Fetal Hemoglobin Improves Cerebral Blood Flow and Decreases Brain Inflammation in Transgenic- Sickle Mice
Authors: Cui Min Hui , Billett Henny H , Suzuka Sandra , Ambadipudi Kamalakar , Minniti Caterina P. , Acharya Seetharama , Branch Craig A 
Published: November 2019

Abstract
Introduction: Fetal hemoglobin (HbF) inhibits the deoxygenation-induced polymerization of sickle hemoglobin (HbS), the underlying pathophysiology of sickle cell disease (SCD). Higher HbF levels are associated with reduced sickle related pathology (Lettre G, Bauer DE. The Lancet 2016; 387(10037): 2554-64.). A major therapeutic approach to SCD has been to try to increase HbF levels, primarily by hydroxyurea administration. While hydroxyurea improves HbF and reduces sickling, its mechanism of action remains controversial, and even less is known regarding its effects on sickle-related cerebral pathology. We studied transgenic Berkeley sickle mice (BERK) (Paszty C et. al., Science 1997; 278(5339): 876-8.) with different HbF levels using in vivo MRI in an effort to estimate the ameliorative effects of HbF on cerebral blood flow and brain inflammation.
Methods: BERK mice, expressing exclusively human a- and bS-globins with low (LF, n=7), medium (MF, n=8) and high (HF, n=6) HbF levels were studied at 9.4 T MRI system under isoflurane anesthesia. C57BL/6J mice were used as controls (n=9). We measured whole-brain cerebral blood flow (CBF-WB) using MRI. Diffusion tensor imaging was used to measure whole-brain mean diffusivity (MD-WB), a measure of brain edema and inflammation, and white matter fractional anisotropy (FA-WM) a biomarker of neuropathology. Tail vein blood was employed to obtain hematological parameters prior to MRI.
Results: Select MRI and related data are shown in Table 1. BERK-LF mice (~3% HbF) exhibited lower mean cell hemoglobin concentration (MCHC) and hematocrit (Hct) compared with C57BL mice. BERK-MF mice (~20% HbF) showed increased MCHC compared with BERK-LF mice. BERK-HF mice (~30% HbF) had the highest MCHC and Hct of all BERK mice, but MCHC was still lower than levels seen in C57BL mice. The low MCHC and Hct levels in BERK-LF mice were associated with high CBF and MD . CBF was significantly elevated in BERK-LF and BERK-MF mice by 130% and 120%, respectively, compared to C57BL mice. BERK-HF mice, on the other hand, exhibited normalized CBF (187.0±27.5 vs. 180.0±30.5 ml/100g/min, BERK-HF vs. C57BL respectively), despite lower MCHC and Hct levels compared to C57BL mice. Brain inflammation (elevated MD levels) were also observed in BERK-LF mice compared to controls. In contrast, BERK-MF and BERK-HF mice showed reduced MD; both were significantly lower than BERK-LF mice and not different from control. Only the BERK-LF demonstrated significantly lower FA values (reduced microstructuraly complexity) compared to controls.
CBF in all mice (BERK and C57BL) was inversely related to Hct (r=-0.433, p=0.019) and MCHC (r=-0.527, p=0.003). However, when controls were excluded from this analysis, only HbF level significantly predicted CBF (r=-0.485, p=0.030) and MD (r=-0.636, p=0.003), shown in Figure 1. HbF level also positively predicted MCHC (r=0.832, p<0.0001) and Hct (r=0.448, p=0.042) in BERK mice.
Conclusions: Cerebral perfusion is markedly elevated in BERK mice compared to C57BL control mice and is inversely correlated with Hct, MCHC and total fetal Hb level. These data suggest that increased HbF can significantly reduce CBF, and in our study CBF levels in high-gamma BERK (~30%) were indistinguishable from control CBF, despite the lower Hct levels. We also demonstrate that brain edema, as reflected in lower MD, is improved when HbF levels reach ~20%.
The improved CBF and reduced edema with higher HbF may be due to several factors: an antisickling effect on HbS, reduced oxidative stress, improved NO activity, and improved blood oxygen carrying capacity. HbF levels approaching 30% improved CBF, improved FA and reduced MD, suggesting that increased HbF levels may reduce cerebral insults stemming from cerebral hyperemia.
These data suggest that new therapeutic approaches to further increase HbF to even higher levels than that achieved with hydroxyurea may provide greater hematologic and neuropathologic improvements in patients with SCD. If hydroxyuyrea induced HbF increases cannot be further improved, additional or combined therapeutic approaches (such as Pegylated Hb) may be combined with HbF therapies to improve oxygen delivery, reduce oxidative stress and reduce CBF toward normal levels.

Disclosures
No relevant conflicts of interest to declare.