Functional and oxygen-metabolic photoacoustic microscopy of the awake mouse brain

Abstract

A long-standing challenge in optical neuroimaging has been the assessment of hemodynamics and oxygen metabolism in the awake rodent brain at the microscopic level. Here, we report first-of-a-kind head-restrained photoacoustic microscopy (PAM), which enables simultaneous imaging of the cerebrovascular anatomy, total concentration and oxygen saturation of hemoglobin, and blood flow in awake mice. Combining these hemodynamic measurements allows us to derive two key metabolic parameters-oxygen extraction fraction (OEF) and the cerebral metabolic rate of oxygen (CMRO₂). This enabling technology offers the first opportunity to comprehensively and quantitatively characterize the hemodynamic and oxygen-metabolic responses of the mouse brain to isoflurane, a general anesthetic widely used in preclinical research and clinical practice. Side-by-side comparison of the awake and anesthetized brains reveals that isoflurane induces diameter-dependent arterial dilation, elevated blood flow, and reduced OEF in a dose-dependent manner. As a result of the combined effects, CMRO₂ is significantly reduced in the anesthetized brain under both normoxia and hypoxia, which suggests a mechanism for anesthetic neuroprotection. The head-restrained functional and metabolic PAM opens a new avenue for basic and translational research on neurovascular coupling without the strong influence of anesthesia and on the neuroprotective effects of various interventions, including but not limited to volatile anesthetics, against cerebral hypoxia and ischemia.

Keywords: Anesthetic neuroprotection; Awake-brain imaging; Head-restrained photoacoustic microscopy; Hemodynamics; Oxygen metabolism.

Figure 1

Schematic of the head-restrained PAM. PBS, polarizing beam splitter; NDF, neutral density filter; BS, beam sampler; SMF, single-mode fiber; AD, achromatic doublet; CL, correction lens; RT, ring-shaped ultrasonic transducer; RM, rotation mount; HP, head plate; RAC, right-angle clamp. Red-boxed inset: photograph of a mouse brain with a thinned-skull window and a nut attached using dental cement. Blue-boxed inset: photograph of the angle- and height-adjustable head-restraint apparatus. Green-boxed inset: the mouse placement during imaging.

Figure 2

Hemodynamic and oxygen-metabolic responses of the normoxic mouse brain to 1.0-MAC isoflurane. (a) Head-restrained PAM of cerebral C_Hb, sO₂, and blood flow speed in the absence (OFF) and presence (ON) of isoflurane. The white arrows in the 2^nd and 3^rd rows highlight the isoflurane-induced changes in s_vO₂ and blood flow speed. (b) 9 feeding arteries and 6 draining veins in the 2.5×2.5 mm² region of interest identified and isolated by vessel segmentation. (c) Quantitative analysis of the isoflurane-induced changes in the average C_Hb, sO₂, diameter, and flow speed of the feeding and draining vessels, from which OEF, CBF, and CMRO₂ of the region of interest under wakefulness and anesthesia were derived. Scale bar, 500 μm.

Figure 3

Statistical comparison (N = 5) of C_Hb, sO₂, vessel diameter, blood flow speed, CBF, OEF, and CMRO₂ in the awake and anesthetized mouse brains. In the paired t-test, ns, **, and **** respectively represent no significance, p<0.01, and p<0.0001. The right panel in the second row shows a strong dependence of isoflurane-induced arterial dilation on the baseline diameter measured under wakefulness. Data are presented as mean ± SD.

Figure 4

Head-restrained PAM of C_Hb, sO₂, and blood flow speed in the normoxic mouse brain in the absence (0 MAC) and presence of different concentrations (0.5, 1.0, and 1.5 MAC) of isoflurane. The white arrows in the 2^nd and 3^rd rows highlight the isoflurane-induced changes in s_vO₂ and blood flow speed. Scale bar, 500 μm.

Figure 5

Dose-dependent effects of isoflurane on cerebral hemodynamics and oxygen metabolism. (a) Statistical comparison (N = 6) of the absolute arterial C_Hb, sO₂, blood flow speed, and diameter, as well as s_vO₂, under wakefulness (0 MAC) and different concentrations (0.5, 1.0, and 1.5 MAC) of isoflurane. (b) Statistical comparison of the relative C_Hb, s_aO₂, OEF, CBF, and CMRO₂ with their corresponding baselines (significance levels, if any, are marked on the top of the columns) and between different concentrations of isoflurane (significance levels, if any, are marked between the two compared columns). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data are presented as mean ± SD.

Figure 6

Cerebral vasodilation in response to different concentrations of isoflurane (0.5, 1.0, and 1.5 MAC) under normoxia. The segmented feeding arteries and draining veins were divided into four different groups: small arteries (SA) and small veins (SV) with diameters less than 40 μm and large arteries (LA) and large veins (LV) with diameters larger than 40 μm. Statistical comparison (39 feeding arteries and 42 draining veins in 6 mice) of the vessel diameters measured under anesthesia with their corresponding baseline values measured under wakefulness shows significant vasodilation for all four groups across all three different anesthetic depths (significance levels are marked on the top of the columns). Moreover, statistical comparison between different groups of vessels under the same concentration of isoflurane shows a strong diameter-dependent vasodilation in the arteries but not veins (significance levels, if any, are marked between the two compared columns). Vasodilation shows no statistically significant dependence on isoflurane concentration. *, p<0.05; **, p<0.01; ****, p<0.0001. Data are presented as mean ± SD.

Figure 7

Head-restrained PAM of C_Hb, sO₂, and blood flow speed in the hypoxic mouse brain in the absence (0 MAC) and presence of different concentrations (0.5, 1.0, and 1.5 MAC) of isoflurane. The white arrows in the 2^nd and 3^rd rows highlight the hypoxia- and isoflurane-induced changes in sO₂ and blood flow speed. Scale bar, 500 μm.

Figure 8

Influence of isoflurane on cerebral hemodynamic and oxygen-metabolic responses to systemic hypoxia. (a) Statistical comparison (N = 5) of the absolute arterial C_Hb, sO₂, blood flow speed, and diameter, as well as s_vO₂, under wakefulness (0 MAC) and different concentrations (0.5, 1.0, and 1.5 MAC) of isoflurane. (b) Statistical comparison of the relative C_Hb, s_aO₂, OEF, CBF, and CMRO₂ with their corresponding baselines (significance levels, if any, are marked on the top of the columns) and between different concentrations of isoflurane (significance levels, if any, are marked between the two compared columns). *, p<0.05; **, p<0.01; ***, p<0.001. Data are presented as mean ± SD.

Figure 9

(a) Cerebral vasodilation in response to different concentrations of isoflurane (0.5, 1.0, and 1.5 MAC) under systemic hypoxia. Statistical comparison (41 feeding arteries and 26 draining veins in 5 mice) of the vessel diameters measured under anesthesia with their corresponding baseline values measured under wakefulness shows significant arterial dilation but only moderate venous dilation across all three different anesthetic depths (significance levels, if any, are marked on the top of the columns). Moreover, statistical comparison between different groups of vessels under the same concentration of isoflurane shows no diameter-dependent dilation in either arteries or veins. Vasodilation shows no statistically significant dependence on isoflurane concentration under hypoxia. (b) Statistical comparison of the vessel diameters measured in the awake mouse brain under hypoxia with their baselines measured under normoxia shows statistically significant vasodilation in small arteries and veins (significance levels, if any, are marked on the top of the columns). Moreover, statistical comparison reveals a strong diameter-dependent dilation in the arteries but not veins (significance levels, if any, are marked between the two compared columns). *, p<0.05; ***, p<0.001; ****, p<0.0001. Data are presented as mean ± SD.