Complement Inhibition Attenuates Early Erythrolysis in the Hematoma and Brain Injury in Aged Rats

Abstract

Background and Purpose- Early erythrolysis in the hematoma contributes to brain injury after intracerebral hemorrhage (ICH). This study investigated the effects of N-acetylheparin, a complement inhibitor, and aurin tricarboxylic acid, a membrane attack complex inhibitor, on early erythrolysis, brain iron deposition, and brain injury in aged rats. Methods- There were 3 parts in the study. First, aged (18 months old) male Fischer 344 rats had an ICH. The time course of erythrolysis in the hematoma was determined by T2* weighted magnetic resonance imaging, and the expression of CD163 was examined. Second, aged rats had an ICH with N-acetylheparin or vehicle. Rats were euthanized at days 1, 3, and 28 after magnetic resonance imaging (T2-, T2*-weighted, and T2* array) and behavioral tests. Brains were used for immunohistochemistry. Third, aged rats had an ICH with avaurin tricarboxylic acid or vehicle. The rats had magnetic resonance imaging and behavioral tests and were euthanized at day 3. Brains were used for immunohistochemistry. Results- Early erythrolysis occurred within the clot in aged F344 rats. There were increased numbers of CD163-positive cells after ICH. Almost all perihematomal CD163-positive cells were microglia/macrophages, while positive neurons were found more distant from the hematoma. Coinjection of N-acetylheparin attenuated erythrolysis, iron accumulation, CD163 expression, microglia activation, brain swelling, and neuronal death in the acute phase, as well as reducing brain atrophy and neurological deficits in the chronic phase. Coinjection of aurin tricarboxylic acid also reduced erythrolysis and ICH-induced brain injury. Conclusions- Inhibiting complement activation resulted in less erythrolysis and brain injury after ICH.

Keywords: CD163; aurin tricarboxylic acid; intracerebral hemorrhage; microglia; n-acetylheparin.

Figure 1.

Time course of early erythrolysis and CD163 expression after ICH in aged rats. A) Representative consecutive T2* MRIs and H&E staining at days 1 and 3 after ICH. Scale bar = 200 μm. The ratio of non-hypo-T2* volume to total T2* lesion volume was quantified. Values are mean±SD, n = 13. B) CD163 immunoreactivity in hematoma, perihematomal tissue, and ipsilateral (Ipsi) and contralateral (Contra) basal ganglia (BG). Scale bar = 20 μm. Values are mean±SD, n = 4. *P<0.05 vs. the other time points. C) Examples of immunofluorescence double labeling for CD163 with either Iba-1 (microglia/macrophage marker), NeuN (neuronal marker) or GFAP (astrocyte marker) at day 3 after ICH. Scale bar = 20 μm. The percentage of CD163 positive cells in the perihematomal and ipsilateral BG areas that were Iba-1- or NeuN-positive is shown. Values are mean±SD, n = 6.

Figure 2.

Effects of N-acetyl heparin (NAH) on early erythrolysis and perihematomal iron after ICH. A) Representative consecutive T2* MRIs including the hematoma at days 1 and 3 after ICH in NAH- and vehicle-treated animals. The ratio of non-hypo-T2* lesion volume to total T2* lesion volume was quantified. Values are mean±SD, n = 20 per group for day 1, n = 13 per group for day 3, # P<0.01 vs. vehicle control. B) Representative consecutive T2* and R2* MRIs at days 1 (n = 20), 3 (n = 13), 7 (n = 8), 14 (n = 8), and 28 (n = 8) after ICH in NAH- and vehicle-treated animals. The ratio of R2* change ((Ipsi-Contra)/Contra) was quantified. Values are mean±SD, * P<0.05, # P<0.01 vs. vehicle control.

Figure 3.

Effects of N-acetyl heparin (NAH) on CD163 expression, brain swelling and neuronal loss at day 3 after ICH. A) CD163 immunoreactivity in hematoma, perihematomal tissue, and ipsilateral (Ipsi) and contralateral (Contra) basal ganglia (BG) 3 days after ICH. Scale bar = 20 μm. Values are mean±SD, n = 6, per group, * P<0.05, # P<0.01 vs. vehicle control. B) Representative T2 MRI at day 3 after ICH in NAH-treated and vehicle-treated animals. Brain swelling was quantified. Values are mean±SD, n = 13, * P<0.05 vs. vehicle control. C) DARPP-32 immunostaining at day 3 after ICH. Scale bar = 1 mm. Neuronal loss was quantified. Values are mean±SD, n = 6, * P<0.05 vs. vehicle control.

Figure 4.

Effects of N-acetyl heparin (NAH) on brain atrophy and neuronal loss at day 28 after ICH and functional recovery after ICH. A) Representative T2 MRI at day 28 after ICH in NAH-treated and vehicle-treated animals. Ipsilateral ventricle enlargement was quantified. Values are mean±SD, n = 8, # P<0.01. B) DARPP-32 immunostaining at day 28 after ICH. Scale bar = 1 mm. Neuronal loss was quantified. Values are mean±SD, n = 8, * P<0.05 vs. vehicle control. C) Forelimb use asymmetry and D) corner turn test scores pre-ICH (n = 20) and at day 1 (n = 20), 3 (n = 14), 7 (n = 8) and 28 (n = 8) after ICH in NAH-treated and vehicle-treated animals. Values are mean±SD, * P<0.05 vs. vehicle control.

Figure 5.

Effects of aurin tricarboxylic acid (ATA) on early erythrolysis, perihematomal iron and CD163 expression after ICH. A) Representative consecutive T2* MRIs including the hematoma at days 1 (n = 18) and 3 (n = 9) after ICH in ATA- and vehicle-treated animals. The ratio of non-hypo-T2* lesion volume to total T2* lesion volume was quantified. Values are mean±SD, # P<0.01 vs. vehicle control. B) Representative consecutive R2* MRIs at days 1 (n = 18) and 3 (n = 9) after ICH in NAH- and vehicle-treated rats. The ratio of R2* change was quantified. Values are mean±SD, * P<0.05, # P<0.01 vs. vehicle control. C) CD163 immunoreactivity in hematoma, perihematomal tissue, ipsilateral and contralateral basal ganglia (BG) at day 3 after ICH. Scale bar=20 μm. Values are mean±SD, n = 9, per group, * P<0.05, # P<0.01 vs. vehicle control.

Figure 6.

Effects of aurin tricarboxylic acid (ATA) on the expression of HO-1 and Iba-1, brain swelling and neuronal loss after ICH. A) HO-1 immunoreactivity in hematoma, perihematomal tissue, ipsilateral and contralateral basal ganglia (BG) at day 3 after ICH. Scale bar = 20 μm. Values are mean±SD, n = 9, per group, * P<0.05 vs. vehicle control. B) Iba-1 immunoreactivity in hematoma, perihematomal tissue, ipsi- and contralateral BG 3 days after ICH. Scale bar = 20 μm. Values are mean±SD, n=9, per group, * P<0.05 vs. vehicle control. C) Representative T2 MRI at day 3 after ICH in ATA- and vehicle-treated animals. Brain swelling was quantified. Values are mean±SD, n=9, * P<0.05 vs. vehicle control. D) DARPP-32 immunostaining at day 3 after ICH. Scale bar = 1 mm. Neuronal loss was quantified. Values are mean±SD, n = 9, * P<0.05 vs. vehicle control.