Mesenchymal Stromal/Stem Cell Therapy Improves Salivary Flow Rate in Radiation-Induced Salivary Gland Hypofunction in Preclinical in vivo Models: A Systematic Review and Meta-Analysis.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) have been suggested for salivary gland (SG) restoration following radio-induced salivary gland damage. This study aimed to determine the safety and effectiveness of MSC therapy on radio-induced SG damage and hypofunction in preclinical in vivo studies. METHODS: PubMed and EMBASE were systematically searched for preclinical in vivo interventional studies evaluating efficacy and safety of MSC treatment following radio-induced salivary gland damage published before 10th of January 2022. The primary endpoint was salivary flow rate (SFR) evaluated in a meta-analysis. The study protocol was published and registered on PROSPERO ( www.crd.ac.uk/prospero ), registration number CRD42021227336. RESULTS: A total of 16 preclinical in vivo studies were included for qualitative analysis (858 experimental animals) and 13 in the meta-analysis (404 experimental animals). MSCs originated from bone marrow (four studies), adipose tissue (10 studies) and salivary gland tissue (two studies) and were administered intravenously (three studies), intra-glandularly (11 studies) or subcutaneously (one study). No serious adverse events were reported. The overall effect on SFR was significantly increased with a standardized mean difference (SMD) of 6.99 (95% CI: 2.55-11.42). Studies reported improvements in acinar tissue, vascular areas and paracrine factors. CONCLUSION: In conclusion, this systematic review and meta-analysis showed a significant effect of MSC therapy for restoring SG functioning and regenerating SG tissue following radiotherapy in preclinical in vivo studies without serious adverse events. MSC therapy holds significant therapeutic potential in the treatment of radio-induced xerostomia, but comprehensive, randomized, clinical trials in humans are required to ascertain their efficacy in a clinical setting.

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.

Comparison of the neuromuscular effects of two infusion rates of rocuronium in anesthetized pigs.

BACKGROUND: Neuromuscular blocking agents are frequently administered to pigs used for research. In humans, administration of the drugs is not without risk and may result in accidental awareness under general anaesthesia and postoperative residual neuromuscular blockade that can lead to serious respiratory complications. Despite the extensive administration, the pharmacodynamics of neuromuscular blocking agents are not thoroughly studied in pigs. Therefore, this study investigates the neuromuscular response of two infusion rates of rocuronium, a commonly used non-depolarizing neuromuscular blocking agent. A group of 14 female Danish Landrace-Yorkshire-Duroc pigs used for supervised surgical training, weighing 40.3 ± 2.1 kg (mean ± SD), were included in the study. They received a loading dose of 0.85 mg/kg rocuronium intravenously followed by infusion of either 2.5 mg/kg/hour (L, low dose) or 5 mg/kg/hour (H, high dose) rocuronium for 30 min. Neuromuscular monitoring was performed with acceleromyography using train-of-four (TOF) stimulation. Onset time, time to reappearance of T1, T4, TOF ratio 90% and 100% were recorded. RESULTS: All pigs in group H experienced loss of T1 throughout rocuronium infusion, whereas six out of seven pigs in group L had reappearance of T1 during rocuronium infusion, with additional reappearance of T4 in three of these pigs. The time to recovery of TOF ratio 90% was 14.0 ± 5.4 (L) and 21.7 ± 6.1 (H) minutes and recovery to TOF ratio 100% was 18.7 ± 6.5 (L) and 27.9 ± 9.2 min (H) (mean ± SD). Substantial inter-animal variation in neuromuscular recovery time was observed. CONCLUSION: The large inter-animal variation in pharmacodynamic profiles emphasizes that individual neuromuscular monitoring and titration to effect should be used routinely in research protocols that include rocuronium. In addition to other important measures, these actions are key in order to avoid overdosing and limit the risk of residual neuromuscular blockade.

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.

Mesenchymal stromal/stem cell therapy for radiation-induced salivary gland hypofunction in animal models: a protocol for a systematic review and meta-analysis.

BACKGROUND: Salivary gland (SG) hypofunction (objectively reduced saliva flow rate) and xerostomia (subjective sensation of dry mouth) are common and burdensome side effects of radiotherapy to the head and neck region. Currently, only sparse symptomatic treatment is available to ease the discomfort of xerostomia. The objective of this study is to assess the effect of mesenchymal stem cell (MSC) therapy on SG function after radiation-induced injury. METHODS: This systematic review will include animal intervention studies assessing efficacy and safety of MSCs in treating radiation-induced SG hypofunction. The primary outcome is the effect of MSC administration on salivary flow rates (SFR), by comparing treated groups to control groups when available. Secondary outcomes are morphological and immunohistochemical effects as well as safety of MSC treatment. Electronic searches in MEDLINE (PubMed) and Embase databases will be constructed and validated according to the peer review of electronic search strategies (PRESS) and assessed by two independent researchers. Data from eligible studies will be extracted, pooled, and analyzed using random-effects models. Risk of bias will be evaluated with the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool. DISCUSSION: Thus far, critical appraisal of MSC therapy as an effective treatment for SG hypofunction caused solely by radiation injury has not been conducted. A summary of the existing literature on preclinical studies concerning this issue can provide valuable information about effectiveness, mode of action, and safety, allowing further optimization of preclinical and clinical trials. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42021227336.

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.

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.

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/.

A Preclinical Study of Casein Glycomacropeptide as a Dietary Intervention for Acute Mania.

Background: Casein glycomacropeptide is a peptide that lacks phenylalanine, tyrosine, and tryptophan. This profile may enable it to deplete phenylalanine, tyrosine, and tryptophan, and subsequently the synthesis of dopamine and serotonin in the brain. Dopamine- and serotonin-depleting amino acid mixtures have shown promise as acute antimanic treatments. In this study, we explore the depleting effects on amino acids, dopamine and serotonin as well as its actions on manic-like and other behavior in rats. Methods: Casein glycomacropeptide and a selection of amino acid mixtures were administered orally at 2, 4, or 8 h or for 1 week chronically. Amino acid and monoamine levels were measured in plasma and brain and behavior was assessed in the amphetamine-hyperlocomotion, forced swim, prepulse inhibition, and elevated plus maze tests. Results: Casein glycomacropeptide induced a time-dependent reduction in tyrosine, tryptophan, and phenylalanine in brain and plasma which was augmented by supplementing with leucine. Casein glycomacropeptide +leucine reduced dopamine in the frontal cortex and serotonin in the hippocampus, frontal cortex, and striatum after 2 and 4 h. Casein glycomacropeptide+leucine also had antimanic activity in the amphetamine-induced hyperlocomotion test at 2 h after a single acute treatment and after 1 week of chronic treatment. Conclusions: Casein glycomacropeptide-based treatments and a branched-chain amino acid mixture affected total tissue levels of dopamine in the frontal cortex and striatum and serotonin in the frontal cortex, striatum, and hippocampus of rats in a time-dependent fashion and displayed antimanic efficacy in a behavioral assay of mania.

Systematic evaluation of skeletal fractures caused by induction of electroconvulsive seizures in rat state a need for attention and refinement of the procedure.

OBJECTIVE: Electroconvulsive therapy (ECT) is one of the most efficient treatments for major depression. Electroconvulsive seizures (ECS), the animal model of ECT, is widely used to study both mechanisms of action and adverse effects of ECT. As the treatment itself serves as an instant anaesthetic and anaesthetic agents may affect memory functions and behaviour, ECS is traditionally administered without muscle relaxation and anaesthesia. A major problem of unmodified ECS, which has only been addressed peripherally in the literature, is that some animals sustain spinal fractures and subsequent hind leg paralysis (paraplegia). This phenomenon leads to a higher degree of suffering and these animals need to be excluded from the studies. To reach sufficient statistical power, the group sizes are therefore often increased and this may lead to a pre-selected study group in risk of skewing the results. Moreover, the study design of the experiments do not comply with the 3R principles, which advocate for both refinement and reduction of animal experiments. The objective of this study is to systematically evaluate injuries caused by ECS. METHODS: We summarise the incidence of spinal fractures from 24 studies conducted during 2009-2015 in six different rat strains and report preliminary findings on scapular fractures following auricular ECS. RESULTS: In total, 12.8% of all tested animals suffered from spinal fractures and we find an increase in spinal fracture incidence over time. Furthermore, X-ray analyses revealed that some animals displayed scapular fractures. CONCLUSION: We discuss consequences of and possible explanations for ECS-induced fractures. Modifications of the method are highly warranted and we furthermore suggest that all animals are thoroughly examined for discrete fractures.

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.

Increased N-terminal CgA in circulation associated with cardiac reperfusion in pigs.

AIM: Acute myocardial infarction causes neurohumoral activation characterized by increased sympathetic activity. CgA is a protein released during sympathoadrenal stress from neuroendocrine tissue. Recently, increased CgA concentrations in circulation have been reported and suggested to be an independent predictor of mortality after acute myocardial infarction. MATERIALS & METHODS: Eighteen pigs underwent 1 h of regional myocardial ischemia followed by 3 h of reperfusion. Blood samples were collected every hour and plasma CgA was measured with two radioimmunoassays. RESULTS: We found a 30% increase in plasma N-terminal CgA 1 h after re-establishment of coronary blood supply. On the other hand, plasma pancreastatin did not change in response to ischemia or reperfusion but decreased during the entire experiment. CONCLUSION: Our results suggest a differentiated CgA response in myocardial reperfusion after local cardiac anoxia that may reflect tissue-specific post-translational processing and release.

Natriuretic peptides as therapy in cardiac ischaemia/reperfusion.

Natriuretic peptides elicit vasodilation, increased sodium excretion and concomitant diuresis, and counteract the RAAS. In the heart itself, natriuretic peptides may also act anti-inflammatory and antifibrotic. This has led to the pursuit of natriuretic peptides and chemically modified peptides as adjunctive therapy in myocardial ischaemia. However, natriuretic peptide infusion may also influence the endogenous natriuretic peptide response and lipid accumulation. We hypothesised that a) natriuretic peptide infusion (BNP and CD-NP) is cardiomyocyte protective, b) affects the endogenous response, and c) facilitate cardiac lipid accumulation. We examined these effects in a minimally invasive porcine model of regional cardiac ischaemia and reperfusion. The studies were supplemented by a 48-hour porcine model of ischemia and reperfusion as well as an in vitro study of BNP administered in a HL-1 cell model of "ischaemia/reperfusion". Infarct size was determined by TTC staining, plasma troponin T release, and total RNA integrity in cardiac tissue samples. The endogenous response was assessed by a processing-independent proANP immunoassay and mRNA quantitation. Lipids in plasma and myocardial tissue were determined by TLC. The studies show that natriuretic peptides decrease cardiomyocyte damage, possibly partly through indirect mechanisms. Furthermore, BNP infusion completely inverts the endogenous response, whereas CD-NP infusion does not. Finally, both natriuretic peptides increase plasma free fatty acids, which is associated with an increased cardiac lipid accumulation in non-ischaemic myocardium. In conclusion, the studies suggest that natriuretic peptides are beneficial in terms of reduced cardiac injury. In addition, the endogenous natriuretic peptide response is inverted. The results advocate for pursuing natriuretic peptide treatment in ischaemia/reperfusion damage. However, the metabolic consequences in a cardiac tissue challenged by ischaemia should be pursued before testing the peptides in patients.