Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter

Abstract

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6 months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5 Gy, two doses per week, for 4 weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO₂) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700 μm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO₂ measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO₂, while the changes of other cerebral capillary PO₂ properties (e.g., capillary mean-PO₂, RBC-PO₂, and InterRBC-PO₂) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

Keywords: Microvascular blood flow; Optical microscopy; Vascular cognitive impairment; White matter; Whole-brain radiation.

Fig. 1

Imaging methods. (a) Representative two-photon angiographic images acquired in the cerebral cortical gray matter (GM; Z = 0.5 mm) and white matter (WM; Z = 1.0 mm). (b) Two 0.5-s-long fluorescence intensity time courses acquired at the locations denoted by the red dots in the panel (a). (c) Two examples of phosphorescence decays (venule in blue and arteriole in red) for PO₂ recording. A 300-μs-long cycle includes a 10-μs-long EOM-gated excitation, followed by a 290-μs-long detection of phosphorescence. (d) A Doppler-OCT image acquired at the cortical surface (left), and the zoomed-in view of a surfacing venule (right) enclosed by the inner circle of the yellow ring. The offset axial flow speed was calculated by averaging over the pixels in the shaded area between the yellow circles. The maximum axial flow speed was calculated by averaging over the pixels enclosed by the blue square in the zoomed-in image. (e) Segmentation of an angiogram (maximum intensity projection of the angiogram across the depth range of 100-200 µm under the brain surface). The segmented centerlines are in green. Scale bars: 100 μm

Fig. 2

Capillary RBC flow parameters. (a–c) Comparisons of capillary RBC flux (a), speed (b), and linear density (LD; c). This analysis was made with the measurements performed in n = 3 control (Ctrl; in total 291 Gy matter capillaries and 163 white matter capillaries) and n = 4 WBI-treated mice (WBI; in total 534 Gy matter capillaries and 160 white matter capillaries). The data were first averaged over the capillaries in each mouse and then over mice. GM and WM stand for gray matter and white matter, respectively. Data are expressed as mean ± STD. The asterisk symbols indicate statistical significance (Student’s t-test, P < 0.05)

Fig. 3

Venular blood flow measured with Doppler-OCT. (a, b) Blood flow (a) and maximum axial flow speed (Max. V_z; b) measured in the surfacing venules within the top 100-µm depth under the cortical surface. This analysis was made with the measurements performed in n = 4 control mice (Ctrl; in total 20 vessels) and n = 5 WBI-treated mice (WBI; in total 27 vessels). The data were first averaged over the vessels in each mouse and then over mice. Data are expressed as mean ± STD. No significant difference was observed in this analysis (Student’s t-test)

Fig. 4

Cerebral vascular oxygenation. (a) Comparison of the PO₂ measurements (a) performed in the arterioles (A) and venules (V) selected within the top 100-µm depth under the cortical surface. This analysis was made with the measurements performed in n = 4 control mice (Ctrl; in total 90 and 166 samples acquired in the arterioles and venules, respectively), and n = 6 WBI-treated mice (WBI; in total 153 and 252 samples acquired in the arterioles and venules, respectively). (b) Comparison of SO₂ calculated based on the PO₂ data from the panel (a). (c) Comparison of OEF calculated based on the SO₂ data from the panel (b). The data were first averaged over the vessels in each mouse and then over mice. Data are expressed as mean ± STD. No significant difference was observed in this analysis (Student’s t-test)

Fig. 5

Cerebral capillary PO₂ properties. (a–d). Comparisons of capillary Mean-PO₂ (a), RBC-PO₂ (b), InterRBC-PO₂ (c), and EATs (d). This analysis was made with the measurements performed in n = 4 control (Ctrl; in total 366 capillaries) and n = 5 WBI-treated mice (WBI; in total 511 capillaries). The measurement depth ranged from the cortical surface down to 300 µm under the cortical surface. The data were first averaged over the vessels in each mouse and then over mice. Data are expressed as mean ± STD. The asterisk symbol indicates statistical significance (Student’s t-test, P < 0.05)

Fig. 6

Cerebral microvascular morphological properties. (a–d) Comparisons of the cerebral microvascular diameter (a), segment length (b), segment density (c), and tortuosity (d) extracted from the two-photon angiograms. The angiograms were acquired in n = 3 control mice (Ctrl; in total 7876 capillaries) and n = 3 WBI-treated mice (WBI: in total 4798 capillaries). The data were first averaged over the angiogram in each mouse and then over mice. Data are expressed as mean ± STD. The asterisk symbol indicates statistical significance (Student’s t-test, P < 0.05)

Fig. 7

Depth-dependent changes of the cerebral microvascular morphological properties. (a–d) Comparisons of the cerebral microvascular diameter (a), segment length (b), segment density (c), and tortuosity (d) in the depth ranges of 100–200 µm, 200–300 µm, 300–400 µm, 400–500 µm, 500–600 µm, and 600–700 µm, with the same data as in Fig. 6. The data were first averaged over the angiogram in each mouse and then over mice. Data are expressed as mean ± STD. No significant difference was observed in this analysis (Student’s t-test)

Fig. 8

Cerebral gray matter and white matter thickness. (a) Two representative OCT B-scan images acquired in a control and a WBI-treated mouse. The red curve in the image delineates the cortical surface, and the two yellow curves delineate the upper and lower boundaries of subcortical white matter, with the green curve representing the midline of the white matter. Scale bars: 100 µm. (b) Comparison of the cerebral gray matter and white matter thickness with the OCT measurements performed in n = 3 control (Ctrl) and n = 3 WBI-treated (WBI) mice. The data were first averaged over the images in each mouse and then over mice. Data are expressed as mean ± STD. No significant difference was observed in this analysis (Student’s t-test)