Real-time monitoring of CBF and cerebral oxygenation is appealing in mouse stroke studies, as it provides a direct and objective way to instantly judge the success of arterial clippings for inducing cerebral ischemia and to continuously assess cerebral hemodynamic responses. The alterations in CBF/cerebral oxygenation during cerebral arterial occlusions reflect the tolerance or injury of brain to hypoperfusion/hypoxia that is closely associated with pathophysiological outcomes of stroke. Quantification of cerebral hemodynamic changes during cerebral ischemia may provide useful information for evaluating the preconditioning/TIA in protection/promotion of stroke. The present study has successfully adapted an innovative DCS flow-oximeter for longitudinally assessing cerebral hemodynamics in mice with transient forebrain ischemia. The portable DCS flow-oximeter (see ) is novel in that it can simultaneously quantify CBF and cerebral oxygenation in deep mouse brain, thus allowing for a comprehensive evaluation of cerebral ischemia and tissue hypoxia.
Several experimental protocols (see Section 2.3) were designed to compare DCS with LDF for CBF measurements and to test the capability and sensitivity of DCS flow-oximeter in detecting cerebral hypoperfusion and hypoxia in mice undergoing repeated transient forebrain ischemia. Several fiber-optic probes (see ) were developed to meet the special needs of the designed experiments. The removal of mouse scalp ensured the good installation of optical probes on the exposed skull and avoided the partial volume effect from the overlaying scalp tissues. The minimally-invasive surgery for removing mouse scalp did not show obvious influence on mouse daily activities. For the validation study, DCS shared the LDF source (see ) to avoid the light interference between the two measurements. This integrated probe design made the fiber arrangement easy and synchronized the two measurements precisely. By placing multiple pairs of S-D fibers at the two sides of mouse head (see ), the regional and global CBFs can be measured simultaneously. For the longitudinal monitoring of repeated ischemic mice over days, the optical fibers were confined in a foam pad that was permanently glued on the mouse skull (see ). The optical fibers can be easily inserted into or removed off the foam pad based on the experimental needs. This unique design for flexibility permits the longitudinal measurements at the same location of mouse brain without significantly interrupting mouse daily activities.
Using Protocol #1, the CBFs measured by DCS and LDF are compared. LDF exhibits a high sensitivity to motions of the probe and animal head (see ) and has difficulty in aligning its tiny probe on a small vessel. By contrast, DCS measurement is more robust and easier to implement. The CBF changes during multiple I-R challenges measured by the two techniques are highly correlated (see ). Interestingly, the CBF reductions during CCA clippings (CBF < 100%) measured by the two techniques are found highly consistent (regression slope = 1.06, see the small inset plot in ) whereas CBF increases after the release of CCA clippings (CBF > 100%) are slightly different (regression slope > 1, see ). The CCA occlusions completely cut off the blood supply to the entire brain, thus reduce the CBF to the same amount in both superficial cortex (detected by LDF) and deep brain (detected by DCS). However, the flow reperfusion after the release of CCA clippings (CBF increase) depends on the vasodilatation capacities of the measured vessels. The deep brain microvasculature may have larger vasodilatation capacity than the superficial single vessel, thus producing higher DCS reperfusion signals originated from deep brain tissues. Other differences between LDF and DCS may also contribute to the CBF measurement discrepancy, including those in analytical models (single scattering versus multiple scattering) [26
], measured vessels (single superficial vessel versus deep microvasculature bed), and probed tissue volumes (tiny spot versus bulk tissues). Similar discrepancy in CBF measurements between DCS and LDF was also observed previously in rat brains under hypocapnia challenges [26
]. Notice however that this pilot study compared the DCS and laser Doppler measurements in only one mouse. Further investigations in a large population are needed to draw solid conclusions.
Protocol #2 tests the capability of dual-wavelength DCS for simultaneously monitoring the regional (left and right hemispheres) and global (across hemispheres) CBF changes in mice during unilateral and bilateral CCA occlusions. The unilateral CCA ligation induces a large CBF decrease (−50.7 ± 4.7%) in ipsilateral (clipped) hemisphere. Meanwhile a relatively small CBF reduction (−8.8 ± 2.9%) in contralateral (unclipped) hemisphere can be simultaneously detected by the DCS flow-oximeter (see ). The small CBF reduction in the unclipped hemisphere is likely due to collateral compensation effects. As expected, bi-CCA occlusion results in further decreases in both regional and global CBFs. The global CBF across the hemispheres is approximately equal to the average value of the regional CBFs in two hemispheres (see ), demonstrating the accuracy of DCS in detecting both regional and global CBF changes. Since neurological injury and cell loss often appear locally in the ischemic brain and are closely associated with the local ischemic status, monitoring of the regional CBF may assist in evaluating in situ neurological infarct and tissue damage.
Protocol #3 is designed to exam the ability of DCS flow-oximeter for simultaneously monitoring CBF and cerebral oxygenation changes in mice undergoing repeated transient forebrain ischemia. As expected, the CCA clipping/unclipping induces instant CBF decrease/increase, leading to cerebral deoxygenation/reoxygenation (see and ). Similar to the results obtained from Protocol #2 (see ), the unilateral CCA ligation at Day 1 induces less CBF changes than bi-CCA occlusions, leading to smaller variations in cerebral oxygenation. DCS flow-oximeter demonstrates high sensitivities in detecting these anticipated hemodynamic changes. Currently, there are only a few published studies measuring both blood flow and oxygenation in rat brains during one-time MCA occlusions [17
]. For comparisons, we calculate the relative oxygenation changes driven by the CBF reduction during bi-CCA occlusions at Day 1 (see ): Δ[HbO2
]/ΔCBF = 0.34 ± 0.06 µM/% and Δ[Hb]/ΔCBF = −0.25 ± 0.08 µM/%. These ratios fall respectively in the ranges of hemodynamic responses extracted from the reported data [28
]/ΔCBF = 0.19 to 0.52 µM/% and Δ[Hb]/ΔCBF = −0.19 to −0.31 µM/%. Since cell death results primarily from the lack of tissue oxygen, nutrients supply and waste exchanges, simultaneous monitoring of cerebral oxygenation and CBF during cerebral ischemia provides the deep insight about the pathophysiology of cerebral tissue damage. Moreover, simultaneous hemodynamic measurements allows for an estimation of cerebral oxygen metabolism [28
]. This metabolic index is potentially a more direct indicator of tissue metabolic activities and provides further insight about tissue physiology/pathophysiology.
Using the special probe design and unique mouse model created in the present study, cerebral hemodynamic changes in mice during repeated 2-minute transient I-R challenges (bi-CCA occlusions) have been successfully monitored over 5 days. Note that two out of ten mice involved in this protocol were excluded from the data analysis due to their severe stroke symptoms in the middle of experiments. The 20% unsuccessful rate is not surprising considering the complexity of multiple I-R circles over days. One important finding from this protocol is that cerebral hemodynamic alternations during transient forebrain ischemia decrease with the progress of repeated short-term I-R challenges (see ). More specifically, the magnitudes of CBF reductions at the last two days (Day 4-5) are significantly smaller than that at the first day (Day 1). Correspondingly, the oxygenation changes at Day 2-5 are also remarkably smaller than those at Day 1. One likely reason for these longitudinal changes is that the repeated short-term intermittent interruptions of CBF (preconditioning) enhance the cerebral blood supply from other branches, thus compensating the flow loss caused by the CCA occlusions. Furthermore, the repeated short-term I-R challenges may also enhance the cerebral ischemic tolerance. These observations imply that mouse adaptation to cerebral ischemia could be influenced by the repeated preconditioning.
For precise evaluation and comparison of ischemic effects in different mice, it is critical to induce a complete forebrain ischemia in all subjects through the bi-CCA occlusions. Although well-trained surgeons may be able to judge the success of a bi-CCA occlusion based on their experience, the heterogeneity of animal responses to arterial occlusion cannot be ruled out.
shows the global CBF reductions during 2-minute bi-CCA occlusions for all 20 mice measured in Protocol #2 and Protocol #3 (at Day 1). Inter-subject variation in CBF reductions exists although all mice receive identical surgical procedures. Particularly, Mouse #1 demonstrates a much less CBF reduction (−65.6%) during the bi-CCA occlusion compared to other mice. The less CBF reduction in this mouse might result from the pre-existed enriched collateral blood flow or improper installation of arterial occluder. Mouse #1 is thus excluded from the data analysis due to its incomplete response to the arterial occlusion. By contrast, the global CBF reductions in all other 19 mice (except Mouse #1) are consistently larger than 75% (see ). Therefore, the 75% CBF reduction may be considered as a threshold to determine a successful bi-CCA occlusion. Further investigations correlating the CBF reductions with behavioral tests and histological outcomes in ischemic mice are needed to verify this ischemic threshold. This threshold is within the range of CBF reductions following bi-CCA occlusions observed in mice [8
] and rats [52
] that were measured by laser Doppler. However, considering the advantages of DCS over laser Doppler as discussed early, DCS is expected to be a robust and easy-to-use tool for objectively assessing the success of cerebral ischemia in mice. In contrast to the consistent large CBF reductions following bi-CCA occlusions, CBF responses to unilateral CCA occlusions vary largely among mice (see -). Following an initial rapid CBF reduction caused by the unilateral CCA ligation, the CBF in the ipsilateral (occluded) hemisphere either recovers towards its baseline (see ) or remains at a lower level than its baseline (see ). The distinct flow responses in different mice are likely due to the compensatory CBFs from the contralateral (non-occluded) hemispheres, which rely on integrity of the circle of Willis [50
]. As a result, the CBFs in the contralateral hemispheres also exhibit variations although they are smaller than the ipsilateral hemispheres (see ). The interruptions of CBF in both hemispheres during unilateral CCA occlusions result in corresponding changes in cerebral oxygenation. The large inter-subject heterogeneities in regional hemodynamic responses further emphasize the necessity to monitor CBF and cerebral oxygenation in both hemispheres for each individual mouse.
Fig. 7 The CBF changes during bi-CCA occlusions in 20 mice involved in Protocol #2 and Protocol #3 at Day 1. Mouse #1-10 and Mouse# 11-20 were measured in Protocol #2 and Protocol #3, respectively. Three mice were eventually excluded from statistical analysis (more ...)
The dual-wavelength DCS flow-oximeter used in the present study has two laser sources (785 and 854 nm), which allow for simultaneous measurements of either regional CBFs in the two hemispheres (see ), or global CBF and oxygenation across the two hemispheres (see ). In order to monitor both CBF and oxygenation in the two hemispheres, a second pair of lasers (785 and 854 nm) must be added into the current dual-wavelength DCS flow-oximeter system, which will be the subject of future work.
To conclude, the novel DCS flow-oximeter has been successfully adapted for simultaneous monitoring of CBF and cerebral oxygenation in mouse brains. DCS for mouse CBF measurement correlates well with LDF, and is less susceptible to motion artifacts. With the unique designs in experimental protocols and corresponding fiber-optic probes, the present study has demonstrated high sensitivities of DCS flow-oximeter in detecting the regional/global changes of CBF and cerebral oxygenation in mice undergoing multi-day repeated transient forebrain ischemia (2-minute bi-CCA occlusion). Monitoring of regional cerebral hemodynamics in each of the two hemispheres provides information for evaluating the collateral compensation effects during unilateral CCA occlusion and for potentially estimating the in situ neurological deficits and tissue damages. Simultaneous measurements of CBF and cerebral oxygenation allow for comprehensively assessing cerebral ischemia and tissue hypoxia during cerebral arterial occlusion. DCS flow-oximeter measurements permit real-time quantification of cerebral hemodynamic responses to transient forebrain ischemia, which can be used to guide proper arterial occlusion and evaluate the preconditioning effects on brain adaptation to cerebral ischemia. More than 75% CBF reductions were found during bi-CCA occlusions in mice, which may be considered as a threshold to determine a successful bi-CCA occlusion. The longitudinal declines in CBF reductions during the 2-minute short-term I-R challenges indicate that mouse adaptation to cerebral ischemia could be influenced by the repeated preconditioning. Future studies will evaluate TIA-induced injury to brain through applying repeated medium-term I-R challenges (e.g., 10-min bi-CCA occlusions) to mice. The cerebral hemodynamic changes during repeated transient/medium I-R challenges will be compared with pathophysiological consequences (e.g., neurologic deficits, stroke volumes) of stroke manipulated by permanent MCA occlusions. The anticipated correlations between the cerebral hemodynamic responses and physiological consequences would provide deep insights about the preconditioning/TIA mechanism in protection/promotion of stroke.