Evaluation of the neonatal streptozotocin model of diabetes in rats: Evidence for a model of neuropathic pain

Abstract

Background: The purpose of this study was to evaluate the participation of satellite glial cells (SGC), microglia and astrocytes in a model of streptozotocin-induced diabetes initiated in neonatal rats (nSTZ) and to determine the pharmacological profile for pain relief.

Methods: nSTZ was used to induce experimental diabetes. Von Frey filaments were used to assess tactile allodynia. Drugs were given by systemic administration. Western blotting and immunohistochemistry were used to determine protein expression and cellular localization.

Results: nSTZ produced mild hyperglycemia, weight loss, glucose intolerance, and reduction of nerve conduction velocity of C fibers. Moreover, nSTZ enhanced activating transcription factor 3 (ATF3) immunoreactivity in dorsal root ganglia (DRG) and sciatic nerve of adult rats. ATF3 was found in SGC (GFAP+ cells) surrounding DRG at week 16. Late changes in ATF3 immunoreactivity in DRG correlated with up-regulation of ATF3 and GFAP protein expression. nSTZ increased GFAP and OX-42 immunoreactivity and percentage of hypertrophied and ameboid microglia in the spinal dorsal horn. These changes correlated with the presence of mechanical hypersensitivity (tactile allodynia). Administration of gabapentin (30-100mg/kg, po) and metformin (200mg/kg/day, po for 2 weeks) alleviated tactile allodynia, whereas morphine (1-3mg/kg, ip) had a modest effect.

Conclusions: Results suggest that nSTZ leads to activation of SGC, microglia and astrocytes in DRG and spinal cord. Pharmacological profile in the nSTZ model resembles diabetic neuropathic pain in humans. Our findings support the conclusion that the nSTZ rat model has utility for the study of a long-lasting diabetic neuropathic pain.

Keywords: Astrocytes; Diabetes; Microglia; Satellite glial cells; nSTZ model.

Figure 1

Characteristics of the neonatal streptozotocin (nSTZ) induced diabetic rat model. (A) Time course of capillary glucose concentrations and (B) body weight from control (CT) and diabetic (DB) rats. Data in panels A and B are the mean of 10 animals ± SEM. Capillary glucose levels (C, E, G, I) and (D, F, H, J) serum insulin concentrations before and during an oral glucose tolerance test in control and diabetic rats. Black arrows indicate the time of glucose (2 g/kg) administration. Data in panels C-J are the mean of 5 animals ± SEM. (A) ***p < 0.001, significantly different from the control-non fasted group and ^&&&p < 0.001, significantly different from the CT-fasted group, as determined by two-way ANOVA followed by the Bonferroni test. (B-J) *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from the CT-group, as determined by two-way ANOVA followed by the Bonferroni test.

Figure 2

Effects of nSTZ on nerve conduction velocity and compound action potential of C fibers (cAP). (A) Illustration of the preparation and recording of the cAP of C fibers. (B) Effect of nSTZ in nerve conduction velocity of C fibers in control (CT) and diabetic (DB) rats. (C) Effect of nSTZ in the C fibers cAP from control (CT) and diabetic (DB) rats. Upper panel: Representative traces of C fibers cAP recorded from control and diabetic rats. Lower panel: Quantification of normalized C fibers cAP area from control and diabetic rats. Data are the mean of 6 animals ± SEM. *p < 0.05, significantly different from the control group, as determined by Student´s t-test.

Figure 3

Effects of the nSTZ on immunoreactivity and expression of ATF3 in lumbar DRG and sciatic nerve. (A-H) Representative images of ATF3 immunoreactivity in L4-L6 DRG from control (CT) and diabetic (DB) rats. (I-J) Representative images of ATF3 immunoreactivity in sciatic nerve in CT and DB rats at week 16. (K-M) representative images of ATF3 immunoreactivity co-localization with satellite glial cells (GFAP+) in L4-L6 DRG at week 16. (N) Time-course of ATF3 and GFAP expression in L4-L6 DRGs by western blot (n=4). (O) Quantification of ATF3-Immunoreactivity in L4-L6 DRG (5 slices from a group of 3 rats). **p < 0.01, ***p < 0.001 Significantly different from the control group (CT), as determined by one-way ANOVA followed by the Bonferroni test.

Figure 4

Effects of nSTZ on glial cells activation and mechanical pain hypersensitivity. (A-E) Representative images of OX-42 staining in the spinal dorsal horn from control (CT) and diabetic (DB) rats. (F-I) Quantification of microglial morphology in the spinal dorsal horn from CT and DB rats (5 slices from a group of 3 rats). (J-N) Representative images of GFAP staining in the spinal dorsal horn from CT and DB rats. (O) Quantification of GFAP fluorescence intensity in CT and DB conditions (5 slices from a group of 3 rats). (P) Time course of mechanical hypersensitivity produced by vehicle or nSTZ administration. Data are the mean of 12 rats ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from the CT group, as determined by one- or two-way ANOVA followed by the Bonferroni test.

Figure 5

Pharmacological effects of drugs. (A) Antinociceptive effects of gabapentin (30–100 mg/kg, po), (B) morphine (1–3 mg/kg, ip), (C) diclofenac (1–10 mg/kg, po), and (D) metformin (200 mg/kg, po, daily from week 14 to week 16) in the mechanical hypersensitivity produced by nSTZ. Data are the mean of 6 rats ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from the CT group, as determined by two-way ANOVA followed by the Bonferroni test.