substance p and neurotensin are up-regulated in the lumbar ...€¦ · von frey hair testing was...

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86 The Canadian Journal of Veterinary Research 2004;68:86–92 2000;64:0–00 The Canadian Journal of Veterinary Research 87

Substance P and neurotensin are up-regulated in the lumbar spinal cord of animals with neuropathic pain

Pascal Vachon, Robert Massé, Bernard F. Gibbs

A b s t r a c tMany neuropeptides have been shown to be up-regulated in response to pain. The purpose of this study was to identify pain-related peptides in a rat model of neuropathic pain induced by sciatic nerve cuff implantation. Rats were tested for touch sen-sitivity prior to and 7 days following cuff implantation or sham surgeries. The lumbar spinal cord was removed on the 8th day post-surgery and the lumbar enlargement was processed for the detection of selected peptides using time-of-flight mass spec-trometry. Only substance P (MW 1346.74) and neurotensin (MW 1673.0) were detected in the lumbar spinal cord of animals with mechanical allodynia (P 0.01) following innocuous tactile stimulation of the affected hind paw. Therefore, substance P and neurotensin may be target candidates for the understanding and treatment of neuropathic pain

R é s u m éL’objectif de cette étude est d’identifier les neuropeptides de la moelle épinière en relation avec la douleur neuropathique en utilisant un modèle animal. La sensibilité au toucher a été évaluée avant et 7 jours suivant la chirurgie consistant en l’application d’un anneau de polyéthylène autour du nerf sciatique chez le rat. Les rats ayant eu la chirurgie sans l’implantation de l’anneau ont servi comme contrôle. La partie lombaire de la moelle épinière a été prélevée au 8ième jour suivant les chirurgies et les tissus ont été préparés afin d’être analysés par spectrométrie de masse. Uniquement la substance P et la neurotensine ont été détectées dans les moelles des animaux ayant démontrés la présence d’une allodynie mécanique (P 0.01) suite à l’application d’un anneau autour du nerf sciatique. La substance P et la neuroten-sine semblent donc des candidats cibles pour le traitement de la douleur neuropathique et la compréhension des mécanismes associés à cette condition.

(Traduit par les auteurs)

I n t r o d u c t i o nNeuropathic pain, defined as aberrant sensory processing without

inflammation in either the peripheral or central nervous system, occurs in animals with trauma and certain diseases (diabetes). Neuropathic pain is revealed by allodynia (pain on light touch) and hyperalgesia (increased perception of pain) (1). Many physiological and anatomical studies have been performed to identify neuropep-tides responsible for hyperalgesia and allodynia in response to neuropathic pain (2). However, no studies have directly detected the presence of pain-related oligopeptides in the spinal cord of animals experiencing neuropathic pain using high-resolution mass spectrom-etry. The presence of certain pain-related peptides in the spinal cord, for example, substance P, vasoactive intestinal peptide, cholecycs-tokinin, neurotensin, met-enkephalin, and bombesin are associated with chronic pain mechanisms (3) and have been measured by liquid chromatography. Immunoreactive methods show that substance P, calcitonin gene-related peptide, somatostatin, met-enkephalin, and neurotensin are up-regulated in rats in response to formalin-induced pain (4). We conducted experiments in which neuropathic pain was induced in rats following surgical implantation of a polyethylene

cuff around the sciatic nerve and evaluated the presence of pain-related peptides in the lumbar spinal cord in relation to mechanical allodynia. Previous studies (5,6) have shown that mechanical allo-dynia develops within 2 d in cuff-implanted animals and may last as long as a few months. In this preliminary study, we searched for the presence of peptides in the lumbar spinal cord at 8 d post-surgery to identify pain-related peptides that may be implicated in chronic pain mechanisms of neuropathic pain in association with normal behavioral responses to touch and allodynia.

M a t e r i a l s a n d m e t h o d s

AnimalsA total of 17 male Sprague Dawley rats (Charles River, St-Constant,

Quebec) weighing between 300 and 350 g were used for these experiments. They were housed in polycarbonate cages (Lab Products, Seaford, Delaware, USA) on hardwood sawdust bedding (PWI, St-Hyacinthe, Quebec) and acclimated for at least 3 d prior to the initiation of the study. Rats received tap water and a standard labo-ratory rodent diet (Ralston Purina Canada, Woodstock, Ontario)

MDS Pharma Services, Montreal, Quebec (Gibbs, Massé); Faculté de Médecine Vétérinaire, Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec (Vachon).

Address all correspondence and reprint requests to Dr. P. Vachon; telephone: (450) 773-8521 ext. 8294; fax: (450) 778-8109; e-mail: [email protected]

Received April 4, 2003. Accepted September 10, 2003.

Article


Table I. Individual and mean von Frey hair testing scores prior to and at day 7 following surgery in cuff-implanted and sham-operated animals

Raw scores (g) (%) cuff-implanted Raw scores (%) of cuff-implanted animals with spinal pain-related animals without detection of Raw scores (%) neuropeptides present pain-related neuropeptides of sham-operated animals Day 7 Day 6 Day 6 Prior to surgery following surgery Prior to surgery following surgery Prior to surgery following surgeryIndividual and 50.0 (100) 4.9 (9.8) 19.7 (100) 14.8 (75.1) 6.2 (100) 9.7 (157)mean von Frey 49.0 (100) 4.7 (9.6) 50.0 (100) 50.0 (100) 21 (100) 21 (100)hair testing scores 49.0 (100) 11.4 (23.3) 9.5 (100) 5.1 (53.7) 50 (100) 50 (100) 12.8 (100) 2.3 (18.0) 9.2 (100) 9.7 (105.4) 14.9 (100) 6.2 (41.6) 19.6 (100) 22.0 (89.1) 9.7 (100) 1.9 (19.6) 16.2 (100) 23.7 (68.4) 14.7 (100) 5.9 (40.1) 14.9 (100) 9.7 (153.6)Mean ± s 100 ± 0 23.1 ± 13.1a 100 ± 0 92.2 ± 32.5b 100 ± 0 119.0 ± 32.9b

s — Standard deviationa P 0.01b P is not significant

Figure 1. Spectrum showing molecular ions of 2 identified peptides using a quadrupole/time-of-flight (QqTOF) mass spectrometer. Detection of substance P (674.4 amu) and neurotensin (836.9 amu) from the lumbar enlargement of the spinal cord taken from a neuropathic rat with mechanical allodynia following a cuff-implantation around the sciatic nerve.


ad libitum. The experimental protocol was approved by the Institutional Animal Care and Use Committee of McGill University prior to animal use and was in accordance with the guidelines of the Canadian Council on Animal Care.

Surgical methods Surgical methods have previously been described (5). Briefly, fol-

lowing anesthesia with 60 mg/kg IP of ketamine (Rogarsetic; Pfizer Canada, Kirkland, Quebec) and 6 mg/kg of xylazine IP (Rompun; Bayer, Toronto, Ontario), the left common sciatic nerve was exposed via blunt dissection at the level of the thigh (biceps femoris). In 14 rats, a 2 mm cuff of PE-90 polyethylene tubing (Fischer Scientific Canada, Nepean, Ontario) was placed around the nerve following its isolation using a ball-tipped glass probe. The skin was closed using stainless steel clips and a 0.2% nitrofurazone ointment (Vétoquinol, Joliette, Quebec) was applied locally. A second group of rats (n = 3) underwent sham operations. In these animals, the same surgical procedures were performed without the placement of the cuff on the sciatic nerve.

Mechanical allodynia testingVon Frey hair testing was performed according to previously

published methods (5). Briefly, paw withdrawal thresholds were tested on the plantar surface of the left hind paw prior to and 7 d following surgery using von Frey monofilaments (0.4 g to 50 g; Stoelting, Wood Dale, Illinois, USA). Monofilament application was to the central region of the plantar surface. The hairs were applied until buckling occurred. Values reported are the percent difference between the threshold force necessary to elicit the mechanical withdrawal of the hind paw evaluated at 7 d post-surgery and the baseline values taken prior to surgery. The threshold was taken as the lowest force that caused at least 3 withdrawals out of 5 consecutive stimuli. On the day following the last von Frey hair testing, the lumbar region of the spinal cord from all animals was rapidly collected following deep anesthesia with halothane. Spinal cord samples were immediately frozen at 50°C in hexane (Sigma-Aldrich, St-Louis, Missourri, USA) and held at 80°C pending analysis.

Figure 2. Typical mass spectrum of lumbar enlargement of the spinal cord from a cuff-implanted animal without any change in mechanical allodynia. Notice absence of 674.3650 and 836.9490 amu, the characteristic protonated molecular ions of substance P and neurotensin. This was observed in all cuff-implanted animals without observable changes in touch sensitivity as well as sham-operated animals.


Peptide extraction and identificationFor peptide extractions, lumbar spinal cord samples were cut into

approximately 1 mm cubes and vortexed with the extracting solu-tion, which consisted of 0.5 mL deionized water, 0.5 mL acetonitrile (Sigma-Aldrich), and 10 L trifluoroacetic acid (Sigma-Aldrich). The mixture was sonicated for 5 min, vortexed for 2 min, and then centrifuged at 27 000 g for 10 min. The supernatant was retained. Two more extractions were performed, the supernatants pooled and filtered through a 0.45 m filter to remove particulate matter. The samples were purified and desalted using cartridges (ZipTip car-tridges; Millipore Corporation, Bedford, Massachusetts, USA) con-taining C18 resin. The final preparation was concentrated to 20 L in a centrifugal concentrator (SpeedVac centrifugal concentrator; ThermoSavant, Holbrook, New York, USA) before infusing into a mass spectrometer (Pulsari QSTAR mass spectrometer; MDS-Sciex, Toronto, Ontario) equipped with a nanospray ion source. Standards were run for the following peptides: substance P, neurotensin, cal-citonin gene-related peptide, met-enkephalin, cholecystokinin, vasoactive intestinal polypeptide, somatostatin, and bombesin (Sigma-Aldrich). Their spectra were in agreement with the Mascot

database (Matrix Net Systems, Austin, Texas, USA). The spectra of the peptides in the extracted tissues were in accordance with those from the standards.

Statistical analysis T-tests were performed on raw data to assess differences in

mechanical hyperalgesia prior to and following surgeries for differ-ent experimental groups. Statistical significance level was set a priori at P 0.05. Statistical analyses were performed using software (Statistica, version 4.3; Statsoft, Tulsa, Oklahoma, USA).

R e s u l t sIn 50% of the cuff-implanted animals (n = 7), a significant hyper-

sensitivity response to touch (allodynia) was observed (P 0.01) (Table I). In these animals, the recorded force values at 7 d post-surgery were decreased to 23.1% ± 13.1% of the baseline values. The mass spectrometry analysis of lumbar spinal cord tissue extracts of these animals demonstrated the presence of substance P (a doubly-charged ion at 674.37 amu with MW 1346.74) and neurotensin

Figure 3. Collision-induced dissociation (CID) spectrum of the ion found at 674.365 amu in the spectrum of the extract of the lumbar spinal cord of neuro-pathic animals. All the circled ions are unique ions characteristic of the fragmentation of substance P.


(a doubly-charged ion at 836.95 amu with MW 1673.0) as shown in Figure 1. Other cuff-implanted animals (n = 7), as well as sham-operated animals (n = 3), did not show a change in touch sensitivity (P was not significant). Recorded values at 7 d post-surgery were 92.2% ± 32.5%, respectively, and 119.0% ± 32.9% compared to base-line values. Substance P and neurotensin were not detected in these animals (Figure 2). The collision-induced dissociation (CID) spectrum of ions found at 674.365 and 836.949 are shown in Figures 3 and 4. These figures show characteristic ion fragmentation of substance P and neurotensin from lumbar spinal cord extracts of neuropathic animals. Therefore at 8 d post-surgery, of all the pain-related neuro-peptides that were looked for, only substance P and neurotensin were detected in the lumbar spinal cord of neuropathic animals identified by the presence of mechanical allodynia, associated with the sciatic nerve-cuff implantation.

D i s c u s s i o nOur results clearly show that mechanical allodynia following

sciatic nerve-cuff implantation in rats is related to a significant

up-regulation in detectable levels of neurotensin and substance P in spinal lumbar tissue. Sham surgery and cuff implantation, by itself, may not be sufficient to induce neuropathic pain in rats. The cuff model has been reported to be very reliable (5); however, our experience with this model shows that only 50% of the animal developed mechanical allodynia following cuff implantation. Following this first publication by Pitcher et al (5), the model has been further investigated and animals do not always develop allodynia (Jennifer Ritchy, personal communication). We do not think that surgical procedures have contributed to our results but rather the weight of the animal used in our study (mean BW : 311 g) as compared to previous studies (375 to 425 g) (5,6) were much smaller. In this case, the sciatic nerve may be less constricted causing a less severe syndrome. No histopathology of the sciatic nerve was performed in this study and, therefore, we cannot confirm this hypothesis. Other well characterized neuropathic pain models (7–10) show that surgical procedures do not neces-sarily produce the neuropathic syndrome, as approximately 80% of the animal show mechanical allodynia. The factors weight differences and intrinsic variability may, therefore, explain in

Figure 4. Collision-induced dissociation (CID) spectrum of the ion found at 836.939 amu in the spectrum of the extract of the lumbar spinal cord of neuro-pathic animals. All the circled ions are unique ions characteristic of the fragmentation of neurotensin.


part why only 50% of the animal developed mechanical allodynia.

Substance P is known to play a prominent role in nociception. Several lines of evidence support a role in neurotransmission of nociceptive stimuli for substance P (2). It is found in the primary afferents fibers that terminate in the substantia gelatinosa of the dorsal spinal cord (11,12) and iontophoretic application excites nociceptors (13). Stimulation of A delta and C fibers cause the release of substance P in cerebrospinal fluid (14). Agents that modify its release produce analgesia following spinal injections (15). Other neurotransmitters, such as glutamate, interact with substance P to excite dorsal horn cells and produce pain (16,17). Hyperexcitability induced by tissue injury can cause the release of substance P follow-ing the activation of NMDA glutamate receptors. Therefore the up-regulation of substance P following cuff implantations could be an indirect effect via other neurotransmitters or neuromodulators.

Neurotensin-containing terminals and cells bodies are found in lamina II of the superficial dorsal horn and have been suggested to play a role in pain transmission. Direct iontophoretic application of neurotensin excite spinal nociceptors (18). Intrathecal administration of neurotensin has a strong inhibitory effect on the nociceptive threshold evaluated with the tail flick test and the writhing response (19). These results suggest that neurotensin is implicated in the modulation of pain transmission in the spinal cord. Neuroanatomical and pharmacological data support roles in pain transmission for these peptides (18,20). Since our results suggest that the presence of both substance P and neurotensin seem important in neuropathic pain, they may be acting to balance excitation and inhibition of nociceptors at the spinal cord level.

Radio immunoassay (RIA) is the most commonly used ana-lytical method to measures neuropeptides. Although sensitive, it is not specific. The antibody may recognize different sequences (metabolites and precursors) of the peptide of interest. For example, cross reactivity has been shown for many different sequences of substance P (21). Using tandem liquid chromatography/micro-electrospray mass spectrometry, Andren and Caprioli (22) have shown that fragments of substance P are present in brain tissue and that substance P (undecapeptide) was not detected. These studies suggest that further work could be performed to charac-terize fragments of substance P in the spinal cords of neuropathic animals and could explain why no substance P was detected in sham-operated rats and cuff implanted animals without allodynia.

Other oligopeptides have been suggested to play a role in pain transmission. In this preliminary study, peptides were detected in full scan mode, which could only show peptides that were clearly up-regulated with cuff-induced allodynia. This may explain the dis-crepancies with other studies that suggest that other peptides may be implicated in pain mechanisms. The high resolution ( 10 000) and mass accuracy (2 ppm) of the new generation of quadrupole/time-of-flight (QqTOF) mass spectrometers, like the Sciex Pulsari, provides a greater specificity than most bioanalytical methods. This preliminary study suggests a correlation between an increase of neuropeptides (substance P and neurotensin) and the development of mechanical allodynia. Further experiments will be required to demonstrate this causal effect. We do not exclude that other peptides may be either

down-regulated or minimally up-regulated following nerve cuff implantation.

A c k n o w l e d g m e n t sThis study was supported by MDS Pharma Services research and

development funds. We greatly acknowledge Jennifer Ritchy and Dr. James Henry for teaching us the surgical approach, as well as the evaluation of touch sensitivity in cuff implanted and sham-operated animals.

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