This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Liu, J. Articles by Xu, J. Search for Related Content PubMed PubMed Citation Articles by Liu, J. Articles by Xu, J. Related Collections Mechanisms Pain Mechanisms Preclinical Pharmacology Pain Pharmacology

---

PAIN MECHANISMS

The Effect of Pentoxifylline on Existing Hypersensitivity in a Rat Model of Neuropathy

From the Department of Anaesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, People's Republic of China.

Address correspondence and reprint requests to Jian Liu, MD, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, People's Republic of China. Address e-mail to liuj_7610{at}msn.com.

Abstract

BACKGROUND: Using a rat L5 spinal nerve transection model we previously showed that pentoxifylline prevents hyperalgesia through antiinflammation in the prefrontal brain. In this study, we examined efficacy when applied after injury.

METHODS: We examined the effect of pentoxifylline on existing mechanical allodynia, observing glial activation and proinflammatory cytokine expression in the lumbar spinal cord, when given 7 days after L5 spinal nerve transection.

RESULTS: There was no effect from pentoxifylline on existing hypersensitivity, glial activation, and cytokine expression when applied after L5 spinal nerve transection.

CONCLUSION: Pentoxifylline administered intraperitoneally on day 7 postsurgery failed to alleviate existing hypersensitivity, or reduce glial activation and cytokine expression.

Chronic neuropathic pain is caused by a lesion or inflammation of the nervous system characterized by spontaneous pain, hyperalgesia, and allodynia. Recently, it has become clear that inflammatory and immune mechanisms in the peripheral and central nervous systems play an important role in the development and maintenance of neuropathic pain.1 Glial cells, such as microglia and astrocytes, are activated in response to nervous system damage with the up-regulation of cell surface markers, including macrophage antigen complex-1 (Mac-1), toll-like receptor-4 (TLR-4), glial fibrillary acidic protein (GFAP),2–4 and subsequently, release several inflammatory mediators.5 Proinflammtory cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6, play a critical role in the development of central sensitization and release of nociceptive mediators such as prostaglandins (PG), substance P, and nitric oxide (NO).6,7 We have previously shown that pentoxifylline dose-dependently attenuates the development of mechanical and thermal hyperalgesia through inhibiting the expression of proinflammatory cytokines and the activation of nuclear factor kappa B in the prefrontal brain.8 The present study explored whether systemic pentoxifylline administered after nerve injury could attenuate existing hypersensitivity (mechanical allodynia) and decrease glial activation and the expression of inflammatory cytokines in the lumbar spinal cord in a rat L5 spinal nerve transection model.

METHODS

Animals
After obtaining approval from the Institutional Animal Care and Use Committee at Jinling Hospital, the experiments were performed on male Sprague-Dawley rats weighing 190 to 220 g at the start of surgery. The animals were housed on a 12-h light/dark cycle with food and water available ad libitum. Efforts were made throughout to minimize animal discomfort and to use the fewest animals needed for statistical significance.

Behavioral Testing
Behavioral studies were performed in a quiet room from 8:00 to 10:00 am. Mechanical allodynia was evaluated by applying 2 and 12 g von Frey filaments to the dorsal surface of the ipsilateral hindpaw (Model 2390CE, IITC Life Science, Woodland Hills, CA).9 We measured mechanical allodynia as the number of paw withdrawals in 3 sets of 10 stimulations each, with sets separated by 10 min from the previous set to avoid sensitization. Mechanical allodynia was assessed before surgery (day 0), and on postnerve transection days 1, 4, 7, 10, 12, and 14.

Experimental Design
After baseline assessment on day 0, rats received an L5 spinal nerve transection or sham surgery as described previously.9 Daily intraperitoneal pentoxifylline (50 or 100 mg/kg) was initiated on day 7 after injury in the evening between 4:00 and 6:00 pm and continued through day 13 after transection. Eight rats were assigned to each treatment group.

Tissue Collection
After behavioral testing on postsurgical day 14, rats were deeply anesthetized with sodium pentobarbital (80 mg/kg intraperitoneal) and decapitated. An 18-gauge needle was inserted into the caudal end of the vertebral column and the spinal cord was expelled with ice-cold phosphate-buffered saline. The spinal cord was frozen immediately and stored on liquid nitrogen until homogenization. The L5 lumbar spinal cord was isolated from the intact frozen cord at the time of mRNA and protein quantification. Total RNA was isolated from the L5 lumbar spinal cord by the TRIzol extraction method (Invitrogen, Carlsbad, CA).

Cytokine Protein Estimation by ELISA
Quantitative determination of TNF, IL-1β, IL-6, and IL-10 cytokines was performed on the L5 spinal cord. Tissue homogenization was prepared as described previously.8 TNF (R & D Systems, Minneapolis, MN), and IL-1β, IL-6, and IL-10 (BioSource International, Camarillo, CA) protein concentrations were determined using the quantitative sandwich enzyme immunoassay according to the manufacturer's protocol.

Real-Time Reverse Transcription-Polymerase Chain Reaction
The primers shown in Table 1 were designed by using the Primer Premier 5.0 program (PREMIER Biosoft International, Silicon Valley) and had a melting temperature of 60°C and 70°C, respectively. The reverse transcription (RT) reaction was performed in a 20-µL total reaction volume containing 4 µL of 5 x RT buffer, 4 µL of 2.5 mM dNTPs, 1 µL of Multiscribe reverse transcriptase (50 U/µL) (Promega, Madison, WI), 1 µl of RNase inhibitor, 5 µL of RNase-free water, and 3 µg of DNase-treated total RNA in a 5 µL volume. The RT reaction was performed at 25°C for 10 min, 37°C for 120 min, and 95°C for 5 min. The real-time polymerase chain reaction (PCR) reactions were performed by using the Rotor-Gene 3000^TM Real-Time PCR detection system (Corbett Research, Sydney, Australia) in a total reaction volume of 25 µL containing the final concentration of 3 U of Platinum TaqDNA polymerase; 1.5 mM MgCl_2; 250 µM dGTP, dCTP, dATP, and dTTP; 400 nM forward and reverse primers; 2.5µl 10 x PCR buffer; SYBR Green I and 1 µL cDNA from the RT step. PCR reagents were from Invitrogen (CA). Relative standard curves were generated by plotting the threshold value versus the log of the amount of total cDNA added to the reaction and used to compare the relative amount of target genes from control to sham groups and L5 nerve-transected animals. Calculation of the threshold value, standard curve preparation and quantification of mRNA in the samples were performed by the Rotor-gene 6.0 software provided with the Corrbett Reasearch.

Statistical Analysis.
Values are expressed as means ± sem. Comparisons between groups were performed using analysis of variance for repeated measurements followed by Tukey-Kramer multiple comparisons test (SPSS 12.0). P < 0.05 was considered significant.

RESULTS

Behavioral Data
Baseline non-noxious mechanical stimuli (2 and 12 g of von Frey filament) did not produce any paw withdrawal response (allodynia). After L5 spinal nerve transection, mechanical allodynia was observed throughout the study period. Sham surgery produced no significant behavioral hypersensitivity. There was no difference in mechanical allodynia between rats who received pentoxifylline and those who received saline (Fig. 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. Changes in the paw withdrawal numbers, 2-g (top) and 12-g (bottom) von Frey filament- induced mechanical allodynia, on day before surgery(day 0) and days 1, 4, 7, 10, 12, and 14 after operation in the postinjury treatment paradigm. Nerve injury resulted in a steady overall statistically significant (P < 0.01) increase of paw withdrawal numbers compared with animals in the sham-operated group. Initiation of pentoxifylline (PTX, 50 and 100 mg/kg i.p.) treatment to rats after day 7 of nerve injury did not show any significant difference in the mechanical allodynia compared with saline treated nerve-injured rats (P > 0.5). Horizontal line indicates the time frame of pentoxifylline administration (i.e., day 7 to day 13). Day 0 mechanical allodynia represents baseline preinjury responses.

Effect of Pentoxifylline on Inflammatory Cytokines
L5 spinal nerve transection increased the expression of TNF, IL-1β, IL-6, and IL-10 in the spinal cord compared with the sham-operated controls (Figs. 2A-C). There was an apparent dose versus response relationship between pentoxifylline and expression for all four cytokines, but only the increase in IL-10 in rats receiving 100 mg/kg reached statistical significance compared with saline-treated controls (Fig. 2D).

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of postinjury treatment of pentoxifylline on the expression of the tumor necrosis factor (TNF) (A), interleukin (IL)-1β (B), IL-6 (C), and IL-10 (D) in the L5 lumbar spinal cord on day 14 after transection. TNF, IL-1β, and IL-6 production was markedly elevated in nerve injury rats compared with the sham-operated animals, initiation of pentoxifylline (PTX, 50 and 100 mg/kg) treatment to rats after day 7 of nerve injury did not attenuated the expression of TNF, IL-1β, and IL-6 compared with saline-treated animals. The level of IL-10 was significantly elevated in the postsurgery pentoxifylline treatment (P < 0.05 for 100 mg/kg) compared with saline-treated animals. *P < 0.05; **P < 0.01 vs sham group; #P < 0.05 vs saline group.

Effect of Pentoxifylline on the Glial Activation Markers
Postinjury administration of pentoxifylline had no effect on the expression of mRNA for Mac-1, TLR-4, and GFAP compared with the saline-treated animals (P > 0.5) (Table 2).

DISCUSSION

Postinjury pentoxifylline treatment had no effect on existing behavioral hypersensitivity in a rat L5 spinal nerve transection model of neuropathic pain. There was a trend towards decreasing levels of TNF, IL-1β, and IL-6 with increasing pentoxifylline dose. However, this trend did not reach statistical significance, and was very modest compared with the increase in cytokine expression associated with L5 spinal nerve transection.

The different effect of pentoxifylline on the development of hypersensitivity versus existing hypersensitivity may have been due to the effect of pentoxifylline on inflammation. In light of a previous study in which pretreatment with pentoxifylline failed to inhibit IL-1β and PGE2-induced hypernociception in the inflammatory pain models,10 the mechanism of action underlying pentoxifylline's effect is upstream of IL-1β release. Elevated levels of TNF, IL-1β, and IL-6 and other cytokines suggest a profound inflammatory cascade in the L5 spinal cord after spinal nerve transection. This inflammatory response may contribute to central sensitization through direct actions on neuronal receptors and indirect actions upon glial cells. For example, IL-1β and TNF are capable of inducing substance P expression and axonal transportation directly.11,12 Indirectly, IL-1β and TNF can feed back on both microglia and astrocytes to sustain activation as well as to up-regulate inducible NO synthase and cyclooxygenase-2 expression.13 Drugs capable of attenuating existing hyperalgesia must have dual abilities to inhibit the production of proinflammatory cytokines as well as to prevent production of downstream nociceptive mediators such as NO and PG. Therefore, initiation of pentoxifylline treatment at day 7 after L5 spinal nerve transection could not attenuate the continuing cytokine cascade induced by nerve injury in the spinal cord.

Propentofylline, which is similar to pentoxifylline, has been shown to be effective in rodent models of neuropathy for both prevention and treatment in previous studies.14,15 Propentofylline exerts its antiallodynic effect through attenuating both microglial and astrocytic activation. These were demonstrated using immunohistochemistry and/or real-time RT-PCR analyses. In the current study, we used only real time RT-PCR to observe the expression of the mRNA of Mac-1, TLR-4, and GFAP. The differences between pentoxifylline and propentofylline in the animal models of neuropathic pain are not fully understood. However, a previous study of the local effect of pentoxifylline and propentofylline on formalin-induced pain demonstrated that pentoxifylline had a better preventive effect, but the propentofylline worked better on established hyperalgesia.16 Differences between pentoxifylline and propentofylline in inflammatory pain might result from their diverse effects on isoforms of phosphodiesterase and on the expression of IL-10.

In summary, this study demonstrated the inability of pentoxifylline to treat the allodynia that developed 7 days after L5 spinal nerve transection when treatment was delayed until a week after the injury. The study also supports the importance of glial activation in the maintenance of neuropathic pain.

ACKNOWLEDGMENT

We thank Feng Genbao for his excellent technical assistance.

Footnotes

Accepted for publication October 3, 2007.

This work was partly supported by Research foundation of Jingling hospital.

REFERENCES

1. Moalem G, Tracey DJ. Immune and inflammatory mechanisms in neuropathic pain. Brain Res Brain Res Rev 2006;51:240–64[Medline]
2. Coyle DE. Partial peripheral nerve injury leads to activation of astroglia and microglia which parallel the development of allodynic behavior. Glia 1998;23:75–83[Web of Science][Medline]
3. Winkelstein BA, Rutkowski MD, Sweitzer SM, Pahl JL, DeLeo JA. Nerve injury proximal or distal to the DRG induces similar spinal glial activation and selective cytokine expression but differential behavioral responses to pharmacologic treatment. J Comp Neurol 2001;439:127–39[Web of Science][Medline]
4. Tanga FY, Nutile-McMenemy N, DeLeo JA. The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy. Proc Natl Acad Sci USA 2005;102:5856–61[Abstract/Free Full Text]
5. Chao CC, Hu S, Ehrlich L, Peterson PK. Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-d-aspartate receptors. Brain Behav Immunol 1995;9:355–65[Web of Science][Medline]
6. Serou MJ, DeCoster MA, Bazan NG. Interleukin-1 beta activates expression of cyclooxygenase-2 and inducible nitric oxide synthase in primary hippocampal neuronal culture: platelet-activating factor as a preferential mediator of cyclooxygenase-2 expression. J Neurosci Res 1999;58:593–8[Web of Science][Medline]
7. Inoue A, Ikoma K, Morioka N, Kumagai K, Hashimoto T, Hide I, Nakata Y. Interleukin-1 beta induces substance P release from primary afferent neurons through the cyclooxygenase-2 system. J Neurochem 1999;73:2206–13[Web of Science][Medline]
8. Liu J, Feng X, Yu M, Xie W, Zhao X, Li W, Guan R, Xu J. Pentoxifylline attenuates the development of hyperalgesia in a rat model of neuropathic pain. Neurosci Lett 2007;412:268–72[Web of Science][Medline]
9. Colburn RW, Rickman AJ, DeLeo JA. The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp Neurol 1999;157:289–304[Web of Science][Medline]
10. Vale ML, Benevides VM, Sachs D, Brito GA, da Rocha FA, Poole S, Ferreira SH, Cunha FQ, Ribeiro RA. Antihyperalgesic effect of pentoxifylline on experimental inflammatory pain. Br J Pharmacol 2004;143:833–44[Web of Science][Medline]
11. Ding M, Hart RP, Jonakait FM. Tumor necrosis factor- induces substance P in sympathetic ganglia through sequential induction of interleukin-1 and leukemia inhibitory factor. J Neurobiol 1995;28:445–54[Web of Science][Medline]
12. Jeanjean AP, Moussaoui SM, Maloteaux JM, Laduron PM. Interleukin-1β induces long term increase of axonally transported opiate receptors and substance P. Neuroscience 1995;68:151–7[Web of Science][Medline]
13. Tonai T, Taketani Y, Ueda N, Nishisho T, Ohmoto Y, Sakata Y, Muraguchi M, Wada K, Yamamoto S. Possible involvement of interleukin-1β in cyclooxygenase-2 induction after spinal cord injury in rats. J Neurochem 1999;72:302–9[Web of Science][Medline]
14. Sweitzer SM, Schubert P, DeLeo JA. Propentofylline, a glial modulating agent, exhibits antiallodynic properties in a rat model of neuropathic pain. J Pharmacol Exp Ther 2001;297:1210–17[Abstract/Free Full Text]
15. Tawfik VL, Nutile-McMenemy N, Lacroix-Fralish ML, Deleo JA. Efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury. Brain Behav Immun 2007;21:238–46[Web of Science][Medline]
16. Dorazil-Dudzik M, Mika J, Schafer MK, Li Y, Obara I, Wordliczek J, Przewlocka B. The effects of local pentoxifylline and propentofylline treatment on formalin-induced pain and tumor necrosis factor-alpha messenger RNA levels in the inflamed tissue of the rat paw. Anesth Analg 2004;98:1566–73[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Liu, J. Articles by Xu, J. Search for Related Content PubMed PubMed Citation Articles by Liu, J. Articles by Xu, J. Related Collections Mechanisms Pain Mechanisms Preclinical Pharmacology Pain Pharmacology