NADPH-diaphorase histochemistry provides evidence for a bilateral, somatotopically inappropriate response to unilateral hindpaw inflammation in the rat

Abstract

Unilateral hindpaw inflammation produces several neurochemical changes in the ipsilateral spinal dorsal horn that have been interpreted as contributing to the associated hyperalgesia. NADPH-diaphorase histochemical staining was used to examine the response of a population of neurons 1, 2, 6 or 24 h following injection of 6 mg carrageenan into the left hindpaw of the rat. The resulting unilateral hindpaw inflammation produced a bilateral, time-dependent, reversible increase in the number of NADPH-diaphorase stained neurons in the lumbar spinal cord that peaked at 6 h. In laminae I–III, there was a significant increase in the number of NADPH-diaphorase stained neurons both ipsilateral (25.9 ± 2.3) and contralateral (26.3 ± 1.3) to the inflamed hindpaw relative to uninflamed, control animals (18.6 ± 1.7, 17.4 ± 1.7, respectively). A smaller but significant increase was observed in laminae IV–VII and X. Under dark field illumination, an increase in the number of densely stained neurons in laminae I–III was also observed to peak at 6 h. A greater percentage of the neurons observed under bright field illumination were visible under dark field illumination at 6 h (47%) compared to control (18%), suggesting an increase in the enzymatic activity of neurons in laminae I–III in addition to the increase in the number of neurons with threshold levels of NADPH-diaphorase activity. There was no consistent increase in this ratio over time in laminae IV–VII or X. Six hours following carrageenan, there was a bilateral 50% increase in the density of NADPH-diaphorase staining in the neuropil in the medial laminae I–III. Both spinal neurons and primary afferent axons contributed to this bilateral increase in staining as the number of NADPH-diaphorase stained dorsal root ganglion cell bodies increased 47% over control. In addition to the increase in staining in the lumbar spinal cord, at 6 h post carrageenan, there was a bilateral 23% increase over control in the number of stained neurons in the cervical dorsal spinal cord. For comparative purposes, the distribution of Fos-immunoreactive nuclei was compared to NADPH-diaphorase 6 h post carrageenan. The distribution of Fos-immunoreactive nuclei differed from the NADPH-diaphorase stained neurons, suggesting that two separate populations of neurons were stained. Unilateral hindpaw inflammation did not result in an increase in NADPH-diaphorase activity in the periaqueductal gray or the ventroposteriolateral thalamic nucleus. The relationship of NADPH-diaphorase to nitric oxide is discussed and it is concluded that NADPH-diaphorase, while labeling nitric oxide-producing neurons, is not an exclusive marker for nitric oxide synthase following unilateral hindpaw inflammation. A role for NADPH-diaphorase in general excitatory mechanisms in the spinal cord is proposed.