Role of cytochrome c (Cyt c) is an apoptogenic agent under certain conditions. The mitochondrial permeability transition pore (MPTP) plays an important role in cell death since it opens, leading to mitochondrial swelling and release of Cyt c, which initiates apoptosis. By inhibiting the opening of MPTP, cyclosporine A (CSA) may contribute to maintaining mitochondrial homeostasis. We investigate the effects of the partial sciatic nerve injury (PSNI)-induced neuropathic pain model on mitochondrial Cyt c release and the effects of CSA on neuroprotection by mitochondrial stabilizing activity in PSNI rats.
Rats were assigned to two groups that received different operations (Group P; PSNI operation, Group S; sham operation). The changes of cyt c and GABAergic neuron were evaluated in the spinal cord tissue. After which, PSNI rats randomly received CSA (Group C) or saline (Group S), and the changes of mechanical thresholds with Cyt c and GABAergic neuron were checked.
PSNI in rats increased the release of cytosolic Cyt c. However, GABAergic cells were not decreased in the spinal cord level on the ipsilateral side to the PSNI. The second experiment reveal a reduction in Cyt c release, using CSA in PSNI model. Rats receiving CSA were afforded the antiallodynia without decrease of GABAergic cell.
The Cyt c probably contributes to nerve dysfunction after PSNI. PSNI induced neuropathic pain was profoundly linked to mitochondrial stabilization. Thus, the potent neuroprotector, CSA, might produce antiallodynia through its capability to inhibit the opening of MPTP.
Cytochrome c (Cyt c) is a ubiquitous, heme-containing protein that normally resides in the space between the inner and outer mitochondrial membranes. The classical function of Cyt c in the intermembrane space is as a component of the electron transport chain and leading to the reduction of oxygen to water. In 1996, another role for Cyt c as an apoptogenic agent was reported [
Neuropathic pain results from pathology in the nervous system. By modulating both synaptic inflow and dorsal horn neuronal excitability, presynaptic and postsynaptic inhibition regulates the transfer of information from the periphery to the CNS. In neuropathic pain, noxious stimulation sensitizes central neurons involved in processing of nociceptive information and this sensitization induces nocuous responsiveness to innocuous peripheral stimulation. This phenomenon is called "central sensitizatio". Reduction or elimination of spinal cord inhibition could be expected and sensitization of higher order neurons is a key mechanism involved in generating pathological excitability, that is to say the development of neuropathic pain.
Partial sciatic nerve injury (PSNI) [
Male Sprague-Dawley rats weighing 180-200 g were used. Rats were housed with free access to food and water under a natural day/night cycle. Rats were acclimated for 7 days before any experimental procedures. All experiments were conducted during the lights-on period in a quiet room. Animal care was provided in adherence to guidelines of the Institutional Care and Use Committee at Pusan National University Hospital.
Two sets of experiments were performed. Firstly, the animals (total n = 30) were randomly assigned to one of the 2 groups and each group received different operations (Group P; rats received PSNI operation, Group S; rats received sham operation, n = 15 per group). PSNI model was performed as described previously by Seltzer et al. [
For western blot and immunohistochemical staining, five rats in each groups were deeply anesthetized and killed at 1, 3, and 7 days after the operation. The spinal cords that included L4-5 were removed and immediately frozen on dry ice. Spinal cord tissue was homogenized in buffer (250 mM sucrose, 20 mM Tris-HCL [pH 7.4], 1.5 mM Na-EDTA, 1.5 mM Na-EGTA, 1 mM MgCl2, 1 mM DTT [Sigma-Aldrich, St. Louis, USA], 20 mM KCl [Samchun Pure Chemical, Pyeongtaek, Korea]) and centrifuged. The supernatant was decanted and used for western blot analyses. The protein concentration of the supernatant was measured using a BCA protein assay kit (Pierce, Rockford, USA). The supernatant, with equal amounts of 50 µg protein, was then fractionated by sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) gel and transferred onto a nitrocellulose membrane (Millipore, Bedford, USA). Membrane were blocked with 2% BSA in Tris-buffered saline containing 0.1% Tween (TBST) at room temperature for 1 h and then incubated overnight at 4℃ with antibody against β-actin and Cyt c. After washing with TBST, anti-rabbit IgG and anti-mouse IgG2B was used as a secondary antibody (1 : 2,000 dilution in 2% BSA in TBST, 1 h 30 min incubation) and the antigen antibody complexes were visualized using an enhanced chemiluminescence detection reagent (Amersham). Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories), and quantification was performed using Multi-Analyst 1.0.2 software (blue color).
The spinal cords (L4-5) were sectioned transversely at a thickness of 6 µm on a freezing microtome for immunohistochemical staining. One tissue from each rats were then dehydrated for 2 h and fixed for 15 min in cold acetone. After washed by PBS and non-specific antibody binding was inhibited by incubation for 1 hour in blocking solution (1% BSA, 0.2% powdered skim milk, 0.3% Triton-X 100 in PBS). Primary antibodies were diluted in PBS blocking buffer and slides were incubated overnight at 4℃ in primary antibodies to GABAergic cells antibody (abCam, Share, UK). After washed by PBS, secondary antibodies Alexa Fluor 633 anti-rabbit IgG (Invitrogen, Oregon, USA; blue color) were used to locate the GABAergic cells in each section. The nuclei were visualized using Leica TCS SP2 (Leica Microsystems Heidelberg GmbH, Germany). For fluorescence intensity analysis, LCS light software (Leica Microsystems Heidelberg GmbH, Germany) was used. The staining was visualized using an optical microscope, the appearance of GABAergic cells was assessed at X1600 and fluorescence intensity was assessed at X400.
In a second set of experiments, the animals (total n = 40) were randomly assigned to one of 2 treatment groups. Depending upon group designation, the animals received CSA (Group C, n = 20) or saline (Group S, n = 20) at pre-operation, postoperative 1 day, 3 day and 7 day. CSA injected intraperitoneally in a solution of polyethylene glycol/sterile saline/cremophor oil solution (20 mg/kg). The rats of group S were treated with the 1 ml of saline in order to serve as controls.
Behavioral tests were conducted blindly by the experimenter who did not know the nature of the experimental manipulation and performed on 5 rats per group at preoperation, postoperative day 1, day 3 and day 7. The foot withdrawal threshold to mechanical stimuli (mechanical threshold) was used as an indicator of mechanical sensitivity of the affected paw. Before drugs injected to rats, behavioral tests were performed. The foot withdrawal threshold to mechanical stimuli was assessed by 8 calibrated von Frey hairs. For each test, the animal was placed in a plastic chamber (15 × 20 × 30 cm) and habituated for at least 15 min. The chamber was placed on top of a mesh screen so that mechanical stimuli could be administered to the plantar surface of left hind paw. Thresholds were determined by the up-down method [
Cyt c and GABAergic neuron were evaluated by the western blot method and immunohistochemical staining as described in first experiment (n = 15 per group).
All data are presented as means ± standard errors of the mean (SEM) and analyzed using the PASW 18K statistical program (SPSS institute, USA). Mechanical thresholds, Cyt c and fluorescence intensities of sections were analyzed with the Mann-Whitney U test to compare values between groups at each time point. In all cases, a P value < 0.05 was considered to be significant.
In the first experiment, postoperative foot drop was not observed. In group P, the nerve damage associated with PSNI elicited behavioral features characteristic of neuropathic pain: the rats developed abnormal gait, posture, guarding and protecting behavior and licking of the hind paw of the ipsilateral side of sciatic ligation after 1 day of operation. The rats did not put weight on the affected side and the hind leg of the affected side was drawn close to the body with distinctive guarding posture. The foot was ventroflexed and the toes were held together tightly. PSNI of the sciatic nerve in the rat produced a profound and prolonged decrease of mechanical thresholds within 1 day of the nerve injury. As shown in
In the second experiment, the mechanical thresholds of group C were high compared with that of group S after 7 days (P = 0.009) and significant anti-allodynic effect occurred on the 7th day after PSNI surgery (
In the present study, we demonstrated that PSNI in rats increased the release of cytosolic Cyt c from mitochondria. However, GABAergic cells were not decreased in the spinal cord level on the ipsilateral side to the PSNI. The results of second experiment reveal a dramatic reduction in Cyt c release using CSA in PSNI model. Rats receiving CSA were afforded the antiallodynia without decrease of GABAergic cell.
A great advance in our understanding of the mechanisms that underlie neuropathic pain states has come from the development of animal models. Since most of these models involve partial nerve injury, some input is preserved. This allows behavioral analysis of changes in thermal and mechanical withdrawal thresholds, in addition to the assessment of spontaneous pain and autotomy. PSNI model not only produces profound behavioral indicators of persistent pain, but it also initiates a cascade of events resulting in altered peptide expression in the superficial dorsal horn [
Recognition that mitochondria could release triggers of cell death, and that at least one of these triggers, Cyt c, was sufficient for the latter stages of cell death, highlighted the potential gatekeeper role of mitochondria in cell death cascades [
Although MPTP inhibitors are suggested to be protective in some models [
The use of CSA is associated with numerous side effects. Neurotoxicity was previously less well-known, but with growing experiences, central nervous system side effects are now reported. The rate of neurotoxicity appears to be dose related and increases with additional doses of chemotherapy delivered [
A permanent decrease or loss of spinal inhibition after nerve injury is a potential mechanism of neuropathic pain. This mechanism that has been proposed to contribute to neuropathic pain is peripheral nerve injury-induced loss of the GABAergic inhibitory interneuronal system in the spinal cord [
In conclusion, the present study suggests that mitochondrial release of Cyt c probably contributes to nerve dysfunction after PSNI. This study provides evidence that PSNI induced neuropathic pain was profoundly linked to mitochondrial stabilization, but it is presumed that pathology of PSNI is not related to the loss of neurons in dorsal horn lamina in the rats. Thus, in agreement with previous evidence, the potent neuroprotector, CSA, might produce antiallodynia through its capability to inhibit the opening of MPTP.
Effects of partial sciatic nerve injury (PSNI) on cytochrome c (Cyt c) in rats. Cyt c of group P was increased compared with group S (P = 0.020, 0.021 and 0.021) in the L4-5 at postoperation 1, 3 and 7 days. PSNI leads to increasing of Cyt c in rats. *P < 0.05 when compared with that of group S. S: sham operation group, P: PSNI operation group, Day1: 1 day after operation, Day3: 3 days after operation, Day7: 7 days after operation.
Effects of partial sciatic nerve injury (PSNI) on GABAergic neuron in rats. The fluorescence intensities of group P were not significantly different from group S in the ipsilateral L4-5 dorsal horn (laminas I-III) at 1, 3, and 7 days after operation. The GABAergic neuron in the ipsilateral L4-5 dorsal horn (laminas I-III) was not reduced by PSNI at 1, 3, and 7 days after operation. S: sham operation group, P: PSNI operation group, Day1: 1 day after operation, Day3: 3 days after operation, Day7: 7 days after operation.
The effects of cyclosporine A (CSA) on mechanical thresholds in partial sciatic nerve injury (PSNI) in rats. The mechanical thresholds of group C were high compared with that of group S after 7 days (P = 0.009) and significant anti-allodynic effect occurred by CSA on the 7th day after PSNI surgery. S: saline intraperitoneal injection group, C: CSA intraperitoneal injection group, Preop: preoperation, Day7: 7 days after operation. *P < 0.05 when compared with that of group S.
The effects of cyclosporine A (CSA) on mitochondrial cytochrome c (Cyt c) release in partial sciatic nerve injury (PSNI) in rats. (A) A reduction of Cyt c by CSA was shown after 7 days on PSNI rats. (B) The Cyt c level of group S was significantly greater than that of group C at 7th day after PSNI surgery (P = 0.021). *P < 0.05 when compared with that of group S. S: saline intraperitoneal injection group, C: CSA intraperitoneal injection group, Day7: 7 days after operation.
The effects of cyclosporine A (CSA) on GABAergic cell in partial sciatic nerve injury (PSNI) in rats. (A) The GABAergic neuron in the ipsilateral L4-5 dorsal horn (laminas I-III) was not reduced by PSNI at 7 days after operation (X1600). (B) The fluorescence intensities of group C were not significantly different from group S in the ipsilateral L4-5 dorsal horn (laminas I-III) at 7 days after PSNI operation. S: saline intraperitoneal injection group, C: CSA intraperitoneal injection group, Day7: 7 days after operation.