Local delivery of nimodipine by prolonged-release microparticles-feasibility, effectiveness and dose-finding in experimental subarachnoid hemorrhage

Abstract

Background and purpose: To investigate the effect of locally applied nimodipine prolonged-release microparticles on angiographic vasospasm and secondary brain injury after experimental subarachnoid hemorrhage (SAH).

Methods: 70 male Wistar rats were categorized into three groups: 1) sham operated animals (control), 2) animals with SAH only (control) and the 3) treatment group. SAH was induced using the double hemorrhage model. The treatment group received different concentrations (20%, 30% or 40%) of nimodipine microparticles. Angiographic vasospasm was assessed 5 days later using digital subtraction angiography (DSA). Histological analysis of frozen sections was performed using H&E-staining as well as Iba1 and MAP2 immunohistochemistry.

Results: DSA images were sufficient for assessment in 42 animals. Severe angiographic vasospasm was present in group 2 (SAH only), as compared to the sham operated group (p<0.001). Only animals within group 3 and the highest nimodipine microparticles concentration (40%) as well as group 1 (sham) demonstrated the largest intracranial artery diameters. Variation in vessel calibers, however, did not result in differences in Iba-1 or MAP2 expression, i.e. in histological findings for secondary brain injury.

Conclusions: Local delivery of high-dose nimodipine prolonged-release microparticles at high concentration resulted in significant reduction in angiographic vasospasm after experimental SAH and with no histological signs for matrix toxicity.

Figure 1. Preparation of in situ forming nimodipine depot system.

Figure 2. Representative digital subtraction angiographies of A: Scham-operated group, B: SAH group and C: Treatment-group (40%) demonstrating the internal carotid artery (ICA) to external carotid artery (ECA).

Figure 3. Angiographic comparison of the sham-operated and the subarachnoid hemorrhage (SAH)-only group, the angiographic comparison of the sham-operated group and the subarachnoid hemorrhage (SAH)-only group revealed a significant difference of arterial filling (p = 0.004, Kruskal-Walis-test).

Figure 4. Graph showing mean value of the total brain cross-sectional vessel area (grey bars) with confidence interval (black lines) in all groups, the treatment group receiving 40% of nimodipine microparticles demonstrated the highest filling intensity, with a statistically significant difference to the treatment group receiving 20% and 30% of nimodipine microparticles (p = 0.014, Mann-Whitney test).

Figure 5. Cortical microinfarct in a control animal.

In the H&E stained section, a small infarct in the deeper cortical layers is visible (a). At higher magnification, a mononuclear infiltrate can be distinguished (b, arrows; inset from a), indicating ongoing resorption. Iba1 immunohistochemistry (c; d inset from c) reveals activated microglia in and around the lesion (d, arrows). Scale bar corresponds to 1000 µm (a,c) and 200 µm (b,d), respectively.

Figure 6. Semiquantitative analysis of MAP2 immunohistochemistry as a marker for subtle ischemic damage reveals no diffrences between the various treatment groups (Kruskal-Wallis test; p<0.05 was considered significant.

Values are means of optical density ± SD presented as % of sham rats).