A single-cell atlas of the normal and malformed human brain vasculature.

Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.

An atlas of cortical arealization identifies dynamic molecular signatures.

The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood1. Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.

COVID-19 and Religious Ethics.

The editors of the JRE solicited short essays on the COVID-19 pandemic from a group of scholars of religious ethics that reflected on how the field might help them make sense of the complex religious, cultural, ethical, and political implications of the pandemic, and on how the pandemic might shape the future of religious ethics.

Prospective 12-month study of intrathecal baclofen therapy for poststroke spastic upper and lower extremity motor control and functional improvement.

OBJECTIVES: To assess the effects of intrathecal baclofen (ITB) therapy for the treatment of poststroke spastic hemiparesis on quality of life, functional independence, and upper, lower extremity (UE, LE) motor functions. MATERIALS AND METHODS: Prospective observational study of adult men and women with a minimum 6-month stroke-related spastic hemiparesis graded as ≥2 in UE and LE on Modified Ashworth Scale (MAS). Patients served as their own controls with measures compared pre-implant with 12 months post ITB including: MAS, manual muscle test (MMT), gait distance/velocity, Functional Independence Measures (FIM), stroke-specific quality of life scale (SSQL), and upper extremity manual activity log. RESULTS: After 12-month ITB therapy, 26 patients (poststroke=6.4±9 years) demonstrated 1) reduced MAS/increased MMT for most LE muscle groups (p≤0.0001); 2) reduced MAS/increased MMT most UE muscle groups (p≤0.01); 3) FIM scores improved (p≤0.05) except bed mobility and lower body dressing; 4) gait distance and velocity improved (p≤0.05); 5) SSQL domains of family roles, mobility, personality, self-care, social roles, thinking, UE function, and work/productivity improved (p≤0.05); 6) amount of use and quality of movement of the spastic UE in performing common activities of daily living increased (p<0.0001). CONCLUSIONS: Regardless of duration of spastic hemiparesis, a reduction in tone with ITB therapy facilitates motor strength improvement and is associated with clinically significant improvements in functional independence and quality of life.

Gamma-aminobutyric acid type A receptor ß3 subunit forebrain-specific knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: ß3 containing γ-aminobutyric acid type A receptors (GABA(A)-Rs) mediate behavioral end points of IV anesthetics such as immobility and hypnosis. A knockout mouse with targeted forebrain deletion of the ß3 subunit of the GABA(A)-R shows reduced sensitivity to the hypnotic effect of etomidate, as measured by the loss of righting reflex. The end points of amnesia and immobility produced by an inhaled anesthetic have yet to be evaluated in this conditional knockout. METHODS: We assessed forebrain selective ß3 conditional knockout mice and their littermate controls for conditional fear to evaluate amnesia and MAC, the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation, to assess immobility. Suppression of conditional fear was assessed for etomidate and isoflurane, and MAC was assessed for isoflurane. RESULTS: Etomidate equally suppressed conditional fear for both genotypes. The knockout showed resistance to the suppression of conditional fear produced by isoflurane in comparison with control littermates. Controls and knockouts did not differ in isoflurane MAC values. CONCLUSIONS: These results suggest that ß3 containing GABA(A)-Rs in the forebrain contribute to hippocampal-dependent memory suppressed by isoflurane, but not etomidate.

Gamma-aminobutyric acid type A receptor alpha 4 subunit knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: General anesthesia produces multiple end points including immobility, hypnosis, sedation, and amnesia. Tonic inhibition via gamma-aminobutyric acid type A receptors (GABA(A)-Rs) may play a role in mediating behavioral end points that are suppressed by low concentrations of anesthetics (e.g., hypnosis and amnesia). GABA(A)-Rs containing the alpha4 subunit are highly concentrated in the hippocampus and thalamus, and when combined with delta subunits they mediate tonic inhibition, which is sensitive to low concentrations of isoflurane. METHODS: In this study, we used a GABA(A) alpha4 receptor knockout mouse line to evaluate the contribution of alpha4-containing GABA(A)-Rs to the effects of immobility, hypnosis, and amnesia produced by isoflurane. Knockout mice and their wild-type counterparts were assessed on 3 behavioral tests: conditional fear (to assess amnesia), loss of righting reflex (to assess hypnosis), and the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation in 50% of subjects (to assess immobility). RESULTS: Genetic inactivation of the alpha4 subunit reduced the amnestic effect of isoflurane, minimally affected loss of righting reflex, and had no effect on immobility. CONCLUSIONS: These results lend support to the hypothesis that different sites of action mediate different anesthetic end points and suggest that alpha4-containing GABA(A)-Rs are important mediators of the amnestic effect of isoflurane on hippocampal-dependent declarative memory.

Isoflurane suppresses stress-enhanced fear learning in a rodent model of post-traumatic stress disorder.

BACKGROUND: A minority of patients who experience awareness and/or pain during surgery subsequently develop post-traumatic stress disorder. In a rodent model of post-traumatic stress disorder, stress-enhanced fear learning (SEFL), rats are preexposed to a stressor of 15 foot shocks. Subsequent exposure to a single foot shock produces an enhanced fear response. This effect is akin to sensitized reactions shown by some post-traumatic stress disorder patients to cues previously associated with the traumatic event. METHODS: The authors studied the effect of isoflurane and nitrous oxide on SEFL. Rats were exposed to the inhaled anesthetic during or after a 15-foot shock stressor. Then, rats were given a single foot shock in a different environment. Their fear response was quantified in response to the 15-foot shock and single-foot shock environments. SEFL longevity was tested by placing a 90-day period between the 15 foot shocks and the single foot shock. In addition, the intensity of the foot shock was increased to evaluate treatment effectiveness. RESULTS: Increasing isoflurane concentrations decreased SEFL when given during, but not after, the stressor. At 0.40 minimum alveolar concentration (MAC), isoflurane given during the stressor blocked SEFL 90 days later. A threefold increase in the stressor intensity increased the isoflurane concentration required to block SEFL to no more than 0.67 MAC. As with isoflurane, nitrous oxide suppressed SEFL at a similar MAC fraction. CONCLUSIONS: These results suggest that sufficient concentrations (perhaps 0.67 MAC or less) of an inhaled anesthetic may prevent SEFL.

Increases in spinal cerebrospinal fluid potassium concentration do not increase isoflurane minimum alveolar concentration in rats.

BACKGROUND: Previous studies demonstrated that MAC for isoflurane directly correlates with the concentration of Na(+) in cerebrospinal fluid surrounding the spinal cord, the primary site for mediation of the immobility produced by inhaled anesthetics. If this correlation resulted from increased irritability of the cord, then infusion of increased concentrations of potassium (K(+)) might be predicted to act similarly. However, an absence of effect of K(+) might be interpreted to indicate that K(+) channels do not mediate the immobility produced by inhaled anesthetics whereas Na(+) channels remain as potential mediators. Accordingly, in the present study, we examined the effect of altering intrathecal concentrations of K(+) on MAC. METHODS: In rats prepared with chronic indwelling intrathecal catheters, we infused solutions deficient in K(+) and with an excess of K(+) into the lumbar space and measured MAC for isoflurane 24 h before, during, and 24 h after infusion. Rats similarly prepared were tested for the effect of altered osmolarity on MAC (accomplished by infusion of mannitol) and for the penetration of Na(+) into the cord. RESULTS: MAC of isoflurane never significantly increased with increasing concentrations of K(+) infused intrathecally. At infused concentrations exceeding 12 times the normal concentration of KCl, i.e., 29 mEq/L, rats moved spontaneously at isoflurane concentrations just below, and sometimes at MAC, but the average MAC in these rats did not exceed their control MAC. At the largest infused concentration (58.1 mEq/L), MAC significantly decreased and did not subsequently return to normal (i.e., such large concentrations produced injury). Infusions of lower concentrations of K(+) had no effect on MAC. Infusion of osmotically equivalent solutions of mannitol did not affect MAC. Na(+) infused intrathecally measurably penetrated the spinal cord. CONCLUSIONS: The results do not support a mediation or modulation of MAC by K(+) channels.

Knockout of the gene encoding the K(2P) channel KCNK7 does not alter volatile anesthetic sensitivity.

The molecular site of action for volatile anesthetics remains unknown despite many years of study. Members of the K(2P) potassium channel family, whose currents are potentiated by volatile anesthetics have emerged as possible anesthetic targets. In fact, a mouse model in which the gene for TREK-1 (KCNK2) has been inactivated shows resistance to volatile anesthetics. In this study we tested whether inactivation of another member of this ion channel family, KCNK7, in a knockout mouse displayed altered sensitivity to the anesthetizing effect of volatile anesthetics. KCNK7 knockout mice were produced by standard gene inactivation methods. Heterozygous breeding pairs produced animals that were homozygous, heterozygous or wild-type for the inactivated gene. Knockout animals were tested for movement in response to noxious stimulus (tail clamp) under varying concentrations of isoflurane, halothane, and desflurane to define the minimum alveolar concentration (MAC) preventing movement. Mice homozygous for inactivated KCNK7 were viable and indistinguishable in weight, general development and behavior from heterozygotes or wild-type littermates. Knockout mice (KCNK7-/-) displayed no difference in MAC for the three volatile anesthetics compared to heterozygous (+/-) or wild-type (+/+) littermates. Because inactivation of KCNK7 does not alter MAC, KCNK7 may play only a minor role in normal CNS function or may have had its function compensated for by other inhibitory mechanisms. Additional studies with transgenic animals will help define the overall role of the K(2P) channels in normal neurophysiology and in volatile anesthetic mechanisms.

Hypothermia decreases ethanol MAC in rats.

Despite the known capacity of hypothermia to increase anesthetic potency (decrease the partial pressure required to produce anesthesia), many in vitro studies examine the effects of ethanol and other anesthetics in oocytes or isolated neurons at room temperature. We tested whether, as predicted for potent inhaled anesthetics, a proportionate increase in solubility with hypothermia matched a decrease in ethanol minimum alveolar concentration (MAC), and thereby made the use of a single anesthetic concentration appropriate regardless of temperature. We determined ethanol MAC in normothermic (37.3 degrees C) and hypothermic (28.5 degrees C) rats, and, at the two temperatures, also determined ethanol solubilities in olive oil and saline. Ethanol MAC decreased, while olive oil/gas and saline/gas partition coefficients increased. However, the increase in the saline/gas partition coefficient did not match the decrease in MAC, and thus the aqueous-phase partial pressure producing absence of movement in 50% of rats (EC50) values for ethanol decreased by 17%. Although this decrease is not large, it may be important for comparative estimates of the in vitro effects of ethanol at different temperatures.

Chirality in anesthesia I: minimum alveolar concentration of secondary alcohol enantiomers.

Most studies of chirality in inhaled anesthetic action have used the enantiomers of isoflurane. These enantiomers are expensive and scarce, which limits studies, such as the preliminary identification of molecular targets of anesthetic action, that can be performed with these isomers. We hypothesized that secondary alcohols (i.e., compounds having a -CH2-CHOH-CH3 group) that are experimental anesthetics would show enantioselectivity. To test this hypothesis, we determined the minimum alveolar anesthetic concentration (MAC) of the enantiomers of the homologous series of 2-alcohols from 2-butanol to 2-heptanol in rats. Because these alcohols are partially metabolized to 2-ketones during the course of study (i.e., having a -CH2-CO-CH3 group), we independently measured the MAC of the 2-ketones. Assuming additivity of MAC of the ketones with the alcohols, we corrected for the anesthetic effect of the ketones in rats to determine the MAC of the alcohols. We found that the 2-butanol and 2-pentanol isomers were enantioselective. S-(+)-2-butanol had a MAC that was 17% larger than for the R-(-)-enantiomer, whereas S-(+)-2-pentanol had a MAC that was 38% larger than the R-(-)- enantiomer. No stereoselectivity was observed for 2-hexanol and 2-heptanol. These findings may permit studies of chirality in anesthesia, particularly in in vitro systems where metabolism does not occur, using inexpensive volatile compounds.

The minimum alveolar anesthetic concentration of 2-, 3-, and 4-alcohols and ketones in rats: relevance to anesthetic mechanisms.

The Meyer-Overton hypothesis predicts that anesthetic potency correlates inversely with lipophilicity; e.g., MAC times the olive oil/gas partition coefficient equals a constant of approximately 1.82 +/- 0.56 atm (mean +/- sd) for conventional inhaled anesthetics. MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. In contrast to conventional inhaled anesthetics, MAC times the olive oil/gas partition coefficient for normal alcohols from methanol through octanol equals a constant one tenth as large as that for conventional inhaled anesthetics. The alcohol (C-OH) group causes a great affinity of alcohols to water, and the C-OH may tether the alcohol at the hydrophobic-hydrophilic interface where anesthetics are thought to act. We hypothesized that the position of the C-OH group determined potency, perhaps by governing the maximum extent to which the acyl portion of the molecule might extend into a hydrophobic phase. Using the same reasoning, we added studies of ketones with similar numbers of carbon atoms between the C=O group and the terminal methyl group. The results for both alcohols and ketones showed the predicted correlation, but the correlation was no better than that with carbon chain length regardless of the placement of the oxygen. The oil/gas partition coefficient predicted potency as well as, or better than, either chain length or oxygen placement. Hydrophilicity, as indicated by the saline/gas partition coefficient, also seemed to influence potency.