Extraocular motoneurons of the adult rat show higher levels of vascular endothelial growth factor and its receptor Flk-1 than other cranial motoneurons

Abstract

Recent studies show a relationship between the deficit of vascular endothelial growth factor (VEGF) and motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). VEGF delivery protects motoneurons from cell death and delayed neurodegeneration in animal models of ALS. Strikingly, extraocular motoneurons show lesser vulnerability to neurodegeneration in ALS compared to other cranial or spinal motoneurons. Therefore, the present study investigates possible differences in VEGF and its main receptor VEGFR-2 or Flk-1 between extraocular and non-extraocular brainstem motoneurons. We performed immunohistochemistry and Western blot to determine the presence of VEGF and Flk-1 in rat motoneurons located in the three extraocular motor nuclei (abducens, trochlear and oculomotor) and to compare it to that observed in two other brainstem nuclei (hypoglossal and facial) that are vulnerable to degeneration. Extraocular motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem motoneurons, and thus these molecules could be participating in their higher resistance to neurodegeneration. In conclusion, we hypothesize that differences in VEGF availability and signaling could be a contributing factor to the different susceptibility of extraocular motoneurons, when compared with other motoneurons, in neurodegenerative diseases.

Fig 1. Antibody characterization.

Confocal microscopy images of rat brain sections through the cerebellum (A-C) or hyppocampus (D-F) showing immunoreactivity to VEGF and Flk-1 in positive controls (A and D) and negative controls consisting in the omission of the primary antibody (Ab; B and E), or the tissue pre-incubation with normal rabbit IgG instead of VEGF (C) and normal mouse IgG instead Flk-1 (F) antibodies. Scale bar: 50 μm.

Fig 2. VEGF immunoreactivity in extraocular motoneurons.

Confocal microscopy images showing the presence of VEGF in the motoneurons of the extraocular system of the adult rat (ABD: abducens nucleus; TRO: trochlear nucleus; OCM: oculomotor nucleus). Extraocular motoneurons, identified by ChAT immunostaining (in red; A, D and G), showed intense immunoreactivity to VEGF (in green; B, E and H). The right column shows the merged images. Insets illustrate higher magnification images of doubly-labeled motoneurons in every nucleus. Scale bars = 100 μm (in C for A-C; in F for D-F; in I for G-I). Inset scale bars = 25 μm.

Fig 3. VEGF immunoreactivity in facial and hypoglossal motoneurons.

Representative confocal microscopy images of facial and hypoglossal (HYPO) motoneurons, immunopositive for ChAT (in red; A and D, respectively), showing weak immunoreactivity to VEGF (in green; B and E, respectively). The right column shows the merged images. Insets illustrate higher magnification images of motoneurons. Scale bars = 100 μm (in C for A-C; in F for D-F). Inset scale bars = 25 μm.

Fig 4. VEGF immunoreactivity is higher in extraocular motoneurons than in non-extraocular brainstem motoneurons.

Quantification of the percentage of VEGF-positive motoneurons in cranial motor nuclei. Note the higher percentage of motoneurons positive for this factor in the ocular motor system, in contrast to the low number observed in the motoneurons of the facial or lingual motor systems. Data represent mean ± SEM. The asterisk represents significant differences between groups (one-way ANOVA test followed by Holm-Sidak method for multiple pairwise comparisons; *, p < 0.001; n = 9 animals).

Fig 5. Flk-1 receptor is extensively distributed in extraocular motor nuclei.

The left panel shows confocal images of the abducens (ABD), trochlear (TRO) and oculomotor (OCM) nuclei, whose motoneurons were identified by means of ChAT immunolabeling (TRICT, red), and expressed high levels of Flk-1 receptor (FITC, green; second column). The third column represents the overlay of ChAT and Flk-1 images. Insets show representative motoneurons of each nucleus at higher magnification. Scale bars = 100 μm (in C for A-C; in F for D-F; in I for G-I). Inset scale bars = 25 μm.

Fig 6. Facial and hypoglossal motoneurons express low levels of Flk-1 receptor.

Confocal microscopy images of facial and hypoglossal (HYPO) motoneurons (A, D; identified as ChAT-immunopositive) showing low levels of Flk-1 immunoreactivity (B, E). C and F represent the merge of VEGF and Flk-1 images. Insets show motoneurons of each nucleus at higher magnification. Scale bars = 100 μm (in C for A-C; in F for D-F). Inset scale bars = 25 μm.

Fig 7. Extraocular motoneurons express higher levels of Flk-1 receptor than non-extraocular brainstem motoneurons.

The bar chart shows the mean and SEM of the percentage of motoneurons expressing Flk-1 receptor in each brainstem nucleus. The three extraocular motor nuclei showed significantly higher percentages of Flk-1-immunopositive motoneurons than the facial or hypoglossal nuclei. Data represent mean ± SEM. The asterisks represent significant differences between groups (one-way ANOVA test followed by Holm-Sidak method for multiple pairwise comparisons; *, p < 0.001; n = 9 animals).

Fig 8. Western blot analysis of VEGF and Flk-1 proteins in rat cranial nuclei.

A. The protein band for VEGF is shown for the oculomotor (OCM), facial and hypoglossal (HYPO) nuclei of the rat. Cerebellum (CB) protein extract was included as a positive control for VEGF protein. GAPDH immunoblotting was used as load control. B. Densitometry data showed a significantly lower amount of VEGF protein in facial and hypoglossal nuclei as compared with the oculomotor complex (one-way ANOVA test followed by Holm-Sidak method for multiple pairwise comparisons; differences with respect to OCM: *, p<0.001; n = 6 animals). C. Western blot for Flk-1 in oculomotor, facial and hypoglossal nuclei of the rat. Hippocampus (HC) protein extract was included as a positive control for Flk-1 protein. GAPDH immunoblotting was used as load control. D. A significantly higher amount of Flk-1 protein was quantified in the oculomotor complex as compared with facial and hypoglossal nuclei (one-way ANOVA test followed by Holm-Sidak method for multiple pairwise comparisons; differences with respect to OCM: *, p<0.001; n = 6 animals).