Lack of evidence for significant neuronal loss in laminae I–III of the spinal dorsal horn of the rat in the chronic constriction injury model

Abstract

Peripheral nerve injury leads to structural and functional changes in the spinal dorsal horn, and these are thought to be involved in the development of neuropathic pain. In the chronic constriction injury (CCI) model, abnormal 'dark' neurons and apoptotic nuclei have been observed in laminae I-III of the dorsal horn in the territory innervated by the injured sciatic nerve. These findings have been taken as evidence that there is significant neuronal death in this model, and it has been suggested that loss of inhibition resulting from death of GABAergic inhibitory interneurons contributes to the neuropathic pain. However, loss of neurons from the dorsal horn has not been directly demonstrated in neuropathic models, even though this issue is of considerable importance for our understanding of the mechanisms that underlie neuropathic pain. In this study, we have looked for evidence of neuronal death by using a stereological method (the optical disector) with NeuN-immunostaining, and examining spinal cords of naïve rats, and of rats that had undergone CCI or sham operations. All of the CCI animals showed clear signs of thermal hyperalgesia. However, the numbers of neurons in laminae I-III of the ipsilateral dorsal horn in these animals did not differ significantly from those on the contralateral side, nor from those of sham-operated or naïve animals. These results do not, therefore, support the suggestion that there is significant neuronal death in the dorsal horn in this model.

Loss of Neurons from Laminas I-III of the Spinal Dorsal Horn Is Not Required for Development of Tactile Allodynia in the Spared Nerve Injury Model of Neuropathic Pain

“…The lengths of this segment in the rats that survived 28 d were measured so that we could determine whether there was any alteration after nerve injury. This was done by photographing the spinal cord in situ and measuring the distance between the points of entry of the most rostral and most caudal rootlets of the L4 dorsal root (Polgár et al, 2004). Spinal cord blocks from all operated animals were notched to allow the two sides to be distinguished, and transverse 60-m-thick sections were cut with a Vibratome, rinsed in buffer, and subsequently treated with 50% ethanol to improve antibody penetration.…”

“…The analysis was performed as described previously (Polgár et al, 2004) by using a modification of the optical disector method (Williams and Rakic, 1988;Coggeshall, 1992;Bjugn and Gundersen, 1993;West, 1999;Guillery, 2002) to provide unbiased counts of neuronal nuclei in laminas I-III. Briefly, merged images representing NeuN and propidium iodide from the z-series obtained with the 60ϫ objective were viewed with Neurolucida for Confocal (MicroBrightField, Colchester, VT).…”

“…Each optical section in the series was examined, and all neuronal nuclei (identified by the presence of both propidium iodide and NeuN) that were present in the reference section or appeared in subsequent sections in the series, but had disappeared by the look-up section, were drawn onto an outline of the gray matter. The borders between laminas I, II, and III were plotted from low-magnification scans of NK1 receptor immunoreactivity (laminas I-II) and those obtained through a darkfield condenser (laminas II-III) (Polgár et al, 2004). Because there is no obvious change in cytoarchitecture at the lamina III/IV border, its position was determined by reference to a standard atlas (Molander et al, 1984).…”

“…By using a greater separation between reference and look-up sections, we were able to obtain a larger sample of neurons from each section. Because tissue shrinkage was found to occur in most sections (see Results), we used the following formula to estimate the number of neurons in each lamina on each side per 10 m length of spinal cord: N ϭ n ϫ T final /T cut ϫ 0.9091, where N is the number of neurons per 10 m of spinal cord, n is the number counted in the 11 m disector, T final is the thickness of the particular section measured after any shrinkage, and T cut the original section thickness cut (60 m) (Polgár et al, 2004).…”

“…However, despite the evidence that neuronal death contributes to neuropathic pain, Polgár et al (2003Polgár et al ( , 2004 reported that there was neither a loss of neurons nor a reduction in the proportion of neurons that were GABA immunoreactive in laminas I-III in the chronic constriction injury (CCI) model of neuropathic pain (Bennett and Xie, 1988). In the present study, we tested the hypothesis that there is neuronal loss in the SNI model (in which there is prominent, long-lasting tactile allodynia) by performing a stereological analysis of neuronal packing density in laminas I-III in rats that had undergone SNI 4 weeks previously.…”