Intranasal guanosine administration presents a wide therapeutic time window to reduce brain damage induced by permanent ischemia in rats

Abstract

In addition to its intracellular roles, the nucleoside guanosine (GUO) also has extracellular effects that identify it as a putative neuromodulator signaling molecule in the central nervous system. Indeed, GUO can modulate glutamatergic neurotransmission, and it can promote neuroprotective effects in animal models involving glutamate neurotoxicity, which is the case in brain ischemia. In the present study, we aimed to investigate a new in vivo GUO administration route (intranasal, IN) to determine putative improvement of GUO neuroprotective effects against an experimental model of permanent focal cerebral ischemia. Initially, we demonstrated that IN [(3)H] GUO administration reached the brain in a dose-dependent and saturable pattern in as few as 5 min, presenting a higher cerebrospinal GUO level compared with systemic administration. IN GUO treatment started immediately or even 3 h after ischemia onset prevented behavior impairment. The behavior recovery was not correlated to decreased brain infarct volume, but it was correlated to reduced mitochondrial dysfunction in the penumbra area. Therefore, we showed that the IN route is an efficient way to promptly deliver GUO to the CNS and that IN GUO treatment prevented behavioral and brain impairment caused by ischemia in a therapeutically wide time window.

Keywords: Guanosine; Intranasal; Ischemia; Neuroprotection; Purines.

Fig. 1

Brain structures. a Dose curve of IN [³H] GUO administration. Radioactivity detected 15 min after administration of 0.05 ml per nostril. b Time curve. Radioactivity detected after administration of 30 mg/ml of IN [³H] GUO solution, 0.05 ml per nostril. Each point represents the mean ± SE. N = 6 animals per group

Fig. 2

Blood plasma. a Dose curve of IN [³H] GUO administration. Radioactivity detected 15 min after administration of 0.05 ml per nostril. b Time curve. Radioactivity detected after administration of 30 mg/ml of IN [³H] GUO solution, 0.05 ml per nostril. Each point represents the mean ± SE. N = 6 animals per group. *P < 0.05 compared to the naïve group

Fig. 3

Comparison of injection routes for [³H] GUO solution administration. The radioactivity in: a different brain structures and b the CSF and blood plasma, five minutes after IN (0.05 ml per nostril) or IP (0.1 ml) 30 mg/ml [³H] GUO administration. Data are expressed as the mean ± SE. N = 6 (brain structures and plasma) or 3–4 (CSF) animals per group. *P < 0.05 compared to IN groups

Fig. 4

Performance in the cylinder test 48 h after ischemia. Symmetry score of rats receiving IN treatment of saline (IS, N = 21) or GUO through a protocol starting immediately (IG 0 h, N = 16), 1 h (GUO 1 h, N = 9) or 3 h (GUO 3 h, N = 19) after ischemia induction. The symmetry score index and the schedule of administration for each treatment are described in material and methods. ^# P < 0.0001 compared to IS, IG 0 h, IG 1 h, IG 3 h groups; *P < 0.05 compared to IS group; **P < 0.001 compared to IS group. Data are expressed as the mean ± SE

Fig. 5

Parameters of brain infarct measured 48 h after cerebral ischemia. a Volume of brain infarct of rats receiving treatment with saline (IS, N = 14) or GUO starting immediately (GUO 0 h, N = 16), 1 h (GUO 1 h, N = 9) or 3 h (GUO 3 h, N = 12) following ischemia induction. b Representative images of brain TTC staining of experimental groups. c Correlation (IS and GUO 0H groups) between cortex infarct volume and forelimb function (symmetry score) measured 48 h after ischemia, Pearson’s R ² = 0.016, P = 0.41. *P < 0.05 compared to IS group

Fig. 6

Dysfunctional mitochondria in the penumbra zone (contralateral and ipsilateral sides) measured 48 h after ischemia induction: a percentage of dysfunctional mitochondria in the penumbra area from rats of SHAM (N = 5 animals), IS (N = 7 animals), and IG 3 h (N = 7 animals). The description of each experimental group is detailed in material and methods. c Correlation between infarct volume and forelimb function (symmetry score; Pearson’s R ² = 0.39, P = 0.006)