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COMPARATIVE EXERCISEPHYSIOLOGY

Wageningen Academic P u b l i s h e r s

ISSN 1755-2540

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Comparative Exercise Physiology, 2017; 13 (1): 45-52 Wageningen Academic P u b l i s h e r s

ISSN 1755-2540 print, ISSN 1755-2559 online, DOI 10.3920/CEP160028 45

1. Introduction

Peripheral nerves are vulnerable to traumatic injuries. Young healthy civilians and military officers are mostly at risk of traumatic injuries (Isaacs et al., 2013). Sciatic nerve injury (SNI) causes motor, autonomic, and sensory alterations in the region supplied by the nerve (Lee and Wolfe, 2000). Many cases of SNI suffer from incomplete recovery which leads to limitations in daily life and work activities (Uzun et al., 2006). Exercise has a great beneficial effect on nerve regeneration and functional recovery on either central or peripheral nervous system injury (Funk et al., 2011; Lee et al., 2014).

During exercise different cytokine concentrations can increase, such as interleukin (IL)-8, macrophage inflammatory protein (1α and 1β) and tumour necrosis factor α (that increases only in prolonged strenuous exercise). However, IL-6 is by far is the most marked cytokine elevated in the circulation and its appearance precedes that of the other cytokines (Pedersen and Febbraio, 2008) IL-6 is known to be produced by T lymphocytes for B cells differentiation and to induce immune response. However, later it was reported that IL-6 had a wide range of activities, including its action as a growth factor (Tadamitsu, 2006). During rest, muscle IL-6 mRNA content is very low, while IL-6 is detectable after 30 min of exercise and can increase up to 100-fold at the end of the exercise bout (Nieman et al., 2015).

IL-6/STAT3 growth signalling induced by exercise conditioning promotes regeneration of

injured rat sciatic nerve: return to an old cytokine

H. Ashour1*, L.A. Rashed2 and M.M. Sabry3

1Department of Physiology, Kasr Alainy, Faculty of Medicine, Cairo University, 11562 Cairo, Egypt; 2Department of Biochemistry, Kasr Alainy, Faculty of Medicine, Cairo University, 11562 Cairo, Egypt; 3Department of Histology, Kasr Alainy, Faculty of Medicine, Cairo University, 11562 Cairo, Egypt; [email protected]

Received: 20 October 2016 / Accepted: 5 December 2016 © 2017 Wageningen Academic Publishers

RESEARCH ARTICLEAbstract

The effects of interleukin-6 (IL-6), a growth promoting myokine through signal transduction, and activator of transcription 3 (STAT3), as a growth promoting signal, in peripheral nerve injury (PNI) are still unclear, including whether exercise pre- and/or post-conditioning are useful in nerve regeneration. Four groups were included in the study: sham for sciatic nerve injury (control), sciatic nerve injury group (SNI), exercise post-conditioning (SNI+Ex) and exercise pre- and post-conditioning (Ex+SNI+Ex). IL-6 levels were measured in serum, muscle, nerve and its surrounding fascia. Elevated levels of IL-6 in serum, nerve, muscle and fascia revealed that IL-6 induced in nerve by exercise were: (1) local nerve tissue expression; (2) serum diffused as a myokine from contracting muscle; and (3) diffused for surrounding peri-tendinous region. Evaluation of nerve functions shows that, exercise post-conditioning significantly improved (P

H. Ashour et al.

46 Comparative Exercise Physiology 13 (1)

IL-6 increases in the dorsal root ganglia (DRG) during early development, but in adults the levels are very low. However, following SNI, its expression increases dramatically, not only in the DRG but also in many sites, such as in the corresponding motor nucleus or sympathetic ganglion (Yang et al., 2015). In the SNI model, IL-6 is potently expressed within axotomised neurons, thus neuronal induction of IL-6 in response to injury probably may be a general protective response. Thus IL-6 may behave like a neurotrophin (Erta et al., 2012). The study of peripheral nerve injury (PNI) and regeneration still needs to be studied in animal models in order to establish a mean for prophylaxis and management of PNI. We hypothesised that, exercise induced nerve IL-6/STAT3 signalling as a growth factor in regeneration of SNI, and elevated levels of nerve IL-6 may originate from different sources. Also exercise pre- and post-conditioning may have a role in promoting nerve regeneration.

2. Materials and methods

Animals and protocol

Thirty-two adult male albino rats (weighing 120-150 g) were included in the study, purchased from the laboratory animal house unit of Faculty of Medicine, Cairo University. The animals were housed 1 week prior to experiments in cages with 12 h light/dark cycles with free access to water and standard rat chow in the institutional animal care unit. All experimental procedures have been approved by the ethical committee of college of medicine. Rats were randomly assigned into one of the following 4 groups (8 rats/group): Sham operated group (control), SNI, nerve injury followed exercise (SNI+Ex), nerve injury group preceded and followed by exercise (Ex+SNI+Ex).

Sciatic nerve crush injury

SNI was induced as described by Bobinski et al. (2011) with minor modifications, under deep anaesthesia using ketamine (80 mg/kg, i.p.) considering aseptic precaution. The left sciatic nerve was exposed by making a 10 mm incision in the gluteal region and crushed twice with a 2-mm-wide non-serrated forceps for 30 s separated by a 5 s interval. The crushed areas of the sciatic nerves looked flattened with preservation of nerve continuity. The limb distally showed flaccid paralysis immediately after induced SNI. Incision was closed in 2 layers by a surgical suture and was left to heal for 2 days. Sham-operated animals underwent the same procedure without crushing the nerves.

Swimming exercise

All swimming animals were adapted to the water depth and temperature 2 days before the start of the exercise for 10 minutes in a glass tank (50×50×70 cm) filled with

up to 30 cm water of room temperature (32±1 °C). This adaptation period is important to familiarise the animals with swimming and to avoid physical and psychological stress. Rats underwent incremental swimming exercise during 5 days as shown in Table 1. This schedule was applied to the (SNI+Ex) group after nerve injury, and two times in the (Ex+SNI+Ex) group, before and after injury.

Sciatic nerve conduction velocity

Measurement of in vivo sciatic nerve conduction velocity (NCV) was done according to Lawrence et al. (2001). Rats were anesthetised using ketamine (50 mg/kg i.p.) and xylazine (10 mg/kg i.p.) (Sigma, Cairo, Egypt). The left sciatic nerve with its innervated gastrocnemius muscle was exposed and the muscle potential recorded with a needle electrode inserted in the muscle. The stimulating electrodes were placed on either sides of the nerve at two points; 4 and 2 cm from the recording muscle electrodes. Stimulating electrodes were connected to a model ML866 PowerLab (AD Instruments, Dunedin, New Zealand) with Lab Chart 7 software (AD Instruments). The stimulator was adjusted to give impulse width: 0.5 msec with increasing intensity to reach maximum nerve compound action potential (CAP) amplitude; digital filter: low pass 30. The latency of the CAP was measured and the NCV was calculated by difference method, in which two different recording positions, proximal and distal, from the stimulating electrodes were used (this gives two distances and two latencies):

(d2–d1)NCV = (latency distal – latency proximal)

where is d2 is the long distance and d1 is the short one (m/s) (Lawrance et al., 2001).

Sample collection

While the animals were under anaesthesia, retro-orbital blood samples were collected for assessment of serum IL-6. The animals then were decapitated, and the fascia surrounding nerve bifurcation was excised and the left gastrocnemius muscles were excised for IL-6 level analysis. Tissue samples from left sciatic nerve were dissected from the popliteal fossa, divided into 2 pieces for histological and IL-6, IL-6R and STAT3 analysis in the nerve tissue.

Table 1. Exercise conditioning schedule.

Days Total exercise time/min Rest period(s)/min

1 20 1×102 30 1×103 45 2×104 60 2×105 90 2×10

IL-6 acts as a growth factor in regeneration of nerve injury

Comparative Exercise Physiology 13 (1) 47

Light microscopic studies

From sciatic nerve, small tissue pieces were fixed in fresh 2.5% glutaraldehyde in sodium phosphate buffer (pH 7.4). Specimens, each of 1 mm in length, were cut from the fixed sciatic nerve and washed in 0.5 mol/l phosphate buffer (pH 7.4) for 2 h (two changes) and postfixed in 1% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon resin. For light microscopy, serial semithin 1 µm sections were cut using a Zeiss 6M ultramicrotome (Carl Zeiss AG, Munich, Germany), stained with 1% toluidine blue, and examined. For electron microscopy, ultrathin sections (50-80 nm) were prepared and stained with uranyl acetate and lead citrate (Hayat, 2000). The sections were examined using a Zeiss 100S transmission electron microscope.

Morphometric studies

Using a Leica Qwin 500 LTD image analyser computer system (Cambridge, UK), the following parameters were measured: mean area % of mylinated nerve fibres and total number of nerve fibres. Five slides of five different specimens were examined; from each slide, 10 non-overlapping fields were measured at a magnification of ×400.

Gene expression of interleukin-6 and STAT-3 in nerve tissue measured by real time PCR

Total RNA extraction

Total RNA was extracted from tissues using TRIzol method according to the manufacturer’s protocol. In brief, RNA was extracted by homogenisation in TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). The homogenate was then incubated for 5 min at room temperature. A 1:5 volume of chloroform was added, and the tube was vortexed and centrifuged at 12,000×g for 15 min. The aqueous phase was isolated, and the total RNA was precipitated with absolute ethanol. After centrifugation and washing, the total RNA was finally eluted in 20 μl of the RNase-free water. The RNA concentrations and purity were measured with an ultraviolet spectrophotometer.

Complementary DNA synthesis

The complementary DNA (cDNA) was synthesised from 1 μg RNA using SuperScript III First-Strand Synthesis System as described in the manufacturer’s protocol (Invitrogen). In brief, 1 μg of total RNA was mixed with 50 μM oligo (dT)20, 50 ng/μl random primers, and 10 mM dNTP mix in a total volume of 10 μl. The mixture was incubated at 56 °C for 5 min, then placed on ice for 3 min. The reverse transcriptase master mix containing 2 μl of 10× RT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, and 1 μl of SuperScript® III RT

(200 U/μl) was added to the mixture and incubated at 25 °C for 10 min, followed by 50 min at 50 °C.

Real-time quantitative PCR

The relative abundance of mRNA species was assessed using the SYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems, Foster City, CA, USA). PCR primers (Table 2) were designed with Gene Runner Software (Hasting Software, Inc., Hasting, NY, USA) from RNA sequences from GenBank. All primer sets had a calculated annealing temperature of 60 °C. Quantitative RT-PCR was performed in a 25 μl reaction volume consisting of 2× SYBR Green PCR Master Mix (Applied Biosystems), 900 nM of each primer and 2-3 μl of cDNA. Amplification conditions were 2 min at 50 °C, 10 min at 95 °C and 40 cycles of denaturation for 15 s and annealing/extension at 60 °C for 10 min. Data from real-time assays were calculated using the v1·7 Sequence Detection Software from PE Biosystems (Foster City, CA, USA). Relative expression of studied gene mRNA was calculated using the comparative CT method. All values were normalised to the β-actin gene and reported as fold change over background levels detected in the diseases group (Livak and Schmittgen, 2001).

Measurement of interleukin 6

Levels of IL-6 in muscle, nerve and fascia tissue was measured by homogenising tissue specimen in 1 ml lysis buffer for protein extraction. This buffer contained 0.0625 mol/l Tris buffer (pH 6.8), 2% sodium dodecyl sulphate, 3% 2-mercaptoethanol, 10% glycerol, 10 lg/ml aprotinin and 1 mmol/l phenyl methyl sulphonyl fluoride (Sigma, St. Louis, MO, USA). After cell lysis the homogenate was centrifuged at 6654×g for 20 min at 4 °C. The supernatant and serum samples were examined for IL-6 using ELISA kit supplied by Quantikine R&D system, Inc. (Minneapolis, MN, USA) according to manufacturer’s instruction (Peters et al., 1998).

Statistical analysis

The data obtained were statistically analysed by comparing the mean values of different groups by one way analysis of variance ANOVA test using SPSS version 20 software

Table 2. Primers sequence for all studied genes.

Gene Primer sequence

IL-6R F: TCACAGAGCAGAGAATGGACTR: GTATGGCTGATAC-CACAAGGT

STAT3 F: 5’-CAAAGAAAACATGGCCGGCA-3’R: 5’-GGGGGCTTTGTGCTTAGGAT-3’

Β-actin F: 5’-TGTTGTCCCTGTATGCCTCT-3’R: 3’-TAATGTCACGCACGATTTCC-5’

H. Ashour et al.


(SPSS, Chicago, IL, USA), with multiple comparisons Tukey post hoc test for comparisons between groups. P-values of



35.63±3.92) (P

H. Ashour et al.


IL-6 can be expressed in the perimuscular tissue which may be force transduced expression caused by contracted muscles. In response to a prolonged run the interstitial IL-6 concentration was elevated markedly in the peritendinous region surrounding the tendoachilles. That increase was 40-folds higher than serum IL-6 and 10-fold higher than exercise-stimulated muscle IL-6 concentration, suggesting a local function of IL-6 (Langberg et al., 2002). Tendon tissue is dominated by fibroblasts, which produce extracellular matrix components, and, in addition to this, are known to release IL-6 (Andersen et al., 2011; Heinemeier et al., 2003). These data suggest that IL-6 could be released from the connective tissue and extracellular matrix, in order to perform certain growth function in a paracrine manner.

Nerve IL-6R as well as STAT3 expression was significantly elevated following SNI, and further elevated in the exercised groups suggesting that damage induced upregulation of IL-6 is necessary to promote recovery.

During development there are growth cones at the termination of the neurites which stimulate nerve growth. Following nerve injury, the proximal axons shows multiple sprouts containing, growth cones aiming to initiate regeneration. The growth cones can be positively guided towards targets through growth factors (Spira et al., 2003). A study done on retinal ganglion cells (RGCs) reported that the IL-6 nerve growth-promoting effect is mediated via the IL-6R. Consistently, RGCs responded within minutes to IL-6 treatment by JAK/STAT3 pathway activation, and an IL-6R antibody can block the IL-6-growth effect. Moreover, a designer cytokine IC7 that exclusively binds to IL-6R, could induce neurite growth. There is negative feedback produced by suppressor of cytokine signalling 3 to regulate STAT signalling (Hirano et al., 2000). Therefore, IL-6R could be an appropriate pharmacological target to initiate axonal regrowth stimulation of injured neurites.

Exercise induced IL-6 is important in the regeneration of nerve injury as we reported an improvement in the NCV with myelination% and muscle (CMAP) with increased number of nerve fibres in the SNI+Ex compared to SNI group. IL-6 was discovered as a B cell growth factor and now it is confirmed that it can act as a growth factor for different cell types, including nerve regeneration (Leibinger et al., 2013). IL-6 belongs to the family of glycoprotein 130

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Figure 5. (A) Physiological nerve conduction velocity (F=95.5); (B) compound muscle action potential amplitude (CMAP) (F=25.9); (C) %myelination (F=82.7); and (D) number of nerve fibres (F=12.7). Values differ significantly at P



(gp130)-activating cytokines. IL-6 acts on the target cell membrane through a hexamer complex (two IL-6, two IL-6R and two gp130) (Hirano et al., 2000). The dimerised gp130 intracellular domain mediates signal transduction. Activation of two molecules of Janus family tyrosine kinases (JAK), cause induction of tyrosine phosphorylation within minutes and recruitment of STAT3, which dimerises and is translocated to the nucleus and where it induces gene expression (Tadamitsu, 2006).

Using a designer cytokine, termed hyper-IL-6, resulted in differentiation of neuronal progenitor cells and gliogenesis, through activation of the mitogen-activated protein kinase/cAMP response element-binding protein signalling pathway (Islam et al., 2009). Furthermore, blockage of the IL-6 receptor signalling pathway, following middle cerebral artery occlusion, resulted in an increase in infarction size and increased levels of apoptosis, suggesting that IL-6 has an anti-apoptotic property in ischemia reperfusion (Yamashita et al., 2005).

Exercise training was accompanied by significant improvement in the nerve function in the form of increased NCV and myelination of the nerve fibre in the SNI+Ex and Ex+SNI+Ex compared to SNI group. The MAP amplitude and number of nerve fibres were significantly increased in the SNI+Ex compared to SNI group. This indicates that exercise training before nerve injury is useful in IL-6 production as a growth factor to increase myelin sheath synthesis with no significant effect on the number of nerve fibres. When physical exercise begins just after nerve injury or even in the early stages of denervation, it leads to resistance to fatigue and to restore muscle contractile and mechanical properties (Teodori et al., 2011).

5. Conclusions

Based on our results we conclude that IL-6/STAT3 growth signalling is induced by exercise training, and is important in nerve regeneration; either by post-conditioning or combined pre- and post-conditioning, and therefore could be a treatment line in clinical trials in nerve injury cases. A limitation of the study was the lack of an exercise pre-conditioned only group. Also, the exercise could be extended for a longer period in another study.

Acknowledgements

We appreciate the help with the revision by professor dr. Maha Gamal, head of Physiology department. The skilful assistance of the technicians of the physiology and histology departments are also highly appreciated.

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