A retinoic acid receptor agonist Am80 rescues neurons, attenuates inflammatory reactions, and improves behavioral recovery after intracerebral hemorrhage in mice

Abstract

Am80 (tamibarotene) is a retinoic acid receptor (RAR) agonist clinically available for treatment of acute promyelocytic leukemia. As intracerebral hemorrhage (ICH) accompanies inflammatory reactions in the brain and also because retinoids may suppress activation of microglia, we investigated the effect of Am80 on collagenase-induced experimental model of ICH in adult mice. Daily oral administration of Am80 (5 mg/kg) starting from 1 day before or from up to 6 hours after intrastriatal injection of collagenase significantly inhibited the decrease in the number of striatal neurons at 3 days after the insult. Am80 showed no significant effect on the hematoma size and the extent of edema associated with hemorrhage. Prominent expression of RARα was observed in activated microglia/macrophages, and the number of activated microglia/macrophages in the perihematoma region was lower in Am80-treated mice than in vehicle-treated mice. Am80 treatment also reduced areas affected by hemorrhage-associated oxidative stress as indicated by nitrotyrosine immunoreactivity, and attenuated heme oxygenase-1 expression in activated microglia/macrophages. Moreover, Am80-treated mice exhibited better recovery from hemorrhage-induced neurologic deficits than vehicle-treated mice. These results suggest that RAR is a promising target of neuroprotective therapy for ICH.

Figure 1

Effect of Am80 on intracerebral hemorrhage (ICH)-induced neuronal damage. (A) A representative image of a NeuN-immunostained coronal section obtained 3 days after collagenase injection. The central region and the peripheral region for cell counts were denoted by rectangles with and without an asterisk, respectively. Scale bar=1 mm. (B–E) Representative images of the area corresponding to the center of hematoma in NeuN-immunostained coronal sections obtained 3 days after induction of ICH. Mice received intrastriatal injection of saline (Sal; B) or collagenase (Col; C–E). Oral administration of vehicle (0.5% carboxymethyl cellulose (CMC); B and C) or Am80 at indicated doses (D and E) was performed once daily for 4 days starting from the day before induction of ICH. Scale bar=50 μm. (F and G) The number of NeuN-positive cells in the central (F) and the peripheral (G) regions of hematoma was quantified. n=6 to 15 for each condition. ^***P<0.001 versus sham group; ^###P<0.001 versus CMC group (analysis of variance results: for E, F_3,36=191.95, P<0.0001; for F, F_3,36=463.92, P<0.0001).

Figure 2

Effect of posttreatment with Am80 on intracerebral hemorrhage (ICH)-induced neuronal damage. Oral administration of vehicle (carboxymethyl cellulose, CMC) or 5 mg/kg Am80 was performed once daily, starting from 2 hours (A and B), 6 hours (C and D), or 24 hours (E and F) after induction of ICH. The number of NeuN-positive cells in the central (A, C, E) and the peripheral (B, D, F) regions of hematoma was assessed 3 days after induction of ICH. n=4 to 7 for each condition. ^***P<0.001 versus sham group; ^##P<0.01 versus CMC group; n.s., not significant (analysis of variance results: for A, F_2,13=58.847, P<0.0001; for B, F_2,13=102.40, P<0.0001; for C, F_2,13=54.271, P<0.0001; for D, F_2,13=122.18, P<0.0001; for E, F_2,14=178.21, P<0.0001; for F, F_2,14=347.83, P<0.0001).

Figure 3

Effect of Am80 on the lesion volume and the increase in brain water content at 3 days after induction of intracerebral hemorrhage (ICH). Vehicle (carboxymethyl cellulose, CMC) or 5 mg/kg Am80 was administered orally for 4 days, once daily from the day before ICH induction by intrastriatal injection of collagenase (Col). (A and B) Representative images of the area invaded by hematoma in CMC-treated (A) and Am80-treated (B) mice, as revealed by Nissl staining. Scale bar=1 mm. (C) Results of quantification of lesion volume in CMC-treated (n=7) and Am80-treated (n=8) mice. n.s., not significant. (D) The region used for the measurement of water content is shown. The black dot indicates the coordinate of collagenase injection. (E) Results of quantification of brain water content. n=4 to 5 for each condition. ^**P<0.01; n.s., not significant.

Figure 4

Immunohistochemical localization of retinoic acid receptor (RAR)α and RARβ at 3 days after induction of intracerebral hemorrhage (ICH). (A–F) Shown are immunoreactivities against RARα (A–C) and RARβ (D–F) in the peripheral region of hematoma (A and D), in the contralateral striatum (B and E) and in the contralateral cerebral cortex (C and F). Scale bar=50 μm. (G) Double staining of isolectin B₄ binding and RARα immunoreactivity in the peripheral region of hematoma. Scale bar=20 μm. (H) Double staining of isolectin B₄ binding and Iba1 immunoreactivity. Scale bar=20 μm. Reproducibility of the results shown in this figure was confirmed in four mice.

Figure 5

Effect of Am80 on the number of microglia/macrophages and the level of oxidative stress at 3 days after induction of intracerebral hemorrhage (ICH). Vehicle (carboxymethyl cellulose, CMC) or 5 mg/kg Am80 was administered orally for 4 days, once daily from the day before ICH induction by collagenase (Col). (A and B) Representative images of cells positive for isolectin B₄ binding in the peripheral region of hematoma of CMC-treated (A) and Am80-treated (B) mice. Scale bar=50 μm. (C and D) Nitrotyrosine immunoreactivity in the brain of CMC-treated (C) and Am80-treated (D) mice. Scale bar=1 mm. (E) Double staining against inducible nitric oxide synthase (iNOS) immunoreactivity and isolectin B₄ binding at 3 days after collagenase injection in control mice. Arrows indicate double-positive cells. Scale bar=50 μm. (F) The number of isolectin B₄ binding-positive cells in CMC-treated (n=6) and Am80-treated (n=7) mice. (G) Results of quantification of nitrotyrosine-positive area in CMC-treated (n=5) and Am80-treated (n=7) mice. ^*P<0.05, ^***P<0.001 versus CMC group.

Figure 6

Effect of Am80 on the number of HO-1-positive cells in the peripheral region of hematoma. Vehicle (carboxymethyl cellulose, CMC) or 5 mg/kg Am80 was administered orally for 4 days, once daily from the day before intracerebral hemorrhage (ICH) induction by collagenase (Col). (A) Double staining of isolectin B₄ binding with HO-1 immunoreactivity at 3 days after ICH. Scale bar=50 μm. (B) HO-1 immunohistochemistry in the peripheral region of hematoma of CMC-treated and Am80-treated mice at 3 days after ICH. Scale bar=50 μm. (C) The number of HO-1-positive cells in CMC-treated (n=5) and Am80-treated (n=8) mice. ^***P<0.001 versus CMC group.

Figure 7

Effect of Am80 on neurologic dysfunctions induced by intracerebral hemorrhage (ICH). (A) Schematic representation of the experimental schedule. Vehicle (carboxymethyl cellulose, CMC) or 5 mg/kg Am80 was administered orally for 4 days as indicated. (B and C) Performance in the beam-walking test in CMC-treated and Am80-treated mice, evaluated by performance score (B) and foot fault rate (C). (D and E) Results of performance in the rotarod test (D) and the modified limb-placing test (E). n=12 for each condition. ^**P<0.01, ^***P<0.001 versus Col+CMC group, by Bonferroni's post hoc comparisons.