Haptoglobin Attenuates Cerebrospinal Fluid Hemoglobin-Induced Neurological Deterioration in Sheep.

Secondary brain injury (SBI) occurs with a lag of several days post-bleeding in patients with aneurysmal subarachnoid hemorrhage (aSAH) and is a strong contributor to mortality and long-term morbidity. aSAH-SBI coincides with cell-free hemoglobin (Hb) release into the cerebrospinal fluid. This temporal association and convincing pathophysiological concepts suggest that CSF-Hb could be a targetable trigger of SBI. However, sparse experimental evidence for Hb's neurotoxicity in vivo defines a significant research gap for clinical translation. We modeled the CSF-Hb exposure observed in aSAH patients in conscious sheep, which allowed us to assess neurological functions in a gyrencephalic species. Twelve animals were randomly assigned for 3-day bi-daily intracerebroventricular (ICV) injections of either Hb or Hb combined with the high-affinity Hb scavenger protein haptoglobin (Hb-Hp, CSL888). Repeated CSF sampling confirmed clinically relevant CSF-Hb concentrations. This prolonged CSF-Hb exposure over 3 days resulted in disturbed movement activity, reduced food intake, and impaired observational neuroscores. The Hb-induced neurotoxic effects were significantly attenuated when Hb was administered with equimolar haptoglobin. Preterminal magnetic resonance imaging (MRI) showed no CSF-Hb-specific structural brain alterations. In both groups, histology demonstrated an inflammatory response and revealed enhanced perivascular histiocytic infiltrates in the Hb-Hp group, indicative of adaptive mechanisms. Heme exposure in CSF and iron deposition in the brain were comparable, suggesting comparable clearance efficiency of Hb and Hb-haptoglobin complexes from the intracranial compartment. We identified a neurological phenotype of CSF-Hb toxicity in conscious sheep, which is rather due to neurovascular dysfunction than structural brain injury. Haptoglobin was effective at attenuating CSF-Hb-induced neurological deterioration, supporting its therapeutic potential.

Blood oxygenation-level dependent cerebrovascular reactivity imaging as strategy to monitor CSF-hemoglobin toxicity.

OBJECTIVES: Cell-free hemoglobin in the cerebrospinal fluid (CSF-Hb) may be one of the main drivers of secondary brain injury after aneurysmal subarachnoid hemorrhage (aSAH). Haptoglobin scavenging of CSF-Hb has been shown to mitigate cerebrovascular disruption. Using digital subtraction angiography (DSA) and blood oxygenation-level dependent cerebrovascular reactivity imaging (BOLD-CVR) the aim was to assess the acute toxic effect of CSF-Hb on cerebral blood flow and autoregulation, as well as to test the protective effects of haptoglobin. METHODS: DSA imaging was performed in eight anesthetized and ventilated sheep (mean weight: 80.4 kg) at baseline, 15, 30, 45 and 60 minutes after infusion of hemoglobin (Hb) or co-infusion with haptoglobin (Hb:Haptoglobin) into the left lateral ventricle. Additionally, 10 ventilated sheep (mean weight: 79.8 kg) underwent BOLD-CVR imaging to assess the cerebrovascular reserve capacity. RESULTS: DSA imaging did not show a difference in mean transit time or cerebral blood flow. Whole-brain BOLD-CVR compared to baseline decreased more in the Hb group after 15 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.01 vs -0.01 ± 0.02) and remained diminished compared to Hb:Haptoglobin group after 30 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.01 vs 0.0 ± 0.01), 45 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.01 vs 0.01 ± 0.02) and 60 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.02 vs 0.01 ± 0.01). CONCLUSION: It is demonstrated that CSF-Hb toxicity leads to rapid cerebrovascular reactivity impairment, which is blunted by haptoglobin co-infusion. BOLD-CVR may therefore be further evaluated as a monitoring strategy for CSF-Hb toxicity after aSAH.

Contractile effects of hemoglobin-free human cerebrospinal fluid on isolated porcine cerebral arteries

To elucidate the mechanism involved in the cerebral vascular spasm following subarachnoid hemorrhage (SAH), the effects of the cerebrospinal fluid (CSF) obtained from the SAH patients on the resting tension and its influence on the contractile responses to various vasoactive agents and to hypoxia were investigated in isolated porcine cerebral arteries. All the CSFs containing hemoglobin (Hb) produced contraction and some Hb-free CSFs also elicited contraction. When the Hb-free CSF was separated by microfilter, the filtrate of < 30,000 MW did not produce contraction, while the fraction above 30,000 MW elicited more marked contractile responses than the unfractionated CSF. The CSF contraction was significantly attenuated in the presence of indomethacin or nimodipine, whereas the contractions induced by KCl, prostaglandin F2alpha (PGF2alpha), or endothelin-1 (ET-1) were not affected by the CSF pretreatment. However, the contractile responses induced by 5-hydroxytryptamine (5-HT) and phenylephrine (PE) were markedly potentiated by the pretreatment. Hypoxia-induced vasoconstriction was significantly potentiated by the pretreatment with either unfractionated CSF or the CSF fraction of above 30,000 MW. These results suggest that unknown vasocontractile substance(s) exists in the Hb-free CSF and that the substance, with its MW above 30,000, is activated by hypoxia and acts synergistically with 5-HT and PE, and that extracellular calcium influx and cyclooxygenase are also involved in the cerebral vasoconstrictory effect of Hb-free CSF.