Erythropoietin both protects from and reverses experimental diabetic neuropathy

Abstract

Erythropoietin (EPO) possesses generalized neuroprotective and neurotrophic actions. We tested the efficacy of recombinant human EPO (rhEPO) in preventing and reversing nerve dysfunction in streptozotocin (STZ)-induced diabetes in rats. Two days after STZ [60 mg/kg of body weight (b.w.), i.p.], diabetic animals were administered rhEPO (40 microg/kg of b.w.) three times weekly for 5 weeks either immediately (preventive) before or after a 5-week delay (therapeutic) after induction of hyperglycemia or at a lower dose (8 microg/kg of b.w. once per week) for 8 weeks (prolonged). Tail-nerve conduction velocities (NCV) was assessed at 5 and 11 weeks for the preventive and therapeutic schedule, respectively. Compared to nondiabetic rats, NCV was 20% lower after 5 weeks in the STZ group, and this decrease was attenuated 50% by rhEPO. Furthermore, the reduction of Na(+),K(+)-ATPase activity of diabetic nerves (by 55%) was limited to 24% in the rhEPO-treated group. In the therapeutic schedule, NCV was reduced by 50% after 11 weeks but by only 23% in the rhEPO-treated group. rhEPO treatment attenuated the decrease in compound muscle action potential in diabetic rats. In addition, rhEPO treatment was associated with a preservation of footpad cutaneous innervation, as assessed by protein gene product 9.5 immunostaining. Diabetic rats developed alterations in mechanical and thermal nociception, which were partially reversed by rhEPO given either in a preventative or therapeutic manner. These observations suggest that administration of rhEPO or its analogues may be useful in the treatment of diabetic neuropathy.

Fig. 1.

Schematic representation of the treatment schedules used.

Fig. 2.

rhEPO prevents and restores changes in thermal and mechanical thresholds in diabetic rats. Control or STZ-diabetic rats were treated with rhEPO according to the preventive (A) or therapeutic (B) schedule. Thermal sensitivity threshold (x axis) is expressed as withdrawal latency in seconds. Mechanical threshold (y axis) is expressed as paw withdrawal latency in grams. Measurements were carried out at 5 (A)or9(B) weeks. Data are the mean ± SEM (number of rats in each group is indicated in parentheses).

Fig. 3.

rhEPO restores the decrease in NCV in diabetic rats. Experimental design was the same as in Fig. 1. Tail NCV was measured at 11 weeks (therapeutic schedule). Data are expressed as m/sec and are the mean ± SEM (number of rats is indicated in parentheses). *, P < 0.001 vs. nondiabetic control by Tukey–Kramer test; †, P < 0.01 vs. STZ.

Fig. 4.

rhEPO prevents the decrease in CMAP in diabetic rats. CMAP was evaluated at week 8. rhEPO was administered according to the prolonged preventive schedule. Data are expressed as mV and are the mean ± SEM (number of rats is indicated in parentheses). *, P < 0.005 vs. nondiabetic control by Tukey–Kramer test; †, P < 0. 005 vs. nondiabetic control; ‡, P < 0.01 vs. diabetic groups.

Fig. 5.

rhEPO restores the loss of intraepidermal fibers in diabetic rats. Hind-paw skin biopsy in control (A), STZ-diabetic at 5 weeks (B), and after 5 weeks of rhEPO treatment initiated after 5 weeks of diabetes (C). Microphotographs are PGP 9.5 immunostaining in 20-μm-thick sections. (Bar = 30 μm.) Small arrows indicate IENF, and large arrows indicate dermal nerve bundles. Note the fragmented and discontinuous PGP 9.5 immunoreactivity in IENF indicative of axonal degeneration. (D) Quantification of IENF density. Data are expressed as the number of linear density of IENF and are the mean ± SEM (number of rats is indicated in parentheses). *, P < 0.05 vs. nondiabetic control by Tukey–Kramer test; †, P < 0. 05 vs. untreated diabetic rats.

Fig. 6.

rhEPO restores the decrease in Na⁺,K⁺-ATPase activity in diabetic rats. Experimental design was the same as in Fig. 1. Na⁺,K⁺-ATPase activity was measured at 11 weeks (therapeutic schedule). Data are expressed as μmol/min per mg protein and are the mean ± SEM (number of rats is indicated in parentheses). *, P < 0.001 vs. nondiabetic control by Tukey–Kramer test; †, P < 0.001 vs. STZ.