Selective activation of microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse

Abstract

Neuronal plasticity along the pathway for sensory transmission including the spinal cord and cortex plays an important role in chronic pain, including inflammatory and neuropathic pain. While recent studies indicate that microglia in the spinal cord are involved in neuropathic pain, a systematic study has not been performed in other regions of the central nervous system (CNS). In the present study, we used heterozygous Cx3cr1GFP/+mice to characterize the morphological phenotypes of microglia following common peroneal nerve (CPN) ligation. We found that microglia showed a uniform distribution throughout the CNS, and peripheral nerve injury selectively activated microglia in the spinal cord dorsal horn and related ventral horn. In contrast, microglia was not activated in supraspinal regions of the CNS, including the anterior cingulate cortex (ACC), prefrontal cortex (PFC), primary and secondary somatosensory cortex (S1 and S2), insular cortex (IC), amygdala, hippocampus, periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Our results provide strong evidence that nerve injury primarily activates microglia in the spinal cord of adult mice, and pain-related cortical plasticity is likely mediated by neurons.

Figure 1

Tactile allodynia post unilateral common peroneal nerve ligation. (A, B) Ipsilateral (A) and contralateral (B) hind paw withdrawals in response to tactile stimulus were plotted against the time. Significant higher scores of allodynia were observed on the nerve injured side than the intact side. (C) Circled area indicates the location receiving von Frey filament touch. * P < 0.05 (n = 4).

Figure 2

Photomicrographs showing microglia in the brain. (A) Montage of a saggittal section showing the distribution of brain microglia in control mice. (a-d) Different types of brain microglial cells observed under confocal laser scanning microscopy. Brain microglial cells were classified as ramified (a), hypertrophied (b), mono-polarized (c) and bipolarized (d). Hypertrophied microglia were defined as having large soma, and short, thick and radially projecting processes. Ramified microglial cells were defined as possessing thin, slender, radially projecting processes with well-developed ramifications. Monopolarized microglial cells were defined as having one thick process with well developed ramifications extending toward one direction. Bipolarized microglial cells were defined as having two thick processes emanating from the opposing poles of the cell and projecting in the opposite directions. Bar = 1 mm in A, and 20 μm in a-d.

Figure 3

Microglia in anterior cingulate cortex (ACC). (A) Microglial cells were evenly distributed in ACC of control mice, the centrifugally projecting ramified processes of neighbor microglial cells didn't overlap. The arrowhead indicates the monopolarized cell. (B) High power image of the microglial cell indicated by arrowhead in A. Bar = 30 μm in A and 12 μm in B.

Figure 4

Microglia in pain-related cortices of control mice. Left column, sham-operated; right column, CPN ligated. The structures are indicated by arrow or enclosed by dashed lines. 1, ACC (Anterior cingulate cortex); 2, Cingulate cortex, area 1; 3, Prelimbic cortex; 4, Infralimbic cortex; 5, Dorsal peduncular cortex; 6, S1 (primary somatosensory cortex); 7, S2 (secondary somatosensory cortex); 8, Insular cortex. Bar = 400 μm.

Figure 5

Microglia in amygdala, hippocampus, thalamus, PAG and RVM of control mice. Left column, sham-operated; right column, CPN ligated. The structures are indicated by arrow or enclosed by dashed lines. 1, Central amygdaloid nucleus; 2, medial amygdaloid nucleus; 3, Posteromedial amydaloid nucleus; 4, Lateral amygdaloid nucleus, basolateral and basomedial amygdaloid nuclei. Bar = 800 μm.

Figure 6

Microglia is activated in spinal segments of nerve injured mice but not control mice. Left column, sham-operated; right column, CPN ligated. Nerve injury evoked an ipsilateral increase of microglial cells in superficial layers of L2 to L4 spinal dorsal horns, and also in deep layers of L3 and L4 spinal segments. No visible change occurred in L5 spinal segment. Asterisks indicate intact side. Bar = 350 um.

Figure 7

Different morphological phenotypes of spinal microglial cells observed with confocal laser scanning microscopy. Four types of spinal microglial cells were detected in control mice. (A, B) Ramified microglial cells have radially projecting processes which are long, thin with fine ramifications. (C) Hypertrophied cell has large soma and thick, short and radially projecting processes with fewer ramifications. (D) Mono-polarized microglia (arrow) has one main process (arrowhead) which is thick and projects toward one direction. (E, F) Bipolarized microglia (arrow) usually has spindle-like cell body and two main processes emanating from the opposing poles of the cell body and projecting in opposite directions. Bar = 10 μm in A-C, 20 μm in D-F.

Figure 8

Activation of microglia in L4 spinal cord segment following nerve injury. (A) Epifluorescence imgage of L4 spinal cord segment following nerve injury. Left half side in the image represents the nerve injured side. Laminae of dorsal and ventral horns are indicated by the numbers. Note the difference of microglia between two sides. (a-f) Confocal laser scanning microscopy observation of microglia from the same laminae as shown by panel A. (a-c) Microglial cells from lamina II, IV and IX of nerve injured side, respectively. (d-f) Microglial cells from lamina II, IV and IX of the intact side, respectively. Bar = 400 μm in A, 20 μm in a – f.

Figure 9

DRG manifest both GFP-labeled neurons and glia. (A-B) GFP-labelling in L4 DRGs of control mice on sham sugery and intact sides, respectively. (C-D) GFP-labelling in L4 DRGs of nerve injured mice on injured and contralateral sides, respectively. Higher density of glia cells in injured DRG (C) contrasts that of intact DRG (D). The GFP-labeled neuron in (D) indicated by arrow is shown in the inset at a higher magnification, arrowheads point to the satellite cells in close apposition to the neuron. (E-F) Confocal laser scanning microscopic images of injured and intact L4 DRGs, respectively. Arrowheads and arrow point to the Schwann cells and satellite cell, respectively. (G) The density of glial cells was significantly higher in injured L4 DRG than in its contralateral counterpart (p < 0.05, n = 3). "Contr." and "CpNL" indicate control and nerve injured mice, respectively. (H) Number of different-sized L4 DRG neurons in intact side of control mice (bin size = 100). (I) the size of L3/L4 DRG neurons with strong labeling showed no change after nerve injury (p > 0.05, n = 3). The area is expressed as Mean ± SEM (μm²/per neuron). "Contr." and "CpNL" indicate control and nerve injured mice, respectively. Bar = 75 μm in A-B; 50 μm in C-D, 20 μm in E-F.