Improving transparency-A call to include social housing information in biomedical research articles involving nonhuman primates.

The social setting of animal subjects in the research environment has known effects on a variety of dependent measures used in biomedical research. Proper evaluation of the robustness of published research is dependent upon transparent, detailed, and accurate reporting of research methods, including the animals' social housing conditions. However, to date, most research articles utilizing nonhuman primates (NHPs) provide only partial data on this topic, hampering transparency, and reproducibility. Therefore, we call for the inclusion of information pertaining to the social aspects of the animals' housing conditions in publications involving NHPs to improve transparency. We argue that including this information in scientific publications is crucial for the interpretation of research findings in the appropriate context and for understanding unexplained variability in study findings. Finally, the inclusion of this information in publications will additionally familiarize scientists with how other researchers conducting similar studies are housing their animals and will encourage them to consider the implications of various housing conditions on their research outcomes.

The Use of Guanfacine to Mediate Anxiety-related Reactivity and Reduce Associated Agonistic Behavior in Two Pigtail Macaques (Macaca nemestrina).

Guanfacine, an α2 adrenoceptor agonist, has been used to successfully treat self-injurious behavior in nonhuman primates, including macaques (Macaca mulatta) and baboons (Papio anubis). It does so by facilitating a correction to the dopaminergic system that mediates a reduction in impulsivity and reactivity. Given this, we assessed the potential efficacy of guanfacine to treat socially directed agonistic behavior in primates with an apparent reactive behavioral phenotype. We present data from 2 pigtail macaques (Macaca nemestrina): an intact adult male housed in a breeding group, and an experimentally naive adult female living in a research setting with her social partner. Baseline behavioral assessments suggested that both macaques showed extreme responses to external stressors that triggered them to aggress social partners often leading to wounding that required veterinary intervention. Both animals were tracked during the course of 1 y. Once treated regularly with guanfacine, both animals showed significant reduction in their agonistic behavior and the rate at which they wounded other animals. Indeed, in the year since the female has been treated with guanfacine she has never wounded her cagemate. By collecting regular and detailed behavioral observations on the male in the breeding colony, we were able to identify triggers for his aggression and to track the behavioral changes evidenced after guanfacine treatment. These data supported our hypothesis that his aggression reflected extreme reactivity to external stressors, rather than general anxiety. Importantly, we saw only a limited and short-lived reduction in the male's affiliative behavioral rates, and thus guanfacine had no sedative effect, but did successfully reduce his reactivity and resultant agonism and wounding.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in rabbits and monkeys. We injected cryopreserved hESC-derived CECs into the anterior chamber of rabbits and monkeys either immediately after mechanical scraping of the central CE or a few days later when corneal edema developed. All preclinical models developed deturgesced and clear corneas following the injection of cryopreserved hESC-derived CECs and remained comparable to the corneas of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues assessed for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet's membrane.

Elucidation of the Central Serotonin Metabolism Pathway in Rhesus Macaques (Macaca mulatta) with Self-injurious Behavior.

Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation. Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.

Serum Cobalamin (Vitamin B12) Concentrations in Rhesus Macaques (Macaca mulatta) and Pigtailed Macaques (Macaca nemestrina) with Chronic Idiopathic Diarrhea.

Chronic diarrhea poses a significant threat to the health of NHP research colonies, and its primary etiology remains unclear. In macaques, the clinical presentation of intractable diarrhea and weight loss that are accompanied by inflammatory infiltrates within the gastrointestinal tract closely resembles inflammatory bowel disease of humans, dogs, and cats, in which low serum and tissue cobalamin (vitamin B12) levels are due to intestinal malabsorption. We therefore hypothesized that macaques with chronic idiopathic diarrhea (CID) have lower serum cobalamin concentrations than do healthy macaques. Here we measured serum cobalamin concentrations in both rhesus and pigtailed macaques with CID and compared them with those of healthy controls. Serum cobalamin levels were 2.5-fold lower in pigtailed macaques with CID than control animals but did not differ between rhesus macaques with CID and their controls. This finding supports the use of serum cobalamin concentration as an adjunct diagnostic tool in pigtailed macaques that present with clinical symptoms of chronic gastrointestinal disease. This use of serum vitamin B12 levels has implications for the future use of parenteral cobalamin supplementation to improve clinical outcomes in this species.

Severity and Distribution of Wounds in Rhesus Macaques (Macaca mulatta) Correlate with Observed Self-Injurious Behavior.

Self-injurious behavior (SIB) occurs within laboratory-housed NHP at low frequency but can have a devastating effect on animal research and wellbeing. One barrier to the study and clinical management of these cases is the cost of equipment and personnel time to quantify the behavior according to the current standard of observation and to score remotely obtained video recordings. In studies of human SIB, in which direct observation is difficult or prohibited, researchers have demonstrated that quantifying the tissue damage resulting from SIB can be a useful proxy to represent the underlying behavior. We hypothesized that the nature of wounds resulting from SIB in NHP could be used in a similar manner to measure the abnormal behavior. Using a cohort of rhesus macaques with high-incidence SIB, we examined severity, distribution, and number of wounds and compared them with observed incidences of SIB during a 12-wk experiment. We found that the number, severity, and distribution of physical wounds were associated with the incidences of biting behavior observed during the 2 wk prior to measurement. We also found that an increased number of wounds was associated with increased severity. Animals with wounds of moderate severity were more likely to also have severe wounds than were macaques with wounds that were lower than moderate in severity. This work is the first representative study in NHP to find that behavioral SIB correlates with physical wounding and that increases in the frequency and number of the body regions affected correlates with the severity of wounding.

A canine socialization and training program at the National Institutes of Health.

Well-socialized and obedient dogs are easier to handle and may make better research models. The authors describe the program they have implemented at the NIH, which has benefited both the animals and their caretakers.

Diagnosis of amyloidosis and differentiation from chronic, idiopathic enterocolitis in rhesus (Macaca mulatta) and pig-tailed (M. nemestrina) macaques.

Amyloidosis is a progressive and ultimately fatal disease in which amyloid, an insoluble fibrillar protein, is deposited inappropriately in multiple organs, eventually leading to organ dysfunction. Although this condition commonly affects macaques, there is currently no reliable method of early diagnosis. Changes in clinical pathology parameters have been associated with amyloidosis but occur in late stages of disease, are nonspecific, and resemble those seen in chronic, idiopathic enterocolitis. A review of animal records revealed that amyloidosis was almost always diagnosed postmortem, with prevalences of 15% and 25% in our rhesus and pig-tailed macaque colonies, respectively. As a noninvasive, high-throughput diagnostic approach to improve antemortem diagnosis of amyloidosis in macaques, we evaluated serum amyloid A (SAA), an acute-phase protein and the precursor to amyloid. Using necropsy records and ELISA analysis of banked serum, we found that SAA is significantly elevated in both rhesus and pig-tailed macaques with amyloid compared with those with chronic enterocolitis and healthy controls. At necropsy, 92% of rhesus and 83% of pig-tailed had amyloid deposition in either the intestines or liver. Minimally invasive biopsy techniques including endoscopy of the small intestine, mucosal biopsy of the colon, and ultrasound-guided trucut biopsy of the liver were used to differentiate macaques in our colonies with similar clinical presentations as either having amyloidosis or chronic, idiopathic enterocolitis. Our data suggest that SAA can serve as an effective noninvasive screening tool for amyloidosis and that minimally invasive biopsies can be used to confirm this diagnosis.

Serum albumin and body weight as biomarkers for the antemortem identification of bone and gastrointestinal disease in the common marmoset.

The increasing use of the common marmoset (Callithrix jacchus) in research makes it important to diagnose spontaneous disease that may confound experimental studies. Bone disease and gastrointestinal disease are two major causes of morbidity and mortality in captive marmosets, but currently no effective antemortem tests are available to identify affected animals prior to the terminal stage of disease. In this study we propose that bone disease and gastrointestinal disease are associated disease entities in marmosets and aim to establish the efficacy of several economical antemortem tests in identifying and predicting disease. Tissues from marmosets were examined to define affected animals and unaffected controls. Complete blood count, serum chemistry values, body weight, quantitative radiographs, and tissue-specific biochemical markers were evaluated as candidate biomarkers for disease. Bone and gastrointestinal disease were associated, with marmosets being over seven times more likely to have either concurrent bone and gastrointestinal disease or neither disease as opposed to lesions in only one organ system. When used in tandem, serum albumin <3.5 g/dL and body weight <325 g identified 100% of the marmosets affected with concurrent bone and gastrointestinal disease. Progressive body weight loss of 0.05% of peak body weight per day predicted which marmosets would develop disease prior to the terminal stage. Bone tissue-specific tests, such as quantitative analysis of radiographs and serum parathyroid hormone levels, were effective for distinguishing between marmosets with bone disease and those without. These results provide an avenue for making informed decisions regarding the removal of affected marmosets from studies in a timely manner, preserving the integrity of research results.

Environmental enrichment during rearing alters corticosterone levels, thymocyte numbers, and aggression in female BALB/c mice.

The goal of environmental enrichment for laboratory animals is to improve welfare, but some enrichment practices may affect research in unintended ways or even be harmful to the animals themselves. We previously found that mice raised at a commercial vendor then given multiple enrichment devices upon arrival at our facilities experienced thymic atrophy and greater variation in measured parameters than did their unenriched counterparts, suggesting that enrichment conditions affected corticosteroid expression in mice. The current study verified and expanded these results, examining 120 female BALB/c mice raised with or without nesting material at a commercial vendor (n = 60 per group) and allocated (n = 20 per group) to receive no enrichment, nesting material, or 'superenrichment' on arrival at our facilities. Nesting material provided prior to weaning was associated with higher levels of urinary corticosteroid, whereas superenrichment and nesting material during the adult period both led to increased thymic atrophy. Paradoxically, mice that never received enrichment, despite having the lowest corticosterone levels and least thymic atrophy, had increased tail wounds resulting from aggressive interactions. Therefore, enrichment devices that are as seemingly innocuous as nesting material, even if only provided in the preweaning period, may lead to significant, lasting changes in behavioral, physical, or immunologic measures with the potential to alter research outcomes.

Effect of fenbendazole on three behavioral tests in male C57BL/6N mice.

Pinworms are highly contagious parasites of laboratory rodents that often are treated with fenbendazole. To our knowledge, the effect of fenbendazole at therapeutic dosages on behavioral tests in mice has not been evaluated. Here we studied 6-wk-old male C57BL/6N mice. We compared the behavior of control mice (fed regular diet) with 3 groups of mice treated with dietary fenbendazole. Treatment groups were 4 wk of fenbendazole, 2 wk of fenbendazole followed by 2 wk of regular diet, and 2 wk of regular diet followed by 2 wk of fenbendazole. At the end of dietary treatment all groups were tested by open field for central, peripheral and vertical activity; elevated plus maze for anxiety; and rotarod for motor ability and then evaluated by clinical pathology and selected histopathology. Treated and control groups showed no differences in open field or elevated plus maze testing, histopathology, or clinical pathology. However mice treated for 4 wk with fenbendazole or 2 wk of fenbendazole followed by 2 wk regular diet stayed on the rotarod for shorter periods than did controls, and mice treated with 2 wk of regular diet followed by 2 wk fenbendazole showed a trend toward shorter rotarod times. In light of this study, we suggest that open field and elevated plus maze testing is unlikely to be affected by 4 wk fenbendazole treatment in male C57BL/6 mice; however, behavioral tests of motor ability such as rotarod tests may be affected during and for at least 2 wk after fenbendazole treatment.