Kazumasu Sasaki

Decreased response to social defeat stress in μ-opioid-receptor knockout mice.

Substantial evidence exists that opioid systems are involved in stress response and that changes in opioid systems in response to stressors affect both reward and analgesia. Reportedly, mice suffering chronic social defeat stress subsequently show aversion to social contact with unfamiliar mice. To further examine the role of opioid systems in stress response, the behavioral and neurochemical effects of chronic social defeat stress (psychosocial stress) were evaluated in μ-opioid-receptor knockout (MOR-KO) mice. Aversion to social contact was induced by chronic social defeat stress in wild-type mice but was reduced in MOR-KO mice. Moreover, basal expression of brain-derived neurotrophic factor (BDNF) mRNA in MOR-KO mice hippocampi was significantly lower than in wild-type mice. Psychosocial stress significantly decreased BDNF mRNA expression in wild-type mice but did not affect BDNF expression in MOR-KO mice; no difference in basal levels of plasma corticosterone was observed. These results suggest that the μ-opioid receptor is involved in the behavioral sequelae of psychosocial stress and consequent regulation of BDNF expression in the hippocampus, and may play an important role in psychiatric disorders for which stress is an important predisposing or precipitating factor, such as depression, posttraumatic stress disorder, and social anxiety disorder.

Value of Three-Dimensional Maximum Intensity Projection Display to Assist in Magnetic Resonance Imaging (MRI)-Based Grading in a Mouse Model of Subarachnoid Hemorrhage

Background Subarachnoid hemorrhage (SAH) is one of the most devastating cerebrovascular disorders. We report on the diagnostic value of three-dimensional (3-D) maximum intensity projection (MIP) reconstruction of T2*-weighted magnetic resonance images (MRI), processed using graphical user interface-based software, to aid in the accurate grading of endovascular-perforation-induced SAH in a mouse model. Material/Methods A total of 30 mice were subjected to SAH by endovascular perforation; three (10%) were scored as grade 0, six (20%) as grade 1, six (20%) as grade 2, eight (27%) as grade 3, and seven (23%) as grade 4 according to T2*-weighted coronal slices. In comparison, none of mice were scored as grade 0, eight (27%) as grade 1, five (17%) as grade 2, nine (30%) as grade 3, and eight (27%) as grade 4 based on subsequent evaluation using reconstructed 3-D MIP images. Results Mice scored as grade 0 (10%; no visible SAH) on T2*-coronal images were categorized as grades 1 (thin/localized SAH) and 3 (thick/diffuse SAH) according to 3-D MIP images. Grades based on T2* 3-D MIP images were more closely correlated with conventional SAH score (r2=0.59; P<0.0001) and neurological score (r2=0.25; P=0.005) than those based on T2*-coronal slices (r2=0.46; P<0.0001 for conventional score and r2=0.15; P=0.035 for neurological score). Conclusions These results suggest that 3-D MIP images generated from T2*-weighted MRI data may be useful for the simple and precise grading of SAH severity in mice to overcome the weakness of the current MRI-based SAH grading system.

Isoflurane postconditioning with cardiac support promotes recovery from early brain injury in mice after severe subarachnoid hemorrhage

AIMS Neurocardiac dysfunction is a life-threatening systemic consequence of subarachnoid hemorrhage (SAH) that contributes to triggering delayed cerebral ischemia (DCI). This study aimed to determine the impact of dobutamine cardiac support during isoflurane postconditioning on post-SAH DCI. MAIN METHODS Male C57BL/6 mice were subjected to SAH, SAH plus isoflurane postconditioning, or SAH plus isoflurane postconditioning with dobutamine. Severity of SAH was graded from 1 to 4 (mild, 1-2; severe, 3-4) based on T2*-weighted magnetic resonance imaging (MRI). Cardiac output (CO) measured by transthoracic pulsed wave Doppler-echocardiography was titrated at a supra-normal level with intravenous dobutamine infusion. Neurological function was examined daily by neurological score and Rotarod tests. DCI was analyzed 3days later by determining new infarction on diffusion-weighted MRI. In a separate experiment, mice were pretreated with hypoxia-inducible factor (HIF) inhibitor 2-methoxyestradiol (2ME2). KEY FINDINGS Clinically relevant CO depression was notable in severe SAH grade mice, in which dobutamine CO management combined with isoflurane postconditioning showed earlier and improved functional recovery than postconditioning with single isoflurane inhalation. Incidence of infarction and volumes on day 3 reduced significantly in this subgroup. All of the effects during preconditioning were attenuated by 2ME2 pretreatment. SIGNIFICANCE Isoflurane postconditioning under dobutamine cardiac support improves recovery from SAH-induced early brain injury, leading to reduced DCI resultant from severe experimental SAH. These results indicate the importance of neuro-cardiac protection, in which HIF may be acting as a critical mediator, as a promising therapeutic approach to SAH.

μ-opioid Receptor-Mediated Alterations of Allergen-Induced Immune Responses of Bronchial Lymph Node Cells in a Murine Model of Stress Asthma

BACKGROUND Psychological stress has a recognized association with asthma symptoms. Using a murine model of allergic asthma, we recently demonstrated the involvement of μ-opioid receptors (MORs) in the central nervous system in the stress-induced exacerbation of airway inflammation. However, the involvement of MORs on neurons and immunological alterations in the stress asthma model remain unclear. METHODS MOR-knockout (MORKO) mice that express MORs only on noradrenergic and adrenergic neurons (MORKO/Tg mice) were produced and characterized for stress responses. Sensitized mice inhaled antigen and were then subjected to restraint stress. After a second antigen inhalation, bronchoalveolar lavage cells were counted. Before the second inhalation, bronchial lymph node (BLN) cells and splenocytes from stressed and non-stressed mice were cultured with antigen, and cytokine levels and the proportions of T cell subsets were measured. RESULTS Stress-induced worsening of allergic airway inflammation was observed in wild-type and MORKO/Tg mice but not MORKO mice. In wild-type stressed mice, IFN-γ/IL-4 ratios in cell culture supernatants and the proportion of regulatory T cells in BLN cell populations were significantly lower than those in non-stressed mice. These differences in BLN cells were not observed between the stressed and non-stressed MORKO mice. Restraint stress had no effect on cytokine production or T cell subsets in splenocytes. CONCLUSIONS Restraint stress aggravated allergic airway inflammation in association with alterations in local immunity characterized by greater Th2-associated cytokine production and a reduced development of regulatory T cells, mediated by MORs.

Inotropic support against early brain injury improves cerebral hypoperfusion and outcomes in a murine model of subarachnoid hemorrhage.

Early brain injury/ischemia is a recent therapeutic target that contributes to triggering delayed cerebral ischemia (DCI) in the setting of subarachnoid hemorrhage (SAH). This study aimed to determine the role of dobutamine for inotropic cardiac support in improving cerebral blood flow (CBF) and outcomes after experimental SAH, mediated by hypoxia-inducible factor (HIF). Thirty-one mice were subjected to SAH by endovascular perforation, and assigned to either 2% isoflurane postconditioning performed between 1 and 2.5h after SAH induction or concomitant intravenous dobutamine infusion (15μg/kg/min) with or without HIF inhibitor 2-methoxyestradiol (2ME2) (10mg/kg) administered intraperitoneally. Neurobehavioral function was assessed daily by neurological scores and open field testing. DCI was defined 3days later by detecting a new infarction on MRI. Global CBF depression was notable early after SAH, but dobutamine showed significant improvement in CBF, lower incidence of DCI, and better recovery of neuroscores and open field test variables compared with isoflurane postconditioning (P<0.05). CBF over the entire brain on day 1 predicted DCI with a cut-off of 36.5ml/100g/min (80% specificity and 67% sensitivity), with a better area under the curve (0.83 versus 0.75) than the hemispheric CBF measured on the perforated side. The dobutamine-mediated outcomes were attenuated (P<0.05) by 2ME2 pretreatment. The data suggest that cardiac support with dobutamine improves global CBF depression induced by early brain injury, leading to reduced prevalence of DCI and better functional outcomes after experimental SAH, in which HIF may be acting as a critical mediator.

Specific regions display altered grey matter volume in μ-opioid receptor knockout mice: MRI voxel-based morphometry

μ Opioid receptor knockout (MOP‐KO) mice display several behavioural differences from wild‐type (WT) littermates including differential responses to nociceptive stimuli. Brain structural changes have been tied to behavioural alterations noted in transgenic mice with targeting of different genes. Hence, we assess the brain structure of MOP‐KO mice.

Neurocardiac protection with milrinone for restoring acute cerebral hypoperfusion and delayed ischemic injury after experimental subarachnoid hemorrhage

BACKGROUND AND PURPOSE Acute cerebral hypoperfusion following subarachnoid hemorrhage (SAH) is highly related to the pathogenesis of delayed cerebral ischemia (DCI), but the therapeutic option is poorly available. This study aimed to clarify the effect of milrinone (MIL) on cerebral blood flow (CBF) and related outcomes after experimental SAH. METHODS Twenty-seven male C57BL/6 mice were assigned to either sham surgery (SAH-sham; n=6), SAH induced by endovascular perforation (control; n=10), or SAH followed by cardiac support with intravenous MIL (n=11) performed 1.5-h after SAH induction. CBF, neurobehavioral function, occurrence of DCI were assessed by MR-continuous arterial spin labeling, daily neurological score testing, and diffusion- and T2-weighted MR images on days 1 and 3, respectively. RESULTS Initial global CBF depression was notable in mice of control and MIL groups as compared to the SAH-sham group (P<0.05). MIL raised CBF in a dose-dependent manner (P<0.001), resulted in lower incidence of DCI (P=0.008) and better recovery from neurobehavioral decline than control (P<0.001). The CBF values on day 1 predicted DCI with a cut-off of 42.5ml/100g/min (82% specificity and 83% sensitivity), which was greater in mice treated with MIL than those of control (51.7 versus 37.6ml/100g/min; P<0.001). CONCLUSION MIL improves post-SAH acute hypoperfusion that can lead to the prevention of DCI and functional worsening, acting as a neurocardiac protective agent against EBI.

Comorbid masticatory impairment delays recovery from acute cerebral ischemia and locomotor hypoactivity after subarachnoid hemorrhage in mice

Tooth loss and related changes in the functionality may lead to worse outcome of stroke patients, but the effect on hemorrhagic stroke remains unclear. This study aimed to determine the impact of impaired masticatory function on acute cerebral oxygenation and locomotor activity after experimental subarachnoid haemorrhage (SAH). Twenty C57BL/6 mice with (MC‐treated group) or without (control group) prior treatment of cutting off the upper molars were subjected to SAH by endovascular perforation. Grading of SAH and acute cerebral infarction were assessed by MR images. Brain tissue oxygen saturation (SbtO2) by photoacoustic imaging and parameters related to locomotor activity by open‐field test were analyzed serially after SAH. In all mice, global SbtO2 depression was notable immediately after SAH induction (P <.001), which recovered close to the baseline levels until day 3. However, MC‐treated mice demonstrated a prolonged relative cerebral hypoxia (<40% of the baseline SbtO2) as compared to the control (3 ± 1 vs 1 ± 1 days; P <.05). The average distance travelled on day 7 and the ratio of central‐area distance/total travelled distance by open‐field test between days 7 and 14 were significantly lower in MC‐treated mice than in the control mice (P <.05), although the occurrences of new infarction were not statistically different (P >.05). These data suggest a possible link between preceding masticatory impairment and early brain injury to deteriorate neurobehavioural function in patients after SAH.This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12874 This article is protected by copyright. All rights reserved. PROF. TATSUSHI MUTOH (Orcid ID : 0000-0001-7770-379X)

Noninvasive stroke volume variation using electrical velocimetry for predicting fluid responsiveness in dogs undergoing cardiac surgery

OBJECTIVE To evaluate the ability of a noninvasive cardiac output monitoring system with electrical velocimetry (EV) for predicting fluid responsiveness in dogs undergoing cardiac surgery. STUDY DESIGN Prospective experimental trial. ANIMALS A total of 30 adult Beagle dogs. METHODS Stroke volume (SV), stroke volume variation (SVV) and cardiac index were measured using the EV device in sevoflurane-anaesthetized, mechanically ventilated dogs undergoing thoracotomies for experimental creation of right ventricular failure. The dogs were considered fluid responsive if stroke volume (SVI; indexed to body weight), measured using pulmonary artery thermodilution, increased by 10% or more after volume loading (10 mL kg-1). Relationships of SVV, central venous pressure (CVP) and pulmonary artery occlusion pressure (PAOP) with SVI were analysed to estimate fluid responsiveness. RESULTS Better prediction of fluid responsiveness, with a significant area under the receiver operating characteristic curve, was observed for SVV (0.85±0.07; p=0.0016) in comparison with CVP (0.65±0.11; p=0.17) or PAOP (0.60±0.12; p=0.35), with a cut-off value of 13.5% (84% specificity and 73% sensitivity). CONCLUSIONS AND CLINICAL RELEVANCE SVV derived from EV is useful for identification of dogs that are likely to respond to fluids, providing valuable information on volume status under cardiothoracic anaesthesia.

MRI-based in vivo assessment of early cerebral infarction in a mouse filament perforation model of subarachnoid hemorrhage

BACKGROUND AND PURPOSE Experimental subarachnoid hemorrhage (SAH) by endovascular filament perforation method is used widely in mice, but it sometimes present acute cerebral infarctions with varied magnitude and anatomical location. This study aimed to determine the prevalence and location of the acute ischemic injury in this experimental model. METHODS Male C57BL/6 mice were subjected to SAH by endovascular perforation. Distribution of SAH was defined by T2*-weighted images within 1h after SAH. Prevalence and location of acute infarction were assessed by diffusion-weighted MR images on day 1 after the induction. RESULTS Among 72 mice successfully acquired post-SAH MR images, 29 (40%) developed acute infarction. Location of the infarcts was classified into either single infarct (ipsilateral cortex, n=12; caudate putamen, n=3; hippocampus, n=1) or multiple lesions (cortex and caudate putamen, n=6; cortex and hippocampus, n=2; cortex, hippocampus and thalamus/hypothalamus, n=3; bilateral cortex, n=2). The mortality rate within 24h was significantly higher in mice with multiple infarcts than those with single lesion (30% versus 0%; P=0.03). Distribution of the ischemic lesion positively correlated with MRI-evidenced SAH grading (r2=0.31, P=0.0002). CONCLUSION Experimental SAH immediately after the vessel perforation can induce acute cerebral infarction in varying vascular territories, resulting in increased mortality. The present model may in part, help researchers to interpret the mechanism of clinically-evidenced early multiple combined infarction.