Quantifying rapidly varying perturbations in cerebral tissue absorption and scattering can potentially help to characterize changes in brain function caused by ischemic trauma. We have developed a platform for rapid intrinsic signal brain optical imaging using macroscopically structured light. The device performs fast, multispectral, spatial frequency domain imaging (SFDI), detecting backscattered light from three-phase binary square-wave projected patterns, which have a much higher refresh rate than sinusoidal patterns used in conventional SFDI. Although not as fast as "single-snapshot" spatial frequency methods that do not require three-phase projection, square-wave patterns allow accurate image demodulation in applications such as small animal imaging where the limited field of view does not allow single-phase demodulation. By using 655, 730, and 850 nm light-emitting diodes, two spatial frequencies ([Formula: see text] and [Formula: see text]), three spatial phases (120 deg, 240 deg, and 360 deg), and an overall camera acquisition rate of 167 Hz, we map changes in tissue absorption and reduced scattering parameters ([Formula: see text] and [Formula: see text]) and oxy- and deoxyhemoglobin concentration at [Formula: see text]. We apply this method to a rat model of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) to quantify hemodynamics and scattering on temporal scales ([Formula: see text]) ranging from tens of milliseconds to minutes. We observe rapid concurrent spatiotemporal changes in tissue oxygenation and scattering during CA and following CPR, even when the cerebral electrical signal is absent. We conclude that square-wave SFDI provides an effective technical strategy for assessing cortical optical and physiological properties by balancing competing performance demands for fast signal acquisition, small fields of view, and quantitative information content.

Abstract:In the present study, we have developed a multi-modal instrument that combines laser speckle imaging, arterial blood pressure, and electroencephalography (EEG) to quantitatively assess cerebral blood flow (CBF), mean arterial pressure (MAP), and brain electrophysiology before, during, and after asphyxial cardiac arrest (CA) and resuscitation. Using the acquired data, we quantified the time and magnitude of the CBF hyperemic peak and stabilized hypoperfusion after resuscitation. Furthermore, we assessed the correlation between CBF and MAP before and after stabilized hypoperfusion. Finally, we examined when brain electrical activity resumes after resuscitation from CA with relation to CBF and MAP, and developed an empirical predictive model to predict when brain electrical activity resumes after resuscitation from CA. Our results show that: 1) more severe CA results in longer time to stabilized cerebral hypoperfusion; 2) CBF and MAP are coupled before stabilized hypoperfusion and uncoupled after stabilized hypoperfusion; 3) EEG activity (bursting) resumes after the CBF hyperemic phase and before stabilized hypoperfusion; 4) CBF predicts when EEG activity resumes for 5-min asphyxial CA, but is a poor predictor for 7-min asphyxial CA. Together, these novel findings highlight the importance of using multimodal approaches to investigate CA recovery to better understand physiological processes and ultimately improve neurological outcome.

. Significance: Quantitative measures of blood flow and metabolism are essential for improved assessment of brain health and response to ischemic injury. Aim: We demonstrate a multimodal technique for measuring the cerebral metabolic rate of oxygen ( ) in the rodent brain on an absolute scale ( ). Approach: We use laser speckle imaging at 809 nm and spatial frequency domain imaging at 655, 730, and 850 nm to obtain spatiotemporal maps of cerebral blood flow, tissue absorption ( ), and tissue scattering ( ). Knowledge of these three values enables calculation of a characteristic blood flow speed, which in turn is input to a mathematical model with a “zero-flow” boundary condition to calculate absolute . We apply this method to a rat model of cardiac arrest (CA) and cardiopulmonary resuscitation. With this model, the zero-flow condition occurs during entry into CA. Results: The values calculated with our method are in good agreement with those measured with magnetic resonance and positron emission tomography by other groups. Conclusions: Our technique provides a quantitative metric of absolute cerebral metabolism that can potentially be used for comparison between animals and longitudinal monitoring of a single animal over multiple days. Though this report focuses on metabolism in a model of ischemia and reperfusion, this technique can potentially be applied to far broader types of acute brain injury and whole-body pathological occurrences.

Despite the huge costs of ischemic neuronal injury, neuroprotective interventions in humans remain elusive, in part reflecting incomplete knowledge of the critical early events. Emerging evidence implicates Zn 2+ as an important early contributor. CA1 pyramidal neurons undergo selective delayed degeneration after transient global ischemia (TGI), and Zn 2+ has been implicated in the injury. In vitro studies have indicated that Zn 2+ enters mitochondria and has potent effects on their function. In addition, Zn 2+ accumulates in CA1 mitochondria after ischemia in hippocampal slice and whole animal models, and appears to contribute to their dysfunction. However, the relationship between mitochondrial Zn 2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo, reflecting the difficulty in assessing dynamics of labile (loosely bound) Zn 2+. We employ a cardiac arrest model of ischemia, combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn 2+ accumulation, and use transmission electron microscopy (TEM) to examine subcellular loci of the Zn 2+ accumulation. In line with prior studies, TGI induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4 than 1 h recovery). Intriguingly, TEM examination of Timm stained sections revealed substantial Zn 2+ accumulation in many post-ischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn 2+ and the degree of their disruption were far greater at 4 than 1 h recovery. These data provide the first direct characterization of Zn 2+ accumulation in CA1 mitochondria after in vivo TGI, and further support the idea that mitochondria constitute an early and potentially targetable locus of Zn 2+ effects in ischemia that contributes to mitochondrial damage and neuronal injury.

Cardiac arrest (CA) affects >550,000 people annually in the United States whereas 80-90% of survivors suffer from a comatose state. Arousal from coma is critical for recovery, but mechanisms of arousal are undefined. Orexin-A, a hypothalamic excitatory neuropeptide, has been linked to arousal deficits in various brain injuries. We investigated the orexinergic system's role in recovery from CA-related neurological impairments, including arousal deficits. Using an asphyxial CA and resuscitation model in rats, we examine neurological recovery post-resuscitation in conjunction with changes in orexin-A levels in cerebrospinal fluid (CSF) and orexin-expressing neurons. We also conduct pharmacological inhibition of orexin post-resuscitation. We show that recovery from neurological deficits begins between 4 and 24 h postresuscitation, with additional recovery by 72 h post-resuscitation. Orexin-A levels in the CSF are lowest during periods of poorest arousal post-resuscitation (4 h) and recover to control levels by 24 h. Immunostaining revealed that the number of orexin-A immunoreactive neurons declined at 4 h post-resuscitation, but increased to near normal levels by 24 h. There were no significant changes in the number of neurons expressing melanin-concentrating hormone, another neuropeptide localized in similar hypothalamus regions. Last, administration of the dual orexin receptor antagonist, suvorexant, during the initial 24 h post-resuscitation, led to sustained neurological deficits. The orexin pathway is critical during early phases of neurological recovery post-CA. Blocking this early action leads to persistent neurological deficits. This is of considerable clinical interest given that suvorexant recently received U.S. Food and Drug Administration approval for insomnia treatment.