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*Multidisciplinary Pain Unit, Ziekenhuis Oost-Limburg, Genk;
R&D, PRD Johnson & Johnson, Beerse; and
Department of Anesthesiology, University Hospital Antwerp, Edegem, Belgium
Address correspondence and reprint requests to Dr. T. Meert, c/o Dr. K. Vissers, Boeyenstraat 2, 3600 Genk, Belgium. Address e-mail to kris.vissers{at}skynet.be
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Abstract |
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IMPLICATIONS: We evaluated the changes in the behavioral reactions and the hormonal effects of a noxious chemical stimulus, i.e., formalin injection in animals with previously induced chronic constriction injury to the sciatic nerve. The effect in animals injected at the ipsilateral and contralateral site, sham-operated and controls, were compared.
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Introduction |
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All these animal models result in hypersensitivity to different noxious and non-noxious stimuli. Less is known about the interference of different pain states over time. Within clinical practice, however, the complaints of a single patient confronted with neuropathic pain are often based on different nociceptive inputs (6). Also, the clinical response to different treatment modalities varies among patients with comparable pain syndromes (7). To study these more complex pain presentations, combinations of different known pain models might be used. The formalin test (8) explores an inflammatory pain condition by subcutaneous injection of diluted formaldehyde into the plantar surface of the hind paw that causes a characteristic pattern of behavioral responses such as lifting and licking or biting of the injected paw. In this test, a first period of increased response lasting from 5 to 10 min, defined as the early phase, is followed by a decreased response for another 5 to 10 min. Between 20 and 50 min after injection, a second response phase can be delineated. This second phase is the reflection of a semi-acute pain stimulus challenging the more central spinal processing neuronal structures.
We describe the impact of formalin testing in rats with a previous chronic constriction injury (CCI), as measured by the presence of cold allodynia noted on a cold plate (2). In this study, the application of different concentrations of formalin to both the ligated- and the nonligated hind paw gives us the opportunity to evaluate the interferences of an inflammatory stimulation on a disturbed nerve transmission and spinal sensitization CCI model. Both behavioral and hormonal variables were monitored.
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Materials and Methods |
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According to the model of Bennett and Xie (2), the rats were anesthetized using Thalamonal® (1 mL per rat subcutaneously) and sodium pentobarbital (40 mg/kg intraperitoneally [IP]), and the common sciatic nerve of the left hind paw was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic trifurcation, approximately 7 mm of nerve was freed, and 4 loose ligatures of 4–0 chromic gut were placed around the sciatic nerve. Great care was taken to tie the ligatures such that the diameter of the nerve was just barely constricted when viewed with a microscope using a 40x magnification. In sham-operated animals, the same surgical procedure was followed until the nerve was exposed, the connective tissue was freed, and no ligatures were applied. After surgery, all animals received 10 mg/kg of naloxone IP as an antagonist for the Thalamonal® anesthesia to hasten the recovery and prevent further cooling of the animals and to prevent respiratory depression in the absence of further surgical stimulation.
Cold plate testing was performed in a cage with transparent acrylic walls (height, 30 cm) and a metal floor of 30 x 30 cm. The surface of the cold plate was cooled by a flow-through cooling apparatus that holds the surface temperature stable at -0.5°C ± 0.5°C (Julabo F25®, Julabo Labortechnik, Seelback, Germany). For testing, the animal was placed on the cold plate and the duration of lifting of both the left and right hind paw was measured over 5 min. For the sciatic nerve ligated animals, only rats with a difference in lifting time >25 s between the ligated and nonligated paw were used for formalin testing. The sham-operated animals and non-operated controls were tested similarly. Because these animals showed no paw lifting at all, these lifting data are not presented.
For formalin testing, the animals were housed individually in standard plastic observation cages with a wire mesh floor. After 1 h of habituation, 50 µL of formalin (0.16%, 0.63%, 2.5%, or 5%) or vehicle was injected in the sub-plantar area of either the left or the right hind paw. Flinching and licking or biting were observed. Flinching is defined as the number of shakings and flinches of the observed hind paw reported per observation period. The duration of licking or biting was recorded as the time the animal was licking or biting per observation period (10).
The behavioral responses were observed from 0 to 5 min, 20 to 25 min, and 40 to 45 min after the formalin injection. For data analysis, the first phase is represented by the first 5 min, whereas the second phase is represented by the sum of T20–25 and T40–45.
At 8 days after surgery, all animals were tested on the cold plate. One hour after cold plate testing, the selected ligated animals were randomly assigned to one of the 10 different experimental conditions (different formalin concentrations in either the left or right hind paw). The sham-operated and control animals were similarly divided over the different experimental conditions. Because no differences were observed between left and right hind paw injected sham controls, these groups were merged to represent the corresponding sham control groups. Ten animals were used for each experimental condition. The observer evaluating the formalin test was blinded to both the surgical intervention (ligated versus sham) and the concentration of formalin given. All experiments were performed between 8 and 12 AM.
Blood sampling was performed on three groups: animals challenged with formalin 5%, with vehicle, and untreated rats (blanco). The hormonal response was tested on a separate group of animals that had undergone CCI ligation and sham operation or no operation.
These rats were subjected to the same experimental conditions as those described in the behavioral experiments. However, no behavioral measurements were recorded except for the cold allodynia. After the normal observation period after formalin injection, these animals were killed, and blood samples were collected for all different groups between 8 and 10 AM. The tubes were kept on ice until centrifugation. Plasma was immediately frozen until assay. For assessment, the commercially available radio-immunoassay kits were used as follows: for the adrenocorticotrope hormone (ACTH) ACTH-Nicholls-RIA®; detection limit, 4.5–1550 pg/mL; corticosterone-Diagnostic Produkt Corporation-RIA®; detection limit 58–5772 nmol/L; Prolactin-DPC (milinea)-ELISA®; detection limit 4.0–80 ng/mL; for thyroid stimulating hormone (TSH): TSH-Amersham-RIA®; detection limit 1.0–64 ng/mL.
All data are expressed as mean ± 1 SEM. Differences between experimental conditions were evaluated using the Mann-Whitney U-test (Siegel; 1956; two tailed). Statistical significance was considered from P < 0.05.
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Results |
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Different concentrations of formalin (5%, 2.5%, 0.63%, and 0.16%) were used for injection in the left operated or right non-operated hind paw. The resulting flinching and licking or biting behavior is illustrated in Figure 1.
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In contrast, for licking or biting behavior, we observed not only a decreased responsiveness of the ipsilateral paw, but also at the contralateral side for both phases, and this was statistically significant (for the early phase; P < 0.05; for the late phase; P < 0.01). Again, the observed reductions were dose related.
For hormonal determinations, only 5% formalin injections were used. Because no differences were observed between the values obtained after formalin injection in the ipsilateral and the contralateral paws, the data were pooled. The different groups therefore included non-operated and sham-operated controls as well as CCI rats.
The baseline plasma levels of ACTH in all three groups of operational conditions were comparable (P > 0.05): 27.00 (± 10.73) pg/mL for non-operated, 29.80 (± 6.09) pg/mL for the sham-operated, and 22.13 (± 1.82) pg/mL for the CCI-operated animals (Table 1). Intradermal vehicle injections caused increases in ACTH in all the groups; this increase was significant for the non-operated and the CCI-operated animals. An injection of formalin 5% further increased (P < 0.05) the ACTH levels in all groups, as compared with the noninjected and vehicle treated groups. However, the increases after formalin in the non-operated animals were statistically significantly larger than those in the sham- (P < 0.01) and CCI-operated rats (P < 0.001).
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Application of 5% formalin to non-operated animals resulted in a decrease (P < 0.05) of TSH plasma levels from 9.63 (± 1.08) for the nontreated animals to 5.61 (± 0.44) ng/mL after formalin (Table 1). Performing a sham operation causes no changes in the basal TSH plasma levels, and neither the injection of formalin or vehicle had any impact on these levels. In contrast, performing a CCI causes a decrease (P < 0.01) in the basal TSH levels from 9.63 (± 1.08) ng/mL in the controls to 6.69 (± 1.13) ng/mL in the CCI rats. Application of formalin or vehicle in the CCI rats did not further affect the average TSH plasma levels. Overall, the changes in plasma TSH during the different manipulations were less pronounced than those observed with either ACTH or corticosterone.
Basal prolactin levels in the untreated groups varied from 4.88 (± 0.43) ng/mL in the CCI rats to 6.08 (± 0.91) ng/mL in the non-operated rats (Table 1). Injections of vehicle and formalin did not result in significantly different plasma levels in either sham- or CCI-operated rats. A vehicle injection resulted in slightly increased prolactin level in non-operated controls (P < 0.05).
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Discussion |
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Different concentrations of formalin, ranging from 0.16% to 5% were used to evaluate the effects of various chemical stimulus intensities. The application of formalin did not result in an increased hypersensitivity to formalin in CCI rats when compared with controls or sham-operated animals.
Although all CCI-operated animals were selected on the presence of hypersensitivity to cold, a reduced reactivity to formalin was present when formalin was injected into the CCI-operated paw. This was true for all behavioral measurements, including flinching and biting or licking, during both the early and the late phase and seemed to be independent of the concentration of formalin used. As such, these results do not confirm the chemical hypersensitivity reported in CCI rats in response to the application of mustard oil (2). The present observations suggest that the hypersensitivity measured in the CCI rat model might be restricted to specific stimulus modalities, such as non-noxious stimuli or specific irritants. Whether a lesion of certain fiber populations (Aß, A
, or C) or a change in functional reactivity and transmission characteristics of some nerves (12) does account for this phenomenon remains to be elucidated. Also, rat strain differences and their consequent different reactivity to inflammatory stimuli and chronic conditions should be checked as a possible explanation. Moreover, from the work of LaBuda et al. (13) and Cesena and Calcutt (14), we conclude that even between the different animal pain models, there may be important differences.
To evaluate whether similar mechanisms will play a role in painful stimuli applied to body regions not affected by a nerve ligation, a formalin test was also performed in the contralateral non-operated hind paw in CCI animals with a well-documented cold allodynia for the operated left hind paw (15).
As such, the present study demonstrates that in animals with a hypersensitivity to non-noxious thermal stimulation caused by a CCI, the responses on an additive inflammatory pain stimulus were decreased for ipsilateral and the non-operated contralateral hind paws for both the early and the late phase independently of the formalin concentration used.
The importance of this contralateral study is emphasized by the work of Sotgiu and Biella (16,17), demonstrating the impact of the contralateral nerve stimulation on the firing properties of the ipsilateral ligated sciatic nerve in rats (18,19), which may be due to changes in the peripheral versus central nervous system. In this study, we focused more on the peripheral impact by the behavioral analysis. The reduced reactivity to formalin in the contralateral paw of CCI-operated animals during the second phase was relatively independent of the concentration of formalin used. Giving formalin to the non-operated CCI hind paw resulted in a normal first phase of the formalin testing but a reduced reactivity in the second phase as compared to controls. As such, these observations clearly demonstrate different modulatory mechanisms involved in both phases of the formalin testing. Apparently, a strong acute nociceptive input, as in the first phase of the formalin test, in an area not affected by nerve ligation seems to result in normal spinal and supraspinal reactions as measured by flinching and licking or biting behavior. As opposed to this, the reactivity of the second phase of formalin in these animals is reduced.
First, the CCI of the sciatic nerve causes a reduction in the total number of nerve fibers (Aß, A, and C fibers) (20,21) and induces a hypersensitivity to non-noxious stimulation. Therefore, the sensitivity of the surviving myelinated and unmyelinated nerve fibers, for different stimulation modalities, might be changed (22). However, the decreased contralateral hypersensitivity to noxious stimulation in a normal nerve conduction system, as measured by the second phase of formalin in the non-operated paw, indicates a more spinal or supraspinal phenomenon (17,23).
Second, animals challenged with formalin in the non-operated CCI-paw might be confronted with a continuous differential and invalidating dilemma. Even during the normal exploration behavior, the animal is regularly lifting the CCI-operated hind paw because of mechanical hyperalgesia. The presence of input of two sites of pain and the possible coping strategies to reduce painful stimuli in those two areas can result in conflicting attentions and body movements resulting in an observable reduction in formalin reactivity.
Finally, because of nerve damage, neuronal fibers of the affected hind paw are firing spontaneously (17), causing continuous stimulation of inhibitory descending pathways. As a result, the transduction of the painful stimuli can be masked. This seems to be especially true in the lesion-affected zone and for mild stimulation in the non-operated contralateral paw. However, strong stimulation in the nonaffected hind paw seems to react normally in the acute phase. Additional research is clearly required to further clarify the exact mechanism(s) playing a role in these changed perceptions and reactivity to the formalin challenge.
The analysis of the plasma hormonal levels of CCI animals challenged with formalin was also resulted in a series of interesting observations.
Injection of 5% formalin increased the plasma levels of ACTH and corticosterone, demonstrating that the injection of formalin in the hind paws of rats is a stressful event. This observation is also confirmed by others (24–26). The degree of augmentation is dependent on the pain state or general condition of the rat. The plasma ACTH and corticosterone levels increased with, respectively, a factor 10 and 5 in non-operated animals. In CCI-operated animals, the increase reached only half that of non-operated animals (Table 1). Because we do not see statistically significant differences in the hormonal responses of non-operated and sham-operated animals, the decreased response in CCI rats suggests an acute noxious stimulus such as a formalin injection, which seems to be less stressful in the latter. Whether the decreased sensitivity to painful stimuli resulting in less stress is responsible for this phenomenon remains to be elucidated. The prolactin plasma levels showed no increase to the stressor formalin in the CCI rats. It is reported that the prolactin levels can increase in acute and chronic pain (27). The TSH levels showed a marked reduction with the application of formalin in control animals. A similar reduction was reported by Marti et al (28). However, in CCI rats, significantly smaller TSH levels were measured, which might have been because of the chronic stimulation and sensitization. We observed no further changes by applying a strong noxious stimulus such as formalin to these animals. This demonstrates that the CCI rats with a clear hypersensitive state can have a reduced sensitivity to noxious chemical stimulation. This reduction may be attributed to a change in peripheral input, but another explanation may be that chronic nociception induces a decreased reactivity to stress or different coping mechanism when different painful and stressful stimuli are presented simultaneously.
In conclusion, a CCI ligation in rats induces a decreased reactivity to particular noxious stimulation such as the application of formalin in the ligated hind paw in terms of both the flinching and the licking or biting behavior. This effect is independent of the formalin concentration used. If formalin is given in the contralateral non-ligated paw of CCI rats, a reduced reactivity with less licking or biting was also observed in the second phase of the formalin test. Whether these reductions in reactivity are caused by decreased pain reactivity at the peripheral, spinal, or supraspinal level, or whether these are caused by changes in the stress reactivity and coping strategies, remains to be elucidated.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This article has been cited by other articles:
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  R. Kaku, M. Yokoyama, H. Kobayashi, Y. Matsuoka, T. Sato, S. Mizobuchi, Y. Itano, and K. Morita Altered Response to Formalin by L5 Spinal Nerve Ligation in Rats: A Behavioral and Molecular Study Anesth. Analg., April 1, 2007; 104(4): 936 - 943. [Abstract] [Full Text] [PDF]
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