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Research Letters |

Autonomic Ganglia Neuronal Density and Synaptic Structure in Chronic Experimental Autoimmune Autonomic Ganglionopathy FREE

Emil Tajzoy; Shalini Mukherjee, PhD; Steven Vernino, MD, PhD
[+] Author Affiliations

Author Affiliations: Department of Neurology, University of Texas Southwestern Medical Center, Dallas.


Arch Neurol. 2011;68(4):540-546. doi:10.1001/archneurol.2011.52.
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Autoimmune autonomic ganglionopathy (AAG) is a severe form of acquired autonomic failure. Symptoms include orthostatic hypotension, gastrointestinal dysmotility, dry mouth and eyes, impaired pupillary constriction, and blunted heart rate responses. While some patients have distal paresthesias or other sensory symptoms, objective evidence of somatic neuropathy is rare. Autoimmune autonomic ganglionopathy is associated with serum antibodies against neuronal acetylcholine receptors in autonomic ganglia (ganglionic AChR). These autoantibodies impair ganglionic synaptic transmission.1,2 Patients with AAG may respond to immunomodulatory treatment even after decades of severe autonomic failure, suggesting that AAG is an antibody-mediated disorder of synaptic transmission without destruction of peripheral autonomic neurons.3 However, some recent clinical studies, based on abnormalities in quantitative sudomotor axon reflex or skin biopsy studies, suggest that postganglionic autonomic fibers are lost.4

Experimental AAG (EAAG) can be produced by active immunization of rabbits against the ganglionic AChR. Rabbits with EAAG have autonomic failure and show a loss of synaptic AChR in autonomic ganglia.5 To investigate further, we examined the superior cervical ganglia of rabbits with chronic EAAG to determine if the disease is associated with any structural changes in autonomic neurons, axons, or ganglionic synapses.

Rabbit EAAG was induced by immunization against the ganglionic AChR, as previously described.2,5 Control rabbits were injected with saline in complete Freund adjuvant. Study rabbits produced ganglionic AChR antibodies starting around 25 days after immunization, and antibody levels peaked 60 to 70 days after immunization. Eight antibody-producing rabbits that showed signs of chronic autonomic dysfunction were killed, and their superior cervical ganglia were harvested for histological studies. These rabbits had high antibody levels (range, 1.3-8.3 nmol/L). Rabbits were studied 100 to 225 days after immunization.

Trimmed tissue sections (0.5-1.0 mm3) were fixed by sequential exposure to glutaraldehyde, osmium tetroxide, uranyl acetate, alcohol, and propylene oxide, and then embedded in epoxy resin. Semithin (1 μm) sections were stained with toluene blue and evaluated by light microscopy to determine neuronal density using AxioVision Imaging System (Carl Zeiss MicroImaging, Thornwood, New York). Neuronal cell bodies were identified and counted only if they contained one or more visible nuclei. Thin tissue sections were prepared on copper grids and examined using a JEOL 1200EX transmission electron microscope (Tokyo, Japan). Electron micrographs were evaluated qualitatively to ascertain any ultra structural difference between control and rabbits with chronic EAAG.

Histologically, superior cervical ganglia from rabbits with chronic EAAG appeared normal (Figure, A). The neuronal density in the superior cervical ganglia from rabbits with chronic EAAG was 14% lower than in control rabbits (Figure). The cross-sectional area of the ganglia did not differ between the groups. Neuronal density did not significantly correlate with antibody level or with duration of disease. At the ultrastructural level (Figure, C), there were no signs of degeneration in neurons or axons. Synapses (presumably on dendrites) were found in interneuronal spaces rather than directly on neuron cell bodies. There was no qualitative difference in ganglionic synaptic morphology between controls and rabbits with EAAG (Figure, D). Because synapses were scattered sparsely in the neuropil, accurate quantitation of synaptic density was not possible.

Place holder to copy figure label and caption
Figure.

Morphology of autonomic ganglia and ganglionic synapses. A, A semithin section of superior cervical ganglia (SCG) from a rabbit with chronic experimental autoimmune autonomic ganglionopathy (EAAG) is shown (scale bar, 100 μm). Large ganglionic neurons are seen without any degenerating forms or inflammatory infiltrates. B, Ganglionic neuronal density in rabbits with EAAG (mean [SD], 63.2 [3.7] neurons per square millimeter; n = 8) was lower than in control rabbits (mean [SD], 73.9 [4.4] neurons per square millimeter; n = 8; t test, P = .04). C, An electron micrograph of SCG from rabbits with EAAG shows a neuronal cell body (*). The neuropil between neurons was composed mainly of groups of unmyelinated fibers (arrow) as well as some groups of small myelinated fibers and synaptic areas (scale bar, 4 μm). D, An electron micrograph shows a ganglionic synapse (scale bar, 200 nm). The presynaptic terminal is characterized by numerous small clear synaptic vesicles clustered near the synapse and rare dense-core vesicles. The apposed postsynaptic membrane density (arrow) is the location of synaptic ganglionic acetylcholine receptor. No ultrastructural differences between control and EAAG ganglia were observed.

Graphic Jump Location

The predominant pathophysiology of EAAG and AAG is antibody-mediated impairment of ganglionic synaptic transmission1,5 rather than destruction of neurons or synapses. This differs from the findings in experimental myasthenia gravis (the other AChR antibody disorder) in which damage and disorganization of the neuromuscular junction membrane are seen. In long-standing disease, however, degeneration of ganglionic neurons may occur.5 This may result from chronic denervation or metabolic stress of the disease process. Our findings help explain the incomplete recovery of autonomic function seen in many patients with AAG. Prompt diagnosis and the early use of immunomodulatory therapy may limit the amount of permanent autonomic dysfunction.

ARTICLE INFORMATION

Correspondence: Dr Vernino, University of Texas Southwestern Medical Center, Department of Neurology, 5323 Harry Hines Blvd, Dallas, TX 75390-9036 (steven.vernino@utsouthwestern.edu).

Author Contributions:Study concept and design: Vernino. Acquisition of data: Tajzoy, Mukherjee, and Vernino. Analysis and interpretation of data: Tajzoy, Mukherjee, and Vernino. Drafting of the manuscript: Tajzoy and Vernino. Critical revision of the manuscript for important intellectual content: Tajzoy, Mukherjee, and Vernino. Statistical analysis: Mukherjee, Vernino. Obtained funding: Vernino. Study supervision: Mukherjee and Vernino.

Financial Disclosure: None reported.

Funding/Support: This study was supported by grant R01NS48077 from the National Institutes of Health (Dr Vernino) and a postdoctoral fellowship from the Myasthenia Gravis Foundation (Dr Mukherjee).

Wang  ZLow  PAVernino  S Antibody-mediated impairment and homeostatic plasticity of autonomic ganglionic synaptic transmission. Exp Neurol 2010;222 (1) 114- 119
PubMed Link to Article
Lennon  VAErmilov  LGSzurszewski  JHVernino  S Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease. J Clin Invest 2003;111 (6) 907- 913
PubMed Link to Article
Schroeder  CVernino  SBirkenfeld  AL  et al.  Plasma exchange for primary autoimmune autonomic failure. N Engl J Med 2005;353 (15) 1585- 1590
PubMed Link to Article
Kimpinski  KIodice  VSandroni  PFealey  RDVernino  SLow  PA Sudomotor dysfunction in autoimmune autonomic ganglionopathy. Neurology 2009;73 (18) 1501- 1506
PubMed Link to Article
Vernino  SLow  PALennon  VA Experimental autoimmune autonomic neuropathy. J Neurophysiol 2003;90 (3) 2053- 2059
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure.

Morphology of autonomic ganglia and ganglionic synapses. A, A semithin section of superior cervical ganglia (SCG) from a rabbit with chronic experimental autoimmune autonomic ganglionopathy (EAAG) is shown (scale bar, 100 μm). Large ganglionic neurons are seen without any degenerating forms or inflammatory infiltrates. B, Ganglionic neuronal density in rabbits with EAAG (mean [SD], 63.2 [3.7] neurons per square millimeter; n = 8) was lower than in control rabbits (mean [SD], 73.9 [4.4] neurons per square millimeter; n = 8; t test, P = .04). C, An electron micrograph of SCG from rabbits with EAAG shows a neuronal cell body (*). The neuropil between neurons was composed mainly of groups of unmyelinated fibers (arrow) as well as some groups of small myelinated fibers and synaptic areas (scale bar, 4 μm). D, An electron micrograph shows a ganglionic synapse (scale bar, 200 nm). The presynaptic terminal is characterized by numerous small clear synaptic vesicles clustered near the synapse and rare dense-core vesicles. The apposed postsynaptic membrane density (arrow) is the location of synaptic ganglionic acetylcholine receptor. No ultrastructural differences between control and EAAG ganglia were observed.

Graphic Jump Location

Tables

References

Wang  ZLow  PAVernino  S Antibody-mediated impairment and homeostatic plasticity of autonomic ganglionic synaptic transmission. Exp Neurol 2010;222 (1) 114- 119
PubMed Link to Article
Lennon  VAErmilov  LGSzurszewski  JHVernino  S Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease. J Clin Invest 2003;111 (6) 907- 913
PubMed Link to Article
Schroeder  CVernino  SBirkenfeld  AL  et al.  Plasma exchange for primary autoimmune autonomic failure. N Engl J Med 2005;353 (15) 1585- 1590
PubMed Link to Article
Kimpinski  KIodice  VSandroni  PFealey  RDVernino  SLow  PA Sudomotor dysfunction in autoimmune autonomic ganglionopathy. Neurology 2009;73 (18) 1501- 1506
PubMed Link to Article
Vernino  SLow  PALennon  VA Experimental autoimmune autonomic neuropathy. J Neurophysiol 2003;90 (3) 2053- 2059
PubMed Link to Article

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