Hemodynamic correlation imaging of the mouse brain for application in unilateral neurodegenerative diseases

: We developed a single-camera two-channel hemodynamic imaging system that uses near-infrared light to monitor the mouse brain in vivo with an exposed, un-thinned, and intact skull to explore the effect of Parkinson’s disease on the resting state functional connectivity of the brain. To demonstrate our system’s ability to monitor cerebral hemodynamics, we first performed direct electrical stimulation of an anesthetized healthy mouse brain and detected hemodynamic changes localized to the stimulated area. Subsequently, we developed a unilaterally lesioned 6-hydroxydopamine (hemi-parkinsonian) mouse model and detected the differences in functional connectivity between the normal and hemi-parkinsonian mouse brains by comparing the hemispheric hemodynamic correlations during the resting state. Seed-based correlation for the oxy-hemoglobin channel from the left and right hemispheres of healthy mice was much higher and more symmetric than in hemi-parkinsonian mice. Through a k-means clustering of the hemodynamic signals, the healthy mouse brains were segmented according to brain region, but the hemi-parkinsonian mice did not show a similar segmentation. Overall, this study highlights the development of a spatial multiplexing hemodynamic imaging system that reveals the resting state hemodynamic connectivity in healthy and hemi-parkinsonian mice.


Introduction
Functional connectivity is an important technique for analyzing the relationships between various regions of the brain and is helpful for understanding the organization of complex brain networks. It is an essential tool because it requires no assumptions about brain regionalization but rather detects correlated patterns directly from the acquired neural signals [1,2]. Functional connectivity of the brain can be observed not only in event-based experiments but also during the resting state [3][4][5]. The study of the brain's functional connectivity can be valuable for investigating the specific effects of neurodegenerative diseases such as Parkinson's disease (PD), which is known to disrupt several neural networks. In addition to a general decrease in functional connectivity in the entire brain [6], the motor cortex has been shown to be particularly affected by PD [7][8][9]. Hemi-parkinsonism is a type of PD that affects only one hemisphere; however, as with many other neurodegenerative diseases, its effects on the functional connectivity of the entire brain has not yet been examined.
Many studies have utilized near-infrared spectroscopy (NIRS) to investigate PD because of its advantages over other imaging modalities. In addition to its greater tolerance of motion artifacts [10], NIRS is capable of imaging deeper tissue regions than those imaged by visiblelight-based imaging methods [11]. This is particularly useful in animal studies, allowing the skull to remain intact while hemodynamic data from deeper regions of the brain are acquired [12]. By acquiring hemodynamic data, we can develop functional connectivity measurements of wide areas of the brain. However, not only can near-infrared light be used to develop functional connectivity measurements as OIS (optical intrinsic signal) but visible light has also been used to show functional connectivity measurements [5].
For this study, we developed a wide-field single-camera imaging system using nearinfrared light to measure the functional connectivity in healthy and PD mice during the resting state. Our system does not rely on switching between wavelengths but rather uses spatial multiplexing of a single camera to record optical density changes from multiple regions of the mouse brain. We also developed a 6-hydroxydopamine-lesioned hemiparkinsonian mouse model for use in testing. In our animal model, the skulls of the mice remained intact while hemodynamic signals were acquired for use in locating functionally similar areas of the brain. In most cases, functional connectivity can be quantified by seedbased calculation of the correlation between regions of the brain over a period of time [13]. Other methods, including independent component analysis, have been proposed for quantifying functional connectivity, but in practice, there is little difference between the results of these approaches [14]. In this paper, we use seed-based calculation of functional connectivity to observe the effects of hemi-parkinsonism in mice during the resting state. To further analyze the resting-state brain activity pattern, we used a k-means clustering algorithm [15][16][17][18] to segment functional regions of the brain based on hemodynamic signals. These analyses demonstrate the applicability of our system to the study of the effects of various neurodegenerative diseases on global brain activity.

Imaging system
A uniform bright-field illumination using a fiber ring light guide (2.00-inch, Edmund Optics) was used to deliver light from two LEDs (780 nm (Thorlabs, M780L3) and 850 nm (Thorlabs, M850L3)). A cranial window in a head-fixed mouse was exposed approximately 10 cm below a 100 mm B-coated (650-1050 nm range) achromatic lens (Thorlabs, AC254-100-B-ML). The reflected signal from the cranial window was focused onto the iris with a 75 mm B-coated achromatic lens (Thorlabs, AC254-075-B-ML), allowing the image size to be controlled using the iris ( Fig. 1(a)).
Two additional 75-mm B-coated achromatic lenses (Thorlabs, AC508-075-B-ML) were also located between the iris and camera. Between these two lenses, the beam was divided into two paths by a dichroic mirror (Semrock, 801 nm long pass). Each beam was filtered using a bandpass filter (Semrock, 780 nm -bandwidth: 12-25 nm and 850 nm -bandwidth: 10-25 nm). The two beams were directed to two different regions of the same camera (Fig.  1). The CCD camera (ORCA Flash 4.0 V2, Hamamatsu, Japan) was set to acquire frames at 10 Hz with a resolution of 1024 x 512 pixels; thus, the image for each of the two wavelength regions occupied 512 × 512 pixels. A 2 × 2 binning was performed for analysis. The camera's field of view of the mouse brain, including the motor cortex and somatosensory cortex, was approximately 1 cm 2 . The resulting resolution of our acquired image was 0.2 mm. Each mouse was experimented on once; one experiment for electrical stimulation (one healthy mouse) and eight separate experiments (four healthy mice/four hemi-parkinsonian mice) for resting state analyses.

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Experimental procedure
Two separate experiments were performed in this study. For the first experiment, we directly stimulated one healthy mouse brain using a bipolar electrode (A320, Isostim) positioned into a hole drilled through the skull, approximately over the motor cortex, for direct brain stimulation. The approximate positioning of the electrode can be seen in Fig. 3(a). Stimulation was initiated after 10 seconds of baseline measurement, at an intensity of 0.1 mA, for a fixed duration of 15 seconds. The stimulation was repeated five times, but only the first trial was taken for analysis due to excessive vasomotion resulting from the first trial. The effect of vasomotion will be discussed in Section 3. The purpose of this test was to provide an example of the system's ability to monitor hemodynamic changes in the mouse brain. For the second experiment, we observed resting state cerebral hemodynamic changes over a period of 7 minutes, in four healthy and four hemi-parkinsonian mice. Resting state means that no other external stimulation was applied to the mouse and it remained in its stereotaxic frame (DJ-308, Daejong Instrument) under constant anesthetic conditions.

System demonstration -hemodynamic changes during electrical stimulation
Electrical stimulation was delivered for 15 s to one of the motor cortex regions, and the hemodynamic response was analyzed for a pixel near the electrode ( Fig. 3(a)). Immediately after the stimulus onset, a small rise in deoxy-hemoglobin and a small decline in oxyhemoglobin, known as the "initial dip," were clearly observable. A large influx of blood followed, and a typical hemodynamic signal was then observed for increasing blood flow [30]. A single trial hemodynamic response is shown in Fig. 3(b). Although the figure does not show a complete return to baseline, it is trending towards baseline. After 50 s, there is continuous fluctuation around baseline levels, indicating additional vasomotion due to the direct stimulation. This continuous fluctuation after the first trial made the analysis of successive trials difficult, and we assume that the exaggerated vasomotion came from the high power of the electrode [31].

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K-means
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