Growth Mixture Models In Longitudinal Research: Fill & Download for Free

I could but I don’t need to do so. As a research scientist, I use the scientific method to analyze data. The data are collected by sampling methods that allow for generalization from the sample to the population under consideration with high levels of confidence. The complex samples are collected as probability samples with appropriate sampling weights.The data collected allow for longitudinal statistical modeling that allows for latent cyclical, latent trend, observed seasonality, on the basis of a local level foundation in either the time domain and the frequency domain. My preference has been for Bayesian structural time series models applying the augmented Kalman Filter (following Andrew C. Harvey and Siem Jan Koopman) and their software written with Jurgen Doornik and Neil Shephard.I also have done longitudinal structural equation modeling of scientific hypotheses about relationships between static as well as dynamic panel variables. Panel analysis allows for periodization the data and comparative analysis of variables between and within different time periods. Other models include both discrete time and parametric survival and cure models as well as joint statistical models. Validation and reliability were usually conducted by repeated measures inter-rater correlation coupled with appropriate power analysis.I have recently been using Bayesian statistical analysis as well as the econometric methods available in the OxMetrics and Stata software. In the past, I have used SAS and SPSS before that.When I have done socio-medical analysis, these have included both clinical samples and national multi-site collections of clinical samples. In these studies I have applied various growth mixture models. These methods are generally accepted among practicing statisticians.

Here’s your detailed answer:BMC Vet Res. 2016; 12: 162.Published online 2016 Aug 3. doi: 10.1186/s12917-016-0786-5PMCID: PMC4973095A mixed grape and blueberry extract is safe for dogs to consumeAnne-Sophie Martineau,1 Véronique Leray,1 Anne Lepoudere,2 Géraldine Blanchard,3 Julien Bensalem,4 David Gaudout,4 Khadija Ouguerram,5 Patrick Nguyen,On behalf of Neurophenols ConsortiumAuthor information ► Article notes ► Copyright and License information ►This article has been cited by other articles in PMC.Go to:AbstractBackgroundGrape and blueberry extracts are known to protect against age-related cognitive decline. However, beneficial effects achieved by mixing grape and blueberry extracts have yet to be evaluated in dogs, or their bioavailability assessed. Of concern to us were cases of acute renal failure in dogs, after their ingestion of grapes or raisins. The European Pet Food Industry Federation (2013) considers only the grape or raisin itself to be potentially dangerous; grape-seed extracts per-se, are not considered to be a threat. Our aim was therefore to evaluate the renal and hepatic safety, and measure plasma derivatives of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) in dogs. Polyphenol expression was analyzed by UHPLC-MS/MS over 8 hours, for dogs given PEGB at 4 mg/kg. Safety was evaluated using four groups of 6 dogs. These groups received capsules containing no PEGB (control), or PEGB at 4, 20, or 40 mg/kg BW/d, for 24 weeks. Blood and urine samples were taken the week prior to study commencement, then at the end of the 24-wk study period. Routine markers of renal and liver damage, including creatinine (Creat), blood urea nitrogen, albumin, minerals, alkaline phosphatase (ALP), and alanine transaminase (ALT) were measured. Biomarkers for early renal damage were also evaluated in plasma (cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL)), and urine (CysC, clusterin (Clu), and NGAL). Ratios of urinary biomarkers to Creat were calculated, and compared with acceptable maximal values obtained for healthy dogs, as reported in the literature.ResultsWhile several PEGB-specific polyphenols and metabolites were detected in dog plasma, at the end of the PEGB consumption period, our biomarker analyses presented no evidence of either renal or liver damage (Creat, BUN, ionogram, albumin and ALT, ALP). Similarly, no indication of early renal damage could be detected. Plasma CysC, urinary CysC/Creat, Clu/Creat, and NGAL/Creat ratios were all beneath reported benchmarked maximums, with no evidence of PEGB toxicity.ConclusionsLong-term consumption of a pet specific blend of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium), was not associated with renal or hepatic injury, and can therefore be considered safe.Keywords: Dog, Neurophenols, Grape, Blueberry, Kidney, Cystatin C, Clusterin, NGAL, FlavonoidsGo to:BackgroundThis work comprises part of a project dedicated to the study of age-related cognitive decline in humans and dogs (the Neurophenols Consortium). We aim to complete a novel study into the efficacy of mixed extracts of grape and blueberry in counteracting age-related deterioration of function. In aged dogs, polyphenol ingestion (including grape pomace), and vitamin use, are both thought to ameliorate the effects of aging on learning ability [1]; similarly beneficial effects have been reported for humans [2]. A popular hypothesis is that protection against oxidative stress explains these effects. In aged mice, the consumption of a mixed grape and blueberry extract, has also been shown to improve spatial navigation; one of the skills that declines with age. In this scenario, increased expression for hippocampal nerve growth factor mRNA [3] may play a causal role [4].Studies reporting grape extract consumption in dogs (using grape seed and skin extracts, or grape seed proanthocyanidins), or grape pomace in aged dogs have, to date, not reported any side effects [1, 5, 6]. However, acute renal failure has been reported in dogs after their consumption of grapes, with kidney histopathology revealing tubular degeneration leading to necrosis, particularly in the proximal tubule [7–9]. In a retrospective study involving a cohort of 43 dogs, all of whom had eaten grapes, raisins, or both, animals presented with clinical signs consistent with kidney deterioration during a window extending from 24 h, until 5 days, after consumption. Vomiting, diarrhea, lethargy, and either olig- or, anuria, were the common clinical signs. A diagnosis of renal damage was supported by biochemical abnormalities showing higher plasma creatinine (Creat), blood urea nitrogen (BUN), an altered ionogram, glycosuria, and proteinuria. Again, histopathology revealed severe diffuse renal tubular degeneration, especially in proximal cells, with glomerular deterioration. Half of the dogs died [9]. In these cases, the precise amount of fruit eaten varied greatly (from 3 g/kg BW of raisin, to 150 g/kg BW of grape), as did the type of fruit (grape, raisin, seedless grape), and the affected breed [7–9]. Hepatic toxicity has also been associated with the consumption of plants such as greater celandine, green tea, valerian, or ayurvedic products. In these cases, higher concentrations of alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and bilirubin, were all demonstrated (reviewed in [10, 11]). Abnormal values of ALT and ALP provoked by grape consumption also point to the liver being a target of grape toxicity [9], although the factors responsible for hepatic damage, as well as the acute renal failure, have yet to be identified.The Neurophenols Consortium is a Europe-North America research collaboration dedicated to the research, and development of natural ingredients and products to prevent age-related cognitive decline in humans and pets. The Consortium brings together scientists in the fields of phytochemistry, neuroscience, psychology and nutrition with companies specialized in the development of active ingredients and food supplements. The specific aims of the program are to characterize and formulate fruit extracts from blueberry and grape, to evaluate their safety and efficacy in pre-clinical and clinical trials.The aim of this study was to assess the safety of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium). We studied the safety of this extract following chronic use in dogs, by monitoring renal and hepatic health, using early biomarkers of renal damage as well as a biochemical approach.Go to:MethodsAnimalsTwenty-four experimental Beagle dogs (4 groups of 5 males, and a single female, body condition score (BCS) 5/9, mean age 31 ± 3 months, mean body weight (BW) 11.4 ± 0.2 kg), originally from CEDS (Centre d’élevage du Domaine des Souches, Mézilles, France), were used. They were fed with a dry maintenance diet (Medium Adult Royal Canin), according to the National Research Council (NRC 2006) [12] recommendation (130 kcal metabolizable energy per kg metabolic body weight).Study designFour groups of 6 dogs (each comprising 5 males and a female) were given a polyphenol-rich extract from grape and blueberry (PEGB) for 24 weeks. The constituents of this PEGB extract were devised by the Neurophenols Consortium; these were grape (Vitis vinifera L.), and blueberry (Vaccinium angustifolium) extracts, containing specific polyphenols with low molecular weight monomers, including catechin (6 % dry matter), oligomers, flavonols (for a total of 0.15 % dry matter), anthocyanins, phenolic acids, and resveratrol formulated in a unique ratio of molecules. The intended dosage was 4 mg/kg BW/d. One group also received a control dose of 0 mg/kg BW/d (control), with two other groups receiving higher doses of the extract; 20 mg/kg BW/d, and 40 mg/kg BW/d. Each dose was given in the daily meal as a gelatin capsule (Cooper, Melun Cedex, France) containing the formulation and maltodextrin. All extracts were prepared in accordance with good laboratory practices.Plasma and urine samplesBlood and urine samples were collected in the week prior to the study commencing, and then at the end of the 24-wk period. Blood samples were obtained by jugular venipuncture into heparin tubes in 24-hour unfed animals. Each blood draw was immediately centrifuged (2124 g for 10 min at 4 °C), and the plasma fraction aliquoted and frozen at -80 °C. Twenty-four-hour urine samples were collected by voiding, following the consumption of the daily meal and the capsules. The expression of specific polyphenols, derived from the PEGB extract, were measured, on the 8th days of exposure, in plasma samples from dogs that were given PEGB at 4 mg/kg/d. For this purpose, plasma samples were taken for 8 h, with polyphenol analyses performed by UHPLC-MS/MS.Chemical analysesConcentrations of Creat, BUN, minerals (sodium (Na+), potassium (K+), calcium (Ca2+), and phosphate (PO43-), albumin, ALT, and ALP, were determined using a VetScan reagent rotor (Comprehensive Diagnostic Profile, VetScan VS2, Abaxis, Ca, USA). Biomarkers of renal damage, including CysC (cystatin C), Clu (clusterin), and NGAL (neutrophil gelatinase-associated lipocalin), were measured by species-specific ELISA (canine cystatin C, Biovendor, Czech Republic; canine clusterin, Biovendor, Czech Republic; dog NGAL, Bioporto, Denmark). Urinary Creat was assayed using an enzymatic colorimetric kit (Creatinine, Randox Laboratories, UK).Data analysesResults are reported as means ± standard error of the mean (SEM). For each early biomarker of renal damage, we compared their maximal values after PEGB consumption with previous maximal values reported for healthy dogs. As replicate datasets were collected, linear mixed-effects model analyses could be undertaken to investigate any interaction between PEGB dose, and time. Moreover, an inter-group analysis was performed using a linear model to compare data for each of the experimental groups (4, 20, and 40 mg/kg/d), with the control group, at the beginning and end of the study. Finally, an intra-group analysis was completed using a linear mixed effects model to compare data from the initiation and end of the study. These analyses were completed using the R software (R Core Team (2013)). The alpha level for determination of significance was 0.05.Go to:ResultsSpecific polyphenols in plasma following PEGB consumptionPolyphenols and their metabolites were detected in plasma samples, and their maximum concentrations (Cmax) determined. These metabolites comprised: hydroxy and dihydroxyphenyl-γ-valerolactone, both derived from flavan-3-ols; the resveratrol derivatives, reseveratrol glucuronide, dihydroresveratrol sulfate, and glucuronide; the flavonol and its metabolite, quercetin and isorhamnetine sulfate; and the anthocyanin metabolite, malvidin. A Cmax for the flavan-3-ol metabolites of 2028 nM was attained after 8 h. Flavonol metabolites reached a Cmax of 5nM, also after 8 h, with malvidin also peaking (7nM) at this timepoint. Peak concentrations for resveratrol metabolites were reached much earlier, after 30 min (Cmax 161 nM).Plasma and urine biomarkersMarkers of liver damagePlasma hepatic biomarker concentrations are shown in Table 1. All ALT and ALP concentrations were within the reference range.Table 1Plasma biomarkers of kidney damage in dogs, at the initiation (Week -1) and the end (Week 24) of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d. (Data are means ± SEM, n = 6/group; minima and ...Markers of renal damagePlasma creatinine, urea, sodium, potassium, calcium, phosphate, and albumin concentrations are shown in Table 2. All values were found to be in the reference range, i.e. the 95 % prediction interval (for a normal population).Table 2Plasma biomarkers of liver damage in dogs, at the initiation (Week -1) and the end (Week 24) of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d. (Data are means ± SEM, n = 6/group; minima and ...The concentration of early renal biomarkers, and their ratios, are presented in Table 3. For each biomarker, interaction analyses failed to identify any difference between the experimental and control groups, either at the beginning, or at the end of the study. No inter-group or intra-group variations could be noted between experimental groups, compared to controls, or between the initiation and the end of the study.Table 3Concentrations, and ratios, of early biomarkers of renal damage in dogs, at the initiation (Week -1) and the end (Week 24) of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d. (Data are means ± SEM, n = 6/group; ...In the experimental groups (PEGB given at 4, 20 or 40 mg/kg/d), the mean plasma CysC concentrations were found to be similar to control group. Mean concentrations ranged from 1.2 to 1.5 μg/mL (Fig. 1a). Intra-group analyses also showed no differences across the experimental groups. Collectively, their mean urinary CysC/Creat ratio varied from 4 to 27 μg/g. These ratios were not significantly different to those determined for the control group, for whom no intra-group difference was noted (Fig. 1b). Mean urinary Clu/Creat ratios varied between 44 and 94 ng/g in groups given PEGB at 4 to 40 mg/kg/d, again with no significant changes compared to control group nor intra-group differences between the initiation and the end of the study (Fig. 2). The mean urinary NGAL/Creat ratios were similar to control group in the experimental groups, varying between 4 and 8 ng/g. Intra-group analyses also failed to determine any significant differences between the initiation and the end of the study (Fig. 3).Fig. 1a Plasma Cystatin C concentration (μg/mL) in dogs at the initiation () and the end () of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d (n = 6 dogs per group). The line indicates the reported maximal value ...Fig. 2Urinary Clusterin/Creatinine ratio (ng/g) in dogs, at the initiation () and the end () of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d (n = 6 dogs per group). The line indicates the reported maximal value in normal ...Fig. 3Urinary NGAL/Creatinine ratio in dogs (ng/g) before at the initiation () and the end () of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d (n = 6 dogs per group). The line indicates the reported maximal value in normal ...Go to:DiscussionOur aim was to assess the safety of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) for dogs, by monitoring early biomarkers of renal damage over a 24-week period. This work considerably extends the previous study periods reported, where platelet effects, and gene expression profiles, were interrogated after 7 days, or 3 months of supplement use [5, 6].After PEGB consumption, biomarker values exceeded the reported maximal limits in no dog, with no differences observed at the end of the 24-week period, compared to beginning, for plasma CysC, and urinary CysC/Creat, Clu/Creat, or NGAL/Creat ratios. When considering these data, we conclude that the dogs neither presented with renal, nor hepatic injury, at the end of the study.While bioavailability of the Neurophenols Consortium PEGB had never been evaluated in dogs, our evaluation of the safety of this supplement necessitated measurement of PEGB derivatives in plasma. The main polyphenols in the extract were flavan-3-ols, resveratrol, anthocyanins (malvidin, petunidin, peonidin, petunidin, cyanidin), and flavonol (quercetin). Some polyphenols and polyphenol metabolites were found in plasma. Malvidin, which is present in blueberry but not in grape, has been the only anthocyan detected, but it is known that anthocyanins are less absorbed than other flavonoids. The finding of resveratrol derivatives (which are grape specific) is in accordance with a study that also showed appearance of resveratrol conjugates (sulfate & glucuronide) in the plasma of dogs after resveratrol administration [13]. The valerolactones detected resulted from the metabolization of flavan-3-ols by gut microflora. Quercetin and isorhamnetin sulfate, which are present in both fruits, were also found. Other compounds may have been absorbed, but either they have not been identified, or their concentration was under the detection threshold, or they were rapidly metabolized and excreted. Very few data on polyphenols pharmacokinetics in dogs are available. Regarding resveratrol, Cmax could not be compared since in previous report [13] it was given to dogs at much higher doses than the intended dose in the present study (200–1200 mg/kg/d, compared to 4 mg/kg/d). When anthocyanins were given to pigs at 1 to 4 % of the diet (w/w), several metabolites were measured in liver, eye and brain while there were not detected in plasma [14], and again the doses were far higher than in the present study. Catechin and epicatechin glucuronides from a grape extract given to mice were measured in plasma [15], which was not the case in our study, but the dose used was still much higher (grape-derived polyphenols: 80 mg/kg/d). When green tea catechins (13 mg/kg/d, [16], 170 mg/kg/d [17]) and epigallocatechin gallate (EGCG; 250 mg/kg/d [18]) were given to dogs, respective metabolites were found in plasma, which was not the case after PEGB consumption where only valerolactones were detected. The difference could be explained either by the catechin sources or higher doses or both. Another possible explanation is that dogs were given the PEGB at the same time of their daily meal, and the plasma measurements were done after a relatively short period of exposure. Indeed in dogs given EGCG at 300 mg/kg/d, plasma area under the curve (AUC) for EGCG was higher in unfed than fed dogs [19]. When EGCG was given at 500 mg/kg/d, authors reported, although the difference did not reach the significance level, that the AUC for EGCG was 1.6 time higher after 28 days of dosing than after 14 days [19]. The data of the present study demonstrated that the polyphenols of the PEGB extract were, at least in part, bioavailable, and this is the first report on the appearance of valerolactones as well as quercetin, isorhamnetin sulfate and malvidin in the plasma of dogs after consumption of a mixture of polyphenols.The origin of the grape toxicity described in the literature for dogs is still obscure, but numerous hypotheses have emerged. Among them, it was reported that exogenous compounds on grapes, such as mycotoxin, pesticides, or herbicide residues, could be responsible for the kidney toxicity, with histopathology indicating that the proximal cells are the primary target [8]. These findings provoked further hypotheses, such as the toxic accumulation of a foreign chemical (a xenobiotic), with a particular affinity for tubular specific transporters. Additionally, the expression of a perinuclear golden brown pigment [8], could imply its cytotoxic accumulation, with failed cellular clearance. Hypercalcemia and renal mineralization induced by the high sugar content of grapes are also current hypotheses.The resveratrol concentration in grapes could also be responsible for renal damage. A previous study described that the no-observed-adverse-effect level of resveratrol consumption was 600 mg/kg BW/d in dogs. Consumption of twice this dose (1200 mg/kg BW/d) induced a loss of appetite, and weight [20]. Given that grapes contain 1.5 to 7.8 μg of total resveratrol per gram of fresh weight [21], it is highly unlikely that resveratrol is responsible for the acute kidney injury observed in clinical cases in dogs.Plasma creatinine and urea are the most frequently measured parameters used to evaluate renal damage. High creatinine concentrations are seen when at least 75 % of renal function has already been lost [22]. In previous studies describing acute renal failure after grape consumption, symptoms appeared rapidly [9]. Therefore, we reasoned that to monitor kidney health, earlier biomarkers of renal damage would be required. In 2010, the Nephrotoxicity Working Group established a consortium between the European Medicines Agency, and the Food and Drug Administration. They listed seven biomarkers needed to detect the early development of renal injury [23]. Among these, we chose to assess CysC and Clu, because of their ease of use in dogs. In addition, NGAL was measured, as a promising early biomarker of drug-induced kidney injury. Collectively, these early biomarkers of renal damage are ideal for monitoring renal health, before irreversible damage, as they survey different renal functions, and compartments of the kidney.Ordinarily, cystatin C, which is a low molecular weight protein produced at a constant rate by all cells, is completely reabsorbed and catabolized in proximal tubular epithelial cells [24]. Following renal injury, CysC concentration increases in the plasma, as the glomerular filtration rate declines [25]; an increased concentration in urine reflects tubular impairment [26]. Plasma CysC has previously been measured in healthy dogs (urea and creatinine concentrations within reference intervals), with the highest reported values of 2 μg/ml [27]. For all dogs that had received PEGB, at any dose, plasma CysC concentrations were beneath this upper limit. To the best of our knowledge, the referenced study [27] is the only one in which plasma CysC concentrations have been measured in healthy dogs by canine ELISA. We therefore conducted the same tests, in our study. In other studies, CysC was measured in serum and/or with a different ELISA kit or technique (i.e. Particle-Enhanced Turbidimetric Immunoassay), which may explain the slightly different reference ranges reported [28–30]. In our study, the maximum urinary CysC/Creat ratio that we measured in dogs following PEGB consumption (regardless of dose) was 79 μg/g, whereas reported urinary CysC/Creat ratios have been as higher as 0.11 ± 0.02 mg/g [31]. Therefore, we conclude that our CysC results revealed no glomerular or tubular impairments.Clusterin is a high molecular weight glycoprotein expressed in epithelial cells (reviewed in [32]); in cases of acute renal failure, clusterin is found at high concentrations in the urine, indicating glomerular damage [33]. The highest urinary Clu/Creat ratio previously reported in healthy dogs was 4.87 μg/g [33]. In this study, the urinary Clu/Creat ratio measured in dogs after PEGB consumption (4 to 40 mg/kg/d) was far lower, ranging from 10 to 437 ng/g. Therefore, clusterin analyses also revealed no evidence of glomerular damage after PEGB consumption.NGAL is a protein that has raised some interest since its mRNA and protein were detected in urine after induction of acute kidney injury in rodents [34]. NGAL mRNA has been found in the ascending limb of Henle, and in collecting duct cells after ischemia-reperfusion [35]. NGAL is ordinarily reabsorbed by the proximal tubule [35, 36]. However, in case of renal injury, reabsorption may decrease, which results in higher urinary concentrations. Tubular damage and reduced filtration may also cause the accumulation of plasma NGAL [37]. The reported ranges of urinary NGAL/Creat ratio have varied greatly in healthy dogs from 10 to 460 ng/g, or from 40 to 3660 ng/g [38, 39]. These variations could reflect reporting from client-owned dogs of various breeds, age, and gender, fed with various diets. In our study, the urinary NGAL/Creat ratios after PEGB consumption (at any dose), ranged from 0.9 to 10 ng/g, leading us to conclude that there was no evidence of tubular damage. Recently, it was found that plasma NGAL was not an absolute criterion with which to discriminate between a healthy dog, versus a dog with either chronic, or acute kidney disease [38] contrary to urinary NGAL [39] and this shows how we must be cautious when interpreting these values. Moreover, increasing plasma NGAL would reflect tubular and filtration dysfunction, data already provided by other early biomarkers of renal damage (Plasma CysC, and urinary CysC/Creat, NGAL/Creat, and Clu/Creat ratios). Therefore, we suggest that plasma NGAL measurements represent redundant data and can be omitted.Intermediate measurements were also taken during the 24-week study period for all biomarkers; these did not reveal any significant differences.The PEGB doses ranged from 4 to 40 mg/kg/d, the intentional dose for dogs facing cognitive decline being 4 mg/kg/d [as recommended by the Neurophenols Consortium]. In studies where dogs were fed supplements with grape seed/skin extract at 20 mg/kg/d [5], or grape seed proanthocyanidins at 5 mg/kg/d [6], symptoms related to acute renal failure were not reported. In the group given the PEGB at 4 mg/kg/d, the dose of grape extract was beneath these previously reported doses. In addition, dogs consuming five or even ten times the intentional PEGB dose, showed no alteration of kidney or hepatic damage at 24 weeks. These data corroborated the 2013 European Pet Food Industry Federation (FEDIAF) advice that dogs could safely consume grape extract.We have considered why our extract, consumed long-term, as described in this study, appears to be entirely safe for consumption by dogs, in stark contrast to reports of acute renal failure in pets following their consumption of whole grapes or raisins. We can envisage some possibilities. The extract developed by our consortium is actually a complex mix of different extracts. How these extracts are derived (i.e. extracted from the grape), may have reduced, denatured, or eliminated, potentially toxic compounds. These factors may underlie the lack of any discernable toxicity when dogs consume the Neurophenols Consortium extract, even at high doses.Go to:ConclusionsFollowing consumption of the PEGB at all doses, conventional biomarkers of renal and liver damage were within the reference range throughout the study, with values of early biomarkers of renal damage CysC, Clu, NGAL unremarkable. To our knowledge, this is the first study demonstrating that chronic consumption of the PEGB extract can be achieved with neither renal, nor hepatic damage, at least based on plasma and urine analyses. Of note, renal health was monitored using a panel of parameters encompassing both early biomarkers of renal damage, as well as conventional biochemistry; this complementary approach is recommended in future studies. To conclude, dogs can safely consume a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium).Go to:AbbreviationsALP, Alkaline phosphatase; ALT, Alanine transaminase; AST, Aspartate aminotransferase; AUC, Area under the curve; BCS, Body condition score; BUN, Blood urea nitrogen; BW, Body weight; Clu, Clusterin; Cmax, maximum concentrations; Creat, Creatinine; CysC, Cystatin C; EGCG, Epigallocatechin gallate; NGAL, Neutrophil gelatinase-associated lipocalin; PEGB, Polyphenol-rich extract from grape and blueberry; UHPLC-MS/MS, Ultra high-performance liquid chromatography coupled to tandem mass spectrometryGo to:AcknowledgementsResearchers also thank Dr Chantal Thorin for assistance with statistical analysis and R software, to Samuel Ninet and the kennel staff as well as Philippe Bleis and Nutrition & Endocrinology Unit for taking good care of the animals and/or for technical assistance. The manuscript has been edited by San Francisco Edit.FundingThis work is part of the Neurophenols project. This project has been selected within the framework of the 12thcall for research projects launched by the French Governmental FUI (Fonds Unique Interministériel), and the present study was then supported by Bpifrance and the Conseil Régional des Pays-de-la-Loire.Availability of data and materialData supporting our conclusions are presented in the Results section of the manuscript.Authors’ contributionsVL, PN and GB conceived and designed the experiments; ASM performed the experiments; ASM, VL, PN, KO analyzed the data; ASM, VL, KO, PN, AL, JB, DG, GB wrote and/or revised the manuscript. All authors read and approved the final manuscript.Competing interestsAuthors’ institutions were affiliated with the Neurophenols Consortium, which produced the combined grape and blueberry extract, which safety is investigated in this study.Consent for publicationNot applicable.Ethics approval and consent to participateExperimental dogs were housed at Oniris (Nantes, France), according to animal welfare regulations of the French Ministry of Agriculture and Fisheries. Our experimental protocols complied fully with European Union guidelines (directive 2010/63 on the protection of animals used for scientific purpose), and our study was approved by the Animal Use and Care Advisory Committee of Pays-de-la-Loire (France), reference CEEA.2012.151.Adherence to ARRIVE guidelinesOur manuscript reporting adheres to the ARRIVE guidelines.Go to:Contributor InformationAnne-Sophie Martineau, Email: [email protected]éronique Leray, Email: [email protected] Lepoudere, Email: [email protected]éraldine Blanchard, Email: [email protected] Bensalem, Email: [email protected] Gaudout, Email: [email protected] Ouguerram, Email: [email protected] Nguyen, Email: [email protected] to:References1. Milgram NW, Head E, Zicker SC, Ikeda-Douglas CJ, Murphey H, Muggenburg B, et al. Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study. 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Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans. J Nutr. 2002;132:3592–3598. [PubMed]6. Salas A, Subirada F, Pérez-Encisco M, Blanch F, Jeussette I, Romano V, et al. Plant polyphenol intake alters gene expression in canine leukocytes. J Nutrigenet Nutrigenomics. 2009;2:43–52. doi: 10.1159/000200018. [PubMed] [Cross Ref]7. Mazzaferro EM, Eubig PA, Hackett TB, Legare M, Miller C, Wingfield WE, et al. Acute renal failure associated with raisin or grape ingestion in 4 dogs. J Vet Emerg Crit Care. 2004;14:203–212. doi: 10.1111/j.1534-6935.2004.00114.x. [Cross Ref]8. Morrow CMK, Valli VE, Volmer PA, Eubig PA. Canine renal pathology associated with grape or raisins ingestion: 10 cases. J Vet Diagn Invest. 2005;17:223–231. doi: 10.1177/104063870501700302. [PubMed] [Cross Ref]9. Eubig PA, Brady MS, Gwaltney-Brant SM, Khan SA, Mazzaferro EM, Morrow CMK. Acute renal failure in dogs after the ingestion of grapes or raisins: a retrospective evaluation of 43 dogs (1992–2002) J Vet Intern Med. 2005;19:663–674. [PubMed]10. Teschke R, Wolff A, Frenzel C, Schulze J, Eickhoff A. Herbal hepatotoxicity: a tabular compilation of reported cases. Liver Int. 2012;32:1543–1556. doi: 10.1111/j.1478-3231.2012.02864.x. [PubMed] [Cross Ref]11. Teschke R, Bahre R. Severe hepatotoxicity by Indian ayurvedic herbal products: a structured causality assessment. Ann Hepatol. 2009;8:258–266. [PubMed]12. National Research Council NRC. Nutrient requirements of dogs and cats. Washington, DC: National Academy Press; 2006.13. Muzzio M, Huang Z, Hu SC, Johnson WD, McCormick DL, Kapetanovic IM. Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC-MS/MS and their pharma-cokinetics in dogs. J Pharm Biomed Anal. 2012;59:201–208. doi: 10.1016/j.jpba.2011.10.023.[PMC free article] [PubMed] [Cross Ref]14. Kalt W, Blumberg JB, McDonald JE, Vinqvist-Tymchuk MR, Fillmore SA, Graf BA, et al. Identifica-tion of anthocyanins in the liver, eye, and brain of blueberry-fed pigs. J Agric Food Chem. 2008;56:705–712. doi: 10.1021/jf071998l. [PubMed] [Cross Ref]15. Wang J, Ferruzzi MG, Ho L, Blount J, Janle EM, Gong B, et al. Brain-targeted proanthocyanidin metabolites for Alzheimer’s disease treatment. J Neurosci. 2012;32:5144–5150. doi: 10.1523/JNEUROSCI.6437-11.2012. [PMC free article] [PubMed] [Cross Ref]16. Mata-Bilbao M, Andrés-Lacueva C, Roura E, Jáuregui O, Escribano E, Torre C, et al. Absorption and pharmacokinetics of green tea catechins in beagles. B J Nutr. 2008;100:496–502. doi: 10.1017/S0007114507898692. [PubMed] [Cross Ref]17. Kapetanovic IM, Crowell JA, Krishnaraj R, Zakharov A, Lindeblad M, Lyubimov A. Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs. Toxicology. 2009;260:28–36. doi: 10.1016/j.tox.2009.03.007. [PMC free article] [PubMed] [Cross Ref]18. Swezey RR, Aldridge DE, LeValley SE, Crowell JA, Hara Y, Green CE. Absorption, tissue distribution and elimination of 4-[3H]-epigallocatechin gallate in beagle dogs. Int J Toxicol. 2003;22:187–193. doi: 10.1080/10915810305101. [PubMed] [Cross Ref]19. Isbrucker RA, Edwards JA, Wolz E, Davidovich A, Bausch J. Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies. Food Chem Toxicol. 2006;44:636–650. doi: 10.1016/j.fct.2005.11.003. [PubMed] [Cross Ref]20. Johnson WD, Morrissey RL, Usborne AL, Kapetanovic I, Crowell JA, Muzzio M, et al. Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity. Food Chem Toxicol. 2011;49:3319–3327. doi: 10.1016/j.fct.2011.08.023. [PMC free article] [PubMed] [Cross Ref]21. Burns J, Yokota T, Ashihara H, Lean MEJ, Crozier A. Plant foods and herbal sources of resveratrol. J Agric Food Chem. 2002;50:337–340. [PubMed]22. Braun JP, Lefèbvre HP, Watson ADJ. Creatinine in the dog: a review. Vet Clin Path. 2003;32:162–179. doi: 10.1111/j.1939-165X.2003.tb00332.x. [PubMed] [Cross Ref]23. Dieterle F, Sistare F, Goodsaid F, Papaluca M, Ozer JS, Webb CP, et al. Renal biomarker qualification submission: a dialog between the FDA-EMEA and predictive safety testing consortium. Nature Biotechnol. 2010;28:455–462. doi: 10.1038/nbt.1625. [PubMed] [Cross Ref]24. Tenstad O, Roald AB, Grubb A, Aukland K. Renal handling of radiolabelled human cystatin C in the rat. Scand J Clin Lab Invest. 1996;56:409–414. doi: 10.3109/00365519609088795. [PubMed][Cross Ref]25. Herget-Rosenthal S, Marggraf G, Hüsing J, Göring F, Pietruck F, Janssen O, et al. Early detection of acute renal failure by serum cystatin C. Kidney Int. 2004;66:1115–1122. doi: 10.1111/j.1523-1755.2004.00861.x. [PubMed] [Cross Ref]26. Conti M, Moutereau S, Zater M, Lallali K, Durrbach A, Manivet P, et al. Urinary cystatin C as a specific marker of tubular dysfunction. Clin Chem Lab Med. 2006;44:288–291. doi: 10.1515/CCLM.2006.050. [PubMed] [Cross Ref]27. Tvarijonaviciute A, Ceron JJ, Holden SL, Biourge V, Morris PJ, German AJ. Effect of weight loss in obese dogs on indicators of renal function or disease. J Vet Intern Med. 2013;27:31–38. doi: 10.1111/jvim.12029. [PubMed] [Cross Ref]28. Miyagawa Y, Takemura N, Hirose H. Evaluation of the measurement of serum cystatin C by an enzyme-linked immunosorbent assay for humans as a marker of the glomerular filtration rate in dogs. J Vet Med Sci. 2009;71:1169–1176. doi: 10.1292/jvms.71.1169. [PubMed] [Cross Ref]29. Wehner A, Hartmann K, Hirschberger J. Utility of serum cystatin C as a clinical measure of renal function in dogs. J Am Anim Hosp Assoc. 2008;44:131–138. doi: 10.5326/0440131.[PubMed] [Cross Ref]30. Almy FS, Christopher MM, King DP, Brown SA. Evaluation of cystatin C as an endogenous marker of glomerular filtration rate in dogs. J Vet Intern Med. 2002;16:45–51. doi: 10.1111/j.1939-1676.2002.tb01605.x. [PubMed] [Cross Ref]31. Sasaki A, Sasaki Y, Iwama R, Shimamura S, Yabe K, Takasuna K, et al. Comparison of renal biomarkers with glomerular filtration rate in susceptibility to the detection of gentamicin-induced acute kidney injury in dogs. J Comp Path. 2014;151:264–270. doi: 10.1016/j.jcpa.2014.06.001.[PubMed] [Cross Ref]32. Jones SE, Jomary C. Clusterin. Int J Biochem Cell Biol. 2002;34:427–431. doi: 10.1016/S1357-2725(01)00155-8. [PubMed] [Cross Ref]33. Garcia-Martinez JD, Tvarijonaviciute A, Ceron JJ, Caldin M, Martinez-Subiela S. Urinary clusterin as a renal marker in dogs. J Vet Diagn Invest. 2012;24:301–306. doi: 10.1177/1040638711435112. [PubMed] [Cross Ref]34. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol. 2003;14:2534–2543. doi: 10.1097/01.ASN.0000088027.54400.C6. [PubMed][Cross Ref]35. Schmidt-Ott KM, Mori K, Kalandadze A, Li JY, Paragas N, Nicholas T, et al. Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia. Curr Opin Nephrol Hypertens. 2006;15:442–449. doi: 10.1097/01.mnh.0000232886.81142.58. [PubMed] [Cross Ref]36. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, et al. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest. 2005;115:610–621. doi: 10.1172/JCI23056. [PMC free article] [PubMed] [Cross Ref]37. Pickering JW, Endre ZH. The clinical utility of plasma neutrophil gelatinase-associated lipocalin in acute kidney injury. Blood Purif. 2013;35:295–302. doi: 10.1159/000351542. [PubMed][Cross Ref]38. Steinbach S, Weis J, Schweighauser A, Francey T, Neiger R. Plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) in dogs with acute kidney injury or chronic kidney disease. J Vet Intern Med. 2014;28:264–269. doi: 10.1111/jvim.12282. [PMC free article] [PubMed][Cross Ref]39. Segev G, Palm C, LeRoy B, Cowgill LD, Westropp JL. Evaluation of neutrophil gelatinase-associated lipocalin as a marker of kidney injury in dogs. J Vet Intern Med. 2013;27:1362–1367. doi: 10.1111/jvim.12180. [PubMed] [Cross Ref]Articles from BMC Veterinary Research are provided here courtesy of BioMed CentralFormats:ArticlePubReaderePub (beta)PDF (1.2M)CitationShareFacebookTwitterGoogle+Save itemsAdd to FavoritesView more optionsSimilar articles in PubMedEffects of dietary supplementation with a mixed blueberry and grape extract on working memory in aged beagle dogs.[J Nutr Sci. 2017]Potentiation of the bioavailability of blueberry phenolic compounds by co-ingested grape phenolic compounds in mice, revealed by targeted metabolomic profiling in plasma and feces.[Food Funct. 2016]Uptake and bioavailability of anthocyanins and phenolic acids from grape/blueberry juice and smoothie in vitro and in vivo.[Br J Nutr. 2015]Recent advances and uses of grape flavonoids as nutraceuticals.[Nutrients. 2014]Final report on the safety assessment of capsicum annuum extract, capsicum annuum fruit extract, capsicum annuum resin, capsicum annuum fruit powder, capsicum frutescens fruit, capsicum frutescens fruit extract, capsicum frutescens resin, and capsaicin.[Int J Toxicol. 2007]See reviews...See all...Cited by other articles in PMCEffects of dietary supplementation with a mixed blueberry and grape extract on working memory in aged beagle dogs[Journal of Nutritional Science...]See all...LinksPubMedTaxonomyRecent ActivityClearTurn OffA mixed grape and blueberry extract is safe for dogs to consumeA mixed grape and blueberry extract is safe for dogs to consumeBMC Veterinary Research. 2016; 12()Discriminatory power of 3.5 MHz convex and 7.5 MHz linear ultrasound probes for ...Discriminatory power of 3.5 MHz convex and 7.5 MHz linear ultrasound probes for the imaging of traumatic splenic lesions: a feasibility study.J Trauma. 2001 Jul;51(1):37-43.PubMedSee more...Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study.[Neurobiol Aging. 2005]Blueberry supplementation improves memory in older adults.[J Agric Food Chem. 2010]Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice.[Front Behav Neurosci. 2016]Hippocampal nerve growth factor levels are related to spatial learning ability in aged rats.[Behav Brain Res. 1992]Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study.[Neurobiol Aging. 2005]Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.[J Nutr. 2002]Plant polyphenol intake alters gene expression in canine leukocytes.[J Nutrigenet Nutrigenomics. 2009]Acute renal failure in dogs after the ingestion of grapes or raisins: a retrospective evaluation of 43 dogs (1992-2002).[J Vet Intern Med. 2005]Review Herbal hepatotoxicity: a tabular compilation of reported cases.[Liver Int. 2012]Severe hepatotoxicity by Indian Ayurvedic herbal products: a structured causality assessment.[Ann Hepatol. 2009]Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.[J Nutr. 2002]Plant polyphenol intake alters gene expression in canine leukocytes.[J Nutrigenet Nutrigenomics. 2009]Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC-MS/MS and their pharmacokinetics in dogs.[J Pharm Biomed Anal. 2012]Identification of anthocyanins in the liver, eye, and brain of blueberry-fed pigs.[J Agric Food Chem. 2008]Brain-targeted proanthocyanidin metabolites for Alzheimer's disease treatment.[J Neurosci. 2012]Absorption and pharmacokinetics of green tea catechins in beagles.[Br J Nutr. 2008]Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs.[Toxicology. 2009]Absorption, tissue distribution and elimination of 4-[(3)h]-epigallocatechin gallate in beagle dogs.[Int J Toxicol. 2003]Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies.[Food Chem Toxicol. 2006]Canine renal pathology associated with grape or raisin ingestion: 10 cases.[J Vet Diagn Invest. 2005]Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity.[Food Chem Toxicol. 2011]Plant foods and herbal sources of resveratrol.[J Agric Food Chem. 2002]Review Creatinine in the dog: a review.[Vet Clin Pathol. 2003]Acute renal failure in dogs after the ingestion of grapes or raisins: a retrospective evaluation of 43 dogs (1992-2002).[J Vet Intern Med. 2005]Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.[Nat Biotechnol. 2010]Renal handling of radiolabelled human cystatin C in the rat.[Scand J Clin Lab Invest. 1996]Early detection of acute renal failure by serum cystatin C.[Kidney Int. 2004]Urinary cystatin C as a specific marker of tubular dysfunction.[Clin Chem Lab Med. 2006]Effect of weight loss in obese dogs on indicators of renal function or disease.[J Vet Intern Med. 2013]Evaluation of the measurement of serum cystatin C by an enzyme-linked immunosorbent assay for humans as a marker of the glomerular filtration rate in dogs.[J Vet Med Sci. 2009]Evaluation of cystatin C as an endogenous marker of glomerular filtration rate in dogs.[J Vet Intern Med. 2002]Comparison of renal biomarkers with glomerular filtration rate in susceptibility to the detection of gentamicin-induced acute kidney injury in dogs.[J Comp Pathol. 2014]Review Clusterin.[Int J Biochem Cell Biol. 2002]Urinary clusterin as a renal marker in dogs.[J Vet Diagn Invest. 2012]Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury.[J Am Soc Nephrol. 2003]Review Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.[Curr Opin Nephrol Hypertens. 2006]Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.[J Clin Invest. 2005]The clinical utility of plasma neutrophil gelatinase-associated lipocalin in acute kidney injury.[Blood Purif. 2013]Plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) in dogs with acute kidney injury or chronic kidney disease.[J Vet Intern Med. 2014]Evaluation of neutrophil gelatinase-associated lipocalin as a marker of kidney injury in dogs.[J Vet Intern Med. 2013]Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.[J Nutr. 2002]Plant polyphenol intake alters gene expression in canine leukocytes.[J Nutrigenet Nutrigenomics. 2009]Support CenterSupport CenterExternal link. 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Can “medical student depression” be viewed as an opportunity as well as a problem? This statement does not intend in any way to minimize the severity of the issue, but invites us to reconsider that alongside managing risk we could also reflect on the opportunities that are offered by a medical school environment to prevent and manage depression in a high-risk group of young people, before they enter the workplace. Opening our minds to the possibilities that this stance might offer could enable us to view things from a slightly different perspective. Perhaps, the unique features of this student group and of the medical program itself could be explored with an eye for spotting “openings” where important “non-drug intervention” skills for depression could be learned and assimilated and stigmatizing attitudes transformed.This review comments on the prevalence and causes of depression and other symptoms of psychological distress in medical students. It then explores some of the key issues that have been shown to contribute to high levels of depression, anxiety, and stress and concludes with recommendations for early identification and support.Prevalence and causes of depression in medical studentsUniversity students face various stressors such as academic requirements, time pressure and social adjustments, and medical students in particular, may face additional challenges such as the large workload, the time commitment and the number of assessments, as well as the pressures of a clinical environment.1 A recent meta-analysis showed that depression affects approximately one third of medical students worldwide,2 and it is also likely that the overall prevalence of depressive symptoms among medical students is higher than that reported in the general population.3 Students with depressive symptoms also suffer from other psychological difficulties, such as anxiety, burnout, suicidal thoughts, and substance abuse.4–6 Research suggests that mental health deteriorates during medical school years and continues to decline when trainees enter the workforce.4 However, it has been shown that medical students can learn to adopt an active coping approach to deal with stress, which may act as a buffer to modulate their perceived stress levels.7 This is important, as students with lower stress levels are less likely to report suicidal thinking.8 A recent study showed that students are more likely to engage in active coping by their final year of medical school than they were in their earlier years.2 It is hard to discern if students learn these skills as they progress due to their medical school experiences, their training, or simply by maturing, but the evidence suggests that junior medical students are more at risk of suicidal thoughts or attempts. This underscores the importance of learning and applying healthy coping mechanisms early on in medical training.9 Research has shown that active coping strategies, such as positive framing, talking to family and friends, leisure activities, and exercising, can reduce the level of perceived stress among college students.10,11The medical curriculum may contribute to the high prevalence of psychological ill-health among medical students. Dyrbye et al5conducted a nationwide survey to compare the prevalence of burnout and other symptoms of psychological distress among medical students, residents, and early career physicians, relative to the general population. Among the medical professionals, being a medical student had the highest odds of depressive symptoms; medical students were also more likely to report depressive symptoms compared to college students of similar age from the general population. Even though certain aspects of mental health (eg, depressive symptoms) improve as students become residents and early career physicians, medical professionals are more likely to have depression and other psychological distress than do their counterparts across different stages of life. The prevalence of psychological distress was shown to be lower among a sample of students entering a medical program in the US than the age-matched sample in a general population.12 This sends a strong warning signal to medical educators that certain aspects of the medical curriculum may not be conducive to students maintaining healthy psychological states.It is also important, when considering the literature reporting the prevalence of depression, to bear in mind the other confounding factors that may influence the picture. It may be, for example, that more support is currently provided for students with psychological difficulties than has been the case in the past, thereby influencing the likelihood of these students succeeding academically,13,14 remaining in their chosen course of study,15 and consequently being available to participate in a study. Furthermore, depression prevalence data are likely to be collected by self-complete questionnaires, which possess an inherent subjective bias. Which students choose to respond and how much they are prepared to disclose may also be affected by the level of stigma in the environment. It is therefore, reasonable to question whether increasing prevalence rates demonstrate a real increase in distress indicators or whether they are also reflecting changes in help-seeking behavior, support services, or reporting practices.16Key issues influencing medical students’ well-beingGiven the high prevalence of psychological distress among medical students, medical educators, ideally, should have a good understanding of some of the key issues to be taken into consideration. These may contribute to the cause of the problems, or they may have the potential to influence what could be done to improve medical students’ mental health. The key issues discussed in this review are the spectrum of well-being including help-seeking behavior and presentations of student distress, assessment, student motivation, and characteristics such as perfectionism and personality type, selection procedures, and the clinical environment.Well-being spectrumIn 1946, the World Health Organization defined health as “a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity”, a definition clearly relevant today.17 It serves to remind us that there is a spectrum of well-being, with illness at one end and optimal well-being at the other, and that merely being “not depressed” is not the end goal. It would be beneficial for all people, including medical students, to be aware of their own place on the well-being spectrum at various times in their lives and to know what they might do to move up the spectrum.The broad wellness spectrum highlights the fact that students are likely to be experiencing a wide range of psychological difficulties, many of which may overlap, such as anxiety and depression, and all of which could contribute to functional impairment.18 It is likely that there will be a high percentage of students attending medical school who have depression or a subthreshold level of distress. This raises the question about how important or not it might be to be aware of the likely “diagnoses” of medical students with psychological distress. Psychological conditions can sometimes be “diagnosed” retrospectively after suicide via psychological autopsy, so using a diagnostic label as a risk marker for serious harm may not be as useful as presumed for prediction, especially when these retrospectively derived figures regarding psychological illness as a risk factor for suicide can include diagnoses such as alcohol or substance abuse.19 While most doctors would argue that when it comes to assessing an individual patient, a diagnosis is important, as it influences decisions regarding management, others believe that categorization of distress is minimally helpful, medicalizes aspects of normal human suffering, and can potentially lead to overdiagnosis and unnecessary treatment.20 We can extrapolate this to the medical school context to consider the fact that there may be a mixture of distress presentations. Students may have depression, stress, or burnout, which present in different ways.4 Some students could have a diagnosable mental health condition and could have an acute or chronic flare of depression, whereas others could be experiencing a short-term burst of distress without a background of psychological ill-health. It is worth reflecting on whether the same approach for both groups of students will work or not. The short-term distress group may not see themselves as “mentally ill” and may have more difficulty identifying with what is happening, even up to the point of suicidal ideation.For example, see the following excerpt taken from a student portfolio, used as assessment of the Personal and Professional Skills (PPS) domain:This last month has been very hard, in the sense that my mood has been repetitively crashing multiple times a day. There were four times where I just broke down, sometimes for a particular reason, others, there was no reason at all. It has been affecting me to the point where I can’t, or won’t, do anything because everything just seems too overwhelming. It’s difficult to describe but it is almost as if I’m fighting to “keep my head above the water” most days. I’ve heard this phrase being thrown around by some of my friends who had experienced depression, but the idea of me having depression is just weird. I have everything to be grateful for, I live an amazing life, have a super supportive family and I love the career that I’m in. [Student 1]Arguably, students who struggle with identifying themselves as being depressed may avoid or delay seeking help,21 which is one of the main barriers to care. The literature clearly shows that although some students seek professional help, many choose not to do so, due to commonly held perceptions about lack of confidentiality or perceptions of their symptoms as a failure or weakness.21 It seems sensible to enhance students’ help-seeking behavior while they are at medical school if possible, as it is thought that poor help-seeking behavior is a likely cause of workplace accidents in doctors.22 This view is supported by research showing that depressed doctors have been shown to make six times as many medication errors as those who are not depressed.23Student motivationGiven the intensity of medical training, it is important to consider additional factors that could influence student learning and well-being. Motivation is an important factor to help medical students maintain a healthy balance between study demand and personal well-being. Lyndon et al24 conducted a systematic review on medical student motivation and well-being. They found significant associations between medical student motivation (eg, intrinsic, extrinsic, “lack of motivation”, self-efficacy, and self-regulation) and well-being, which was further moderated by demographic characteristics and curricular factors. Specifically, medical students who are more intrinsically motivated also experience better well-being.25 Motivation also contributes to the way students approach learning and to the outcome of study, such that highly motivated students also tend to perform better academically and adopt deep learning strategies.25As mentioned previously, the extent to which motivation influences medical student well-being and learning could be moderated by curriculum variables. For instance, Henning et al26 found a positive relationship between preclinical medical students’ self-reported motivation and their written academic achievement. In contrast, no such relationship was found between motivation and students’ self-estimated clinical competence. It is critical for future research to continue to examine the different pathways that motivation contributes to student well-being and learning.An example of the interplay between motivation and psychological health can be seen in a Year 3 student’s reflective diary entry:[…] high levels of stress and low levels of motivation, confidence and self-esteem are rife among medical students, and I was no exception. Coming into my second year I found myself wondering whether I wanted to pursue medicine as a career, while being very interested in engineering, physics, maths and teaching. As a result, many of my experiences were tempered by this uncertainty, and I found it difficult to motivate myself to commit fully to my studies, and began to feel detached and lost. [Student 2]In terms of motivating students to take steps toward improving their own psychological health to manage or prevent depression, it can be helpful to adhere to some of the principles from the health behavior literature. The spirit of motivational interviewing is composed of autonomy, collaboration, and evocation,27 and therefore it is important to encourage students to take ownership of the issue, to enhance their self-awareness, and to contemplate what improvements they might be prepared to make in their own health. This can be done through experiential exercises such as self-care diaries, well-being goals, or reflective writing as part of a portfolio. Spoon-feeding well-being information without engagement and “buy-in” from students is unlikely to be helpful. It is also critical to have staff on board for well-being initiatives aimed at addressing depression in students. Staff will need to know how to identify students in trouble, how to talk with those who are distressed, and where to refer students to should the need arise. They are also an integral part of any destigmatizing initiatives.AssessmentThere are multiple factors that seem to affect the depression and anxiety rates of medical students, as outlined by the authors earlier. One aspect included in the literature is the highly competitive environment of medical programs. While students entering the MBChB program have proven themselves to be high performing, academically capable, and motivated through their achievements before being accepted into the program, once they begin medical study, an assessment system that mathematically ranks students using a grading system from D to A+ creates a perception of spread that may be artificial and is distressing for those students not achieving their previous “A” scores. This may lead to coping strategies that are detrimental to emotional and physical well-being. According to Wilkinson, where[…]decisions about a test are driven primarily by mathematical methods […] students will be ranked even if real differences in achievement are small”. This ranking is at odds with a standards-based model of education that assumes that “all students can reach the required standard, albeit some taking longer than others.28Several authors maintain that pass/fail grading promotes collaborative learning while de-emphasizing examination scores, reduces competition and anxiety, encourages cooperation among students, and enhances relations with teachers.4,29–31 Rohe et al30 cite Forsythe, who maintains that letter grades “transform intrinsically motivated learners into extrinsically motivated learners, precisely the opposite of currently espoused medical education principles”. Dyrbye et al,4 in their study about sources of distress for medical students, stated that: “The A-F grading scheme, used to classify performance, often creates a competitive environment that promotes anxiety and peer competition rather than collaborative learning.” Patel and David, in their study about prizes and distinctions that are the outcomes of a ranking system, noted that: “Rather than creating a stimulating and productive educational environment, rewards ultimately alienate students because they cause far more disappointment to those who tried but were unsuccessful than pleasure to the student who wins.”32 Furthermore, they argue that such a system engenders feelings of failure, self-criticism, and poor self-esteem in the majority of students, to the degree that they may not feel capable of undertaking tasks within their abilities. Instead of being encouraged to strive for success, they are forced to “adopt strategies (notably non-attendance) to avoid failure, public humiliation, and confirmation that they are less able than other students”.32Bloodgood et al31 maintained that: “The principal attraction in moving toward a pass/fail grading system lies in the expectation that it will improve students’ psychological well-being (reduce stress and anxiety), decrease competitiveness, and promote cooperative learning.” These authors, along with Rohe et al30 and Robins et al29 carried out studies to measure the impact of the grading system on medical students. These three studies found that students graded using a pass–fail (or pass–borderline pass–fail variation) were significantly more satisfied with their evaluation and examination system and with the learning environment, exhibited a significant increase in well-being, had greater satisfaction with their personal lives, and perceived less stress and greater group cohesion. Furthermore, they found that student motivation to achieve excellence remained intact and that student performance (in courses, clerkships, licensing examinations, and residency placements) and attendance did not decline.29–31In the MBChB program at the University of Auckland, Years 1–3 use the standard university 11-point grading scale, while the final 3 years are graded as pass, fail, or distinction. Moves to change this have not been successful in the early years of the program. However, the introduction of progress testing and year-long courses that allow for longitudinal assessment across multiple assessment points have been implemented to reduce the high-stakes nature of previous assessment methods.Characteristics of studentsThe desire to be excellent coupled with a competitive learning environment engages the classic “Type A” medical students in learning behavior that may be detrimental to psychological health. Type A behavior pattern is defined by Lohse et al33 as an action–emotion complex, with individuals characterized by ambition, display of highly competitive attitudes toward achievement, and feeling compelled to “work harder than Type B individuals to accomplish tasks, regardless of external stressors”.34 They are said to have an exaggerated sense of urgency with regard to time, preferring to spend it on things they deem as priorities, and possibly becoming aggressive, hostile, or impatient in frustrating situations.34 These authors suggest that Type A characteristics begin with a predisposition for competitiveness and then lead to the manifestation of characteristics such as ambitiousness, aggressiveness, impatience, and physiological behaviors such as muscle tension, a hurried pace, and alertness.While anecdotally it is recognized that medical students exhibit Type A characteristics, there appears to be little evidence to support this, although a study by Alfulaij and Alnasir35 found that of 77 Year 1 medical students in Bahrain in 2013, 76.6% had Type A personality, as opposed to 23.4% with Type B. They also found that Type A personality was more prevalent among female medical students (63.6% as compared to 36.4%). These authors linked Type A personality with endeavor for perfectionism. While the drive exhibited by this personality type may result in extraordinary achievements, pressure to achieve may result in inappropriate self-expectations and psychologically related stress disorders such as anxiety and depression. Jackson,6 in the study about the perfectionist tendencies of dentists, maintained that an inability to settle for “good enough” results in inflexible rigidity and limited the person to only the two options of perfection or failure. This may create an untenable long-term work pressure. Alfulaij and Alnasir35 commented that Type A personality doctors may fail to schedule time for relaxation and, since they have been shown to have a more internalized locus of control, they may internalize the burden related to their patients. They recommended that medical students are educated about the implications of Type A behavior early in their program, including ways of improving their psychological health. This recommendation is in accord with the introduction of the Health and Well-being theme within the PPS domain in the Auckland medical program, although Type A personality has not explicitly been introduced as a topic within the domain.Students have described the link between perfectionism and depression in their portfolios, for example:My mother asked me what was wrong. I said “nothing.” I felt I had to maintain a veneer of strength, I saw anxiety as a flaw, and in front of my parents I had to maintain this image of perfection because this is what I thought they expected of me, that is what they pride themselves on – a perfect child. [Student 3]In the context of the notion that many doctors are attracted to the profession through their own experiences of physical or psychological suffering, psychological health and resilience need to be considered at the entry point to medical program in both their positive and negative aspects. The notion of the “wounded healer” is referred to by Jackson as “[…]the inner ‘woundedness’ of a healer – the healer’s own suffering and vulnerability, which have been said to contribute crucially to the capacity to heal”.36 Jackson36 is referring to the way a person’s experience of illness, when worked through in a meaningful way, may result in attitudes and sensitivities that enhance the capacity to work empathically with others. This is acknowledged as a significant factor in both vocational choice and in contribution to healing the patient, particularly in many of the helping professions, such as psychotherapy, psychiatry, and professions that involve counseling and pastoral care. As such, it is important to consider the motivations of students in applying to study medicine – if they identify past illness, this could be considered beneficial if this has led to a deepening of understanding, while at the same time, it is important to establish current levels of health and likely future resilience.Selection processRecent efforts to quantify characteristics that establish “higher or lower risk” for psychological difficulties in medical students have led to the suggestion by some authors that more resilient students could be chosen at the point of medical school selection.37 However, there are multiple factors to be taken into consideration with selection procedures, such as equity admission pathways, to ensure that a broad range of students are selected to reflect cultural and economic diversity and potentially reduce health inequities. Students selected via these pathways may bring with them levels of complexity, including pastoral and socioeconomic issues,38 factors that may impact on their psychological health. Similarly, graduate-entry students and undergraduate students may have different coping mechanisms, which may also influence their psychological health.39Tyssen et al40 found the highest risk group for deterioration in psychological health during medical school was formed by those students with high conscientiousness and high neuroticism, while the group most protected from worsening psychological health was formed from those with high extroversion, low neuroticism, and low conscientiousness. However, conscientiousness is the trait most significantly associated with better medical school performance.41Therefore, it could be argued that selecting students at low risk of psychological ill-health at medical school entry does not necessarily lead to the selection of those with the characteristics considered best for a doctor to possess (eg, empathy formed through personal experience). Instead, the focus might be better placed on reducing the elements of medical school that contribute to psychological distress. In fact, it could be suggested that this is simply an extension to the victim-blaming culture that creates psychological distress within the health care system. Certainly, doctors do need a certain level of resilience and psychological stability to be able to capably operate while under pressure, make critical decisions, problem solve, and communicate clearly. They also need to be able to withstand the demands of an emotionally and sometimes physically demanding job and handle responsibility, uncertainty, and heavy workloads. However, there is a balance to be found that encompasses some elements of selecting for more resilient students without this being at the expense of selecting for the best doctors. In addition, Faculty and the profession could also look at what might be done to reduce some of these demands and to tackle the flaws in the health care system.Clinical environmentIn the last few years, there has been an increased focus on the issue of bullying and harassment of medical trainees in New Zealand and internationally.42–44 It is clear that this practice is widespread and can have a dramatic impact on students’ psychological health and ability to learn.45 In one study, approximately one-sixth of study participants stated that a bullying or harassment experience had made them consider leaving medical school.45 The Medical Council of New Zealand and the Royal Australasian College of Surgeons have shown leadership in beginning to tackle this issue. The World Medical Association has recently adopted a policy statement put forward by The New Zealand Medical Association and has issued a position statement condemning bullying and harassment and stating that international action is required.46 In order to tackle this problem, it will take a whole system approach that aims to influence the culture at all levels for bystanders, perpetrators, and victims and includes changes in reporting systems, medical training, and awareness raising.42 Positive role modeling by all clinicians is key, as the hidden curriculum strongly influences students’ personal and professional development47,48 to enable a change in culture away from intimidation and toward collegiality and the acceptance of vulnerability. This could create a different sort of environment, where students are not fearful of admitting ignorance or distress, and these are viewed as a normal part of growth and learning, not as a weakness.For example, a student expressed his/her views about medical students’ mental health in his/her reflective diary:[…] as medical students, we are so out of touch sometimes. We can’t really share our concerns or worries because we have to appear to be at our best all the time. There is this underlying pressure to be perfect. And there is no way that can be conducive to good mental health I reckon […]. [Student 4]RecommendationsStrategic frameworkWe believe that improving rates and levels of depression in medical students requires a comprehensive approach and that addressing specific aspects of this issue in isolation is less likely to be successful. Overarching guidelines for tertiary institutions have been developed to enable organizations to pinpoint strategic gaps in their systems.49Models have been specifically developed for medical schools, such as the “three pillars” from The Vanderbilt School of Medicine, which recommends the implementation of a well-being curriculum, student-led support, and faculty services.50 Another good example of a strategic support framework developed for medical students is the Four-Tier Continuum of Academic and Behavioural Support integrated model from the Nether-lands. This model targets students at all levels and emphasizes the importance of the provision of academic and behavioral support and evidence-based early intervention.51Well-being curriculumThere are strong arguments for the inclusion of well-being in a medical curriculum, such as the impact of doctors’ personal health practices on their communication and patient care.23,52,53 Improving doctors’ well-being also has been shown to enhance their empathy, communication skills, and reflective practice.54 In 2014, when The Medical Council of New Zealand introduced the New Zealand Curriculum Framework for newly graduated doctors, “Personal Well-being” was part of it.55 This section of the framework included learning objectives such as “balance availability to others with care for personal health, managing fatigue, stress and illness” and emphasized that it was important for doctors to have their own general practitioner.56There is a wide range of possible content that could be included in a well-being curriculum for medical students, and developers need to choose carefully as they are likely to be “competing” with other disciplines for space in the curriculum. Content can also be assessed if required, bearing in mind that for many students, assessment drives learning. Topics such as sleep, exercise, problem-solving, and an ability to manage stress and worried thinking can be taught, and many of these skills are the first “step” of non-drug interventions for managing depression.57 Active coping is a key skill to be included, as passive coping mechanisms have been linked to poorer long-term psychological health.58 It may also be possible to target specific personality traits that can be common in this student group. For example, optional sessions could be included to assist students with managing perfectionist tendencies, such as the opportunity to learn self-compassion exercises. This could enable students to be able to optimize the benefits of perfectionism while minimizing the detriments.When considering topics to include in a health and well-being curriculum, it may be worth exploring whether there are unique “medical student or medical school factors” that lend themselves to learning useful skills for preventing and managing depression. To have gained successful entry to medical school, students in the program will be academically capable of assimilating a relevant knowledge base. It is also likely that they will be motivated to succeed, which may inspire them to assimilate skills that might enable them to be more “successful”. For example, increased emotional intelligence has been cited as a leadership quality,59 which may be attractive to some students, while at the same time, self-awareness and self-management can be useful stress management skills that may prevent a depressive spiral.60 Mindfulness, a state of mind where one chooses to focus one ’s mind on the present moment in a non-judgmental manner, is a good example of a topic that can be included as a useful component of medical training on both a professional and a personal level. The ability to step back, tune out distractions, and choose what to pay attention to is clearly a skill that will be useful for a doctor, for instance, if they are required to focus on carrying out a procedure in a high-stress situation, where it can function as a “technique” and on the spot de-stressor. Mindfulness meditation training can also improve focus, productivity, and effective learning skills, and for some, it is a spiritual practice and way of being. In addition, mindfulness has also been shown to prevent the recurrence of depression.57The core components of the University of Auckland SAFE-DRS curriculum are “Self-Care Skills”, “Accessing Help”, “Focused Attention”, “Emotional Intelligence”, “Doctor as Patient and Colleague”, “Reflective Practice”, and “Stress Resistance”. There are some practical lessons to be learnt in developing and implementing a health and well-being curriculum in a medical program, such as the importance of referring to available evidence to impress upon others the significance of well-being initiatives. Although the recent New Zealand Health and Safety at Work Act 201561 may assist in this regard in the future, there may be some people who will not see “well-being” as a priority or who will brush it off as being “fluffy” or optional. The best position to shift this mind-set is by clearly and consistently drawing links to service quality and safety and by encouraging autonomy and collaboration, integrating others’ ideas whenever possible. While there needs to be governance to set up systems, an exclusively top–down approach is unlikely to be as successful. Well-being is “personal” and is seen by many people as a sensitive topic. There is a range of views on where the boundary lies between peoples’ personal and professional lives, and this needs to be taken into consideration.A well-being curriculum is the fence at the top of the cliff, not the ambulance at the bottom. In searching for better metaphors, perhaps in time we can hope that students will move away from the cliff edge altogether and turn instead to face the mountain of possibilities, thriving on challenge and flourishing. There is a spectrum of well-being, and being “not depressed” is only the midpoint, not the top end of the scale.Peer-led initiativesStudent-led approaches may include multiple types of well-being activities, for instance, social and supportive networking activities. This is important, as strong social ties are a protective factor for depression,62 and being connected to other people is a basic human need that impacts on well-being. One of the key recommendations of The Youth Development Strategy Aotearoa is to involve young people in a meaningful way in both the development and the delivery of new initiatives.63 There is also evidence to show that medical students prefer to approach peers for support rather than seeking help from health professionals or faculty members.64 In the extreme situation of suicide, suicidal young adults prefer to talk to a peer rather than a parent, a staff member, or a counsellor.65 One New Zealand study showed that students were willing to seek help for a peer who expressed suicidal concerns, while often being less willing to seek help for themselves.66 Peer-led interventions have been shown to be feasible and acceptable,67 for example, the Oxford Peer Support program, which has been run successfully out of the counseling service at Oxford University for over 20 years.68 Peer leader training will need to be provided, as students may feel that they do not have the skills or resources to provide this support.69 In the USA, peer support programs in medical schools have been shown to often go hand in hand with peer teaching,70 which is another possibility to be considered. Peer support programs do not have to be face-to-face; the Internet could be another medium to deliver support programs as Shaw and Gant71 showed that participants reported significant decreases in loneliness and depression after five chat sessions with an anonymous partner online.For example, the following excerpt from a peer leader illustrates that there are benefits for the leaders as well as the students they support:Not sure how psychologists and counsellors do this for a living. It’s exhausting listening to someone share their concerns, feelings, crises, and I think in particular because I still haven’t mastered the art of not feeling for them, as if I’m taking on board their feelings as my own. I do feel like I have helped her in a way. Like, it was good to see her walk away looking that bit lighter and that bit more purposeful and determined. But extending from that, I think it also helped me as well. Having that human connection, that interaction, made me feel in touch and made me feel human. [Student 5]Faculty-led initiativesIn terms of preventing and managing depression, early identification and management are vital and encompass independent university services providing support to students and some aligned faculty support. One possibility is to employ an independent person in a student support role, who is ideally not also involved in assessment, as it is possible that a dual role can result in a conflict of interest and unhelpful power dynamics. When considering the provision of student support, there are several key issues such as giving students a choice of services and people to approach, along with transparency regarding roles and documentation, flexible services, and practicalities such as cost and location.Anecdotally, some students have expressed a belief that perhaps Faculty view students with psychological difficulties as a process of “natural selection” and have no desire or impetus to help those struggling through the program, perhaps preferring that those students would leave medical school as they appear to not be suited to the environment. Although this view is at odds with the many initiatives that have been put in place in medical schools worldwide, such as well-being training and support services,72 if this perception persists, it could enable stigma to flourish and have a negative impact on students’ help-seeking behavior. It could be helpful to dispel this belief by providing information about periods during the year where it may be appropriate to take some additional time off for ill-health if required, with faculty approval, so that students may feel they can “hold on” until this point. It is also important to have appropriate deferral policies in place, so that students can take extended time, such as a year or even two, away from the program, without negative implications for their career. Many students appear to believe that anything that jeopardizes their progression through the program is undesirable and that this is to be avoided, regardless of the severity of psychological deterioration or the misery they may be experiencing.ConclusionDepression in medical students is a complex issue, compounded by many factors including selection procedures, the likely personalities of those attracted to medicine, assessment methods, and the clinical environment. Students’ motivation to learn and their willingness and ability to take ownership and manage their own health also play a part. While certain groups of students may be more at risk of becoming unwell,73 students are likely to experience a broad range of psychological difficulties across the whole well-being spectrum, and therefore medical schools will need wide-ranging strategies to assist with the different concerns and levels of distress. It is clear that many of the skills that may be helpful in com-batting depression and preventing its recurrence will also be useful competencies for a medical practitioner, as well as having clear links to patient safety and quality of care, and this provides a rationale for their inclusion in the curriculum. Additionally, it could be useful to strengthen support provided by the faculty and the students and to normalize “well-being” to combat stigma. Ideally, wellness initiatives should form part of a comprehensive model, be core business for all staff and students, and be integrated into the values and day-to-day operation of the organization.If what we currently have is a medical culture rife with exhaustion, learned helplessness, bullying, and psychological distress, we need to persevere until we discover how to improve it. The recent addition to the Hippocratic Oath74 of doctors attending to their own health is encouraging.75 Perhaps, it is not an unrealistic aspiration to aim for a culture which focuses on practitioner well-being, self-compassion and collegiality and strive for this to become “the new normal” for our next generation of doctors. By adopting a proactive, strength-based approach to wellness, which involves meaningful participation by students and staff, we might be able to see an improvement in the prevalence of depression in medical students. There might be advantages in viewing this issue not just as a risk to be managed or a problem to be solved, but as a clear message that things need to change in the profession. As well as being centers of teaching and learning, medical programs are spheres of influence. They are places where students develop their strategies, attitudes, and thinking and have multiple opportunities to engage, inspire, and role model a new way of being, both in self-care and in professional behaviors. Medical schools could hold the key to effect change and may be in the best position to enable medical students, educators, and clinicians to work together to build a healthier workplace culture.