Peripheral neuropathy in mice with neuronal nitric oxide synthase gene deficiency

Abstract

Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neuropathy and neuropathic pain is emerging. This study evaluated the contribution of neuronal nitric oxide synthase (nNOS) to diabetes-induced nitrosative stress in peripheral nerve and dorsal root ganglia, and peripheral nerve dysfunction and degeneration. Control and nNOS-/- mice were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) immunoreactivities. Peripheral diabetic neuropathy was evaluated by measurements of sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia, tactile allodynia, and intraepidermal nerve fiber density. Control nNOS-/- mice displayed normal motor nerve conduction velocity and thermal response latency, whereas sensory nerve conduction velocity was slightly lower compared with non-diabetic wild-type mice, and tactile response threshold and intraepidermal nerve fiber density were reduced by 47 and 38%, respectively. Both diabetic wild-type and nNOS-/- mice displayed enhanced nitrosative stress in peripheral nerve. In contrast to diabetic wild-type mice, diabetic nNOS-/- mice had near normal nitrotyrosine and poly(ADP-ribose) immunofluorescence in dorsal root ganglia. Both diabetic wild-type and nNOS-/- mice developed motor and sensory nerve conduction velocity deficits and thermal hypoalgesia although nNOS gene deficiency slightly reduced severity of the three disorders. Tactile response thresholds were similarly decreased in control and diabetic nNOS-/- mice compared with non-diabetic wild-type mice. Intraepidermal nerve fiber density was lower by 27% in diabetic nNOS-/- mice compared with the corresponding non-diabetic group, and by 20% in diabetic nNOS-/- mice compared with diabetic wild-type mice. In conclusion, nNOS is required for maintaining the normal peripheral nerve function and small sensory nerve fibre innervation. nNOS gene deficiency does not protect from development of nerve conduction deficit, sensory neuropathy and intraepidermal nerve fiber loss.

Figure 1

Sciatic motor nerve conduction velocities (A) and hind-limb digital sensory nerve conduction velocities (B) in control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=8–10 per group. C, control mice; D, diabetic mice. ^*p<0.05 and ^**p<0.01 vs corresponding non-diabetic groups.

Figure 2

Paw withdrawal latencies in response to radiant heat (A) and tail-flick test response latencies (B) in control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=8–11 per group. C, control mice; D, diabetic mice. ^**p<0.01 vs corresponding non-diabetic groups; ^##p<0.01 vs diabetic wild-type mice.

Figure 3

Tactile response thresholds in response to stimulation with flexible von Frey filaments in control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=8–11 per group. C, control mice; D, diabetic mice. ^**p<0.01 vs non-diabetic control mice.

Figure 4

Intraepidermal nerve fiber profiles in control and diabetic wild-type and nNOS^−/− mice. (A) Representative images of intraepidermal nerve fiber profiles, magnification x200; (B) skin fiber density. Mean ± SEM, n=8–11 per group. C, control mice; D, diabetic mice. ^*p<0.05 vs control mice.

Figure 5

(A) Representative microphotographs of immunofluorescent staining of nitrotyrosine in sciatic nerves of control and diabetic wild-type and nNOS^−/− mice. Magnification x40. (B) Nitrotyrosine fluorescence counts in sciatic nerves of control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=7–11 per group. C, control mice; D, diabetic mice. ^**p<0.01 vs non-diabetic control mice.

Figure 6

(A) Representative microphotographs of immunofluorescent staining of nitrotyrosine in dorsal root ganglia of control and diabetic wild-type and nNOS^−/− mice. Magnification x40. (B) Nitrotyrosine fluorescence counts in dorsal root ganglia of control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=7–11 per group. C, control mice; D, diabetic mice. ^**p<0.01 vs non-diabetic control mice. ^##p<0.01 vs diabetic wild-type mice.

Figure 7

(A) Representative microphotographs of immunofluorescent staining of poly(ADP-ribose) in dorsal root gangia of control and diabetic wild-type and nNOS^−/− mice. Magnification x40. (B) Poly(ADP-ribose) fluorescence counts in dorsal root ganglia of control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=7–11 per group. C, control mice; D, diabetic mice. ^**p<0.01 vs non-diabetic control mice. ^##p<0.01 vs diabetic wild-type mice.

Figure 8

(A) Representative microphotographs of immunofluorescent staining of poly(ADP-ribose) in dorsal root gangion neurons of control and diabetic wild-type and nNOS^−/− mice. Magnification x100. (B) Percentage of dorsal root ganglion neurons with weak, moderate, and intense poly(ADP-ribose) immunofluorescence in experimental groups. The number of dorsal root ganglion neurons with weak, moderate and intense poly(ADP-ribose) immunofluorescence was expressed as a percentage of neurons with identifiable poly(ADP-ribose) immunofluorescence in the dorsal root ganglia of control and diabetic wild-type and nNOS^−/− mice. Mean ± SEM, n=8–11 per group. C, control mice; D, diabetic mice. ^**p<0.01 vs non-diabetic control mice; ^#,##p<0.01 vs diabetic wild-type mice.