Degeneration Of Adrenergic Nerves Research Articles

Dependence on Schwann Cell-Derived Laminin-γ1 Differentiates Early Lymphoid and Myeloid Development

Blood cell production is regulated by peripheral nerve fibers that innervate the bone marrow. However, little is known about the development or maintenance of hematopoietic innervation. Schwann cells (SCs) are the primary axon 'support cells' of the peripheral nervous system (PNS), and abnormal SC development is sufficient to impair peripheral nerve function. SCs are also the primary repair cell for the PNS which makes them an attractive therapeutic target for normalization of drug or malignancy-induced 'hematopoietic neuropathy'. We hypothesized that neural regulation of hematopoiesis is dependent on SC development. To test this hypothesis, we used the Myelin Protein Zero-Cre (MP0-Cre); Lamc1fl/fl mouse line in which laminin-γ1 expression is deleted from SC precursors and their progeny1. Early SC maturation is dependent on autocrine SC precursor-derived molecules such as laminin-γ1. SC differentiation arrests prior to axon sorting and ensheathment in MP0-Cre; Lamc1fl/fl mice, and causes a global peripheral neuropathy that persists throughout the lifetime of the animal. Preliminary hematopoietic analysis of 'steady state' MP0-Cre; Lamc1fl/fl and littermate control mice has shown the following: (1) MP0-Cre; Lamc1fl/fl bone marrow is innervated, and Cre-mediated gene recombination occurs in cells immunophenotypically consistent with SCs throughout the peripheral nervous system, including those in the bone marrow; (2) MP0-Cre; Lamc1fl/fl mice are lymphopenic but not neutropenic; (3) MP0-Cre; Lamc1fl/fl mice have significantly reduced spleen size and cellularity; and (4) MP0-Cre; Lamc1fl/fl bone marrow has an ~50% reduction in Lin-Sca-1+Kit+(LSK) cells (measured as a percentage of the Lin- compartment of the bone marrow). These results are consistent with earlier work by our groups in which we found that global Lamc1 gene deletion in adult mice induced peripheral blood lymphopenia, reduced spleen size, and a niche-dependent reduction of lymphoid progenitor and precursor cells that was secondary to increased lymphoid precursor cell apoptosis and reduced proliferation (UBC-CreERT2; Lamc1fl/fl mouse line). As with the SC-specific laminin-γ1 deficient mice, myelopoiesis was preserved in the UBC-CreERT2; Lamc1fl/fl mice. Based on results from MP0-Cre; Lamc1fl/fl and UBC-CreERT2; Lamc1fl/fl mice, we conclude that early lymphoid but not myeloid development requires laminin-γ1 expression by MP0-Cre-targetted niche cells, i.e. Schwann Cells. Our results are consistent with reports from other labs that hematopoietic sympathetic neuropathy promotes aberrant myeloid expansion at the expense of lymphopoiesis2. Going forward, we will determine whether lymphopoietic development is dependent on global versus laminin-specific SC-derived cues, and whether these signals are transmitted directly between SCs and lymphoid biased HSPCs or indirectly via other components of the hematopoietic niche. We anticipate that this line of investigation will provide molecular insights and pharmacologic targets for prevention and or normalization of the 'hematopoietic neuropathy' induced by diabetes, aging, neurotoxic chemotherapies and myeloid malignancies. REFERENCES: 1 Yu, W. M., Feltri, M. L., Wrabetz, L., Strickland, S. & Chen, Z. L. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci25, 4463-4472, doi:10.1523/JNEUROSCI.5032-04.2005 (2005). 2 Maryanovich, M. et al. Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche. Nat Med24, 782-791, doi:10.1038/s41591-018-0030-x (2018). Disclosures No relevant conflicts of interest to declare.

3103 - Adrenergic Nerve Degeneration in the Bone Marrow Drives Aging of the Hematopoietic Stem Cell Niche

Aging of hematopoietic stem cells (HSCs) is associated with a decline in their regenerative capacity and multi-lineage differentiation potential, leading to the development of blood disorders. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, but the underlying mechanisms still remain largely unknown. HSCs reside in a specialized microenvironment in the BM (referred to as niche), which represents a critical regulatory unit essential to maintain healthy hematopoiesis. HSC niches have recently been identified as perivascular, where subsets of quiescent HSCs are closely associated with arteriolar perivascular Nestin-GFP+ mesenchymal stem cells (MSCs), adrenergic nerves and megakaryocytes, supported by a network of reticular Lepr+ Nestin-GFP+ stromal cells. The sympathetic nervous system (SNS) represents a major component of the HSC niche regulating the proliferative state of MSCs, HSC mobilization, and hematopoietic regenerative capacity. Here, we show that HSC aging critically depends on BM innervation by the SNS, in that aging revealed significant reductions in innervating adrenergic nerve fibers in the BM. The SNS neuropathy of aged mice was accompanied with niche remodeling characterized by an increase in total vascular density, loss of arteriolar structures and expansion of poorly functional MSCs. Importantly, premature loss of SNS nerves (following surgical denervation) or loss of adrenoreceptor 3 signaling (Adrb3-/- mice) in young mice led to accelerated HSC aging, marked with expansion of myeloid biased HSCs, lymphopenia and diminished repopulation capacity, phenotypes reminiscent of physiological aging. As HSCs dont express ADR3 receptors and dont respond to ADR3 signals, our data demonstrate that extrinsic factors within the niche regulate HSC longevity and that perturbation of SNS signaling drives niche and HSC aging. Strikingly, supplementation of adrenergic agonist, acting selectively on ADR3, to old mice significantly rejuvenated the in vivo function of aged HSCs, suggesting that the preservation or restitution of BM SNS innervation during aging may hold the potential for new HSC rejuvenation strategies.

Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

Aging of hematopoietic stem cells (HSCs) is associated with a decline in their regenerative capacity and multi-lineage differentiation potential, contributing to the development of blood disorders. The bone marrow microenvironment has recently been suggested to influence HSC aging, but the underlying mechanisms remain largely unknown. Here, we show that HSC aging critically depends on bone marrow innervation by the sympathetic nervous system (SNS), as loss of SNS nerves or adrenoreceptor β3 (ADRβ3) signaling in the bone marrow microenvironment of young mice led to premature HSC aging, as evidenced by appearance of HSC phenotypes reminiscent of physiological aging. Strikingly, supplementation of a sympathomimetic acting selectively on ADRβ3 to old mice significantly rejuvenated the in vivo function of aged HSCs, suggesting that the preservation or restitution of bone marrow SNS innervation during aging may hold the potential for new HSC rejuvenation strategies.

Degeneration of Adrenergic Nerves in the Urinary Bladder During Pregnancy

The adrenergic innervation of the urinary bladder of normal female and pregnant rats has been studied using a fluorescence histochemical method. The bladder is richly innervated by adrenergic nerve fibres as is evidenced by the presence of numerous adrenergic nerves in the adventitia, musculosa and submucosa. However, adrenergic nerve cells could not be observed. During pregnancy, adrenergic nerve fibres showed signs of degeneration, as most of the nerve fibres disappeared and the surviving fibres were much swollen. 10 days after parturition the pattern and density of adrenergic innervation became almost similar to those of the control animals.

Degeneration of Adrenergic Nerves in the Gail-Bladder of Cholecystitis Patients

The adrenergic innervation of the gall-bladder of healthy persons and patients suffering from cholecystitis has been studied following fluorescence histochemical methods. The normal gall-bladder is moderately innervated by adrenergic nerve fibres. Fine nerve fibres have been observed in the lamina propria, fibromuscularis and serosa. Catecholamine-containing fluorescent cells have been observed at different sites and their possible role as chemoreceptors and their endocrine nature have been reaffirmed. Adrenergic nerve fibres show signs of degeneration in cholecystitis as is evidenced by swelling of the nerve fibres and enhanced intensity of fluorescence in fluorescent cells.

Reduced uptake of noradrenaline into storage vesicles from the pregnant uterus

A noradrenaline (NA) storage vesicle fraction was isolated from the uteri of virgin as well as pregnant guinea-pigs. The uptake of 3H-NA into respective vesicle fractions was compared. Total uptake of 3H-NA in the presence of Mg2+ (2.5mM) and ATP (2.5 mM) at 30 degrees C after 20 min was 6.0 +/- 1.4 pmol . mg-1 in uterus fractions from virgin guinea-pigs as compared to 1.87 +/- 0.41 pmol . mg-1 in uterus fractions from pregnant animals. When desipramine (5mM) was added in order to block neuronal uptake the corresponding figures were 4.75 +/- 0.92 and 0.51 +/- 0.18 pmol . mg-1, respectively. Thus in the presence of desipramine (5mM) uptake was reduced by 21.4% in fractions from virgin uteri but by 72.7% in fractions from pregnant uteri. Reserpine (0.1 microM) inhibited uptake of 3H-NA by 34% in fractions from virgin uteri and by 46% in fractions from pregnant uteri. Our results indicate a decreased amount of functional storage vesicles in uteri of guinea-pigs at term pregnancy and are consistent with the proposed degeneration of adrenergic nerves during pregnancy.

Chlorhexidine-induced degeneration of adrenergic nerves.

Possible toxic effects of chlorhexidine (CHX) on the sympathetic adrenergic ground plexus were studied in whole mounts of albino rat irides using Falck-Hillarp fluorescence histochemistry. CHX dissolved in an isotone , buffered sodium-acetate solution or in 70% alcohol was injected into the anterior chamber of eye. CHX caused a marked and dose-dependent degeneration of adrenergic nerves. Two days after the lowest dose, 0,25 micrograms (5 microliters of a 0.05% CHX solution), approximately 30% of the nerves had disappeared. Almost complete degeneration was observed after the same time with higher doses (2.5 micrograms, 5.0 micrograms, and 7.5 micrograms corresponding to 0.5, 1.0, and 1.5% CHX respectively). Two weeks after the lowest dose, the nerves had regenerated almost completely. With the highest dose used, only some 40% of the normal adrenergic nerve plexus had reformed after 51 days. Alcohol as a solvent did not have an additive effect on the neurotoxic action caused by CHX. The results demonstrate yet another aspect of chlorhexidine neurotoxicity, degeneration of peripheral adrenergic nerve terminals. This suggests that neurotoxic actions on thin unmyelinated fiber systems should be looked for also in the central nervous system (CNS).

The adenylate cyclase activity in heart membranes from normotensive and spontaneously hypertensive rats, after chemical sympathectomy, suggests the presence of presynaptic secretin receptors

Normotensive (WKY) and spontaneously hypertensive (SHR) male adult rats were sacrified 2 and 3 weeks after 6-hydroxydopamine treatment. Untreated WKY and SHR rats served as controls. In rat heart membranes from WKY rats, 6-hydroxydopamine treatment increased guanosine 5′-O-(2–3-imido)-triphosphate (Gpp(NH)p)-, NaF-, D,L-isoproterenol- and glucagon-stimulated adenylate cyclase activities by 18–38% while secretin stimulation was unaffected. In heart membranes from SHR rats, Gpp(NH)p, NaF, D,L-isoproterenol, or glucagon stimulation of the enzyme was similarly increased by 14–38% whilst the low secretin responsiveness which is characteristic of these animals decreased even further (by 24–47%). These results are consistent with: (1) an up regulation of postsynaptic β-adrenergic receptors coupled to adenylate cyclase after degeneration of adrenergic nerves, and (2) a differential response of secretin receptors coupled to adenylate cyclase in the two strains of rats: there was no change in WKY rats and a decreased response in SHR rats. The possible presence and contribution of presynaptic secretin cardiac receptors is considered.

Hexachlorophene-induced degeneration of adrenergic nerves: application of quantitative image analysis to Falck-Hillarp fluorescence histochemistry.

Possible toxic effects of hexachlorophene (HCP) on sympathetic adrenergic nerves were studied using Falck-Hillarp fluorescence histochemistry on whole-mounts of albino rat irides. HCP dissolved in 5 microliters DMSO, or DMSO alone, was injected into the anterior chamber of the eye. HCP caused a dose-dependent degeneration of adrenergic nerves, first observable after 7 micrograms and profound after 21 micrograms. One and 3 days after 35 micrograms of HCP there was an almost total loss of adrenergic nerves. Regeneration from remaining non-terminal axons led to an almost complete reformation of the adrenergic nerve plexus after 18 days. The results demonstrate a new aspect of hexachlorophene neurotoxicity, degeneration of peripheral adrenergic nerve terminals and suggest that neurotoxic actions on thin unmyelinated fiber systems should be looked for also in the central nervous system (CNS).

The cholinergic innervation of human pancreatic islets

The cholinergic innervation of pancreatic islets was investigated in the human using operatory samples. In order to analyze the nature of stained cholinergic nerve fibers some specimens were incubated in a solution containing 6-hydroxydopamine (6-HDA) to obtain a selective degeneration of adrenergic nerves. Cholinergic nerve fibers are present in human pancreatic islets, and appear to be organized in an external peri-insular plexus. Some nerve fibers from the peri-insular plexus enter the islets and seem to innervate directly various types of endocrine insular elements. The 6-HDA treatment does not alter the distribution pattern of cholinergic nerve fibers within pancreatic islets.

Delay by drugs of adrenergic nerve degeneration after proximal or distal sympathectomy of the submaxillary gland

1. 1. Degeneration of nerve terminals of the submaxillary gland of the rat proceeds at a faster rate after crushing adrenergic nerves close to the gland than after ganglionectomy. 2. 2. Bretylium, pargyline, nialamide and clorgyline delayed degeneration to the same extent after either type of denervation. 3. 3. Chlorpromazine and pentobarbitone also delayed adrenergic degeneration, effect related to the hypothermai induced by these drugs. 4. 4. Colchicine applied on the nerve trunk innervating the right gland delayed not only degeneration of the ipsilateral nerve terminals but the contralateral gland as well. 5. 5. From the data obtained it seems probable that the drugs tested delay adrenergic nerve degeneration at the levels of nerve terminals. This effect is not related to lysosomal stabilization.

The Effect of Surgical Procedures on Blood Vessel Innervation

Four commonly used surgical procedures have been shown to cause extensive damage to the sympathetic nerves which supply and accompany blood vessels. One of these procedures, which produced a localised crush lesion, was shown by fluorescence microscopy to cause extensive degeneration of adrenergic nerves in two densely innervated blood vessels of different character in the guinea pig: the elastic carotid artery, and the muscular mesenteric artery. The extent of denervation was different in the two vessels: in the carotid artery the crush lesion destroyed all the nerves near the lesion, but left undamaged sparse nerves which reached the vessel wall along its length with the vasa vasorum; in the mesenteric artery all the nerves ran with the artery, consequently denervation could be extensive (at least 2 cm distal to the crush), although the largest nerve bundles were resistant to crush injury. The regrowth rate of nerves in the mesenteric artery was faster than that found in the carotid artery. A plexus of normal density and appearance was re-established between 3 and 8 weeks, and hyperinnervation was observed in some specimens. In the carotid artery this process was usually not complete by 8 weeks and involved both the regrowth of injured fibres and collateral sprouting distal to the lesion of the sympathetic nerve fibres that had not been injured. Axon sprouting was observed in both vessels near the lesion site. Explanations for these differences are discussed.

Electron microscopic evidence that bretylium and pargyline delay adrenergic nerve degeneration after sympathectomy of the pineal gland.

Seventeen and twenty four hours after sympathetic denervation, noradrenaline stores of the rat pineal gland were depleted to 50% and 10% of controls, respectively. Electron microscopic studies showed the coexistence of normal and altered nerve endings 17 h after denervation, while 24 h after denervation, only degenerated nerve terminals were observed. Treatment with pargyline (512 mumoles/kg) or bretylium (24 mumoles/kg) significantly delayed the loss of noradrenaline from denervated glands. In pargyline treated rats, 17 h after denervation, noradrenaline stores were 90% of control glands. After bretylium, values obtained 24 h after denervation, declined to 36% of innervated glands. Persistence of neurotransmitter coincided with the presence of normal nerve endings as observed electron microscopically. It is concluded that both, pargyline and bretylium, prolonged the survival of nerve endings severed from the cell body.

Differential morphological changes in sympathetic nerve fibers elicited by lead, cadmium, and mercury

Effects of intraocular injection of lead acetate, cadmium chloride, or mercury chloride on the sympathetic adrenergic ground plexus of the iris were studied using monoamine fluorescence histochemistry. Injection of 5 μl of a 1.4 m m lead solution into the anterior eye chamber caused a significant hyperinnervation as compared to sodium acetate-injected control irides. This hyperinnervation persisted 2 weeks after injection. Increasing the dose to 42 m m did not increase the extent of the hyperinnervation. Cadmium chloride solutions (1.4 and 7 m m) did not affect the adrenergic nerve fiber density as compared to sodium chloride-injected controls. Injection of mercury chloride caused a marked and dose-dependent degeneration of adrenergic nerves. Two weeks after injection of a 3.5 m m mercury chloride solution, the nerves had regenerated almost completely. Our results demonstrate that intraocular injections of neurotoxic heavy metals, and subsequent analysis with Falck-Hillarp fluorescence microscopy, is a rapid and sensitive method to evaluate adrenergic neurotoxicity.

Pregnancy is associated with extensive adrenergic nerve degeneration in the uterus. An electronmicroscopic study in the guinea-pig

The uterus of virgin guinea-pigs is supplied with a well-developed system of adrenergic nerves, characterized by 50 nm diameter synaptic vesicles whose electron-density is enhanced following administration of 5-hydroxydopamine. In the course of pregnancy, an increasing number of nerves with signs of various stages of degeneration are seen, varying from a slight condensation of the axonal content or myelin figures, to highly osmiophilic dense bodies lacking plasma membrane and with no organelles discernible. Incipient degeneration in a small number of axons is evident already in early pregnancy (up to 10 days post coitum). At late pregnancy (40–50 days post coitum), almost all adrenergic nerve terminals have disappeared as a consequence of degeneration, so that only a few isolated heavily degenerated axon terminals remain visible. Neither normal nor degenerating axons are found in the myometrium at full term. The degenerative changes occur earlier and are, at the various pregnancy stages, more pronounced in that part of the uterus surrounding and expanded by the fetus, compared to those segments of the uterine wall separating the fetuses. Thus, the normal neuromuscular relationship in the uterus during such an entirely physiological condition as pregnancy is lost entirely, in contrast to other sympathetically innervated organs, where the nerve plexus usually keeps pace with volume changes in the effector tissue.

Cholinergic nerves in the human liver

The cholinergic innervation of the human liver was studied. Slices (150--200 micrometer thick) of human liver and of the greater hepatic blood vessels (hepatic artery and vein, portal vein) were incubated in a solution of 6-hydroxydopamine (6-HDA) in order to obtain a selective degeneration of adrenergic nerves. Controls were prepared from samples incubated with buffer alone. The slices were cut on a cryostat into 15-20 micrometer thick sections and processed for the histochemical detection of cholinesterases. Cholinergic nerve fibres innervate the extra hepatic and the intrahepatic branches of the hepatic artery, the portal vein as well as the hepatic vein. Fewer cholinergic fibres innervate the hepatocytes and the hepatic sinusoids. The 6-HDA treatment does not seem to alter the pattern of the cholinergic innervation of the liver. The findings indicate the presence of a cholinergic parasympathetic innervation in the human liver.

Ultrastructural changes in blood vessels of perinephritic hypertensive dogs.

Mesenteric arteries and saphenous veins of control and perinephritic hypertensive dogs were studied with light and electron microscopy. 2 weeks after surgery there are ultrastructural changes, both in arteries and veins which are thought to be of hydroelectrolytic origin. These alterations are more marked 7 weeks after surgery and disappear thereafter. There are signs of increased protein synthesis activity in the smooth muscle of the mesenteric arteries, but not of the saphenous veins obtained 7 and 14 weeks after surgery, leading to a progressive hypertrophy of the muscular cells. The connective tissue augmented proportionally. Degeneration of adrenergic nerves in the arteries, accompanied by a parallel decrease in their noradrenaline content, constituted an unexpected finding clearly deserving further study.

Electrical field stimulation of myometrial strips from non‐pregnant and pregnant guinea‐pigs

Myometrial tissue strips from term pregnant and non‐pregnant guinea‐pigs were studied in vitro by electrical field stimulation, and the adrenoceptor sensitivity to exogenously applied noradrenaline was determined. The induced response was characterized by various sympatholytic and nerve blocking drugs. In previously contracted strips from non‐pregnant animals field stimulation provoked a relaxation, probably mediated via postganglionic adrenergic nerve terminals and myometrial β‐receptors. In strips from myometrial tissue surrounding fetuses, no response was seen after field stimulation. Relaxation induced by exogenous noradrenaline was more pronounced and provoked at lower concentrations in pregnant than in non‐pregnant tissue. However, in non‐fetus‐carrying uterine horns in unilateral pregnancies field stimulation still induced a relaxation, and the sensitivity to exogenous noradrenaline was less than in the contra‐lateral fetus‐carrying horn. The physiological findings were compared with the endogenous level of uterine noradrenaline transmitter and the presence of adrenergic nerves, demonstrated by the Falck‐Hillarp fluorescence technique. It is suggested that the altered effects at term pregnancy are due to a pregnancy induced postganglionic denervation of the adrenergic terminals in the fetus‐carrying uterine horn. This is accompanied by an increased postsynaptic adrenoceptor sensitivity to the transmitter in analogue with a denervation supersensitivity after adrenergic denervation. Functionally, the present results verify the recent structural and biochemical findings of an adrenergic nerve degeneration in fetus‐carrying myometrial tissue during pregnancy, probably representing a very special neuroendocrine neuron‐target relationship.

Delay by bretylium of adrenergic nerve degeneration after sympathectomy of the submaxillary gland

Administration of 24 μmol/kg of bretylium 10 h after ganglionectomy delayed the loss of endogenous norepinephrine and the impairment of neuronal uptake of 3H-metaraminol ( 3H-MA) that follow sympathetic denervation. This delay was evident 16 and 20 h after denervation. Twenty four h after ganglionectomy, when NE stores and uptake of 3H-MA were reduced to their lowest values in untreated rats, in bretylium-treated ones these values were approximately 40% of those in normal glands. The onset of degeneration secretion in treated rats was delayed by about 9 h. The development of prejunctional supersensitivity was also delayed. The subcellular distribution of NE in normal and 16 h denervated glands showed that denervation reduced the neurotransmitter to the same extent in the 3 fractions: coarse, supernatant and microsomal. Treatment with bretylium and pargyline prevented the loss of NE from the microsomal fraction. Previous administration of pargyline antagonized thep protection of 3H-MA uptake seen in 28 h denervated rats treated with bretylium. However, this drug combination induced a greater retention of endogenous NE 24 h after denervation. Bretylium inhibited intraneuronal MAO by 40%. It is concluded that bretylium treatment can delay the degeneration of adrenergic nerve terminals separated from the cell bodies by a phamacological effect probably not related to MAO inhibition or to its neurone blocking action.

Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy.

In the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40-45 days) than in an early one (20-25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.