Inhibition of carbonic anhydrase reduces brain injury after intracerebral hemorrhage

Abstract

Carbonic anhydrase-1 (CA-1) is a metalloenzyme present at high concentrations in erythrocytes. Our previous studies showed that erythrocyte lysis contributes to brain edema formation after intracerebral hemorrhage (ICH) and a recent study indicates that CA-1 can cause blood-brain barrier disruption. The present study investigated the role of CA-1 in ICH-induced brain injury.There were three groups in the study. In the first, adult male Sprague-Dawley rats received 100 μl autologous blood injection into the right caudate. Sham rats had a needle insertion. Rat brains were used for brain CA-1 level determination. In the second group, rats received an intracaudate injection of either 50 μl CA-1 (1 μg/μl) or saline. Brain water content, microglia activation and neuronal death (Fluoro-Jade C staining) were examined 24 hours later. In the third group, acetazolamide (AZA, 5 μl, 1 mM), an inhibitor of carbonic anhydrases, or vehicle was co-injected with 100 μl blood. Brain water content, neuronal death and behavioral deficits were measured. We found that CA-I levels were elevated in the ipsilateral basal ganglia at 24 hours after ICH. Intracaudate injection of CA-1 induced brain edema (79.0 ± 0.6 vs. 78.0±0.2% in saline group, p<0.01), microglia activation and neuronal death (p<0.01) at 24 hours. AZA, an inhibitor of CA, reduced ICH-induced brain water content (79.3 ± 0.7 vs. 81.0 ± 1.0% in the vehicle-treated group, p<0.05), neuronal death and improved functional outcome (p<0.05).These results suggest that CA-1 from erythrocyte lysis contributes to brain injury after ICH.

Figure 1

(A): Western blot analysis showing CA-1 content in the ipsilateral basal ganglia 1 day (lanes 1-3), 3 days (lanes 4-6) and 7 days (lanes 7-9) after 100 μl blood injected into right caudate. Equal amounts of protein (50 μg) were used. CA-1 levels were expressed as % of the sham control. Values are mean ± S.D., * p< 0.05 versus day 7. (B): CA-1 contents in the ipsilateral (ipsi-) basal ganglia 24 hours after sham operation (lanes 1-3), and the ipsilateral basal ganglia (lanes 4-6) and the contralateral (contra-) basal ganglia (lanes 7-9) after ICH. Equal amounts of protein (50 μg) were loaded. CA-1 levels were expressed as % of the sham control. Values are mean ± S.D.; #p< 0.01 versus sham and ICH-contra.

Figure 2

Bar graphs showing brain water (A) and sodium (B) ion content 24 hours after intracaudate injection of CA-1 or saline. Values are mean ± SD; *p<0.05 versus saline group.

Figure 3

Fluoro-Jade C staining showing degenerated neurons in the ipsilateral basal ganglia 1 day and 3 days after intracaudate injection of either saline or CA-1.Values are mean ± SD; #p< 0.01 versus saline group.

Figure 4

OX-42 immunohistochemistry in the ipsilateral basal ganglia 3 days after intracaudate injection of either saline or CA-1.Values are mean ± SD; #p< 0.01 versus saline group.

Figure 5

Bar graphs showing the effects AZA on brain water (A) and sodium ion (B) contents 24 hours after ICH. Values are mean ± S.D.; *p< 0.05 versus the vehicle group.

Figure 6

Bar graph showing the effects of AZA on ICH-induced neuronal death. Values are mean ± S.D. #p< 0.01 versus the vehicle group.

Figure 7

Bar graphs displaying AZA improved behavioral outcome including forelimb placing (A), forelimb use asymmetry (B), and corner turn (C) scores 24 hours after ICH. Values are mean ± S.D.; #p< 0.01 or *p< 0.05 versus the vehicle group.