Cerebral Metabolic Alterations in Transgenic and Hemizygous Murine Models of Sickle Cell Disease

BACKGROUND: In SCD, the mechanisms leading to the high incidence of acute stroke in both children and adults remain controversial, while the effects of chronic hypoxia, hyperemia and oxidative stress upon the sickle brain are poorly understood. In limited studies, elevation of brain N-acetyl-aspartate (NAA) has been observed in SCD inflicted children (Steen RG and Ogg RJ, AJNR, 2005). We recently presented evidence that NAA is elevated in one mild murine model (NY1DD) of SCD (Cui MH, NMR Biomed., 2017); we proposed that alterations in NAA are linked to neuronal metabolism and may be indicative of a compensatory increase in mitochondrial energy utilization. Here we present data obtained in additional animal models of SCD that further support these hypotheses.

METHODS: Control animals (C57BL6) were compared to sickle animals; the BERK mouse expressing various levels of fetal hemoglobin (HbF, BERK-G) ; the BERK-TX mouse, based on C57BL6 mice irradiated to lethal levels and subsequently infused with nucleated cells from the bone-marrow of BERK mice; and the BERK-HEMI (Hemizygous sickle transgenic animals with murine a knockout, human HbS (a^Hb^S) and a mouse b human S transgene (mb/hS). BERK-HEMI exhibit low O₂ affinity hemoglobin (P₅₀ ~ 57).

Cerebral measurements were conducted using MRI/MRS methods at 9.4 Tesla. We measuredcerebral blood flow (CBF), cerebral oxygen extraction (using blood level oxygen dependent MRI during a short bolus of hyperoxia [BOLD-RHO]), brain microstructural integrity (from Diffusion Tensor Imaging [DTI] fractional isotropy [FA]) and tissue inflammation (from DTI mean diffusivity [MD]). Magnetic Resonance Spectroscopy (MRS) in the thalamus provided NAA, glutamate (Glu), myoinositial (Ins), and other metabolite measures.

RESULTS: MRI and MRS findings exhibit increasing NAA, Glu and BOLD-RHO which seem to suggest a metabolic adjustment not just to anemia but to some other factor(s) (Figure 1); BERK-HEMI animals exhibit the most significant metabolic changes while the BERK-G animals (with low gamma) had the highest CBF and worst anemia, although NAA in the BERK-G appeared similar to WT levels. Evolving changes in the BERK-TX animal after transplant (Figures 2 and 3) suggest a phase of increasing NAA while HbSS and CBF peak. These changes inversely correlate with decreasing Ins. Oxygen extraction and brain inflamation appear to rise after NAA stabilizes or drops. Full BERK-like systemic pathology is established by 12 weeks post transplant but in brain > 12 weeks are required.

CONCLUSIONS: NAA was not elevated where the anemia and CBF were the highest (BERK-G and advanced BERK-TX). Instead, NAA was highest in NY1DD and BERK-HEMI (both without significant anemia), in early BERK-TX, where CBF was mildly elevated. This suggests that something other than anemia was driving the NAA elevations. Possibilities include chronic mild hypoxia (from low Hb O₂ affinity, reperfusion injury, or ROS). The changes evident in the early post-transplant BERK-TX brain suggest that CBF is driven by HbSS expression and not anemia, since hematocrit remains relatively stable (after an initial drop). This finding suggests likely candidates to be ROS activity, Hb O₂ affinity, blood products or degredation in oxygen carrying capacity. The compensatory alterations in energy homeostasis (increased NAA and Glu, decreased Ins and other metabolite changes) may reflect a shift toward increased neuronal glycolysis or increased GSH production and recycling, both of which require increased mitochondrial energy utilization. Decreases in Ins may be related to altered astrocyte/neuronal homeostasis, or the increased cerebrovascular activity. In the advanced stages of the BERK-TX transition, NAA and glutamate levels drop, CBF plateaus, oxygen extraction is high, cerebral inflammation increases and neuronal structure is degraded, all of which are consistent with a system failure, which further predispose the brain to cerebrovascular injury and may explain why NAA increases have been observed only in children with SCD and not adults. If so, the decline of pediatric NAA levels to its "normalization" in adult patients may signal emergence of cerebral dysfunction and impaired cognition (Strouse JJ et al., Blood, 2006) and are consistent with elevations in oxygen extraction fraction and exhaustion of cerebrovascular reserves (Kosinski PD et al; Br. J Haematol., 2017).

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