Calcitonin Gene-related Peptide Immunostaining Research Articles

The expression of substance P and calcitonin gene-related peptide is associated with the severity of tendon degeneration in lateral epicondylitis

BackgroundIn this study, we investigated whether substance P (SP) or calcitonin gene-related peptide (CGRP) expression is associated with tendon degeneration in patients with lateral epicondylitis.MethodsTwenty-nine patients who underwent surgical treatment for lateral epicondylitis were enrolled in the final analyses. Extensor carpi radialis brevis tendon origins were harvested for histological analysis.ResultsSP and CGRP immunostaining were negative in healthy tendons but positive in degenerative tendons; moreover, their immunoreactivity increased with degeneration severity. Univariate analysis indicated that variables such as the preoperative visual analog scale (VAS) score or SP or CGRP expression levels were significantly associated with the Movin score. However, multivariate analysis revealed that only higher SP and/or CGRP signals were associated with higher Movin scores. Elevations in SP or CGRP expression were also linked with significantly severe preoperative VAS scores.ConclusionWe demonstrated that tendon degeneration severity is associated with increased SP and CGRP expression in the biopsy samples of lateral epicondylitis.

Loss of α-Calcitonin Gene-Related Peptide (αCGRP) Reduces Otolith Activation Timing Dynamics and Impairs Balance.

Calcitonin gene-related peptide (CGRP) is a neuroactive peptide that is thought to play a role at efferent synapses in hair cell organs including the cochlea, lateral line, and semicircular canal. The deletion of CGRP in transgenic mice is associated with a significant reduction in suprathreshold cochlear nerve activity and vestibulo–ocular reflex (VOR) gain efficacy when compared to littermate controls. Here we asked whether the loss of CGRP also influences otolithic end organ function and contributes to balance impairments. Immunostaining for CGRP was absent in the otolithic end organs of αCGRP null (-/-) mice while choline acetyltransferase (ChAT) immunolabeling appeared unchanged suggesting the overall gross development of efferent innervation in otolithic organs was unaltered. Otolithic function was assessed by quantifying the thresholds, suprathreshold amplitudes, and latencies of vestibular sensory-evoked potentials (VsEPs) while general balance function was assessed using a modified rotarod assay. The loss of αCGRP in null (-/-) mice was associated with: (1) shorter VsEP latencies without a concomitant change in amplitude or thresholds, and (2) deficits in the rotarod balance assay. Our findings show that CGRP loss results in faster otolith afferent activation timing, suggesting that the CGRP component of the efferent vestibular system (EVS) also plays a role in otolithic organ dynamics, which when coupled with reduced VOR gain efficacy, impairs balance.

Expression of neurotransmitters and neurotrophins in neurogenic inflammation of the rat retina.

Antidromic stimulation of the rat trigeminal ganglion triggers the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from sensory nerve terminals of the capsaicin sensitive C-fibers. These pro-inflammatory neuropeptides produce a marked hyperemia in the anterior segment of the eye, accompanied by increased intraocular pressure, breakdown of the blood-aqueous barrier and myosis. To assess the effects of neurogenic inflammation on the retina, specifically on the immunostaining of neurotransmitters and neurotrophins, as well as on the expression of neurotrophin receptors in the retina. RT-PCR was also accomplished in control and stimulated animals to confirm the immunohistochemical results. In the electrically stimulated eyes, immunostaining for SP, CGRP, VIP and nNOS demonstrated a marked increase in the RPE/POS (Retinal Pigment Epithelium/Photoreceptor Outer Segments), in the inner and outer granular layers and in the ganglion cells in comparison to the control eyes. CGRP and SP were found increased in stimulated animals and this result has been confirmed by RT- PCR. Changes in neurotrophin immunostaining and in receptor expression were also observed after electric stimulation of trigeminal ganglia. Decrease of BDNF and NT4 in the outer and inner layers and in ganglion cells was particularly marked. In stimulated rat retinas immunostaining and RT-PCR showed a NGF expression increase. Neurotrophin receptors remained substantially unchanged. These studies demonstrated, for the first time, that antidromic stimulation of the trigeminal ganglion and subsequent neurogenic inflammation affect immunostaining of retinal cell neurotransmitter/neuropeptides and neurotrophins as well as the expression of neurotrophin receptors.

Calcitonin gene-related peptide affects synaptic and membrane properties of bronchial parasympathetic neurons

Calcitonin gene-related peptide (CGRP) is located with substance P in nerve varicosities in close apposition to principal neurons in airway parasympathetic ganglia. Substance P has multiple effects on airway parasympathetic neurons but the role of CGRP is unknown. Using intracellular current clamp recording of ganglionic neurons, stimulation of vagal afferent nerves in the presence of neurokinin receptor antagonists evoked hyperpolarization of the membrane potential which was blocked by the CGRP-1 receptor antagonist, CGRP(8-37). Exogenous application of alpha-CGRP (0.001-0.1 microM) hyperpolarized the membrane potential, which was either blocked or reversed to depolarization in the presence of CGRP(8-37), whereas higher concentrations of alpha-CGRP (1.0-10.0 microM) caused depolarization. Action potential accommodation in phasic-type neurons decreased in the presence of alpha-CGRP (0.1-10 microM). The co-localization of substance P- and CGRP-immunoreactivity was observed in nerve varicosities within ganglia; prolonged exposure to capsaicin in vitro depleted substance P and CGRP immunostaining in nerve varicosities. These results demonstrate that CGRP has multiple effects on the excitability of airway parasympathetic neurons and may alter their activity, ultimately affecting parasympathetic tone in the lower airways.

Cardiac Nociception

Cardiac Nociception

Genetic Manipulation of Intraspinal Plasticity after Spinal Cord Injury Alters the Severity of Autonomic Dysreflexia

Severe spinal cord injuries above mid-thoracic levels can lead to a potentially life-threatening hypertensive condition termed autonomic dysreflexia, which is often triggered by painful distension of pelvic viscera (bladder or bowel) and consequent sensory fiber activation, including nociceptive C-fibers. Interruption of tonically active medullo-spinal pathways after injury causes disinhibition of thoracolumbar sympathetic preganglionic neurons, and intraspinal sprouting of nerve growth factor (NGF)-responsive primary afferent fibers is thought to contribute to their hyperactivity. We investigated spinal levels that are critical for eliciting autonomic dysreflexia using a model of noxious colorectal distension (CRD) after complete spinal transection at the fourth thoracic segment in rats. Post-traumatic sprouting of calcitonin gene-related peptide (CGRP)-immunoreactive primary afferent fibers was selectively altered at specific spinal levels caudal to the injury with bilateral microinjections of adenovirus encoding the growth-promoting NGF or growth-inhibitory semaphorin 3A (Sema3a) compared with control green fluorescent protein (GFP). Two weeks later, cardio-physiological responses to CRD were assessed among treatment groups before histological analysis of afferent fiber density at the injection sites. Dysreflexic hypertension was significantly higher with NGF overexpression in lumbosacral segments compared with GFP, whereas similar overexpression of Sema3a significantly reduced noxious CRD-evoked hypertension. Quantitative analysis of CGRP immunostaining in the spinal dorsal horns showed a significant correlation between the extent of fiber sprouting into the spinal segments injected and the severity of autonomic dysreflexia. These results demonstrate that site-directed genetic manipulation of axon guidance molecules after complete spinal cord injury can alter endogenous circuitry to modulate plasticity-induced autonomic pathophysiology.

Morphological evidence for functional capsaicin receptor expression and calcitonin gene-related peptide exocytosis in isolated peripheral nerve axons of the mouse

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles—presumably on axonal transport—as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10−6 M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures—the ω structures—lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.

Morphological evidence for functional capsaicin receptor expression and calcitonin gene-related peptide exocytosis in isolated peripheral nerve axons of the mouse

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca 2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles—presumably on axonal transport—as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10 −6 M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures—the ω structures—lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.

Possible role of bioactive peptides in the regulation of human detrusor smooth muscle - functional effects in vitro and immunohistochemical presence.

Results from basic research implicate a role for bioactive peptides in controlling the mammalian lower urinary tract. Although various peptides are assumed to be involved in the potentiaton or inhibition of cholinergic or purinergic activity in the urinary bladder, there is still much controversy regarding the mode of action and functional significance of such peptides in detrusor smooth muscle. Thus, we evaluated the functional effects of atrial natriuretic peptide (ANP), calcitonin gene related peptide (CGRP), endothelin 1 (ET-1), substance P (SP) and vasoactive intestinal polypeptide (VIP) on isolated strip preparations of human detrusor smooth muscle and determined the presence of those peptides in the human detrusor by means of immunohistochemistry. The effects of peptides on isometric tension of isolated detrusor strip preparations and on tissue levels of cyclic nucleotides cAMP and cGMP were compared to those of adenylyl cyclase activator forskolin (F), nitric oxide donor Na(+)-nitroprusside (SNP) and non-specific phosphodiesterase (PDE) inhibitor papaverine (P). The effects of the compounds on isometric tension of isolated human detrusor smooth muscle were examined using the organ bath technique. To determine time- and dose-dependent effects on cyclic nucleotide levels, bladder strips were exposed to increasing doses of F, SNP, P, ANP, CGRP and VIP, then rapidly frozen in liquid nitrogen and homogenised in the frozen state. cAMP and cGMP were extracted and assayed using specific radioimmunoassays. The presence of peptides was investigated by light microscopy using the Avidin-Biotin-Complex (ABC) method. F, P and VIP most effectively reversed the carbachol-induced tension of isolated human detrusor strips. Relaxing effects of ANP, CGRP and SNP were negligible. In contrast, ET-1 and SP elicited dose-dependent contractions of the tissue. The relaxing effects of F, P and VIP were accompanied by an increase in cAMP and cGMP levels, respectively. Light microscopy revealed positive immunostaining for CGRP, ET 1, VIP and SP in sections of the detrusor muscle coat. Our results suggest a possible importance of ET 1, SP and VIP in regulating detrusor smooth muscle contraction and relaxation. Even if a peptide is not synthesised, stored or released in a smooth muscle tissue and is, therefore, unable to reach its target cells under physiologic conditions, a functional effect on the tissue might be mediated by peptide-binding to specific cell surface receptors.

A resorbable nerve conduit as an alternative to nerve autograft in nerve gap repair

Poly-3-hydroxybutyrate (PHB) occurs within bacterial cytoplasm as granules and is available as bioabsorbable sheets. Previously, the advantage of PHB in primary repair has been investigated while in this study the same material has been used to bridge an irreducible gap. The aim was to assess the level of regeneration in PHB conduits compared to nerve autografts.The rat sciatic nerve was exposed, a 10 mm nerve segment was resected and bridged with either an autologous nerve graft or a PHB conduit. The grafted segments were harvested up to 30 days. Immunohistochemical staining was performed and computerised quantification of penetration distance and volume of axonal regeneration was estimated by protein gene product (PGP) immunostaining and calcitonin gene-related peptide (CGRP) positive fibres. Penetration and proliferation density of Schwann cells into the conduit was measured by quantifying S-100 staining. The inflammatory response was quantified with ED-1 staining for macrophages. Antibodies to vWf provided an assessment of angiogenesis and capillary infiltration.Percentage immunostaining for PGP in autograft and PHB groups showed a progressive increase up to 30 days with a significant linear trend with time and an increase in the volume of axonal regeneration. A similar pattern of progressive increase with time was observed with CGRP immunostaining for both groups and with S-100 in the PHB group. Good angiogenesis was present at the nerve ends and through the walls of the conduit. The results demonstrate good nerve regeneration in PHB conduits in comparison with nerve grafts.

Increased Calcitonin Gene-Related Peptide Immunoreactivity in Gracile Nucleus after Partial Sciatic Nerve Injury: Age-Dependent and Originating from Spared Sensory Neurons

Following a unilateral chronic constriction injury of the sciatic nerve, calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fiber density increases in the ipsilateral gracile nucleus, and this is more pronounced in aged (16-month) rats where the fibers are dystrophic. In this study we show that a second type of partial sciatic nerve injury, a half-transection, also induces CGRP-IR fibers in the gracile nucleus, but this effect is strongly age-dependent, being much more pronounced in 8- to 10-month-old rats than in 2- to 3-month-old rats. Dystrophic CGRP-IR fibers were rarely observed in 8- to 10-month-old animals, so the increased reaction in aged animals and axonal dystrophy are separate phenomena. Using double-labeling with fluorescent dye tracing for 8- to 10-month-old rats, we showed that neuron profiles in the dorsal root ganglion (DRG) with peripheral axons spared by the partial sciatic nerve injury were 10 times more likely to be CGRP mRNA-positive than profiles with injured peripheral axons, suggesting that spared neurons are more likely to contribute to the increase in CGRP-IR fibers in the gracile nucleus. Using combined fluorescent dye tracing with in situ hybridization for CGRP mRNA or CGRP immunostaining, we further showed that CGRP-expressing DRG neuron profiles with central projections to the gracile nucleus had peripheral axons spared by the partial nerve injury. We conclude that the increased CGRP immunoreactivity in the gracile nucleus following partial sciatic nerve injury originates from primary sensory neurons with axons spared by the injury. These neurons may still transmit cutaneous sensory information and thus the increased CGRP immunoreactive fibers in the gracile nucleus may be involved in the mechanical allodynia characteristic of neuropathic pain syndromes following partial nerve injury.

Stereological analysis of galanin and CGRP synapses in the dorsal horn of neuropathic primates

The present study investigates the nature of the galanin (GAL) increase and the calcitonin gene-related peptide (CGRP) decrease in the dorsal horn following peripheral nerve injury. These two peptides are known to colocalize in primary afferent terminals. Primates which had a tight ligation of the L7 spinal nerve demonstrated a variety of neuropathic symptoms 2 weeks postsurgery, including mechanical and cold allodynia, and heat hyperalgesia. Computer-enhanced image analyses of L7 spinal cord sections demonstrated an increase in GAL immunostaining and a decrease in CGRP immunostaining in the experimental compared to the control dorsal horn. Stereological analyses demonstrated that neither the numbers of GAL-labeled synapses nor the numbers or diameters of the dense-core vesicles in each GAL terminal changed after the lesion. However, there was a significant increase in the number of GAL-labeled glial cell bodies and processes on the experimental side, which accounted for the increased staining density observed at the light microscopic level. In contrast, the number of CGRP-labeled terminals was decreased on the experimental side, accounting for the decreased staining density seen at the light level. Thus, the decrease in number of CGRP synapses combined with the stable number of GAL synapses suggests that many GAL terminals no longer colocalize with CGRP after peripheral nerve lesion. This may indicate increased antinociceptive activity after nerve lesions. If so, there is less of a morphologic and more of a functional and chemical plasticity for GAL than may be presently envisioned. The possible role of GAL in neuropathic pain is discussed.

Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.

Chemical codes of sensory neurons innervating the guinea-pig adrenal gland

Retrograde neuronal tracing in combination with double-labelling immunofluorescence was applied to distinguish the chemical coding of guinea-pig primary sensory neurons projecting to the adrenal medulla and cortex. Seven subpopulations of retrogradely traced neurons were identified in thoracic spinal ganglia T1-L1. Five subpopulations contained immunolabelling either for calcitonin gene-related peptide (CGRP) alone (I), or for CGRP, together with substance P (II), substance P/dynorphin (III), substance P/cholecystokinin (IV), and substance P/nitric oxide synthase (V), respectively. Two additional subpopulations of retrogradely traced neurons were distinct from these groups: neurofilament-immunoreactive neurons (VI), and cell bodies that were nonreactive to either of the antisera applied (VII). Nerve fibers in the adrenal medulla and cortex were equipped with the mediator combinations I, II, IV and VI. An additional meshwork of fibres solely labelled for nitric oxide synthase was visible in the medulla. Medullary as well as cortical fibres along endocrine tissue apparently lacked the chemical code V, while in the external cortex some fibre exhibited code III. Some intramedullary neuronal cell bodies revealed immunostaining for nitric oxide synthase, CGRP or substance P, providing an additional intrinsic adrenal innervation. Perikarya, immunolabelled for nitric oxide synthase, however, were too few to match with the large number of intramedullary nitric oxide synthase-immunoreactive fibres. A non-sensory participation is also supposed for the particularly dense intramedullary network of solely neurofilament-immunoreactive nerve fibres. The findings give evidence for a differential sensory innervation of the guinea-pig adrenal cortex and medulla. Specific sensory neuron subpopulations suggest that nervous control of adrenal functions is more complex than hitherto believed.

Effect of subarachnoid haemorrhage on trigeminovascular calcitonin-gene-related peptide and substance P of the rat dura mater versus cerebral vasculature.

While the presence of a robust perivascular neural network accompanying cerebral and dural blood vessels that contain various neuropeptides is well documented, the functional significance of this innervation is unclear. Following experimentally induced subarachnoid haemorrhage (SAH) in animal models, immunocytochemical studies have revealed that changes occur in the staining intensity of some of these neuropeptides. This study compared the immunostaining intensity of calcitonin-gene-related peptide (CGRP) and substance P (SP) in cerebral and dural perivascular nerve fibers after SAH in the rat. Subarachnoid haemorrhage was produced by injecting 0.3 ml of autologous blood into the cisterna magna of male Sprague Dawley rats. Sham operated animals received an equal volume of buffered lactated Ringer's solution (pH 7.4). Changes in the immunostaining intensity of cerebral and dural vessels were evaluated by independent observers at 6, 24, and 48 hours after SAH. Immunostaining of CGRP was reduced in cerebral vessels at 6 hours and returned to normal by 48 hours. In contrast, CGRP immunostaining of dural perivascular nerve fibers was unchanged at all time periods examined. A marked decrease in SP immunostaining was documented at 6 hours in both the cerebral and dural vessels in all animals; at 48 hours, the staining intensity had returned to control levels. These results support the idea that several subpopulations of trigeminovascular neurons containing CGRP, SP, or both project to cerebral and dural vessels. Since these subpopulations may be differentially activated in pathologic conditions, such as SAH or vascular headache, the potential exists for pharmacologic intervention of specific neuropeptides with the resultant abatement of a pathologic process.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in motoneurons of the human spinal cord following injury.

Changes that occur in the localization of calcitonin gene-related peptide-like-immunoreactivity (CGRP-LI) in motoneurons, following injury to the human spinal cord, were examined. CGRP-LI above and below the level of injury was compared to normal human spinal cord. Vibratome sections were cut and processed for immunostaining using the avidin-biotin immunoperoxidase method. Whereas motoneurons above the lesion contained CGRP-LI, below the lesion the CGRP-LI was reduced. In 3 spinal cords from patients who had survived between 1 and 8 years after injury, CGRP-LI in motoneurons of lumbar spinal cord was absent or considerably diminished. In the spinal cord from one patient who had died several years after injury, there was a unilateral augmentation of CGRP immunostaining. The results suggest that CGRP levels in motoneurons are affected by spinal cord injury in humans and that CGRP levels in motoneurons may be regulated either by supraspinal afferent inputs or by muscle activity.

Dural neuropeptide changes after subarachnoid hemorrhage in rats

The effect of subarachnoid hemorrhage (SAH) on the neuropeptides and mast cells of the rat dura mater has not been reported. We examined the outcome of SAH on the rat supratentorial dura mater to determine whether durai nerves undergo effects similar to those of nerves accompanying cerebral blood vessels after SAH. Following the injection of fresh autologous arterial blood into the cisterna magna, animals were sacrificed at 6, 24, and 48 h, and 6 days post-SAH. Dural whole mounts were immunohistochemically reacted with antibodies to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and serotonin (5-HT). SP-like immunostaining was substantially reduced after SAH and subsequently returned to control levels at 6 days. NPY-like fiber innervation of the dura was markedly reduced after SAH; although immunostaining intensity increased, it had not returned to control levels at 6 days. The 5-HT content of dural mast cells identified by immunostaining markedly decreased at 6 and 24 h and returned to control levels at 48 h. In contrast, CGRP immunostaining was unchanged in all experimental groups. One possible explanation for this differential response is that sub-populations of trigeminovascular neurons containing SP, CGRP, or CGRP and SP respond differently to various stimuli, including SAH. Another possibility is a differential release of SP or CGRP from the same fiber. To the best of our knowledge this is the first documentation that the dura is also a target for intracranial pathological processes, such as SAH. Because the dura mater contains a well-represented neuropeptidergic innervation similar to the perivascular innervation of the major vessels at the base of the brain, the dura may serve as a model to understand central neural responses following pure intracranial pathological events.

CGRP-immunoreactive primary afferent nerve fibers in the rat urinary bladder: Effects of dorsal rhizotomy and MK-801

A transection lesion of the suprasacral spinal cord results in a decreased density of calcitonin gene-related peptide (CGRP)-immunoreactive (I) primary afferent nerve fibers in the rat urinary bladder. The fiber density can be restored by postsurgical treatment with the N-methyl- d-aspartate receptor antagonist MK-801. We are attempting to determine the level of the primary afferent neuron at which MK-801 might have a restorative effect on CGRP immunostaining. Thus, the purpose of this study was to determine if MK-801 had a similar restorative effect on immunostaining for CGRP in bladder nerves after a direct lesion of the sacral afferent system, i.e., rhizotomy of the L 6 and S 1 dorsal roots. To assess the effect of the lesion, the mean length and number of bladder CGRP-I nerve fibers, as well as the number of CGRP-I perikarya in the L 6 and S 1 dorsal root ganglia (DRG), were measured following bilateral L 6 and S 1 dorsal rhizotomies. Both the mean length and the numbers of CGRP-I bladder fibers were significantly decreased by the lesion. However, the number of CGRP-I primary afferent perikarya in the L 6 and S 1 DRG was unchanged from control values. Rats which received rhizotomies and subsequent treatment with MK-801 did not exhibit restoration of the density of CGRP-I bladder fibers nor an alteration in the number of CGRP-I primary afferent perikarya. These data suggest that MK-801-induced restoration of bladder CGRP-I primary afferent nerve fibers may rely on an intact central process.

CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis

Calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerve fibers are abundant in the rat penis. In addition, NADPH-diaphorase, which stains for nitric oxide synthase, has been localized within both autonomic and sensory dorsal root ganglia (DRG) and may be part of an important biochemical pathway involved in penile tumescence. The purpose of this study was: 1) to examine the circuitry of afferent nerves that are CGRP immunoreactive from the L6 DRG, 2) to examine the possibility that there are NADPH-diaphorase-positive afferent fibers from the L6 DRG to the rat penis, and 3) to examine the localization and colocalization of CGRP and NADPH-diaphorase within L6 DRG afferent perikarya. Calcitonin gene-related peptide immunostaining in the penis was eliminated following a bilateral transection of the pudendal nerves, but was unchanged following a bilateral transection of the pelvic splanchnic or hypogastric nerves. The NADPH-diaphorase staining was not altered by any of the nerve transections. Injection of the retrograde axonal tracer fluorogold (FG) into the dorsum penis labeled perikarya in the L6 DRG. Although the majority of FG-labeled perikarya contained neither CGRP nor NADPH-diaphorase, small subpopulations of perikarya contained either CGRP immunoreactivity, NADPH-diaphorase, or both. A unilateral pudendal nerve transection virtually eliminated (>99%) FG labeling in the ipsilateral L6 DRG. These data suggest that NADPH-diaphorase and CGRP are present, either together or separately, within a subpopulation of penile afferent perikarya. In addition, CGRP-immunoreactive afferent nerve fibers reach the penis primarily via the pudendal nerves. Finally, NADPH-diaphorase-positive penile afferents may be another important source of nitric oxide (NO) for penile tumescence.

Effects of spinal cord transection and MK-801 on CGRP immunostaining in the rat urinary bladder

Numerous studies have demonstrated that lesions in the CNS can alter the density of sensory nerve processes in peripheral organs. In the present study, rat spinal cords were transected at the second lumbar segmental level and the density of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the urinary bladder was examined. Additional rats had spinal cord transections followed by 12 days of treatment with the N-methyl- d-aspartate receptor antagonist, MK-801. In the bladders of control rats, CGRP-immunoreactive fibers were present as thick nerve trunks, perivascular plexi, and a fine meshwork of varicose nerve fibers. Twelve days following a spinal cord transection, the density of CGRP-immunoreactive nerve fibers was markedly reduced; occasional fibers appeared primarily as nonvaricose fine fibers. In bladders from rats receiving a spinal cord transection and MK-801 treatment, CGRP-immunoreactive fibers were abundantly distributed throughout the detrusor muscle; these fibers exhibited numerous varicosities as well as some enlarged terminal varicosities. These data demonstrate that (i) an upper motor neuron-type lesion markedly decreases the density of CGRP-immunoreactive peripheral afferent nerve processes and (ii) following a spinal cord transection, MK-801 appears to enhance the density of CGRP immunostaining in the bladder.