β-carbolines that enhance GABAA receptor response expressed in oligodendrocytes promote remyelination in an in vivo rat model of focal demyelination

Abstract

Demyelination is typically followed by a remyelination process through mature oligodendrocytes (OLs) differentiated from precursor cells (OPCs) recruited into the lesioned areas, however, this event usually results in uncompleted myelination. Potentiation of the remyelination process is an important target for designing effective therapeutic strategies against white matter loss. Here, it was evaluated the remyelinating effect of different β-carbolines that present differential allosteric modulation on the GABA_A receptor expressed in OLs. For this, we used a focalized demyelination model in the inferior cerebellar peduncle (i.c.p.) of rats (DRICP model), in which, demyelination by ethidium bromide (0.05%) stereotaxic injection was confirmed histologically by staining with Black-Gold II (BGII) and toluidine blue. In addition, a longitudinal analysis with diffusion-weighted magnetic resonance imaging (dMRI) was made by computing fractional anisotropy (FA), apparent diffusion coefficient (ADC) and diffusivity parameters to infer i.c.p. microstructural changes. First, dMRI analysis revealed FA decreases together with ADC and radial diffusivity (RD) increases after demyelination, which correlates with histological BGII observations. Then, we evaluated the effect produced by three allosteric GABA_A receptor modulators, the N-butyl-β-carboline-3-carboxylate (β-CCB), ethyl 9H-pyrido [3,4-b]indole-3-carboxylate (β-CCE), and 4-ethyl-6,7-dimethoxy-9H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester (DMCM). The results indicated that daily systemic β-CCB (1 mg/Kg) or β-CCE (1 mg/Kg) administration for 2 weeks, but not DMCM (0.35 mg/Kg), in lesioned animals increased FA and decreased ADC or RD, suggesting myelination improvement. This was supported by BGII staining analysis that showed a recovery of myelin content. Also, it was quantified by immunohistochemistry both NG2⁺ and CC1⁺ cellular population in the different experimental sceneries. Data indicated that either β-CCB or β-CCE, but not DMCM, produced an increase in the population of CC1⁺ cells in the lesioned area. Finally, it was also calculated the g-ratio of myelinated axons and observed a similar value in those lesioned animals treated with β-CCB or β-CCE compared to controls. Thus, using the DRICP model, it was observed that either β-CCB or β-CCE, positive modulators of the GABA_A receptor in OLs, had a potent promyelinating effect.

Keywords: GABAergic signaling; OPC; diffusion tensor imaging; myelin; oligodendrocyte; white matter; β-carbolines.

Figure 1

Demyelination lesion followed by BGII and MRI in the inferior cerebellar peduncle. (A) Representative T2-weighted images of an animal injected with EB (0.05%) in the inferior cerebellar peduncle (i.c.p.); the yellow dotted line indicates the i.c.p. region hyperintensity. (B) T2 signal quantification in the i.c.p. (ipsilateral/contralateral) from 0 to 28 dpl (n = 4–5 for each point). (C) Representative images of sections in the panoramic coronal plane stained with BGII of the cerebellum. Amplifications for both control (top insert) and lesioned (bottom insert) i.c.p. are also shown. Bar = 200 μM. (D) Relative intensity of staining (RIS) quantification in i.c.p. sections as illustrated in C (n = 4 for each case). (E) Images in the parasagittal plane stained with BGII including superior (s.c.p.), inferior (i.c.p.) and medial (m.c.p.) cerebellar peduncles from both contralateral and ipsilateral side to EB injection, as indicated. (F) RIS quantification for each peduncle. The RIS decrease in the lesioned i.c.p. was evident, while the RIS for m.c.p. or s.c.p. remained unaffected (n = 4 for each case). *p < 0.05. Data are the mean ± S.D., t-test was used.

Figure 2

Toluidine blue staining of the i.c.p. lesioned by EB injection in vivo. (A) Image illustrates lesioned i.c.p. stained with toluidine blue, the yellow line signals the boundary between a low stained region that coincided with the lesioned i.c.p. area (center of image), while the adjacent area showed a strong staining. Rectangle B indicate boundary areas corresponding to amplified image to the right. (B) Amplification to illustrate that the lesioned i.c.p. exhibited a great number of demyelinated axons (DA, white arrows) compare with the adjacent area which mainly contained myelinated axons (MA, yellow arrows). (C,D) shows the myelin thickness and g-ratio quantification in MA and DA zones. More than 50 axons were quantified from sections obtained from different animals (n = 4 animals). Each group represents the mean (± SD) of the counted axons. ^*p < 0.05, t-test was used.

Figure 3

Longitudinal analysis of DTI metrics evaluated over the DRICP model time-course and the effect of β-carboline administration. (A) Images are representative FA maps acquired at 28 dpl in regions that include the i.c.p. as indicated in the diagram and delineated by the yellow dotted line in the images. Five experimental groups are illustrated: control group, lesioned (i.e., animals injected with EB in the ipsilateral i.c.p.) without treatment, and the lesioned group that was treated (L +) with β-CCB (1 mg/Kg), β-CCE (1 mg/Kg) or DMCM (0.35 mg/Kg). (B) The graphs represent the values of the laterality index for FA’ (B), ADC’ (C), RD´ (D) and AD´ (E) for the images captured on the indicated days of the experiment. Each data point represents the mean ± SD (n = 4 for each point. (ANOVA)) of the respective group (* or ^# indicates differences of the values at 28 dpl vs. 14 dpl or 28 dpl vs. 7 dpl, respectively, within the same group identified with the corresponding color, ^*p < 0.05, ANOVA).

Figure 4

DTI metrics of the DRICP model and the effect of β-carboline administration. Graphs show quantification of the FA’ (A), ADC’ (B), RD´ (C) and AD´ (D) metrics expressed as the laterality index calculated for each subject and every parameter at 28 dpl as defined in the methods section. Data represent the mean ± SD (n = 4) for each experimental group as illustrated in Figure 3 (^*p < 0.05, ANOVA).

Figure 5

DRICP model evaluated using BGII staining and analysis of the effect of β-carboline administration. (A) Diagram of the i.c.p area analyzed by BGII staining at 28 dpl. B-F. Representative amplified images of the i.c.p. region for the distinct experimental conditions indicated in Figure 3: control (B), lesioned (C), and lesioned group that was treated with β-CCB [1 mg/Kg, (D)], β-CCE [1 mg/Kg, (E)], or DMCM [0.35 mg/Kg, (F)]. (G) Graph shows the i.c.p. relative intensity stain (RIS) for each experimental group at 28 dpl (n = 4 for each case). ^*p < 0.05 vs. control group (ANOVA).

Figure 6

Axonal effect and myelin morphometric analysis at 28 dpl and β-carboline effect on the DRICP model. (A–D) Images illustrate i.c.p. semithin sections (600 nm thickness) stained with toluidine blue in control animals (A), lesioned animals (B), and lesioned animals treated with 1 mg/Kg β-CCB (C) or β-CCE (D). (E,F) Show the myelin thickness and g-ratio quantification in all the experimental groups analyzed. More than 100 axons were quantified from sections obtained from different animals for each group. Bar = 20 μm. ^*p < 0.05(n = 3–4; ANOVA).

Figure 7

Status of CC1⁺ and NG2⁺ cells population in the i.c.p. at 28 dpl under the DRICP model and the effect of β-carbolines treatment. Diagram that indicates sagittal sections of the i.c.p. region that were immunolabeled and visualized using confocal microscopy. Antibodies against CC1 (specific for mature oligodendrocytes, in green) and NG2 (to label OPCs, in red) were co-labeled with DAPI (in blue). Representative images correspond to sections from the distinct experimental groups as indicated (bar = 400 μm). Graphs show quantification of the percentage of CC1⁺ cells or NG2⁺ cells labeled in each of the experimental groups analyzed: control, lesioned, and lesioned that was treated with one of the β-carbolines studied, as indicated. Columns represent the mean ± SD obtained from the analysis of sections from 3 subjects in each group (^*p < 0.05).