Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

Abstract

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4(+) Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4(+) Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3(-/-) mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2(-/-) (recombination activating gene-2-deficient) mice adoptively transferred CD4(+) T-cells isolated from CCR3(-/-) mice, but not in CCR3(-/-) mice adoptively transferred CD4(+) T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4(+) T-cell- and CCR3-mediated neuroprotection after FMN injury.

Figure 1. Th2-associated chemokine receptor mRNA levels in mouse facial motor nucleus 7 DPA

Chemokine receptor mRNA levels are displayed as the fold-change between the axotomized side and the uninjured control side.

Figure 2. CCR3 mRNA levels in axotomized mouse facial motor nucleus 7, 14 and 30 DPA

CCR3 mRNA levels in the facial motor nucleus at 7, 14 and 30 DPA (values are means± S.E.M.). CCR3 levels are displayed as the percentage change in facial motor nuclei in the axotomized relative to control facial motor nucleus within each animal. *P≤0.01.

Figure 3. FMN survival levels 4 weeks after facial nerve axotomy in mice that are WT, CCR3^−/−, CCR3^−/− adoptively transferred WT mouse CD4⁺ T-cells or Rag2^−/− adoptively transferred CCR3^−/− mouse CD4⁺ T-cells

(A) Thionin-stained FMNs in control or axotomized facial motor nuclei of mice that are WT, CCR3^−/−, CCR3^−/− adoptively transferred WT mouse CD4⁺ T-cells or Rag2^−/− adoptively transferred CCR3^−/− mouse CD4⁺ T-cells (original magnification, ×10). (B) Average percentage of FMN survival after facial nerve injury (values are means±S.E.M.). *P≤0.05.

Figure 4. CCR3 immunoreactivity in control mouse facial motor nucleus

(A) Low- (original magnification, ×100) and high- (original magnification, ×600) power photomicrographs of mouse facial motor nucleus. Sections were processed for CCR3 immunohistochemistry. Control experiments include omission of CCR3 primary antibody. (B) High-power immunofluorescence photomicrographs of mouse facial motor nucleus immunoreactive for anti-NeuN (green) and anti-CCR3 (red) antibodies. Photomicrographs are based on the typical result of at least four independent experiments.

Figure 5. CCR3 immunoreactivity in 14 DPA mouse facial motor nucleus

(A) Low- (original magnification, ×100) and high- (original magnification, ×600) power photomicrographs of mouse facial motor nucleus. Sections were processed for CCR3 immunohistochemistry. (B) High-power immunofluorescence photomicrographs of mouse facial motor nucleus immunoreactive for anti-CD68 (green) and anti-CCR3 [red; left-hand panel)] or anti-GFAP (green) and anti-CCR3 [red; right-hand panel)] antibodies. Photomicrographs are based on the typical result of at least four independent experiments.

Figure 6. Quantitative immunoreactivity for CCR3 in control and 14 DPA mouse facial motor nucleus

(A) Low- (original magnification, ×100) and high- (original magnification, ×600) photomicrographs of control (left-hand panels) and axotomized (right-hand panels) mouse facial motor nucleus. (B) Average percentage of CCR3⁺ FMN with nucleus only, cytoplasm only or both nucleus and cytoplasm immunoreactivity in the control (open bars) and axotomized (grey bars) mouse facial motor nucleus (values are means±S.E.M.). (C) Average percentage total of CCR3⁺ FMN in the control (open bars) and axotomized (grey bars) mouse facial motor nucleus (values are means±S.E.M.). (D) Mean fluorescence intensity of the control (open bars) and axotomized (grey bars) mouse facial motor nucleus (values are means±S.E.M.). *P≤0.01. Con, control; Axo, axotomized.