Remote ischemic postconditioning for neuroprotection after newborn hypoxia-ischemia: systematic review of preclinical studies.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a major contributor to death and disability worldwide. Remote ischemic postconditioning (RIPC) may offer neuroprotection but has only been tested in preclinical models. Various preclinical models with different assessments of outcomes complicate interpretation. The objective of this systematic review was to determine the neuroprotective effect of RIPC in animal models of HIE. METHODS: The protocol was preregistered at The International Prospective Register of Systematic Reviews (PROSPERO) (CRD42020205944). Literature was searched in PubMed, Embase, and Web of Science (April 2020). A formal meta-analysis was impossible due to heterogeneity and a descriptive synthesis was performed. RESULTS: Thirty-two papers were screened, and five papers were included in the analysis. These included three piglet studies and two rat studies. A broad range of outcome measures was assessed, with inconsistent results. RIPC improved brain lactate/N-acetylaspartate ratios in two piglet studies, suggesting a limited metabolic effect, while most other outcomes assessed were equally likely to improve or not. CONCLUSIONS: There is a lack of evidence to evaluate the neuroprotective effect of RIPC in HIE. Additional studies should aim to standardize methodology and outcome acquisition focusing on clinically relevant outcomes. Future studies should address the optimal timing and duration of RIPC and the combination with therapeutic hypothermia. IMPACT: This systematic review summarizes five preclinical studies that reported inconsistent effects of RIPC as a neuroprotective intervention after hypoxia-ischemia. The heterogeneity of hypoxia-ischemia animal models employed, mode of postconditioning, and diverse outcomes assessed at varying times means the key message is that no clear conclusions on effect can be drawn. This review highlights the need for future studies to be designed with standardized methodology and common clinically relevant outcomes in models with documented translatability to the human condition.

The evidence for the physiological effects of lactate on the cerebral microcirculation: a systematic review.

Lactate's role in the brain is understood as a contributor to brain energy metabolism, but it may also regulate the cerebral microcirculation. The purpose of this systematic review was to evaluate evidence of lactate as a physiological effector within the normal cerebral microcirculation in reports ranging from in vitro experiments to in vivo studies in animals and humans. Following pre-registration of a review protocol, we systematically searched the PubMed, EMBASE, and Cochrane databases for literature covering themes of 'lactate', 'the brain', and 'microcirculation'. Abstracts were screened, and data extracted independently by two individuals. We excluded studies evaluating lactate in disease models. Twenty-eight papers were identified, 18 of which were in vivo animal experiments (65%), four on human studies (14%), and six on in vitro or ex vivo experiments (21%). Approximately half of the papers identified lactate as an augmenter of the hyperemic response to functional activation by a visual stimulus or as an instigator of hyperemia in a dose-dependent manner, without external stimulation. The mechanisms are likely to be coupled to NAD+ /NADH redox state influencing the production of nitric oxide. Unfortunately, only 38% of these studies demonstrated any control for bias, which makes reliable generalizations of the conclusions insecure. This systematic review identifies that lactate may act as a dose-dependent regulator of cerebral microcirculation by augmenting the hyperemic response to functional activation below 5 mmol/kg, and by initiating a hyperemic response above 5 mmol/kg. OPEN SCIENCE BADGES: This article has received a badge for *Pre-registration* because it made the data publicly available. The data can be accessed at www.radboudumc.nl/getmedia/53625326-d1df-432c-980f-27c7c80d1a90/THollyer_lactate_protocol.aspx. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Reporting of 3Rs Approaches in Preclinical Animal Experimental Studies-A Nationwide Study.

The 3Rs aim to refine animal welfare, reduce animal numbers, and replace animal experiments. Investigations disclose that researchers are positive towards 3Rs recommendations from peers. Communication of 3Rs approaches via primary preclinical animal experimental literature may become a fast-forward extension to learn relevant 3Rs approaches if such are reported. This study investigates 3Rs-reporting in peer-reviewed preclinical animal research with at least one author affiliated to a Danish university. Using a systematic search and random sampling, we included 500 studies from 2009 and 2018. Reporting was low and improvement over time limited. A word search for 3R retrieved zero results in 2009 and 3.2% in 2018. Reporting on 3Rs-related sentences increased from 6.4% in 2009 to 18.4% in 2018, "reduction" increased from 2.4% to 8.0%, and "refinement" from 5.2% to 14.4%. Replacement was not reported. Reporting of the methodology was missing. For "reduction", methodology was mentioned in one study in 2009 and 11 studies in 2018, and for "refinement" in 9 and 21, respectively. Twenty-one studies stated compliance with ARRIVE-guidelines or similar without disclosure of details. Reporting of 3Rs approaches in preclinical publications is currently insufficient to guide researchers. Other strategies, e.g., education, interdisciplinary collaboration, and 3Rs funding initiatives, are needed.

Reporting quality in preclinical animal experimental research in 2009 and 2018: A nationwide systematic investigation.

Lack of translation and irreproducibility challenge preclinical animal research. Insufficient reporting methodologies to safeguard study quality is part of the reason. This nationwide study investigates the reporting prevalence of these methodologies and scrutinizes the reported information's level of detail. Publications were from two time periods to convey any reporting progress and had at least one author affiliated to a Danish University. We retrieved all relevant animal experimental studies using a predefined research protocol and a systematic search. A random sampling of 250 studies from 2009 and 2018 led to 500 publications in total. Reporting of measures known to impact study results estimates were assessed. Part I discloses a simplified two-level scoring "yes/no" to identify the presence of reporting. Part II demonstrates an additional three-level scoring to analyze the reported information's level of detail. Overall reporting prevalence is low, although minor improvements are noted. Reporting of randomization increased from 24.0% in 2009 to 40.8% in 2018, blinded experiment conduct from 2.4% to 4.4%, blinded outcome assessment from 23.6% to 38.0%, and sample size calculation from 3.2% to 14.0%. Poor reporting of details is striking with reporting of the random allocation method to groups being only 1.2% in 2009 and 6.0% in 2018. Reporting of sample size calculation method was 2.4% in 2009 and 7.6% in 2018. Only conflict-of-interest statements reporting increased from 37.6% in 2009 to 90.4%. Measures safeguarding study quality are poorly reported in publications affiliated with Danish research institutions. Only a modest improvement was noted during the period 2009-2018, and the lack of details urgently prompts institutional strategies to accelerate this. We suggest thorough teaching in designing, conducting and reporting animal studies. Education in systematic review methodology should be implemented in this training and will increase motivation and behavior working towards quality improvements in science.

Transition of Farm Pigs to Research Pigs using a Designated Checklist followed by Initiation of Clicker Training - a Refinement Initiative.

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in experiments. We improved the checklist during the development. By using the checklist each individual pig's progress was documented. This aids the caretakers so they remain focused on the actions necessary to socialize the animals and limit the animal's stress and anxiety level. During this phase, the pigs become calm and ready to commence additional clicker training to further limit stress during the experimental period. The pigs will readily allow basic outcome measures to be collected without sedation or fixation. The protocol is divided in two. First the transition phase that begins as the pigs arrive at the research facility. Introducing treats and gradually closer contact to the caretakers are the first steps. Then, the caretaker introduces actions to be able to touch the head and hind, to mimic e.g. manipulating the ear to take a blood sample or the hind to obtain a rectal temperature. A strong foundation is established in the animal-caretaker relationship and this swiftly minimizes the stress the pigs express after arrival. Secondly, the positive reinforcement training commences to teach the animal how to carry out more complex tasks without becoming stressed. A clicker is introduced as a secondary reinforcer. Time, structure, education and thorough communication seem to be the most important criteria to succeed in the application of this protocol. In addition to having calm and compliant pigs, the caretakers report that they enjoy the focus they are allowed to give each individual pig, adding to their overall job satisfaction. Having caretakers with a strong commitment, and a focus on the transition progress and welfare of each pig, additionally strengthens the department's Culture of Care.

High doses of ANP and BNP exacerbate lipolysis in humans and the lipolytic effect of BNP is associated with cardiac triglyceride content in pigs.

Drugs facilitating the cardioprotective effects of natriuretic peptides are introduced in heart failure treatment. ANP and BNP also stimulate lipolysis and increase circulating concentrations of free fatty acids (FFAs); an aspect, however, thought to be confined to primates. We examined the lipolytic effect of natriuretic peptide infusion in healthy young men and evaluated the effect in a porcine model of myocardial ischemia and reperfusion. Six young healthy normotensive men underwent infusion with ANP, BNP, or CNP for 20 min. Blood samples were collected before, during, and after infusion for measurement of FFAs. In a porcine model of myocardial ischemia and reperfusion, animals were infused for 3 h with either BNP (n = 7) or saline (n = 5). Blood samples were collected throughout the infusion period, and cardiac tissue was obtained after infusion for lipid analysis. In humans, ANP infusion dose-dependently increased the FFA concentration in plasma 2.5-10-fold (baseline vs. 0.05 µg/kg/min P < 0.002) and with BNP 1.6-3.5-fold (P = 0.001, baseline vs. 0.02 µg/kg/min) 30 min after initiation of infusion. Infusion of CNP did not affect plasma FFA. In pigs, BNP infusion induced a 3.5-fold increase in plasma FFA (P < 0.0001), which remained elevated throughout the infusion period. Triglyceride content in porcine right cardiac ventricle tissue increased ∼5.5 fold in animals infused with BNP (P = 0.02). Natriuretic peptide infusion has similar lipolytic activity in human and pig. Our data suggest that short-term infusion increases the cardiac lipid content, and that the pig is a suitable model for studies of long-term effects mediated by natriuretic peptides.

Natriuretic peptides in cardiometabolic regulation and disease.

In the 30 years since the identification of the natriuretic peptides, their involvement in regulating fluid and blood pressure has become firmly established. Data indicating a role for these hormones in lifestyle-related metabolic and cardiovascular disorders have also accumulated over the past decade. Dysregulation of the natriuretic peptide system has been associated with obesity, glucose intolerance, type 2 diabetes mellitus, and essential hypertension. Moreover, the natriuretic peptides have been implicated in the protection against atherosclerosis, thrombosis, and myocardial ischaemia. All these conditions can coexist and potentially lead to heart failure, a syndrome associated with a functional natriuretic peptide deficiency despite high circulating concentrations of immunoreactive peptides. Therefore, dysregulation of the natriuretic peptide system, a 'natriuretic handicap', might be an important factor in the initiation and progression of metabolic dysfunction and its accompanying cardiovascular complications. This Review provides a summary of the natriuretic peptide system and its involvement in these cardiometabolic conditions. We propose that these peptides might have an integrating role in lifestyle-related metabolic and cardiovascular disorders.