A hypothalamomedullary network for physiological responses to environmental stresses.

Various environmental stressors, such as extreme temperatures (hot and cold), pathogens, predators and insufficient food, can threaten life. Remarkable progress has recently been made in understanding the central circuit mechanisms of physiological responses to such stressors. A hypothalamomedullary neural pathway from the dorsomedial hypothalamus (DMH) to the rostral medullary raphe region (rMR) regulates sympathetic outflows to effector organs for homeostasis. Thermal and infection stress inputs to the preoptic area dynamically alter the DMH → rMR transmission to elicit thermoregulatory, febrile and cardiovascular responses. Psychological stress signalling from a ventromedial prefrontal cortical area to the DMH drives sympathetic and behavioural responses for stress coping, representing a psychosomatic connection from the corticolimbic emotion circuit to the autonomic and somatic motor systems. Under starvation stress, medullary reticular neurons activated by hunger signalling from the hypothalamus suppress thermogenic drive from the rMR for energy saving and prime mastication to promote food intake. This Perspective presents a combined neural network for environmental stress responses, providing insights into the central circuit mechanism for the integrative regulation of systemic organs.

Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease.

cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.

Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Two Ascending Thermosensory Pathways from the Lateral Parabrachial Nucleus That Mediate Behavioral and Autonomous Thermoregulation.

Thermoregulatory behavior in homeothermic animals is an innate behavior to defend body core temperature from environmental thermal challenges in coordination with autonomous thermoregulatory responses. In contrast to the progress in understanding the central mechanisms of autonomous thermoregulation, those of behavioral thermoregulation remain poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates cutaneous thermosensory afferent signaling for thermoregulation. To understand the thermosensory neural network for behavioral thermoregulation, in the present study, we investigated the roles of ascending thermosensory pathways from the LPB in avoidance behavior from innocuous heat and cold in male rats. Neuronal tracing revealed two segregated groups of LPB neurons projecting to the median preoptic nucleus (MnPO), a thermoregulatory center (LPB→MnPO neurons), and those projecting to the central amygdaloid nucleus (CeA), a limbic emotion center (LPB→CeA neurons). While LPB→MnPO neurons include separate subgroups activated by heat or cold exposure of rats, LPB→CeA neurons were only activated by cold exposure. By selectively inhibiting LPB→MnPO or LPB→CeA neurons using tetanus toxin light chain or chemogenetic or optogenetic techniques, we found that LPB→MnPO transmission mediates heat avoidance, whereas LPB→CeA transmission contributes to cold avoidance. In vivo electrophysiological experiments showed that skin cooling-evoked thermogenesis in brown adipose tissue requires not only LPB→MnPO neurons but also LPB→CeA neurons, providing a novel insight into the central mechanism of autonomous thermoregulation. Our findings reveal an important framework of central thermosensory afferent pathways to coordinate behavioral and autonomous thermoregulation and to generate the emotions of thermal comfort and discomfort that drive thermoregulatory behavior.SIGNIFICANCE STATEMENT Coordination of behavioral and autonomous thermoregulation is important for maintaining thermal homeostasis in homeothermic animals. However, the central mechanism of thermoregulatory behaviors remains poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates ascending thermosensory signaling that drives thermoregulatory behavior. In this study, we found that one pathway from the LPB to the median preoptic nucleus mediates heat avoidance, whereas the other pathway from the LPB to the central amygdaloid nucleus is required for cold avoidance. Surprisingly, both pathways are required for skin cooling-evoked thermogenesis in brown adipose tissue, an autonomous thermoregulatory response. This study provides a central thermosensory network that coordinates behavioral and autonomous thermoregulation and generates thermal comfort and discomfort that drive thermoregulatory behavior.

Effects of halogen atom substitution on luminescent radicals: a case study on tris(2,4,6-trichlorophenyl)methyl radical-carbazole dyads.

A series of halogen-substitute carbazole TTM radicals was synthesized. The effect of halogen substituents on radical luminescence was systematically evaluated. It was found that the well-known heavy atom effect does not work in the emission of radicals and that halogen substitution of the donor carbazole can change the HOMO and alter the absorption and emission wavelengths. In addition, the photostability was found to be improved with respect to TTM but not significantly different from that of closed-shell fluorescent molecules.

Auto-Lectin Dotcoding by Two Octopuses: Rapid Analysis of Fluorescence-Labeled Glycoproteins by an 8-channel Fully-Automatic Bead Array Scanner with a Rolling-Circle Detector.

Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.

Natural variations of wheat EARLY FLOWERING 3 highlight their contributions to local adaptation through fine-tuning of heading time.

KEY MESSAGE: We identified a large chromosomal deletion containing TaELF-B3 that confers early flowering in wheat. This allele has been preferred in recent wheat breeding in Japan to adapt to the environment. Heading at the appropriate time in each cultivation region can greatly contribute to stabilizing and maximizing yield. Vrn-1 and Ppd-1 are known as the major genes for vernalization requirement and photoperiod sensitivity in wheat. Genotype combinations of Vrn-1 and Ppd-1 can explain the variation in heading time. However, the genes that can explain the remaining variations in heading time are largely unknown. In this study, we aimed to identify the genes conferring early heading using doubled haploid lines derived from Japanese wheat varieties. Quantitative trait locus (QTL) analysis revealed a significant QTL on the long arm of chromosome 1B in multiple growing seasons. Genome sequencing using Illumina short reads and Pacbio HiFi reads revealed a large deletion of a ~ 500 kb region containing TaELF-B3, an orthologue of Arabidopsis clock gene EARLY FLOWERING 3 (ELF3). Plants with the deleted allele of TaELF-B3 (ΔTaELF-B3 allele) headed earlier only under short-day vernalization conditions. Higher expression levels of clock- and clock-output genes, such as Ppd-1 and TaGI, were observed in plants with the ΔTaELF-B3 allele. These results suggest that the deletion of TaELF-B3 causes early heading. Of the TaELF-3 homoeoalleles conferring early heading, the ΔTaELF-B3 allele showed the greatest effect on the early heading phenotype in Japan. The higher allele frequency of the ΔTaELF-B3 allele in western Japan suggests that the ΔTaELF-B3 allele was preferred during recent breeding to adapt to the environment. TaELF-3 homoeologs will help to expand the cultivated area by fine-tuning the optimal timing of heading in each environment.

Carbazole-Dendronized Luminescent Radicals.

A series of carbazole-dendronized tris(2,4,6-trichlorophenyl)methyl (TTM) radicals have been synthesized. The photophysical properties of dendronized radicals up to the fourth generation were compared systematically to understand how structure-property relationships evolve with generation. The photoluminescence quantum yield (PLQY) was found to increase with the increasing generation, and the fourth generation (G4TTM) in cyclohexane solution showed a PLQY as high as 63 % at a wavelength of 627 nm (in the deep-red region) from the doublet state. The dendron modification strategy also showed a blue-shift of the emission on increasing the generation number, and the photostability was also increased compared to the bare TTM radical.

Application study of infrared free-electron lasers towards the development of amyloidosis therapy.

Amyloidosis is known to be caused by the deposition of amyloid fibrils into various biological tissues; effective treatments for the disease are little established today. An infrared free-electron laser (IR-FEL) is an accelerator-based picosecond-pulse laser having tunable infrared wavelengths. In the current study, the irradiation effect of an IR-FEL was tested on an 11-residue peptide (NFLNCYVSGFH) fibril from ß2-microglobulin (ß2M) with the aim of applying IR-FELs to amyloidosis therapy. Infrared microspectroscopy (IRM) and scanning electron microscopy showed that a fibril of ß2M peptide was clearly dissociated by IR-FEL at 6.1 µm (amide I) accompanied by a decrease of the ß-sheet and an increase of the α-helix. No dissociative process was recognized at 6.5 µm (amide II) as well as at 5.0 µm (non-specific wavelength). Equilibrium molecular dynamics simulations indicated that the α-helix can exist stably and the probability of forming interchain hydrogen bonds associated with the internal asparagine residue (N4) is notably reduced compared with other amino acids after the ß-sheet is dissociated by amide I specific irradiation. This result implies that N4 plays a key role for recombination of hydrogen bonds in the dissociation of the ß2M fibril. In addition, the ß-sheet was disrupted at temperatures higher than 340 K while the α-helix did not appear even though the fibril was heated up to 363 K as revealed by IRM. The current study gives solid evidence for the laser-mediated conversion from ß-sheet to α-helix in amyloid fibrils at the molecular level.

Allelic variations of Vrn-1 and Ppd-1 genes in Japanese wheat varieties reveal the genotype-environment interaction for heading time.

The timing of heading is largely affected by environmental conditions. In wheat, Vrn-1 and Ppd-1 have been identified as the major genes involved in vernalization requirement and photoperiod sensitivity, respectively. To compare the effects of Vrn-1 and Ppd-1 alleles on heading time under different environments, we genotyped Vrn-1 and Ppd-1 homoeologues and measured the heading time at Morioka, Tsukuba and Chikugo in Japan for two growing seasons. A total of 128 Japanese and six foreign varieties, classified into four populations based on the 519 genome-wide SNPs, were used for analysis. Varieties with the spring alleles (Vrn-D1a or Vrn-D1b) at the Vrn-D1 locus and insensitive allele (Hapl-I) at the Ppd-D1 locus were found in earlier heading varieties. The effects of Vrn-D1 and Ppd-D1 on heading time were stronger than those of the other Vrn-1 and Ppd-1 homoeologues. Analysis of variance revealed that heading time was significantly affected by the genotype-environment interactions. Some Vrn-1 and Ppd-1 alleles conferred earlier or later heading in specific environments, indicating that the effect of both alleles on the timing of heading depends on the environment. Information on Vrn-1 and Ppd-1 alleles, together with heading time in various environments, provide useful information for wheat breeding.

Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats.

CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

Genetic mechanisms determining grain number distribution along the spike and their effect on yield components in wheat.

The number of wheat grains is one of the major determinants of yield. Many quantitative trait loci (QTLs) and some causal genes such as GNI-A1 and WAPO-A1 that are associated with grain number per spike (GNS) have been identified, but the underlying mechanisms remain largely unknown. We analyzed QTLs for grain number and other related traits using 188 doubled haploid lines derived from the Japanese high-yield variety, Kitahonami, as a parent to elucidate the genetic mechanism determining grain number. The major QTLs for grain number at the apical, central, and basal parts of the spike were identified in different chromosomal regions. We considered GNI-A1 and WAPO-A1 as candidate genes controlling grain number at the central and basal parts of the spike, respectively. Kitahonami had the favorable 105Y allele of GNI-A1 and WAPO-A1b allele and unfavorable alleles of QTLs for grain number at the apical part of spikes. Pyramiding the favorable alleles of these QTLs significantly increased GNS without significantly reducing thousand-grain weight (TGW). In contrast, the accumulation of favorable alleles of QTLs for TGW significantly decreased GNS, whereas days to heading positively correlated with GNS. Late heading increased the spikelet number per spike, resulting in a higher GNS. Pyramiding of the QTLs for TGW and days to heading also altered the GNS. In conclusion, GNS is a complex trait controlled by many QTLs, and it is essential for breeding to design. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01255-8.

Hunger and Satiety Signaling: Modeling Two Hypothalamomedullary Pathways for Energy Homeostasis.

The recent discovery of the medullary circuit driving "hunger responses" - reduced thermogenesis and promoted feeding - has greatly expanded our knowledge on the central neural networks for energy homeostasis. However, how hypothalamic hunger and satiety signals generated under fasted and fed conditions, respectively, control the medullary autonomic and somatic motor mechanisms remains unknown. Here, in reviewing this field, we propose two hypothalamomedullary neural pathways for hunger and satiety signaling. To trigger hunger signaling, neuropeptide Y activates a group of neurons in the paraventricular hypothalamic nucleus (PVH), which then stimulate an excitatory pathway to the medullary circuit to drive the hunger responses. In contrast, melanocortin-mediated satiety signaling activates a distinct group of PVH neurons, which then stimulate a putatively inhibitory pathway to the medullary circuit to counteract the hunger signaling. The medullary circuit likely contains inhibitory and excitatory premotor neurons whose alternate phasic activation generates the coordinated masticatory motor rhythms to promote feeding.

Lectin Bead Array in a Single Tip Facilitates Fully Automatic Glycoprotein Profiling.

A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan-lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.

[Cognitive Dysfunction in Children who Underwent Surgery after a Traumatic Brain Injury:A Single Institute Investigation].

The cognitive function of children who underwent surgical therapy after a traumatic brain injury is poorly studied. In this study, we investigated the characteristics of 27 children who received surgical therapy at our institution. The children were between 1 and 16 years of age, of which 15 had cognitive dysfunction. Their Glasgow Coma Scale score at the acute stage of dysfunction was worse than in children who did not have cognitive dysfunction. Acute subdural hematoma was more frequent in the cognitive dysfunction group. Moreover, all children in this group showed brain injury by imaging analysis. Differences in imaging characteristics and the association with cognitive dysfunction could not be readily associated with a specific injury. Memory and verbal disorder were the most common cognitive dysfunctions:these symptoms were present among children of all ages;conversely, behavior disorder, impaired attention, and infeasibility were limited to the children under 9 years of age. Since the immature brain is developing, the acquisition of new abilities may be blocked by the injury;thus, we speculate that brain injury at a younger age causes greater cognitive dysfunction.

Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.

KEY POINTS: Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN-RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. ABSTRACT: The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN-RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN-RVLM neurons. Male rats received microinjections into the PVN with an adeno-associated virus (AAV) vector that encoded a hybrid of channelrhodopsin-2/1 with the reporter tdTomato (ChIEF-tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2-GFPretro ). Under anaesthesia with urethane and α-chloralose, photostimulation (473 nm wavelength) of PVN-RVLM neurons, achieved by laser illumination of either RVLM of ChIEF-tdTomato rats (n = 8) or PVN of ChR2-GFPretro rats (n = 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF-tdTomato rats were closely associated with tdTomato-labelled, PVN-derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF-tdTomato rats (n = 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN-RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons.

Central regulation of brown adipose tissue thermogenesis and energy homeostasis dependent on food availability.

Energy homeostasis of mammals is maintained by balancing energy expenditure within the body and energy intake through feeding. Several lines of evidence indicate that brown adipose tissue (BAT), a sympathetically activated thermogenic organ, turns excess energy into heat to maintain the energy balance in rodents and humans, in addition to its thermoregulatory role for the defense of body core temperature in cold environments. Elucidating the central circuit mechanism controlling BAT thermogenesis dependent on nutritional conditions and food availability in relation to energy homeostasis is essential to understand the etiology of symptoms caused by energy imbalance, such as obesity. The central thermogenic command outflow to BAT descends through an excitatory neural pathway mediated by hypothalamic, medullary and spinal sites. This sympathoexcitatory thermogenic drive is controlled by tonic GABAergic inhibitory signaling from the thermoregulatory center in the preoptic area, whose tone is altered by body core and cutaneous thermosensory inputs. This circuit controlling BAT thermogenesis for cold defense also functions for the development of fever and psychological stress-induced hyperthermia, indicating its important role in the defense from a variety of environmental stressors. When food is unavailable, hunger-driven neural signaling from the hypothalamus activates GABAergic neurons in the medullary reticular formation, which then block the sympathoexcitatory thermogenic outflow to BAT to reduce energy expenditure and simultaneously command the masticatory motor system to promote food intake-effectively commanding responses to survive starvation. This article reviews the central mechanism controlling BAT thermogenesis in relation to the regulation of energy and thermal homeostasis dependent on food availability.

Dissociation of ß-Sheet Stacking of Amyloid ß Fibrils by Irradiation of Intense, Short-Pulsed Mid-infrared Laser.

Structure of amyloid ß (Aß) fibrils is rigidly stacked by ß-sheet conformation, and the fibril state of Aß is profoundly related to pathogenesis of Alzheimer's disease (AD). Although mid-infrared light has been used for various biological researches, it has not yet been known whether the infrared light changes the fibril structure of Aß. In this study, we tested the effect of irradiation of intense mid-infrared light from a free-electron laser (FEL) targeting the amide bond on the reduction of ß-sheet content in Aß fibrils. The FEL reduced entire contents of proteins exhibiting ß-sheet structure in brain sections from AD model mice, as shown by synchrotron-radiation infrared microscopy analysis. Since Aß1-42 fibril absorbed a considerable FEL energy at amide I band (6.17 µm), we irradiated the FEL at 6.17 µm and found that ß-sheet content of naked Aß1-42 fibril was decreased using infrared microscopic analysis. Consistent with the decrease in the ß-sheet content, Congo-red signal is decreased after the irradiation to Aß1-42 fibril. Furthermore, electron microscopy analysis revealed that morphologies of the fibril and proto-fibril were largely changed after the irradiation. Thus, mid-infrared light dissociates ß-sheet structure of Aß fibrils, which justifies exploration of possible laser-based therapy for AD.

[Thermoregulatory behavior and its central circuit mechanism-What thermosensory pathway drives it?]

Thermoregulatory behavior in homeothermic animals is an instinctive behavior to reduce the cost of their homeothermy, such as warm-seeking behavior to reduce metabolic energy for heat production in cold environments. A recent study identified the central neural pathway that transmits cutaneous thermosensory information on environmental temperature to drive thermoregulatory behavior. This study proposed that the brain mechanism that drives thermoregulatory behavior does not require conscious"feeling"(i.e., perception)of skin temperature mediated by the spinothalamocortical pathway, whereas thermosensory information that ascends through the spinal cord and the lateral parabrachial nucleus of the pons generates thermal comfort and discomfort that drive thermoregulatory behavior.

Acute cardiac support with intravenous milrinone promotes recovery from early brain injury in a murine model of severe subarachnoid haemorrhage.

Early brain injury/ischaemia (EBI) is a serious complication early after subarachnoid haemorrhage (SAH) that contributes to development of delayed cerebral ischaemia (DCI). This study aimed to determine the role of inotropic cardiac support using milrinone (MIL) on restoring acute cerebral hypoperfusion attributable to EBI and improving outcomes after experimental SAH. Forty-three male C57BL/6 mice were assigned to either sham surgery (SAH-sham), SAH induced by endovascular perforation plus postconditioning with 2% isoflurane (Control), or SAH plus isoflurane combined with MIL with and without hypoxia-inducible factor inhibitor (HIF-I) pretreatment. Cardiac output (CO) during intravenous MIL infusion (0.25-0.75 µg/kg/min) between 1.5 and 2.5 hours after SAH induction was monitored with Doppler echocardiography. Magnetic resonance imaging (MRI)-continuous arterial spin labelling was used for quantitative cerebral blood flow (CBF) measurements. Neurobehavioral function was assessed daily by neurological score and open field test. DCI was analyzed 3 days later by determining infarction on MRI. Mild reduction of cardiac output (CO) and global cerebral blood flow (CBF) depression were notable early after SAH. MIL increased CO in a dose-dependent manner (P<.001), which was accompanied by improved hypoperfusion, incidence of DCI and functional recovery than Control (P<.05). The neuroprotective effects afforded by MIL or Control were attenuated by hypoxia-inducible factor (HIF) inhibition (P<.05). These results suggest that MIL improves acute hypoperfusion by its inotropic effect, leading to neurobehavioral improvement in mice after severe SAH, in which HIF may be acting as a critical mediator.