Previous presentation in conferences: This work has been presented in part at the 51st Annual Scientific Meeting of the Korean Society of Pain, November 2010, Grand Hilton, Seoul, Korea.
Studies investigating the correlation between spinal adenosine A1 receptors and vincristine-induced peripheral neuropathy (VIPN) are limited. This study explored the role of intrathecal N6-(2-phenylisopropyl)-adenosine R-(-)isomer (R-PIA) in the rat model of VIPN.
Vincristine (100 μg/kg) was intraperitoneally administered for 10 days (two 5-day cycles with a 2-day pause) and VIPN was induced in rats. Pain was assessed by evaluating mechanical hyperalgesia, mechanical dynamic allodynia, thermal hyperalgesia, cold allodynia, and mechanical static allodynia. Biochemically, tumor necrosis factor-alpha (TNF-α) level and myeloperoxidase (MPO) activity were measured in the tissue from beneath the sciatic nerve.
Vincristine administration resulted in the development of cold allodynia, mechanical hyperalgesia, thermal hyperalgesia, mechanical dynamic allodynia, and mechanical static allodynia. Intrathecally administered R-PIA (1.0 and 3.0 μg/10 μl) reversed vincristine-induced neuropathic pain (cold and mechanical static allodynia). The attenuating effect peaked 15 min after intrathecal administration of R-PIA after which it decreased until 180 min. However, pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 μg/10 μl) 15 min before intrathecal R-PIA administration significantly attenuated the antiallodynic effect of R-PIA. This antiallodynic effect of intrathecal R-PIA may be mediated through adenosine A1 receptors in the spinal cord. Intrathecally administered R-PIA also attenuated vincristine-induced increases in TNF-α level and MPO activity. However, pretreatment with intrathecal DPCPX significantly reversed this attenuation.
These results suggest that intrathecally administered R-PIA attenuates cold and mechanical static allodynia in a rat model of VIPN, partially due to its anti-inflammatory actions.
Chemotherapeutic agents such as vincristine, paclitaxel, and oxaliplatin are widely used to treat several types of malignant tumors. However, these anti-cancer agents are also associated with peripheral neuropathic pain [
Adenosine is an endogenous purine compound, and it functions as an extracellular signaling molecule in the peripheral and central nervous systems [
However, studies investigating the correlation between spinal adenosine A1 receptors and VIPN are limited. Therefore, the present study was designed to investigate the antinociceptive and anti-inflammatory effects of an intrathecally administered adenosine A1 receptor agonist, R-PIA, in a rat model of VIPN.
A total of 50 male Sprague-Dawley rats (200–250 g, Orient, Seoul, Korea) were used across all experiments. Animals were housed in groups of 2–3 rats per cage in a room maintained at 22 ± 0.5°C with an alternating 12 h light-dark cycle. Food and water were available ad libitum. The animals were allowed to adapt to the laboratory environment for at least 2 h before testing. Experiments were performed during the light phase of the cycle (10 am–5 pm). All animal procedures and study protocols were approved by the Institutional Animal Care and Use Committee of the Asan Institute of Life Sciences, Seoul, Korea (IACUC Number: 2010-13-155).
Vincristine, the adenosine A1 receptor agonist R-PIA, and the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. All drugs were dissolved in sterilized saline (0.9% sodium chloride solution).
Vincristine sulfate (100 μg/kg/day) was intraperitoneally administered for a period of 10 days (two 5-day cycles with a 2-day pause in between) and peripheral neuropathy was induced [
Cold allodynia was measured using the acetone drop method at the hind paw as described by Choi et al. [
Mechanical hyperalgesia of the paw was evaluated using the pin-prick test, as previously described by Erichsen and Blackburn-Munro [
The thermal nociceptive threshold, employed as an index of thermal hyperalgesia, was assessed by the hot plate method, using a plate maintained at a temperature of 52.5 ± 1.0°C. The rat was placed on the hot plate, and the withdrawal latency with respect to licking of the hind paw was recorded in seconds. A cut-off time of 15 s was used [
The ‘paint brush’ behavioral test is well-established for investigating dynamic responses to a mechanical stimulus. Normal rats never withdraw from a smooth paint brush, so a response to this stimulus is described as allodynia. The rat was placed in a cylinder with a wire mesh floor and a smooth paint brush was used to rub the plantar surface of the hind paw from the heel to the toes. This stimulus was applied five times, with intervals of 5 s, and the number of withdrawals was noted (between 0 and 5). The same procedure was repeated twice, with a gap of 5 min, and the number of withdrawals across the three tests was summed to obtain a single cumulative score of mechanical dynamic allodynia. The minimum and maximum values were 0 and 15, respectively [
For assessment of mechanical state allodynia, the rat was placed in an individual plastic cage with a wire mesh bottom. After 20 min, the mechanical threshold was measured by applying a series of eight calibrated von Frey filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, and 15.0 g; Stoelting Co., USA) to the mid-plantar surface of the hind paw, ipsilateral to the nerve injury. This was conducted until a positive sign of pain behavior was elicited, at which point it was then held for 6 s. A brisk withdrawal or paw flinching was considered a positive response, in which case the next lower force filament was then applied. In the absence of such a response, the next greater force filament was then applied. In the absence of a response at 15.0 g of pressure, this cut-off value was assigned to the animal. The mechanical stimulus producing a 50% likelihood of withdrawal was determined using the up-down method [
Drugs were injected in 10 µl volumes by a direct lumbar puncture method between the L5 and L6 vertebrae [
On the 28th day, after behavioral examination (cold allodynia, mechanical hyperalgesia, thermal hyperalgesia, mechanical dynamic allodynia, and mechanical static allodynia), the antinociceptive and anti-inflammatory effects of intrathecal R-PIA were estimated as follows. A pretreatment dose of saline (10 μl) or DPCPX (10 μg/10 μl) was intrathecally administered 15 min before intrathecal administration of R-PIA (0.3, 1.0, and 3.0 μg/10 μl). Measurements of cold and mechanical static allodynia were performed 15, 30, 45, 60, 90, 120, and 180 min after intrathecal R-PIA administration. Thirty minutes after the final measurements of cold and mechanical static allodynia (i.e., at 210 min), the animals were sacrificed. The sciatic nerve was then obtained, and biochemical estimations were conducted (
After euthanasia, sciatic nerve samples were utilized for the determination of the tumor necrosis factor-alpha (TNF-α) level. TNF-α levels (sensitivity: 25 pg/ml) were determined using a rat TNF-α ELISA kit (R&D Systems, Inc., USA), following the manufacturer’s instructions. Testing of sciatic nerve homogenate samples was performed in duplicate. Recombinant anti-rat TNF-α was used to generate a standard curve (range: 0–20,000 pg/ml) as per the diagnostic kit. Absorbance was determined spectrophotometrically at 450 nm. The results were expressed as pg of TNF-α per mg of total protein in the supernatant.
Myeloperoxidase (MPO) activity was measured using a method described by Jain, Jaggi, and Singh [
Eight groups were involved in the present study (
The rats were subjected to no treatment and kept for 14 days. Behavioral tests were conducted on days 0, 7, 14, and 28. On the 28th day, after behavioral testing (cold and mechanical static allodynia), the animals were sacrificed. The sciatic nerve was then obtained and biochemical estimations were conducted.
After induction of VIPN, behavioral examinations were conducted on days 0 (before treatment), 14, and 28. On the 28th day, a 10 μl pretreatment dose of saline was intrathecally administered 15 min before intrathecal administration of 10 μl saline. Measurements of cold and mechanical static allodynia were performed 15, 30, 45, 60, 90, 120, and 180 min after the intrathecal doses of saline. Thirty min after the final measurements of cold and mechanical static allodynia (i.e., at 210 min), the animals were sacrificed. The sciatic nerve was then obtained and biochemical estimations were conducted.
Groups III, IV, and V: R-PIA-administered (0.3 μg/10 μl, 1.0 μg/10 μl, and 3.0 μg/10 μl, intrathecal) vincristine-treated animals (n = 6 /each group)
After induction of VIPN, same behavioral examinations were conducted. On the 28th day, a 10 μl pretreatment dose of saline was intrathecally administered 15 min before intrathecal administration of R-PIA (0.3, 1.0, or 3.0 μg/10 μl). After measurements of cold and mechanical static allodynia, the animals were sacrificed, and the sciatic nerve was obtained for biochemical estimations.
Groups VI, VII, and VIII: DPCPX- (10 μg/10 μl) and R-PIA-administered (0.3, 1.0, or 3.0 μg/10 μl, intrathecal) vincristine-treated animals (n = 6 /each group)
All procedures were performed in the same serial order as the Groups III, IV, and V. However, a pretreatment dose of DPCPX (10 μg/10 μl) was intrathecally administered instead of saline.
Data are expressed as mean ± standard error of mean. Data from the behavioral tests were analyzed using the one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, using SigmaPlot® Version 11 software (Systat Software Inc., USA). A P value < 0.05 was considered to be statistically significant.
Compared with the normal group, administration of vincristine resulted in a significant increase in acetone- and pin prick-evoked paw withdrawal duration, indicating the development of cold allodynia (
Administration of vincristine resulted in a significant increase in acetone-evoked paw withdrawal duration (13.2 ± 0.4 s) compared with the normal group (0.5 ± 0.1 s) on the 28th day. Treatment with intrathecal R-PIA (1.0 and 3.0 μg/10 μl) resulted in a dose-dependent antiallodynic effect against cold allodynia. The antiallodynic effect was higher at the 3.0 μg/10 μl dose of R-PIA (0.7 ± 0.3 s) than at the 1.0 μg/10 μl dose (4.2 ± 0.7 s) (
Administration of vincristine resulted in a significant decrease in the paw withdrawal threshold in response to von Frey filaments (1.8 ± 0.3 g), compared with the normal group (15.0 ± 0.0 g). Treatment with intrathecal R-PIA resulted in a dose-dependent antiallodynic effect against mechanical static allodynia. This antiallodynic effect was higher at the 3.0 μg/10 μl dose of R-PIA (14.5 ± 0.3 g) compared with the 1.0 μg/10 μl dose (10.8 ± 0.8 g) (
Administration of vincristine resulted in a significant increase in TNF-α level in tissue from the sciatic nerve (12.3 ± 0.6 pg/mg) compared with the normal group (4.1 ± 0.3 pg/mg). Intrathecal administration of R-PIA significantly reversed this increase in TNF-α level in a dose-dependent manner (9.0 ± 0.3 and 6.4 ± 0.4 pg/mg for R-PIA 1.0 and 3.0 μg/10 μl, respectively). Intrathecal administration of 0.3 μg/10 μl R-PIA did not affect the TNF-α level (
Administration of vincristine also resulted in a significant increase in MPO activity in tissue from the sciatic nerve (0.9 ± 0.1 U/mg) compared with the normal group (0.2 ± 0.0 U/mg). Intrathecal administration of R-PIA significantly reversed this increase in MPO activity in a dose-dependent manner (0.5 ± 0.0 and 0.3 ± 0.1 U/mg for R-PIA 1.0 and 3.0 μg/10 μl, respectively) (
Vincristine is a well-known chemotherapeutic agent widely used for the treatment of several malignancies such as breast cancer, leukemia, lymphoma, and primary brain tumors [
In the present investigation, treatment with intrathecal R-PIA after the development of vincristine-induced neuropathy significantly reversed behavioral changes, including paw cold allodynia and mechanical static allodynia. The antiallodynic effect of intrathecal R-PIA was significantly reversed by pretreatment with intrathecal DPCPX. Therefore, it is thought that the antiallodynic effect of intrathecal R-PIA may be mediated through adenosine A1 receptors in the spinal cord.
Since initial observations in the 1970s, numerous studies have demonstrated that systemic administration of adenosine A1 receptor agonists causes pain-reducing effects in animal models, such as inflammatory and neuropathic models exhibiting hyper-responsiveness. Additionally, the potential of adenosine A1 receptor agonists as therapeutic agents for pain has been considered in previous reports [
Adenosine A1 receptors are located on peripheral sensory nerve endings [
Several studies have reported that intrathecal administration of adenosine A1 receptor agonists produces pain-reducing effects in animal models such as the spinal nerve ligation model of neuropathic pain and the formalin model of inflammation [
In the present investigation, the anti-inflammatory effect of intrathecally administered R-PIA was also investigated. There exist a limited number of studies that have investigated the anti-inflammatory effects of R-PIA [
In the present study, we measured TNF-α levels in tissue surrounding the sciatic nerve as a biochemical surrogate marker of vincristine-induced inflammation. We found that vincristine treatment increased the level of TNF-α in this tissue. Inflammatory stimuli release reactive species such as NO• and •O2-, proinflammatory factors such as TNF-α, and pronociceptive mediators such as cytokines [
In conclusion, the results of the present study suggest that intrathecally administered R-PIA attenuates neuropathic cold and mechanical static allodynia in the VIPN model of rats, partially due to its anti-inflammatory actions. Further studies are required to evaluate the involvement of the anti-inflammatory effects of R-PIA in VIPN.
No potential conflict of interest relevant to this article was reported.
Kyungmi Kim (Data curation; Formal analysis; Investigation; Methodology; Software; Validation; Writing – original draft)
Wonyeong Jeong (Data curation; Formal analysis; Investigation; Methodology; Software; Validation; Writing – original draft)
In Gu Jun (Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Validation; Visualization)
Jong Yeon Park (Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision; Validation)
Diagrammatic representation of the experimental protocol. DPCPX: 1,3-dipropyl-8-cyclopentylxanthine = adenosine A1 receptor antagonist; R-PIA: N6-(2-phenylisopropyl)-adenosine R-(-)isomer = adenosine A1 receptor agonist.
Behavioral examinations following vincristine administration. (A) Cold allodynia assessed by the acetone drop test, (B) Mechanical hyperalgesia assessed by the pin-prick test, (C) Thermal hyperalgesia assessed by the hot plate test, (D) Mechanical dynamic allodynia assessed by the paint brush test, (E) Mechanical static allodynia assessed by the von Frey filament test. Values are presented as mean ± standard error of mean, n = 6 rats per group. One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 vs. behavioral examination on day 0, †P < 0.05 vs. behavioral examination on day 14.
The effect of different pharmacological interventions on TNF-α level (A) and myeloperoxidase activity (B) in tissue from beneath the sciatic nerve. Peripheral neuropathy was induced by the administration of vincristine (100 μg/kg, i.p.) for 10 days. On the 28th day, saline or DPCPX (10 μg/10 μl) was intrathecally administered 15 min before intrathecal R-PIA administration (1.0 μg/10 μl or 3.0 μg/10 μl). Values are presented as mean ± standard error of mean, n = 6 rats per group. One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 vs. normal control group, †P < 0.05 vs. vincristine control group, ‡P < 0.05 vs. R-PIA 1.0 μg/10 μl group, §P < 0.05 vs. R-PIA 3.0 μg/10 μl group. DPCPX: 1,3-dipropyl-8-cyclopentylxanthine = adenosine A1 receptor antagonist; R-PIA: N6-(2-phenylisopropyl)-adenosine R-(-)isomer = adenosine A1 receptor agonist.
Antiallodynic Effect of Intrathecally Administered R-PIA against Cold Allodynia
Time (min) | 0 | 15 | 30 | 45 | 60 | 90 | 120 | 180 |
---|---|---|---|---|---|---|---|---|
Normal | 0.5 ± 0.1 | 0.7 ± 0.1 | 0.6 ± 0.1 | 0.6 ± 0.1 | 0.5 ± 0.1 | 0.7 ± 0.1 | 0.6 ± 0.1 | 0.5 ± 0.1 |
V + S + S | 13.2 ± 0.4 |
13.4 ± 0.4 |
12.9 ± 0.4 |
12.9 ± 0.3 |
13.4 ± 0.4 |
12.9 ± 0.4 |
13.2 ± 0.4 |
13.0 ± 0.4 |
V + S + R-PIA 0.3 | 12.6 ± 0.3 |
11.7 ± 0.6 |
11.5 ± 0.4 |
11.8 ± 0.5 |
12.5 ± 0.3 |
11.6 ± 0.4 |
12.2 ± 0.4 |
12.7 ± 0.3 |
V + DPCPX + R-PIA 0.3 | 12.7 ± 0.4 |
12.6 ± 0.6 |
12.3 ± 0.5 |
12.1 ± 0.5 |
11.8 ± 0.4 |
12.4 ± 0.4 |
12.2 ± 0.4 |
12.7 ± 0.3 |
V + S + R-PIA 1.0 | 13.2 ± 0.4 |
4.2 ± 0.7 |
4.9 ± 0.9 |
6.8 ± 0.7 |
6.6 ± 0.4 |
7.7 ± 0.6 |
10.0 ± 0.3 |
11.0 ± 0.3 |
V + DPCPX + R-PIA 1.0 | 13.1 ± 0.3 |
11.4 ± 0.4 |
11.7 ± 0.4 |
12.2 ± 0.6 |
12.9 ± 0.2 |
12.4 ± 0.5 |
13.0 ± 0.2 |
13.3 ± 0.4 |
V + S + R-PIA 3.0 | 13.4 ± 0.4 |
0.7 ± 0.3 |
1.3 ± 0.6 |
2.0 ± 0.8 |
3.2 ± 0.8 |
3.7 ± 0.7 |
5.6 ± 0.6 |
6.7 ± 0.4 |
V + DPCPX + R-PIA 3.0 | 13.0 ± 0.4 |
11.1 ± 0.3 |
10.6 ± 0.3 |
11.2 ± 0.5 |
11.2 ± 0.4 |
11.7 ± 0.3 |
12.7 ± 0.4 |
12.9 ± 0.3 |
Peripheral neuropathy was induced by the administration of vincristine (100 μg/kg, i.p.) for 10 days. On the 28th day, saline or DPCPX (10 μg/10 μl) was intrathecally administered 15 min before intrathecal R-PIA administration (1.0 μg/10 μl or 3.0 μg/10 μl). Cold allodynia was then assessed using the acetone drop test (seconds). Results are expressed as mean ± standard error of mean, n = 6 rats per group. One-way ANOVA followed by Tukey’s post hoc test.
P < 0.05 vs. normal control group,
P < 0.05 vs. vincristine control group,
P < 0.05 vs. R-PIA 1.0 μg/10 μl group,
P < 0.05 vs. R-PIA 3.0 μg/10 μl group. V: vincristine, S: saline, DPCPX: 1,3-dipropyl-8-cyclopentylxanthine = adenosine A1 receptor antagonist, R-PIA: N6-(2-phenylisopropyl)-adenosine R-(-)isomer = adenosine A1 receptor agonist.
Antiallodynic Effect of Intrathecally Administered R-PIA against Mechanical Static Allodynia
Time (min) | 0 | 15 | 30 | 45 | 60 | 90 | 120 | 180 |
---|---|---|---|---|---|---|---|---|
Normal | 15.0 ± 0.0 | 14.9 ± 0.1 | 15.0 ± 0.0 | 14.9 ± 0.1 | 14.9 ± 0.1 | 15.0 ± 0.0 | 15.0 ± 0.0 | 14.9 ± 0.1 |
V + S + S | 1.8 ± 0.3 |
1.6 ± 0.4 |
2.2 ± 0.3 |
2.1 ± 0.4 |
1.6 ± 0.3 |
2.1 ± 0.4 |
1.8 ± 0.4 |
2.0 ± 0.4 |
V + S + R-PIA 0.3 | 2.4 ± 0.3 |
2.3 ± 0.5 |
2.5 ± 0.3 |
3.2 ± 0.3 |
3.6 ± 0.3 |
4.5 ± 0.3 |
2.8 ± 0.2 |
2.3 ± 0.2 |
V + DPCPX + R-PIA 0.3 | 2.3 ± 0.3 |
3.4 ± 0.5 |
2.7 ± 0.4 |
3.9 ± 0.4 |
4.2 ± 0.3 |
3.6 ± 0.3 |
2.8 ± 0.2 |
2.3 ± 0.2 |
V + S + R-PIA 1.0 | 1.8 ± 0.3 |
10.8 ± 0.8 |
10.1 ± 0.7 |
8.2 ± 0.5 |
8.4 ± 0.4 |
7.3 ± 0.4 |
5.0 ± 0.2 |
4.0 ± 0.2 |
V + DPCPX + R-PIA 1.0 | 1.9 ± 0.2 |
3.6 ± 0.4 |
3.4 ± 0.3 |
2.8 ± 0.5 |
2.1 ± 0.1 |
2.6 ± 0.4 |
2.0 ± 0.3 |
1.7 ± 0.2 |
V + S + R-PIA 3.0 | 1.6 ± 0.3 |
14.5 ± 0.3 |
13.9 ± 0.7 |
13.1 ± 0.8 |
11.9 ± 0.8 |
11.3 ± 0.7 |
9.4 ± 0.4 |
8.3 ± 0.4 |
V + DPCPX + R-PIA 3.0 | 2.0 ± 0.2 |
3.9 ± 0.3 |
4.4 ± 0.3 |
3.8 ± 0.4 |
3.8 ± 0.3 |
3.3 ± 0.3 |
2.3 ± 0.2 |
2.1 ± 0.2 |
Peripheral neuropathy was induced by the administration of vincristine (100 μg/kg, i.p.) for 10 days. On the 28th day, saline or DPCPX (10 μg/10 μl) was intrathecally administered 15 min before intrathecal R-PIA administration (1.0 μg/10 μl or 3.0 μg/10 μl). Mechanical static allodynia was then assessed using the von Frey filament test (gram). Results are expressed as mean ± standard error of mean, n = 6 rats per group. One-way ANOVA followed by Tukey’s post hoc test.
P < 0.05 vs. normal control group,
P < 0.05 vs. vincristine control group,
P < 0.05 vs. R-PIA 1.0 μg/10 μl group,
P < 0.05 vs. R-PIA 3.0 μg/10 μl group. V: vincristine, S: saline, DPCPX: 1,3-dipropyl-8-cyclopentylxanthine = adenosine A1 receptor antagonist, R-PIA: N6-(2-phenylisopropyl)-adenosine R-(-)isomer = adenosine A1 receptor agonist.