Animals
In the experiments, female New Zealand white rabbits with a weight of approximately 4 kg (age ranging from 6 to 9 months) were utilized. In total, the investigation included evaluations of 16 animals. All the animals were subjected to an exercise protocol leading to marked overuse of the triceps surae muscle. The muscle overuse was combined with injection treatment (see below).
Exercise procedure
The animals were exposed to an exercise procedure that was performed according to previously described procedures [28], with some modifications [4–6, 23]. The rabbits were kept under anaesthesia, induced by i.m. injections of diazepam (5 mg/ml; 0.2 ml/kg) and fentanylfluanison (0.2-0.3 ml/kg), throughout the experiment. Fentanylfluanison (0.1 ml/kg) was injected each 30-45 min during the experiment in order to maintain the anaesthesia. The experiments lasted for 2 hours. For analgesia, buprenorphine (0.01-0.05 mg/kg) was given s.c. after each experimental session. The experiment was repeated every second day for one week (4 exercise sessions in total).
In order to achieve passive repetitive flexions and extensions of the right ankle joint, an apparatus (“kicking machine”) was used. A pneumatic piston attached to the foot produced the movements. An active contraction was furthermore induced by electrical stimulation via surface electrodes (Pediatric electrode 40 426A, Hewlett Packard, Andover, MA, USA) placed 2 cm apart over the triceps surae muscle of the right leg. This active contraction occurred during the plantar flexion phase. The stimulation was synchronized with the plantar flexion movement of the piston by a microswitch, which trigged the stimulator unit (Disa stimulator Type 14E, Disa Elektronik A/S, Herlev, Denmark). An impulse of 0.2 ms duration was delivered 85 ms after the initiation of the plantar flexion at an amplitude of 35-50 V. The movement frequency was 150 movements per minute. The left leg was not attached to the kicking machine and the pelvis was strapped down. In-between the experimental periods, the animals were kept in ordinary cages allowing good freedom of movement. For further details about the apparatus and the exercise protocol, see [4–6, 23].
Injection treatments
Injections were given in the loose connective tissue outside the Achilles tendon, i.e. in the paratenon region, of the right leg. The injections were given directly after each of the exercise regimens. The substances injected were Captopril (C) (c4042; Sigma), DL-Thiorphan (Th) (T 6031; Sigma), substance P (SP) (S6883; Sigma) and NaCl. Captopril is an angiotensin-converting enzyme inhibitor (ACE inhibitor) and DL-Thiorphan inhibits the activity of neutral endopeptidase (NEP). NEP is also called enkephalinase and neprilysin or common acute lymphoblastic leukemia antigen or CD10 [29].
Injections were performed as follows: a) Substance P (10-8 μmol/ml) and Captopril (30 μmol/kg) both in distilled water (volume: 1 ml) + Dl-Thiorphan (500 μg/ml; 0.02 ml) (5 animals), b) Captopril (30 μmol/kg, dissolved in distilled water, volume 1 ml) + DL-Thiorphan (500 μg/ml, 0.02 ml) (6 animals), c) NaCl (volume: 1 ml) (5 animals). The groups are further referred to as the SP + C + Th group, the C + Th group and the NaCl group.
Collection of muscle samples, fixation and sectioning
The animals were sacrificed, using an overdose of Pentobarbital, the day after the last exercise. The triceps surae muscles were dissected out. Directly thereafter the muscle samples were transported on ice to the laboratory. The distal parts of the soleus and gastrocnemius muscles (5-8 × 10 mm) were dissected out. Specimens from all samples were collected and processed in three ways: Immersion fixed, directly embedded/frozen or directly frozen in liquid nitrogen. For all types of specimens, specimens from all the various muscles were obtained. Thus, for both the soleus and gastrocnemius muscles, 5-6 specimens were generated for each experimental condition.
For fixation, specimens were fixed by immersion overnight at 4°C in an ice-cold solution of 4% formaldehyde in 0.1 M phosphate buffer (pH 7.0). These specimens were thereafter thoroughly washed in Tyrode’s solution containing 10% sucrose at 4°C overnight, mounted on thin cardboard in OCT embedding medium (Miles Laboratories, Naperville, Ill.), and frozen in propane chilled with liquid nitrogen. Other specimens were, as described above, directly embedded and frozen. Both types of specimens were stored at -80°C and from both types, series of 7-8 μm thin sections were cut using a cryostat. The sections were mounted on slides precoated with chrome-alum gelatine and were then processed for morphology (chemically unfixed samples) or immunohistochemistry and morphology (chemically fixed samples). Other specimens weighing approximately 30 mg were directly frozen in liquid nitrogen. They were further used for EIA analyses.
From one of the experimental groups (the SP + C + Th group) specimens, conforming to all three types of specimens as described above, were collected from both the right and left legs.
Processing for morphology (haematoxylin & eosin)
Sections in the series were stained in Harris Haematoxylin solution for 2 min. They were then rinsed in distilled water, dipped in 0.1% acetic acid for a few seconds, followed by washing in running water. Counterstaining was achieved by immersion in eosin for 1 min. The sections were dehydrated in ethanol and mounted in Permount.
Morphological semiquantitative evaluations
Evaluations with respect to the magnitude of morphological changes were made. The evaluations were based on the haematoxylin & eosin (H&E) stainings. The evaluations were made blinded.
The degree of inflammatory infiltrates in the tissue and the degree of presence of abnormal connective tissue areas, necrotic muscle fibers and variations in muscle fiber sizes as well as the degree of occurrence of internal nuclei were all assessed. An overall estimation for all these parameters was performed. The evaluations included an overall estimation of the entire sections from both the chemically unfixed and fixed specimens of each muscle.
The following pattern of scoring was used: 0 = no changes; morphology resembling that seen in normal muscle tissue, 1 = mild degree of changes, 2 = moderate degree of changes, 3 = marked changes, 4 = very marked changes. The scoring was relative, i.e. the magnitude of changes in one muscle was related to those in others. The mean value for scores for the different groups was then calculated. The significance level was set at the 0.05 level. The scoring is a simplified scoring method in relation to the one used in our previous morphological study on the effects of 1,3 and 6 weeks of experiment [4]. Based on the experience we obtained from that study, we consider that the scoring procedures that were presently used are reliable. Thus, the degree of occurrence of the different abnormal features analyzed parallelled each other.
Immunohistochemistry
Staining procedures for demonstration of substance P (SP) and NK-1R
Two antibodies for staining for demonstration of SP were used. One of the SP antibodies and the NK-1 R antibody were goat polyclonal antibodies (see below). The staining procedures for these antibodies conformed to those previously described [5, 6]. This included the use of 5% normal donkey serum (code no: 017-000-121, Jackson ImmunoResearch Lab. Inc., PA) as normal serum and FITC-conjugated AffiniPure donkey antigoat IgG (705-095-147; Jackson ImmunoResearch Lab Inc, dilution 1:100) as secondary antiserum. Mounting was performed in Vectashield Mounting Medium (H-1000) (Vector Laboratories, Burlingame, CA) and examination was carried out in a Zeiss Axioscope 2 plus microscope equipped with an Olympus DP70 digital camera.
Staining using a monoclonal SP antibody was also performed. In this case, an initial incubation with Triton X-100 was done, followed by incubation in 5% normal donkey serum and incubation with the primary antibody, diluted in PBS with BSA, in a humid environment. Incubation was performed for 60 min at 37°C. Another incubation in normal donkey serum followed, after which the sections were incubated with secondary antiserum. As secondary antiserum, tetramethylrhodamine isothiocyanate (TRITC)-conjugated AffiniPure donkey anti-rat IgG (712-025-150) (Jackson ImmunoResearch, PA), diluted 1:40 in PBS supplemented with 0.1% BSA, was used. Incubation with secondary antiserum proceded for 30 min at 37°C. The procedures for mounting and examination were as described above.
As an alternative procedure for demonstration of SP and NK-1R immunoreactions, sections were initially pretreated with acid potassium permanganate for 2 min, a procedure found to enhance specific immunofluorescence reactions [30]. After this pretreatment, the procedures described above followed. Both procedures (with or without acid potassium permanganate treatment) gave in principle similar results. There were, however, some differences in staining intensities and levels of background reactions, why it was useful to analyse sections by using both types of procedures.
Stainings for demonstration of desmin and CD31
Immunostaining for desmin was used in order to verify the occurrence of muscle fiber necrosis since it is well-known that necrotic muscle fibers show no or negligible desmin immunoreactivity [31, 32]. CD31 is a well-known marker of endothelia of blood vessels.
The CD31 staining was performed using visualization with FITC-conjugated donkey anti-mouse IgG (715-095-15) (Jackson ImmunoResearch), dilution used 1:100, and as normal serum, normal donkey serum with PBS in BSA was utilized. For the evaluation of desmin reaction patterns, double staining desmin/NK-1R was made. FITC-conjugated AffiniPure donkey anti-goat IgG (705-095-147) was used as secondary antibody for visualization of NK-1R and 5% normal donkey serum in PBS was used as normal serum. Concerning desmin immunodetection, rabbit anti-mouse immunolobulins/TRITC (R0276) (Dako, Denmark) was used as secondary antiserum, 1:40 dilution being used, and 5% normal rabbit serum in PBS with BSA as normal serum. Mounting was made in Vectashield Mounting Medium (H-1000).
Antibodies and control stainings
SP antibodies
An affinity purified goat polyclonal SP antibody was utlized. It was obtained from Santa Cruz Biotechnology (code: sc-14104) and was used at a dilution of 1:25-1:50. This antibody is raised against a peptide mapping within an internal region of preprotachykinin 1 of human origin and is recommended for detection of mature SP and all isoforms of the protachykinin 1 precursor of various species [mouse, rat and human origin]. According to “Human Protein Reference Database” and “BLAST-Basic Local Alignment Search Tool”, the protein sequence of rabbit protachykinin 1 shows at least 85% identity with human protachykinin 1.
A rat monoclonal SP antibody was also used. It was obtained from Biogenesis, Poole, UK (code: 8450-0505). It was used at a dilution of 1:50-1:100. The antibody is reported to recognize the COOH terminal end of SP and has been previously used for immunohistochemical detection of SP in experimental animals and man [5, 33].
Control stainings included the use of SP blocking substance (code: sc14104P; Santa Cruz) (20-50 μg/ml antiserum) and SP peptide (full length SP peptide; S6883; Sigma) (50 μg/ml antiserum). Ordinary stainings with the SP antisera were performed in parallel. Other control stainings conformed to stainings when the primary antibodies were excluded (buffer instead of the antibody).
NK-1R antibody
A NK-1R antibody produced in goats was used (code: sc-5220, Santa Cruz). It is an affinity purified polyclonal antibody raised against a peptide mapping within an internal region of NK-1R of human origin. It was used at a dilution of 1:50-1:100 in 0.1% PBS. It has been previously used in studies on immunoexpression patterns in rabbit muscle tissue [6]. Control stainings included the use of NK-1R blocking substance (sc-5220P) (50 μg/ml antiserum). Ordinary stainings for NK-1R were made in parallel. As was the case concerning stainings for SP, other control stainings conformed to stainings when the primary antibody was excluded.
CD31 and desmin antibodies
Mouse monoclonal antibodies against CD31 and desmin were used. The CD31 antibody was obtained from Dako, Glostrup, Denmark (code: M0823) and was used at a dilution of 1:100. It is a marker of the endothelial cells of blood vessel walls and has previously been used with reliable results in our laboratory [23]. The desmin antibody was also obtained from Dako, Glostrup, Denmark (code: Ab D33) and was utilized at a dilution of 1:100. It is by the supplier reported to be specific for desmin and to not show reactivity with other types of intermediate filaments. This antibody has been evaluated in previous studies [4].
Processing for EIA
Tissue homogenization
After being frozen in liquid nitrogen (see above), the samples were homogenised, by mechanical homogenization using a Precellys 24 tissue homogenizer (Bertin Technologies, Saint Quentin en Yvelines, Cedex, France), in a 100 mM Tris-HCl buffer, pH 7.0, containing 1 M NaCl, 2% BSA, 4 mM EDTA, 0.2% Triton X-100 (pH 7.0), 0.02% Na-azide and the protease inhibitors Pepstatin A (0.1 μg/ml), Aprotinin (5 μg/ml), Antipain (0.5 μg/ml), Benzamidin (167 μg/ml) and PMSF (5.2 μg/ml). All the protease inhibitors were purchased from Sigma, Germany. Tissue and buffer were mixed in a 1:20 ratio. The procedure was performed on ice. Directly after the homogenisation procedure, the tissue samples were centrifuged in +4°C, 13 000 g, for 15 min. The supernatant was then transferred to a new Eppendorf tube and stored at -80°C.
EIA procedure
The concentration of SP in the muscle samples was evaluated using commercially available enzyme immunoassay SP kits (Phoenix Pharmaceuticals, Burlingame, CA, USA). The sequence of the peptide detected conforms to the full-length of SP. 100% cross-reactivity is also reported for SP 2-11 – SP 5-11. Less than 0.01% cross-reactivity is reported for SP 7-11, neuropeptide K and neurokinin A and 0% for neurokinin B. The assays were performed in accordance with the instructions from the manufacturer. In order to obtain comparable results between different plates, reference samples were included in the various analyses. The levels of SP were normalized to the weights of the tissue samples, i.e. the values are expressed as pg/mg tissue.
Statistics
All data are expressed as mean and standard deviations (SD) are given. A one way ANOVA in combination with post hoc comparisons by use of Least Square Difference (LSD) was used for analysis of differences between the different groups. The paired t test was used for the evaluation of possible differencees in SP concentration between the experimental and non-experimental sides (concerning SP + C + Th group). The normality for the data for each group was examined and the distribution was found to be normal or approximately normal. All the statistical analysis was performed by software SPSS (PASW Statistics 20). A p-value < 0.05 was considered to be significant.
Ethics
The study protocol was approved by the local ethical committee at Umeå University (A34/07, A95/07). A licensed breeder had bred all the animals for the sole purpose of being used in animal experiments. All efforts were made to minimize animal suffering.