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.

Fig. 1

Schematic showing the sampling paradigm for the stereological analysis of microglia within the dorsal horn (a). Examples of images within optical dissectors for naïve and SNI rats are shown in (b). Extended focus images of each full z-series are shown in (C). Yellow asterisks indicate the emergence of a soma coming into focus to be counted. Scale bar = 15 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 2

Quantitative analysis of microglia in the dorsal horn of naïve and SNI rats (a). Following SNI there is a marked significant increase in the ipsilateral dorsal horn (p < 0.005). (b) Examples of naïve and SNI sections showing the characteristic microglial response (blue). CTB-labelled central terminals are shown in red and IB4 labelling in green. Scale bar = 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 3

Somatotopic dorsal view illustration of the central terminal fields of indicated hindlimb nerves in lamina II of the spinal cord (adapted from Molander and Grant, 1986, with permission).

Fig. 4

(a) Labelling of injured primary afferents with CTB (red) and uninjured terminals with IB4 (green). Following SNI (right) there is a clear delineation between the terminal fields of the two groups of afferents (white arrow). (b) Spread of microglia (red) out of the terminal fields of injured primary afferents and into the more lateral, uninjured, sural territory. Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 5

Top panel: Reconstructions of naïve and SNI lumbar dorsal horns showing the somatotopic nature of the microglial response to PNI. Microglia are indicated in blue, CTB in red and IB4 in green. Bottom panel: SNI dorsal horn with the microglial labelling (right) separated from the primary afferent labelling (left). While the majority of microglial activity is restricted to the injured central terminal fields, shown by overlap of the blue and red labelling, there is a small but distinct spread of microglial labelling into the intact sural territory. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)