Nerve injury induces a rapid efflux of nitric oxide (NO) detected with a novel NO microsensor

Abstract

An early step in repair of the leech CNS is the appearance of endothelial nitric oxide synthase (eNOS) immunoreactivity and NOS activity, but coincident generation of NO at the lesion after injury has not been shown. This is important because NO can regulate microglial cell motility and axon growth. Indirect measurement of NO with the standard citrulline assay demonstrated that NO was generated within 30 min after nerve cord injury. A polarographic NO-selective self-referencing microelectrode that measures NO flux noninvasively was developed to obtain higher spatial and temporal resolution. With this probe, it was possible to demonstrate that immediately after the leech CNS was injured, NO left the lesion with a mean peak efflux of 803 +/- 99 fmol NO cm(-2) sec(-1). NO efflux exponentially declined to a constant value, as described through the equation f(t) = y(o) + ae(-t/tau), with tau = 117 +/- 30 sec. The constant y(o) = 15.8 +/- 4.5 fmol cm(-2) represents a sustained efflux of NO. Approximately 200 pmol NO cm(-2) is produced at the lesion (n = 8). Thus, injury activates eNOS already present in the CNS and precedes the accumulation of microglia at the lesion, consistent with the hypothesis that NO acts to stop the migrating microglia at the lesion site.

Fig. 1.

Diagram of the leech, showing the site of crushing the nerve cord, and the recording arrangement. A, The CNS of Hirudo medicinalis consists of head and tail ganglia and 21 segmental ganglia. Each segmental ganglion contains >400 cell bodies and is linked to its neighbors by thousands of axons that form the connectives. Connectives were injured by crushing with forceps (Materials and Methods). B, C, Photographs of an injured leech nerve cord and the self-referencing NO probe. During measurements, the probe was moved orthogonally to the cord over a distance of 30 μm at a frequency of 0.3 Hz in a plane parallel to the surface of the culture dish. The probe was positioned at the injury site in B, whereas in C it was displaced 30 μm. Scale bar, 100 μm.

Fig. 2.

Construction and calibration of the NO-selective microelectrode. A, The electrode body was constructed by pulling a heated glass microcapillary around a 5 μm carbon fiber. The fiber was then sealed into the micropipette with epoxy. A copper wire was connected to the fiber by filling the back of the electrode with graphite paste. The tip was finished by beveling it, coating with Nafion, and plating with o-phenylenediamine.B, Before each experiment, the electrode was calibrated with a series of known concentrations of NO, and its selectivity was tested against ascorbic acid. The slope S of this calibration curve was used to calculate the flux of NO released at the injury site according to the Fick equation (see Materials and Methods).

Fig. 3.

Injury to the leech CNS causes an increase in citrulline, reflecting NO production. In the experimental group, cords were injured and incubated in Ringer's for 30 min before being processed in the citrulline assay. Uncrushed controls were exposed and incubated for 30 min before being processed in the citrulline assay. Citrulline counts from blank controls were subtracted from each treatment. Statistical analysis of five experiments indicated that there was a statistical difference, with 95% confidence, according to Scheffe's test, between the citrulline counts of crushed and uncrushed cords [DF_(2,12) = 6.436, p < 0.01]. KCPM, 1000 cpm.

Fig. 4.

Injury causes NO efflux generated by NOS at the lesion. A, Representative response of the nerve cord to injury in physiological saline at time indicated byarrows. Differential current measured by the self-referencing probe, converted to flux (vertical axis, see Materials and Methods), jumped abruptly after injury. The mean peak efflux was 803 ± 99 fmol cm⁻²sec⁻¹(n = 8). All data were modeled to the equation y =Y₀ ±ae^−bt. Modeled data are represented by the solid line. B, Representative response to injury (arrow) in saline containing 1 mml-NAME. C, Same asB, but in 1 mmd-NAME. Treatment with 1 mml-NAME reduced the injury response to a mean peak efflux of 199 ± 20 fmol NO cm⁻²sec⁻¹ _{(n = 11)}, whereas treatment with the inert enantiomer (d-NAME) resulted in a mean peak efflux of 1888 ± 773 fmol cm⁻² sec⁻¹(n = 6). Results with l-NAME andd-NAME controls illustrate that the injury-induced response is caused specifically by the efflux of NO that is generated by NOS.

Fig. 5.

Injury-induced NO is blocked by NOS inhibition. Integration over time of the flux versus time graphs for each treatment gives the amount of NO produced at the lesion expressed as fmol NO cm⁻² after crushing. Treatments were with physiological saline alone (control), in 1 mml-NAME (a concentration that inhibits NOS isoforms), and ind-NAME as another control. Comparisons between treatments indicated that l-NAME significantly reduced (**) the NO produced during control injury experiments in physiological saline alone [one-way ANOVA, 95% confidence, Scheffe's test,F_(2,11) = 15.271; p< 0.001] or during treatment with 1 mmd-NAME [one-way ANOVA, 95% confidence, Scheffe's test,F_(2,11) = 15.271; p< 0.001].