOVA) followed by Duncan's test for multiple comparisons. Values for the Wallerian degeneration, number of myelinated axons, degree of muscle atrophy and MDA level were analyzed using one-way ANOVA followed by Duncan's test for multiple comparisons. Significance at p < 0.05 has been given receptive in all tests.

Results

Groups one (intact) and two (sham surgery) showed no significant differences. Therefore the results belong to sham surgery have not been shown in figures. We did not observe any significant differences among groups 3 (crush + paraffin), 7 (crush + rice bran oil) and 9 (crush + normal saline). These results showed that liquid paraffin and rice bran oil did not affect sciatic nerve function. Therefore, the results belong to groups 3, 7 and 9 have been presented as only a crush group in figures.

Intact group showed near zero SFI scores throughout the study. The SFI scores in crush group showed a dramatic decline near to -100 on day 7 after crush followed by a gradual increase to the end of the experiment. Safranal at a dose of 0.05 mg/kg did not alter the SFI scores pattern induced by crush, whereas, at doses of 0.2 and 0.8 mg/kg, it significantly (p<0.05) recovered SFI scores nearly to intact group. No significant differences were observed between the effects of safranal (0.8 mg/kg) and vitamin E on SFI (Fig. 2).

Weak paw withdrawal frequencies were observed after paw spray of acetone in intact group. Crush group respond vigorously to acetone spray test during the three weeks evaluation period. Safranal at a dose of 0.05 mg/kg had no effect on paw withdrawal frequencies, whereas at doses of 0.2 and 0.8 mg/kg, it significantly (p<0.05) decreased acetone spray-induced paw response. Vitamin E at a dose of 100 mg/kg produced the same effect as 0.2 and 0.8 mg/kg of safranal did (Fig. 3).

Plantar surface application of von Frey filaments showed paw withdrawal threshold around 35 and 5 g for intact and crush groups. Paw withdrawal threshold was not influenced with 0.05 mg/kg of safranal, whereas safranal at doses of 0.2 and 0.8 mg/kg significantly (p<0.05) increased paw withdrawal threshold during the three weeks evaluation period. No significant differences were observed between safranal (0.8 mg/kg) and vitamin E (100 mg/kg) in recovering of paw withdrawal threshold (Fig. 4).

Figs. 5 and 6 show the effect of safranal and vitamin E on histopathological changes in the sciatic nerve induced by crush injury. In intact group, no histopathological changes were observed (Fig. 5A). Crush injury produced severe changes in the sciatic nerve, including swelling of myelin sheet, vacuolization and myelin ellipsoids (Figs. 5B and 6). Safranal at a dose of 0.05 mg/kg produced no significant effect (Figs. 5C and 6). Safranal at doses of 0.2 (Figs. 5D and 6) and 0.8 (Figs. 5E and 6) mg/kg and vitamin E at a dose of 100 (Figs. 5F and 6) mg/kg significantly (p < 0.05) alleviated all the histological changes consequent to the crush injury in the sciatic nerve.

Figs. 7 and 8 show the effect of safranal and vitamin E on the muscle atrophy severity in the sciatic nerve induced by crush injury. In intact group, no muscular atrophy was observed (Fig. 7A). Crush injury produced severe atrophy (Figs. 7B and 8). Safranal at a dose of 0.05 mg/kg produced no significant effect (Figs. 7C and 8). Safranal at doses of 0.2 (Figs. 7D and 8) and 0.8 (Figs. 7E and 8) mg/kg and vitamin E at a dose of 100 (Figs. 7F and 8) mg/kg significantly (p < 0.05) prevented the muscular atrophy induced by sciatic nerve injury.

Blood levels of MDA in intact and crush groups were 0.94 [+ or -] 0.06 and 1.57 [+ or -] 0.14nmol/ml, respectively. Safranal at a dose of 0.05 mg/kg did not alter blood level of MDA, whereas at doses of 0.2 and 0.8 mg/kg it significantly (p < 0.05) recovered the increased level of blood MDA. The same lowering effect of blood MDA level was observed in vitamin E (100 mg/kg) treated group (Fig. 9).

Discussion

In the present study, safranal and vitamin E accelerated the retune of SFI. The SFI is a quite useful tool for the evaluation of functional recovery of the sciatic nerve of rats in a number of experimental injuries and treatments (Varejao et al., 2001; Tamaddonfard et al., 2013a). There are no evidences showing the effect of safranal on motor impairment due to peripheral nerve injury. Other constituent of saffron, crocin, has been reported to accelerate the return of SFI in sciatic nerve crush injured rats (Tamaddonfard et al., 2013a). Al Moutaery et al. (1998) reported a correlation between functional recovery and vitamin E content of sciatic nerve on 10th day after sciatic nerve crush in normal and diabetic rats.

In the present study, safranal and vitamin E suppressed cold and mechanical allodynia due to sciatic nerve crush injury. Our data are in accord with Lancellota et al. (2003) who recently published that rats subjected to crush lesion display hyperalgesia to both mechanical and cold stimuli. In chronic constriction injury model of neuropathic pain in rats, intraperitoneal injection of safranal, attenuated cold and mechanical allodynia as well as thermal hyperalgesia (Amin and Flosseinzadeh, 2012). Moreover, in carrageenan-induced inflammatory pain, we showed suppression of cold and mechanical allodynia and mechanical hyperalgesia by safranal (Tamaddonfard et al., 2013a). Early co-administration of vitamin E and methylcobalamin after induction of sciatic nerve crush lesion ameliorated neuropathic pain responses by improving paw withdrawal latency and motor nerve conduction velocity in rats (Morani and Bodhankar, 2010).

The results of present study showed swelling of myelin sheet, vacuolization and myelin ellipsoids in the distal segment of sciatic nerve as well as gastrocnemius muscle atrophy after crush injury. When an axon is crushed or severed, changes occur in proximal and distal segments of the lesion. Distally, Wallerian degeneration takes place, resulting in axonal degeneration. The myelin sheet of axon detaches, degrades and converts into ellipsoidal segments (Burnett and Zager, 2004; Hall, 2005; Tamaddonfard et al., 2013a). Following sciatic nerve crush injury or resection, gastrocnemius muscle weight loss and atrophy were reported (Zhang et al., 2006; Liu et al., 2007). There are no report showing the effects of safranal on peripheral nerve degeneration and regeneration processes and muscular atrophy. However, safranal produced protective effects on hippocampal neurons in ischemic brain and on skeletal muscle in lower limb ischemia-reperfusion injury in rats (Hosseinzadeh and Sadeghnia, 2005; Hosseinzadeh et al., 2009). In vitamin E deficient rats, two months after crush denervation of sciatic nerve, the number of myelinated fibers was lower than in the corresponding control suggesting the involvement of vitamin E in nerve regeneration processes (Cecchini et al., 1990).

In the present study, plasma MDA level was increased after crush. It has been reported that MDA level increases in ischemia/reperfusion injury of sciatic nerve in rats (Bagdatoglu et al., 2002). In addition, an elevation of MDA level was reported in rats with crush injury in sciatic nerve (Tamaddonfard et al., 2013a). Moreover, Senoglu et al. (2009) reported an elevation of sciatic nerve tissue MDA levels after sciatic nerve crush injury in rats. It has been reported that mitochondrial reactive oxygen species involve in the mechanism underlying denervation-induced skeletal muscle atrophy (Muller et al., 2007). Safranal reduced the level of MDA in the hippocampus and skeletal muscle in rats underlying cerebral and lower limb ischemia-reperfusion injuries, respectively (Hosseinzadeh and Sadeghnia, 2005; Hosseinzadeh et al., 2009). In denervation induced by right leg sciatic nerve axotomy, treatment with vitamin E reduced the increased level of MDA in gastrocnemius muscle in rats (Demiryurek and Babul, 2004).

In conclusion, the results of present study showed that safranal and vitamin E produced same improving effects on motor impairment, pain hypersensitivity, Wallerian degeneration, and muscular atrophy induced by sciatic nerve crush injury. These improving effects of safranal and vitamin E may be mediated through their antioxidant effects by reducing MDA level.

http://dx.doi.org/10.1016/j.phymed.2013.10.031

ARTICLE INFO

Article history:

Received 25 August 2013

Received in revised form 30 September 2013

Accepted 27 October 2013

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Esmaeal Tamaddonfard (a), *, Amir Abbas Farshid (b), Shirin Maroufi (b), Sharare Kazemi-Shojaei (a), Amir Erfanparast (a), Siamak Asri-Rezaei (c), Mina Taati (a), Milad Dabbaghi (a), Mona Escort (a)

(a) Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia 57153-1177, Iran

(b) Division of Pathology, Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia 57153- 1177, Iran

(c) Division of Clinical Pathology. Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia 57153-1177, Iran

* Corresponding author. Tel.: +98 441 2770508; fax:+98 441 2771926.

E-mail addresses: etamaddonfard@yahoo.com, e.tamaddonfard@urmia.ac.ir (E. Tamaddonfard).
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Author:Tamaddonfard, Esmaeal; Farshid, Amir Abbas; Maroufi, Shirin; Kazemi-Shojaei, Sharare; Erfanparast, A
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
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