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 Ϯ 99 fmol NO cm Ϫ2 sec Ϫ1 . NO efflux exponentially declined to a constant value, as described through the equation f(t) ϭ y o ϩ ae Ϫt/ , with ϭ 117 Ϯ 30 sec. The constant y o ϭ 15.8 Ϯ 4.5 fmol cm Ϫ2 represents a sustained efflux of NO. Approximately 200 pmol NO cm Ϫ2 is produced at the lesion (n ϭ 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.

Physiology of Microglia

“…When the leech nerve cord is injured, microglia migrate towards the lesion and accumulate at the damage site (145,482,556,970). In response to injury, leech connective glia and some microglia express endothelial nitric oxide synthase (eNOS) as revealed by immunolabeling.…”

Three-dimensional culture of leech and snail ganglia for studies of neural repair

“…Leech microglia resemble vertebrate microglia in migrating in response to tissue damage, in their phagocytic properties and in their changing morphology, as do the microglia in two species of snail and an insect described by Sonetti et al (1994). The behaviour and function of microglial cells after injury to leech CNS has been studied by Muller, Sahley and colleagues (Masuda-Nakagawa et al 1990;von Bernhardi and Muller 1995;Chen et al 2000;Kumar et al 2001). When the leech nerve cord is injured in vivo, microglia move towards the lesion and accumulate at the damage site (McGade-McColloh et al 1989).…”

“…When the leech nerve cord is injured in vivo, microglia move towards the lesion and accumulate at the damage site (McGade-McColloh et al 1989). Their migration to and stopping at the site of injury appears to be regulated by nitric oxide (Chen et al 2000;Kumar et al 2001). Collagen gels offer a way of isolating this migrating population of cells for molecular analysis, or for in vitro study of the effect of conditioned medium on microglial morphology and interactions with damaged axons.…”

Metabolic oxygen consumption measurement with a single-cell biosensor after particle microbeam irradiation

“…Self-referencing polarographic ( SERP ) microsensor technology was developed at the BioCurrents Shared Resource (Land et al 1999; Porterfield et al 2001; Kumar et al 2001). These sensors are made of borosilicate glass capillaries (1B150, WPI) which are pulled to outer tip diameters of ~3 μm using a Sutter P-97 (Sutter Instrument, CA).…”