Neuroprotective effects of the MAO-B inhibitor, PF9601N, in an in vivo model of excitotoxicity

Abstract

Background: PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] is an inhibitor of monoamine oxidase B (MAO-B), which has shown to possess neuroprotective properties in several in vitro and in vivo models of Parkinson's disease (PD). As there is evidence that excitotoxicity may be implicated in the pathophysiology of several neurodegenerative diseases, the aim of the present work was to investigate the effects of PF9601N in an acute in vivo model of excitotoxicity induced by the local administration of kainic acid during striatal microdialysis in adult rats.

Methods: The basal and evoked release of neurotransmitters was monitored by HPLC analysis of microdialysate samples and tissue damage was evaluated histologically "ex vivo."

Results: PF9601N (40 mg/kg, single i.p. administration) reduced the kainate-evoked release of glutamate and aspartate and increased taurine release, but it had no effect on the release of dopamine, DOPAC, and HVA. PF9601N pretreatment also resulted in a significant reduction in the kainate-induced astrocytosis, microgliosis, and apoptosis.

Conclusions: The results suggest PF9601N to be a good candidate for the treatment of neurodegenerative diseases mediated by excitotoxicity.

Keywords: Amino acids; Dopamine; Glia; Kainate; Microdialysis.

Scheme 1

Chemical structure of PF9601N [N‐(2‐propynyl)‐2‐(5‐benzyloxy‐indolyl) methylamine].

Figure 1

Time course of the output of aspartate (A), glutamate (B), and taurine (C) evoked by local administration of K ⁺ and kainate (KA). The extracellular concentrations were expressed as percentage of their respective basal levels for illustrative purposes. Original concentration values, expressed as fmol/μL of perfusate (nM), were used for statistical analysis. The time course reported on the x‐axis shows the consecutively collected 20‐min fractions, where −60 min, −40 min, and −20 min represent those collected under basal conditions, followed by those corresponding to the K ⁺‐ and KA‐stimulated period (from +20 min to +180 min), where +20 min represents the fraction collected immediately after the last basal fraction of −20 min, during which K ⁺ was administered through the probe. The mean net stimulated output ± SEM, obtained from the area under the K ⁺, from −20 min to +60 min, or kainate‐evoked concentration time curve (AUC), from +80 min to +160 min, normalized to one time interval of 20 min, with basal output subtracted, are shown, together with their statistical analysis parameters, in Table 1. The concentration peaks of aspartate, glutamate, and taurine evoked by K ⁺ and KA were statistically significant over basal levels. Concentration peaks were not statistically significant, either in the absence of stimulation or when the stimulation was applied to PF9601N pretreated animals.

Figure 2

Time course of the output of dopamine (A), DOPAC (B), and HVA (C) evoked by local administration of K ⁺ and kainate (KA). The extracellular concentrations were expressed as percentage of their respective basal levels for illustrative purposes. Original concentration values, fmol/μL of perfusate (nM), were used for statistical analysis. The mean net stimulated output ± SEM, obtained from the area under the K ⁺, from −20 min to + 60 min, or kainate‐evoked concentration time curve (AUC), from +80 min to 160 min, normalized to one time interval of 20 min, with basal output subtracted, are shown in Table 2, together with their statistical analysis parameters. The concentration increases in dopamine evoked by K ⁺ and KA were statistically significant, and they were not affected by PF9601N pretreatment. The concentration of the metabolites of dopamine, DOPAC and HVA, was significantly reduced by K ⁺ and KA and was not affected by PF9601N pretreatment. PF9601N pretreatment, in the absence of K ⁺ and KA stimulation, did not affect the basal levels of dopamine and metabolites.

Figure 3

Glial activation in response to kainate (KA) in the presence and absence of PF9601N. (A) Representative microphotographs of microglial reactivity (upper panels; lectin histochemistry) and astrocytosis (lower panels; GFAP immunostaining) in the striatum of animals after local kainate (KA) administration. A significant increase in microglial activation and astrocytosis was apparent in the striatum of KA‐treated rats (center panels) shown by both the number of positive cells and their staining intensity. PF9601N pretreatment was able to prevent the activation of microglial cells and astrocytosis (right panels). (B) Quantification of microglial activation (each treatment group: n = 4). (C) Quantification of astrogliosis (each treatment group: n = 4). One‐way ANOVA followed by post hoc Bonferroni test for multiple comparisons. KA treated group: *significantly different from control (P < 0.05), ***highly significantly different from control (P < 0.001), ^###highly significantly different from KA treated + PF9601N (P < 0.001).

Figure 4

Apoptosis in response to kainate (KA) in the presence and the absence of PF9601N. Representative microphotographs of TUNEL immunohistochemistry are shown in the upper panel with the quantitative analysis of apoptotic nuclei in the lower panel. The local administration of kainate (KA) in the striatum (n = 6) led to significant number of apoptotic nuclei, 70 ± 13% (mean ± SEM) of total counts (apoptotic + healthy nuclei). Pretreatment with PF9601N (n = 6) was able to prevent this, as the percentage of apoptotic nuclei found, 3.2 ± 1.1%, was similar to the control group, 4.2 ± 4.2% (n = 5). One‐way ANOVA followed by post hoc Bonferroni test for multiple comparisons. KA treated group: ***highly significantly different from control (P < 0.001), NS, not significantly different from control, ^###Highly significantly different from KA treated + PF9601N (P < 0.001).