Influence of Gabapentin and Atorvastatin, or its Association, in Mechanical Hyperalgesia and Motor Function Assessed on Crushing Nerve Injury Model

Nerve lesions causes hyperalgesia and loss of motor function. Gabapentin (GABAP) is used in this condition for pain relief, while atorvastatin (ATORV) has demonstrated neuroprotective effects in preclinical studies. We have investigated the influence of GABAP and ATORV on nerve injury. Mice (25-35 g) were subjected to partial ligation of the sciatic nerve. Influence of the drugs on hyperalgesia and grip force was assessed before injury, 7, 14 and 21 days thereafter. Data evaluated by 1 or 2-way ANOVA (P < 0.05). GABAP (26.67  4.21% of response) and ATORV (36.67  10.85% of response), alone or in combination, reduced hyperalgesia (vehicle: 76.00  11.66% of response); there was an important effect for the association of these drugs on the grip force. The two agents augmented levels of brain derived neurotrophic factor, BDNF, (vehicle: 105.30  12.53 pg/mg of protein; GABAP: 34.92  5.92 pg/mg of protein; ATORV: 33.77  4.20 pg/mg of protein) and insulin-like growth factor-1, IGF-1, (vehicle: 399.60  61.30 pg/mg of protein; GABAP: 388.30  38.57 pg/mg of protein; ATORV: 306.50  16.72 pg/mg of protein). Association of these substances of different pharmacological classes, may bring benefits on hyperalgesia and motor function associated with nerve lesions.


Introduction
Peripheral nerve lesions are frequently observed in the medical clinic and are a global health problem, which results in functional deficits in approximately 2.8% of all trauma patients1 [1] and are the main reason for absenteeism in Brazil [2]. These patients suffer from muscle weakness, loss of touch sensation, and increased likelihood of developing neurotrophic ulcers. In most cases surgical repair of the injured peripheral nerve is necessary for functional recovery and, in the United States, this represents an annual expenditure of $7 billion [3]. The main consequences of nerve damage are sensory changes, including neuropathic pain, as well as functional loss related to motor changes. Neuropathic pain leads to common symptoms such as pain in shock or burning, tingling and altered sensations [4,5], and its neurobiological mechanism is quite different from that observed in acute pain, been related to central and peripheral sensitization mechanisms, from which the sensory neurons are sensitized by different compounds and present a decrease in their activation threshold for the nontoxic stimuli [6].
These sensitizing compounds are produced by different cells, both centrally and locally, leading to a paradigm shift in the understanding of neuropathic pain, which has been based on the perception that neurons are not the only type of cell involved in the etiology of this condition. Thus, immune cells such as macrophages, neutrophils and lymphocytes, as well as glial cells such as microglia, astrocytes and endothelial cells, contribute to sensitization in pain pathways, both in the periphery and Central Nervous System (CNS) [7]. Inflammation also contributes to the painful phenomena during the neuropathy, being able to generate the phenomena of allodynia and hyperalgesia; allodynia being the pain that arises after mild stimuli that do not usually cause pain, whereas hyperalgesia refers to the intensification of pain before a painful stimulus [8,9]. In this context, pro-inflammatory or anti-inflammatory cytokines, as well as neurotrophic factors exert influence on painful phenomena and nerve regeneration, which in turn influence the motor capacity of the affected limb. Among Interleukins (IL), IL-10 is an antiinflammatory cytokine that can inhibit the production of other proinflammatory cytokines, and its expression is increased in the distal portion of the sciatic nerve seven days after crushing [10,11]. Sciatic nerve crushing model is a model of axonotmosis, produced by the application of a compressive force to a given nerve, in which the myelin sheath and axons are interrupted, but the basal lamina of Schwann Cells (SC) remains intact [12].
The fibers distal to the lesion undergo degeneration 48 to 96 h after nerve transection, through a process known as Wallerian Degeneration, the macrophages enter the endoneurium to remove cellular debris, and recovery occurs when the axons send growth cones down from the basal lamina tubes originally preserved toward the final organ [13,14]. In this type of model, evaluations of histomorphometric and functional parameters (muscle contraction force) as well as sensory parameters (allodynia and mechanical hyperalgesia) are common [15]. These aspects may be influenced by factors such as Brain-Derived Neurotrophic Factor (BDNF) [16], and Insulin-like Growth Factor-1 (IGF-1) [17], which contribute to nerve regeneration by having functions such as cell-cell adhesion, cytoskeletal reorganization, neurite growth, synaptic plasticity, neuronal survival and neurogenesis, among others [18].
The use of gabapentin (GABAP) to treat painful conditions associated with nerve damage is a consensus in clinical practice, and it has been shown that the drug has improved aspects of nerve regeneration in an animal model of crushing followed by stretching in rats [19]. On the other hand, atorvastatin (ATORV) also promoted neuroprotection in different animal models [20]. Despite this, the possible benefit of the association of these two drugs in different outcomes associated with nerve injury by crushing in mice has not been investigated in preclinical studies. In view of this, the objective of the present study was to evaluate the possible influence of the treatment with GABAP and ATORV, alone or in association, on different aspects observed in a sciatic nerve crush model in mice.

Animals
All experiments were conducted using male Swiss mice (25-

Crushing-Induced Nerve Injury
Mice were kept anesthetized by the inhalation of 2% isoflurane plus 100% oxygen. To obtain nerve injury, the sciatic nerve was located through a 2 cm long rectilinear cutaneous incision on the lateral side of the thigh, from the major trochanter to the knee.
After exposure, the nerve was crushed in an area of 5 mm in length proximal to its trifurcation, using a clamp specially made for this purpose, calibrated for an approximate static load of 5.0 g. After this, the nerve was reattached to its bed of origin and the surgical wound was closed by planes with isolated 5-0 gauge nylon stitches (Ethicon, Edimburg, UK). In the control group, the nerve was exposed but not crushed (sham group).

Drug Infusions
From days 2 to 21 after crushing of the nerve animals were treated with GABAP (10, 30 and 100 mg/kg, intragastric [i.g]), ATORV (3 or 10 mg/kg, i.g) or sterile saline (0.9% NaCl solution) and mechanical hyperalgesia or grip force were evaluated at different time points after its administration. Sham animals were subjected to the same procedures but were only treated with saline (0.9% NaCl solution).

Study Outlines
The main outcomes of this study were the mechanical hyperalgesia evaluated by the von Frey test and the grip force. For the evaluation of mechanical hyperalgesia, mice were individually placed in clear Plexiglas boxes (9 cm × 7 cm × 11 cm) on elevated wire mesh platforms to allow access to the ventral surface of the right hand paw. The withdrawal response frequency was measured following 10 applications (manually, duration of 3 s each) of a 0.6 g von Frey hair (VFH, Stoelting, Chicago, IL, USA), responsible to produce a mean withdrawal frequency of about 20% [21].
Data are presented as the percentage of the animal's response in 10 applications, with 100% being 10/10 and 0% being 0/10 responses. A significant increase in the number of responses, at the different periods of times as mentioned above, was interpreted as mechanical hyperalgesia. Indirect evaluation of motor function was performed by the grip force test of the limb subjected to crushing.
The apparatus consists of a force transducer connected to a small metallic support, from which the difference (∆) of the force (in g) that the animal exerted during the holding of this support with the member that suffered the injury was recorded to the member that has not undergone the crushing procedure. The apparatus was calibrated prior to each experiment and the animals were habituated to the experiment room for at least 1 h prior to the test.

Evaluation of the Effectiveness of the Association of GABAP + ATORV on Hyperalgesia and Motor Function in the Sciatic Nerve Crush Model of Paw Edema
In order to confirm the antihyperalgesic effect of GABAP in this model, different animals were submitted to the nerve injury procedure as described above and were treated daily between day 2 and day 21 after the sciatic nerve crush procedure with i.e. vehicle (saline solution, 10 ml/kg) or GABAP administration at doses of 10, 30 and 100 mg/kg for selection of the dose to be used in this study. These doses of GABAP and observation periods were chosen from a study conducted in our laboratory [22], also considering the minimum time required for the installation of the neuropathic pain process in the model. After chosen better dose of GABAP to be used in this study, the possible benefit of ATORV alone (3 mg / kg) or in combination with GABAP (30 mg/kg) was investigated at doses of 3 or 10 mg/kg, these doses were selected from previous work [23][24][25][26][27][28]. At days zero (basal), 7, 14 and 21 days after injury, the animals were evaluated in relation to mechanical hyperalgesia and grip force, as previously described.

Comparison of the Levels of Anti-Inflammatory Cytokine (Il-10) and Neurotrophins (BDNF and IGF-1) from the Sciatic Nerve in Animals Under Different Treatments
The

Statistical Analysis
Results are presented as the mean ± standard errors of the mean (S.E.M.) for each group. A statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni's test or one-way ANOVA followed by Newman-Keuls's test when appropriated. P-values less than 0.05 were considered to be statistically significant.

Results
In the present study crushing of the sciatic nerve induced a behavior of mechanical hyperalgesia in relation to the sham group, already observed from the 7 th day after the procedure ( Figure   1      Data for BDNF and IGF-1 are expressed as mean ± standard error of the mean (n= 8 animals), while for IL-10 are presented as median ± interquartile range. (*) represents the comparison with the sham group; (#) represents the comparison with the vehicle group. One-way ANOVA followed by Dunnets's test; P  0.05.

Discussion
The mechanisms responsible for nerve regeneration and Thus, the fact that in the present study, the levels of BDNF in the nerve have reduced after treatment with ATORV and GABAP may suggest that by treatment with them there is a control of the inflammatory process that decreases the need for expression of this factor, even occurring for IGF-1 when treated with the combination of ATORV and GABAP. Confirmation for a possible anti-inflammatory effect of treatments in the present study was not possible since no changes were observed in the levels of IL-10 for any treatment in relation to the control group (sham). This is an anti-inflammatory cytokine that can inhibit the production of other proinflammatory cytokines, and its expression is increased in the distal portion of the sciatic nerve seven days after its crush injury.

Conclusion
the findings of present study demonstrate the beneficial effect of GABAP or ATORV, alone, on mechanical hyperalgesia in the sciatic nerve crush model in mice. In addition, an important effect for the association of these drugs was observed on the grip strength in this model, and the influence of the two agents on the inflammatory process following the nerve injury may serve as a possible mechanism of action for the observed effects. As a perspective, it is suggested that the association of substances of different pharmacological classes, such as GABAP and ATORV, in products aimed at the treatment of comorbidities associated with nerve lesions can be evaluated in humans, mainly because they are already used in the population that is more neuropathic conditions, and epidemiological studies such as clinical trials may confirm the potential benefit raised here.