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Phoneutria nigriventer spider venom (PNV) contains ion channels-acting neuropeptides that in rat induces transitory blood-brain barrier breakdown (BBBb) in hippocampus in parallel with VEGF upregulation. We investigated whether VEGF has a neuroprotective role by inhibiting its binding to receptor Flk-1 by itraconazole (ITZ). FT-IR spectroscopy examined the biochemical status of hippocampus and evaluated BBBb in rats administered PNV or ITZ/PNV at periods with greatest toxicity (1-2h), recovery (5h) and visual absence of symptoms (24h), and compared to saline and ITZ controls. The antifungal treatment before venom intoxication aggravated the venom effects and increased BBB damage. FT-IR spectra of venom, hippocampi of controls, PNV and ITZ-PNV showed a 1400 cm−1 band linked to symmetric stretch of carboxylate and 1467 cm−1 band (CH2 bending: mainly lipids) that were considered biomarker and reference bands, respectively. Inhibition of VEGF/Flk-1 binding produced marked changes in lipid/protein stability at 1-2h. The largest differences were observed in spectra regions assigned to lipids, both symmetric (2852 cm−1) and asymmetric (2924 and 2968 cm−1). Quantitative analyses showed greatest increases in the 1400 cm−1/1467 cm−1 ratio also at 1h. Such changes at period of rats' severe intoxication referred to wavenumber region from 3106 cm−1 to 687 cm−1 assigning for C-H and N-H stretching of protein, Amide I, C=N cytosine, N-H adenine, Amide II, CH2 bending: mainly lipids, C-O stretch: glycogen, polysaccharides, glycolipids, z-type DNA, C-C, C-O and CH out-of-plane bending vibrations. We conclude that VEGF has a neuroprotective role and can be a therapeutic target in PNV envenomation. FT-IR spectroscopy showed to be instrumental for monitoring biochemical changes in this model of P. nigriventer venom-induced BBB disruption.VEGF neuroprotection is investigated in PNV-induced blood-brain barrier breakdown.Blockade of VEGF-Flk-1 binding by itraconazole (was analyzed by FT-IR spectroscopy.Blockade of VEGF-Flk-1 aggravated rats' toxic condition and increased BBB damage.FT-IR data revealed profound disturbance in biochemical homeostasis of hippocampus.FT-IR spectra data revealed a PNV biomarker and a neuroprotective role for VEGF.