Jeremy B. Tuttle

Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function.

Urethral obstruction produces increased voiding frequency (0.7 +/- 0.06 to 1.1 +/- 0.08 h-1) and hypertrophy of the urinary bladder (89 +/- 1.7 to 708 +/- 40 mg) with profound increments in the dimensions of afferent (4, 6) and efferent neurons (299 +/- 4.7 to 573 +/- 8.6 microns2) supplying this organ in the rat. We discovered that hypertrophied bladders of rat and human contain significantly more nerve growth factor (NGF) per milligram wet weight, protein, and DNA than normal bladders. The temporal correlation between NGF content, neuronal hypertrophy, and bladder weight was consistent with a role for this growth factor in the neurotrophic effects associated with obstruction. Autoimmunity to NGF abolished the hypertrophy of NGF-sensitive bladder neurons in the pelvic ganglion after obstruction. Relief of urethral obstruction reduced bladder size (349 +/- 78 mg), but neuronal hypertrophy (460.2 +/- 10.2 microns2) and elevated NGF levels were only partially reversed. Bladder hypertrophy (133 +/- 4.3 mg) induced by osmotic diuresis slightly increased ganglion cell area (365.2 +/- 6.1 microns2) and only doubled NGF content of the bladder. These findings provide important new evidence that parenchymal cells in the hypertrophied bladder can synthesize NGF and possibly other molecular messengers that act to alter the size and function of neurons in adult animals and man.

Mitochondria in Sporadic Amyotrophic Lateral Sclerosis

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.

Mechanisms of Disease: the role of nerve growth factor in the pathophysiology of bladder disorders

The case is compelling for the involvement of nerve growth factor (NGF) in the pathogenesis of lower urinary tract disease, especially in conditions with altered neural function. Remodeling of the micturition pathways occurs following experimental bladder-outlet obstruction, denervation, spinal cord injury, cystitis, and diabetes mellitus. Clinically, NGF levels are elevated in the bladders of men with benign prostatic hyperplasia, women with interstitial cystitis and in patients with idiopathic overactive bladder. Blockade of NGF, using either an endogenous antibody or an antibody against the NGF receptor, prevents neural plasticity and bladder overactivity in experimental models of these conditions. The ability of NGF to trigger bladder overactivity might rely on altering the properties of sodium or potassium channels (or their expression) in bladder afferent fibers. Therapies based on altered NGF levels, or changes in channel properties in afferent nerves, represent an intriguing avenue of investigation for the management of detrusor overactivity or diabetic cystopathy.

IMMUNITY TO NERVE GROWTH FACTOR PREVENTS AFFERENT PLASTICITY FOLLOWING URINARY BLADDER HYPERTROPHY

PURPOSE The goal of this investigation was to examine the effect of immunity to nerve growth factor (NGF) on alterations in sensory nerves from the urinary bladder in the dorsal root ganglia (DRG) and their projections to the L6/S1 spinal cord following urethral obstruction in the rat. MATERIALS AND METHODS Female Wistar rats were immunized to murine 2.5S NGF, then obstructed by partial urethral ligation for 6 weeks. Retrograde axonal tracing with FluoroGold and WGA-HRP was used to measure areas of bladder DRG cells and afferent projections in the sacral spinal cord. Multiunit activity on bladder nerves allowed recording of micturition reflexes. Immunohistochemical staining for growth associated protein (GAP)-43 in the sacral parasympathetic nucleus (SPN) was used to assess potential growth or activity of axons in the spinal cord. Voiding frequencies were then measured in awake obstructed and NGF immune-obstructed rats. RESULTS Immunity to NGF prevented obstruction-induced hypertrophy of DRG neurons, reduced retrograde axonal labeling of sacral afferent projections, eliminated enhancement of a spinal micturition reflex and abolished the increased GAP-43 expression in the SPN. Immunity to NGF prevented the urinary frequency that accompanies obstruction. CONCLUSIONS Our results demonstrate that obstruction of the bladder elicits structural and functional plasticity in afferents as a result of ongoing neurotrophic interactions. Neurotrophic interactions offer a potential mechanism whereby symptoms and bladder hyperactivity develop with obstruction associated with benign prostatic hyperplasia.

Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats

The influence of noradrenergic mechanisms involved in micturition in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was investigated using continuous cystometry in in vivo and in vitro studies on isolated bladder and urethral tissues. Compared with WKY rats, SHR had a significantly lower bladder capacity (SHR: 0.7 +/- 0. 05 ml; WKY rats: 1.3 +/- 0.06 ml; P < 0.001), micturition volume (SHR: 0.4 +/- 0.04 ml, WKY rats: 1.2 +/- 0.05 ml; P < 0.001), and an increased amplitude of nonvoiding (unstable) bladder contractions. The effects of intrathecal and intra-arterial doxazosin on cystometric parameters were more pronounced in SHR than in WKY rats. There was a marked reduction in nonvoiding contractions after intrathecal (but not intra-arterial) doxazosin in SHR. Norepinephrine (0.1 microM-1 mM) failed to evoke contractions in bladder strips from WKY rats, in contrast to a weak contractile response in SHR. The response to electrical field stimulation was significantly less in bladder strips from SHR than from WKY rats. In WKY rats, norepinephrine produced concentration-dependent inhibition (87 +/- 5%, n = 6) of nerve-evoked bladder contractions. Almost no inhibition (11 +/- 8%, n = 6) was found in SHR. Alterations in bladder function of SHR appear to be associated with changes in the noradrenergic control of the micturition reflex, in addition to an increased smooth muscle and decreased neuronal responsiveness to norepinephrine. The marked reduction in nonvoiding contractions after intrathecal doxazosin suggests that the bladder hyperactivity in SHR has at least part of its origin in supraspinal and/or spinal structures.

HISTOLOGICAL AND NEUROTROPHIC CHANGES TRIGGERED BY VARYING MODELS OF BLADDER INFLAMMATION

PURPOSE We determined whether bladder inflammation causes elevated expression of nerve growth factor by bladder parenchymal cells, leading to alterations in neurons innervating the bladder. To answer this question biochemical, histological and neuronal size data were obtained in rats following various experimental models of bladder inflammation. MATERIALS AND METHODS Chemical (2.5% formalin), immune (lipopolysaccharide 2 x 104 cfu/ml.) and mechanical (chromic catgut) inflammation was evaluated at various times and compared to control bladders. Hematoxylin and eosin, and Giemsa staining was done to characterize inflammation and quantify mast cells in the bladder. Nerve growth factor protein and messenger RNA were assayed in the bladder and major pelvic ganglion using 2-site enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction, respectively. Retrograde axonal tracing was done to size bladder neurons in the major pelvic and dorsal root ganglia. RESULTS All forms of inflammation increased bladder weight and produced diffuse hyperplasia, intramural edema, acute and chronic inflammatory cells, infiltration and mastocytosis. Generally bladder inflammation resulted in a 50% increase in nerve growth factor and 52% to 58% enlargement of peripheral neurons. CONCLUSIONS Inflammation results in altered nerve growth factor content of the bladder, and morphological changes in sensory and motor neurons innervating the bladder. Such neuroplasticity may be a possible explanation for the association of bladder inflammation with long-term symptoms and pain after inflammation subsides.

Neural input regulates tissue NGF and growth of the adult rat urinary bladder.

To gain insight into the effect of innervation on neurotrophin production, NGF levels in the urinary bladder were measured following unilateral ganglionectomy (bladder denervation) or separation of the post-ganglionic bladder neurons from the central nervous system of the adult rat (bladder and ganglion decentralization). These interruptions of the neural input to half of the bladder caused histological evidence of smooth muscle growth, increased bladder weight (denervation-3 weeks: 98.6 +/- 6 mg; decentralization-3 weeks: 94.0 +/- 7 mg vs. control: 79.6 +/- 4 mg, P < 0.05), transient increases in tissue NGF up to 10-fold (1.99 +/- 0.65 pg NGF/bladder control vs. 20.24 +/- 0.53 (P < 0.05) denervated, ipsilateral, 1 week) and hypertrophy of the neurons in the pelvic ganglia supplying the bladder (control: 340 +/- 4.4 microns2; denervated-3 weeks: 530 +/- 6.8 microns2, P < 0.05; decentralized-3 weeks: 463 +/- 6.8 microns2, P < 0.05). These data suggest that neural input has a significant role in regulating growth of the bladder. Furthermore, the findings show that innervation influences tissue levels of NGF in the bladder.

The spontaneously hypertensive rat: insight into the pathogenesis of irritative symptoms in benign prostatic hyperplasia and young anxious males.

Recent epidemiological studies have shown that hypertensive men are more likely to undergo surgical intervention for irritative voiding symptoms from BPH than age‐matched controls. Indeed, noradrenergic nerves which regulate vascular tone also participate in the functional component of bladder outlet obstruction due to BPH. Newer, less invasive therapies for BPH such as thermal therapy can relieve symptoms yet do not eliminate obstruction based on urodynamic studies. Coincidentally, drugs such as α‐adrenoceptor antagonists, which have been thought to relieve obstruction due to a peripheral effect, can be given intrathecally in animals to relieve urinary frequency due to obstruction. Taken together these observations implicate both peripheral and central sympathetic pathways in the motor control of the urinary bladder especially with disease states.

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Abstract: Adenosine formation and release were studied in 48‐h‐old cultured ciliary‐ ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2‐deoxygIucose and 2 μg/ml oli‐gomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15–40% of the fall in ATP. Dilazep (3 × 10−6M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80–90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of α,β‐methylene ADP that inhibited ecto‐5′‐nucleotidase by 80–90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto5′‐nucleotidase was excluded.

MPP+ induced apoptotic cell death in SH-SY5Y neuroblastoma cells: An electron microscope study

PD is a common, late‐onset neurodegenerative disorder that results in part from the gradual loss of dopaminergic neurons in the substantia nigra pars compacta. The neurotoxin MPTP can induce PD‐like clinical symptomatology and neuropathological destruction and, thus, has been used as a PD model. The human neuroblastoma cell line SH‐SY5Y possesses many of the qualities of human neurons and, as such, has served as a model for them. Apoptosis is the mode of cell death induced in SH‐SY5Y cells by MPTP, and this was confirmed with nick end labeling and bisbenzimide staining. Transmission electron microscopic analysis of the ultrastructural changes occurring in neurotoxin exposed SH‐SY5Ys revealed many morphological characteristics consistent with apoptosis. These changes included plasmalemmal blebbing, altered cytosolic density, nuclear condensation and fragmentation, pronounced vacuole formation, ribosomal dispersion, and the disappearance of the golgi complex, microtubules, and smooth endoplasmic reticulum. Limited amounts of rough endoplasmic reticulum and mitochondria exhibited normal morphology throughout the apoptotic changes but then were disrupted during secondary necrotic changes.