Epidural administration of neostigmine-loaded nanofibers provides extended analgesia in rats

Abstract

Background: In this study, neostigmine-loaded electrospun nanofibers were prepared and then their efficacy and duration of analgesic action were studied after epidural administration in rats by repeated tail flick and formalin tests.

Methods: The neostigmine poly vinyl alcohol (PVA) nanofibers were fabricated by electrospinning methods. The nanofibers (1 mg) were injected into the lumbar epidural space (L5-L6) of rats (n = 6). Cerebrospinal fluid samples of rats were collected 1, 5 and 24 hours after injection and then were sampled once weekly for 4 weeks. Free-neostigmine concentration was measured in the samples spectrophotometrically. Rat nociceptive responses were evaluated by repeated tail-flick and formalin tests for 5 weeks after the nanofibers (1 mg) injection. Locomotor activity of rats was measured in the open-field at the same period.

Results: The cerebrospinal fluid concentration of free neostigmine reached 5 μg/ml five hours after injection and remained constant until the end of the experiments. The tail-flick latency of treated rats was significantly (p < 0.01) increased and remained constant up to 4 weeks. Pain scores of the rats in both phases of formalin test were significantly (p < 0.01) reduced during the same periods, Epidural injection of the nanofibers had no effect on locomotor activity of rats in an open-field.

Conclusions: Our results indicate that the neostigmine nanofibers can provide sustained release of neostigmine for induction of prolonged analgesia after epidural administration. High tissue distribution and penetration of the nanofibers in dorsal horn can increase thermal and chemical analgesia duration without altering locomotor activity in rats for 4 weeks.

Figure 1

Schematic representation of an electrospinning setup for production of neostigmine-PVA loaded nanofibers [12].

Figure 2

The Scanning electron microscope photomicrograph of Polyvinyl Alcohol nanofibers before (A) and after neostigmine loading (B). In this figure the arrows show the loading position of neostigmine in the core of nanofibers. Magnification: 20,000 × .

Figure 3

Percentage activity remaining during inhibition of acetylcholinesterase after addition of nanofibers. (A) The measurement of acetylcholinesterase activity following addition of the nanofibers were fabricated with different proportions of poly vinyl alcohol and neostigmine (0.00125 v/v); (B) the acetylcholinesterase activity after addition of the neostigmine nanofibers that fabricated by %6 PVA and two different concentrations of neostigmine (pH = 7/4, 22°C).

Figure 4

Verification of injection site and evaluation the distribution of methylene blue injected into the epidural space. (A) The exposed lumbar spinal cord region of control rats and (B) drug extension evaluation by epidural injection of 50 μL 1% methylene blue.

Figure 5

Measurement of neostigmine concentration in CSF in rats. CSF concentrations of neostigmine were measured, (A) up to 24 hours and (B) for 4 weeks after epidural injection of neostigmine loaded nanofibers. Values presented are means ± SEM.

Figure 6

The effect of epidural injection of neostigmine loaded nanofibers (NLN) on the cumulative nociceptive scores of rats in the early phase (A) the late phase (B) of formalin test. Sham group treated with free PVA nanofibers (Mean ± SEM, n = 6, **p < 0.01, Repeated-two way ANOVA followed by Bonferroni test).

Figure 7

Effect of epidural neostigmine loaded nanofibers (NLN) administration on tail-flick latencies of rats (Mean ± SEM, n = 6, **p < 0.01, repeated two way ANOVA followed by Bonferroni test), Sham group treated with epidural free PVA nanofibers.

Figure 8

Measurement of rats total traveled distance (A) and average velocity (B) for 3 weeks after epidural injection of neostigmine loaded nanofibers (NLN). Sham group treated with free PVA nanofibers (Mean ± SEM, n = 6, **p < 0.01, Repeated-two way ANOVA followed by Bonferroni test).