Functional and anatomical evidence of cerebral tissue hypoxia in young sickle cell anemia mice

Abstract

Cerebral ischemia is a significant source of morbidity in children with sickle cell anemia; however, the mechanism of injury is poorly understood. Increased cerebral blood flow and low hemoglobin levels in children with sickle cell anemia are associated with increased stroke risk, suggesting that anemia-induced tissue hypoxia may be an important factor contributing to subsequent morbidity. To better understand the pathophysiology of brain injury, brain physiology and morphology were characterized in a transgenic mouse model, the Townes sickle cell model. Relative to age-matched controls, sickle cell anemia mice demonstrated: (1) decreased brain tissue pO₂ and increased expression of hypoxia signaling protein in the perivascular regions of the cerebral cortex; (2) elevated basal cerebral blood flow , consistent with adaptation to anemia-induced tissue hypoxia; (3) significant reduction in cerebrovascular blood flow reactivity to a hypercapnic challenge; (4) increased diameter of the carotid artery; and (5) significant volume changes in white and gray matter regions in the brain, as assessed by ex vivo magnetic resonance imaging. Collectively, these findings support the hypothesis that brain tissue hypoxia contributes to adaptive physiological and anatomic changes in Townes sickle cell mice. These findings may help define the pathophysiology for stroke in children with sickle cell anemia.

Introduction

Stroke occurs in up to 10% of children with sickle cell anemia and is a common cause of morbidity in these patients.¹ Silent cerebral infarctions are more prevalent and may reflect microvascular pathology.² Impaired learning and cognitive function are also observed in these pediatric patients, suggesting that anemia causes impairment in neurological function.^3,4 For decades, chronic blood transfusion has been used as a therapy to reduce the level of sickle hemoglobin (HbS) to less than 30%. Recent clinical trials have demonstrated that targeted red blood cell (RBC) transfusion therapy provides a significant reduction of stroke occurrence in these children, with high risk of stroke identified by elevated transcranial Doppler cerebral blood flow (CBF) velocity.^5,6 Surprisingly, little is known about the mechanism of increased stroke and neurological injury in children with sickle cell anemia.

Potential candidate mechanisms for stroke include: (1) the vaso-occlusive properties of sickled red cells and their increased adherence to the endothelial lining of blood vessels;^7,8 (2) a lack of vascular reactivity or vasodilatory reserve to physiological stimuli including hypercarbia, hypoxemia, and increased neuronal activity,^9,10 and (3) anemia-mediated tissue hypoxia.¹¹ Animal studies have clearly demonstrated that anemia-induced tissue hypoxia contributes to the compensatory increase in CBF, which is required to maintain oxygen homeostasis in the brain.^12,13 Interruption of the cardiovascular responses to increase CBF has been associated with cerebral hypoxia^14,15 and stroke.¹⁶

Exposure to hypoxia increased the mortality rate in a mouse model of sickle cell disease,¹⁷ suggesting that these mice are susceptible to hypoxia-induced organ failure and death. In humans, adult sickle cell patients demonstrate impairment in cerebrovascular reserve (CVR) capacity⁹ and autoregulation of CBF,¹⁸ suggesting a deficiency in cerebrovascular reactivity in the brain of these patients. Elevated cerebral CBF and reduced CVR have also been reported in the pediatric population.^19–24 We hypothesize that sickle cell anemia leads to inadequate cerebral oxygen delivery and tissue hypoxia, resulting in a compensatory increase in basal cerebral vasodilation (blood flow) and reduced capacity for further adaptive cerebral vasodilation to additional physiological stimuli (CO₂). This basal condition may make patients more susceptible to stroke. The demonstration that RBC transfusion can reduce CBF and stroke risk in children with sickle cell disease supports the hypothesis that disrupted oxygen homeostasis contributes to cerebral injury.^2,5

To better understand the pathophysiology and mechanism of stroke and brain injury in sickle cell anemia, a transgenic mouse model of sickle cell disease was utilized.²⁵ This knock-in sickle cell mouse model only expresses human α and βs globin genes in the mouse locus, resulting in chronically mild to moderate anemia (Hb ∼ 70–90 g/L).^25,26 These mice demonstrate sickled red cell properties and splenomegaly, which can be reversed by replacing the defective β-globin gene (βs) with a normal copy of the gene (βA) in the embryonic stem cell.²⁵ In the context of brain injury, a recent study demonstrated increased infarct size with a middle cerebral arterial occlusion model in these mice.²⁷ The goal of this study was to characterize clinical features and brain physiology of sickle cell disease in this mouse model, including abnormally high CBF, impaired CVR, and magnetic resonance imaging (MRI)-detectable changes in brain anatomy. This work validates the use of this mouse model for further investigation into the pathophysiology and mechanism of stroke and brain injury in sickle cell anemia.

Materials and methods

Animal model

Female homozygous Townes (B6; 129-Hba^tm1(HBA)TowHbb^{tm2(HBG1,HBB*)Tow}/Hbb^{tm3(HBG1,HBB)Tow}/J) transgenic sickle cell mice of C57BL/6J background (with both human α- and β- (βA and βs form) globin genes knocked into the mouse locus) from the Jackson Laboratory (Bar Harbor, ME, USA) were used.²⁵ Female C57BL/6J mice from the Jackson Laboratory were used as controls. Vendor health reports indicated that the mice were free of viral and parasitic pathogens; however, the Townes sickle cell mice were from an area that tested positive for the organism Klebsiella. In total, 21 sickle cell mice and 27 control mice were used in the study; a subset of mice was used for more than one of the four experimental assays. The number of animals studied using each assay is described in the experimental subsections below. Mice were co-housed in a standard cage, with ad libitum access to food and water, in a pathogen-free environment on a 12-h light:dark cycle. All of the experimental procedures were performed during the light phase. All animal experiments were approved by the Animal Care Committee at the Toronto Centre for Phenogenomics, conducted in accordance with the Canadian Council on Animal Care's Guide to the Care and Use of Experimental Animals and complied with the ARRIVE guidelines. For a subset of mice, blood samples were collected for blood smears (Supplementary Figure 1) and Hb concentration (83 ± 8 g/L in the sickle cell mice) was measured using a hemoglobin analyzer (HemoCue).

Measurement of CBF using continuous arterial spin labelling

Experimental set-up and hypercapnia protocol

Mice were anesthetized using isoflurane (4% for induction and 1% for maintenance) in 100% O₂. Mice were then endotracheally intubated (22 gauge catheter), placed in a purpose-built holder, and mechanically ventilated using a pressure-controlled ventilator (TOPO small animal ventilator, Kent Scientific, Torrington, CT, USA). Ventilation parameters were the same for each animal (135 cycles per minute) and the respiration rate was monitored continuously throughout the scan. Lactate Ringer's was injected intraperitoneally to maintain hydration. Additionally, 0.1 mg/kg pancuronium bromide (Sigma-Aldrich, St Louis, MO, USA) was injected intraperitoneally for muscle relaxation, ensuring optimal breathing control throughout the experiment. The partial pressure of carbon dioxide (pCO₂) was monitored throughout the experiment using a transcutaneous blood gas analyzer (TCM4, Radiometer, Cleveland, OH, USA). Temperature was maintained at 37℃ using warmed air and was monitored using a rectal probe. The gas mixture inhaled by the mice cycled between 30% O₂/70% N₂ (normocapnic) and 5% CO₂/30% O₂/65% N₂ (hypercapnic) for a total of two cycles. After the CO₂ challenge, there was a six-minute break between measurements to allow the animal to return to normal physiologic conditions.

MRI

Two-dimensional (2D) continuous arterial spin labelling (CASL) measurements were performed at 6–7 weeks of age in 11 sickle cell mice (19.3 ± 1.9 g) and 11 control mice (18.2 ± 1.1 g) using a multi-channel 7.0-T, 40 cm horizontal bore magnet (Varian Inc. Palo Alto, CA, USA), and a custom-built saddle coil. If the transcutaneous blood gas analyzer failed during the hypercapnia protocol, the cerebrovascular reactivity data had to be excluded (total of three mice (two sickle cell and one control) were excluded).

To identify the landmarks needed for positioning the imaging and labeling planes, a mid-sagittal localizer scan was acquired using a 2D spin echo sequence with TR = 500 ms, TE = 7 ms, slice thickness = 3 mm, NEX = 1, field-of-view (FOV) = 7.0 × 3.5 cm and matrix size = 350 × 176 and an imaging time of 1.5 min/scan. A coronal imaging slice was positioned to transect the hippocampus with the labeling plane positioned 0.8 cm posterior of this location. The control label condition was created by positioning the labeling plane an equivalent distance anterior of this plane. A 3 second 9-μT RF labeling pulse accompanied by a 1.3-G/cm gradient was used to label blood passing through the common carotid. Control and label images were interleaved and a post-label delay of 500 ms was applied to reduce transit time dispersion artifacts and to clear the intravascular signal. The CASL images were acquired using a 2D fast spin echo pulse sequence with TR = 6000 ms, echo train length = 16, TE_eff = 15 ms, 90° hard excitation and 180° hard refocusing pulses, slice thickness = 2 mm, FOV = 5.0 × 10.0 cm, matrix size = 200 × 384, giving an in-plane resolution of 250 μm and a total CASL imaging time of approximately 5 min. Inversion efficiency was measured in six adult C57BL/6J mice using a 2D flow compensated spoiled gradient echo sequence with the inversion and imaging planes positioned on the common carotid arteries and separated by 5 mm (TR = 3200 ms, TE = 4.5 ms, FOV = 2.5 × 2.5, matrix size = 256 × 256, slice thickness = 1 mm, post-label delay = 0). The measured inversion efficiency was 0.80 ± 0.03. CBF was quantified using a single-compartment biophysical model and corrected for the enhancement of T₁ relaxation due to magnetization transfer, as described previously.²⁸ One experimenter (LSC) performed the data acquisition and a second experimenter (SP), blinded to the genotype of the animals, manually drew regions-of-interest around the whole brain, the cortex, and the hippocampus.

High-frequency ultrasound imaging

Ultrasound measurements were performed at 10–13 weeks of age in 9 sickle cell mice (22.3 ± 3.0 g) and 10 control mice (21.4 ± 1.3 g). A high-frequency ultrasound imaging system (Vevo 2100, VisualSonics Inc., Toronto, Canada) with a 30 MHz linear array transducer was used.²⁹ During ultrasound imaging, mice were anesthetized using isoflurane at 1.5% in 21% O₂, and body temperature was maintained at 36–37℃. The heart rate and respiration rate were continuously monitored. For evaluating the cardiac function, M-mode recordings of the left ventricle were made and analyzed for wall thicknesses and chamber dimensions. Fractional shortening was calculated as a measure of left ventricular systolic function.³⁰ For measuring the aortic blood flow, the diameter of the aortic annulus was measured at peak systole in two-dimensional imaging at the aortic orifice, and the Doppler velocity was recorded at the same level. Then the left ventricular stroke volume and cardiac output were derived. For assessing the blood flow to the brain, M-mode traces and pulsed Doppler velocity spectra were recorded from the left common carotid artery (LCCA), approximately 3 mm proximal to its bifurcation, and from the internal carotid artery (ICA). M-mode recordings were made with the ultrasound beam perpendicular to the vessel, and pulsed Doppler velocity was measured with the sample volume adjusted to cover the entire vascular lumen and the smallest intercept angle possible (<60°) between the flow direction and the ultrasound beam. The intensity-weighted mean velocity of Doppler spectrum was traced as a function of time to measure the velocity-time integral (VTI).³¹ The flow was calculated by multiplying the VTI with the vessel area derived from the diameter as well as the heart rate. All parameters were averaged for three cardiac cycles.

Brain tissue pO₂

The brain O₂ tension was measured at 10–13 weeks of age in 7 sickle cell mice and 10 control mice using Oxyphor G4 phosphorescence as described previously,^13,32 utilizing a novel oxygen microsensor.³³ Briefly, mice were anesthetized using isoflurane (3% induction and 2% maintenance) and temperature was maintained at 37℃ using a heating pad and monitored using a rectal probe. A small hole (1–2 mm in diameter) was made in the skull and a microsensor filled with the oxyphor and connected to a fiber optic thread was threaded through a 20-gauge needle and inserted into the brain. Tissue pO₂ was recorded in real-time and, once the signal had stabilized, averaged over 10 min. Two of the sickle cell mice had to be excluded because of instrumentation failure.

Brain sample preparation

At 13 weeks of age, eight sickle cell mice (23.7 ± 1.3 g) and eight control mice (22.7 ± 1.2 g) were anesthetized and a transcardiac perfusion was performed as described previously.^34,35 Briefly, animals were anesthetized with a combination of Ketamine/Xylazine (150 mg/kg/10 mg/kg) via intraperitoneal injection. The thoracic cavity was opened and animals were perfused through the left ventricle with 30 mL of phosphate-buffered saline (PBS) containing 1 μL/mL heparin (1000 USP units/mL) and 2 mM ProHance (Bracco Diagnostics, Inc., NJ, USA) followed by 30 mL of 4% paraformaldehyde (PFA) with 2 mM ProHance in PBS for fixation. After perfusion, mice were decapitated and the skin, lower jaw, and ears were removed. The brain within the skull was incubated in 4% PFA containing 2 mM ProHance overnight at 4℃ and then transferred to PBS containing 2 mM ProHance and 0.02% sodium azide for at least seven days prior to imaging.

Ex vivo MRI

A custom-built 16-coil solenoid array was used to image 16 samples concurrently.³⁶ Parameters used in the scans were optimized for gray-white matter contrast: T2-weighted 3D fast spin echo sequence with TR = 2000 ms, echo train length = 6, TE_eff = 42 ms, FOV = 2.5 × 2.8 × 1.4 cm, and matrix size = 450 × 504 × 250, producing an image with 56-μm isotropic nominal spatial resolution. In the first phase-encode dimension, consecutive k-space lines were assigned to alternating echoes to move discontinuity-related ghosting artifacts to the edges of the FOV.³⁷ This scheme necessitates oversampling in the phase-encode direction to avoid interference of the ghosts with the main image. This first phase-encode was oversampled by a factor of 2 (504 phase-encode points) giving a FOV of 2.8 cm that was subsequently cropped to 1.4 cm after reconstruction. Total imaging time was 11.7 h.

Volume analysis

An automated image registration-based approach using the advanced normalization tools (ANTs) deformation algorithm³⁸ was used to assess anatomical differences related to sickle cell disease.³⁹ The images were registered together through a process of linear and nonlinear registration steps to allow calculation of an average image. The registration yielded deformation fields for each individual brain, and the Jacobian determinants of these deformation fields provided an estimate of local volume expansion/contraction at every voxel in the brain.⁴⁰ Using the results of the linear alignment, multiple templates of a segmented anatomical atlas with 62 labelled structures⁴¹ were created (the MAGeT procedure).⁴² From the final voted segmentation, volume changes were calculated and expressed in absolute (mm³) volumes.

HIF-1α immunofluorescence staining

PFA-fixed brain tissues were paraffin-blocked and sectioned (10 µm thick) as previously described.^13,43 Immunofluorescence staining was performed by incubating tissue slides overnight at 4℃ with a 1:100 dilution of specific primary antibodies for HIF-1α (Novus Biologicals; NB100-131). A fluorescently labeled secondary antibody (Abcam ab150113) was used to detect specific binding of the primary antibody at a dilution of 1:200. Microscopy was performed utilizing a fluorescence confocal microscope (Nikon ECLIPSE 90i).

Statistical analysis

All statistical tests were performed using RMINC (https://github.com/mcvaneede/RMINC) and R statistical software (www.r-project.org). CBF, CVR, ultrasound data, and brain tissue pO₂ measurements are presented as box and whisker plots and analyzed using t-tests to compare sickle cell and control mice. Data from each group of animals are reported as the mean ± standard deviation. Test for normality was performed using a Shapiro–Wilk test. CVR was expressed as percentage change in CBF per mmHg change in pCO₂ between normocapnia and hypercapnia. A value of p < 0.05 was taken to be significant. For the MR volumetric data, a t-test was computed for each structure and at every voxel comparing sickle cell to control mice. Multiple comparisons were controlled for using the false discovery rate (FDR).⁴⁴ Statistical significance was defined at an FDR threshold of 10%.

Results

Changes in CBF and CO₂ vascular reactivity and carotid anatomy

Basal CBF in sickle cell mice (n = 11) was significantly elevated relative to age-matched C57BL/6J control mice (n = 11) (3.1 ± 0.5 vs. 2.2 ± 0.4 ml/g/min; p < 0.00005) (Figure 1). Cerebrovascular reactivity to CO₂ was reduced by 55% in the whole brain (p < 0.0005), 40% in the cerebral cortex (p < 0.05) and 73% in the hippocampus (p < 0.001) of sickle cell mice (n = 9), relative to control mice (n = 10) (Figure 2). This CVR deficit was not attributed to a difference in a change in pCO₂, which was comparable between the two groups (23 ± 14 vs. 18 ± 9 mmHg for sickle cell mice and controls, respectively). Ultrasound measurements demonstrated that sickle cell mice (n = 9) had significantly increased LCCA diameter (0.58 ± 0.04 vs. 0.43 ± 0.04 mm; p < 0.000001), ICA diameter (0.35 ± 0.08 vs. 0.29 ± 0.03 mm; p < 0.03), LCCA blood flow (1.09 ± 0.34 vs. 0.80 ± 0.17 ml/min; p < 0.03), and a trend towards increased ICA blood flow (0.52 ± 0.24 vs. 0.38 ± 0.08; p < 0.1), relative to control mice (n = 10) (Figure 3).

Figure 1.

Cerebral blood flow (CBF) under normoxic condition in sickle cell mice (n = 11) versus controls (n = 11) at 6–7 weeks of age. The basal blood flow in the entire brain slice is increased by approximately 41% in sickle cell mice (***p < 0.00005).

Figure 2.

Cerebrovascular reserve (CVR) in sickle cell mice (n = 9) versus controls (n = 10) at 6–7 weeks of age. The CVR is decreased in sickle cell mice by approximately 55% in the entire brain slice (***p < 0.0005), 40% in the cortex (*p < 0.05), and 73% in the hippocampus (**p < 0.001) relative to controls.

Figure 3.

Left common carotid artery (LCCA) and internal carotid artery (ICA) diameter and blood flow as measured by ultrasound at 10–13 weeks of age in sickle cell mice (n = 9) and controls (n = 10). The LCCA diameter is 33% larger in sickle cell mice compared to controls (**p < 0.000001), resulting in a 37% increase in blood flow through this vessel (*p < 0.03). The ICA diameter is 23% larger in sickle cell mice compared to controls (*p < 0.03), resulting in a 36% increase in blood flow through this vessel (p < 0.1).

Cardiac function in sickle cell mice

The results of cardiac ultrasound imaging performed in sickle cell and control mice are shown in Table 1. The diameter of the aortic orifice was significantly elevated in sickle cell mice relative to age-matched control mice. The left ventricular stroke volume, cardiac output, and average left ventricular wall thickness were significantly increased in the sickle cell mice relative to controls. However, there was no difference in fractional shortening between the sickle cell mice and control mice. Five of the nine sickle cell mice imaged using ultrasound showed evidence of aortic valve regurgitation. These cardiovascular adaptations and evidence of aortic regurgitation are consistent with findings in humans with sickle cell anemia.⁴⁵

Table 1.

Cardiac parameters measured by high-frequency ultrasound.

Parameter	Control (n = 10)	Sickle cell (n = 9)
Aortic flow
Heart rate (bpm)	431 ± 73	444 ± 75
AO diameter (mm)	1.09 ± 0.07	1.23 ± 0.05**
LV SV (μl)	18.6 ± 2.0	29.6 ± 12.0*
CO (ml/min)	8.0 ± 1.4	13.0 ± 5.2*
LV chamber dimensions by M mode
Average wall thickness (mm)	1.02 ± 0.05	1.37 ± 0.14**
LV FS (%)	31.3 ± 4.3	28.3 ± 7.6

AO: aortic orifice; CO: cardiac output; FS: fractional shortening; LV: left ventricle; SV: stroke volume.

Note: Data are mean ± standard deviation.

^*p < 0.01,

^**p < 0.0001.

Morphological assessment of sickle cell anemia brains

Assessment of brain morphology was performed using ex vivo MRI. A voxelwise comparison between sickle cell (n = 8) and control mice (n = 8) demonstrated significant volume differences in specific regions in the gray and white matter of the brain (Figure 4). Areas of the brain that demonstrated a decrease in volume were regions in the striatum, amygdala, cortex, posterior hippocampus, thalamus, lateral septum, and fimbria. Other areas of the brain were larger in sickle cell anemia mice, including regions in the anterior hippocampus, hypothalamus, cerebellum, periaqueductal gray, superior colliculus, inferior colliculus, and anterior commissure. Brain morphology was also evaluated based on segmented structure volumes. While there was no difference in whole brain volume, 13 of the segmented structures showed a significant volume difference in sickle cell mice compared to control mice (Supplementary Table 1). In addition to the regions identified by the voxelwise analysis, two white matter structures, the habenular commissure and the mammilothalamic tract, were found to be significantly larger in sickle cell mice compared to controls.

Figure 4.

Anatomical differences in the brains of sickle cell mice (n = 8) versus controls (n = 8) at 13 week of age. (a) Average ex vivo MR image overlaid with color maps indicating regions that are significantly smaller (cool colors) or significantly larger (hot colors) in sickle cell mice compared to controls (10% FDR). Focal significant differences are seen bilaterally throughout the brain in gray matter regions including the motor cortex (white arrowhead in 1), the lateral septum (green arrowhead in 1), the striatum (black arrowhead in 2), the basolateral complex of the amygdala (black arrowhead in 3), the thalamus (white arrowhead in 3), the hippocampus (white arrowhead in 4 and in 5), the medial dorsal nucleus of the thalamus (black arrowhead in 4), the hypothalamus (green arrowhead in 4), the superior and inferior colliculus (black arrowhead in 6), the periaqueductal gray (green arrowhead in 6) and regions of the cerebellum (cerebellar paravermis – white arrowhead in 6, central lobule – black arrowhead in 7, paramedian lobule and copula of the pyramis – white arrowhead in 7, crus I and II of the ansiform lobule – green arrowhead in 7), as well as in some white matter regions including the anterior commissure (black arrowhead in 1) and the fimbria (white arrowhead in 2). (b) Volume of indicated voxels to give an indication of the scale of differences illustrated in (a). The vertical axis shows the value of the Jacobian determinant, which represents a volume scale factor relative to the volume of the voxel in the average image.

Assessment of tissue hypoxia and hypoxic cell signaling (HIF-1α)

Brain tissue pO₂ in sickle cell mice (n = 5) was significantly reduced relative to age-matched control mice (n = 10) (30 ± 14 vs. 57 ± 14 mmHg; p < 0.005) (Figure 5). In addition, increased HIF-1α staining was clearly evident in the perivascular regions of sickle cell mice relative to controls (Figure 6). The pattern of HIF-1α staining is strikingly similar to animals exposed to acute hemodilutional anemia.^13,43

Figure 5.

Brain tissue oxygen tension in sickle cell mice (n = 5) versus controls (n = 10) at 10–13 week of age. The pO₂ is decreased by approximately 46% in sickle cell mice (*p < 0.005) relative to controls.

Figure 6.

Increased HIF-1α perivascular staining in the cerebral cortex of sickle cell anemia mice versus controls. Two representative images from each group demonstrate increased perivascular HIF-1α protein expression (green) in sickle cell mice. This HIF-1α staining pattern is similar to that in acute hemodilutional anemia (13, 43). Scale bar = 100 µm.

Discussion

This study has demonstrated a number of interrelated physiological and anatomical features that suggest that tissue hypoxia may contribute to altered cerebrovascular responses, cardiovascular remodeling, and anatomical changes within the brain of sickle cell mice. We have demonstrated decreased brain tissue pO₂ and increased expression of HIF-1α with a perivascular distribution within the cerebral cortex under basal conditions. This suggests that anemia-induced tissue hypoxia is present in these mice^13,46 and that hypoxia may contribute to the mechanism of cerebral injury. This was associated with an elevated basal CBF, an increased carotid artery diameter and an increased left ventricular cardiac output and wall thickness, suggesting that adaptive cardiovascular responses had occurred in an attempt to optimize cerebral oxygen delivery. Reduced cerebrovascular CO₂ reactivity in sickle cell mice suggests that adaptive capacity or cerebrovascular response is impaired. Evidence in humans has supported the idea that a lack of cerebrovascular reactivity to CO₂ may predict subsequent cerebral injury.^47,48 Finally, anatomical differences in cerebral structures suggest that adaptive responses to maintain cerebral oxygen delivery may be insufficient to provide adequate perfusion for normal neurological development. In combination, these experimental data support the idea that tissue hypoxia contributes to the pathophysiology of cerebral injury in sickle cell anemia. Further investigation of the mechanisms involved may provide a means to improve cerebral perfusion in developing children with sickle cell anemia with the goal of improving cerebral function and minimizing the risk of stroke in these patients.

As a master regulator of genes involved in maintaining cellular oxygen homeostasis, HIF-1α is regulated at the protein level in response to changes in oxygen levels. In hypoxia, HIF-1α protein is stabilized due to the inhibition of a degradation pathway involving von Hippel–Lindau protein.⁴⁹ Accumulated HIF-1α binds to nuclear HIF-1β to activate genes important in oxygen homeostasis, such as those involved in glycolysis, erythropoiesis, and iron metabolism.⁴⁹ Thus, HIF-1α can serve as a marker of tissue hypoxia. Evidence of tissue hypoxia was observed with increased HIF-1α protein expression in perivascular regions in the brain of sickle cell mice. The pattern of brain HIF-1α staining in sickle cell mice is very similar to that of mice exposed to acute hemodilutional anemia who experience demonstrable tissue hypoxia.^13,43 This finding support the hypothesis that cerebral tissue hypoxia is present in the brains of young sickle cell mice prior to clear evidence of cerebral injury. In acute hemodilutional anemia, quantitative assessment of tissue oxygen tension demonstrated a significant drop in brain pO₂ at Hb levels as high as 90 g/L, with more profound hypoxia at Hb levels of 50–60 g/L.^14,15,43,50 This occurred despite a compensatory increase in cardiac output and CBF to optimize brain O₂ delivery. The decrease in brain oxygen tension was accompanied by an increase in brain HIF-1α protein expression and downstream HIF-dependent genes as early as 6–24 h following hemodilution,^13,46 which returned to baseline after three days when Hb recovered to near 90 g/L. Sickle cell mice demonstrated increased brain HIF-1α expression and decreased brain pO₂ at comparable Hb levels (Hb∼70–90 g/L), likely due to the fact that they have been chronically anemic since birth. The consequence of anemia-induced brain tissue hypoxia has been linked to cognitive dysfunction and memory loss in animal⁵¹ and human studies,^52,53 consistent with reports of neurological impairment in sickle cell children.^3,4 Correcting anemia has been shown to improve cognitive capacity.⁵⁴ Reduction in tissue hypoxia may contribute to normalization of CBF and reduced incidence of stroke in sickle cell anemia children who undergo proactive transfusion of RBCs.^2,5

The importance of maintaining cerebrovascular reactivity to maintain adequate cerebral perfusion has been demonstrated in animal and human studies.⁵⁵ In humans, Fierstra et al.⁵⁶ and others have demonstrated that a loss of CO₂ reactivity is associated with cerebral cortical atrophy⁵⁷ and stroke. It has also been demonstrated that loss of cerebrovascular reactivity is an indication for surgical revascularization and that such treatments reduce the incidence of cerebral injury.⁵⁸ Thus, demonstration of a reduction in CVR in sickle cell patients may be a means of predicting children at risk of subsequent cerebral injury.

The interaction between cerebrovasodilation in response to anemia (brain tissue hypoxia) and CO₂ reactivity suggests that both stimuli utilize a common pool of CVR. In anemia, the ability to regulate CBF and maintain brain oxygen delivery is critical to brain function and organism survival.¹³ Animal studies have demonstrated that acute anemia can lead to increased CBF that is primarily dependent on blood oxygen content.⁵⁹ Conversely, CO₂ reactivity likely contributes to the requirement for enhanced cerebral oxygen delivery to brain regions with high metabolic requirements and to washout metabolites. Despite these two distinct physiological requirements (the response to decreased Hb and increased CO₂), they both utilize or borrow from the same pool of CVR. This reserve has a finite capacity; therefore, enhanced vasodilation due to anemia may limit CO₂ reactivity. This interaction may make the brain unable to respond adequately to additional stimuli, such as increased neurological activity, and make the brain susceptible to injury. For example, in anemic humans, the impact of CO₂ on CBF responsiveness is reduced in proportion to the degree of anemia.¹¹

Sickle cell anemia leads to structural changes within the brain. The chronic reduction in tissue oxygen delivery in sickle cell mice can be compensated for by the remodeling of the vasculature to optimize oxygen delivery to the brain. Larger carotid arteries and increased carotid blood flow reflect increased perfusion to the brain and provide an example of this.

Remodeling of the brain's anatomical structure can also occur. Localized anatomical differences were seen bilaterally in many structures throughout the brain, while the whole brain volume was unaffected. Cellular atrophy may occur secondary to inadequate brain perfusion and tissue hypoxia. Several specific regions involved in learning and memory were smaller in the sickle cell mice, which may be due to the chronic deficit of oxygen delivery to the brain. This is consistent with reports of brain dysfunction, cognitive impairment, and memory deficit in anemic patients with sickle cell disease,⁶⁰ acute hemodilution,⁶¹ and chronic kidney disease.⁶² The distribution and specificity of the anatomical differences suggest a variable sensitivity among brain structures to hypoxia and blood flow changes. Interestingly, assessment of CBF changes may identify brain abnormalities in children with sickle cell disease.⁶² Paradoxically, hypoxia has also been associated with astrocytic hypertrophy,⁶³ providing evidence that hypoxia can have divergent effects on different cell populations within the brain and possibly explaining the finding of enlarged brain regions. Investigation into the development of these anatomical differences may help elucidate the underlying mechanism behind neurocognitive deficits in sickle cell disease. Collectively, cerebral, vascular, and molecular adaptation to chronic anemia in sickle cell mice enables them to survive by optimizing tissue oxygen delivery.

There are limitations to the current study. Despite similar physiological changes (elevated CBF, reduced CVR), the failure to clearly identify MRI detectable stroke in Townes sickle cell mice may suggest a differential susceptibility to the disease between mice and humans. The role of anemia-induced tissue hypoxia in cognitive impairment for sickle cell disease needs to be further addressed in animal and human studies. Despite the fact that blood transfusion has had a positive impact in children with sickle cell disease, the ability of RBC transfusion to reduce tissue hypoxia and improve brain function requires further investigation.

In conclusion, we have provided evidence that the adaptive physiological and anatomical changes in sickle cell mice may be a result of chronic tissue hypoxia. These structural modifications allow oxygen delivery to be optimized in this chronic oxygen-deprived condition. The use of MRI and ultrasound in this study facilitates comparison with findings in humans. In particular, our observations of elevated CBF,^19–23 reduced CVR²⁴ and increased diameter of the carotid artery,⁶⁴ mirror the clinical features of sickle cell children, whereas tissue pO₂ and HIF-1α expression levels are not known. Thus, this study has demonstrated novel and confirmatory evidence for the use of this mouse model to further investigate the pathophysiology in sickle cell disease.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the Canadian Institutes of Health Research Grant MOP231389 and the Ontario Research Fund.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors' contribution

LSC designed and carried out the MRI experiments and performed the data analysis. LMG completed the brain pO₂ measurements, the MRI morphometry analysis and prepared the figures. AKYT and GMTH wrote the manuscript. YQZ performed the ultrasound experiments. SP contributed to optimization of the CASL imaging protocol and manually drew ROIs for data analysis. EL and CDM carried out the HIF-1α staining. AK and JGS conceived of the study and are responsible for the overall design of the research. All of the authors approved the submitted version of the manuscript.

Supplementary material

Supplementary material for this paper can be found at http://jcbfm.sagepub.com/content/by/supplemental-data