Neuritic dystrophy and neuronopathy in Akita (Ins2Akita) diabetic mouse sympathetic ganglia☆
Introduction
Autonomic neuropathy is an increasingly recognized problem in human diabetes which may result in a variety of complaints involving cardiovascular, genitourinary, sudomotor and alimentary symptoms (Rundles, 1945) or result in subclinical disease. Studies of prevertebral sympathetic ganglia in autopsied diabetic human subjects demonstrate neuroaxonal dystrophy (NAD) (Duchen et al., 1980, Schmidt et al., 1993), an axonopathy represented by marked enlargement of distal axons containing a distinctive admixture of cytoskeletal, autophagic, vesicular and membranous elements. Immunohistochemical studies are consistent with the origin of NAD from other sympathetic neurons (Schmidt, 2002), possibly as the result of intraganglionic sprouting. These axonopathic changes are accompanied by a mild, poorly characterized decrease in neuronal density (Schmidt et al., 1993). Nerve terminal damage is likely to dis- or misconnect ganglionic neurons and, particularly for prevertebral ganglia serving the viscera, contribute to the loss of integrated reflexes. Rat models of diabetic sympathetic autonomic neuropathy show correspondence with human pathology, developing dystrophic axons in prevertebral ganglia (Schmidt, 2002) in the absence of significant neuronal loss (Schmidt, 2001). The fidelity of animal models to the neuropathology of aged and diabetic humans suggests that similar pathogenetic mechanisms may be involved with a comparable response to experimental therapeutic approaches.
Previously we have shown that non-obese diabetic (NOD) mice or streptozotocin-treated NOD/severe combined immune deficient (STZ-Rx NOD/SCID) mice develop dramatic axonal as well as dendritic pathology (thus designated “neuritic dystrophy”) within a few weeks of onset of diabetes (Schmidt et al., 2003). Our recent studies with these models (Schmidt et al., 2008b) have shown that there is a continuum of ultrastructural changes in identified presynaptic axon terminals in diabetic mouse sympathetic ganglia which begin with early alterations in synaptic vesicle content and morphology and culminate in the development of anastomosing tubulovesicular membranous aggregates in swollen preterminal axons and formation of multivesicular autophagic bodies. Using STZ-treated NOD/SCID mice, we were able to show that erythropoietin and carbamylated erythropoietin prevented the development of experimental diabetic autonomic neuropathy (Schmidt et al., 2008a). Unfortunately, NOD and STZ-treated NOD/SCID mice, once diabetic, do not survive for extended periods needed to determine the ability of therapeutic agents to correct established neuropathy, clinically a more relevant paradigm than prevention of the neuropathy. We have previously shown that STZ-treatment of B6D2F1, C57BL6, DBA/2J and BL6/NCR strains of mice resulted in neuritic dystrophy identical in ultrastructural appearance and anatomic distribution to that in NOD or STZ-Rx NOD/SCID mice (Schmidt et al., 2003). However, the pace of development of neuritic dystrophy in these strains is significantly slower and the ultimate severity of neuritic dystrophy limited in comparison to NOD and STZ-Rx NOD/SCID mice (e.g., 5 months of STZ-induced diabetes in C57BL6 mice produced 7-fold fewer dystrophic neurites than NOD mice diabetic for 5 weeks).
In search of a better mouse model we examined the Akita (Ins2Akita) mouse in which a spontaneous dominant mutation in the insulin 2 gene on a C57BL6 mouse background results in replacement of cysteine with tyrosine at position 96. This substitution disrupts a disulfide bridge required for proper insulin folding, resulting in initiation of the unfolded protein response and pancreatic β-cell apoptosis in the absence of obesity, insulitis or insulin resistance (Yoshioka et al., 1997, Mathews et al., 2002, Ron, 2002, Izumi et al., 2003). Heterozygous Akita mice become reproducibly and severely hyperglycemic and hypoinsulinemic at 3–4 weeks of age but remain fertile and viable in the absence of exogenous insulin treatment. The diabetic phenotype is more severe and progressive in the Akita male than in the female. Akita mice are relatively hardy and maintain marked hyperglycemia comparable in severity to that of NOD and STZ-Rx NOD/SCID mice for durations up to 8 months, far longer than 5–8 weeks of severe hyperglycemia which can be maintained in NOD and NOD/SCID models in which animals become debilitated and fragile. Recent studies of chronically diabetic Akita mice have shown diabetic complications including gait disturbance and sensory neuropathy as well as progressive retinopathy (Choeiri et al., 2005, Barber et al., 2005). In anticipation of initiating studies of novel therapeutic agents, in this manuscript we have characterized the neuropathology of sympathetic autonomic ganglia in Akita diabetic and age-matched controls diabetic for 2–8 months. We demonstrate that male Akita-diabetic mice show marked neuritic dystrophy in prevertebral sympathetic ganglia identical in appearance and anatomic distribution to that which develops in other mouse models. The frequency of neuritic dystrophy is substantial in Akita mice after only 2 months of diabetes and is progressive over the next 6 months. In addition, neurons in Akita mouse prevertebral sympathetic ganglia show an unusual perikaryal alteration characterized by the accumulation of membranous aggregates and minute mitochondria and loss of rough endoplasmic reticulum culminating in neuronal degeneration.
Section snippets
Animals
The C57BL/6J-Ins2Akita animals used in the currently reported studies were obtained from the Jackson Laboratory. All animals were housed and cared for in accordance with the guidelines of the Washington University Committee for the Humane Care of Laboratory Animals and with National Institutes of Health guidelines on laboratory animal welfare. All mice were allowed standard rat chow and water ad libitum and maintained on a 12/12 h light/dark cycle.
Tissue preparation
Animals were anesthetized with
Metabolic parameters
Akita mice were received at 6–8 weeks of age and initial blood glucose values were determined showing all the animals were diabetic at that point (blood glucose values > 300 mg/dl). Diabetic animals and age-matched C57BL6 control mice were killed at 2, 4, and 8 months of diabetes. Body weights of diabetics were significantly less than controls at 4 and 8 months and blood glucose values were significantly increased (Table 1).
Light microscopic examination
Examination of 1 μm thick plastic sections of SMG and CG of 2, 4 and
Comparison of mouse models of diabetic sympathetic autonomic neuropathy
We have examined the development of sympathetic autonomic neuropathy in several different mouse models. Although the NOD mouse rapidly develops dramatic neuropathology within 3–5 weeks of onset of diabetes, NOD mice develop diabetes spontaneously over a time course which may range from 12–30 weeks of age; therefore, NOD mice with the same duration of diabetes may actually represent animals which differ significantly in age. Unfortunately, the necessity of using the NOD background inbred strain
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Support: NIH awards R37 DK19645 and AG10299; Juvenile Diabetes Research Foundation Grants 1-2005-1085 and 1-2008-193