The experimental procedures followed the guidelines for the Care and Use of Experimental Animals of the Canadian Council on Animal Care and the International Association for the Study of Pain (IASP). All protocols were approved by the McGill University Faculty of Medicine Animal Care Committee.
Forty male Sprague–Dawley rats (Charles River, QC, Canada), weighing between 220 and 230 g, were used. The number of animals used and their suffering was kept to the minimum necessary for the conduction of the study. Animals were exposed to 12 h light/dark cycles with food and water available ad libitum and were housed four animals to a cage fitted with soft bedding and a plastic tube for an enriched environment.
Animal preparation
Surgeries
Animals were anesthetized with 5 % isoflurane in oxygen. Unilateral injections were carried out on the left sciatic nerve. Using blunt dissection, the left biceps femoris and gluteus superficialis muscles were separated. Care was taken to minimize the stretching of the sciatic nerve when it was separated from the surrounding connective tissue. Experimental animals received a total of 6 µL of an 800 μg/mL solution of IB4-saporin (Advanced Targeting Systems, San Diego CA, USA) in 0.2 M Phosphate Buffered Saline (PBS) and Fast Green Dye (Sigma, MO, USA) injected at three injection sites into the sciatic nerve proximal to its branching point using calibrated glass micropipettes (Wiretrol II, Drummond Scientific Company, Broomall, PA, USA). The Fast Green dye was used to monitor the accuracy of the injection. The control group was injected with vehicle solution of 6 µL 0.2 M PBS in Fast Green dye using the same method. The incision was sutured in two layers using 4-0 Vicryl sutures (Ethicon Inc, New Jersey, USA). Animals were returned to their cages to recover. No difference in weight gain between experimental and sham groups was observed at any time point throughout the study.
Injection of tracer
To retrogradely trace projection neurons, animals were first anesthetized using 5 % isoflurane in oxygen, placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) and the head stabilized with non-perforating ear bars. The coordinates for the parabrachial nucleus (rostral/caudal: −9.12 mm; medial/lateral: −2.1 mm; dorsal/ventral: −6.3 mm) were calculated, with Bregma as the reference point, from the Paxinos & Watson Rat Brain Atlas [36]. 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, USA) was lowered, through the dura mater, to the stereotaxic position of the parabrachial nucleus and 2 µL of 1.0 % solution of cholera toxin subunit B (CTb) (List, Campbell, CA, USA) was slowly injected over a period of 20 min. To minimize leakage of the tracer, a 10 min waiting period was imposed before the micropipette was retracted from its position. CTb was injected 7 days prior to sacrificing the animals.
Behavior testing
Animals were always habituated to the testing apparatus for 30 min prior to testing. Baseline behavioral thresholds were measured for 2 consecutive days prior to surgical treatments.
Testing for mechanical allodynia
Animals were placed in 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 previously described by Chaplan et al. [37]. Filaments of increasing stiffness were applied to the mid-plantar area of the hind paw, avoiding the foot pads, until an obvious withdrawal or flicking/licking behavior occurred. In case of a positive response, the next weaker filament was presented, however, in the case of a negative response; the next stronger filament was applied. This process was repeated until six responses were recorded and a mean threshold was calculated. The testing was performed on the right paw of all the rats followed by the testing of the left paw in the same manner.
Testing for mechanical hyperalgesia
Mechanical hyperalgesia was assessed using the pin prick method described by Coderre et al. [38]. The point of a blunted 23 gauge needle was applied to the skin of the heel (touching, but not penetrating). Behavioral responses to the pin prick were rated according to the following scale: 0 = no response; 1 = rapid paw flicking, stamping, or shaking (less than 1 s); 2 = repeated paw stamping, shaking, or paw lift less than 3 s; 3 = above behaviors or hind paw licking for more than 3 s; 4 = above behaviors for more than 3 s and hind paw licking for more than 3 s. An additional point was added if any vocalizations occurred. The mean for reaction for each paw was calculated.
Testing for thermal hyperalgesia
The Hargreaves test [39] was used to measure thermal nociceptive thresholds. Clear plastic enclosures were set on top of a glass floor. The light source was directed onto skin area of the paw in contact with the glass. The time from turning on of light source until withdrawal was noted. Testing included three trials per paw with each trial being completed for all the animals before the start of the next trial. This ensured there was a 30 min wait before the start of the next trial to minimize desensitization effects. The average of the three trials per paw was calculated.
Animal perfusion
At the end of the experiment (21 days after the injection IB4-saporin or sham injection), the animals were deeply anesthetized with Equithesin (6.5 mg chloral hydrate and 3 mg sodium pentobarbital in a volume of 0.3 mL, i.p., per 100 g body weight). They were then perfused through the left cardiac ventricle with perfusion buffer (for composition see [40]) for 1 min, followed by 30 min of 4 % paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brain and spinal cord segments L4–L5 were extracted and postfixed for 4 and 2 h, respectively, in 4 % paraformaldehyde in PB. The specimens were then cryoprotected in 30 % sucrose in PB overnight at 4 °C.
Immunohistochemistry
Serial 100 µm-thick coronal sections of the brain were obtained, to examine the site of injection at the level of the parabrachial nucleus. L4–L5 spinal cord segments were trimmed and cut serially at 50 µm thickness. Both horizontal and coronal sections were cut using a freezing-sledge microtome (Leica, Richmond Hill, ON, Canada). Sections were collected as free-floating in phosphate-buffered saline (PBS) with 0.2 % Triton-X 100 (PBS + T).
To examine lamina I projection neurons, horizontal sections were incubated in 10 % normal donkey serum (Jackson, West Grove, PA, USA) in PBS + T for 1 h at room temperature to block unspecific staining. Subsequently, spinal cord sections were incubated, for 48 h at 4 °C, with the primary antibodies: goat anti-CTb at 1:5000 dilution (List Biological), rabbit anti-NK-1r, raised against residues 376–407 of the C-terminal sequence of the rat receptor, at 1:10,000 dilution (Sigma) and IB4 conjugated to AlexaFluor 647 (Molecular Probes) in PBS + T containing 5 % normal donkey serum. Then, the sections were washed several times with PBS + T and incubated for 2 h at room temperature with secondary antibodies preabsorbed with 10 mg/mL acetone rat brain powder: donkey anti-goat Rhodamine Red X (1:200, Jackson, West Grove, PA, USA) and donkey anti-rabbit AlexaFluor 488 (Molecular Probes). Finally, sections were washed with PBS, mounted on gelatin-subbed slides and coverslipped with an anti-fading mounting medium (Aqua Polymount; Polysciences, Warrington, PA, USA). Slides were stored at −4 °C. Control sections were processed by omitting the primary antibody which resulted in complete loss of immunoreactivity. The above protocol was also followed for the comparison of NK-1 receptor immunoreactivity in sham- and IB4-saporin-treated groups in transverse sections and the labeling of non-peptidergic afferents in horizontal sections, however in these two cases IB4 was conjugated to AlexaFluor 568 (Molecular Probes) instead of AlexaFluor 647.
To examine the primary afferent populations in the spinal dorsal horn, sections were processed as described above except that they were incubated for 48 h with IB4 conjugated to AlexaFluor 568, at 1:200 dilution (Molecular Probes) and rabbit anti-CGRP, at 1:2000 dilution (Sigma) followed by incubation with goat anti-rabbit AlexaFluor 488 (Molecular Probes).
Brainstem sections of the injection site were incubated with anti-CTb antibody (List Biological) followed by biotinylated donkey anti-goat IgG (Jackson, West Grove, PA, USA) and streptavidin conjugated to AlexaFluor 568 (Molecular Probes). All sections were mounted and coverslipped as described above.
Antibody specificity
As controls for immunocytochemistry, some sections were processed by omitting the primary antibodies or by pre-absorption with the peptide used to generate the antibody, in both cases resulting in a complete loss of immunoreactivity.
The goat anti-CTb antibody (List Biological; 703, lot 7032A5) was raised against purified CTb and its specificity was demonstrated by the lack of any staining in animals not injected with CTb. The rabbit anti-NK-1r antibody (Sigma; S8305, lot 084K4845) was generated against a synthetic peptide corresponding to amino acids 393–407 of the C-terminus region of the rat NK-1r conjugated to KLH as the immunogen and purified by ion-exchange chromatography. In Western blots from rat brain, the antibody was found to label only a specific 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 [41]. The rabbit anti-CGRP antibody (Sigma; C8198, lot 070M4835) was generated against synthetic rat CGRP conjugated to KLH as the immunogen. Using dot-blot immunoassay, the antibody was found to recognize rat CGRP conjugated to bovine serum albumin (BSA); it only shows cross-reactivity with CGRP (human) and β-CGRP (human) (data supplied by the manufacturer). Specific staining was abolished by pre-incubating the antiserum with rat CGRP. This antibody was previously used by us as a marker for peptidergic fibers in rat skin, dorsal root ganglia and spinal cord [25, 42–44].
Morphological characterization 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. [24]). In brief, in the current study, six serial, 50 µm-thick horizontal sections were cut from the dorsal part of the L4–L5 spinal segments. Six rats were used per group. Sections were examined under a PlanFluotar 40× oil immersion objective on a Zeiss Axioplan 2e imaging fluorescence microscope. Only neurons with visible nuclei, ipsilateral to the injection side, and with the cell body entirely located within the plane of the section, as assessed with the fine focus of the microscope, were included in our quantifications. Lamina I neurons were classified according to the shape of their cell body and their dendritic arborization in the horizontal plane. Fusiform neurons have two primary dendrites with one arising from each end of an elongated, spindle-shaped soma. Multipolar neurons have an irregularly-shaped cell body with four or more primary dendrites arising from the cell body. Pyramidal neurons have a triangularly-shaped soma with three primary dendrites arising from each of the cell body’s corners, in some cases, a fourth primary dendrite, oriented toward the white matter, was visible by confocal reconstruction or by adjusting the fine focus of a conventional fluorescence microscope. Neurons were classified as “unclassified” if they exhibited features transitional between any of these types, as they did not meet the required criteria.
To obtain images for the illustrations and to confirm the data obtained with conventional fluorescence microscopy, some sections were examined using a Zeiss LSM 510 confocal scanning laser microscope, using a multi-track scanning method and appropriate filters for the separate detections of AlexaFluor 488, AlexaFluor 568 or Rhodamine Red X and AlexaFluor 647. Low magnification images represent single optical section, whereas images of individual neurons represent serial optical sections obtained along the z-axis (z-stacks), using a 63× plan-apochromatic oil-immersion objective.
Quantitative analysis of CGRP immunolabeling and IB4-binding
Four sham animals and 5 IB4-SAP animals were used for the quantitative analysis. For quantification IB4-positive and CGRP-ir varicosities, single-plane confocal images were obtained in the confocal microscope using the 63× objective, equidistantly from the lateral and medial limits of the dorsal horn. This region corresponded to an area of maximum depletion of IB4 binding ipsilateral to the IB4-SAP injection. The images, originally in the Zeiss file format, were exported to TIFF and quantified using the ImageJ software. For each animal, 8–11 spinal cord cross sections were used, and two images (one from the ipsi- and the other from contralateral side) were obtained per section. In each image, a rectangle of 125 × 110 µm and 125 × 100 µm for IB4 and CGRP labeling, respectively, was placed with longer axis centered on the middle third of lamina II (for IB4) or with the dorsal longer side of the rectangle at on the white matter-lamina I border (for CGRP). To compensate overlapping or clustered varicosities, a correction was performed by the ImageJ software. The average number of varicosities per section’s side (ipsilateral or contralateral), per µm2, for each animal was calculated in the two conditions (Sham or IB4-SAP) ± SEM.
Statistics
Two-way ANOVA followed by Bonferroni correction were applied to compare differences in pain-related behavior at each time point between the sham and experimental groups. One-way analyses of variance (ANOVA) was applied to compare the differences between sham and experimental groups within each neuronal population at the 21 day time point post-IB4-saporin injection. To compare differences in densities of IB4 or CGRP-labeled varicosities, t-tests were used. Values were expressed as mean ± SEM. The significance level was set at P < 0.05. All data were analyzed using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA).
Figure preparation
All immunofluorescence images were obtained with the confocal microscope. They were saved in the Zeiss LSM format, exported as TIFF files and prepared for publication using Adobe Photoshop 7.0 (San Jose, CA, USA). The original images were optimized for brightness and contrast, and pseudo colors were assigned to the markers (green to NK-1r and CGRP and red to IB4 and CtB), to ensure uniformity throughout the paper, but no other image manipulation was done.
