The experimental groups are schematized in figure 12. One animal was subjected to the electrodes implantation surgery, as a Sham control of this surgical approach, but did not receive any DRG neurostimulation protocol (Sham-DRG-S). SNI was induced in different sets of animals, as a model of peripheral neuropathic pain. In one group of SNI rats the disease was allowed to develop until 14 days after induction and the animals were only behaviorally tested to monitor the development of neuropathic pain hypersensitivity (SNI 14d). Two groups of SNI animals, each maintaining the neuropathic condition for 7 or 14 days after induction, received DRG neurostimulation protocols (SNI+DRG-S 7d and SNI+DRG-S 14d groups). One SNI animal was allowed to develop the neuropathic condition until 21 days after induction and received DRG neurostimulation protocols (SNI+DRG-S 14d-21d). Mechanical allodynia and hyperalgesia, cold thermal allodynia and motor coordination were assessed in all animals through adequate standard behavioral tests. In addition, spontaneous pain behaviors were also evaluated in SNI animals that were allowed to maintain the neuropathic condition for longer than 7 days.
Figure 12: Distribution of the animals by the different experimental groups.
The experimental design of behavioral testing in each set of animals is schematized in figures 13-17, and is briefly described as follows:
a) Sham-DRG-S animal: on day -1 before the surgery for electrodes implantation (baseline), and on days 7, 14, and 21 after surgery, the animal was submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. After nociceptive assessment, the animal was sacrificed by perfusion (figure 13);
b) SNI 14d group: on day -1 before SNI induction (baseline), and on days 7 and 14 after neuropathic pain model induction, the animals were submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. Between days 9 and 12, the animals went through the conditioning days of Conditioned Place Preference (CPP), without any conditioning. On day 14, after nociceptive assessment, the animals were sacrificed by perfusion (figure 14);
c) SNI+DRG-S 7d group: On days -8, and on day 7, the animals were submitted to von-Frey, Pin-Prick, and Acetone tests to evaluate the nociceptive behavior and motor coordination. On day 7, the animals were also tested for the motor threshold induced by neurostimulation and received a session of 30 minutes of neurostimulation. The animals were sacrificed by perfusion, after the behavioral assessment (figure 15).
d) SNI+DRG-S 14d group: On days -8, and every 7 days up to 14 days, the animals were submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. On days 7 and 14, the animals were also tested for the motor threshold induced by neurostimulation and received a session of 30 minutes of neurostimulation. Between the day 9 and 12, the animals were conditioned on the CPP, receiving a daily neurostimulation session in these days. On day 14, the SNI+DRG-S 14d animals were sacrificed by perfusion (figure 16).
e) SNI+DRG-S 14d-21d animal: On days -8, and on 14 and 21 days, the animal was submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the
nociceptive behavior and motor coordination. On days 14 and 21, the animal was also tested for the motor threshold induced by neurostimulation and received a session of 30 minutes of neurostimulation. Between the day 16 and 19, the animal was conditioned on the CPP, receiving a daily neurostimulation session in these days. On day 21, the animal was sacrificed by perfusion (figure 17).
Figure 13: Timeline for behavioral analyses of Sham-DRG-S animal (n=1) (animal with electrodes without neurostimulation protocol). On days -1, and every 7 days up to 21 days, the animal was submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. On day 21 the animal was sacrificed by perfusion.
Figure 14: Timeline for behavioral analyses of the SNI 14d group (n=5). On days -1, and every 7 days up to 14 days, the animals were submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. Between days 9 and 12, the animals were placed on the CPP apparatus but without any conditioning. On day 14 the SNI animals were sacrificed by perfusion.
Figure 15: Timeline for behavioral analyses of SNI+DRG-S 7d group (n=3) (SNI animals with neurostimulation protocol). On days -8 and day 7, the animals were submitted to von-Frey, Pin-Prick, and Acetone tests to evaluate the nociceptive behavior and motor coordination. On day 7
the animals were also tested for the motor threshold induced by neurostimulation and received a session of 30 minutes of neurostimulation. After behavior evaluation, the SNI+DRGS 7d animals were sacrificed by perfusion.
Figure 16: Timeline for behavioral analyses of SNI+DRG-S 14d group (n=3) (SNI animals with neurostimulation protocol). On days -8 and every 7 days up to 14 days, the animals were submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. On days 7 and 14, the animals were also tested for the motor threshold induced by neurostimulation and received a session of 30 minutes of neurostimulation. Between the day 9 and 12, the animals were conditioned on the CPP, receiving a daily neurostimulation session in these days. On day 14 the animals were sacrificed by perfusion.
Figure 17: Timeline for behavioral analyses of SNI+DRG-S 14d-21d animal (n=1) (SNI animal with neurostimulation protocol). On days -8 and days 14 and 21, the animal was submitted to von-Frey, Pin-Prick, Acetone, and Rotarod tests to evaluate the nociceptive behavior and motor coordination. On days 14 and 21 the animal was also tested for the motor threshold induced by neurostimulation and received a session of 30 minutes of neurostimulation. Between the day 16 and 19, the animal was conditioned on the CPP, receiving a daily neurostimulation session in these days. On day 21 the animal was sacrificed by perfusion.