Optimization of mouse kidney digestion protocols for single-cell applications.

Single-cell technologies such as flow cytometry and single-cell RNA sequencing have allowed for comprehensive characterization of the kidney cellulome. However, there is a disparity in the various protocols for preparing kidney single-cell suspensions. We aimed to address this limitation by characterizing kidney cellular heterogeneity using three previously published single-cell preparation protocols. Single-cell suspensions were prepared from male and female C57BL/6 kidneys using the following kidney tissue dissociation protocols: a scRNAseq protocol (P1), a multi-tissue digestion kit from Miltenyi Biotec (P2), and a protocol established in our laboratory (P3). Following dissociation, flow cytometry was used to identify known major cell types including leukocytes (myeloid and lymphoid), vascular cells (smooth muscle and endothelial), nephron epithelial cells (intercalating, principal, proximal, and distal tubule cells), podocytes, and fibroblasts. Of the protocols tested, P2 yielded significantly less leukocytes and type B intercalating cells compared with the other techniques. P1 and P3 produced similar yields for most cell types; however, endothelial and myeloid-derived cells were significantly enriched using P1. Significant sex differences were detected in only two cell types: granulocytes (increased in males) and smooth muscle cells (increased in females). Future single-cell studies that aim to enrich specific kidney cell types may benefit from this comparative analysis.NEW & NOTEWORTHY This study is the first to evaluate published single-cell suspension preparation protocols and their ability to produce high-quality cellular yields from the mouse kidney. Three single-cell digestion protocols were compared and each produced significant differences in kidney cellular heterogeneity. These findings highlight the importance of the digestion protocol when using single-cell technologies. This study may help future single-cell science research by guiding researchers to choose protocols that enrich certain cell types of interest.

Neuromodulation for the management of chronic pelvic pain syndromes: A systematic review.

BACKGROUND: Chronic pelvic pain is a burdensome condition that involves multiple medical sub-specialties and is often difficult to treat. Sacral stimulation for functional bladder disease has been well established, but little large-scale evidence exists regarding utilization of other neuromodulation techniques to treat chronic pelvic pain. Emerging evidence does suggest that neuromodulation is a promising treatment, and we aim to characterize the use and efficacy of such techniques for treating chronic pelvic pain syndromes. MATERIALS AND METHODS: A systematic review of the literature demonstrating the treatment of chronic pelvic pain syndromes with neuromodulation. Abstracts were reviewed and selected for inclusion, including case series, prospective studies, and randomized controlled trials (RCTs). Case studies and publications in abstract only were not included. The reporting for this systematic review follows Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The literature search was performed using MEDLINE, Embase, Cochrane Library, PubMed, CINAHL, and Scopus. RESULTS: A total of 50 studies were included in this review, three of which were randomized controlled trials, and the remaining were prospective and retrospective case series. The range of pelvic pain conditions treated included interstitial cystitis, peripheral neuralgia, pudendal neuralgia, gastrointestinal pain, urogenital pain, sacroiliac joint pain, and visceral chronic pelvic pain. We reported on outcomes involving pain, functionality, psychosocial improvement, and medication reduction. CONCLUSIONS: Neuromodulation is a growing treatment for various chronic pain syndromes. Peripheral nerve stimulation was the least studied form of stimulation. Posterior tibial nerve stimulation appears to offer short-term benefit, but long-term results are challenging. Sacral nerve stimulation is established for use in functional bladder syndromes and appears to offer pain improvement in these patients as well. Dorsal root ganglion stimulation and spinal cord stimulation have been used for a variety of conditions with promising results. Further studies of homogeneous patient populations are necessary before strong recommendations can be made at this time, although pooled analysis may also be impactful.

Sympathetic Nervous System and Atherosclerosis.

Atherosclerosis is characterized by the narrowing of the arterial lumen due to subendothelial lipid accumulation, with hypercholesterolemia being a major risk factor. Despite the recent advances in effective lipid-lowering therapies, atherosclerosis remains the leading cause of mortality globally, highlighting the need for additional therapeutic strategies. Accumulating evidence suggests that the sympathetic nervous system plays an important role in atherosclerosis. In this article, we reviewed the sympathetic innervation in the vasculature, norepinephrine synthesis and metabolism, sympathetic activity measurement, and common signaling pathways of sympathetic activation. The focus of this paper was to review the effectiveness of pharmacological antagonists or agonists of adrenoceptors (α1, α2, ß1, ß2, and ß3) and renal denervation on atherosclerosis. All five types of adrenoceptors are present in arterial blood vessels. α1 blockers inhibit atherosclerosis but increase the risk of heart failure while α2 agonism may protect against atherosclerosis and newer generations of ß blockers and ß3 agonists are promising therapies against atherosclerosis; however, new randomized controlled trials are warranted to investigate the effectiveness of these therapies in atherosclerosis inhibition and cardiovascular risk reduction in the future. The role of renal denervation in atherosclerosis inhibition in humans is yet to be established.

AIM2 inflammasome mediates hallmark neuropathological alterations and cognitive impairment in a mouse model of vascular dementia.

Chronic cerebral hypoperfusion is associated with vascular dementia (VaD). Cerebral hypoperfusion may initiate complex molecular and cellular inflammatory pathways that contribute to long-term cognitive impairment and memory loss. Here we used a bilateral common carotid artery stenosis (BCAS) mouse model of VaD to investigate its effect on the innate immune response-particularly the inflammasome signaling pathway. Comprehensive analyses revealed that chronic cerebral hypoperfusion induces a complex temporal expression and activation of inflammasome components and their downstream products (IL-1ß and IL-18) in different brain regions, and promotes activation of apoptotic and pyroptotic cell death pathways. Polarized glial-cell activation, white-matter lesion formation and hippocampal neuronal loss also occurred in a spatiotemporal manner. Moreover, in AIM2 knockout mice we observed attenuated inflammasome-mediated production of proinflammatory cytokines, apoptosis, and pyroptosis, as well as resistance to chronic microglial activation, myelin breakdown, hippocampal neuronal loss, and behavioral and cognitive deficits following BCAS. Hence, we have demonstrated that activation of the AIM2 inflammasome substantially contributes to the pathophysiology of chronic cerebral hypoperfusion-induced brain injury and may therefore represent a promising therapeutic target for attenuating cognitive impairment in VaD.

Large-Scale Multivariate Analysis to Interrogate an Animal Model of Stroke: Novel Insights Into Poststroke Pathology.

Background and Purpose: Preclinical stroke studies endeavor to model the pathophysiology of clinical stroke, assessing a range of parameters of injury and impairment. However, poststroke pathology is complex and variable, and associations between diverse parameters may be difficult to identify within the usual small study designs that focus on infarct size. Methods: We have performed a retrospective large-scale big data analysis of records from 631 C57BL/6 mice of either sex in which the middle cerebral artery was occluded by 1 of 5 surgeons either transiently for 1 hour followed by 23-hour reperfusion (transient middle cerebral artery occlusion [MCAO]; n=435) or permanently for 24 hours without reperfusion (permanent MCAO; n=196). Analyses included a multivariate linear mixed model with random intercept for different surgeons as a random effect to reduce type I and type II errors and a generalized ordinal regression model for ordinal data when random effects are low. Results: Analyses indicated that brain edema volume was associated with infarct volume at 24 hours (ß, 0.52 [95% CI, 0.45­0.59]) and was higher after permanent MCAO than after transient MCAO (P<0.05). A more severe clinical score was associated with a greater infarct volume but not with the animal's age or edema volume. Further, a more severe clinical score was observed for a given brain infarct volume after transient MCAO versus permanent MCAO. Remarkably the animal's age, which corresponded with the period of young adulthood (6­40 weeks; equivalent to ≈18­35 years in humans), was positively associated with severity of lung infection (ß, 0.65 [95% CI, 0.42­0.88]) and negatively with spleen weight (ß, −0.36 [95% CI, −0.63 to −0.09]). Conclusions: Large-scale analysis of preclinical stroke data can provide researchers in our field with insight into relationships between variables not possible if individual studies are analyzed in isolation and has identified hypotheses for future study.

Behavioral, axonal, and proteomic alterations following repeated mild traumatic brain injury: Novel insights using a clinically relevant rat model.

A history of mild traumatic brain injury (mTBI) is linked to a number of chronic neurological conditions, however there is still much unknown about the underlying mechanisms. To provide new insights, this study used a clinically relevant model of repeated mTBI in rats to characterize the acute and chronic neuropathological and neurobehavioral consequences of these injuries. Rats were given four sham-injuries or four mTBIs and allocated to 7-day or 3.5-months post-injury recovery groups. Behavioral analysis assessed sensorimotor function, locomotion, anxiety, and spatial memory. Neuropathological analysis included serum quantification of neurofilament light (NfL), mass spectrometry of the hippocampal proteome, and ex vivo magnetic resonance imaging (MRI). Repeated mTBI rats had evidence of acute cognitive deficits and prolonged sensorimotor impairments. Serum NfL was elevated at 7 days post injury, with levels correlating with sensorimotor deficits; however, no NfL differences were observed at 3.5 months. Several hippocampal proteins were altered by repeated mTBI, including those associated with energy metabolism, neuroinflammation, and impaired neurogenic capacity. Diffusion MRI analysis at 3.5 months found widespread reductions in white matter integrity. Taken together, these findings provide novel insights into the nature and progression of repeated mTBI neuropathology that may underlie lingering or chronic neurobehavioral deficits.

Celiac plexus block for chronic flank pain: a case series.

Non-obstructive, chronic flank pain in urologic patients can be a challenging problem to manage. In this series, we examined the efficacy of celiac plexus blockade in providing pain relief and reducing opiate use in 14 adult urology patients with non-obstructive flank pain for > 1 year. Demographic, clinical, and procedural variables were collected from the medical record for retrospective analysis. Subjective improvement in pain occurred in 11 individuals (79%), and 5 (50%) were able to reduce their daily morphine equivalent dose (MED). Celiac plexus blockade is a viable option for symptomatic relief in urologic patients with non-obstructive chronic flank pain.

Transcriptome analysis reveals intermittent fasting-induced genetic changes in ischemic stroke.

Genetic changes due to dietary intervention in the form of either calorie restriction (CR) or intermittent fasting (IF) are not reported in detail until now. However, it is well established that both CR and IF extend the lifespan and protect against neurodegenerative diseases and stroke. The current research aims were first to describe the transcriptomic changes in brains of IF mice and, second, to determine whether IF induces extensive transcriptomic changes following ischemic stroke to protect the brain from injury. Mice were randomly assigned to ad libitum feeding (AL), 12 (IF12) or 16 (IF16) h daily fasting. Each diet group was then subjected to sham surgery or middle cerebral artery occlusion and consecutive reperfusion. Mid-coronal sections of ipsilateral cerebral tissue were harvested at the end of the 1 h ischemic period or at 3, 12, 24 or 72 h of reperfusion, and genome-wide mRNA expression was quantified by RNA sequencing. The cerebral transcriptome of mice in AL group exhibited robust, sustained up-regulation of detrimental genetic pathways under ischemic stroke, but activation of these pathways was suppressed in IF16 group. Interestingly, the cerebral transcriptome of AL mice was largely unchanged during the 1 h of ischemia, whereas mice in IF16 group exhibited extensive up-regulation of genetic pathways involved in neuroplasticity and down-regulation of protein synthesis. Our data provide a genetic molecular framework for understanding how IF protects brain cells against damage caused by ischemic stroke, and reveal cellular signaling and bioenergetic pathways to target in the development of clinical interventions.

Maintaining High-Quality Multidisciplinary Pain Medicine Fellowship Programs: Part I: Innovations in Pain Fellows' Education, Research, Applicant Selection Process, Wellness, and ACGME Implementation During the COVID-19 Pandemic.

BACKGROUND: Pain fellowship programs are facing unique challenges during the COVID-19 pandemic. Restrictions from state governments and the Centers for Disease Control and Prevention have resulted in a rapidly changing and evolving learning environment for todays' fellows. Innovative solutions must be sought to guarantee that proper education is maintained and to ensure the well-being of our trainees. METHODS: We assembled a panel of pain program directors who serve as officers/board members of the Association of Pain Program Directors to provide guidance and formulate recommendations to pain fellowship directors nationally. This guidance is based on reviewing current changes to the Accreditation Council for Graduate Medical Education (ACGME) and American Board of Anesthesiology policies and best available evidence and expert opinion on the use of remote educational activities, research endeavors, and trainee wellness. CONCLUSIONS: The country is in the midst of an unprecedented pandemic. The impact on pain management fellowships has been severe and will likely last for months, resulting in extraordinary challenges to the administration of pain fellowship programs and the education of our fellows. Understanding revisions to ACGME policies, using technology to promote remote learning opportunities, and providing trainees with opportunities to alleviate their anxiety and encourage mental health are beneficial strategies to implement. Together, we can implement innovative solutions to help overcome these challenges.

Maintaining High-Quality Multidisciplinary Pain Medicine Fellowship Programs: Part II: Innovations in Clinical Care Workflow, Clinical Supervision, Job Satisfaction, and Postgraduation Mentorship for Pain Fellows During the COVID-19 Pandemic.

BACKGROUND: Pain fellowship programs are facing unique challenges during the COVID-19 pandemic. Restrictions by state governments and the Centers for Disease Control and Prevention have resulted in a rapidly changing and evolving learning environment for today's fellows. Innovative solutions must be sought to maintain proper education and ensure the well-being of our trainees. METHODS: We assembled a panel of pain program directors who serve as officers/board members of the Association of Pain Program Directors, offering guidance and recommendations to pain fellowship directors nationwide. Panel members evaluate the best available evidence and expert opinion on use of remote and virtual platforms in clinical care, adaptability to alterations in clinic and referral management, and provide guidance on postgraduate impact. CONCLUSIONS: The country is in the midst of an unprecedented pandemic. The impact on pain management fellowships has been significant and will likely last for months, resulting in extraordinary challenges to the administration of pain fellowship programs and the education of our fellows. Several strategies will help address these challenges, including employing telehealth capabilities to continue clinical experiences and providing trainees with opportunities to continue their professional growth beyond fellowship completion. Together, we can implement innovative solutions to overcome these challenges.

Aged rats have an altered immune response and worse outcomes after traumatic brain injury.

Initial studies suggest that increased age is associated with worse outcomes after traumatic brain injury (TBI), though the pathophysiological mechanisms responsible for this remain unclear. Immunosenescence (i.e., dysregulation of the immune system due to aging) may play a significant role in influencing TBI outcomes. This study therefore examined neurological outcomes and immune response in young-adult (i.e., 10 weeks old) compared to middle-aged (i.e., 1 year old) rats following a TBI (i.e., fluid percussion) or sham-injury. Rats were euthanized at either 24 h or one-week post-injury to analyze immune cell populations in the brain and periphery via flow cytometry, as well as telomere length (i.e., a biomarker of neurological health). Behavioral testing, as well as volumetric and diffusion-weighted MRI, were also performed in the one-week recovery rats to assess for functional deficits and brain damage. Middle-aged rats had worse sensorimotor deficits and shorter telomeres after TBI compared to young rats. Both aging and TBI independently worsened cognitive function and cortical volume. These changes occurred in the presence of fewer total leukocytes, fewer infiltrating myeloid cells, and fewer microglia in the brains of middle-aged TBI rats compared to young rats. These findings indicate that middle-aged rats have worse sensorimotor deficits and shorter telomeres after TBI than young rats, and this may be related to an altered neuroimmune response. Although further studies are required, these findings have important implications for understanding the pathophysiology and optimal treatment strategies in TBI patients across the life span.

Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke.

Stroke is the second leading cause of death in the world and a major cause of long-term disability. Recent evidence has provided insight into a newly described inflammatory mechanism that contributes to neuronal and glial cell death, and impaired neurological outcome following ischemic stroke - a form of sterile inflammation involving innate immune complexes termed inflammasomes. It has been established that inflammasome activation following ischemic stroke contributes to neuronal cell death, but little is known about inflammasome function and cell death in activated microglial cells following cerebral ischemia. Microglia are considered the resident immune cells that function as the primary immune defense in the brain. This study has comprehensively investigated the expression and activation of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in isolates of microglial cells subjected to simulated ischemic conditions and in the brain following ischemic stroke. Immunoblot analysis from culture media indicated microglial cells release inflammasome components and inflammasome activation-dependent pro-inflammatory cytokines following ischemic conditions. In addition, a functional role for NLRC4 inflammasomes was determined using siRNA knockdown of NLRC4 and pharmacological inhibitors of caspase-1 and -8 to target apoptotic and pyroptotic cell death in BV2 microglial cells under ischemic conditions. In summary, the present study provides evidence that the NLRC4 inflammasome complex mediates the inflammatory response, as well as apoptotic and pyroptotic cell death in microglial cells under in vitro and in vivo ischemic conditions.

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

BACKGROUND AND PURPOSE: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. METHODS: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. RESULTS: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. CONCLUSIONS: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

Treatment with an interleukin-1 receptor antagonist mitigates neuroinflammation and brain damage after polytrauma.

Traumatic brain injury (TBI) and long bone fracture are common in polytrauma. This injury combination in mice results in elevated levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) and exacerbated neuropathology when compared to isolated-TBI. Here we examined the effect of treatment with an IL-1 receptor antagonist (IL-1ra) in mice given a TBI and a concomitant tibial fracture (i.e., polytrauma). Adult male C57BL/6 mice were given sham-injuries or polytrauma and treated with saline-vehicle or IL-1ra (100mg/kg). Treatments were subcutaneously injected at 1, 6, and 24h, and then once daily for one week post-injury. 7-8 mice/group were euthanized at 48h post-injury. 12-16 mice/group underwent behavioral testing at 12weeks post-injury and MRI at 14weeks post-injury before being euthanized at 16weeks post-injury. At 48h post-injury, markers for activated microglia and astrocytes, as well as neutrophils and edema, were decreased in polytrauma mice treated with IL-1ra compared to polytrauma mice treated with vehicle. At 14weeks post-injury, MRI analysis demonstrated that IL-1ra treatment after polytrauma reduced volumetric loss in the injured cortex and mitigated track-weighted MRI markers for axonal injury. As IL-1ra (Anakinra) is approved for human use, it may represent a promising therapy in polytrauma cases involving TBI and fracture.

Advanced atherosclerosis is associated with inflammation, vascular dysfunction and oxidative stress, but not hypertension.

Although hypertension may involve underlying inflammation, it is unknown whether advanced atherosclerosis - a chronic inflammatory condition - can by itself promote hypertension. We thus tested if advanced atherosclerosis in chronically hypercholesterolemic mice is associated with systemic and end-organ inflammation, vascular dysfunction and oxidative stress, and whether blood pressure is higher than in control mice. Male ApoE-/- and wild-type (C57Bl6J) mice were placed on a high fat or chow diet, respectively, from 5 to 61 weeks of age. Expression of several cytokines (including IL-6, TNF-α, IFN-γ and/or IL-1ß) was elevated in plasma, brain, and aorta of ApoE-/- mice. Aortic superoxide production was ∼3.5-fold greater, and endothelium-dependent relaxation was markedly reduced in aorta and mesenteric artery of ApoE-/- versus wild-type mice. There was no difference in blood pressure of aged ApoE-/- (104±3mmHg, n=13) and wild-type mice (113±1mmHg, n=18). To clarify any effects of aging alone, findings from 61 week-old wild-type mice were compared with those from young (8-12 weeks old) chow-fed wild-type mice. The data indicate that aging alone increased renal and aortic expression of numerous cytokines (including CCL2, CCL7 and IL-1ß). Aging had no effect on blood pressure, systemic inflammation, oxidative stress or endothelial function. Despite systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not necessarily result in elevated blood pressure.

The opposing roles of NO and oxidative stress in cardiovascular disease.

Nitric oxide (NO) plays a pivotal role in the maintenance of cardiovascular homeostasis. A reduction in the bioavailability of endogenous NO, manifest as a decrease in the production and/or impaired signaling, is associated with many cardiovascular diseases including hypertension, atherosclerosis, stroke and heart failure. There is substantial evidence that reactive oxygen species (ROS), generated predominantly from NADPH oxidases (Nox), are responsible for the reduced NO bioavailability in vascular and cardiac pathologies. ROS can compromise NO function via a direct inactivation of NO, together with a reduction in NO synthesis and oxidation of its receptor, soluble guanylyl cyclase. Whilst nitrovasodilators are administered to compensate for the ROS-mediated loss in NO bioactivity, their clinical utility is limited due to the development of tolerance and resistance and systemic hypotension. Moreover, efforts to directly scavenge ROS with antioxidants has had limited clinical efficacy. This review outlines the therapeutic utility of NO-based therapeutics in cardiovascular diseases and describes the source and impact of ROS in these pathologies, with particular focus on the interaction with NO. Future therapeutic approaches in the treatment of cardiovascular diseases are highlighted with a focus on nitroxyl (HNO) donors as an alternative to traditional NO donors and the development of novel Nox inhibitors.

Anakinra reduces blood pressure and renal fibrosis in one kidney/DOCA/salt-induced hypertension.

OBJECTIVE: To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. METHODS: Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. RESULTS: By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ∼20mmHg (n=16, P<0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced (∼30%) renal expression of some (CCL5, CCL2; n=7-8; P<0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P>0.05). Anakinra reduced renal collagen content (n=6, P<0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P<0.001) that accompanied 1K/DOCA/salt-induced hypertension. CONCLUSION: Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain.

Pin1 promotes neuronal death in stroke by stabilizing Notch intracellular domain.

OBJECTIVE: Stroke is a leading cause of mortality and disability. The peptidyl-prolyl cis/trans isomerase Pin1 regulates factors involved in cell growth. Recent evidence has shown that Pin1 plays a major role in apoptosis. However, the role of Pin1 in ischemic stroke remains to be investigated. METHODS: We used Pin1 overexpression and knockdown to manipulate Pin1 expression and explore the effects of Pin1 in cell death on ischemic stress in vitro and in a mouse stroke model. We also used Pin 1 inhibitor, γ-secretase inhibitor, Notch1 intracellular domain (NICD1)-deleted mutant cells, and Pin1 mutant cells to investigate the underlying mechanisms of Pin1-NICD1-mediated cell death. RESULTS: Our findings indicate that Pin1 facilitates NICD1 stability and its proapoptotic function following ischemic stroke. Thus, overexpression of Pin1 increased NICD1 levels and enhanced its potentiation of neuronal death in simulated ischemia. By contrast, depletion or knockout of Pin1 reduced the NICD1 level, which in turn desensitized neurons to ischemic conditions. Pin1 interacted with NICD1 and increased its stability by inhibiting FBW7-induced polyubiquitination. We also demonstrate that Pin1 and NICD1 levels increase following stroke. Pin1 heterozygous (+/-) and knockout (-/-) mice, and also wild-type mice treated with an inhibitor of Pin1, each showed reduced brain damage and improved functional outcomes in a model of focal ischemic stroke. INTERPRETATION: These results suggest that Pin1 contributes to the pathogenesis of ischemic stroke by promoting Notch signaling, and that inhibition of Pin1 is a novel approach for treating ischemic stroke.

Ghrelin-related peptides do not modulate vasodilator nitric oxide production or superoxide levels in mouse systemic arteries.

The ghrelin gene is expressed in the stomach where it ultimately encodes up to three peptides, namely, acylated ghrelin, des-acylated ghrelin and obestatin, which all have neuroendocrine roles. Recently, the authors' reported that these peptides have important physiological roles in positively regulating vasodilator nitric oxide (NO) production in the cerebral circulation, and may normally suppress superoxide production by the pro-oxidant enzyme, Nox2-NADPH oxidase. To date, the majority of studies using exogenous peptides infer that they may have similar roles in the systemic circulation. Therefore, this study examined whether exogenous and endogenous ghrelin-related peptides modulate NO production and superoxide levels in mouse mesenteric arteries and/or thoracic aorta. Using wire myography, it was found that application of exogenous acylated ghrelin, des-acylated ghrelin or obestatin to mouse thoracic aorta or mesenteric arteries failed to elicit a vasorelaxation response, whereas all three peptides elicited vasorelaxation responses of rat thoracic aorta. Also, none of the peptides modulated mouse aortic superoxide levels as measured by L-012-enhanced chemiluminescence. Next, it was found that NO bioactivity and superoxide levels were unaffected in the thoracic aorta from ghrelin-deficient mice when compared with wild-type mice. Lastly, using novel GHSR-eGFP reporter mice in combination with double-labelled immunofluorescence, no evidence was found for the growth hormone secretagogue receptor (GHSR1a) in the throracic aorta, which is the only functional ghrelin receptor identified to date. Collectively these findings demonstrate that, in contrast to systemic vessels of other species (e.g. rat and human) and mouse cerebral vessels, ghrelin-related peptides do not modulate vasodilator NO production or superoxide levels in mouse systemic arteries.