Red Blood Cell Microparticles Limit Hematoma Growth in Intracerebral Hemorrhage

Abstract

Background: Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype with no effective therapies. Limiting hematoma expansion is a promising therapeutic approach. Red blood cell-derived microparticles (RMPs) are novel hemostatic agents. Therefore, we studied the potential of RMPs in limiting hematoma growth and improving outcomes post-sICH.

Methods: sICH was induced in rats by intrastriatal injection of collagenase. RMPs were prepared from human RBCs by high-pressure extrusion. Behavioral and hematoma/lesion volume assessment were done post-sICH. The optimal dose, dosing regimen, and therapeutic time window of RMP therapy required to limit hematoma growth post-sICH were determined. We also evaluated the effect of RMPs on long-term behavioral and histopathologic outcomes post-sICH.

Results: RMP treatment limited hematoma growth following sICH. Hematoma volume (mm³) for vehicle- and RMP- (2.66×10¹⁰ particles/kg) treated group was 143±8 and 86±4, respectively. The optimal RMP dosing regimen that limits hematoma expansion was identified. RMPs limit hematoma volume when administered up to 4.5-hour post-sICH. Hematoma volume in the 4.5-hour post-sICH RMP treatment group was lower by 24% when compared with the control group. RMP treatment also improved long-term histopathologic and behavioral outcomes post-sICH.

Conclusions: Our results demonstrate that RMP therapy limits hematoma growth and improves outcomes post-sICH in a rodent model. Therefore, RMPs have the potential to limit hematoma growth in sICH patients.

Keywords: behavior; brain; collagenases; erythrocytes; hemostasis; histology.

Figure 1:

The time course of hematoma expansion post-collagenase-induced sICH: (A) experimental design; (B) hematoma volume * p<0.05, vs previous time group; and (C) hematoma frequency maps at 7 coronal levels. n=4 for 2, 4, 12, 24, 48 h post-sICH groups and n=5 for 6 h post-sICH group * p<0.05 vs immediate previous time group.

Figure 2:

The effect of different RMP doses on hematoma expansion post-collagenase-induced sICH: (A) experimental design; (B) hematoma volume; (C) neurological score; and (D) hematoma frequency maps at 7 coronal levels (bregma +6 mm to −6 mm). Bottom row depicts the difference in hematoma expansion between vehicle and treatment groups. n=19, and 10 for vehicle all RMP-treated groups, respectively * p<0.05, ** p<0.01 and *** p<0.001.

Figure 3:

The effect of vehicle and three different RMP treatment paradigms on hematoma expansion post-collagenase-induced sICH: (A) experimental design; (B) hematoma volume; (C) neurological score; and (D) hematoma frequency maps at 7 coronal levels (bregma +6 mm to −6 mm). Bottom row depicts the difference in hematoma expansion between vehicle and treatment groups. n=35, 10, 15, and 15 for vehicle, paradigm A, B, and C groups, respectively. * p<0.05 and *** p<0.001.

Figure 4:

The effect of vehicle, RMP, and rFVIIa treatment on long-term brain damage and behavioral deficits following collagenase-induced sICH: (A) experimental design; (B) neurological score ⁑ p<0.01 vs vehicle, ⁂ p<0.001 vs vehicle. n=17, 16, and 18 for vehicle-, RMP-, and rFVIIa-treated groups, respectively. (C) ladder rung walking test * p<0.05 vs vehicle, ⁑ p<0.01 vs vehicle, ǀ p<0.05 vs pre-sICH, † p<0.01 vs pre-sICH, and ‡ p<0.001 vs pre-sICH. n=17, 15, and 16 for vehicle-, RMP-, and rFVIIa-treated groups, respectively. (D) Corner turn test. ǀ p<0.05 vs pre-sICH, ‡ p<0.001 vs pre-sICH. n=17, 16, and 18 for vehicle-, RMP-, and rFVIIa-treated groups, respectively. ‡ p<0.001 vs pre-sICH, * p<0.05 vs vehicle. (E) Cylinder test n=17, 14, and 16 for vehicle-, RMP-, and rFVIIa-treated groups, respectively. ǀ p<0.05 vs pre-sICH, † p<0.01 vs pre-sICH, ‡ p<0.001 vs pre-sICH. (F) Representative example images showing lesion volume following sICH. Presented histology example images are photoshop processed. Lesion volume following sICH. n=10, 14, and 15 for vehicle-, RMP-, and rFVIIa-treated groups, respectively * p<0.05 vs vehicle.

Figure 5:

The effect of vehicle and four different RMP treatment time points on hematoma expansion post-collagenase-induced sICH: (A) experimental design; (B) hematoma volume; (C) neurological score; and (D) hematoma frequency maps at 7 coronal levels (bregma +6 mm to −6 mm). Right row depicts the difference in hematoma expansion between vehicle and treatment groups. n=20, and 10 for vehicle and RMP treated groups, respectively * p<0.05, ** p<0.01 and *** p<0.001.