Neuroprotective effect of Lovastatin on motor deficit induced by sciatic nerve crush in the rat

Author’s Accepted Manuscript Neuroprotective effect of Lovastatin on motor deficit induced by sciatic nerve crush in the rat Mohammad B. Ghayour, Arash Abdolmaleki, Morteza. B. Rassouli www.elsevier.com/locate/ejphar

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S0014-2999(17)30458-2 http://dx.doi.org/10.1016/j.ejphar.2017.07.018 EJP71310

To appear in: European Journal of Pharmacology Received date: 22 April 2017 Revised date: 3 July 2017 Accepted date: 5 July 2017 Cite this article as: Mohammad B. Ghayour, Arash Abdolmaleki and Morteza. B. Rassouli, Neuroprotective effect of Lovastatin on motor deficit induced by sciatic nerve crush in the rat, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.07.018 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 galley proof before it is published in its final citable 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.

Neuroprotective effect of Lovastatin on motor deficit induced by sciatic nerve crush in the rat Mohammad B. Ghayour, Arash. Abdolmaleki*1, Morteza. B. Rassouli *

Corresponding author: Arash Abdolmaleki, Tel.: +98(0)9183705217.

[email protected]

Abstract Following severe peripheral nerve injury (PNI), regeneration is often insufficient and functional recovery is incomplete. Any agents that limit the spread of neural tissue damage may enhance the nerve regeneration. In this regard, statins have been shown to have neuroprotective properties. We investigated the effects of Lovastatin against sciatic nerve crush injury in male Wistar Rats. Lovastatin or vehicle was given parenteraly to rats for 7 days postoperative. In Lovastatin treatment groups, a single dose of agent (2 and 5 mg/kg) was administered daily. The control group was given vehicle in the same manner. The rats were subjected to crush injury in the left sciatic nerve with non-serrated clamp for 30 seconds. Behavioural, electrophysiological and morphological alterations were evaluated during the experimental period. Results showed that Lovastatin in a dose of 5 mg/kg could significantly (P < 0.05) accelerate regeneration process and functional recovery. Also results demonstrated that morphometric parameters such as mean axonal number and myelin thickness were significantly higher in Lovastatin (5 mg/kg) treatment groups compared to controls (P < 0.05). These findings suggest that a short-term course treatment with Lovastatin can protect against sciatic nerve injury. Findings indicate that postoperative administration of Lovastatin led to accelerate regeneration process and motor function recovery in nerve crush model in rats. This effect might be due to the anti-inflammatory, immunomodulatory or antioxidative properties of Lovastatin. It is clear that more research is needed to confirm these findings. Keywords: Lovastatin, motoneuron, peripheral nerve injury, regeneration, sciatic functional index, sciatic nerve

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Postal address: Department of Biology, Ferdowsi University, Azadi square, Mashhad, Iran. Zip code: 9177948974.

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1. Introduction Peripheral nerve injury (PNI) is a common traumatic injury. Despite the innate capacity of peripheral nervous system to regenerate, following severe PNI, regeneration is often insufficient and functional recovery is incomplete (Kim et al., 2011). Primary damage initiates a series of secondary events such as oxidative stress, inflammation and excitotoxicity that lead to the expanding damage zone (Tator and Fehlings, 1991),(Umebayashi et al., 2014). Therefore, any agents that attenuate above-mentioned mechanisms may enhance the efficacy of nerve regeneration process and improve functional recovery, especially if the continuity of the nerve is still intact. Statins such as lovastatin, are potent cholesterol-lowering agents that clinically used for prevention of cardiovascular events due to atherosclerosis (Vollmer et al., 2004). Also, statins have pleiotropic effects including anti-inflammatory, antioxidant, immunomodulatory and neuroprotective properties (Hayashi et al., 2005),(van der Most et al., 2009),(Li et al., 2009). Several studies indicate that statins are effective against a variety of neurological disorders. Some evidences showed that statins may be effective in the treatment of neurodegenerative diseases such as Alzheimer’s disease (AD) (Zamrini et al., 2004), Parkinson’s disease (PD) (Wolozin et al., 2007) and multiple sclerosis (MS) (Ciurleo et al., 2014). Also, some studies showed that administration of statins can attenuate traumatic brain and spinal cord injury (Pannu et al., 2005),(Chen et al., 2007b). Although several studies have investigated the effects of statins on the central nervous system (CNS), there is little scientific information about their effects on the peripheral nerve regeneration. The results of some studies indicated that Simvastatin and Atorvastatin can protect against sciatic nerve crush injury in rats (Pan et al., 2010; Xavier et al., 2012). Also, 2

Atorvastatin is effective against neuropathic pain in rat neuropathy model (Pathak et al., 2014). In contrast, the results of another study show that Simvastatin delay regeneration as shown in histological studies but still there was no influence on electrophysiological measurements (Daglioglu et al., 2010). In this regard, Lovastatin as a lipophilic statins is capable of crossing the blood–brain barrier (BBB) (Botti et al., 1991). Some studies show that Lovastatin can increase neurite outgrowth (van der Most et al., 2009), attenuate glutamate excitotoxicity (Dolga et al., 2009) and has immunomedulatory effects (Paintlia et al., 2006). Due to the lack of sufficient information about neuroprotective effects of Lovastatin against peripheral nerve injury, this experimental study was designed to evaluate the effect of Lovastatin on behavioral, electrophysiological and morphological parameters during sciatic nerve regeneration process in Wistar rats. 2. Materials and Methods: 2.1. Chemicals Lovastatin and dimethyl sulfoxide (DMSO) were purchased from Sigma (USA) while ketamine and xylazine were obtained from Alfasan Pharmaceutical Co. (Holland) in injectable form. Lovastatin was dissolved in DMSO and fresh drug solutions were prepared each day of the experiments. All drugs were injected intraperitoneally. 2.2. Animals All experiments were performed on adult male Wistar rats (weighing 250–300 g, aged 3 months). Animals were maintained under standardized housing conditions (temperature, 22 ± 2 °C, 12-h light/dark cycle light on from 7 a.m. and 60 ± 5% humidity) in plexiglas cages with free access to food (standard laboratory rodent chow) and tap water ad libitum. 3

Experiments were carried out between 9 a.m. and 12 p.m. Ten rats were used for each treatment group. All animal experiments were carried out in accordance with the European Communities Council directive of 24 November 1986 (86/609/EEC). 2.3. Surgical procedures All experiments were performed under an operating microscope in sterile conditions by the same investigator. All animals were deeply anesthetized using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). The skin was shaved and disinfected using 10% povidone iodine. Then, rats were fixed in the prone position on the operating table under sterile conditions. The left sciatic nerve was exposed through a longitudinal incision extending from the greater trochanter to the mid-thigh. Then, a 3 mm-long segment of the sciatic nerve was crushed by maximally clamping the nerve with non-serrated hemostatic forceps (Belge de Gembloux, Belgium) for 30 s at 1 cm below sciatic notch. This procedure causes axonal interruption but preserves the connective sheaths (axonotmesis). For limiting the inter-animal variability in the postoperative outcome followed by microsurgical neurorrhaphy, the crush model was used. The crush model is appropriate to assess the roles of different agents in the nerve regeneration process and for pharmacological investigation (Tos et al., 2009). All surgeries were done using the same forceps. The nerves were kept moist with 37 °C sterile saline solution throughout the surgical intervention. The crush site was marked with a 10-0 nylon suture (Alcon). In the sham-operated group, the left sciatic nerve was treated in the same way except for the crush. Finally, the muscle and skin were sutured with 6-0 nylon and rats were allowed to recover spontaneously from anesthesia. After recovery, each rat has been housed separately per cage. All animals received buprenorphine (1mg/kg) for three days after surgery in order to control pain. To prevent autotomy, bitter nail

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polish was applied to each rat’s left foot. During the study, animals were examined for signs of autotomy and contracture. 2.4. Experimental groups Fifty rats with sciatic nerve crush were randomly allocated into five groups (n = 10). In the two experimental groups, the animals were treated daily with Lovastatin at the doses of 2 or 5 mg/kg within 7 days after surgery. These selected doses of Lovastatin had neuroprotective effect in CNS and experimental autoimmune encephalomyelitis (Aguirre-Vidal et al., 2015; Paintlia et al., 2008). Controls were injected with vehicle (DMSO) and the sham-operated group was subjected to the surgical procedure without the nerve crush. 2.5. Functional evaluation The recovery of motor function was assessed by calculating the sciatic functional index (SFI) at 1, 3, 5, 7 and 9 weeks after crush injury. 2.6. Sciatic functional index (SFI) The SFI test was performed in a confined corridor (100 × 10 × 20 cm) with a dark box at the end. A white paper was placed on the floor of the corridor. Before the surgery, all rats were trained to walk in the corridor. Once the animals had learned to walk along the runway without stopping, their footprints were recorded. To record the footprints, the hind paws of rats were pressed down onto a finger paint-soaked sponge. Then animals were allowed to walk down the corridor leaving their hind footprints on the paper. The SFI value was calculated by putting the obtained data in the formula: SFI = -38.3[(EPL-NPL)/NPL] + 109.5[(ETS-NTS)/NTS] + 13.3 [(EIT-NIT)/NIT] - 8.8, where EPL: the experimental paw length, NPL: the normal paw length, ETS: the experimental toe spread, NTS: the normal toe spread, EIT: the experimental intermediary toe spread and, NIT: the normal intermediary toe 5

spread (Bain et al., 1989). The SFI value varies from 0 to -100, with 0 corresponding to normal function and -100 indicating total impairment. When no footprints were measurable, the index score of −100 was given (Dijkstra et al., 2000). In each walking track, three footprints were analyzed by a single observer and the average of the measurements was used in SFI calculations. 2.7. Electrophysiological evaluation At before and immediately after sciatic nerve crush, 5 and 9 weeks postoperative, noninvasive compound muscle action potential (CMAP) recording was performed in all animals following anaesthesia by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). CMAPs were recorded in the gastrocnemius muscle by surface stimulation via the tendon–belly method (Oğğuzhanoğğlu et al., 2010), using an electrophysiological apparatus (CEPTU, England) with PicoScope System Software. The sciatic nerve was stimulated by bipolar stimulating electrodes, which were placed on the skin premoistened with gel electrode, just in between ischial tuberosity and major trochanter and parallel to the sciatic nerve. The active and reference monopolar needle electrodes were inserted into the mid-belly and muscle tendon surface, respectively. A ground electrode was clamped to the skin, between the stimulating and recording electrodes. Stimulations with durations of 0.02 ms were given at gradually increasing intensity until a maximal CMAP response was obtained. The recording was repeated three times, and the amplitude and latency of CMAP were averaged for each rat. Normal CMAPs were measured from the contralateral uninjured sides. All acquired data were entered into the computer to calculate the electrophysiological parameters of the regenerated nerve. 2.8. Histomorphometry analysis

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At 9 weeks postoperative, following the electrophysiology study, the animals were deeply anaesthetized with an intraperitoneal injection of ketamine and xylazine cocktail, distal parts of the crushed site of the left sciatic nerves were harvested from every group. Nerve samples were fixed in 4% paraformaldehyde and post-fixed in 1% osmium tetroxide. After dehydration with ascending ethanol passages, the specimens were embedded in paraffin and serial cross-sections of the distal zone for each nerve were cut at 5μm thickness. Cross sections of nerves were conducted starting 1 mm distally to the distal nylon suture (where the distal stump had been originally sutured) in order to allow visualization of regenerated fibers entering the distal nerve stump. For histomorphometric analysis, paraffin sections were stained with 1% toluidine blue (Raimondo et al., 2009). Axon counts, axon diameter and myelin thickness were calculated using the Image J program. The contralateral sciatic nerves were used as controls. 2.9. Wet gastrocnemius muscle weights Muscle weight was measured to assess denervation atrophy at 9 weeks postoperative and after the electrophysiological test. The gastrocnemius muscles were harvested from both the experimental and contralateral (control) sides in each group. After blood stain removal, the muscle was weighed while still wet through a digital scale (Sartorius, Germany). Then the gastrocnemius muscle mass ratio of the operated side to the contralateral side was calculated (muscle mass ratio = weight of experimental muscle/weight of contralateral muscle). The index percentage produced represented the recovery in denervation atrophy of the gastrocnemius muscle on the operated side, with approximately 100% gastrocnemius muscle index (GMI) indicating full recovery of the operated side (Lewin et al., 1997). 2.10. Statistical analysis

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Data were analyzed with SPSS Statistics 16.0 software (SPSS Inc., Chicago, Illinois, USA). Statistical analysis was carried out using a one-way analysis of variance (ANOVA) to determine the significant differences among five groups. Intergroup comparison of means was performed using a Tukey-Post hoc analysis. All data are expressed as mean ± standard error of mean (S.E.M.) and values of P < 0.05 were considered statistically significant. 3. Results Immediately after crushing the sciatic nerves, the compression areas were flattened but epineurium sheath continuity was preserved (Fig. 1). All animals developed flaccid paralysis of the operated foot and survived with no wound infection. 3.1. Motor function recovery The SFI values decreased dramatically to the lowest level within 1 week after nerve crush in all groups, indicating complete loss of function. One week post-surgery, all sciatic deficit groups showed a time-dependent increase in SFI value with a significant difference between Lovastatin (5 mg/kg) treatment, control and sham groups at 3, 5 and 7 weeks post-injury (P < 0.05; Fig. 2). At 9 week, SFI returned to the baseline values of the preoperatively period in all crushed groups (Fig. 2). These results indicated that the rate of regeneration was significantly faster in Lovastatin (5 mg/kg) treatment than control group. However, there was no significant difference between Lovastatin (2 mg/kg) treatment and control groups in any weeks (Fig. 2). 3.2. Electrophysiological evaluation To assess the regeneration process, electrophysiological recordings were conducted at before and immediately after sciatic nerve crush, 5 and 9 weeks end point. For this purpose, the CMAPs peak amplitude and CMAPs onset latency were measured. At 5 weeks post-injury, 8

CMAPs amplitude values showed significant difference between Lovastatin (5 mg/kg) treatment, control and sham groups (P < 0.05; Fig. 3). In all crushed groups, the CMAPs amplitude increased and CMAPs onset latencies decreased progressively with time. At the end of 9 week, there was no significant difference between the CMAP amplitude in Lovastatin treatment and control groups (P < 0.05; Fig. 3). Also, CMAPs onset latencies have a significant difference between Lovastatin (5 mg/kg) treatment with control and sham groups at the same time (P < 0.05; Fig. 4). 3.3 Histomorphometry analysis Table shows quantitative morphometric analyses of regenerated nerves for each of the experimental groups. At the 9 week end point, morphometric parameter such as mean axonal number and myelin thickness were significantly higher in Lovastatin (5 mg/kg) treatment groups compared to controls (P < 0.05; Table). Totally, at 9 weeks after injury, regenerating myelinated axons in the distal to injury site were found to be densely populated, with thinner myelin in comparison with sham group. 3.4. Muscle mass At post-operative week 9, the mean ratios of gastrocnemius muscles weight were measured. The experimental gastrocnemius muscles exhibited atrophy compared with the contralateral side in all groups (Fig. 5). The results showed that in Lovastatin (5 mg/kg) treatment groups, muscle weight ratio was higher than control group. Results indicated that muscle atrophy was significantly reduced by systemic administration of Lovastatin (5 mg/kg) (Fig. 5). 4. Discussion Following nerve trauma, administration of neuroprotective agents is an appropriate strategy to control damage and promoting nerve regeneration process (Terenghi et al., 2011). In this 9

study, we evaluated the efficacy of parenteral administration of lovastatin within 7 days on sciatic nerve regeneration. Our results showed that chronic administration of Lovastatin (5 mg/kg) immediately after sciatic nerve crush could accelerate the regeneration process and motor function recovery. These effects were not seen in Lovastatin (2 mg/kg) treatment group. It seems that Lovastatin concentration at the doses of 2 mg/kg not to be enough to influence functional recovery. We utilized several parameters to evaluate nerve regeneration process and functional recovery. Behavioral assessment showed that sciatic nerve crush result in decrease in SFI values immediately after injury. Also, electrophysiological parameters indicated that crush injury lowered the evoked CMAP amplitude and prolonged its latency. Furthermore, morphometric analyses showed increase in nerve fiber density and decrease in axon diameter and myelin sheath thickness in control groups compared to sham group. According to our results, Lovastatin in a dose of 5 mg/kg could improve the above-mentioned parameters. Scince CMAP amplitude is correlated to the density of motor nerve fibers re-innervating the muscle, and the latency reflects the maturation of motor nerve fibers (Hüseyinoğlu et al., 2012),(Wolthers et al., 2005), it seems that Lovastatin (5 mg/kg) could accelerate the nerve regeneration and recovery. Also, the increased number of axons might result from the regenerated nerves or likely axonal bifurcation. The pharmacokinetic properties of statins may differ substantially between humans and rodents (Black et al., 1998; Stern et al., 2000). Use of statins as a neuroprotective agent is not without controversy. Some evidence suggest that long-term exposure to statins may be associated with some adverse effects such as rhabdomyolysis (Thompson et al., 2003) and hepatotoxicity (Chalasani, 2005) Although we have no explanation for this finding, we propose that these might be due to neuroprotective properties of Lovastatin. Statins have both cholesterol-dependent and

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independent neuroprotective effects and can activate several neuroprotective pathways. It seems that some neuroprotective effects of statins may be associated with its immunomodulatory and anti-inflammatory properties (Blanco-Colio et al., 2003),(Arnaud et al., 2005). Regeneration process requires the sequential expression of specific growthassociated and function-associated genes in both sheath cells and neurons (Chen et al., 2007a). In this regard, statins can augment regeneration-associated gene expression in the early stage of the regenerative process. Pretreatment with atorvastatin increased expression of axonal growth cone-associated GAP43 and Schwann-related remyelination associated MBP in injured nerves 7 days after operation (Pan et al., 2010). Statins such as Lovastatin can down-regulate the leukocyte functional antigen (intercellular adhesion molecule ligands that mediates T cell adhesion) and inhibit the release of proinflammatory cytokines (Ehrenstein et al., 2005; Pahan et al., 1997). Inflammatory and immunological responses play an important role during wallerian degeneration and axonal regeneration (Boivin et al., 2007),(Perrin et al., 2005). Also, statins can induce neuroprotection by promoting the release of neurotrophic factors (Chen et al., 2005),(Wu et al., 2008). For axonal regeneration, neurotrophic factors play a crucial role (Gordon, 2009). In addition, statins show potential to promote neurite outgrowth, neurogenesis and angiogenesis in CNS (Chen et al., 2003; Lu et al., 2007; Pooler et al., 2006). It is likely these mechanisms also effective in peripheral nerve. Pan and his colleagues showed oral pretreatment of atorvastatin for 7 consecutive days attenuated crush injury in rat sciatic nerves and promoted regeneration, this findings is consistent with our results (Pan et al., 2010). Moreover, some evidence support the hypothesis that statins, in particular lovastatin, have antioxidant activity and can reduce levels of free radicals (Kim et al., 2004),(Stoll et al., 2004). Oxidative stress promote neural tissue injury (Feldman, 2003). Although other mechanisms may contribute to the observed neuroprotection, further studies are necessary to 11

elucidate the exact protective mechanism and possible protective effect of Lovastatin on other forms of neuronal injury. In conclusion, our findings indicate that postoperative administration of Lovastatin led to accelerate regeneration process and motor function recovery in nerve crush model in rats. This effect might be due to the anti-inflammatory, immunomodulatory and anti-oxidative properties of Lovastatin. It is clear that more research is needed to confirm these findings. We suggest to study the effect of Lovastatin on recovery from a sciatic nerve transection. Conflict of interest The authors report no conflicts of interest. Acknowledgments This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. The authors would like to thank Dr. Ann Paterson for assisting with the English. References Aguirre-Vidal, Y., Montes, S., Tristan-López, L., Anaya-Ramos, L., Teiber, J., Ríos, C., Baron-Flores, V., Monroy-Noyola, A., 2015. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology 48, 166-170. Arnaud, C., Braunersreuther, V., Mach, F., 2005. Toward immunomodulatory and antiinflammatory properties of statins. Trends in cardiovascular medicine 15, 202-206. Bain, J., Mackinnon, S., Hunter, D., 1989. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plastic and reconstructive surgery 83, 129-136. Black, A.E., Sinz, M.W., Hayes, R.N., Woolf, T.F., 1998. Metabolism and excretion studies in mouse after single and multiple oral doses of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin. Drug metabolism and disposition 26, 755-763. Blanco-Colio, L.M., Tuñón, J., Martín-Ventura, J.L., Egido, J., 2003. Anti-inflammatory and immunomodulatory effects of statins. Kidney international 63, 12-23. Boivin, A., Pineau, I., Barrette, B., Filali, M., Vallières, N., Rivest, S., Lacroix, S., 2007. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. The Journal of Neuroscience 27, 12565-12576.

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Figure legends Figure 1. Picture show the crushing step of the sciatic nerve, intact nerve (A), crushing step with non-serrated hemostatic forceps (B),arrow shows the crush site, epineurium sheath continuity was preserved (C).

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Figure 2. Sciatic function index (SFI) measured every other week after injury. in each experimental group. Data were expressed as mean ± S.E.M. (n = 10). # P < 0.001 and *P < 0.05 vs. DMSO and control group respectively at the same time point. Figure 3. Representative results of CMAP amplitude measurements after proximal stimulation of operated and unoperated sciatic nerve at 5th and 9th weeks post-injury. The data are shown as mean ± S.E.M. (n = 10). # P < 0.001 and *P < 0.05 vs. DMSO and control group respectively. Figure 4. Representative results of CMAP delay measurements at 5th and 9th weeks postinjury. The data are shown as mean ± S.E.M. (n = 10). # P < 0.001 and *P < 0.05 vs. DMSO and control group respectively. Figure 5. Gastrocnemius muscle mass ratio measurement. The gastrocnemius muscles of operated and unoperated sides were excised and weighed in the experimental groups at 9 weeks post-operatively. The data are shown as mean ± S.E.M. (n = 10). # P < 0.001 and *P < 0.05 vs. DMSO and control group respectively.

Table: Morphometric analyses of transverse sections at the sciatic nerve distal to injury for each of the experimental groups 9 weeks post-injury. Values are shown as mean ± S.D. statistically significant differences from control. a P < 0.05.

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Groups

Sham

Diameter of

Diameter of

Thickness of

fibers

axon

myelin sheath

7.8 ± 0.04

5.1 ± 0.05

1.35 ± 0.02

5.7 ± 0.05

4.02 ± 0.06

0.75 ± 0.03

5.75 ± 0.05

4.22 ± 0.05

0.77 ± 0.02

Number of fibers

7752.75 ± 85.21 9848.75 ±

Control

124.31 Lovastatin 2

9824.37 ±

mg/kg

132.38

Lovastatin 5

10205.87 ± a

mg/kg

154.88

DMSO

9794 ± 107.29

5.86 ± 0.04

a

5.61 ± 0.05

4.19 ± 0.05

4.2 ± 0.06

a

0.84 ± 0.03

a

0.7 ± 0.01

Morphometric analyses of transverse sections at the sciatic nerve distal to injury for each of the experimental groups 9 weeks post-injury. Values are shown as mean ± S.D. statistically significant differences from control. a P < 0.05.

17

18

19

20

21