Global transcriptional programs in peripheral nerve endoneurium and DRG are resistant to the onset of type 1 diabetic neuropathy in Ins2 mice

Abstract

While the morphological and electrophysiological changes underlying diabetic peripheral neuropathy (DPN) are relatively well described, the involved molecular mechanisms remain poorly understood. In this study, we investigated whether phenotypic changes associated with early DPN are correlated with transcriptional alterations in the neuronal (dorsal root ganglia [DRG]) or the glial (endoneurium) compartments of the peripheral nerve. We used Ins2(Akita/+) mice to study transcriptional changes underlying the onset of DPN in type 1 diabetes mellitus (DM). Weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Ins2(Akita/+) and control mice during the first three months of life in order to determine the onset of DPN. Based on this phenotypic characterization, we performed gene expression profiling using sciatic nerve endoneurium and DRG isolated from pre-symptomatic and early symptomatic Ins2(Akita/+) mice and sex-matched littermate controls. Our phenotypic analysis of Ins2(Akita/+) mice revealed that DPN, as measured by reduced MNCV, is detectable in affected animals already one week after the onset of hyperglycemia. Surprisingly, the onset of DPN was not associated with any major persistent changes in gene expression profiles in either sciatic nerve endoneurium or DRG. Our data thus demonstrated that the transcriptional programs in both endoneurial and neuronal compartments of the peripheral nerve are relatively resistant to the onset of hyperglycemia and hypoinsulinemia suggesting that either minor transcriptional alterations or changes on the proteomic level are responsible for the functional deficits associated with the onset of DPN in type 1 DM.

Figure 1. Phenotypic characterization of Ins2^Akita/+ mice.

Body weight (A), tail vein blood glucose (B) and motor nerve conduction velocity (MNCV, C) were measured at depicted time-points in Ins2^Akita/+ and Ins2^+/+ mice. All results are expressed as the mean ± standard error of the mean (S.E.M.). (Ins2^+/+: n = 4; Ins2^Akita/+: n = 4).

Figure 2. Characterization of motor and sensory behavior in Ins2^Akita/+ mice.

Sciatic nerve motor (MNCV, A) and tail sensory (SNCV, B) nerve conduction velocities were compared in diabetic (Ins2^Akita/+) and control (Ins2^+/+) mice at two and five months of age. Results are expressed as the mean ± standard error of the mean (S.E.M). (Ins2^+/+: n = 4; Ins2^Akita/+: n = 4). C) Sensory performances were evaluated in two month old Ins2^Akita/+ and control littermates using the hot plate test at 49, 52 and 55°C. Results are expressed as the mean±standard error of the mean (S.E.M.) and analyzed by Student's t test (Ins2^+/+: n = 8; Ins2^Akita/+: n = 7). (*) p<0.05.

Figure 3. Morphometric evaluation of peripheral nervous system of Ins2^Akita/+ mice.

A) Semi-thin toluidin blue stained cross sections of sciatic nerves from three months old Ins2^Akita/+ and control mice show well preserved nerve structures in both genotypes (scale bar: 15 µm). B) The scatter plot displays g ratios (g ratio = axon area/axon+myelin area) of individual axons as a function of the respective axonal diameters determined using sciatic nerves of three month old wild-type and Ins2^Akita/+ mice. Each point corresponds to one fiber. Thin dark-grey and thick light-grey lines represent the trend-lines for Ins2^Akita/+ and wild-type mice respectively. The two lines are superimposed reflecting close similarity of the two data sets. C) Axonal distribution represented as the percentage of axons for each class of sizes does not reveal any differences between wild-type and Ins2^Akita/+ mice.

Figure 4. Design of the microarray experiment.

A) Eight time-points were selected between 20 and 56 days of age. This time window covers the pre-symptomatic situation (P20, 20 days old), the onset of hyperglycemia (P24–P28), the onset of DPN (P32–P36) and later time-points with a clear symptomatic situation (P36–P56) in Ins2^Akita/+ mice. Squares: blood glucose; circles: MNCV. B) Schematic view of the microarray data analysis.

Figure 5. Expression profiles of selected genes involved in sensory neuron development and function.

Normalized levels of expression at eight analyzed time-points (P20–P56) in both diabetic (Ins2^Akita/+) and control (Ins2^+/+) mice are shown. A) Ntrk1, B) Ret, C) Scn7a, D) Scn8a, E) Scn10a, F) Scn11a. All values were normalized by the median of the intensity obtained for a probe throughout the chips.

Figure 6. Expression profiles of selected genes involved in Schwann cell myelination.

Normalized levels of expression at eight analyzed time-points (P20–P56) are shown for control (Ins2^+/+) mice. In diabetic (Ins2^Akita/+) mice, the expression was analyzed only at 7 time-points (data point P36 was excluded, see methods for more explanations). A) Scip, B) Krox20, C) Pmp22, D) Mbp, E) Mag, F) Plp. All values are normalized by the median of the intensity obtained for a probe throughout the chips.