Nanoelectronics enabled chronic multimodal neural platform in a mouse ischemic model
Graphical abstract
Introduction
Because brain function and dysfunction depend on the delicate balance between substrate delivery through blood flow and energy demands imposed by neural activity (Attwell et al., 2010), simultaneous mapping of neural activity and hemodynamics in behaving brain is crucial to the understanding of brain functionality in health (Fox and Raichle, 1986), as well as the damaging mechanism and recovery of neurovascular diseases (Bundo et al., 2002). In particular, the characteristics and the impacts of ischemic stroke are multifaceted in nature, in which disrupted cerebral vascular blood flow negatively impacts the neuronal activity and tissue outcome (Zhang and Murphy, 2007, Strong et al., 2007, Nakamura et al., 2010). Moreover, although the effects of ischemic stroke in both patients and experimental animal models are apparent only minutes after blood flow is reduced (Zhang et al., 1997, Hainsworth and Markus, 2008), the progression of ischemia lasts for several days after the initial insult (Hartings et al., 2003, Fabricius et al., 2006), and the functional recovery of the injured brain continues for months and longer (Murphy and Corbett, 2009). While extensive research has been done on the progression of brain injury in focal stroke at the acute phases in small animal models (Zhang and Murphy, 2007, Shin et al., 2006, Jones et al., 2008, Brown et al., 2009), the progression of ischemic conditions and recovery into chronic time scales are understudied (Dirnagl et al., 1999), in large part due to a lack of methods capable of quantifying multiple neurophysiological parameters simultaneously in behaving brains with sufficient spatial resolution over periods of weeks to months.
In vivo optical imaging has been a major tool for studying stroke models in small animals (Zhang and Murphy, 2007, Nakamura et al., 2010, Jones et al., 2008, Strong et al., 2006, Brown et al., 2007, Sakadzic et al., 2010) owing to its unique strength including high spatial resolution, reasonable penetration depth, and high specificity and sensitivity to various structural and functional imaging parameters. For example, two-photon (2P) microscopy have been routinely used for imaging subsurface microvascular structures (Nishimura et al., 2006, Schaffer and et al., 2006), neuron (Li and Murphy, 2008) and glial (Davalos et al., 2005) morphology, for quantitative, depth-resolved measurement of red blood cell (RBC) flux and velocities (Kamoun et al., 2010), for phosphorescence lifetime imaging of pO2 that determines the absolute oxygen concentration with subcellular resolution (Rumsey et al., 1988), and for voltage-sensitive dye (Brown et al., 2009) and calcium imaging (Winship and Murphy, 2008) of individual neuron activities. In particular, laser speckle flowmetry (LSF) is used to measure cortical perfusion (Strong et al., 2006) and cerebral blood flow (CBF) (Dunn et al., 2001) with high temporal and spatial resolution. Laser speckle contrast imaging (LSCI) is used as a cost-effective method for visualizing and quantifying neurovascular blood flows particularly in small animals (Dunn et al., 2001, Li et al., 2006). Multi-exposure speckle imaging (MESI), a refined method of LSCI to eliminate artifacts, allows for quantitative measurement of CBF for longitudinal studies and cross-animal comparisons (Kazmi et al., 2013, Kazmi et al., 2015, Schrandt et al., 2015). In contrast, electrical recording in stroke models mostly relies on techniques developed decades ago that offers one or few recording sites either subdural (Nakamura et al., 2010, Strong et al., 2002, Dohmen et al., 2008, Dreier et al., 2006) or intra-cortical (Jeffcote et al., 2014), with electrode dimensions and distance from the infarct often both on millimeter scales, lacking the necessary spatial resolution and specificity. In the effort of multi-modality investigation, transparent electrode arrays were used for combined neuroimaging and recording from the surface of the brain (Park et al., 2014) or on tissue slices (Kuzum et al., 2014). The spatiotemporal relationship between cortical slow potential shifts and CBF changes in response to peri-infarct depolarizations (PIDs) was studied using one or a few electrodes simultaneously with LSF or LSCI in rodents (Shin et al., 2006) and cats (Strong et al., 2007). However, the study was only carried out acutely without the ability to record and track single-unit neural activity.
The challenge for integrating high-resolution electrical recording with optical techniques chronically lies on the fundamental challenges of tissue long-term biocompatibility using intracortical microelectrodes, which is the only method to record action potentials from individual neurons at sub-milliseconds temporal resolution. Conventional electrodes generate substantial tissue damage both acutely (Potter et al., 2012, Kozai et al., 2014) and chronically (Rousche and Normann, 1998, Polikov et al., 2005), resulting in sustained tissue reaction near the implants including continuous leakage of blood-brain barrier, neuronal death and glial scar formation (Seymour and Kipke, 2007, Zhong and Bellamkonda, 2008, Jeong et al., 2015). These reactions generate a probe-induced damage zone in brain, which affects the viability of experimental models if the electrodes were placed within or in close vicinity of the ischemic penumbra. Furthermore, conventional microelectrodes are constructed on rigid material such as metal and silicon. Their long-term implantation and skull fixation geometrically affect chronic optical access to the same brain region (Kozai et al., 2016).
We successfully resolved both the challenges of tissue-compatibility and chronic optical access by our recent development of a novel type of ultraflexible neural electrodes, the nanoelectronic thread (NET) (Luan et al., 2017). We demonstrated that NETs form reliable, glial scar free neural-probe interface, which was verified by chronic neural recordings and comprehensive tissue-probe interface characterizations. Longitudinal in vivo two-photon imaging and postmortem histological analysis revealed seamless integration of NET probes with the local cellular and vasculature networks. In particular, we observed fully recovered capillaries with intact blood brain barrier, and complete absence of chronic neuronal degradation and glial scar (Luan et al., 2017). In this study, we combine LSCI of relative CBF (rCBF) with electrical recording of neural activity using NETs at different locations and cortical depths in a mouse stroke model, in which we are also able to induce targeted photothrombotic occlusions within individual vessels with a fine control over lesion location and size (Ponticorvo and Dunn, 2010, Sullender et al., 2017). We demonstrate simultaneous mapping of neural activity and rCBF beyond the acute phase of stroke, including the progression of ischemia, and the reperfusion and revival of neural activity over days and longer.
Section snippets
Ultraflexible NET electrodes fabrication and preparation
The NET brain probes were fabricated using specialized fabrication methods similar to previously reported (Luan et al., 2017, Tian et al., 2012, Xie et al., 2015). The multi-layer probes were fabricated using photolithography on a nickel metal release layer deposited on a silicon substrate (900 nm SiO2, n-type 0.005 V cm, University Wafer, Inc. MA, USA). SU-8 photoresist (SU-8 2000.5, MicroChem Corp. MA, USA), which offers excellent tensile strength, ease of fabrication and demonstrated durability
Experimental
During cranial surgery, we implanted NETs on the surface of and/or into the cortical regions of somatosensory and motor cortices (Fig. 1C) for n = 4 mice. After allowing the animal to recover after cranial surgery for eight weeks, we performed baseline imaging to confirm the recovery of vasculature. As shown in the representative images in Fig. 1F,G, LSCI shows the normal surface vasculature and rCBF near the NETs. Consistently, 2P imaging shows the normal morphology and density of vasculature
Results
The progression of ischemia induced by targeted photothrombosis within one or few branches of descending arteriole was recorded with LSCI while the neural activity near the thrombosis was recorded with NET electrodes. Fig. 2 shows the hemodynamic and neural consequences of the targeted photothrombosis on one animal in which a 4-shank NET with 22 working electrodes was implanted intracortically in the mouse motor cortex. In Fig. 2A, the green overlay in the pre-photothrombotic frame depicts the
Discussion
Comparing with conventional microelectrodes that are made of opaque, rigid materials, the NET electrodes enable facile optical access to the same brain region for longitudinal in vivo studies due to their ultra-flexibility and optical transparency. Moreover, we demonstrate in this study that intracortical implantation of NET arrays in the vicinity of the lesion sites does not qualitatively affect the induction and the progression of the ischemic insult, nor the progressive reperfusion over days
Conclusions
We have presented a chronic multimodal neural platform that simultaneously maps relative cerebral blood flow with LSCI and neural activity using NET electrode array, both can be repeated for longitudinal studies over weeks and longer. We demonstrated the ability to induce targeted photothrombotic strokes within individual vessels in mouse cortex, to simultaneously map the change of neural activity and blood flow during an ischemic event, and to chronically track neural activity and blood flow
Acknowledgements
We thank the Microelectronics Research Center at UT Austin for the microfabrication facility and support, and the Animal Resources Center at UT Austin for animal housing and care. This work was funded by National Institute of Neurological Disorders and Stroke through R21NS102964 (L.L.), R01NS102917 (C.X.), R01NS082518 (A.K.D.) and R01NS078791 (A.K.D.), by National Institute of Biomedical Imaging and Bioengineering through R01EB011556 (A.K.D.), by the American Heart Association through
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