PARP inhibition or gene deficiency counteracts intraepidermal nerve fiber loss and neuropathic pain in advanced diabetic neuropathy

Abstract

Evidence that poly(ADP-ribose) polymerase (PARP) activation plays an important role in diabetic complications is emerging. This study evaluated the role of PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methylpiperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15427; formulated as a mesilate salt, 30 mg kg(-1) day(-1) in the drinking water for 10 weeks after the first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, most importantly, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP -/- mice. Furthermore, whereas diabetic PARP +/+ mice displayed approximately 46% intraepidermal nerve fiber loss, diabetic PARP -/- mice retained completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support a rationale for the development of potent and low-toxicity PARP inhibitors and PARP inhibitor-containing combination therapies.

Fig.1

A. Paw withdrawal latencies in response to radiant heat and tail-flick test response latencies in control and diabetic mice with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D - diabetic rats. T- GPI-15427 treatment. ** – p < 0.01 vs non-diabetic control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. B. Paw withdrawal latencies in response to radiant heat and tail-flick test response latencies in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice. C. Mechanical withdrawal thresholds in rigid von Frey filament test and paw-pressure Randall-Selitto test in control and diabetic rats with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D – diabetic rats. T - GPI-15427 treatment. ** – p < 0.01 vs control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. D. Mechanical withdrawal thresholds in tail-pressure Randall-Selitto test in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice. E. Left - tactile response thresholds in flexible von Frey filament test (left) in control and diabetic rats with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D – diabetic rats. T - GPI-15427 treatment. ** – p < 0.01 vs control rats; ^## – p < 0.01 vs untreated diabetic rats. Right - tactile response thresholds in flexible von Frey filament test in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice.

Fig.1

A. Paw withdrawal latencies in response to radiant heat and tail-flick test response latencies in control and diabetic mice with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D - diabetic rats. T- GPI-15427 treatment. ** – p < 0.01 vs non-diabetic control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. B. Paw withdrawal latencies in response to radiant heat and tail-flick test response latencies in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice. C. Mechanical withdrawal thresholds in rigid von Frey filament test and paw-pressure Randall-Selitto test in control and diabetic rats with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D – diabetic rats. T - GPI-15427 treatment. ** – p < 0.01 vs control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. D. Mechanical withdrawal thresholds in tail-pressure Randall-Selitto test in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice. E. Left - tactile response thresholds in flexible von Frey filament test (left) in control and diabetic rats with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D – diabetic rats. T - GPI-15427 treatment. ** – p < 0.01 vs control rats; ^## – p < 0.01 vs untreated diabetic rats. Right - tactile response thresholds in flexible von Frey filament test in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice.

Fig.1

A. Paw withdrawal latencies in response to radiant heat and tail-flick test response latencies in control and diabetic mice with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D - diabetic rats. T- GPI-15427 treatment. ** – p < 0.01 vs non-diabetic control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. B. Paw withdrawal latencies in response to radiant heat and tail-flick test response latencies in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice. C. Mechanical withdrawal thresholds in rigid von Frey filament test and paw-pressure Randall-Selitto test in control and diabetic rats with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D – diabetic rats. T - GPI-15427 treatment. ** – p < 0.01 vs control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. D. Mechanical withdrawal thresholds in tail-pressure Randall-Selitto test in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice. E. Left - tactile response thresholds in flexible von Frey filament test (left) in control and diabetic rats with or without the PARP inhibitor GPI-15427 treatment. Mean ± SEM, n = 6–12 per group. C – control rats, D – diabetic rats. T - GPI-15427 treatment. ** – p < 0.01 vs control rats; ^## – p < 0.01 vs untreated diabetic rats. Right - tactile response thresholds in flexible von Frey filament test in control and diabetic PARP+/+ and PARP−/− mice. Mean ± SEM, n = 13–18 per group. C – control mice, D - diabetic mice. ** – p < 0.01 vs non-diabetic control mice; ^## – p < 0.01 vs diabetic wild-type mice.

Fig.2

Total number of flinches in the first and second phases of the formalin pain test in control and diabetic rats with and without the PARP inhibitor GPI-15427 treatment. C – control rats, D – diabetic rats. T- GPI-15427 treatment. * – p < 0.05 vs control rats.

Fig.3

A. Intraepidermal nerve fiber profiles in control and diabetic rats with and without the PARP inhibitor GPI-15427 treatment. Left – representative image of intraepidermal nerve fiber profiles, magnification × 40; Right – skin fiber density. Mean ± SEM, n = 6–9 per group. C – control rats, D – diabetic rats. T- GPI-15427 treatment. * – p < 0.05 vs control rats; ^# – p < 0.05 vs untreated diabetic rats. B. Intraepidermal nerve fiber profiles in control and diabetic PARP+/+ and PARP−/− mice. Left – representative image of intraepidermal nerve fiber profiles, magnification × 80; Right – skin fiber density. Mean ± SEM, n = 8–11 per group. C – control mice, D – diabetic mice. ** – p < 0.01 vs control mice; ^## – p < 0.01 vs diabetic PARP+/+ mice.

Fig.4

Left - representative microphotographs of immunofluorescent staining of poly(ADP-ribose) in sciatic nerves (A) and DRG (B) in control and diabetic rats with and without the PARP inhibitor GPI-15427 treatment. C – control rats, D – diabetic rats. T-GPI-15427 treatment. Magnification × 100, and × 200, respectively. Right - the numbers of poly(ADP-ribose)-positive nuclei in sciatic nerves (A) and DRG (B) of control and diabetic rats with and without the PARP inhibitor GPI-15427 treatment. C – control rats, D – diabetic rats. T - GPI-15427 treatment. Mean ± SEM, n = 6–17 per group. ** – p < 0.01 vs control rats; ^{#, ##} – p < 0.05 and < 0.01 vs untreated diabetic rats. Left - representative microphotographs of immunofluorescent staining of poly(ADP-ribose) in sciatic nerves of control and diabetic PARP+/+ and PARP−/− mice (C). C – control mice, D – diabetic mice. Magnification × 100. Right - the numbers of poly(ADP-ribose)-positive nuclei in sciatic nerves of control and diabetic PARP+/+ and PARP−/− mice. C – control mice, D – diabetic mice. Mean ± SEM, n = 6–13 per group. ** – p < 0.01 vs control mice; ^## – p < 0.01 vs diabetic PARP+/+ mice.