In vivo PET imaging of the α4β2 nicotinic acetylcholine receptor as a marker for brain inflammation after cerebral ischemia

Abstract

PET imaging of nicotinic acetylcholine receptors (nAChRs) could become an effective tool for the diagnosis and therapy evaluation of neurologic diseases. Despite this, the role of nAChRs α4β2 receptors after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. To investigate this, we performed in parallel in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 at 1, 3, 7, 14, 21, and 28 d after middle cerebral artery occlusion (MCAO) in rats. In the ischemic territory, PET with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 showed a progressive binding increase from days 3-7, followed by a progressive decrease from days 14-28 after cerebral ischemia onset. Ex vivo immunohistochemistry for the nicotinic α4β2 receptor and the mitochondrial translocator protein (18 kDa) (TSPO) confirmed the PET findings and demonstrated the overexpression of α4β2 receptors in both microglia/macrophages and astrocytes from days 7-28 after experimental ischemic stroke. Likewise, the role played by α4β2 receptors on neuroinflammation was supported by the increase of [(11)C]PK11195 binding in ischemic rats treated with the α4β2 antagonist dihydro-β-erythroidine hydrobromide (DHBE) at day 7 after MCAO. Finally, both functional and behavioral testing showed major impaired outcome at day 1 after ischemia onset, followed by a recovery of the sensorimotor function and dexterity from days 21-28 after experimental stroke. Together, these results suggest that the nicotinic α4β2 receptor could have a key role in the inflammatory reaction underlying cerebral ischemia in rats.

Keywords: 2[18F]-fluoro-A85380; PET; TSPO; [11C]PK11195; cerebral ischemia; α4β2.

Figure 1.

Serial images of [¹¹C]PK11195 and 2-[¹⁸F]-fluoro-A85380 PET at day 0 (control), days 1, 7, 14, and 28 after MCAO. Normalized coronal and axial PET images of [¹¹C]PK11195 (A, B) and 2-[¹⁸F]-fluoro-A85380 (C, D) signals before and after cerebral ischemia are coregistered with a MRI (T2W) rat template to localize anatomically the PET signal from left to right. Images correspond to the same representative animal for each time and condition and radiotracer.

Figure 2.

Time course of the progression of the 2-[¹⁸F]-fluoro-A85380 PET signal before and after cerebral ischemia. BP_ND (mean ± SD) of 2-[¹⁸F]-fluoro-A85380 was quantified in eight VOIs. The entire ipsilateral cerebral hemisphere (A), contralateral cerebral hemisphere (B), ipsilateral cortex (C), contralateral cortex (D), ipsilateral striatum (E), contralateral striatum (F), ipsilateral thalamus (G), and contralateral thalamus (H) are shown. The upper right panels of each figure show the selected brain ROIs for the quantification defined on a slice of a MRI (T2W) template. Rats (n = 6) were repeatedly examined by PET before (day 0) and at 1, 3, 7, 14, 21, and 28 d after ischemia. *p < 0.05 and **p < 0.01 compared with control.

Figure 3.

Time course of the progression of the [¹¹C]PK11195 PET signal before and after cerebral ischemia. The BP_ND (mean ± SD) of [¹¹C]PK11195 was quantified in eight VOIs. The entire ipsilateral cerebral hemisphere (A), contralateral cerebral hemisphere (B), ipsilateral cortex (C), contralateral cortex (D), ipsilateral striatum (E), contralateral striatum (F), ipsilateral thalamus (G), and contralateral thalamus (H) are shown. The upper right panels of each figure show the selected brain ROIs for the quantification defined on a slice of a MRI (T2W) template. Rats (n = 6) were repeatedly examined by PET before (day 0) and at 1, 3, 7, 14, 21, and 28 d after ischemia. *p < 0.05 and **p < 0.01 compared with control.

Figure 4.

Immunofluorescent labeling of α4β2 (blue), CD11b (green), GFAP (white), and TSPO (red) in the ischemic area. The data show temporal evolution of both α4β2 and TSPO expression in microglial and astrocytic cells at day 0 (control, n = 1, left column), day 7 (n = 1, middle column), and day 28 (n = 1, right column) after cerebral ischemia. A–C, G–I, Number of CD11b-reactive microglia/macrophages (arrowheads) increase at 7 d. D–F, J–L, Number of GFAP-positive astrocytes (arrows) increase in the ischemic area over time. α4β2 (A–F) and TSPO (G–L) immunoreactivities colocalize with the temporal activation of CD11b and GFAP after cerebral ischemia. Scale bars, 5 μm.

Figure 5.

Temporal profiles of α4β2+/CD11b⁺ microglial cells (A), microglial/α4β2 immunoreactivity (B), α4β2+/GFAP⁺ astrocytic cells (C), and astrocytic/α4β2 immunoreactivity (D) at day 0, 7, and 28 after cerebral ischemia. α4β2+/CD11b⁺ cells increase versus control at day 7 followed by a dramatic decline at day 28 (A, n = 15, 5 rats/time point). Intensity of microglial/α4β2 receptors increases versus control at day 7, followed by a dramatic decline at day 28 (B, n = 12, 4 rats/time point). The number of α4β2⁺/GFAP⁺ cells increases versus control at days 7 and 28 after ischemia (C, n = 15, 5 rats/time point). Finally, the intensity of astrocytic α4β2 receptors increases versus control at days 7 and 28 (D, n = 12, 4 rats/time point). *p < 0.05 and **p < 0.01 compared with control.

Figure 6.

Normalized coronal and axial PET images of [¹¹C]PK11195 for control (A, B) and DHβE-treated (C, D) rats at day 7 after cerebral ischemia are coregistered with a MRI (T2W) rat template to localize anatomically the PET signal from left to right. The BP_ND (mean ± SD) of [¹¹C]PK11195 was quantified in the entire ipsilateral cerebral hemisphere. Vehicle (n = 8) and DHβE-treated (n = 6) rats were examined by PET 7 d after ischemia (E). *p < 0.05 compared with control.

Figure 7.

Neurologic and behavioral outcomes before (day 0) and 1, 3, 7, 14, 21, and 28 d after cerebral ischemia. The neurologic score (A), the time to contact (B), and the time to remove (C) tape tests showed an improvement over time. For A, **p < 0.01 compared with control; ##p < 0.01 compared with day 1. For B and C, #p < 0.05 compared with corresponding time to ipsilateral and **p < 0.01 and ***p < 0.001 compared with control.