Background
Diabetic sensory neuropathies are a common, clinically observed sequelae of hyperglycemia
and are characterized by a progressive degradation of primary afferent function [[1],[2]]. Functional and structural evidence suggest an early and frequent involvement of
small diameter primary sensory neurons leading to nociceptive abnormalities [[2],[3],[4]]. In order to examine basic mechanisms underlying this disorder, we utilized the
OVE26 transgenic mouse model of diabetes mellitus [[5],[6]] to examine the effects of long-standing hyperglycemia on enzyme histochemical indicators
of sensory neuron metabolism and evaluate the potential utility of this model for
future studies of diabetic neuropathy. The OVE26 mouse line uses cell-specific overexpression
of calmodulin to destroy pancreatic β-cells and the result is a viable diabetic mouse
(>1 year survival) that displays both early-onset (<1 week after birth) and chronic
elevated blood glucose (>500 mg/dl) and decreased serum and pancreatic insulin (<50%
of normal) [[5],[6]]. Enzyme histochemical techniques demonstrated to be sensitive to neuronal perturbation
[[7]] were used to examine the impact of long-standing hyperglycemia and hypoinsulinemia
on the distribution and activity of lysosomal acid β-glycerophosphatase (AP), cytochrome
oxidase (CO), and NADPH-diaphorase (NADPH-d; a correlate of nitric oxide synthase
in aldehyde fixed tissue [[8]]) in both sensory ganglia and the spinal cord. It has been previously demonstrated
[[7],[11]] that the metabolic status of sensory neurons, as reflected by the endogenous activity
of specific homeostatic enzymes, is sensitive to injury and perturbation. Therefore,
these enzymes were selected as putatively reflective of mitochondrial function (CO),
lysosomal or degradative activity (AP) and primary sensory neuron injury or repair
(NADPH-diaphorase).
The OVE26 transgenic mouse line (characterized by the insulin promoter-linked overexpression
of calmodulin in pancreatic β-cells) used in this study displays a well-characterized
chronic hyperglycemia and hypoinsulinemia within days after birth. [[5],[6]]. Ten (five OVE26 transgenic and five age-matched, control FVB animals) aged (>365
days old) mice were anesthetized with pentobarbital, perfused with 4% paraformaldehyde
and the lumbar spinal cord and sensory ganglia removed, sectioned and processed for
AP, CO or NADPH-d enzyme histochemistry as previously described [[7],[9],[10],[11],[12]]. Counts of primary sensory somata were conducted on toluidine blue counterstained
sections of L5 spinal ganglia and quantified using previously published methodologies
[[7],[12]].
Quantitative analysis of CO, AP and NADPH-d staining was undertaken on both the dorsal
horn of the L5 segment of the spinal cord and the large and small cells of L5 sensory
ganglion using previously described densitometric analysis [[7],[12]]. The entire mediolateral extent of lamina I to III was selected for staining intensity
measurement. Statistical analyses (t-test, Mann-Whitney Rank Sum test, one way analysis of variance, Kruskal-Wallis analysis
of variance on ranks, z-test of proportions) were conducted using SigmaStat (Jandel).
Controls for densitometric analysis consisted of: 1) simultaneous sectioning and mounting
of diabetic and control tissue on the same slide to ensure identical histological
processing; 2) statistical analysis to verify consistency of staining between animals
within control and experimental groups; 3) correction for small fluctuations in tissue
opacity/thickness by subtractive illumination whereby the density value of white matter
was subtracted from the immediately adjacent ventral horn; and 4) manual adjustment
and calibration of the video camera parameters and microscope illumination and acquisition
of all images using identical settings. All experiments were conducted in accordance
with the guidelines of our institutions and the National Institutes of Health regarding
the care and use of animals for experimental procedures.
Prior to fixative perfusion, the phenotypic status of OVE26 diabetic mice were confirmed
by their characteristic small eyes caused by the GR19 gene in their transgenic construct
[[5]]. All adult OVE26 mice maintained fed blood glucose levels of at least 400 mg/dl.
At the histological level, a survey [[13]] of the ratio of small (50 and 500 μm2 area) to large (500 and 1950 μm2 area) primary sensory somata in the fifth lumbar spinal ganglia revealed a significant
decrease in the proportion of small to large cells in diabetic (1.29:1 small:large
perikarya) compared to control (1.94:1 small:large perikarya) mice (P < 0.05 by z-test of proportions; 417 cells measured). Quantitative densitometric analysis of
the abundance and distribution of enzyme histochemical reaction product in dorsal
root ganglia (DRG) revealed substantive differences between diabetic and control mice
(720 cells were quantified for both densitometry and cell size; 240 cells for each
enzyme). Small somata from the ganglia of diabetic mice exhibited lower levels of
AP (13.4% decrease; P < 0.001) and CO ([Fig 1]; 9% decrease; P < 0.001) reaction product and an increase in the density of the reaction product
for NADPH-d ([Fig. 1]; 13.2% increase; P < 0.001) in comparison to control animals. No differences were observed in large
diameter neurons from diabetic as compared to control animals.
Figure 1
Enzyme histochemical reaction product in the fifth lumbar dorsal root ganglion from
control and diabetic mice. (A,B) Cytochrome oxidase reaction product in sections from
control (A) and the diabetic (B) mice. Diabetic mice display reduced levels of CO
reaction product compared to control. Quantitative analysis revealed a decrease in
CO reaction product density in small neuronal somata. (C,D) NADPH-diaphorase reaction
product in sections from control (C) and diabetic (D) mice. Diabetic mice display
an increase in NADPH-diaphorase reaction product density compared to control. Scale
bar in microns.
In the spinal cord, all observed differences were confined to lamina I to III. Motoneuron
somata in the ventral horn appeared both qualitatively and quantitatively similar
in diabetic and control animals. In diabetic animals, there was an observable loss
of AP reaction product in lamina I and II of the dorsal horn ([Fig. 2]) as compared to control mice ([Fig. 2]). Similarly, these laminae appeared to have qualitatively fewer NADPH-d labeled
fibers and neuronal somata in diabetic ([Fig. 2]) as compared to control animals. ([Fig. 2]). The decrease of both AP and NADPH-d labeling was most profound in the medial portion
of lamina I and II. Quantitative densitometric analysis supported the qualitative
observations and revealed significantly reduced levels of AP (P = 0.026; 27 sections quantified) and NADPH-d (P < 0.001; 27 sections quantified) reaction product in lamina I and II of the dorsal
horn of control and diabetic mice ([Table 1]). No significant differences were observed in qualitative staining appearance or
intensity of CO reaction product labeling in the dorsal horn of diabetic, compared
to non-diabetic animals (31 sections quantified).
Table 1
Quantitative histochemical reaction product in the dorsal horn of control and diabetic
mice.
|
Enzyme
|
Control Mean ± S.D.
|
Diabetic Mean ± S.D
|
t-test significance level
|
|
AP
|
6.058 ± 0.254
|
5.949 ± 0.210
|
P = 0.026*
|
|
CO
|
3.997 ± 0.172
|
3.989 ± 0.137
|
P = 0.800
|
|
NADPH-d
|
1.320 ± 0.354
|
0.733 ± 0.228
|
P < 0.001*
|
* indicates significant difference. Standard deviation (S.D.). Following densitometric
measurements, the numbers were transformed to a 0 to 10 scale for clarity of presentation
and comparison. Optical density values fall along a range of 0 representing minimal
staining intensity (no staining) and 10 representing extremely dense staining (black).
Figure 2
Enzyme histochemical reaction product in the dorsal horn of the fifth lumbar spinal
cord from control and diabetic mice. (A,B) Acid phosphatase reaction product in sections
from control (A) and the diabetic (B) mice. Diabetic mice display reduced levels of
AP reaction product in medial lamina I and II of the dorsal horn. (C,D) NADPH-diaphorase
reaction product in lamina I and II of control (C) and diabetic (D) mice. A reduction
in the number of labeled fibers and somata can be observed in the superficial dorsal
horn of diabetic animals. Scale bar in microns and dorsal horn is to top in all sections.
Here we have demonstrated that chronic hyperglycemia has an impact on both the survival
and metabolic profile of primary sensory neurons. The observed decrease in the ratio
of small to large diameter primary sensory somata in diabetic animals most likely
represents a loss of unmyelinated or small myelinated primary sensory neurons although
a relative increase in the number of large myelinated neurons, however, unlikely,
cannot be discounted. Nonetheless, the former interpretation is supported by the observed
decrease in AP labeling in the dorsal horn of the spinal cord. The observed decrease
in AP intensity in the surviving small neuronal somata from the DRG of the OVE26 animals,
as compared to small neurons from control DRG, suggests that acid phosphatase activity
in those cells is depressed. As FRAP containing sensory neurons represent a subpopulation
of unmyelinated C-fibers [[14]], our results suggest that there is a loss, or at least a metabolic disruption of,
unmyelinated neurons in the dorsal horn and in DRG. These results are consistent with
the possibility of apoptosis and cell loss in the DRG of rodent models of diabetes
[[15],[16]] although differences in model, species and duration of hyperglycemia must be considered
[[17],[18]] along with the likelihood that there are a spectrum of different diabetic neuropathies
including painful (small fiber involvement) and non-painful (large-fiber involvement)
syndromes [[2],[3]]. In light of this, it should not be entirely unexpected that our observations of
a putative small fiber disorder complements findings of large fiber disorders in alternate
animal models of diabetes [[19]].
Cytochrome oxidase is the terminal enzyme in the electron transport chain, and is
therefore considered to be a strong indicator of somatic mitochondrial activity. The
decrease in CO staining within small DRG neurons, as compared to a similar cohort
of small neurons from control animal ganglia, suggests a disruption of oxidative metabolism
which corresponds well with results from other animal models of diabetes that demonstrate
diminished CO activity or disruptions in mitochondrial morphology or function [[16],[20],[21],[22]]. Alternatively, our observed decrease in CO staining may reflect a simple decrease
in mitochondrial number, as DRG neurons exposed to high glucose in vitro exposure contain fewer mitochondria [[23]]. The lack of an observed change in CO activity in the dorsal horn is not unexpected
as both physical (axotomy) and functional (tetrodotoxin) disconnection have previously
been shown to leave CO activity in the dorsal horn unaltered [[7]].
In DRG and the dorsal horn of the spinal cord, NADPH-d activity levels have been previously
shown to be responsive to peripheral neuronal injury or attenuation of electrical
activity [[7]]. Our results suggest that in addition to the pathological state that led to our
observed loss of sensory neurons (and diminished NADPH-d labeling in the dorsal horn),
there is a ongoing perturbation resulting in increased NADPH-d labeling in small DRG
neurons from hyperglycemic animals as compared to the primary sensory somata from
normoglycemic animals. As utilized here, NADPH-d enzyme histochemical reaction product
represents nitric oxide synthase activity [[8]]. The elevated ganglionic NADPH-diaphorase and diminished CO labeling in the DRG
of hyperglycemic mice is consistent with previously proposed inhibition of CO activity
[[24]] by the product of nitric oxide synthase, nitric oxide. Although the reported statistically
significant changes may appear to be quantitatively modest, these percent changes
represent group averages. Qualitatively and quantitatively, the changes are more pronounced
in some animals and tissues sections, and less obvious in others. This is not unexpected
as chronic diseases processes impact individuals with profound variability in both
severity and temporal progress.
Our results suggest that the OVE26 model of chronic hyperglycemia does alter the overall
neurochemical profile of the sensory nervous system through cell loss and/or altered
enzyme activity and that this pathology seems to specifically impact unmyelinated
and/or small myelinated primary sensory neurons.
Declaration of competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
RZ completed this work as part of his doctoral dissertation and was involved in the
writing of this manuscript and contributed both intellectually and practically to
the content. PE created, characterized and supplied the transgenic mice and was also
involved in the writing of this manuscript and contributed both intellectually and
practically to the content. PC provided the lab, supervision, and support for this
work, exclusive of that associated with generation and characterization of the mouse
model. PC was also involved in the design and coordination of this study and participated
in the writing of this manuscript and contributed both intellectually and practically
to the content. All authors read and approved the final manuscript.
