Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BACKGROUND: The presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies - a question raised frequently due to their large size. MATERIAL AND METHODS: Rats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594. RESULTS: In rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections. DISCUSSION: Our inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

Evaluation of effect of Acacia jacquemontii Benth. on blood pressure in normotensive and fructose induced hypertensive sprague dawley rats: An ethnopharmacological approach.

Acacia jacquemontii Benth. is used traditionally to treat hypertension but no scientific literature supports this claim. So, this study was aimed at validating this claim. This was done by injecting various doses of crude extract of Acacia jacquemontii, AJC (5, 10, 20, 30mg/kg) and all fractions (hexane, ethyl acetate, n-butanol and aqueous) (3, 5, 10, 20mg/kg) intravenously in anaesthetized rat. Based on the results, butanol fraction (AJB) at 20mg/kg was found to be the most potent, so it was selected for exploring mechanisms of action. For this purpose, different groups were injected with various pharmacological inhibitors (L-NAME, atropine, captopril, propranolol and hexamethonium) prior to AJB administration. Also, AJB at 20mg/kg was evaluated for prolonged hypotensive effect for the period of 40 min. Results showed a significant dose dependent reduction in BP in normotensive and in hypertensive rats. AJC and AJB produced a decline in SBP, DBP and MAP with p<0.05 - p<0.001 and p<0.001 respectively in normotensive animals. Whereas in hypertensive animals, AJC showed significant reduction at 5mg/kg with p<0.01 and at 10, 20 and 30 mg/kg with p<0.001. AJB produced a decline in hypertensive animals at all tested doses with p<0.001. AJB resulted in hypotensive effect mediated by ß receptors, ganglionic block operating central sympathetic neural responses and renin angiotensin aldosterone system (RAAS). This study supports the ethnomedicinal claim of Acacia jacquemontii Benth. in treating hypertension.

New basic insights on the potential of a chitosan-based medical device for improving functional recovery after radical prostatectomy.

OBJECTIVES: To evaluate: (i) the neuro-regenerative potential of chitosan membrane (CS-Me) on acutely axotomised autonomic neurones in vitro; (ii) to exclude the possibility that a pro-regenerative biomaterial could interfere with the proliferation activity of prostate cancer cell lines; (iii) to provide an in vivo proof of the biocompatibility and regeneration promoting effect of CS-Me in a standardised rat model of peripheral nerve injury and repair; (iv) finally, to evaluate the tissue reaction induced by the degrading material; as previous studies have shown promising effects of CS-Me for protection of the neurovascular bundles for potency recovery in patients that undergo nerve-sparing radical prostatectomy (RP). MATERIALS AND METHODS: Addressing aim (i), the neuro-regenerative potential, organotypic cultures derived from primary sympathetic ganglia were cultured on CS-Me over 3 days and neurite extension and axonal sprouting were evaluated. Addressing aim (ii), effects of CS on cancer cells, different human prostate cancer cell lines (PC3, DU-145, LN-Cap) were seeded on CS-coated plates or cultured in the presence of CS-Me dissolution products. Addressing aims (iii) and (iv), functional recovery of peripheral nerve fibres and tissue reaction with the biomaterial, CS-Me and CS nerve guides were used to repair a median nerve injury in the rat. Functional recovery was evaluated during the post-recovery time by the behavioural grasping test. RESULTS: CS-Me significantly stimulated axon elongation from autonomic ganglia in comparison to control conditions in organotypic three-dimensional cultures. CS coating, as well as the dissolution products of CS-Me, led to a significantly lower proliferation rate of prostate cancer cell lines in vitro. Tissue reaction towards CS-Me and standard CS nerve guides was similar in the rat median nerve model, as was the outcome of nerve fibre regeneration and functional recovery. CONCLUSION: The results of this study provide the first experimental evidence in support of the clinical safety of CS-Me and of their postulated effectiveness for improving functional recovery after RP. The presented results are coherent in demonstrating that acutely axotomised autonomic neurones show increased neurite outgrowth on CS-Me substrate, whilst the same substrate reduces prostate cancer cell line proliferation in vitro. Furthermore, CS-Me do not demonstrate any disadvantage for peripheral nerve repair in a standard animal model.

Plastics and cardiovascular health: phthalates may disrupt heart rate variability and cardiovascular reactivity.

Plastics have revolutionized medical device technology, transformed hematological care, and facilitated modern cardiology procedures. Despite these advances, studies have shown that phthalate chemicals migrate out of plastic products and that these chemicals are bioactive. Recent epidemiological and research studies have suggested that phthalate exposure adversely affects cardiovascular function. Our objective was to assess the safety and biocompatibility of phthalate chemicals and resolve the impact on cardiovascular and autonomic physiology. Adult mice were implanted with radiofrequency transmitters to monitor heart rate variability, blood pressure, and autonomic regulation in response to di-2-ethylhexyl-phthalate (DEHP) exposure. DEHP-treated animals displayed a decrease in heart rate variability (-17% SD of normal beat-to-beat intervals and -36% high-frequency power) and an exaggerated mean arterial pressure response to ganglionic blockade (31.5% via chlorisondamine). In response to a conditioned stressor, DEHP-treated animals displayed enhanced cardiovascular reactivity (-56% SD major axis Poincarè plot) and prolonged blood pressure recovery. Alterations in cardiac gene expression of endothelin-1, angiotensin-converting enzyme, and nitric oxide synthase may partly explain these cardiovascular alterations. This is the first study to show an association between phthalate chemicals that are used in medical devices with alterations in autonomic regulation, heart rate variability, and cardiovascular reactivity. Because changes in autonomic balance often precede clinical manifestations of hypertension, atherosclerosis, and conduction abnormalities, future studies are warranted to assess the downstream impact of plastic chemical exposure on end-organ function in sensitive patient populations. This study also highlights the importance of adopting safer biomaterials, chemicals, and/or surface coatings for use in medical devices.NEW & NOTEWORTHY Phthalates are widely used in the manufacturing of consumer and medical products. In the present study, di-2-ethylhexyl-phthalate exposure was associated with alterations in heart rate variability and cardiovascular reactivity. This highlights the importance of investigating the impact of phthalates on health and identifying suitable alternatives for medical device manufacturing.

Sildenafil promotes neuroprotection of the pelvic ganglia neurones after bilateral cavernosal nerve resection in the rat.

OBJECTIVE: To determine the gene expression profile of pelvic ganglia neurones after bilateral cavernosal nerve resection (BCNR) and subsequent treatment with sildenafil in relation to neurotrophic-related pathways. MATERIALS AND METHODS: Fisher rats aged 5 months were subjected to BCNR or sham operation and treated with or without sildenafil (20 mg/kg body-weight in drinking water) for 7 days. Total RNA isolated from pelvic ganglia was subjected to reverse transcription and then to quantitative reverse transcriptase-polymerase chain reaction (PCR) with the RAT-neurotrophic array. Results were corroborated by real-time PCR and western blotting. Another set of animals were injected with a fluorescent tracer at the base of the penis, 7 days before BCNR or sham operation, and were sacrificed 7 days after surgery. Sections of pelvic ganglia were used for immunohistochemistry with antibodies against neurturin, neuronal nitric oxide synthase, tyrosine hydroxylase and glial cell line-derived neurotrophic factor receptor α2. RESULTS: A down-regulation of the expression of neuronal nitric oxide synthase accompanied by changes in the level of cholinergic neurotrophic factors, such as neurturin and its receptor glial cell line-derived neurotrophic factor receptor α2, artemin, neurotrophin-4 and cilliary neurotrophic factor, was observed 7 days after BCNR in pelvic ganglia neurones. Treatment with sildenafil, starting immediately after surgery, reversed all these changes at a level similar to that in sham-operated animals. CONCLUSIONS: Sildenafil treatment promotes changes in the neurotrophic phenotype, leading to a regenerative state of pelvic ganglia neurones. The present study provides a justification for the use of phosphodiesterase 5 inhibitors as a neuroprotective agent after BCNR.

Sonic hedgehog is neuroprotective in the cavernous nerve with crush injury.

INTRODUCTION: The cavernous nerve (CN) is commonly injured during prostatectomy, resulting in erectile dysfunction (ED). Although peripheral nerves have a limited ability to regenerate, a return of function typically does not occur due to irreversible down stream morphological changes in the penis that result from CN injury. We have shown in previous studies that sonic hedgehog (SHH) is critical for CN regeneration and improves erectile function after crush injury. AIMS: Examine a new direction, to determine if SHH is neuroprotective to the pelvic ganglia (PG)/CN after crush injury. A secondary focus is to examine if SHH signaling decreases with age in the PG/CN. METHODS: Sprague-Dawley rats underwent bilateral CN crush and SHH and glial fibrillary acidic protein were quantified by western analysis of the PG/CN (N = 6 rats at each time point) at 1, 2, 4, 7, and 14 days, and the apoptotic index was measured in the penis. SHH was quantified by western in the PG/CN with blockade of anterograde transport (N = 4 rats) in comparison to mouse IgG (N = 4 rats). If SHH is neuroprotective was examined at 4 (N = 14 rats) and 7 days (N = 16 rats) of treatment after CN crush. SHH protein was quantified in aging (P200-300, N = 5 rats) PG/CN in comparison to normal adult (P115-120, N = 3 rats) PG/CN. Main Outcome Measures. SHH pathway was examined in PG via immunohistochemistry, in situ, western, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS: SHH is neuroprotective in the PG/CN with injury. SHH localization in the PG/CN suggests SHH interaction in neuronal/glial signaling. SHH protein is significantly decreased in the PG/CN after crush injury and in the aged PG/CN. Signals from the PG are required to maintain SHH in the CN. CONCLUSIONS: There is a window of opportunity immediately after nerve insult in which manipulation of SHH signaling in the nerve microenvironment can affect long-term regeneration outcome.

Ganglion impar blockade: a review.

Since its original description in 1990, blockade of the ganglion impar (ganglion of Walther) has become a widely used intervention in the management of pelvic and perineal pain. We review the indications, techniques, and evidence for this pain intervention.

Neuromodulation targets intrinsic cardiac neurons to attenuate neuronally mediated atrial arrhythmias.

Our objective was to determine whether atrial fibrillation (AF) results from excessive activation of intrinsic cardiac neurons (ICNs) and, if so, whether select subpopulations of neurons therein represent therapeutic targets for suppression of this arrhythmogenic potential. Trains of five electrical stimuli (0.3-1.2 mA, 1 ms) were delivered during the atrial refractory period to mediastinal nerves (MSN) on the superior vena cava to evoke AF. Neuroanatomical studies were performed by injecting the neuronal tracer DiI into MSN sites that induced AF. Functional studies involved recording of neuronal activity in situ from the right atrial ganglionated plexus (RAGP) in response to MSN stimulation (MSNS) prior to and following neuromodulation involving either preemptive spinal cord stimulation (SCS; T(1)-T(3), 50 Hz, 200-ms duration) or ganglionic blockade (hexamethonium, 5 mg/kg). The tetramethylindocarbocyanine perchlorate (DiI) neuronal tracer labeled a subset (13.2%) of RAGP neurons, which also colocalized with cholinergic or adrenergic markers. A subset of DiI-labeled RAGP neurons were noncholinergic/nonadrenergic. MSNS evoked an ∼4-fold increase in RAGP neuronal activity from baseline, which SCS reduced by 43%. Hexamethonium blocked MSNS-evoked increases in neuronal activity. MSNS evoked AF in 78% of right-sided MSN sites, which SCS reduced to 33% and hexamethonium reduced to 7%. MSNS-induced bradycardia was maintained with SCS but was mitigated by hexamethonium. We conclude that MSNS activates subpopulations of intrinsic cardiac neurons, thereby resulting in the formation of atrial arrhythmias leading to atrial fibrillation. Stabilization of ICN local circuit neurons by SCS or the local circuit and autonomic efferent neurons with hexamethonium reduces the arrhythmogenic potential.

Antiarrhythmic effects of vasostatin-1 in a canine model of atrial fibrillation.

BACKGROUND: We examined the antiarrhythmic effects of vasostatin-1, a recently identified cardioregulatory peptide, in canine models of atrial fibrillation (AF). METHODS AND RESULTS: In 13 pentobarbital-anesthetized dogs bilateral thoracotomies allowed the attachment of multielectrode catheters to superior and inferior pulmonary veins and atrial appendages (AA). Rapid atrial pacing (RAP) was maintained for 6 hours. Each hour, programmed stimulation was performed to determine the window of vulnerability (WOV), a measure of AF inducibility, at all sites. During the last 3 hours, vasostatin-1, 33 nM, was injected into the anterior right (AR) ganglionated plexus (GP) and inferior right (IR) GP every 30 minutes (n = 6). Seven dogs underwent 6 hours of RAP only (controls). At baseline, acetylcholine, 100 mM, was applied on the right AA and AF duration was recorded before and after injection of vasostatin-1, 33 nM, into the ARGP and IRGP. In separate experiments (n = 8), voltage-sinus rate response curves (surrogate for GP function) were constructed by applying high-frequency stimulation to the ARGP with incremental voltages with or without vasostatin-1. Vasostatin-1 significantly decreased the duration of acetylcholine-induced AF (11.0 ± 4.1 vs 5.5 ± 2.6 min, P = 0.02). The cumulative WOV (the sum of individual WOVs) significantly increased (P < 0.0001) during the first 3 hours and decreased toward baseline in the presence of vasostatin-1 (P < 0.0001). Cumulative WOV in controls steadily increased. Vasostatin-1 blunted the slowing of sinus rate with increasing stimulation voltage of ARGP. CONCLUSIONS: Vasostatin-1 suppresses AF inducibility, likely by inhibiting GP function. These data may provide new insights into the role of peptide neuromodulators for AF therapy.

Autonomic denervation with magnetic nanoparticles.

BACKGROUND: prior studies indicated that ablation of the 4 major atrial ganglionated plexi (GP) suppressed atrial fibrillation. METHODS AND RESULTS: superparamagnetic nanoparticles (MNPs) made of Fe(3)O(4) (core), thermoresponsive polymeric hydrogel (shell), and neurotoxic agent (N-isopropylacrylamide monomer [NIPA-M]) were synthesized. In 23 dogs, a right thoracotomy exposed the anterior right GP (ARGP) and inferior right GP (IRGP). The sinus rate and ventricular rate slowing responses to high-frequency stimulation (20 Hz, 0.1 ms) were used as the surrogate for the ARGP and IRGP functions, respectively. In 6 dogs, MNPs carrying 0.4 mg NIPA-M were injected into the ARGP. In 4 other dogs, a cylindrical magnet (2600 G) was placed epicardially on the IRGP. MNPs carrying 0.8 mg NIPA-M were then infused into the circumflex artery supplying the IRGP. The hydrogel shell reliably contracted in vitro at temperatures ≥ 37°C, releasing NIPA-M. MNPs injected into the ARGP suppressed high-frequency stimulation-induced sinus rate slowing response (40 ± 8% at baseline; 21 ± 9% at 2 hours; P=0.006). The lowest voltage of ARGP high-frequency stimulation inducing atrial fibrillation was increased from 5.9 ± 0.8 V (baseline) to 10.2 ± 0.9 V (2 hours; P=0.009). Intracoronary infusion of MNPs suppressed the IRGP but not ARGP function (ventricular rate slowing: 57 ± 8% at baseline, 20 ± 8% at 2 hours; P=0.002; sinus rate slowing: 31 ± 7% at baseline, 33 ± 8 % at 2 hours; P=0.604). Prussian Blue staining revealed MNP aggregates only in the IRGP, not the ARGP. CONCLUSIONS: intravascularly administered MNPs carrying NIPA-M can be magnetically targeted to the IRGP and reduce GP activity presumably by the subsequent release of NIPA-M. This novel targeted drug delivery system can be used intravascularly for targeted autonomic denervation.

Tetrodotoxin- and resiniferatoxin-induced changes in paracervical ganglion ChAT- and nNOS-IR neurons supplying the urinary bladder in female pigs.

The aim of the present study was to establish the effect of intravesical administration of resiniferatoxin (RTX) and tetrodotoxin (TTX) on the chemical coding of paracervical ganglion (PCG) neurons supplying the urinary bladder in the pig. In order to identify the PCG neurons innervating the bladder, retrograde tracer Fast Blue was injected into the bladder wall prior to intravesical RTX or TTX administration. Consequent application of immunocytochemical methods revealed that in the control group 76.82% of Fast Blue positive PCG neurons contain nitric oxide synthetase (nNOS), and 66.92% contain acetylcholine transferase (ChAT). Intravesical infusion of RTX resulted in a reduction of the nNOS-IR neurons to 57.74% and ChAT-IR to 57.05%. Alternative administration of TTX induced an increase of nNOS-IR neurons up to 79.29% and a reduction of the ChAT-IR population down to 3.73% of the Fast Blue positive PCG cells. Our data show that both neurotoxins affect the chemical coding of PCG cells supplying the porcine urinary bladder, but the effects of their action are different. Moreover, these results shed light on the possible involvement of NO-ergic and cholinergic neurons in the mechanisms of therapeutic action exerted by RTX and TTX in curing the overactive bladder disorder.

Administration of noradrenaline in the autonomic ganglia modifies the testosterone release from the testis using an ex vivo system.

The male gonad receives nerve fibres from the autonomic ganglionic system. These fibres converge on the testis along two pathways, the superior and the inferior spermatic nerves. The superior spermatic nerve runs from the superior mesenteric ganglion alongside the testicular artery, whereas the inferior spermatic nerve originates in inferior mesenteric ganglion, accompanies the vas deferens and penetrates the inferior pole of the testis. The aim of this work was to evaluate androgen release after the addition of noradrenaline or adrenoreceptor antagonists (propranolol or phentolamine) to the ganglionic compartment. An ex vivo system used in a previous work was incubated in two separate containers, one for the testis and the other for the ganglion. Both organs remain interconnected (as in vivo) by the respective spermatic nerve. When noradrenaline was added to the inferior mesenteric ganglion, testosterone release in the gonad container underwent a progressive and significant increment. Propranolol diminishes and phentolamine increases the androgen release. When using the superior mesenteric ganglion, no changes were observed. These results indicate that the ganglionic stimulation of the autonomic system clearly participates in testosterone release from the testis. This effect depends on the ganglion involved. These results make it evident that not only the classical and well-known hypothalamus-hypophysial axis, but also the peripheral nervous system, via the autonomic ganglia, are directly involved in the endocrine control of the testis.

Deuterium oxide: direct action on sympathetic ganglia isolated in culture.

Immature ganglia from chicks and rodents were maintained as organized, developing cultures for 2 months or more, during which time they were continuously exposed to deuterium oxide in their medium. Observations of the living cell communities with the light microscope indicated that deuteration within viable limits (up to 25 percent) accelerates and increases the growth of sympathetic neurons and favors their repeated subdivision as a very large size is attained, thus inducing them to recapitulate cyclically the early stages of neurogenesis. Living deuterated cells appear more opaque and heteromerous than control neurons; furthermore, electron micrographs reveal an unusual abundance of granular and fibrillar elements in the nuclei of both neurons and supporting cells. Sheaves of complexly organized fibrillar components appear in the neuronal perikaryon; and ribosomes, Golgi elements, and microtubules are conspicuously numerous. Both fine structure and function of these ganglia therefore appear to have been modified directly by action of the deuterium isotope.

Generation of adrenergic and cholinergic potentials in sympathetic ganglion cells.

Norepinephrine elicited a hyperpolarizing response, and acetylcholine (during nicotinic blockade) elicited a depolarizing one. Both responses showed no increase in membrane conductance. The norepinephrine response was suppressed by initial depolarization; the acetylcholine response (frog cells); by hyperpolarization. These neurotransmitters apparently can activate electrogenic mechanisms which do not involve movement of ions down their electrochemical gradients.

Neurotransmitters increase cyclic nucleotides in postganglionic neurons: immunocytochemical demonstration.

Dopamine increases adenosine 3',5'-monophosphate (cyclic AMP) but not guanosine 3',5'-monophosphate (cyclic GMP) in slices of bovine sympathetic ganglion; this increase is localized to the postganglionic neurons. Conversely, acetylcholine increases cyclic GMP but not cycle AMP in the ganglion; this increase also occurs within postganglionic neurons. Thus, different neurotransmitters can selectively alter cyclic nucleotide levels within the same neuronal population.

6-Hydroxydopamine potentiates acute herpes simplex virus infection of the superior cervical ganglion in mice.

Treatment of mice with 6-hydroxydopamine increased herpes simplex virus replication in the superior cervical ganglion while it decreased the subsequent prevalence of latent infection. Preganglionic neurectomy failed to block this effect. These observations suggest that intrinsic neural events modify the outcome of viral infections of the nervous system.

Cyclic nucleotides injected intracellularly into rat superior cervical ganglion cells.

Intracellular iontophoresis of either adenosine 3',5'-monophosphate or guanosine 3',5'-monophosphate produces a membrane depolarization and an increased membrane conductance in sympathetic ganglion cells of the rat superior cervical ganglion. Since adenosine 3',5'-monophosphate did not cause a membrane hyperpolarization, it is difficult to assign it a second messenger role in the mediation of the slow inhibitory postsynaptic potential. However, these results do not rule out the possibility that the cyclic nucleotides, at the intracellular concentrations attained in these experiments, participate in cellular processes that contribute to conductance changes which result in depolarization of the ganglion cell membrane.

Parasympathetic ganglia: activation of an adrenergic inhibitory mechanism by cholinomimetic agents.

Electrical stimulation of the sympathetic nerves to the urinary bladder or the intraarterial administration of the cholinomimetic substances acetylcholine or methacholine produced adrenergic inhibition in parasympathetic ganglia on the surface of the bladder. The inhibition appeared to be mediated, at least in part, via adrenergic inhibitory neurons located in the pelvic plexus. Atropine blocked the inhibitory response to injected cholinomimetic agents but did not alter the response to stimulation of the sympathetic nerves. Thus, the inhibitory neurons can be activated via both muscarinic and nonmuscarinic receptors, the latter being of primary physiological importance.

Batrachotoxin: chemistry and pharmacology.

Batrachotoxin has been shown to be a pyrrolecarboxylic ester of a novel steroidal base with unique and selective actions on a variety of electrogenic membranes. The effects of batrachotoxin in neuromuscular preparations both pre- and postsynaptically, in nerve axons, in superior cervical ganglion, in heart Purkinje fibers, and in brain slices appear to be due to the selective and irreversible increase in permeability of membranes to sodium ions. The subsequent effects of this increase in Na(+) permeability evoked by batrachotoxin-such as membrane depolarization, enhanced spontaneous transmitter release, muscle contracture, and enhanced formation of cyclic AMP in brain slices-may be blocked reversibly by tetrodotoxin.

Norepinephrine inhibits calcium-dependent potentials in rat sympathetic neurons.

Norepinephrine reversibly antagonizes three calcium-dependent potentials recorded from rat postganglionic neurons. Norepinephrine inhibits the development of a shoulder on the aciton potential, the magnitude of the hyperpolarizing afterpotential, and the rate of rise and amplitude of the calcium spike. The action of norepinephrine is antagonized by the alpha-adrenergic antagonist phentolamine, but not by MJ 1999, a beta-adrenergic antagonist. These results suggest that activation of an alpha-adrenergic receptor may antagonize a voltage-sensitive calcium current.