BACKGROUND: Erythrolysis releases free Hb (hemoglobin), which is one of the most upstream and important molecules causing early brain injury and cerebral vasospasm after subarachnoid hemorrhage (SAH). The purpose of this study was to investigate if a Hb scavenger protein Hp (haptoglobin) supplementation prevents early brain injury and cerebral vasospasm and influences the lymphatic drainage mechanism in an established SAH model of mice by a blood injection into the prechiasmatic cistern. METHODS: This study consisted of 4 parts, with a total of 317 C57BL/6 male mice undergoing sham or SAH modeling, randomly followed by 24-hour intracerebroventricular infusion of no vehicle, 30.1 mg/mL BSA, 30.1 mg/mL Hp (100%; a mixture of Hp1-1, Hp2-1, and Hp2-2), 50% Hp, 25% Hp, and 12.5% Hp solutions from 30 minutes postmodeling. The effects were evaluated at 24 and 48 hours postmodeling. RESULTS: The 100%Hp decreased mortality and brain edema until 48 hours post-SAH and suppressed post-SAH neurological impairments, Hb infiltrations into the perivascular spaces and brain tissues, fibrinogen-positive microthrombi formation, microglial activation, and caspase-dependent neuronal apoptosis, as well as large-vessel vasospasm on India-ink angiography at 24 hours compared with vehicle-treated SAH mice. The Hp solutions up to 50% concentrations also prevented post-SAH neurological impairments, and those up to 25% concentrations suppressed post-SAH neuronal apoptosis and vasospasm development until 48 hours. Immunohistochemical staining of deep cervical and mandibular lymph nodes demonstrated that Hb was increased in the lymphatic sinus after SAH and was further increased by Hp administration in SAH animals. CONCLUSIONS: This study first showed that an Hp concentrate prevented early brain injury and cerebral vasospasm by inhibiting Hb penetration into brain tissues and increasing lymphatic drainage of free Hb in an established SAH model of mice.

Aneurysmal subarachnoid hemorrhage (SAH) is a poor-outcome disease with a delayed neurological exacerbation. Fibulin-5 (FBLN5) is one of matricellular proteins, some of which have been involved in SAH pathologies. However, no study has investigated FBLN5’s roles in SAH. This study was aimed at examining the relationships between serially measured plasma FBLN5 levels and neurovascular events or outcomes in 204 consecutive aneurysmal SAH patients, including 77 patients (37.7%) with poor outcomes (90-day modified Rankin Scale 3–6). Plasma FBLN5 levels were not related to angiographic vasospasm, delayed cerebral ischemia, and delayed cerebral infarction, but elevated levels were associated with severe admission clinical grades, any neurological exacerbation and poor outcomes. Receiver-operating characteristic curves indicated that the most reasonable cut-off values of plasma FBLN5, in order to differentiate 90-day poor from good outcomes, were obtained from analyses at days 4–6 for all patients (487.2 ng/mL; specificity, 61.4%; and sensitivity, 62.3%) and from analyses at days 7–9 for only non-severe patient (476.8 ng/mL; specificity, 66.0%; and sensitivity, 77.8%). Multivariate analyses revealed that the plasma FBLN5 levels were independent determinants of the 90-day poor outcomes in both all patients’ and non-severe patients’ analyses. These findings suggest that the delayed elevation of plasma FBLN5 is related to poor outcomes, and that FBLN5 may be a new molecular target to reveal a post-SAH pathophysiology.

Early brain injury (EBI) is an important cause that determines outcomes after aneurysmal subarachnoid hemorrhage (SAH). Our recent clinical study reported that a high concentration of plasma fibulin-5 (FBLN5), one of matricellular proteins, was associated with poor outcomes after SAH. The aim of this study was to investigate whether and how FBLN5 was associated with EBI during an acute phase of SAH in mice. C57BL/6 male mice underwent sham or filament perforation SAH modeling, and vehicle or four dosages (0.001, 0.01, 0.1, and 1 μg) of short or long recombinant FBLN5 (rFBLN5) were randomly administrated by an intracerebroventricular injection. Neurobehavioral test, measurements of brain water content, immunohistochemical staining, and Western blotting were performed to evaluate EBI 24 h after SAH. Short rFBLN5 had no significant effects on EBI, but administration of long rFBLN5 containing an arginine-glycine-aspartic acid motif improved neurobehavior functions depending on the dosages, without affecting brain edema. Administration of long rFBLN5 also reduced cleaved caspase-3-dependent neuronal apoptosis, associated with the inhibition of post-SAH upregulation of transforming growth factor-β1, but no significant changes in the expression level of Smad 2/3, mitogen-activated protein kinases, and another matricellular protein tenascin-C. Although further research is required to clarify the detailed mechanism, this study demonstrated for the first time that FBLN5 played a protective role against neuronal apoptosis in an acute phase of experimental SAH.

Aneurysmal subarachnoid hemorrhage (SAH) has increased with the aging of the population, but the outcome for elderly SAH patients is very poor. Therefore, predicting the outcome is important for determining whether to pursue aggressive treatment. Pigment epithelium-derived factor (PEDF) is a matricellular protein that is induced in the brain, and the plasma levels could be used as a biomarker for the severity of metabolic diseases. This study investigated whether acute-phase plasma PEDF levels could predict outcomes after aneurysmal SAH in the elderly. Plasma samples and clinical variables were collected over 1–3 days, post-SAH, from 56 consecutive elderly SAH patients ≥75 years of age registered in nine regional stroke centers in Japan between September 2013 and December 2016. The samples and variables were analyzed in terms of 3-month outcomes. Acute-phase plasma PEDF levels were significantly elevated in patients with ultimately poor outcomes, and the cutoff value of 12.6 µg/mL differentiated 3-month outcomes with high sensitivity (75.6%) and specificity (80.0%). Acute-phase plasma PEDF levels of ≥12.6 µg/mL were an independent and possibly better predictor of poor outcome than previously reported clinical variables. Acute-phase plasma PEDF levels may serve as the first biomarker to predict 3-month outcomes and to select elderly SAH patients who should be actively treated.

Chronic hydrocephalus following aneurysmal subarachnoid hemorrhage (SAH) is a complication that can lead to deterioration in neurological status and cognitive impairment. Our recent clinical study reported that a high concentration of plasma fibulin-5 (FBLN5), a matricellular protein, was associated with the occurrence of chronic hydrocephalus after SAH. This study aimed to investigate whether and how FBLN5 was associated with hydrocephalus during acute to later phases of SAH in mice. C57BL/6 male mice underwent sham or filament perforation SAH modeling, and vehicle or two dosages (0.01 and 0.1 μg) of short or long recombinant FBLN5 (rFBLN5) were randomly administrated by an intracerebroventricular injection. Neurobehavioral tests, measurements of the degree of ventricular enlargement, Western blotting, and immunohistochemical staining were performed to evaluate hydrocephalus 24 and 48 h after SAH. After SAH, ventricular dilatation did not occur at 24 h but developed at 48 h, and both doses of long rFBLN5 with an arginine–glycine–aspartic acid domain suppressed ventricular dilatation at 48 h after SAH. Long rFBLN5 also decreased phosphorylated p38 in the brain parenchyma and prevented post-SAH increases in perivascular macrophages, as well as microglia activation in the brain parenchyma at 48 h after SAH. Although further research is required to clarify the detailed mechanism, this study demonstrated for the first time that exogenous administration of FBLN5 may have a protective effect against ventricular dilatation after experimental SAH.