To systematically compare the drug delivery efficiency of three administration routes—intracalvariosseous administration (ICO), intrathecal administration (ICA), and intranasal administration (INA)—and to evaluate the therapeutic effects of Y3 solution delivered via these routes in a mouse model of permanent middle cerebral artery occlusion (pMCAO).

Male C57BL/6J mice were randomly assigned to the blank control group, ICO-EB group, ICA-EB group, and INA-EB group. In the ICO-EB group, 5 μL of 10% Evans blue (EB) solution was administered via ICO at the lambda point of the parietal bone. In the ICA-EB group, 5 μL of 10% EB solution was injected into the cerebellomedullary cistern. In the INA-EB group, 5 μL of 10% EB solution was administered intranasally into both nostrils in four divided doses. EB concentrations in various tissues (skull, meninges, cerebral cortex, basal ganglia, hippocampus, cerebellum, and serum) were measured at 10, 30, and 60 min after administration using a microplate reader. Male mice were further randomized into the sham group, model group, ICO-Y3 group, ICA-Y3 group, and INA-Y3 group. A pMCAO model was established using the intraluminal filament method. One hour after modeling, the sham group was divided into three subgroups (n=2 each) receiving 5 μL normal saline via ICO, ICA, or INA once daily for 7 days. The ICOY3, ICAY3, and INAY3 groups received 5 μL of Y3 solution (2 mg/mL) via the corresponding route once daily for 7 days. Body weight and survival were recorded daily. Neurological function was assessed on days 1, 3, 5, and 7 postischemia using the modified neurological severity score. Infarct volume was measured with 2,3,5triphenyltetrazolium chloride staining on day 7. EB concentrations, body weight changes, and neurological scores were analyzed by twoway ANOVA followed by Dunnett's test; infarct volume was compared by oneway ANOVA; survival was analyzed with the Kaplan-Meier method.

From 10 to 60 min after EB administration, EB concentrations in the cerebral cortex decreased over time in the ICA-EB group [(12.04±8.23), (11.55±2.94), (4.15±2.37) μg/mL] and the INA-EB group [(16.52±1.19), (8.20±0.82), (4.59±1.11) μg/mL], but increased in the ICO-EB group [(50.92±2.99), (52.33±9.96), (53.89±11.52) μg/mL]. At 60 min after administration, the EB concentration in the cerebral cortex of the ICO-EB group was significantly higher than that in the ICA-EB and INA-EB group [(53.89±11.52) μg/mL vs (4.15±2.37), (4.59±1.11) μg/mL; t=16.165, 13.256, both P＜0.001]. At 60 min after ICO, EB concentrations in brain tissues were highest in the cerebellum, followed by the cerebral cortex and hippocampus, and lowest in the basal ganglia, the EB concentrations were (63.55±11.94), (53.89±11.52), (53.02±5.94) and (40.83±12.03) μg/mL, respectively. On day 7 post-ischemia, compared with the model group, the ICO-Y3 group showed a increased survival rate (60% vs 40%, df=4.000, P=0.228), improved neurological function scores (14.86±1.07 vs 9.00±0.82, t=10.752, P＜0.001), and reduced cerebral infarct volume [(28.07±4.92) mm³vs (38.62±6.18) mm³, t=3.551, P=0.004].

Intracalvariosseous administration demonstrates superior drug-delivery efficiency and therapeutic efficacy in ischemic stroke compared with ICA and INA, suggesting its potential as a novel treatment strategy.