Journal of Neuropathology and Experimental Neurology
Copyright q 2004 by the American Association of Neuropathologists
Vol. 63, No. 5
May, 2004
pp. 450 460
Experimental Rat Models of Types 1 and 2 Diabetes Differ in Sympathetic
Neuroaxonal Dystrophy
ROBERT E. SCHMIDT, MD, PHD, DENISE A. DORSEY, AB, LUCIE N. BEAUDET, CURTIS A. PARVIN, PHD,
WEIXIAN ZHANG, MD, AND ANDERS A. F. SIMA, MD, PHD
Abstract. Dysfunction of the autonomic nervous system is a recognized complication of diabetes, ranging in severity from
relatively minor sweating and pupillomotor abnormality to debilitating interference with cardiovascular, genitourinary, and
alimentary dysfunction. Neuroaxonal dystrophy (NAD), a distinctive distal axonopathy involving terminal axons and synapses,
represents the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in man and several insulinopenic
experimental rodent models. Although the pathogenesis of diabetic sympathetic NAD is unknown, recent studies have suggested that loss of the neurotrophic effects of insulin and/or insulin-like growth factor-I (IGF-I) on sympathetic neurons rather
than hyperglycemia per se, may be critical to its development. Therefore, in our current investigation we have compared the
sympathetic neuropathology developing after 8 months of diabetes in the streptozotocin (STZ)-induced diabetic rat and BB/
Wor rat, both models of hypoinsulinemic type 1 diabetes, with the BBZDR/Wor rat, a hyperglycemic and hyperinsulinemic
type 2 diabetes model. Both STZ- and BB/Wor-diabetic rats reproducibly developed NAD in nerve terminals in the prevertebral
superior mesenteric sympathetic ganglia (SMG) and ileal mesenteric nerves. The BBZDR/Wor-diabetic rat, in comparison,
failed to develop superior mesenteric ganglionic NAD in excess of that of age-matched controls. Similarly, NAD which
developed in axons of ileal mesenteric nerves of BBZDR/Wor rats was substantially less frequent than in BB/Wor- and STZrats. These data, considered in the light of the results of previous experiments, argue that hyperglycemia alone is not sufficient
to produce sympathetic ganglionic NAD, but rather that it may be the diabetes-induced superimposed loss of trophic support,
likely of IGF-I, insulin, or C-peptide, that ultimately causes NAD.
Key Words:
BB rats; Diabetes; Neuronal dystrophy; Sympathetic ganglia.
INTRODUCTION
Clinical presentations of diabetic neuropathy include a
distally accentuated symmetrical sensory polyneuropathy,
asymmetric mononeuropathies involving cranial and somatic nerves, and autonomic neuropathy (1). Although
sensory polyneuropathy (resulting in classical ‘‘stockingglove’’ limb anesthesia) is pathologically the most extensively studied form, diabetic autonomic neuropathy results in increased patient morbidity and mortality (2–4).
Symptoms of diabetic autonomic neuropathy range widely from minor pupillary and sweating problems to debilitating disturbances in cardiovascular, alimentary, and
genitourinary function, and involve both the sympathetic
and parasympathetic nervous systems. Autopsy studies of
diabetic patients (5) have established that the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy is the reproducible development of markedly
From Department of Pathology and Immunology (RES, DAD, LNB),
Divisions of Neuropathology and Laboratory Medicine (CAP), Washington University School of Medicine, St. Louis, Missouri; Departments
of Pathology and Neurology and Morris Hood Jr. Comprehensive Diabetes Center (WZ, AAFS), Wayne State University, Detroit, Michigan.
Correspondence to: Robert E. Schmidt, MD, PhD, Department of
Pathology and Immunology, Division of Neuropathology (Box 8118),
Washington University School of Medicine, 660 South Euclid Avenue,
St. Louis, MO 63110. E-mail: [email protected]
Support: Grants from the NIH (R37 DK19645, P60 DK20579, P30
DK56341), Juvenile Diabetes Research Foundation and the THOMAS
Foundation.
swollen distal axons and nerve terminals (‘‘neuroaxonal
dystrophy’’ [NAD]), perhaps representing aberrant intraganglionic sprouting (5, 6), in prevertebral sympathetic
ganglia in the absence of significant neuron loss. Degenerating, regenerating, and pathologically distinctive dystrophic axons and nerve terminals also develop in prevertebral superior mesenteric sympathetic ganglia (SMG)
and noradrenergic ileal mesenteric nerves in streptozotocin (STZ)-induced and BB/Wor-diabetic rats (7) in the
absence of neuronal or axon loss (8, 9).
The pathogenesis of diabetic autonomic neuropathy is
not established. A large body of investigation has determined that hyperglycemia directly results in a variety of
abnormal metabolic reactions in nerve (e.g. disordered
polyol and phosphoinositide metabolism, exaggerated oxidative stress, alteration in signaling pathways or apoptotic mediators, increase in glycated proteins) that may
contribute to the development of diabetic neuropathy (5),
although other processes may also participate (10). For
some time, investigators have considered that there is a
role for the neurotrophic action of insulin or insulin-like
growth factor-I (IGF-I), independent of the glycemic effects, in the pathogenesis of diabetic autonomic neuropathy (11–13). Therefore, in our current investigation we
compared the sympathetic neuropathology developing after 8 months of diabetes in the pancreatic b-cell toxin
STZ-induced diabetic rat and BB/Wor spontaneously diabetic rat (both hyperglycemic and hypoinsulinemic models of type 1 diabetes) with the BBZDR/Wor rat, a hyperglycemic and hyperinsulinemic type 2 model. BB/Wor
450
SYMPATHETIC DYSTROPHY IN TYPES 1 AND 2 DIABETES
and BBZDR/Wor rats are closely related genetically, decreasing the likelihood of a strain difference being misinterpreted as diabetes-related. In the studies reported
here both STZ- and BB/Wor-diabetic rats reproducibly
develop NAD in nerve terminals in the prevertebral sympathetic ganglia and in the distal portions of noradrenergic ileal mesenteric nerves. The BBZDR/Wor-diabetic
rat, in comparison, failed to develop NAD in the SMG
and ileal mesenteric nerves in excess of that observed in
age-matched controls.
MATERIALS AND METHODS
Animals
451
Tissue Collection
The SMG and ileal mesenteric nerves were dissected,
cleaned of extraneous tissue, and fixed by immersion in 3%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, overnight.
Tissue samples were postfixed in phosphate-buffered 2% OsO4
containing 1.5% potassium ferricyanide, dehydrated in graded
concentrations of alcohol, and embedded in Epon with propylene oxide as an intermediary solvent. One-mm-thick plastic sections were examined by light microscopy after staining with
toluidine blue. Ultrathin sections of individual SMG and ileal
mesenteric nerve pedicles were cut onto formvar-coated slot
grids, which permits visualization of entire ganglionic and
nerve cross sections, and stained with uranyl acetate and lead
citrate and examined with a JEOL 1200 electron microscope.
Male pre-diabetic BB/Wor- and BBZDR/Wor-rats and their
non-diabetic controls, i.e. non-diabetes prone lean, age-matched
BB control rats (Biomedical Research Models, Worcester, MA)
were obtained and maintained in metabolic cages with free access to rat chow and water. Blood sugar, urine volume, and
glucosuria were monitored daily to determine the time of spontaneous onset of diabetes. Diabetic BB/Wor rats were subsequently supplemented with small titrated doses of protamine
zinc insulin (a combination of beef and pork insulins, 1.3–3.0
U/day; IDEXX Pharmaceuticals, Westbrook, ME) to prevent
ketoacidosis from developing. The type 2 diabetic BBZDR/Wor
rats do not require insulin to survive and maintain levels of
hyperglycemia comparable to BB/Wor rats. Male SpragueDawley rats (;300 grams; Charles River, Belmont, MA) received a single dose of STZ (65 mg/kg, i.v.; Upjohn, Kalamazoo, MI) and within a few days developed plasma glucose
levels $350 mg/dl. The frequency of NAD in this group of
STZ-induced diabetic rats formed part of a previously reported
investigation (14) and is included in the current study for comparison only.
Animals were housed and cared for in accordance within the
guidelines of the NIH, the Wayne State University Animal Investigation Committee, and Washington University Committee
for the Humane Care of Laboratory Animals. All rats were
killed 8 months after the onset of diabetes. HbA1c levels were
determined at 2-month intervals and at the time of death.
HbA1c values were determined using the Glycogel B kit
(Pierce, Rockford, IL) in the STZ experiment or Bayer DCA
20001 analyzer in the BB/Wor and BBZDR/Wor experiments.
Ganglionic dystrophic elements are typically intimately related to neuronal perikarya and are expressed as the ratio of
numbers of lesions to nucleated neuronal cell bodies. This
method, used in our previous studies (14), substantively reduces
the variance in assessments of intraganglionic lesion frequency
and its simplicity permits the rapid quantitative ultrastructural
examination of relatively large numbers of ganglia. In our current animal studies, entire cross sections of the SMG were
scanned at 312,000 magnification and the number of dystrophic
neurites and synapses was determined. Dystrophic neurites
were divided into several morphologic classes based on their
content of 1) tubulovesicular aggregates; 2) admixed normal
and degenerating subcellular organelles, multivesicular and
dense bodies (‘‘mixed organelles’’); 3) neurofilament accumulations; and, 4) coarse tubules typically arising from dendritic
or perikaryal spines (‘‘dendritic forms’’). The number of nucleated neurons (range: 50–200 neurons examined in each ganglionic cross section) was then determined by recounting at
36,000 magnification. The frequency of ganglionic NAD was
expressed as the number of dystrophic neurites per nucleated
neuron in the same cross section. The frequency of NAD in
ileal mesenteric nerves was expressed as a percentage of total
mesenteric nerve axon number and as lesions per ileal mesenteric nerve fascicle (14). Each vascular arcade contains 2 main
paravascular fascicles, largely destined for alimentary targets.
Metabolic Studies
All results are expressed as means 6 SEM. Statistical comparisons between groups used unpaired 2-tailed t-tests.
Acid/ethanol extracted rat serum IGF-I was measured in the
Washington University Diabetes Research and Training Center
(DRTC) RIA Core laboratory at the time of death by double
antibody radioimmunoassay using antibody produced in rabbits
against human IGF-I and human IGF-I calibrators. Serum insulin was measured at death using 2 methods. The first method
is a double antibody radioimmunoassay (CrystalChem, Inc.,
Downer’s Grove, IL), which is pan-insulin reactive, using an
antibody produced in guinea pigs and rat insulin calibrators and
will detect rat insulin as well as beef and pork insulin, the
constituents of protamine zinc insulin (IDEXX Pharmaceuticals). A second method uses a rat insulin-specific ELISA (Linco
Research, St. Charles, MO). Rat proinsulin C-peptide levels
were determined using a kit from Linco Research.
Quantitative Ultrastructural Studies
Statistical Analysis
RESULTS
Metabolic Parameters
Diabetic animals in all 3 models exhibited marked hyperglycemia in comparison to their respective controls.
Although BB/Wor and BBZDR/Wor rats developed comparable levels of hyperglycemia, as measured by plasma
glucose and HbA1c values, plasma glucose values and
HbA1c in STZ-rats indicated a more severe diabetic state
(Table 1A). HbA1c values in STZ-diabetic rats were,
however, measured using different techniques and in different laboratories than BB/Wor and BBZDR/Wor rats.
J Neuropathol Exp Neurol, Vol 63, May, 2004
452
SCHMIDT ET AL
TABLE 1A
Metabolic Parameters
Model
STZ
BB Control
BB/Wor
BBZDR/Wor
Status
n
Control
Diabetic
Control
Diabetic
Diabetic
6
8
11
18
8
Plasma glucose
(mg%)
116
668
96
427
521
6
6
6
6
6
5
41*
2
8*
24*
Body weight (gm)
642
418
496
384
610
6
6
6
6
6
22
26*
7
2*
51†
HbA1c (%)
3.8
18.2
3.3
6.4
7.4
6
6
6
6
6
0.2
1.7*
0.04
0.1*
0.3*
Values represent the means 6 SEM of n rats.
Statistical comparison: * p # 0.001; † p # 0.05 vs control.
TABLE 1B
Metabolic Parameters: Serum Levels of Insulin, IGF-I, and Proinsulin C-Peptide
Group
n
Serum insulin
(pan-insulin) (pg/ml)
Serum insulin
(rat specific) (pM)
BB Control
BB/Wor
BBZDR/Wor
8
8
7
2,301 6 159
723 6 113†
4,103 6 1,695
430 6 20
52 6 5*
586 6 25†
Serum IGF-I
(ng/ml)
1,758 6 102
1,322 6 70*‡
1,617 6 110
Serum C-peptide
(pM)
1,506 6 41
,25*
1,516 6 37
Values represent the means 6 SEM of n rats.
Statistical comparison: * p # 0.001; † p # 0.01 vs controls; ‡ p # 0.05 vs BBZDR/Wor rats.
STZ- and BB/Wor-diabetic rats were significantly lighter
than age-matched controls (by 35% and 23%, respectively). As expected for a type 2 diabetes model, BBZDR/
Wor diabetic rats were significantly heavier (23%) than
age-matched non-diabetic controls (Table 1A).
Plasma insulin levels determined using a rat insulin
specific assay showed nearly a 90% decrease in BB/Wor
rats in comparison to BB control rat serum, a result that
paralleled serum C-peptide levels (Table 1B). BBZDR/
Wor rats, in contrast, showed a 36% increase in rat-specific serum insulin and normal serum levels of C-peptide.
Since BB/Wor rats received daily injections of a commercial preparation consisting of a mixture of pork and
beef insulin in order to survive, we also measured serum
insulin using a pan-insulin method that detects rat, pork,
and beef insulins. Using this assay, serum insulin levels
in BB/Wor were decreased approximately 70% in comparison to BB controls and were increased approximately
80% in BBZDR/Wor rats compared to controls. Serum
IGF-I levels were significantly but modestly decreased
(30%) in BB/Wor rats compared to controls. In contrast,
serum IGF-I levels in BBZDR/Wor rats were significantly
increased in comparison to BB/Wor values but were not
significantly different from BB rat controls.
Superior Mesenteric Ganglion
Ultrastructural examination of the SMG of the STZ,
BB/Wor, and BBZDR/Wor diabetic rats demonstrated
neuroaxonal dystrophy (Fig. 1), the neuropathologic hallmark of sympathetic autonomic neuropathy, which we
J Neuropathol Exp Neurol, Vol 63, May, 2004
have described in detail previously in STZ- and BB/Wordiabetic rats (7, 15). Swollen dystrophic axons and synapses were typically found intimately apposed to principal sympathetic neurons often within their satellite cell
sheaths (Fig. 1A), or in the immediately adjacent neuropil. Although the contours of neuronal perikarya were
distorted by large swellings (Fig. 1A), the appearance of
the cell body was otherwise unremarkable. Specifically,
degenerating or chromatolytic neuronal cell bodies were
not encountered. Neuroaxonal dystrophy in sympathetic
ganglia in all diabetic rats consisted of swollen preterminal axons and synapses containing tubulovesicular elements and compact membranous aggregates (Fig. 1B),
loose ‘‘fingerprint’’ forms (Fig. 1C), or neurofilaments
(Fig. 1D), often in disorganized skeins, and a variety of
subcellular organelles (Fig. 1E). Rare collections of
coarse tubular structures (Fig. 1F) occasionally involved
perisomal perikaryal projections or dendrites, often with
synaptic specializations (Fig. 1F, arrow), although frequently it was difficult to confidently establish the dendritic or axonal origin of these forms. The range of ultrastructural appearances of individual dystrophic axons
and dendritic elements was similar in all diabetic and
control groups.
Since all groups of ganglia exhibited NAD, quantitative ultrastructural studies were performed to permit comparison between control and diabetic animals within and
between STZ-, BB/Wor-, and BBZDR/Wor-groups.
Quantitative ultrastructural studies demonstrated a 2.5fold and 4-fold increase in the frequency of neuroaxonal
SYMPATHETIC DYSTROPHY IN TYPES 1 AND 2 DIABETES
dystrophy in the STZ-diabetic and BB/Wor-diabetic rat
groups, respectively, in comparison to their age-matched
controls (Table 2A). BBZDR/Wor diabetic rats, however,
failed to show a significant difference in the frequency
of NAD in comparison to age-matched control ganglia
(Table 2A).
Ultrastructural classification of dystrophic neurites (Table 2B) in diabetic STZ rats showed similar subpopulations of axons containing neurofilaments (38% of the total) or tubulovesicular elements (39%) and fewer mixed
organelle and dendritic forms, distributions roughly similar to their age-matched controls. On the other hand, the
BB/Wor rat showed a preponderance of neurofilamentladen dystrophic axons (60%) and relatively fewer axons
containing tubulovesicular elements (10%), mixed organelles, and dendritic forms. BBZDR/Wor rats showed
equal numbers of neurofilament (41%) and mixed organelle (40%) containing dystrophic axons and very few tubulovesicular element-containing dystrophic axons (8%).
A similar variety of ultrastructural appearances of dystrophic axons have been encountered in all diabetic species
examined without the emergence of a single theme; dystrophic axons followed in sequential sections in mesenteric nerves frequently vary significantly in ultrastructural
appearance from level to level.
Neuroaxonal Dystrophy in Ileal Mesenteric Nerves
Axons with the ultrastructural appearance of neuroaxonal dystrophy also represent the hallmark of experimental diabetic autonomic neuropathy in ileal mesenteric
nerves (Fig. 2). Swollen axons (Fig. 2A, arrows) may
overwhelm the tiny fascicles in which they reside and
displace adjacent, otherwise normal, axons. Dystrophic
axons include markedly enlarged forms (Fig. 2A), containing an admixture of tubulovesicular elements, mitochondria, dense core vesicles, and disorganized microtubules (Fig. 2B), as well as more modestly (Fig. 2C) and
minimally (Fig. 2D) enlarged axons.
The ultrastructural content of individual dystrophic axons was similar in all diabetic and control groups, although dystrophic axons in BB/Wor rats and BBZDR/
Wor rats were frequently smaller than those in STZ rats
(Fig. 2D). The frequency of NAD was expressed as a
percentage of the total number of axons in each mesenteric fascicle or as the number of dystrophic axons in
each mesenteric nerve fascicle with comparable results
(Table 3). The absolute frequency of NAD in ileal mesenteric nerves was more than 2-fold greater in diabetic
STZ-rats, although significantly more variable from animal to animal in comparison to BB/Wor rats, which may
reflect the necessity of administration of significant
amounts of exogenous insulin to permit BB/Wor rat survival. The total number of axons in each fascicle (Table
3) did not differ between diabetics and controls in STZ
453
or BBZDR/Wor rats but was mildly (18%), although not
statistically significantly, decreased in the BB/Wor group.
DISCUSSION
The present results demonstrate that the type 2 diabetic
BBZDR/Wor rat model does not develop sympathetic
NAD as seen in the type 1 diabetic BB/Wor or STZ rat
models, despite comparable hyperglycemia. A series of
recent comparative studies of BB/Wor and BBZDR/Wor
rats (16–19), focusing on myelinated fiber alterations in
diabetic somatic sensory neuropathy, have also demonstrated significant differences between these 2 models.
Specifically, somatic neuropathy in BB/Wor rats is characterized by severe nerve conduction defects, axonal atrophy, node of Ranvier-based pathology with molecular
alterations, defects in axonal regeneration (10, 17, 19, 20)
and, eventually, significant loss of myelinated axons (17).
In comparison, diabetic somatic neuropathy in the
BBZDR/Wor rat demonstrates only mild atrophy of myelinated axons, minimal nodal pathology with no molecular changes, segmental demyelination, and active Wallerian degeneration in the absence of axon loss (10, 17).
The relative preservation of myelinated axon number in
BBZDR/Wor rat sural nerve in the presence of ongoing
Wallerian degeneration is thought to reflect a 4-fold increase in axonal regeneration that replaces lost axons (17,
21). In contrast, impairment of nerve regeneration following sciatic nerve crush has been reported in STZ (22,
23) and BB/Wor (19, 24) rats. Delayed and attenuated
upregulation of IGF-I, NGF, p75, or asynchronous changes in IGF-I receptor message in distal segments of injured
sciatic nerve of BB/Wor rats contrasts with significantly
milder changes in BBZDR/Wor rats and may account for
the more efficient regenerative response (21). Comparable studies of response to injury have not been performed
in the autonomic nervous system of the BB/Wor and
BBZDR/Wor rat models. Nonetheless, NAD is thought
to represent the structural outcome of frustrated axon regeneration or abnormal turnover of intraganglionic nerve
terminals (6) and differences in regenerative capability.
Insight into the Pathogenesis of Diabetic Autonomic
Neuropathy
Do marked differences in sympathetic NAD between
STZ, BB/Wor and BBZDR/Wor rats provide insights into
possible pathogenetic mechanisms? Comparison of the 3
rat models makes it clear that hyperglycemia alone is not
sufficient for the development of sympathetic NAD. The
findings in the hyperglycemic and hyperinsulinemic
BBZDR/Wor rat are in striking contrast to the STZ (15)
and BB/Wor rat (7) models in which NAD develops in a
setting of hyperglycemia and decreased circulating levels
of insulin, C-peptide, and IGF-I (7, 23, 25–29).
Sympathetic NAD does not develop in other type 2
diabetic animal models such as the ZDF-rat (30), which
J Neuropathol Exp Neurol, Vol 63, May, 2004
454
SCHMIDT ET AL
Fig. 1. Neuroaxonal dystrophy in diabetic rat SMG. A, B: Eight-month STZ-diabetic rat. The diabetic SMG is shown consisting of a normal appearing principal sympathetic neuron and an intimately apposed swollen dystrophic nerve ending (arrow,
A, shown at higher magnification in B). The dystrophic axon contains tubulovesicular elements and compact membrane aggregates
(arrow, B), scattered dense neurotransmitter containing granules, and exhibits synaptic specialization with a perisomal or dendritic
spine (arrowhead, B). C–E: C: STZ-rat; D, E: BBZDR/Wor rat. Other dystrophic axons contain ‘‘fingerprint-like’’ membranous
aggregates (C), neurofilaments (D), or mixed collections of subcellular organelles, including mitochondria, dense and multivesicular bodies (E). F: STZ-rat. Coarse tubulovesicular profiles often exhibit synaptic specializations (arrow), in which they typically
represent the postsynaptic element. Original magnifications: A, 34,080; B, 313,910; C, 333,500; D, 316,570; E, 35,050. F,
316,240.
J Neuropathol Exp Neurol, Vol 63, May, 2004
SYMPATHETIC DYSTROPHY IN TYPES 1 AND 2 DIABETES
Fig. 1.
455
(Continued)
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456
SCHMIDT ET AL
TABLE 2A
NAD in STZ-, BB/Wor-, and BBZDR/Wor-Rat SMG
Model
STZ
BB Control
BB/Wor
BBZDR/Wor
n
Status
6
6
11
17
5
Control
Diabetic
Control
Diabetic
Diabetic
A large study of human subjects with type 1 and 2 diabetes showed a 40% to 50% decrease in serum IGF-I in
both groups (36), which is consistent with most studies
(37–42), although no change (43) or increased IGF-I levels have also been reported (44). Moreover, other studies
have reported that serum IGF-I levels are preferentially
decreased in patients with sensory and autonomic neuropathic symptoms compared to either non-neuropathic
diabetics or controls (45).
These findings suggest that a likely candidate in the
pathogenesis of sympathetic NAD is impaired direct or
indirect neurotrophic support mediated via impaired insulin action. We (9, 10, 18, 19, 30) and others (11–13)
have previously proposed that impaired insulin action
may play an important role in the development of the
usually more severe diabetic polyneuropathies accompanying type 1 diabetes.
One major candidate for a critical role in the pathogenesis of NAD is insulin itself, serving as a neurotrophic
factor independent of its glucose regulatory functions. A
neurotrophic role for insulin has been previously proposed in the pathogenesis of diabetic sensory and autonomic neuropathies (11–13, 30). This is based on its ability to bind to specific receptors on sensory and
sympathetic neuronal perikarya axons and nerve terminals (46–48) and to support their survival in cell cultures
(13, 49). Serum levels of insulin are increased in
BBZDR/Wor rats and significantly decreased in BB/Wor
rats in our current experiments. STZ-diabetic rats survive
for extended intervals without exogenous insulin supplementation, maintaining serum concentration at approximately 25% of normal circulating levels of control rats
(23, 25–29, Schmidt et al, unpublished data). In contrast,
BB/Wor rats require administration of small maintenance
doses of insulin in order to survive, while remaining hyperglycemic. This small insulin dose failed to prevent the
development of NAD in the SMG and mesenteric nerves
in these studies, although the absolute frequency of NAD
in ileal mesenteric nerves is more than 2-fold greater in
Ratio
(NAD/neuron)
0.12
0.30
0.15
0.57
0.21
6
6
6
6
6
0.02
0.03*
0.02
0.06†‡
0.05
Comparison of the frequency of dystrophic axons (expressed
as a ratio of number of dystrophic axons per nucleated neuron)
in the SMG of 8-month diabetic STZ, BB/Wor and BBZDR/
Wor rats in comparison to non-diabetic age-matched controls.
Values represent the means 6 SEM.
Statistical comparison: † p # 0.0001; * p # 0.001 vs control;
‡ p # 0.001 vs BBZDR/Wor rats.
shows chronic hyperglycemia, hyperinsulinemia, and
normal levels of serum IGF-I, or the hyperinsulinemic
type 2 db/db mouse (31). On the other hand, sympathetic
NAD is a characteristic finding in several hypoinsulinemic type 1 diabetic models examined to date, including
insulinopenic rat models such as the BB/Wor rat (7),
STZ-induced Lewis rats (32, 33), and STZ-induced
Sprague Dawley rats (15) and insulinopenic mice-models
such as STZ-induced diabetes in C57BL6, DBA-2,
B6D2F1, BL6/NCR, and autoimmune NOD-mice (31)
and Chinese hamsters (34). These findings suggest that
the development of sympathetic NAD is somehow associated with insulin and/or C-peptide deficiency or their
consequences such as altered levels or action of IGF-I or
NGF.
On the other hand, humans with type 2 diabetes do
develop sympathetic NAD (35), which seems to be at
odds with the current findings in the BBZDR/Wor rat and
other hyperinsulinemic type 2 diabetic murine models.
However, autopsy-based studies of type 2 diabetic human
subjects (35) are confounded by the fact that chronically
type 2 diabetic patients frequently become dependent on
exogenous insulin as pancreatic b-cell exhaustion occurs.
TABLE 2B
Subpopulations of Dystrophic Neurites in the SMG of STZ-, BB/Wor-, and BBZDR/Wor-Diabetic Rats and Non-Diabetic,
Age-Matched Controls
Model
STZ
BB Control
BB/Wor
BBZDR/Wor
n
Status
6
6
11
17
5
Control
Diabetic
Control
Diabetic
Diabetic
Tubulovesicular
elements (%)
54
39
16
10
8
6
6
6
6
6
7
9
3
1
6
Neurofilaments
(%)
22
38
14
60
41
6
6
6
6
6
7
7
7
4*
6*
Mixed organelles Dendritic forms
(%)
(%)
19
17
56
20
40
6
6
6
6
6
10
6
6
5*
12
4
10
14
10
11
6
6
6
6
6
2
3
3
2
8
Dystrophic neurites of 8-month diabetic rats were separated into subpopulations for each animal based on ultrastructural content
of dystrophic neurites and expressed as a percentage of total numbers calculated for each animal. Values represent the means 6
SEM.
Statistical comparison: * p # 0.001 vs control.
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SYMPATHETIC DYSTROPHY IN TYPES 1 AND 2 DIABETES
457
Fig. 2. Neuroaxonal dystrophy in the ileal mesenteric nerves of STZ, BB/Wor and BBZDR/Wor diabetic rats. A, B: STZdiabetic rat. A mesenteric nerve fascicle with a markedly swollen axon (arrow, A) containing an admixture of mitochondria,
dense core and coated vesicles, as well as disorganized microtubules (B). C: BB/Wor rat. Dystrophic axons (arrow) containing
compact tubulovesicular elements. D: BBZDR/Wor rat. Although containing the same subcellular constituents, a typical dystrophic
axon (arrow) in the ileal mesenteric nerves of BBZDR/Wor rats was significantly smaller than those of STZ and BB/Wor rats.
Original magnifications: A, 31,560; B, 314,960; C, 33,710; D, 332,000.
J Neuropathol Exp Neurol, Vol 63, May, 2004
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SCHMIDT ET AL
TABLE 3
Neuroaxonal Dystrophy in Rat Ileal Mesenteric Nerves
Model
STZ
BB Control
BB/Wor
BBZDR/Wor
Status
n
(rats)
Control
Diabetic
Control
Diabetic
Diabetic
6
8
11
18
7
Axons/fascicle
478
512
569
466
505
6
6
6
6
6
30
34
47
17‡
34
NAD
(lesions/fascicle)
0.14
2.2
0.06
0.93
0.14
6
6
6
6
6
0.06
0.7‡
0.03
0.17*
0.04
NAD
(% total axons)
0.03
0.45
0.009
0.23
0.03
6
6
6
6
6
0.01
0.13‡
0.004
0.05*
0.01‡
The frequency of NAD in ileal mesenteric nerves is shown as a function of number of dystrophic axons per ileal mesenteric
nerve fascicle and as a percentage of total axon number. Values represent the means 6 SEM of n rats.
Statistical comparison: * p # 0.001; ‡ p # 0.05 vs control.
STZ-rats compared to BB/Wor rats. The inability of small
doses of insulin to prevent the development of NAD in
BB/Wor rats may reflect a threshold effect, since salutary
effects of higher doses of insulin on the frequency of
NAD in the SMG and ileal mesenteric nerves of the STZrat have been described in the absence of tight metabolic
control (32, 33), which may reflect a direct insulin neurotrophic effect. However, in the DCCT trial in which
type 1 diabetic patients were controlled with intensive
insulin treatment to achieve close to normal glycemic levels, diabetic neuropathy was only prevented by 50% over
a 5-year period (50), suggesting that replacement of insulin and dose to euglycemic control alone are not sufficient. We have previously suggested that replacement of
C-peptide may also be necessary to optimize prevention
(20, also see below).
A second candidate neurotrophic factor in diabetes-induced sympathetic NAD is IGF-I, which is typically altered in parallel with insulin levels in types 1 and 2 diabetes, complicating the identification of the critical
pathogenetic element as either insulin or IGF-I. Our current results show a modest (30%) but statistically significant decrease in IGF-I levels in BB/Wor rats compared
to controls and preservation of serum IGF-I levels in
BBZDR/Wor rats. A large number of studies have shown
more robust decreases (50%–86%) in IGF-I in STZ rats
at various durations of diabetes (23, 25–29). We have
found that administration of pharmacologic doses of
rhIGF-I reverse established NAD within 2 months in
chronically diabetic STZ-diabetic rats (9). In this study,
a separate group of 6-month diabetic rats received small
daily doses of regular insulin (Humulin, 0.3 U/kg/day,
s.c. to mimic the transient hypoglycemic effect of high
doses of rhIGF-I), which did not affect glycated hemoglobin nor did it influence the frequency of established
NAD in the SMG and ileal mesenteric nerves (9). Together with the present results these findings are consistent with the contention that IGF-I has a direct neurotrophic effect on sympathetic NAD. The effect of insulin
on the frequency of NAD may therefore reflect an insulin-induced increase in hepatic synthesis of circulating
J Neuropathol Exp Neurol, Vol 63, May, 2004
IGF-I (46, 51). Decreased levels of mRNA transcripts for
IGF-I and its receptor as well as diminished 125I-IGF-I
binding to crude membrane preparations have been reported in the SCG of 3-month diabetic STZ-rats (52),
although precise cellular localization was not determined.
STZ-diabetic rats also showed a 50% to 86% decrease in
the levels of circulating IGF-I and .75% loss of circulating insulin (25–30), as well as increased levels of
IGFBP-1 (53), one of several IGF-I binding proteins that
may sequester IGF-I, reducing its activity or control its
local availability (11). Although there is evidence to suggest that circulating IGF-I plays a role in the genesis of
diabetic polyneuropathy, there is also evidence indicating
that perturbed endogenous neuronal IGFs are of pathogenetic significance (54).
Circulating C-peptide cleaved from the proinsulin molecule is secreted in concentrations that parallel endogenous insulin levels. C-peptide has been identified as another neurotrophic factor with a pathogenetic role in the
development of the more severe diabetic polyneuropathy
in type 1 diabetes (10, 20, 55, 56). C-peptide has insulinomimetic effects and enhances insulin-signaling activities in the presence of low insulin levels (57, 58). Pertinent to the discussion of the present data, C-peptide has
been shown to normalize the expression of several neurotrophic factors such as IGF-I, IGF-IR, IGF-II, and the
insulin-receptor itself in dorsal root ganglia, somatic peripheral nerve, and hippocampus in type 1 BB/Wor rats
(18, 59). Whether this also occurs in peripheral sympathetic nerves has not been examined, however, it appears
that in somatic nerves, C-peptide deficiency plays an important role in the perturbed regulation of several neurotrophic factors, including IGF-I, and that insulin supplementation alone is not sufficient to maintain the
integrity of these neurotrophic influences (18, 49). In support of this supposition, the beneficial effect of C-peptide
was demonstrated in a recent clinical trial in which replacement of C-peptide in a well-controlled type 1 population resulted in a significant improvement of sensory
nerve conduction velocity and vibration perception as
compared to patients who received insulin alone (60).
SYMPATHETIC DYSTROPHY IN TYPES 1 AND 2 DIABETES
These findings may explain the suboptimal outcome of
the DCCT-trial.
Summary
The diabetic milieu is an exceedingly complex environment in which many pathogenetic mechanisms may
exist and interplay to result in the varied presentations of
diabetic neuropathy in man and experimental animal
models. The use of several animal models has shown that
hyperglycemia alone is not sufficient for the development
of NAD and suggests a possible role for the superimposed deficiency of IGF-I, insulin or C-peptide, acting as
neurotrophic substances, as critical elements in the pathogenesis of sympathetic NAD. Indeed, the development
of NAD in this model may require deficiency of one or
more neurotrophic substances in the presence of hyperglycemia with its attendant metabolic alterations (e.g. increased sorbitol pathway flux, exaggerated oxidative
stress, mitochondriopathy, or formation of advanced glycosylation endproducts).
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Received November 11, 2003
Revision received January 28, 2004
Accepted January 30, 2004