Stereological and somatotopic analysis of the spinal microglial response to peripheral nerve injury

Abstract

The involvement of glia, and glia-neuronal signalling in enhancing nociceptive transmission has become an area of intense scientific interest. In particular, a role has emerged for activated microglia in the development and maintenance of neuropathic pain following peripheral nerve injury. Following activation, spinal microglia proliferate and release many substances which are capable of modulating neuronal excitability within the spinal cord. Here, we the investigated the response of spinal microglia to a unilateral spared nerve injury (SNI) in terms of the quantitative increase in cell number and the spatial distribution of the increase. Design-based stereological techniques were combined with iba-1 immunohistochemistry to estimate the total number of microglia in the spinal dorsal horn in naïve and peripheral nerve-injured adult rats. In addition, by mapping the central terminals of hindlimb nerves, the somatotopic distribution of the microglial response was mapped. Following SNI there was a marked increase in the number of spinal microglia: The total number of microglia (mean ± SD) in the dorsal horn sciatic territory of the naïve rat was estimated to be 28,591 ± 2715. Following SNI the number of microglia was 82,034 ± 8828. While the pattern of microglial activation generally followed somatotopic boundaries, with the majority of microglia within the territory occupied by peripherally axotomised primary afferents, some spread was seen into regions occupied by intact, ‘spared’ central projections of the sural nerve. This study provides a reproducible method of assaying spinal microglial dynamics following peripheral nerve injury both quantitatively and spatially.

Introduction

Chronic neuropathic pain following peripheral nerve injury represents a major problem in health care and persists in often being refractory to conventional treatment (Scholz and Woolf, 2002, Tsuda et al., 2005). Multiple mechanisms have been proposed to underlie the long-term changes associated with pain states that persist long after any initial injury has healed. It is likely that considerable redundancy exists and that multiple mechanisms both in the peripheral and central nervous systems contribute to the maintenance of neuropathic pain. It is generally accepted that there is a considerable central component and that increased spinal neuronal excitability mediates, at least in part, the pathological pain state. In recent years much attention has been focused on the role of glial cells and in particular their interaction with first and second order neurons in the central nervous system through the release of cytokines, neurotrophic factors and other pro-inflammatory molecules as direct modulators of neuronal excitability. Microglia in particular have been implicated in this role.

The role of microglia as sensors of CNS damage is well described in many neuropathological conditions. It is becoming increasingly clear that their function goes beyond that of sensor and that they represent a key causative component of certain pathologies such as neuropathic pain. Peripheral nerve injury often results in a chronic pain conditions that have a strong central component. Recently it has been shown that microglia mediate this central response. PNI induces a marked microglial reaction in the dorsal horn of the spinal cord. Microglia respond with a stereotypical ‘activation’—their numbers swell through proliferation and infiltration and they change their morphology from the characteristic ramified ‘resting’ state to the more amoeboid ‘activated’ state, a process also associated with considerable change in gene expression (Perry, 1994, Kreutzberg, 1996, Stoll and Jander, 1999, Nakajima and Kohsaka, 2001). This ‘activation’ response has been reported in all experimental models of neuropathic pain involving peripheral nerve injury (Liu et al., 1995, Coyle, 1998, Colburn et al., 1999, Herzberg and Sagen, 2001, Tsuda et al., 2003, Zhang and De Koninck, 2006). In all cases the response is typified by a marked increase in the number of microglia in the ipsilateral dorsal horn of the spinal cord.

Activation of microglia is characterized by the production and secretion of proinflammatory cytokines and other mediators that could potentially contribute to the initiation and maintenance of pain hypersensitivity. Other proteins indicative of activation include members of the complement cascade; complement receptor 3 (CR3), Toll-like receptor 4 (TLR4), CD14, CD4 and major histocompatibility complex (MHC) class I and II. A correlation between an upregulation of many of these markers in the spinal cord and peripheral nerve injury has long been reported (Gehrmann et al., 1991, Streit et al., 1988, Eriksson et al., 1993, Liu et al., 1995). The coincident onset of pain behavior with increases in microglial activity has also been well documented (Colburn et al., 1997, Coyle, 1998, Colburn et al., 1999), but it is only recently that a causal role of microglial activation in nerve injury-induced pain behaviors has been established (Tsuda et al., 2003, Jin et al., 2003) and the key mediator released by microglia identified as BDNF (Coull et al., 2005).

There is a wealth of research detailing the production and secretion of many cytokines, chemokines and other proinflammatory mediators from microglia within the CNS following peripheral nerve injury (Wieseler-Frank et al., 2004, Tsuda et al., 2005, Moalem and Tracey, 2006) and many of these factors are also capable of contributing to the changes in second order neuronal excitability seen following nerve injury that underlie neuropathic pain behaviours (Watkins et al., 2001, Ji et al., 2003, Salter, 2005). The relative spatial distribution of injured peripheral nerve central terminals, microglial cells and second order dorsal horn cells within the spinal dorsal horn is, therefore, of key importance when considering a signalling pathway that involves these three elements. The activation of spinal microglia involves the production and secretion of the many proinflammatory factors from a proliferating population. The concentration of these factors and by extension the effect on second order neurons and spinal excitability will be determined by both the amount of proliferation and the spatial location within the spinal cord. It is important, therefore, to take into consideration these two phenomena when examining the central microglial response to peripheral injury.

In contrast to other CNS areas, where considerable effort has been made to stereologically quantify the microglial response to a wide range of stimuli (Long et al., 1998, Ayoub and Salm, 2003, Wirenfeldt et al., 2003), few reports have attempted to quantify the microglial response to injury to peripheral sensory nerves (Eriksson et al., 1993, Melzer et al., 1997, Fu et al., 1999) and none has used design-based counting techniques. The use of immunoreactive profiles as a measure of cell density will likely introduce an inherent error due to the change in size of the microglial soma upon activation (Melzer et al., 1997). Other studies have described the somatotopic distribution of the central microglial reaction to peripheral nerve injury (Melzer et al., 1997), yet no reports have attempted to characterize the details or accuracy of this somatotopy within the spinal cord.

Here we use design-based stereological counting techniques to make accurate estimates of the number of microglia in the dorsal horn of the spinal cord following spared nerve injury. The optical fractionator technique (West et al., 1991, West et al., 1996) allows stereological principles to be used for counting tissue preparations that were previously unsuitable to such techniques. The advantages are that estimates of total cell number are unaffected by uniform tissue shrinkage, often a confounding factor in previous estimates. The fixation, cutting and mounting procedures necessary for optimal immunohistochemistry inevitably result in significant tissue shrinkage. For the optional fractionator, assuming that the immunostaining penetrates the full thickness of the tissue, and that the thickness of sections is measurable, these issues are overcome. Similarly, measurements of the volume or area of the tissue being investigated are also unnecessary, again an advantage when looking at a region of the CNS that changes both shape and area from section to section, such as the spinal cord dorsal horn. The optional fractionator relies on the cell type under investigation to be identifiable, as well as the anatomical boundaries within which those cells of interest lie. In essence the optional fractionator estimates the total number of cells by directly counting the number of those cells in a known fraction of the total volume of tissue of interest, in this case the grey matter of the lumbar dorsal horn.