Changes of substance P-immunoreactive nerve fiber ... - Springer Link

Osteoporos Int (2008) 19:559–569 DOI 10.1007/s00198-007-0481-2

ORIGINAL ARTICLE

Changes of substance P-immunoreactive nerve fiber innervation density in the sublesional bones in young growing rats at an early stage after spinal cord injury D. Liu & H. Li & C.-Q. Zhao & L.-S. Jiang & L.-Y. Dai

Received: 28 June 2007 / Accepted: 24 August 2007 / Published online: 9 October 2007 # International Osteoporosis Foundation and National Osteoporosis Foundation 2007

Abstract Summary Spinal cord injury (SCI) causes osteoporosis (OP), and the neuropeptide substance P (SP) may play important roles in the pathogenesis of OP after SCI. Our study confirmed SCI-induced sublesional bone loss in young rats at an early stage is associated with a significant increase of SP-immunoreactive nerve fiber innervation density. Introduction Spinal cord injury (SCI) causes osteoporosis (OP), and neuropeptides may play important roles in the pathogenesis of OP after SCI. However, few data exist concerning the relationship between neural factors and OP following SCI. Methods One hundred and eight SCI and hindlimb cast immobilization (HCI) rats were studied for skeletal innervation of substance P (SP) and neurofilament 200 (NF200) with immunocytochemistry. Bone and serum SP levels were also assessed using enzyme immunoassay. Results Developing bone loss was successfully induced by SCI at 3 wks and by HCI at 6 wks. We observed a significant increase of SP-immunoreactive (IR) nerve fibers and decrease of NF200-IR nerve fibers in the tibiae of SCI rats compared with HCI and control (CON) rats at all time points. SP in the proximal tibiae in SCI rats was significantly higher than that in HCI and CON rats at all time points, but no difference was found in the serum. D. Liu : H. Li : C.-Q. Zhao : L.-S. Jiang Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China L.-Y. Dai (*) Department of Orthopaedic Surgery, Xinhua Hospital, 1665 Kongjiang Road, Shanghai 200092, China e-mail: [email protected]

Conclusion SCI-induced sublesional bone loss in young rats at an early stage is associated with a significant increase of nerve fiber innervation density of SP-IR and decrease of NF200-IR. We speculated that neural factors may play an important role in pathogenesis of OP after SCI. Keywords Innervation . Osteoporosis . Spinal cord injury . Substance P

Introduction Osteoporosis (OP) is one of the common and important complications of spinal cord injury (SCI), characterized with low bone mass and bone ultrastructure deterioration. SCI results in more bone loss than bed rest and weightlessness [1]. Because mechanical stimulus is a directory agent for bone modeling and remodeling, disuse is generally thought to be one of the major causes for OP in SCI [2–4]. In these cases, bone resorption is accelerated and bone loss cannot be completely reversed by functional exercise and drug therapy. Moreover, the location of SCIinduced OP is different from that of disuse OP. We speculated that the mechanisms of OP induced by SCI are more complicated and neural lesion itself may be involved in the bone loss [5, 6]. Neuropeptides, including substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide, pituitary adenylate cyclase activating peptides and neuropeptide Y, are released from sensory and sympathetic nerve fibers and identified in skeletal tissues [7–9]. Functional receptors for these neuropeptides are expressed by bone cells, and many in vitro studies have shown that neuropeptides affect their biological functions [9, 10]. Both sensory and sympathetic nerve fibers form dense networks around blood vessels

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adjacent to bone trabeculae [11–13]. Experimental and clinical studies have shown increasing evidence for the neural control of bone development, growth, turnover and repair [14–19]. In a word, normal bone metabolism requires a coordinated interaction between sensory/sympathetic nervous system and cells within the bone tissues. SP is a neuropeptide released from axons of sensory neurons, belongs to the tachykinin family and plays important roles in many physiological and pathological processes. It activates signal transduction cascades by acting on the neurokinin-1 receptor (NK1-R) [20]. Previous studies have confirmed that the SP-immunoreactive (IR) axons innervate bone and adjacent tissues, and that their densities vary depending on the stage of skeletal development, location and the presence or absence of pathological conditions [11, 21, 22]. Over the past few years, it has been found that SP takes part in the stimulation of bone resorption, and its receptors are located in osteoclasts [11]. Notably, in studies of skeletal ontogeny SP-IR axons have been shown to appear at an early stage, mostly coinciding with the sequence of long bone mineralization [21]. These findings, together with data obtained from chemically or surgically targeted nerve deletion experiments, strongly suggest that SP is a potent regulator of skeletal physiology. The specific distributions of SP-IR nerve fibers, the different amounts of SP within regions, and the various levels of expression of NK1-R in targeted cells are presumably related to bone metabolism. Neurofilament 200 (NF200) is specific of neurofilaments which are structural proteins found in all nerve processes and plays an important role in cell architecture [23]. It may be used to study the changes in the level of neurofilament associated with differentiation or neuronal damage. All the nerve fibers contain NF200, which is not capable of discriminating the differences between sympathetic and sensory nerve fibers. Therefore, the density of NF200 immunoreactivity represents a general innervation density. SP is released from sensory nerve axons, and its density specially accounts for the amount of sensory nerve fibers. Although numerous in vitro studies have shown roles for neuropeptides in bone cell functions [9, 10, 24–33], the relationship between the presence of these nerve fibers and bone cell metabolism has been poorly investigated in vivo and very few in vivo studies about the changes of innervation associated with the changes in bone modeling or remodeling were obtained. To our knowledge, no data exist concerning the quantitative changes of SP-IR nerve fibers and NF200-IR nerve fibers during bone loss associated with SCI and hindlimb cast immobilization (HCI) rats. In this experiment, rats with SCI produced by complete transection and with simple hindlimb immobilization were used to identify the relationship between SP-IR nerve density and bone loss after SCI. We supposed that SCI-

Osteoporos Int (2008) 19:559–569

induced bone loss might relate to the modulations of SP-IR nerve fibers and bone general innervation. Therefore, we evaluated the changes using immunohistochemistry and enzyme immunoassay assay (EIA) method in SCI and HCIinduced OP rats. Our results indicated that SCI resulted in a significant increase of SP-IR nerve fiber density and decrease of NF200-IR nerve fiber density in the proximal tibiae in young rats at an early stage. Moreover, the SP content in the same region was significantly different between SCI and HCI rats.

Materials and methods Animals Rats were purchased and raised at the centralab of our institution. All experimental procedures were performed in accordance with the principle defined by the university animal welfare and ethical review committees. One hundred and eight male Sprague-Dawley (SD) rats, aged 6 wks and weighing 139 to 163 grams, were randomly assigned into nine groups according to the treatments and duration after treatments, with each group treated by SCI, HCI, or untreated control (CON) and killed at 1 wk, 3 wks or 6 wks interval, respectively. Rats in SCI group were anaesthetized by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). The spinal cord was exposed by laminectomy at the T10–12 level and then completely transected with a sharp knife. All rats received penicillin G (40,000 U/kg; i.p.) for the first week after surgery. The urinary bladders of SCI rats were emptied manually at least three times a day for the first two weeks until spontaneous micturition recovered. Hindlimb immobilization of the rats was performed according to the previously described procedures [34]. Rats were lightly anaesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (6 mg/kg) for attachment of plaster of Paris cast. Briefly, both hindlimbs were fixed from the waist down with the hips at extension, the knees at 110° flexion and ankles at 150° plantarflexion. The cast were extended 1 cm below the feet, making weight bearing impossible [35]. Post-treatment care also included housing the rats in pairs (to reduce isolation-induced stress) in a controlled environment at 22°C with a 12-hour light/dark cycle and using highly absorbent bedding. All rats were maintained on commercial rat chow available ad libitum with 0.95% calcium and 0.67% phosphate. Experimental design Six animals in each group were randomly selected for histomorphometry analysis. The 3 wks and 6 wks groups


were injected with tetracycline hydrochloride (30 mg/kg; Sigma, St. Louis, MO, USA) 4 and 14 days before being killed. At designed time points, the rats were anaesthetized and sacrificed by exsanguinations from the abdominal aorta. The serum was collected and stored, and then the right tibiae and humeri were dissected, measured for bone mineral density (BMD), and processed for bone histomorphometric analysis. The left tibiae and humeri of another six animals in each group were immediately dissected and preserved in liquid nitrogen to examine the content of SP in bone tissue with EIA method. Then the animals were perfused transcardially with heparinized saline (pH 7.4, 4°C), followed by a cold fixative containing 4% paraformaldehyde plus 0.2% picric acid in 0.1 M phosphate buffer (PB; pH 7.4) for immuno-light microscopy. The right tibiae and humeri were immediately dissected out and immersed in the same fixative for 3 h at 4°C. All specimens were then decalcified in a 5% EDTA solution (pH 7.4) for 3 wks. BMD measurement The BMD of the right tibiae and humeri in SCI, HCI, and CON rats was measured by dual-energy X-ray absorptiometry (DEXA) on a Hologic Discovery-A QDR bone densitometer (Bedford, MA, USA) with software modified for small animals (Regional High Resolution version 4.76; Hologic). The percentage coefficient of variations (%CVs) for BMD measurements was 1.1%. Histomorphometry The right tibiae and humeri were dehydrated in graded alcohols, embedded in methylmethacrylate without decalcification and sectioned longitudinally with a Leica/Jung 2065 microtome at 4 and 8 μm thickness. Histomorphometry was performed on the regions between 0.5 mm and 2.0 mm distal to the growth plate in the proximal tibiae and humeri in order to exclude the primary trabecular spongiosa. The 4 μm thick sections were stained with Toluidine Blue for the collection of bone architecture data with light microscope, whereas the 8 μm sections were left unstained for the measurement of fluorochrome-based indices. Histomorphometric variables of cancellous bone structure, bone formation and bone resorption were measured using a morphometric program (OsteoMeasure, OsteoMetrics, Atlanta, GA, USA) at a magnification of 100×. The structural variables included cancellous bone volume (BV/ TV), trabecular number (Tb.N), trabecular thickness (Tb. Th) and trabecular separation (Tb.Sp). Bone formation was expressed by the mineral apposition rate (MAR), mineralizing surface (MS/BS) and surface-based bone formation rate (BFR/BS). Bone resorption was expressed as the eroded surface (ES/BS). Nomenclature and units were used

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according to the recommendation of the Nomenclature Committee of the American Society for Bone and Mineral Research [36]. Quantitative histomorphometric analyses were conducted in a blind fashion. The %CVs of interand intra-analyser variations for all histomorphometric parameters were less than 5% in our laboratory. Immunohistochemistry After decalcification, the tissue specimens were rinsed in 20% sucrose in 0.1 M PB overnight and sectioned at a thickness of 15–30 μm on a cryostat, and then were mounted on gelatin-coated slides. The sections were processed immunohistochemically to demonstrate the presence of SP or NF200 by means of the avidin-biotin-peroxidase complex method. After treatment in phosphate-buffered saline (PBS) with 0.3% Triton-X 100 (pH 7.4) for 30 min and 0.3% H2O2 for 1 h in order to block endogenous peroxidase, the sections were then incubated with polyclonal SP rabbit antirat antibody (Chemicon, Temecula, CA) diluted 1:2000 or with monopoly NF200 mouse anti-rat antibody (Sigma, St. Louis, MO, USA) diluted 1:400 in a solution consisting of 1% bovine serum albumin and 0.05% sodium azide in 0.1 M PBS for 24 h at 4°C. After three washings in PBS, the specimens were then exposed to biotinylated goat antirabbit or -mouse IgG (Changdao Co., Shanghai, China) diluted 1:200 in PBS for 4 h at room temperature. Three washings in PBS were followed by treatment with ABC (Changdao Co., Shanghai, China) diluted 1:100 in PBS for 4 h at room temperature. The peroxidase reaction was then developed for 10 min in 0.05 M TRIS buffer (pH 7.6) containing 0.02% 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA) and 0.006% H2O2. The sections were photographed with a Nikon Optiphoto microscope. The positive staining of SP and NF200 in bone metaphyses was visible as brown punctums and bundles which were distributed in the bone marrow, periosteum, and bone trabeculae. We used spinal cord tissues as a positive control that showed positive brown staining and the samples without primary antibody as a negative control without positive signals. Quantitative analysis of immunostaining Quantitative image analyses of immunostaining intensity of SP and NF200 from SCI, HCI, and CON groups were performed in secondary spongiosa of rat tibiae and humeri. It is important to emphasize that all the sections were performed at the same time for the quantitative study. The images obtained were analyzed for positive staining at a magnification of 400× using quantitative immunohistochemical analysis software Image Pro Plus (Media Cybernetics, Baltimore, MD, USA). The total area of each tissue

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analyzed was approximately the same. We evaluated the integrated optical density (IOD), in which “integrated” refers to the sum of all the pixel intensity or density values in a given region [37]. The numerical values obtained from the ten different regions in each tissue were then averaged as representing of the specified marker in the given tissue. Unlike the standard optical density that measures the absorption value of light by the chromogen, the quantitative immunohistochemical analysis software used by us measures the scattering value by the chromogen. Therefore, the values we measured are opposite to standard values. Higher values in the bars represent weaker staining and vice-versa. The %CVs for immunostaining measurements were 4.1 and 4.5% for SP and NF200, respectively. EIA for SP concentration of bone and serum The left proximal tibiae metaphyses (3–5 mm, 60.3– 87.6 mg) and humeri metaphyses (4–5 mm, 45.6– 63.4 mg) were collected, immediately frozen, and weighed. The samples were minced in 2 ml of 3:1 ethanol/0.7 M HCl and homogenized for 20 s, shaken for 2 h and centrifuged at 3000 g for 20 minutes at 4°C. The supernatant was frozen and lyophilized, and the lyophilized product was stored at −80°C. Blood sampling (5 mL) was carried out between 8 and 10 Am after overnight fasting. To avoid disintegration of SP, the blood was drawn into chilled serum tubes containing 500 KIU/mL aprotinin as proteinase inhibitor, and immediately centrifuged at 1600 g at 4 C, then the supernatants were stored at −80°C until assay (no longer than 2 months). The values were assayed in duplicate using an enzyme immunoassay kit (QRCT34827, ADL, Fremont, CA) in this study. The %CVs for the values of SP was 3.7%. Statistical analysis All data values are presented as means±SD. Comparisons of data were performed by a two-factor analysis of

variance (ANOVA) with treatments (SCI, HCI, and CON) and time courses (1 wk, 3 wks, and 6 wks) as the factors and with Student-Newman-Keuls (SNK) test for multiple comparisons. All statistical analyses were performed using SPSS 11.5 software (SPSS Inc., Chicago, IL, USA). A significance level of P