Initiation, Expansion, and Cryopreservation of Human Primary Tissue-Derived Normal and Diseased Organoids in Embedded Three-Dimensional Culture.

Organoids are primary patient-derived micro tissues grown within a three-dimensional extracellular matrix that better represents in vivo physiology and genetic diversity than existing two-dimensional cell lines. Organoids rely on the self-renewal and differentiation of tissue-resident stem cells that expand in culture and self-organize into complex three-dimensional structures. Depending on the tissue, organoids typically lack stromal, vascular, neural, and immune cells but otherwise can contain cells from all the respective tissue-specific cell lineages found in vivo. Established organoids can be initiated from cryopreserved material, cultured using largely traditional cell culture techniques and equipment, and then expanded and cryopreserved for future use. Organoid models have been developed from a variety of diseased and normal tissues including small intestine, colon, mammary, esophagus, lung, prostate, and pancreas. © 2018 by John Wiley & Sons, Inc.

Surgical Management of Cervical Spondylotic Myelopathy.

Cervical spondylotic myelopathy (CSM) is a common cause of neurologic impairment in adults worldwide. Numerous studies have investigated the pathophysiology of CSM, which has provided surgeons with insight on the important factors that lead to the symptoms and deficits observed in patients who have CSM. However, further analysis of many unknown aspects of CSM is required to fully understand the disease and potential alternative treatment modalities. The diagnosis of CSM is based on a patient's history and physical examination and then confirmed with imaging studies. Progression, symptomatology, and imaging findings may vary by patient. Because of the variability of CSM, the disease course and a patient's response to treatment are difficult to predict. CSM can be managed either nonsurgically or it can be managed surgically via posterior or anterior cervical approaches, each of which has its own indications and possible complications.

MicroRNA 138, let-7b, and 125a inhibitors differentially alter sleep and EEG delta-wave activity in rats.

Sleep deprivation was previously reported to alter microRNA (miRNA) levels in the brain; however, the direct effects of any miRNA on sleep have only been described recently. We determined miRNA 138 (miR-138), miRNA let-7b (let-7b), and miRNA 125a-5p (miR-125a) levels in different brain areas at the transitions between light and dark. In addition, we examined the extent to which inhibiting these miRNAs affects sleep and EEG measures. We report that the levels of multiple miRNAs differ at the end of the sleep-dominant light period vs. the end of the wake-dominant dark period in cortical areas, hippocampus, and hypothalamus. For instance, in multiple regions of the cortex, miR-138, let-7b, and miR-125a expression was higher at the end of the dark period compared with the end of the light period. Intracerebroventricular injection of a specific inhibitor (antiMIR) to miR-138 suppressed sleep and nonrapid eye movement sleep (NREMS) EEG delta power. The antiMIR to let-7b did not affect time in state but decreased NREMS EEG delta power, whereas the antiMIR to miR-125a failed to affect sleep until after 3 days and did not affect EEG delta power on any day. We conclude that miRNAs are uniquely expressed at different times and in different structures in the brain and have discrete effects and varied timings on several sleep phenotypes and therefore, likely play a role in the regulation of sleep.

5'-Ectonucleotidase-knockout mice lack non-REM sleep responses to sleep deprivation.

Adenosine and extracellular adenosine triphosphate (ATP) have multiple physiological central nervous system actions including regulation of cerebral blood flow, inflammation and sleep. However, their exact sleep regulatory mechanisms remain unknown. Extracellular ATP and adenosine diphosphate are converted to adenosine monophosphate (AMP) by the enzyme ectonucleoside triphosphate diphosphohydrolase 1, also known as CD39, and extracellular AMP is in turn converted to adenosine by the 5'-ectonuleotidase enzyme CD73. We investigated the role of CD73 in sleep regulation. Duration of spontaneous non-rapid eye movement sleep (NREMS) was greater in CD73-knockout (KO) mice than in C57BL/6 controls whether determined in our laboratory or by others. After sleep deprivation (SD), NREMS was enhanced in controls but not CD73-KO mice. Interleukin-1 beta (IL1ß) enhanced NREMS in both strains, indicating that the CD73-KO mice were capable of sleep responses. Electroencephalographic power spectra during NREMS in the 1.0-2.5 Hz frequency range was significantly enhanced after SD in both CD73-KO and WT mice; the increases were significantly greater in the WT mice than in the CD73-KO mice. Rapid eye movement sleep did not differ between strains in any of the experimental conditions. With the exception of CD73 mRNA, the effects of SD on various adenosine-related mRNAs were small and similar in the two strains. These data suggest that sleep is regulated, in part, by extracellular adenosine derived from the actions of CD73.

Brain-specific interleukin-1 receptor accessory protein in sleep regulation.

Interleukin (IL)-1ß is involved in several brain functions, including sleep regulation. It promotes non-rapid eye movement (NREM) sleep via the IL-1 type I receptor. IL-1ß/IL-1 receptor complex signaling requires adaptor proteins, e.g., the IL-1 receptor brain-specific accessory protein (AcPb). We have cloned and characterized rat AcPb, which shares substantial homologies with mouse AcPb and, compared with AcP, is preferentially expressed in the brain. Furthermore, rat somatosensory cortex AcPb mRNA varied across the day with sleep propensity, increased after sleep deprivation, and was induced by somnogenic doses of IL-1ß. Duration of NREM sleep was slightly shorter and duration of REM sleep was slightly longer in AcPb knockout than wild-type mice. In response to lipopolysaccharide, which is used to induce IL-1ß, sleep responses were exaggerated in AcPb knockout mice, suggesting that, in normal mice, inflammation-mediated sleep responses are attenuated by AcPb. We conclude that AcPb has a role in sleep responses to inflammatory stimuli and, possibly, in physiological sleep regulation.

Delta wave power: an independent sleep phenotype or epiphenomenon?

Electroencephalographic (EEG) δ waves during non-rapid eye movement sleep (NREMS) after sleep deprivation are enhanced. That observation eventually led to the use of EEG δ power as a parameter to model process S in the two-process model of sleep. It works remarkably well as a model parameter because it often co-varies with sleep duration and intensity. Nevertheless there is a large volume of literature indicating that EEG δ power is regulated independently of sleep duration. For example, high amplitude EEG δ waves occur in wakefulness after systemic atropine administration or after hyperventilation in children. Human neonates have periods of sleep with an almost flat EEG. Similarly, elderly people have reduced EEG δ power, yet retain substantial NREMS. Rats provided with a cafeteria diet have excess duration of NREMS but simultaneously decreased EEG δ power for days. Mice challenged with influenza virus have excessive EEG δ power and NREMS. In contrast, if mice lacking TNF receptors are infected, they still sleep more but have reduced EEG δ power. Sleep regulatory substances, e.g., IL1, TNF, and GHRH, directly injected unilaterally onto the cortex induce state-dependent ipsilateral enhancement of EEG δ power without changing duration of organism sleep. IL1 given systemically enhances duration of NREMS but reduces EEG δ power in mice. Benzodiazepines enhance NREMS but inhibit EEG δ power. If duration of NREMS is an indicator of prior sleepiness then simultaneous EEG δ power may or may not be a useful index of sleepiness. Finally, most sleep regulatory substances are cerebral vasodilators and blood flow affects EEG δ power. In conclusion, it seems unlikely that a single EEG measure will be reliable as a marker of sleepiness for all conditions.

Biochemical regulation of sleep and sleep biomarkers.

Symptoms commonly associated with sleep loss and chronic inflammation include sleepiness, fatigue, poor cognition, enhanced sensitivity to pain and kindling stimuli, excess sleep and increases in circulating levels of tumor necrosis factor α (TNF) in humans and brain levels of interleukin-1 ß (IL1) and TNF in animals. Cytokines including IL1 and TNF partake in non-rapid eye movement sleep (NREMS) regulation under physiological and inflammatory conditions. Administration of exogenous IL1 or TNF mimics the accumulation of these cytokines occurring during sleep loss to the extent that it induces the aforementioned symptoms. Extracellular ATP associated with neuro- and glio-transmission, acting via purine type 2 receptors, e.g., the P2X7 receptor, has a role in glia release of IL1 and TNF. These substances in turn act on neurons to change their intrinsic membrane properties and sensitivities to neurotransmitters and neuromodulators such as adenosine, glutamate and GABA. These actions change the network input-output properties, i.e., a state shift for the network. State oscillations occur locally within cortical columns and are defined using evoked response potentials. One such state, so defined, shares properties with whole animal sleep in that it is dependent on prior cellular activity--it shows homeostasis. The cortical column sleep-like state is induced by TNF and is associated with experimental performance detriments. ATP released extracellularly as a consequence of cellular activity is posited to initiate a mechanism by which the brain tracks its prior sleep-state history to induce/prohibit sleep. Thus, sleep is an emergent property of populations of local neural networks undergoing state transitions. Specific neuronal groups participating in sleep depend upon prior network use driving local network state changes via the ATP-cytokine-adenosine mechanism. Such considerations add complexity to finding biochemical markers for sleepiness.

Involvement of cytokines in slow wave sleep.

Cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1ß) play a role in sleep regulation in health and disease. TNFα or IL1ß injection enhances non-rapid eye movement sleep. Inhibition of TNFα or IL1ß reduces spontaneous sleep. Mice lacking TNFα or IL1ß receptors sleep less. In normal humans and in multiple disease states, plasma levels of TNFα covary with EEG slow wave activity (SWA) and sleep propensity. Many of the symptoms induced by sleep loss, for example, sleepiness, fatigue, poor cognition, enhanced sensitivity to pain, are elicited by injection of exogenous TNFα or IL1ß. IL1ß or TNFα applied unilaterally to the surface of the cortex induces state-dependent enhancement of EEG SWA ipsilaterally, suggesting greater regional sleep intensity. Interventions such as unilateral somatosensory stimulation enhance localized sleep EEG SWA, blood flow, and somatosensory cortical expression of IL1ß and TNFα. State oscillations occur within cortical columns. One such state shares properties with whole animal sleep in that it is dependent on prior cellular activity, shows homeostasis, and is induced by TNFα. Extracellular ATP released during neuro- and gliotransmission enhances cytokine release via purine type 2 receptors. An ATP agonist enhances sleep, while ATP antagonists inhibit sleep. Mice lacking the P2X7 receptor have attenuated sleep rebound responses after sleep loss. TNFα and IL1ß alter neuron sensitivity by changing neuromodulator/neurotransmitter receptor expression, allowing the neuron to scale its activity to the presynaptic neurons. TNFα's role in synaptic scaling is well characterized. Because the sensitivity of the postsynaptic neuron is changed, the same input will result in a different network output signal and this is a state change. The top-down paradigm of sleep regulation requires intentional action from sleep/wake regulatory brain circuits to initiate whole-organism sleep. This raises unresolved questions as to how such purposeful action might itself be initiated. In the new paradigm, sleep is initiated within networks and local sleep is a direct consequence of prior local cell activity. Whole-organism sleep is a bottom-up, self-organizing, and emergent property of the collective states of networks throughout the brain.

MicroRNA 132 alters sleep and varies with time in brain.

MicroRNA (miRNA) levels in brain are altered by sleep deprivation; however, the direct effects of any miRNA on sleep have not heretofore been described. We report herein that intracerebroventricular application of a miRNA-132 mimetic (preMIR-132) decreased duration of non-rapid-eye-movement sleep (NREMS) while simultaneously increasing duration of rapid eye movement sleep (REMS) during the light phase. Further, preMIR-132 decreased electroencephalographic (EEG) slow-wave activity (SWA) during NREMS, an index of sleep intensity. In separate experiments unilateral supracortical application of preMIR-132 ipsilaterally decreased EEG SWA during NREMS but did not alter global sleep duration. In addition, after ventricular or supracortical injections of preMIR-132, the mimetic-induced effects were state specific, occurring only during NREMS. After local supracortical injections of the mimetic, cortical miRNA-132 levels were higher at the time sleep-related EEG effects were manifest. We also report that spontaneous cortical levels of miRNA-132 were lower at the end of the sleep-dominant light period compared with at the end of the dark period in rats. Results suggest that miRNAs play a regulatory role in sleep and provide a new tool for investigating sleep regulation.

ATP and the purine type 2 X7 receptor affect sleep.

Sleep is dependent upon prior brain activities, e.g., after prolonged wakefulness sleep rebound occurs. These effects are mediated, in part, by humoral sleep regulatory substances such as cytokines. However, the property of wakefulness activity that initiates production and release of such substances and thereby provides a signal for indexing prior waking activity is unknown. We propose that extracellular ATP, released during neuro- and gliotransmission and acting via purine type 2 (P2) receptors, is such a signal. ATP induces cytokine release from glia. Cytokines in turn affect sleep. We show here that a P2 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), increased non-rapid eye movement sleep (NREMS) and electroencephalographic (EEG) delta power while two different P2 receptor antagonists, acting by different inhibitory mechanisms, reduced spontaneous NREMS in rats. Rat P2X7 receptor protein varied in the somatosensory cortex with time of day, and P2X7 mRNA was altered by interleukin-1 treatment, by sleep deprivation, and with time of day in the hypothalamus and somatosensory cortex. Mice lacking functional P2X7 receptors had attenuated NREMS and EEG delta power responses to sleep deprivation but not to interleukin-1 treatment compared with wild-type mice. Data are consistent with the hypothesis that extracellular ATP, released as a consequence of cell activity and acting via P2 receptors to release cytokines and other sleep regulatory substances, provides a mechanism by which the brain could monitor prior activity and translate it into sleep.