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Keywords:

  • chronic constriction injury;
  • polyethylene cuff;
  • confocal microscopy;
  • substance P;
  • nociception;
  • retrograde labeling;
  • neurokinin 1 receptor

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

Lamina I of the spinal dorsal horn is a major site of integration and transmission to higher centers of nociceptive information from the periphery. One important primary afferent population that transmits such information to the spinal cord expresses substance P (SP). These fibers terminate in contact with lamina I projection neurons that express the SP receptor, also known as the neurokinin-1 receptor (NK-1r). Three types of lamina I projection neurons have been described: multipolar, fusiform, and pyramidal. Most neurons of the first two types are thought to be nociceptive and express the NK-1r, whereas most pyramidal neurons are nonnociceptive and do not express the NK-1r. In this immunocytochemical and behavioral study, we induced a neuropathic pain-like condition in the rat by means of a polyethylene cuff placed around in the sciatic nerve. We document that this lesion led to a de novo expression of NK-1r on pyramidal neurons as well as a significant increase in SP-immunoreactive innervation onto these neurons. These phenotypic changes were evident at the time of onset of neuropathic pain-related behavior. Additionally, we show that, after a noxious stimulus (intradermal capsaicin injection), these NK-1r on pyramidal neurons were internalized, providing evidence that these neurons become responsive to peripheral noxious stimulation. We suggest that the changes following nerve lesion in the phenotype and innervation pattern of pyramidal neurons are of significance for neuropathic pain and/or limb temperature regulation. J. Comp. Neurol. 521:1915–1928, 2013. © 2012 Wiley Periodicals, Inc.

Chronic neuropathic pain occurs as a consequence of damage to either the central (e.g., spinal cord injury or multiple sclerosis) or the peripheral (e.g., postherpetic neuralgia or painful diabetic neuropathy) nervous systems. However, in spite of considerable progress, the mechanisms that trigger and maintain neuropathic pain are still poorly understood. After lesions to peripheral nerves, some changes thought to be related to neuropathic pain occur at the level of the peripheral nervous system (Grelik et al.,2005; Peleshok and Ribeiro-da-Silva,2011; Taylor and Ribeiro-da-Silva,2011; Yen et al.,2006), but some develop in the dorsal horn of the spinal cord, where the modulation of incoming pain-related signals occurs (Abbadie et al.,1996; Bailey and Ribeiro-da-Silva,2006; Castro-Lopes et al.,1993; Coull et al.,2003; Keller et al.,2007).

Spinal lamina I projection neurons play a major role in the forwarding of pain-related information to higher centers. Therefore, changes in the properties of these neurons may occur in neuropathic pain states and may be important for the triggering and/or maintenance of the pain-related condition. These lamina I neurons project mainly to the lateral parabrachial nucleus (LPb; Bernard et al.,1995; Cechetto et al.,1985; Craig,1995; Feil and Herbert,1995; Wiberg and Blomqvist,1984) and to the thalamus (Carstens and Trevino,1978; Craig and Burton,1981; Giesler et al.,1979,1981; Willis et al.,1979). Lamina I neurons were originally classified into four types in the rat, independently of being projection or local circuit neurons (Lima and Coimbra,1983,1986). Most recent studies focus on lamina I projection neurons, which have been classified into three populations, based on their morphological properties: fusiform, multipolar, and pyramidal (Yu et al.,1999,2005; Zhang and Craig,1997; Zhang et al.,1996). An intracellular physiological study allowed the correlation of morphological and physiological characteristics. Fusiform neurons were nociceptive-specific (NS) and responded to noxious heat and pinch; multipolar neurons were either NS or responded to noxious heat, pinch, and noxious and innocuous cold (HPC); in contrast, pyramidal neurons were nonnociceptive and responded to innocuous cooling (COOL; Han et al.,1998). In agreement with their responsiveness to noxious stimuli, most fusiform and pyramidal neurons were shown to be immunoreactive for the main substance P (SP) receptor, the neurokinin-1 receptor (NK-1r). However, in agreement with their lack of response to noxious stimuli, most pyramidal neurons did not express NK-1r (Almarestani et al.,2007; Yu et al.,1999,2005).

We found that NK-1r was expressed de novo by pyramidal neurons, in an animal model of inflammatory arthritis (Almarestani et al.,2009). However, such expression started only at 2 weeks after the injection of complete Freund's adjuvant (CFA) into the plantar surface of the rat hind paw (Almarestani et al.,2009), coincident with the onset of extensive joint and bone damage, as assessed by imaging approaches (Almarestani et al.,2011). We detected that, in normal animals, most pyramidal neurons were hardly innervated by peptidergic nociceptive primary afferents, as revealed by SP immunoreactivity, but became abundantly innervated as from 2 weeks post-CFA injection (Almarestani et al.,2009). This is an important observation, because in past studies in the cat, combining intracellular physiology and injection of a marker with immunocytochemistry, it was shown that dorsal horn neurons that were abundantly innervated by SP were nociceptive, whereas nonnociceptive neurons were scarcely innervated by SP (De Koninck et al.,1992; Ma et al.,1996). In agreement with this, a study in the trigeminal subnucleus caudalis of the owl monkey showed a specific thermoreceptive region of lamina I, which was devoid of SP innervation and had clusters of nonnociceptive COOL cells of the pyramidal type projecting to the thalamus (Craig et al.,1999). It was also shown that, in the rat, neurons expressing the NK-1r are abundantly innervated by SP-immunoreactive (-IR) primary afferents and display c-fos immunoreactivity following noxious stimulation (Todd et al.,2002). Based on these findings, there seems to be a correlation between the presence of nociceptive responses and the abundance of SP innervation and expression of NK-1r by these neurons. Therefore, the lack of SP innervation of lamina I pyramidal neurons and the lack of expression of NK-1r by most of them in naïve animals and the drastic change in NK-1r expression and SP innervation in these neurons in the presence of arthritis would suggest the switch from a nonnociceptive to a nociceptive phenotype (Almarestani et al.,2009). In this study, we investigated whether the induction of neuropathic pain would trigger the de novo expression of NK-1r by lamina I pyramidal cells and alter their innervation by SP-IR nociceptive afferents.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

The guidelines of the Canadian Council on Animal Care for the care and use of experimental animals were thoroughly followed in all the experiments described. Furthermore, the studies were conducted with approval by the McGill University Faculty of Medicine Animal Care Committee and followed the guidelines of the International Association for the Study of Pain.

In total 74 male Sprague Dawley rats (Charles River, Quebec, Canada) weighing 215–225 g were used for the experiments. The number of animals used and their suffering were kept to the minimum necessary for the study. The number of animals per group varied between four and eight animals. All animals were exposed to a 12-hour light/dark cycle and given food and water ad libitum. The cages housed four animals each and were fitted with soft bedding and a plastic tube for an enriched environment.

Animal preparation

Surgeries

Animals were anesthetized with 5% isoflurane in oxygen in a gas chamber. The left femoral muscle of the thigh was exposed by blunt dissection. The sciatic nerve was exposed and freed from surrounding connective tissue with a glass pipette. In the experimental group, a 2-mm PE60 polyethylene tubing (Intramedic; Fisher Scientific, Whitby, Ontario, Canada) was loosely placed on the sciatic nerve with 90° bent tip tweezers, ensuring minimal manipulation of the nerve. In the sham group, the sciatic nerve was exposed in the same manner but was not manipulated in any way. The incision was then sutured in two layers (muscles and skin). Animals were returned to their cages to recover. There was no difference in weight gain between experimental and sham groups at any time point studied.

Injection of tracers

For all experiments requiring retrograde tracing, animals were anesthetized with 5% isoflurane in oxygen, placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), and stabilized with nonperforating ear bars. With Bregma as the reference point, the coordinates for the parabrachial nucleus (rostral/caudal −9.12, medial/lateral −2.1, dorsal/ventral −6.3) were calculated from the Paxinos and Watson (2005) rat brain atlas. A small hole was drilled through the skull at the target point, exposing the dura mater. A glass micropipette (Wiretrol II; Drummond Scientific Company, Broomall, PA) was lowered to the stereotaxic position of the parabrachial nucleus. Two microliters of 1.0% solution of cholera toxin subunit B (CTb; List, Campbell, CA) was slowly injected into the parabrachial nucleus over a period of 20 minutes. A 10-minute waiting period was imposed before the micropipette was retracted from its position to minimize leakage of the tracer. CTb was injected 7 days prior to killing the animals.

Capsaicin injection

As a noxious stimulus, a 20-μl intradermal injection of a 0.1% capsaicin solution (Sigma/Aldrich, St. Louis, MO) in ethanol, Tween 80, and physiological saline was given between the interdigital pads of the left hind paw. The sham group received an intradermal vehicle injection of ethanol, Tween 80, and physiological saline. Animals were killed at 12 minutes postinjection.

Behavior testing

Animals were always habituated to the testing apparatus for 30 minutes prior to testing. Baseline behavioral thresholds for both tests were measured for 2 consecutive days prior to surgical treatments. For behavioral testing, the investigator was blinded to the experimental groups; all animals were coded, and the codes were broken only at the end of the experiments.

Testing for mechanical allodynia

The testing apparatus consisted of clear plastic enclosures elevated on a mesh grid, which allowed complete access to the ventral side of the animal. Animals were tested using the up–down method described by Chaplan et al. (1994). In brief, von Frey filaments of increasing stiffness were applied with a 5-second delay between all presentations. The filament was applied to the midplantar area of the hind paw, avoiding the pads of the foot, until it buckled and was maintained for 10 seconds or until an obvious behavior (paw withdrawal, flicking, or licking) occurred. A positive response prompted the next weaker filament to be presented, whereas a negative response prompted the presentation of the next stronger filament. This process was repeated six times, and the number of positive reactions was noted and an average threshold calculated. After the right paw for all the animals was tested, the left paw was tested in the same manner.

Testing for thermal hyperalgesia

The Hargreaves test (Hargreaves et al.,1988) was employed to measure heat nociceptive thresholds. Clear plastic enclosures were set on top of a glass floor. The light source was directed onto the skin area of the paw in contact with the glass. When the paw was lifted, the light source automatically turned off. Testing included three trials per paw, with each trial completed for all the animals before the start of the next trial, ensuring a 30-minute wait before the start of the next trial to minimize desensitization effects. The time from turning on of the light source until withdrawal was noted, and an average of the three trials per paw was calculated.

Animal perfusion

At the end of each period (7, 10, 15, 21, 28 days), the animals were deeply anesthetized with 0.3 ml/100 g body weight of Equithesin (6.5 mg chloral hydrate and 3 mg sodium pentobarbital i.p.). They were then perfused through the left cardiac ventricle with perfusion buffer (for composition see Côté et al.,1993) for 1 minute, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 30 minutes. The brain and spinal cord segments L4–L5 were extracted and postfixed for 4 hours and 2 hours, respectively, in 4% paraformaldehyde in PB. The specimens were then cryoprotected in 30% sucrose in PB overnight at 4°C.

Immunohistochemistry

To examine the injection site at the level of the parabrachial nucleus, serial 100-μm-thick coronal sections of the relevant brain region were obtained. Furthermore, the dorsal aspect of the L4–L5 spinal cord segment was cut into serial 50-μm-thick horizontal sections, to examine lamina I. All specimens were cut using a freezing sledge microtome (Leica, Richmond Hill, Ontario, Canada) and collected free-floating in phosphate-buffered saline (PBS) with 0.2% Triton X-100 (PBS + T).

Sections were incubated in 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA) in PBS + T for 1 hour at room temperature to block nonspecific staining. Then, spinal cord sections were incubated with the primary antibodies: goat anti-CTb (List; product 703, lot 7032A6) at 1:5,000 dilution and rabbit anti-NK-1 r (Sigma/Aldrich; product S8305, lot 084K4845) at 1:10,000 dilution in PBS + T containing 5% normal donkey serum for 48 hours at 4°C. Next, sections were washed several times with PBS + T and incubated for 2 hours at room temperature with a biotinylated donkey anti-goat IgG (1:250; Jackson Immunoresearch) and, after further washing in PBS + T, incubated with streptavidin conjugated to AlexaFluor 568 (Molecular Probes) and donkey anti-rabbit AlexaFluor 488 in 5% normal donkey serum and PBS + T for 2 hours at room temperature. Finally, sections were washed with PBS, mounted on gelatin-subbed slides, and coverslipped with an antifading mounting medium (Aqua Polymount; Polysciences, Warrington, PA). Slides were stored in the dark at −20°C.

Brainstem sections of the injection site were incubated with anti-CTb antibody followed by biotinylated donkey anti-goat IgG and streptavidin conjugated to AlexaFluor 568. They were mounted and coverslipped as described above.

Spinal cord sections for the quantification of SP-IR input to lamina I neurons originated from a different cohort of sham and lesioned animals at the 21-day time point (n = 6). Sections were cut and processed as described above, but the primary antibody mixture included a rat anti-SP monoclonal antibody [spent tissue culture supernatant diluted 1:10, kindly supplied by A. Claudio Cuello (Cuello et al.,1979); Medimabs, Montreal, Quebec, Canada]. Furthermore, the secondary antibody mixture also included a donkey anti-rat IgG conjugated to Cy5 (Jackson Immunoresearch).

Antibody specificity

See Table 1 for a list of all primary antibodies used. The goat anti-CTb antibody was generated against CTb, and its specificity was demonstrated by the lack of any staining in animals not injected with CTb. The rabbit anti-NK-1r antibody was generated against a synthetic peptide corresponding to amino acids 393–407 of the C-terminus region of the rat NK-1r and purified by ion-exchange chromatography. In Western blots from rat brain, it recognizes a single band at 46 kDa, whose staining is specifically inhibited by incubation with the blocking peptide (data supplied by the manufacturer). Furthermore, it was shown that it does not produce any staining in NK-1r knockout mice, although it recognizes the receptor in wild-type mice (Ptak et al.,2002).

Table 1. Primary Antibodies Used in This Study
AntigenImmunogenSourceDilution
Cholera toxin subunit B (CTb)Purified CTb isolated from Vibrio choleraeList, Campbell, CA; goat polyclonal; product 703, lot 7032A61:5,000
Neurokin 1 receptor (NK-1r)Synthetic peptide, aa 393–407 from C-terminus of rat NK-1rSigma/Aldrich, St. Louis, MO; rabbit polyclonal; product S8305; lot 084K48451:10,000
Substance PEntire peptide sequence of substance PGift from Dr. A.C. Cuello, McGill University; rat monoclonal; NC1/34, clone NC1/34.HL1:10

The anti-substance P antibody has been extensively used by us (e.g., Almarestani et al.,2009) and by others. Although we obtained it directly from Prof. Cuello, it is available commercially (e.g., MediMabs, BD Biosciences Pharmingen, Abcam). It is a monoclonal antibody (coded NC1/34) generated in the rat by immunization against the entire sequence of SP conjugated to bovine serum albumin with carbodiimide and fusing the spleen cells from these animals with a myeloma cell line (Cuello et al.,1979). One clone (NC1/34.HL) was chosen and characterized by radioimmunoassay; it did not recognize enkephalins, somatostatin, or β-endorphin and did recognize the C-terminal sequence of the SP molecule (Cuello et al.,1979). The characterization of this antibody was described in detail in previous publications from our laboratory (Almarestani et al.,2009; McLeod et al.,2000). In short, although this antibody should not be able to differentiate SP from the other two mammalian tackykinins, neurokinin A and neurokinin B, it does not recognize neurokinin B in the concentrations used for immunocytochemistry.

Morphological identification and quantification of lamina I neurons

Our criteria of identification and quantification of lamina I neurons have been described extensively in previous publications from our laboratory (see, e.g., Almarestani et al.,2009). In brief, in the current study, six 50-μm-thick serial horizontal sections were cut from the dorsal part of the L4–L5 spinal segments. Four rats were used per time point. Sections were examined with a PlanFluotar ×40 oil immersion objective on a Zeiss Axioplan 2e imaging fluorescence microscope. We counted only neurons ipsilateral to the lesion side that had visible nuclei and with the cell body entirely located within the plane of the section, as assessed with the fine focus of the microscope. Lamina I neurons were classified according to their cell body shape and dendritic arborization into multipolar, fusiform, pyramidal, and unclassified types (Almarestani et al.,2009). For all morphological studies and quantifications, the microscope slides were coded, and the researcher was completely blinded with regard to the animal experimental groups.

Quantification of SP-IR boutons on pyramidal neurons

Sections were examined with a Zeiss LSM 510 confocal scanning laser microscope. We used a multitrack scanning method and appropriate filters for the separate detections of AlexaFluor 488, AlexaFluor 568, and Cy5. Images used for quantification represented serial optical sections obtained along the z-axis (z-stacks) with a ×63 plan-apochromatic oil-immersion objective. Furthermore, for an unbiased representation of the images taken, all the parameters of laser power, pinhole size, and image detection were kept constant for all samples. All neurons classified as pyramidal neurons based on the criteria described above were scanned, independently of being immunoreactive for the NK-1r or labeled with the retrograde tracer only. Because the CTb labeled reliably and consistently only the cells bodies and primary dendrites, the analysis of the SP-IR innervation was restricted to these compartments of the cells. The images obtained were converted to TIFF files. To calculate the density of SP-IR boutons per unit length of neuronal membrane, the length of membrane in the soma and proximal dendrites was measured with the help of an MCID Elite Image Analysis System (Imaging Research, St. Catharines, Ontario, Canada), and the number of appositions from SP-IR boutons onto it was counted. This was performed on alternate optical sections from the z-stack, to avoid counting the same boutons twice. However, as we did not perform any correction, there is a potential bias in that we did not correct our data for elongated boutons that may be present in alternate sections and for potential changes in bouton size with experimental groups.

Statistical analysis

GraphPad Prism 5 (GraphPad Software, San Diego, CA) was used to perform all statistical tests. Two-way ANOVA followed by Bonferroni correction was applied to compare differences in pain-related behavior and lamina I cell populations between sham group and the experimental time points studied. Unpaired t-test was used to compare the SP-IR boutons in direct apposition to lamina I neurons following vehicle or capsaicin intradermal injection. All values are expressed as means ± SEM. Significance level for all the tests was set at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

Development of mechanical allodynia and thermal hyperalgesia in neuropathic animals

We confirmed the development of neuropathic pain in our polyethylene cuff model of chronic constriction injury (“cuff”) by means of behavioral testing. Animals in the cuff group developed mechanical allodynia, as revealed by testing with the von Frey filaments (Fig. 1A). This allodynia developed at 10 days after cuff placement and was still present at 28 days. These results were significantly different from results for the sham group.

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Figure 1. Behavioral changes following the cuff lesion. A: Assessment of tactile allodynia using von Frey filaments in cuff and sham-operated animals. Neuropathic animals showed a decrease in withdrawal threshold by 10 days postcuff. No changes from baseline were detected in the sham group. N = 10, two-way ANOVA with Bonferroni correction (***P < 0.001). B: Assessment of thermal hyperalgesia using the Hargreaves test in cuff and sham groups. Neuropathic animals displayed a significant decrease in withdrawal latencies starting at 10 days postsurgery compared with sham. No significant reduction in withdrawal latencies was detected for the sham group. N = 10, two-way ANOVA with Bonferonni correction (**P < 0.01, ***P < 0.001).

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With regard to changes in thermal sensitivity, we detected a decrease in withdrawal latency to heat compared with the sham group, beginning at 10 days and still present at 28 days (Fig. 1B). There was no difference from baseline in the contralateral side data, either in sham-operated or in cuff animals (data not shown).

CTb injection into the parabrachial nucleus

The microscopic observation of the CTb injection site revealed a distribution of the marker similar to what we observed in previous studies (Almarestani et al.,2007,2009). The injection site covered most of the parabrachial complex, including the lateral parabrachial nucleus (LPb). Retrogradely labeled lamina I neurons were found mostly on the contralateral side, although a few were also present on the side ipsilateral to the CTb injection. CTb labeling of spinoparabrachial lamina I neurons included the cell body and primary dendrites (Fig. 2).

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Figure 2. Confocal images showing the morphology and NK-1r expression of lamina I pinoparabrachial neuronal populations, in sham-operated and cuff groups, at the 21 days time point. Unlike multipolar and fusiform neurons, pyramidal neurons normally did not express NK-1r (A,C,E). However, neuropathic animals developed a de novo expression of NK-1r on pyramidal neurons (F), whereas multipolar and fusiform neurons maintained their normal levels of expression of these receptors (B,D). Scale bar = 20 μm.

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Classification of lamina I cells

As in previous publications from our laboratory, the different lamina I spinoparabrachial neuronal populations were identified based on their dendritic arborization and cell body shape when viewed in the horizontal plane. Multipolar neurons possess four or more primary dendrites arising from the cell body, independently of the shape of the soma (Fig. 2A). Fusiform neurons have a spindle-shaped soma, with one dendrite arising from each end of the cell body (Fig. 2C). In the horizontal plane, pyramidal neurons have a triangular soma, with a primary dendrite originating from each corner (Fig. 2E). A relatively small number of neurons displayed features that were transitional between two of the cell types described above and did not meet the criteria for the main types because of their atypical appearance. Furthermore, some neurons had a portion of the cell body and/or proximal dendritic tree truncated by sectioning. All these cells were not classifiable and were considered as “unclassified” for the purposes of our quantitative analyses.

Changes in lamina I neurons following cuff application

As previously described (Almarestani et al.,2007,2009), lamina I spinoparabrachial neurons, in control animals, identified by CTb retrograde labeling, often expressed NK-1r. Indeed, most lamina I spinoparabrachial neurons of the multipolar and fusiform types expressed the receptor (Figs. 2A–D, 3B,C). However, pyramidal neurons seldom expressed NK-1r in sham-operated animals (Figs. 2E, 3A). After cuff application, pyramidal neurons showed a de novo expression of NK-1r in a high proportion of cells (Figs. 2F, 3A), whereas there were no changes in NK-1r expression by multipolar and fusiform neurons (Figs. 2B,D, 3B,C). This de novo expression of NK-1r was detected from day 10 postcuff (Fig. 3A).

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Figure 3. Comparison of the distribution of neurons of each morphological type that were immunoreactive for the NK-1r (NK1 only), retrogradely labeled from the parabrachial nucleus (CTb only), or double labeled (NK1 + CTb) ipsilateral to the lesion or sham operation at 7, 10, 15, 21, and 28 days postsurgery. A: Pyramidal neurons. B: Fusiform neurons. C: Multipolar neurons. D: Unclassified neurons. In A, note that no change was detected at any time point in the number of neurons that expressed only the NK-1r; at 7 days, the number of pyramidal neurons that expressed the NK-1r and were also CTb positive was very low, but, at and after the 10-day time point, there was a marked increase in the number of neurons that were immunoreactive for both the NK-1r and CTb, accompanied by a parallel decrease in the number of neurons that were not immunoreactive for NK-1r and were positive or not for CTb. In B–D, note that there were no changes at any time point in the number of fusiform, multipolar, and unclassified neurons that were immunoreactive or not for the two markers that we used. Only cells with visible nuclei were counted. Values represent average number of neurons (±SEM) counted per animal. N = 4, two-way ANOVA with Bonferroni post hoc tests (***P < 0.001).

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Quantification of SP innervation

Our observations of SP innervation were carried at 21 days postlesion. With low magnification, we did not detect any noticeable change in the density or pattern of SP innervation of laminae I–II in lesioned animals compared with sham. In sham animals, we confirmed that fusiform and multipolar neurons receive abundant innervation from SP-IR fibers, whereas pyramidal neurons were scarcely innervated, as we had previously observed (Almarestani et al.,2009). For cuff animals, we observed a significant increase in the number of appositions from SP-IR fibers at 21 days compared with pyramidal neurons of sham animals (Figs. 4, 5). This quantification was carried out in the cell bodies and proximal dendrites of neurons, independently of NK-1r expression. No significant change in SP-IR innervation was observed in the multipolar or fusiform neuronal populations (data not shown).

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Figure 4. Confocal triple-labeling images showing increased substance P innervation of pyramidal neurons and de novo NK-1r expression following a cuff lesion at the 21 day time point. We observed a very substantial increase in the number substance P boutons apposed to lamina I spinoparabrachial pyramidal neurons that had de novo NK-1r expression (B; arrowheads) compared with neurons in the sham-operated group with no NK-1r expression (A). SP in blue, NK-1r in green, CTb in magenta. Scale bar = 20 μm.

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Figure 5. Quantification of substance P-IR boutons in direct apposition to lamina I spinoparabrachial pyramidal neurons. At 21 days, there was a significant increase in the number of substance P boutons in close apposition with the pyramidal neurons compared with the sham-operated group, independent of the presence or absence of NK-1r staining. Because of limitations in the CTb filling of neurons, boutons counted were only those apposed to the soma membrane and membranes of the primary dendrites. N = 6, unpaired t-test (***P < 0.001).

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NK-1 receptor internalization in pyramidal cells following capsaicin injection

Injection of capsaicin intradermally into the hind paw of animals with cuff injury at the 21 day time point led to the internalization of the NK-1r from the cell surface to the cytosol of pyramidal neurons (Fig. 6B). However, solvent injection did not induce NK-1r internalization (Fig. 6A). NK-1r on multipolar and fusiform neurons was also internalized following capsaicin but not solvent injections (data not shown).

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Figure 6. Demonstration of NK-1r internalization following noxious stimulation. Intradermal injection of capsaicin in the ipsilateral hind paw at the 21-day point caused internalization of surface NK-1r (B). No internalization was detected with vehicle injections at the same time point in cuff animals (A). N = 4. Scale bar = 20 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

We show here that, in the cuff model of neuropathic pain, considerable numbers of lamina I spinoparabrachial pyramidal neurons develop a de novo expression of functional NK-1r. Furthermore, we found a substantial increase in the number of appositions from axons immunoreactive for SP onto pyramidal neurons, raising the possibility of a functional switch of these neurons.

Properties of pyramidal neurons

There is evidence from the literature that there are three main functional types of lamina I neurons in the cat and primate. These have been identified on the basis of their response to cutaneous inputs: 1) nociceptive-specific neurons (NS) are responsive only to noxious heat and pinch; 2) polymodal nociceptive neurons (HPC) are responsive to noxious pinch and heat as well as to innocuous and noxious cold; and 3) innocuous thermoreceptive neurons (COLD) are responsive only to innocuous cooling (Craig and Bushnell,1994; Craig and Kniffki,1985; Craig et al.,1999; Dostrovsky and Craig,1996; Han et al.,1998). For the cat, a correlation was found between the physiological and morphological properties of lamina I projection neurons, in a study combining intracellular recording from lamina I cells with intracellular labeling (Han et al.,1998). Indeed, in the Han et al. study, fusiform cells were all NS, pyramidal cells were all COLD, and multipolar cells were divided among HPC and NS neurons. In agreement with the finding that pyramidal cells are nonnociceptive, a study from our laboratory in the primate revealed that spinothalamic pyramidal neurons seldom expressed NK-1r, whereas fusiform and multipolar cells were normally NK-1r immunopositive (Yu et al.,1999). Subsequent studies from our laboratory demonstrated that, in rat, most spinothalamic and spinoparabrachial pyramidal neurons were not immunoreactive for the NK-1r, in contrast to fusiform and multipolar neurons, which were mostly immunoreactive for this receptor (Almarestani et al.,2007; Yu et al.,2005). There is ample evidence that nociceptive cells express NK-1r (Mantyh et al.,1995,1997). Therefore, the observation that in naïve animals most pyramidal neurons do not express NK-1r concurs with a nonnociceptive role for these cells. However, we found that 22% of the pyramidal neurons expressed NK-1r in control animals. These NK-1r-positive pyramidal neurons were mostly of larger diameter and displayed abundant innervation by SP (data not shown), suggesting that they represent a separate population of neurons, likely with nociceptive properties. However, our data on the absence of expression of NK-1r by most pyramidal neurons in the rat spinal cord contrasts with the results from another laboratory. Indeed, the group of A.J. Todd did not find any differences among the three types of lamina I projection neuron in the expression of NK-1r (Todd et al.,2002). This group also found that pyramidal neurons should be nociceptive, as they not only display NK-1r immunoreactivity but also respond to noxious stimulation with c-fos expression (Todd et al.,2002). The reasons for this discrepancy were discussed at length in one of our previous publications (Almarestani et al.,2009). In short, the differences were certainly not caused by the antibody used, because we obtained the same results with three different antibodies, nor by the animal strain. One possible explanation for the different results obtained by our group compared with Todd's laboratory is the application of different cell classification criteria. A key element used by our group to distinguish pyramidal from multipolar neurons is the number of primary dendritic trunks when viewed in the horizontal plane. Indeed, although pyramidal neurons have four primary dendrites, only three of them are visible on a confocal optical section cut to show the cell nucleus. This is due to the fact that the fourth dendrite is oriented toward the white matter. We consider as multipolar all cells that unequivocally display four or more main dendritic trunks in the horizontal plane. We stress this point, because some main dendrites branch very close to the cell body (Almarestani et al.,2009). One difference between our work and Todd's data is that we limited our analysis to the cells of the middle one-third of the lateromedial extent of lamina I. We did this for the following reasons: 1) because sciatic nerve afferents terminate in this region, cells would be affected by the lesion; and 2) the white-matter/lamina I border is in the horizontal plane in this region for the segments analyzed, so that cell types are easily recognizable in a conventional fluorescence microscope. Todd's group looked at all of lamina I. This may explain some differences between our data and Todd's, because it has been shown that pyramidal neurons are more abundant in the lateral part of lamina I (Lima and Coimbra,1983). Indeed, according to Todd's group (Spike et al.,2003), the percentage of lamina I projection neurons belonging to the pyramidal type is about 30% (the same as for multipolar and fusiform), with only a small percentage being unclassified. In contrast, in our study, pyramidal neurons were just 22% of the total in control animals, suggesting that Todd's group included in the counts pyramidal neurons from more lateral regions of lamina I. Within the fusiform and multipolar types, we found a considerably higher proportion than did Todd that was NK1r-IR (93% and 92%, respectively). One interesting issue is that, after lesion, the only cells that changed in NK-1r expression were the pyramidal (84% positive in lesioned animals compared with 22% in the sham group). Also, independent of the presence or absence of NK-1r immunoreactivity, the overall number of neurons of each type remained the same, excluding the possibility of one type in sham being classified differently in the lesioned animals. These data are consistent with those from a previous study from our laboratory, in which we detected a de novo expression of NK-1r by pyramidal neurons in an arthritis model (Almarestani et al.,2009). However, in spite of the differences in cell classification, Spike et al. (2003) found that, overall, 23% of the retrogradely labeled lamina I cells were negative for the NK-1r, a value very similar to the value 24% that we found in the current study for control animals.

Our current study expands our previous observations in an arthritis model (Almarestani et al.,2009) to a model of neuropathic pain. In the current study, we observed that, at 10 days postcuff, but not at 7 days, there were significantly more pyramidal neurons expressing NK-1r than in sham-operated animals. This increased receptor expression persisted for at least 28 days postcuff, the last time point examined, and was strictly ipsilateral (contralateral data not shown).

Significance of increased peptidergic fiber innervation on pyramidal neurons

In the current study, we confirmed quantitatively previous qualitative data from our laboratory revealing that non-NK-1r-IR pyramidal neurons in control animals were almost devoid of SP innervation (Almarestani et al.,2009). This information is in agreement with the concept that these cells are nonnociceptive. Indeed, electrophysiological studies have provided evidence that nonnociceptive dorsal horn cells do not respond to SP (Henry,1976), and studies combining intracellular recording and injection with HRP of neurons with ultrastructural immunocytochemistry in the cat have shown that nonnociceptive neurons are scarcely innervated by SP (De Koninck et al.,1992; Ma et al.,1996). In contrast, the same studies have shown that spinal dorsal horn neurons with strong nociceptive responses were abundantly innervated by SP-IR fibers (De Koninck et al.,1992; Ma et al.,1996). In agreement with this, the current study confirmed our previous observation that fusiform and multipolar neurons, which have been considered as nociceptive, were abundantly innervated by SP (Almarestani et al.,2009). We also confirmed that the few pyramidal neurons that were NK-1r-IR in control rats were abundantly innervated by SP-IR fibers. If we exclude the discrepancy on what should be considered a pyramidal neuron, our data concur with the results from Todd's laboratory in that lamina I neurons that are immunoreactive for the NK-1r are abundantly innervated by SP and respond to noxious stimulation (Todd et al.,2002).

Importantly, as shown in Figure 5, we detected that, after a cuff lesion, there was a very significant increase of SP-IR appositions on pyramidal neurons. Because we did not discriminate between pyramidal neurons that were NK-1r positive and those that were not when performing our counts, we cannot confirm whether this high innervation by SP of pyramidal neurons was restricted to the population that became NK-1r-IR. However, our visual assessment would suggest that NK-1r expression and abundant innervation by SP are associated. Our data support our previous qualitative-only observation in the CFA arthritis model of a de novo innervation of pyramidal neurons by SP-IR fibers (Almarestani et al.,2009).

This increase in SP innervation of a specific population of neurons in lamina I may appear surprising, as peripheral nerve lesions are known to reduce SP expression (for reviews see Hökfelt et al.,1994; McMahon and Priestley,2005). However, the situation is different after partial nerve lesions such as a partial tight ligation of the sciatic nerve (Seltzer model) and a chronic constriction injury of the sciatic nerve (Bennett model; Ma and Bisby,1998). In those two models, no changes in SP immunoreactivity were found ipsilateral to the lesion. These data concur with what we previously observed with the cuff model, in which no loss of calcitonin gene-related peptide (CGRP) immunoreactivity was detected at any time point ipsilateral to the lesion (Bailey and Ribeiro-da-Silva,2006). With a tracing method, a significant increase in SP immunoreactivity was detected in the unlesioned dorsal root ganglion neurons, which explains the lack of overall changes in dorsal horn (Ma and Bisby,1998). This raises the interesting possibility that the increased SP innervation of pyramidal neurons originates from the sprouting of undamaged primary afferents. However, we cannot rule out entirely that these SP-IR fibers do not represent primary afferents but rather axons from SP-containing local circuit neurons that also contain enkephalin (for review see Ribeiro-da-Silva and De Koninck,2008). A colocalization study of SP and CGRP immunoreactivities would have to be performed to exclude this possibility.

Significance of NK-1 receptor internalization

NK-1r internalization has been used as a measure of the receptor's activation (Abbadie et al.,1997; Adelson et al.,2009; Allen et al.,1997; Mantyh et al.,1995; Marvizon et al.,2003). In the current study, we tested the functionality of the NK-1r expressed on pyramidal neurons, following development of neuropathic pain, by injecting capsaicin intradermally in the hind paw. We used capsaicin because it has been described previously as a potent noxious stimulus (Afrah et al.,2001; Go and Yaksh,1987; Lever and Malcangio,2002; Marvizon et al.,2003). With neuropathic animals, we observed the internalization of NK-1r in all three lamina I neuronal populations following capsaicin injection; however, vehicle injection did not induce NK-1r internalization.

Role in nociception

As mentioned above, there is evidence indicating that SP-positive innervation is abundant on neurons with nociceptive responses compared with neurons lacking such responses (De Koninck et al.,1992; Ma et al.,1996) and that neurons that express the NK-1r are selectively innervated by SP (McLeod et al.,1998; Todd et al.,2002) and are nociceptive (Todd et al.,2002). The integration of this information with the data from this study would suggest that, following neuropathic pain development, the population of pyramidal neurons with de novo NK-1r expression might have undergone a functional change.

There is evidence from the literature that, under normal physiological conditions, pyramidal neurons represent COOL cells, activated by innocuous cooling of the skin but unresponsive to other stimuli (Craig and Bushnell,1994; Craig and Hunsley,1991; Craig and Kniffki,1985; Craig and Serrano,1994; Craig et al.,1999,2001; Dostrovsky and Craig,1996; Han et al.,1998). Our results suggest that in situations accompanied by a chronic pain state, such as in arthritis (Almarestani et al.,2009), and in neuropathic pain (current study), a considerable number of pyramidal cells would be converted into HPC cells and therefore would be responsive to noxious pinch and heat as well as to innocuous and noxious cold (Han et al.,1998). This hypothesis is at present based only on speculation, as it would have to be confirmed electrophysiologically. It would also require that pyramidal cells have direct and/or indirect connections with thalamic areas involved in nociception, which is far from clear for the thermoreceptive-specific population of pyramidal neurons (meaning those that normally do not express NK-1r). Indeed, it has been shown that, in cat and primate, pyramidal cells project in the thalamus to a region involved in innocuous thermal sensation (Han et al.,1998). However, the pyramidal neurons that we describe here for the rat project to the parabrachial nucleus, and it is possible that in this species they connect also to thalamic areas involved in nociception (see Yu et al.,2005). Further studies are required to clarify this issue.

Role in thermoregulation and homeostasis

Given the findings described above, it is possible that pyramidal cells in chronic pain states, although activated by noxious stimuli, might still be involved only in innocuous pain sensation, through either an indirect thalamic connection (see above) or a direct or indirect connection from the parabrachial nucleus to the preoptic area (POA), where the thermoregulatory center resides (Nakamura and Morrison,2008). We can speculate that, after neuropathy, the increased activity of the pyramidal neurons would be perceived as cooling, causing a shift from homeostatic temperatures, which would activate the thermoregulatory center, leading to thermogenesis (Nakamura and Morrison,2008) and cutaneous vasoconstriction (Osborne and Kurosawa,1994). This possibility would have to be investigated by measuring peripheral and core temperatures and documenting any increase in food consumption. At present, it is more speculative than based on facts.

We have limited our study to changes in NK-1r immunoreactivity and SP innervation of lamina I neurons. However, pain-related projection neurons expressing NK-1r and responding to noxious stimulation also occur in deeper dorsal horn regions such as laminae III–IV (Mantyh et al.,1995; Naim et al.,1997). It is possible that there will be a population of projection neurons in the deep dorsal that will start expressing NK-1r after lesion. With regard to lamina I neurons only, other phenotypic changes have been reported after nerve lesion. For instance, a physiological study showed that, after a cuff lesion, nociceptive spinoparabrachial lamina I neurons start to respond to innocuous stimuli such as touch or brushing and that the same changes can be reproduced in naïve animals by means of ATP-activated microglia (which induces changes in chloride homeostasis leading to disinhibition; Keller et al.,2007). Also, a study using a crush nerve lesion in the rat provided evidence that normally innocuous mechanical stimuli leads to pain-related behavior and c-fos activation in both lamina I and the parabrachial nucleus (Bester et al.,2000). An electrophysiological study of rat lamina I spinoparabrachial neurons provided evidence of their sensitization to heat following a chronic constriction injury of the sciatic nerve (Andrew,2009). Another study using transgenic mice and a nerve lesion model provided important evidence suggesting that unmyelinated low-threshold mechanoreceptors terminating in lamina I are required for the development of the injury-induced mechanical hypersensitivity (Seal et al.,2009). Data from this study also suggest that the information conveyed by these afferents innervating lamina I neurons would switch from touch to pain (Seal et al.,2009). Therefore, there is considerable evidence of extensive phenotypic changes in lamina I after a nerve lesion creating a neuropathic pain-like condition. If we accept the view that pain is a homeostatic emotion (Craig,2003), we can suggest that these changes likely lead to profound alterations in homeostasis, with modifications not only in sensations but also in the homeostatic regulation, not limited to thermoregulation.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

We have described a de novo expression of NK-1r on pyramidal neurons and an associated increase in SP-IR innervation of these cells in a model of chronic neuropathic pain. We have also demonstrated the internalization of these receptors following a noxious stimulus, indicating that these cells become responsive to noxious stimuli. These data, together with the results from our work on an arthritis model, suggest that in chronic pain states the properties of most lamina I pyramidal neurons change and may contribute to the triggering and maintenance of the chronic pain condition. Our results may aid in better understanding the development of cold allodynia or changes in homeostatic basal levels following nerve injury.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

The authors thank Manon St. Louis for laboratory expertise and Jacynthe Laliberté for confocal microscopy assistance. We are also particularly grateful to Dr. Claudio Cuello for helpful discussions and the supply of the anti-substance P monoclonal antibody and to Dr. Terence Coderre for the use of his animal behavior facility.

CONFLICT OF INTEREST STATEMENT

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED

The authors have no known or potential conflict of interest with respect to this work.

ROLE OF AUTHORS

Both authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: AS and ARS. Acquisition of data: AS. Analysis and interpretation of data: AS and ARS. Drafting of the manuscript: AS. Critical revision of the manuscript for important intellectual content: ARS. Statistical analysis: AS. Obtained funding: ARS. Administrative, technical, and material support: ARS. Study supervision: ARS.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. CONFLICT OF INTEREST STATEMENT
  9. LITERATURE CITED