Research PaperSynaptic loss and firing alterations in Axotomized Motoneurons are restored by vascular endothelial growth factor (VEGF) and VEGF-B
Graphical abstract
Introduction
The vascular endothelial growth factor (VEGF) was initially characterized by its vasculogenic, angiogenic properties and capacity to increase the permeability of blood vessels (Ferrara and Henzel, 1989; Senger et al., 1983; Yancopoulos et al., 2000). However, recent evidence indicates that VEGF likely exerts direct effects on neurons so that, at present, VEGF is also considered a neuroprotective factor (Lambrechts and Carmeliet, 2006; Lange et al., 2016; Storkebaum et al., 2004).
Interestingly, a causal link between low levels of VEGF and amyotrophic lateral sclerosis (ALS) has been established (Sathasivam, 2008). In particular, the design and investigation of the VEGFδ/δ mutant mice (characterized by low levels of VEGF) have shown that these animals develop an adult-onset motoneuron disease resembling ALS (Oosthuyse et al., 2001). Also in humans, post-mortem studies in ALS patients have demonstrated low levels of VEGF and its receptor VEGFR-2 in the spinal cord (Brockington et al., 2006; Sathasivam, 2008). Moreover, when mice that overexpress VEGF (VEGF+/+) are crossed with the mutant mice G93A superoxide dismutase 1 (SOD1G93A), a classical model of ALS, the double-transgenic mice show delayed motoneuron loss and prolonged survival as compared to the SOD1G93A single-transgenic mice (Wang et al., 2007). It has been also demonstrated that the viral delivery of VEGF to SOD1G93A mice delays the onset of the disease, slows the progression of motoneuronal degeneration and increases life expectancy (Azzouz et al., 2004; Wang et al., 2016). Similarly, the chronic administration of VEGF in the spinal cord prevents paralysis and motoneuronal death in rats exposed to excitotoxic motoneuron degeneration (Tovar-y-Romo et al., 2007). In addition to motoneurons, neuroprotective effects of VEGF have also been described in other neuronal types and following different types of injury, such as ischemia, epileptic stages, or neurological diseases (Carmeliet and Storkebaum, 2002; Lange et al., 2016; Matsuzaki et al., 2001; Nicoletti et al., 2008; Storkebaum et al., 2004).
VEGF is also known as VEGF-A and is the prototypical member of a related group of trophic factors, that also includes VEGF-B. VEGF-B shares a high degree of homology with VEGF, but, in contrast to VEGF, has low angiogenic activity and is not pro-inflammatory (Ruiz de Almodovar et al., 2009). The role of VEGF-B remains enigmatic, but recent evidence points to a direct neuroprotective role in degenerating motoneurons, with the advantage, as compared to VEGF, of not producing vascular side effects, such as inflammation and tissue edema (Manoonkitiwongsa et al., 2004; Poesen et al., 2008).
In addition to the survival-promoting effects of VEGF and VEGF-B, behavioral experiments have described motor improvement in different models of motoneuron degeneration (Azzouz et al., 2004; Poesen et al., 2008; Storkebaum et al., 2005; Tovar-y-Romo et al., 2007). However, there are currently no physiological studies regarding the functional state of those neurons that have been rescued from cell death. We have pursued this question using the chronic alert cat preparation to record the discharge activity of motoneurons, an approach that allows the correlation of neuronal firing with motor performance, before and after the administration of the factor (VEGF or VEGF-B) to injured motoneurons. Abducens motoneurons offer several advantages: their discharge pattern is well characterized, and both their afferents and the signals they carry have been described in detail (Büttner-Ennever, 2006; Davis-López de Carrizosa et al., 2011; Delgado-García et al., 1986a; Escudero et al., 1992). In addition to the physiological study, we have also carried out a confocal immunofluorescence analysis of the synaptic boutons impinging on injured and VEGF-treated motoneurons.
Section snippets
Animals and surgical procedures
Experiments were performed on adult female cats weighing 2.0–2.5 kg that were obtained from authorized suppliers (Universidad de Córdoba, Spain). All procedures were performed in accordance with the guidelines of the European Union (2010/63/EU) and Spanish legislation (R.D. 53/2013, BOE 34/11370–421) for the use and care of laboratory animals, and were approved by the ethics committee. All efforts were made to reduce the number of animals used.
A total of 12 animals was used for the present
Abducens motoneurons express VEGFR-1 and VEGFR-2
Since VEGF binds to two tyrosine-kinase receptors, VEGFR-1 and VEGFR-2, which mediate its biological activities, whereas VEGF-B binds only to VEGFR-1 (Ferrara et al., 2003; Lange et al., 2016), we aimed at evaluating the presence of these two receptors in abducens motoneurons of the cat. This study was needed to demonstrate whether abducens motoneurons were responsive to these factors in order to be treated according to our experimental design and protocols (Fig. 1A, B). For this purpose,
Discussion
We have shown that the axotomy-induced changes in the discharge characteristics and synaptic inputs of motoneurons can be completely recovered by VEGF and VEGF-B. These factors restored both the tonic and the phasic components of motoneuronal firing and during different types of eye movements. Since the different signals encoded by abducens motoneurons are provided preferentially by particular afferents (Büttner-Ennever, 2006; Escudero and Delgado-García, 1988), these findings indicate that
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Acknowledgements
This work was supported by the Ministerio de Economía y Competitividad-FEDER (Grant reference: BFU2015-64515-P) in Spain. Confocal and electron microscopy images were performed in the Central Research Services of University of Sevilla (CITIUS). P.M.C. was a scholar of MEC (BES-2016-077912) in Spain.
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