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Rapid proliferation of CGRP-IR nerves during healing of rat tibial fracture suggests neural involvement in bone growth and healing
NEUROPEPTIDES:
203
FUNCTION AND PHARMA COLOGY
phocytes. This appeared to affect sensory and sympathetic nerve fibres equally. The data support the hypothesis that the mflammatory infiltrate is toxic to the nerve supply. The depletion of nerves is not seen in areas that are not in&need nor in a purely macrophage response, the presence of a mixed macrophage and lymphocyte inflammatory infiltrate is a requirement. P41 Distribution
and Frequency of Peptidecontaining Nerves During the Development of Human Articular Tissues
M. Hukkanen, Y. T. Konttinen*, S. Sautavirtat, G. Terenghi, G. Moscoso$, and J. M. Polak Department of Histochemistry, Royal Postgraduate Medical School, London, *Department of Anatomy, Helsinki University, TGrthopaedic Hospital of the Invalid Foundation, Helsinki, Finland and SDepartment Morbid Anatomy, King’s College School of Medicine, London. UK Human fetuses as young as 8-9 weeks of gestational age will respond to external stimuli with purposeful movements. For the development of movements a functional reflex arc, including sensory receptors in joints, is needed. The aim of this study was to delineate the developmental patterns of human articular innervation. The nerve supply to the joint, as detected by neuronal markers, was already well developed by 9 weeks of gestation, with nerve fibres being present in synovial stroma and periosteum. In these tissues, substance P-immunoreactive nerve fibres first appeared by 11 weeks of gestation, while sympathetic CPON-and tyrosine hydroxylase-immmunoreactive fibres were found in close proximity to vascular structures at 13 weeks, and it was not until 20 weeks of gestational age, when CGRP-immunoreactive varicosities were observed in synovial stroma and subintima, suggesting a differential expression of substance P and CGRP in sensory ganglia. Image analysis quantification revealed a rapid increase in total nerve number, nerve length and intercepts starting from 9 weeks, and progressing up to 17-18 weeks, followed by a decrease of all parameters. This may indicate that the peripheral afferent pathways of reflex arc are already developed early in fetal life following soon afterwards a selective targeting of their endings and natural regression of non-targeted terminals.
P42 Positive Correlation Between Substance P Serum Levels and Pain Threshold in Ankylosing Spondylitis G. Bemat&*, A. Sariat, A. H. Grafs, and G. Leinerg *Department of Zoology, University of Salzburg, Hellbrunner Str. 34, A-5020 Salzburg, tNeurochemica1 Unit, Department of Psychiatry, University Hospital Innsbruck, $General Hospital Salzburg, Austria and 5Badehospiz Badgastein, Austria
Substance P (SP) has been reported to be increased in plasma and synovial fluid of patients with rheumatic arthritis. We measured SP serum levels and pain threshold in patients with ankylosing spondylitis as pain threshold in rheumatic diseases may be changed. 33 male patients with ankylosing spondylitis undergoing a spa treatment in Austria were inch&d. The pain threshold of the thenar of the right hand and the actual pain intensity was measured with the Path Tester MP 1 100 and a visual analog scale, respectively. SP-immunoreactivity (IR) was determined in pre-extracted venous blood samples drawn immediately after pain testing. The SP-IR levels at the beginning and at the end of the spa treatment were slightly different (23.2 vs 19.0 fmol/ml, p < 0.05) and similar to the normal values in our laboratory. The thermal pain threshold varied considerably (44“C to 52”C), but the mean values at the beginning and at the end of the spa treatment were not significantly different. However, correlation analysis indicated a significant positive correlation between pain threshold and SP serum levels at both days of investigation. No correlation between SP-IR and actual pain intensity was observed. Our results suggest that SP-lR levels and pain threshold in ankylosing spondylitis are interrelated or are independent indicators of this disease.
P43 Rapid Proliferation of CGRP-IR Nerves During Eealing of Rat Tibial Fracture Suggests Neural Involvement in Bone Growth and Healing M. Hukkanen, Y. T. Konttinen*, S. Sat&vu-tat, P. Paavolainent, X. H. Gu, G. Terenghi and J. M. Polak Department of Histochemistty, Royal Postgraduate Medical School, London, *Dept. Anatomy, Helsinki University and torthopaedic Hospital of the Invalid Foundation, Helsinki, Finland The nervous system may be actively involved in bone repair and in remodelling of callous tissue in bone fractures. The aim of this study was to assess the distribution and nature of the innervation of the periosteum of normal bone and during bone healing subsequent to fracture of rat tibiae at 7, 14 and 2 1 days after experimental fracture using immunocytochemistry and image analysis quantification of protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). At 7 days, periosteal PGP 9.5-and CGRP-immunoreactive fibres showed dense ramifications and terminal sprouting. In addition toperiosteum, the nerve fibres were found in the middle of the callus interspersed with inflammatory cells and penetrating into secondary minor fractures. At days 14 and 21 many tortuous nerves were found in the periosteum but not in mid-callus. Image analysis quantification revealed a uniform increase of nerves after 7 days. At 2 1 days, the intercept countings showed in excess of a three-fold increase of CGRP-immunoreactive nerve fibres compared with the
204 normal control group (P < 0.0001) and were almost as numerous as PGP 9.5~immunoreactive fibres (P < 0.005). It is postulated that CGRP-containing sensory innervation may have a potential importance in the fracture vascular control, angiogenesis and osteogenesis in addition to a protective role against excessive fracture movement. The results are consistent with the neural involvement in bone growth and remodelling.
P44 Osteoblasts Express Nitric Oxide Synthase (NOS) in Response to Stimulation with Cytokines and Lipopolysaccharide (LPS) M. Hukkanen, F. Hughes*, G. Terenghi, D. R. Springall, Y. T. Konttinent, J. M. Polak, Department of Histochemistry, Royal Postgraduate Medical School, London, Department of Oral Medicine, London Hospital Medical School and tDept. Anatomy, Helsinki University, Finland Osteoclastic bone resorption is regulated by systemic mechanisms (calcitonin and PTH) and by locally produced factors including eicosanoids, cytokines and growth factors. It is well established that most local factors interact with osteoblasts which in response modulate osteoclastic resorption activity by an unknown mechanism. Nitric oxide has recently been shown to act as a potent inhibitor of osteoclast activity. In this study, we have attempted to demonstrate whether nitric oxide enzymes are expressed by osteoblasts. Primary cultures of rat osteoblasts, rat and human osteoblast-like cultures and rat skin fibroblast cultures were exposed to human IL-l l3, IL-6 or LPS for 6-24 h prior to fixation. hnmunocytochemistry was subsequently performed for inducible and constitntive forms of NOS and a histochemical method was used for the demonstration of NADPH-diaphorase. Strong cytoplasmic immunoreactivity for inducible NOS was observed in all osteoblast cultures. Immunostaining of the constitutive form of NOS was weakly positive but no apparent changes between cell stimulations could be observed. In addition, a moderate positive reaction for NADPH-diaphorase was observed in many cells. Fibroblasts were negative for both inducible and constitutive forms of NOS. These initial results show that osteoblasts express NOS-immunoreactivity and this expression can be regulated by the presence of stimulatory factors. The ability of osteoblasts to express NOS and therefore nitric oxide itself, provides a novel mechanism for the local control of bone resorption that may have implications in diseases with excessive resorption activity.
P45 The Effect of Intra-articular Capsaicin on Passive Synovial Anaphylaxis (PSA) and Blood Flow in the Rat Knee Joint H. Cambridge and S. D. Brain Pharmacology Group, Biomedical Sciences Division, Ring’s College London SW3 6LX, UK
NEUROPEPTIDES
The acute and long-term effects of capsaicin on PSA and blood flow in the rat knee joint were examined. Joints were sensitised by intra-articular injection of monoclonal IgE against dinitrophenol and PSA was induced by i.v. injection of the antigen a minimum of 24 h later. Plasma extravasation was measured by accumulation of lz51human serum albumin in the infrapatella fat pad synovium. Blood flow was measured by a 133Xenonclearance technique.’ Acute injection of capsaicin into the synovial space (330 nmol in 100 pl, 30 min prior to antigen) significantly inhibited plasma extravasation into the joint tissues (0.27 1 f 0.048 plmg’ versus 0.581 f 0.065 plmg’, treated versus control, meanf S.E.M., n= 13,p
P46 Inflammatory Influence on the Density of CGRP- and SP-Immunoreactive Nerve Endings in Rat Skeletal Muscle A. Reinert Institut fiir Heidelberg, Heidelberg,
and S. Merise Anatomie und Zellbiologie, Universitat Im Neuenheimer Feld 307, D-6900 Germany
The aim of the present study was to compare quantitatively neuropeptide-containing nerve endings in intact, inflamed and sympathectomized skeletal muscle. Serial frozen sections (thickness 12 pm) were cut from medial gastrocnemius muscles. 6 intact muscles, 6 persistently inflamed muscles (myositis induced by a single injection of Freund’s adjuvant into the muscle, 12 d survival time) and 2 chemically sympathectomized muscles (repeated injections of guanethidine i.p.) were examined. Sections were processed for CGRP- (peroxidase-DAB) and SP-(Texas Red) immunohistochemistry. All
203
FUNCTION AND PHARMA COLOGY
phocytes. This appeared to affect sensory and sympathetic nerve fibres equally. The data support the hypothesis that the mflammatory infiltrate is toxic to the nerve supply. The depletion of nerves is not seen in areas that are not in&need nor in a purely macrophage response, the presence of a mixed macrophage and lymphocyte inflammatory infiltrate is a requirement. P41 Distribution
and Frequency of Peptidecontaining Nerves During the Development of Human Articular Tissues
M. Hukkanen, Y. T. Konttinen*, S. Sautavirtat, G. Terenghi, G. Moscoso$, and J. M. Polak Department of Histochemistry, Royal Postgraduate Medical School, London, *Department of Anatomy, Helsinki University, TGrthopaedic Hospital of the Invalid Foundation, Helsinki, Finland and SDepartment Morbid Anatomy, King’s College School of Medicine, London. UK Human fetuses as young as 8-9 weeks of gestational age will respond to external stimuli with purposeful movements. For the development of movements a functional reflex arc, including sensory receptors in joints, is needed. The aim of this study was to delineate the developmental patterns of human articular innervation. The nerve supply to the joint, as detected by neuronal markers, was already well developed by 9 weeks of gestation, with nerve fibres being present in synovial stroma and periosteum. In these tissues, substance P-immunoreactive nerve fibres first appeared by 11 weeks of gestation, while sympathetic CPON-and tyrosine hydroxylase-immmunoreactive fibres were found in close proximity to vascular structures at 13 weeks, and it was not until 20 weeks of gestational age, when CGRP-immunoreactive varicosities were observed in synovial stroma and subintima, suggesting a differential expression of substance P and CGRP in sensory ganglia. Image analysis quantification revealed a rapid increase in total nerve number, nerve length and intercepts starting from 9 weeks, and progressing up to 17-18 weeks, followed by a decrease of all parameters. This may indicate that the peripheral afferent pathways of reflex arc are already developed early in fetal life following soon afterwards a selective targeting of their endings and natural regression of non-targeted terminals.
P42 Positive Correlation Between Substance P Serum Levels and Pain Threshold in Ankylosing Spondylitis G. Bemat&*, A. Sariat, A. H. Grafs, and G. Leinerg *Department of Zoology, University of Salzburg, Hellbrunner Str. 34, A-5020 Salzburg, tNeurochemica1 Unit, Department of Psychiatry, University Hospital Innsbruck, $General Hospital Salzburg, Austria and 5Badehospiz Badgastein, Austria
Substance P (SP) has been reported to be increased in plasma and synovial fluid of patients with rheumatic arthritis. We measured SP serum levels and pain threshold in patients with ankylosing spondylitis as pain threshold in rheumatic diseases may be changed. 33 male patients with ankylosing spondylitis undergoing a spa treatment in Austria were inch&d. The pain threshold of the thenar of the right hand and the actual pain intensity was measured with the Path Tester MP 1 100 and a visual analog scale, respectively. SP-immunoreactivity (IR) was determined in pre-extracted venous blood samples drawn immediately after pain testing. The SP-IR levels at the beginning and at the end of the spa treatment were slightly different (23.2 vs 19.0 fmol/ml, p < 0.05) and similar to the normal values in our laboratory. The thermal pain threshold varied considerably (44“C to 52”C), but the mean values at the beginning and at the end of the spa treatment were not significantly different. However, correlation analysis indicated a significant positive correlation between pain threshold and SP serum levels at both days of investigation. No correlation between SP-IR and actual pain intensity was observed. Our results suggest that SP-lR levels and pain threshold in ankylosing spondylitis are interrelated or are independent indicators of this disease.
P43 Rapid Proliferation of CGRP-IR Nerves During Eealing of Rat Tibial Fracture Suggests Neural Involvement in Bone Growth and Healing M. Hukkanen, Y. T. Konttinen*, S. Sat&vu-tat, P. Paavolainent, X. H. Gu, G. Terenghi and J. M. Polak Department of Histochemistty, Royal Postgraduate Medical School, London, *Dept. Anatomy, Helsinki University and torthopaedic Hospital of the Invalid Foundation, Helsinki, Finland The nervous system may be actively involved in bone repair and in remodelling of callous tissue in bone fractures. The aim of this study was to assess the distribution and nature of the innervation of the periosteum of normal bone and during bone healing subsequent to fracture of rat tibiae at 7, 14 and 2 1 days after experimental fracture using immunocytochemistry and image analysis quantification of protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). At 7 days, periosteal PGP 9.5-and CGRP-immunoreactive fibres showed dense ramifications and terminal sprouting. In addition toperiosteum, the nerve fibres were found in the middle of the callus interspersed with inflammatory cells and penetrating into secondary minor fractures. At days 14 and 21 many tortuous nerves were found in the periosteum but not in mid-callus. Image analysis quantification revealed a uniform increase of nerves after 7 days. At 2 1 days, the intercept countings showed in excess of a three-fold increase of CGRP-immunoreactive nerve fibres compared with the
204 normal control group (P < 0.0001) and were almost as numerous as PGP 9.5~immunoreactive fibres (P < 0.005). It is postulated that CGRP-containing sensory innervation may have a potential importance in the fracture vascular control, angiogenesis and osteogenesis in addition to a protective role against excessive fracture movement. The results are consistent with the neural involvement in bone growth and remodelling.
P44 Osteoblasts Express Nitric Oxide Synthase (NOS) in Response to Stimulation with Cytokines and Lipopolysaccharide (LPS) M. Hukkanen, F. Hughes*, G. Terenghi, D. R. Springall, Y. T. Konttinent, J. M. Polak, Department of Histochemistry, Royal Postgraduate Medical School, London, Department of Oral Medicine, London Hospital Medical School and tDept. Anatomy, Helsinki University, Finland Osteoclastic bone resorption is regulated by systemic mechanisms (calcitonin and PTH) and by locally produced factors including eicosanoids, cytokines and growth factors. It is well established that most local factors interact with osteoblasts which in response modulate osteoclastic resorption activity by an unknown mechanism. Nitric oxide has recently been shown to act as a potent inhibitor of osteoclast activity. In this study, we have attempted to demonstrate whether nitric oxide enzymes are expressed by osteoblasts. Primary cultures of rat osteoblasts, rat and human osteoblast-like cultures and rat skin fibroblast cultures were exposed to human IL-l l3, IL-6 or LPS for 6-24 h prior to fixation. hnmunocytochemistry was subsequently performed for inducible and constitntive forms of NOS and a histochemical method was used for the demonstration of NADPH-diaphorase. Strong cytoplasmic immunoreactivity for inducible NOS was observed in all osteoblast cultures. Immunostaining of the constitutive form of NOS was weakly positive but no apparent changes between cell stimulations could be observed. In addition, a moderate positive reaction for NADPH-diaphorase was observed in many cells. Fibroblasts were negative for both inducible and constitutive forms of NOS. These initial results show that osteoblasts express NOS-immunoreactivity and this expression can be regulated by the presence of stimulatory factors. The ability of osteoblasts to express NOS and therefore nitric oxide itself, provides a novel mechanism for the local control of bone resorption that may have implications in diseases with excessive resorption activity.
P45 The Effect of Intra-articular Capsaicin on Passive Synovial Anaphylaxis (PSA) and Blood Flow in the Rat Knee Joint H. Cambridge and S. D. Brain Pharmacology Group, Biomedical Sciences Division, Ring’s College London SW3 6LX, UK
NEUROPEPTIDES
The acute and long-term effects of capsaicin on PSA and blood flow in the rat knee joint were examined. Joints were sensitised by intra-articular injection of monoclonal IgE against dinitrophenol and PSA was induced by i.v. injection of the antigen a minimum of 24 h later. Plasma extravasation was measured by accumulation of lz51human serum albumin in the infrapatella fat pad synovium. Blood flow was measured by a 133Xenonclearance technique.’ Acute injection of capsaicin into the synovial space (330 nmol in 100 pl, 30 min prior to antigen) significantly inhibited plasma extravasation into the joint tissues (0.27 1 f 0.048 plmg’ versus 0.581 f 0.065 plmg’, treated versus control, meanf S.E.M., n= 13,p
P46 Inflammatory Influence on the Density of CGRP- and SP-Immunoreactive Nerve Endings in Rat Skeletal Muscle A. Reinert Institut fiir Heidelberg, Heidelberg,
and S. Merise Anatomie und Zellbiologie, Universitat Im Neuenheimer Feld 307, D-6900 Germany
The aim of the present study was to compare quantitatively neuropeptide-containing nerve endings in intact, inflamed and sympathectomized skeletal muscle. Serial frozen sections (thickness 12 pm) were cut from medial gastrocnemius muscles. 6 intact muscles, 6 persistently inflamed muscles (myositis induced by a single injection of Freund’s adjuvant into the muscle, 12 d survival time) and 2 chemically sympathectomized muscles (repeated injections of guanethidine i.p.) were examined. Sections were processed for CGRP- (peroxidase-DAB) and SP-(Texas Red) immunohistochemistry. All