Calcitonin Gene-related Peptide-immunoreactive Innervation Research Articles

Time course and localization of nerve growth factor expression and sensory nerve growth during progression of knee osteoarthritis in rats

Nerve growth factor (NGF) and sensory nerves are key factors in established osteoarthritis (OA) knee pain. We investigated the time course of NGF expression and sensory nerve growth across early and late stages of OA progression in rat knees. Knee OA was induced by medial meniscectomy in rats. OA histopathology, NGF expression, and calcitonin gene-related peptide immunoreactive (CGRP-IR) nerves were quantified pre-surgery and post-surgery at weeks 1, 2, 4 and 6. Pain-related behavior was evaluated using dynamic weight distribution and mechanical sensitivity of the hind paw. NGF expression in chondrocytes increased from week 1 and remained elevated until the advanced stage. In synovium, NGF expression increased only in early stages, whereas in osteochondral channels and bone marrow, NGF expression increased in the later stages of OA progression. CGRP-IR nerve density in suprapatellar pouch peaked at week 4 and decreased at week 6, whereas in osteochondral channels and bone marrow, CGRP-IR innervation increased through week 6. Percent ipsilateral weight-bearing decreased throughout the OA time course, whereas reduced paw withdrawal thresholds were observed only in later stages. During progression of knee OA, time-dependent alterations of NGF expression and CGRP-IR sensory innervation are knee tissue specific. NGF expression increased in early stages and decreased in advanced stage in the synovium but continued to increase in osteochondral channels and bone marrow. Increases in CGRP- IR sensory innervation followed increases in NGF expression, implicating that NGF is a key driver of articular nerve growth associated with OA pain.

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

Contribution of nerves within osteochondral channels to osteoarthritis knee pain in humans and rats

Subchondral bone may contribute to knee osteoarthritis (OA) pain. Nerve growth factor (NGF) can stimulate nerve growth through TrkA. We aimed to identify how sensory nerve growth at the osteochondral junction in human and rat knees associates with OA pain. Eleven symptomatic chondropathy cases were selected from people undergoing total knee replacement for OA. Twelve asymptomatic chondropathy cases who had not presented with knee pain were selected post-mortem. OA was induced in rat knees by meniscal transection (MNX) and sham-operated rats were used as controls. Twice-daily oral doses (30mg/kg) of TrkA inhibitor (AR786) or vehicle were administered from before and up to 28 days after OA induction. Joints were analysed for macroscopic appearances of articular surfaces, OA histopathology and calcitonin gene-related peptide-immunoreactive (CGRP-IR) sensory nerves in medial tibial plateaux, and rats were assessed for pain behaviors. The percentage of osteochondral channels containing CGRP-IR nerves in symptomatic chondropathy was higher than in asymptomatic chondropathy (difference: 2.5% [95% CI: 1.1-3.7]), and in MNX-than in sham-operated rat knees (difference: 7.8% [95%CI: 1.7-15.0]). Osteochondral CGRP-IR innervation was significantly associated with pain behavior in rats. Treatment with AR786 prevented the increase in CGRP-IR nerves in osteochondral channels and reduced pain behavior in MNX-operated rats. Structural OA was not significantly affected by AR786 treatment. CGRP-IR sensory nerves within osteochondral channels are associated with pain in human and rat knee OA. Reduced pathological innervation of the osteochondral junction might contribute to analgesic effects of reduced NGF activity achieved by blocking TrkA.

Role of Calcitonin Gene-Related Peptide in Inhibitory Neurotransmission to the Pig Bladder Neck

We studied the role of calcitonin gene-related peptide in nonadrenergic, noncholinergic neurotransmission to the pig bladder neck. We used immunohistochemical techniques to determine the distribution of calcitonin gene-related peptide immunoreactive fibers as well as organ baths for isometric force recording. We investigated relaxation due to endogenously released or exogenously applied calcitonin gene-related peptide in urothelium denuded phenylephrine precontracted strips treated with guanethidine, atropine and NG-nitro-L-arginine to block noradrenergic neurotransmission, muscarinic receptors and nitric oxide synthase, respectively. Rich calcitonin gene-related peptide immunoreactive innervation was found penetrating through the adventitia and distributed in the suburothelial and muscle layers. Numerous, variable size, varicose calcitonin gene-related peptide immunopositive terminals were seen close below the urothelium. In the muscle layer calcitonin gene-related peptide immunopositive nerves usually appeared as varicose terminals running along muscle fibers. Electrical field stimulation (2 to 16 Hz) and exogenous calcitonin gene-related peptide (0.1 nM to 0.3 μM) evoked frequency and concentration dependent relaxation, respectively. Nerve responses were potentiated by capsaicin, decreased by calcitonin gene-related peptide (8-37) and abolished by tetrodotoxin, capsaicin sensitive primary afferent blockers, calcitonin gene-related peptide receptors and neuronal voltage gated Na+ channels. Calcitonin gene-related peptide-induced relaxation was potentiated by the neuronal voltage gated Ca2+ channels blocker ω-conotoxin-GVIA and decreased by calcitonin gene-related peptide (8-37). Calcitonin gene-related peptide relaxation was not modified by blockade of endopeptidases, nitric oxide synthase, guanylyl cyclase and cyclooxygenase. Results suggest that calcitonin gene-related peptide is involved in the nonadrenergic, noncholinergic inhibitory neurotransmission of the pig bladder neck, producing relaxation through neuronal and muscle calcitonin gene-related peptide receptors. Nitric oxide/cyclic guanosine monophosphate and cyclooxygenase pathways do not seem to be involved in such responses.

A morphometric analysis of protein gene product 9.5-, substance P-, and calcitonin gene-related peptide immunoreactive innervation in the shoulder joint of the Japanese macaque

The shoulder capsule and labrum of Japanese macaque monkeys were studied immunohistochemically with the use of antisera against protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), and substance P (SP) to further characterize the innervation of the supporting structures of the shoulder joint. With PGP 9.5 immunohistochemistry thick nerve fibers (diameter ≥ 10 μm) presumed to be proprioceptive based on topographic location were found to be abundant in the posterior half of the capsule. Thinner fibers (diameter < 10 μm) presumed to contain nociceptive and autonomic fibers were located in the posterior half of the capsule. Ruffini-like corpuscles were predominantly located in the inferior portion of the capsule. SP and CGRP immunoreactive thin fibers presumed to be nociceptive were abundant in the posterior half of the capsule. Thin fibers that appeared to be nociceptive fibers were found in the marginal portion and the parenchyma of the labrum, although the number was small. The predominant distribution of the Ruffini-like corpuscles in the inferior portion of the capsule suggest an important role of the inferior portion in generation of the proprioceptive output, which should be advantageous in stabilization of the joint in motion. The abundance of nociceptive fibers in the posterior half of the capsule may be responsible for the pathophysiological transmission of pain around the shoulder joint.

Calcitonin gene-related peptide in the hepatic and splanchnic vascular systems of the rat.

The distribution and origin of calcitonin gene-related peptide immunoreactive structures in hepatic and splanchnic vasculature of the rat were investigated by the immunofluorescent technique. Calcitonin gene-related peptide immunoreactive fibers were dissociated from compact thick calcitonin gene-related peptide immunoreactive fiber bundles located around the hepatic and splanchnic vasculature, and reached the tunica adventitia of the vasculature. Some fibers penetrated further into the tunica media of the vasculature. In the tunica adventitia of the vasculature, calcitonin gene-related peptide immunoreactive fibers formed a fine meshwork which could be traced to the branches and arterioles. In the liver, calcitonin gene-related peptide immunoreactive innervation extended from porta hepatis to portal triads, running with branches of the hepatic artery and portal vein. Some calcitonin gene-related peptide immunoreactive fibers separated from thinner calcitonin gene-related peptide immunoreactive fiber bundles in portal triads and coursed into the immediately adjacent parenchyma. In the dorsal spinal ganglia (T8-10, L1), calcitonin gene-related peptide immunoreactive cells accounted for about 60% of the total number of ganglion cells. Bilateral vagotomy just below the diaphragm or bilateral transection of the greater splanchnic nerve resulted in a marked decrease of the number of calcitonin gene-related peptide immunoreactive fibers in the vasculature, whereas a combined operation was followed by the complete depletion of calcitonin gene-related peptide immunoreactive fibers. These results indicate that calcitonin gene-related peptide immunoreactive innervation in hepatic and splanchnic vasculature may have a dual origin. In conclusion, the widespread distribution of calcitonin gene-related peptide immunoreactive fibers in the hepatic and splanchnic vasculature suggest that calcitonin gene-related peptide innervation may be involved in sensory transmission.