A systematic review of the development and application of home cage monitoring in laboratory mice and rats.

BACKGROUND: Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside of their home cages at selected time points. However, the outcome of such tests can be influenced by various factors and valuable information may be missed when the animals are only monitored for short periods. These issues can be overcome by longitudinally monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state-of-the-art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. RESULTS: Both the absolute (~ × 26) and relative (~ × 7) number of HCM-related publications increased from 1974 to 2020. There was a clear bias towards males and individually housed animals, but during the past decade (2011-2020), an increasing number of studies used both sexes and group housing. In most studies, animals were kept for short (up to 4 weeks) time periods in the HCM systems; intermediate time periods (4-12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 h, while 24-h measurements have been more frequent since the 2000s. The systematic review demonstrated that manual monitoring is decreasing in relation to automatic techniques but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the year of publication, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters have been investigated in the home cage more recently. CONCLUSIONS: Over the period covered in this study, techniques for HCM of mice and rats have improved considerably. This development is ongoing and further progress as well as validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.

Hair glucocorticoids are not a historical marker of stress - Exploring the time-scale of corticosterone incorporation into hairs in a rat model.

Hair glucocorticoids are increasingly popular biomarkers, used across numerous research fields, and studied species, as a measure of stress. Although they are suggested to be a proxy of the average HPA axis activity spanning a period of weeks or months into the past, this theory has never been tested. In the present study, adrenalectomized rats with no endogenous (adrenal) glucocorticoid production were used to study how circulating glucocorticoid levels would be reflected in the glucocorticoid levels found in hair samples. By dosing the animals daily with high levels of corticosterone for seven days, while sampling hairs before, during, and after treatments, a timeline for glucocorticoid uptake into hairs was constructed. This kinetic profile was compared to two hypothetical models, and the theory that hair glucocorticoids are a record of historical stress had to be rejected. Corticosterone concentrations in hairs were found to increase within three hours of the first injection, the highest concentrations were found on the seventh day of treatments, and the decrease in concentrations post-treatment suggests rapid elimination. We speculate that hair glucocorticoid levels can only be used to characterize a stress-response for a few days following a postulated stressor. An updated model, where glucocorticoids diffuse into, along, and out of hairs needs to be adopted to reconcile the experimentally obtained data. The inescapable consequence of this updated model is that hair glucocorticoids become a marker of - and can only be used to study - recent, or ongoing, stress, as opposed to historical events, weeks or months in the past.

An autologous blood-derived patch as a hemostatic agent: evidence from thromboelastography experiments and a porcine liver punch biopsy model.

Perioperative bleeding is a common complication in surgeries that increases morbidity, risk of mortality, and leads to increased socioeconomic costs. In this study we investigated a blood-derived autologous combined leukocyte, platelet, and fibrin patch as a new means of activating coagulation and maintaining hemostasis in a surgical setting. We evaluated the effects of an extract derived from the patch on the clotting of human blood in vitro, using thromboelastography (TEG). The autologous blood-derived patch activated hemostasis, seen as a reduced mean activation time compared to both non-activated controls, kaolin-activated samples, and fibrinogen/thrombin-patch-activated samples. The accelerated clotting was reproducible and did not compromise the quality or stability of the resulting blood clot. We also evaluated the patch in vivo in a porcine liver punch biopsy model. In this surgical model we saw 100% effective hemostasis and a significant reduction of the time-to-hemostasis, when compared to controls. These results were comparable to the hemostatic properties of a commercially available, xenogeneic fibrinogen/thrombin patch. Our findings suggest clinical potential for the autologous blood-derived patch as a hemostatic agent.

Autologous Blood-Derived Patches Used as Anti-adhesives in a Rat Uterine Horn Damage Model.

BACKGROUND: Intra-abdominal adhesions are frequent side effects of surgery, associated with risks of serious complications such as abdominal pain, infertility, and small bowel obstruction. This study investigated a new autologous blood-based approach to adhesion prophylaxis. MATERIALS AND METHOD: Two autologous blood-derived patches (whole-blood-derived, n = 20, and plasma-derived, n = 20) were evaluated as anti-adhesives. The patches were tested in a rat uterine horn damage model. We simulated an intraabdominal surgery by cauterizing and suturing the uterine horns and created an opposing damage by denuding a part of the abdominal wall. Each rat served as its own control with one treated uterine horn and one untreated. After 14 d of post-surgical recovery, the adhesions were assessed and graded macroscopically and microscopically. Statistical analyses were performed with Wilcoxon signed rank and Mann-Whitney U tests. RESULTS: Both whole-blood and plasma-derived patches resulted in significantly less macroscopic adhesions than were found in untreated uterine horns (P = 0.001 and P = 0.002, respectively). Unpaired analysis found no significant differences between the whole-blood and plasma-derived patch outcomes in this study design. Histopathological evaluation of inflammation and fibrosis did not reveal significant differences between the patches and their matched controls. CONCLUSIONS: The autologous blood-derived patches reduced macroscopic adhesion formation significantly compared with no treatment. There were no adverse events and no histological differences between treatment and control, suggesting that the treatments were feasible and safe. In summary, this study confirms the potential of autologous anti-adhesives for the use in intraabdominal surgery.

Markers of HPA-axis activity and nucleic acid damage from oxidation after electroconvulsive stimulations in rats.

OBJECTIVE: Oxidative stress has been suggested to increase after electroconvulsive therapy (ECT), a treatment which continues to be the most effective for severe depression. Oxidative stress could potentially be mechanistically involved in both the therapeutic effects and side effects of ECT. METHODS: We measured sensitive markers of systemic and central nervous system (CNS) oxidative stress on DNA and RNA (urinary 8-oxodG/8-oxoGuo, cerebrospinal fluid 8-oxoGuo, and brain oxoguanine glycosylase mRNA expression) in male rats subjected to electroconvulsive stimulations (ECS), an animal model of ECT. Due to the previous observations that link hypothalamic-pituitary-adrenal (HPA)-axis activity and age to DNA/RNA damage from oxidation, groups of young and middle-aged male animals were included, and markers of HPA-axis activity were measured. RESULTS: ECS induced weight loss, increased corticosterone (only in middle-aged animals), and decreased cerebral glucocorticoid receptor mRNA expression, while largely leaving the markers of systemic and CNS DNA/RNA damage from oxidation unaltered. CONCLUSION: These results suggest that ECS is not associated with any lasting effects on oxidative stress on nucleic acids neither in young nor middle-aged rats.

The distorting effect of varying diets on fecal glucocorticoid measurements as indicators of stress: a cautionary demonstration using laboratory mice.

The physiological stress response is frequently gauged in animals, non-invasively, through measuring glucocorticoids in excreta. A concern with this method is, however, the unknown effect of variations in diets on the measurements. With an energy dense diet, leading to reduced defecation, will low concentrations of glucocorticoids be artificially inflated? Can this effect be overcome by measuring the total output of glucocorticoids in excreta? In a controlled laboratory setting we explored the effect in mice. When standard mouse chow - high in dietary fiber - was replaced with a 17% more energy-dense diet, fecal mass was significantly reduced. As circulating levels of corticosterone and the total output of corticosterone metabolites over time remained unaffected, the result was an overestimation - more than a doubling - of the corticosterone metabolite excretion if expressed as concentrations. Similar results were obtained for testosterone metabolites. Although measuring the total output is not feasible in, for example, wildlife studies, the present findings highlight the perilousness of relying on concentrations of hormones in excreta with no associated information of the dietary intake as even moderate changes can exert a great influence.

Reliability of sucrose preference testing following short or no food and water deprivation-a Systematic Review and Meta-Analysis of rat models of chronic unpredictable stress.

The sucrose preference test is a popular test for anhedonia in the chronic unpredictable stress model of depression. Yet, the test does not always produce consistent results. Long food and water deprivation before the test, while often implemented, confounds the results by introducing unwanted drives in the form of hunger and thirst. We assessed the reliability of the test when only short or no fasting was used. We searched PubMed, Embase, and Web of Science for studies in rats exposed to chronic unpredictable stress that used no more than 6 h of food and/or water deprivation before the test. Sweet consumptions, for stressed and control/antidepressant-treated animals, in 132 studies were pooled using random effects models. We found a decrease in sweet consumption in stressed rats, compared to controls, that was halved when a non-caloric sweetener was used and significantly reduced when sucrose consumption was corrected for body weight. What is more, the length of food and water deprivation was found to confound the effect. The effect was reversed when the stressed rats were treated with antidepressants. Methodological strategies meant to control for recognized sources of bias when conducting the test were often missing, and so was a clear and complete report of essential study information. Our results indicate that not only is food and water deprivation before the test unnecessary, but not recommended. Even in absence of long fasting, we found evidence of an additional effect on sweet consumption that is unrelated to anhedonia. Without properly controlling for non-hedonic drivers of consumption, the test is unreliable as a proxy measure of anhedonia. Strengthening the methodological rigor and addressing the confounding effect of metabolic factors in the sucrose preference test prevents misleading conclusions that harm the translatability of the associated research and perpetuates the use of animals for little gain.

Rethinking data treatment: The sucrose preference threshold for anhedonia in stress-induced rat models of depression.

BACKGROUND: Exposing rats to repeated unpredictable stressors is a popular method for modelling depression. The sucrose preference test is used to assess the validity of this method, as it measures a rat´s preference for a sweet solution as an indicator of its ability to experience pleasure. Typically, if stressed rats show a lower preference compared to unstressed rats, it is concluded they are experiencing stress-induced anhedonia. METHODS: While conducting a systematic review, we identified 18 studies that used thresholds to define anhedonia and to distinguish "susceptible" from "resilient" individuals. Based on their definitions, researchers either excluded "resilient" animals from further analyses or treated them as a separate cohort. We performed a descriptive analysis to understand the rationale behind these criteria. RESULTS: we found that the methods used for characterizing the stressed rats were largely unsupported. Many authors failed to justify their choices or relied exclusively on referencing previous studies. When tracing back the method to its origins, we converged on a pioneering article that, although employed as a universal evidence-based justification, cannot be regarded as such. What is more, through a simulation study, we provided evidence that removing or splitting data, based on an arbitrary threshold, introduces statistical bias by overestimating the effect of stress. CONCLUSION: Caution must be exercised when implementing a predefined cut-off for anhedonia. Researchers should be aware of potential biases introduced by their data treatment strategies and strive for transparent reporting of methodological decisions.

Rapid ammonia build-up in small individually ventilated mouse cages cannot be overcome by adjusting the amount of bedding.

We sought to investigate if varying levels of bedding had an effect on intra-cage ammonia levels in individually ventilated mouse cages (Euro Standard Types II and III). Employing a routine 2 week cage-changing interval, our goal is to keep ammonia levels under 50 ppm. In smaller cages used for breeding or for housing more than four mice, we measured problematic levels of intra-cage ammonia, and a considerable proportion of these cages had ammonia levels at more than 50 ppm toward the end of the cage-change cycle. These levels were not reduced significantly when the levels of absorbent wood chip bedding was either increased or decreased by 50%. The mice in both cage types II and III were housed at comparable stocking densities, yet ammonia levels in larger cages remained lower. This finding highlights the role of cage volume, as opposed to simply the floor space, in controlling air quality. With the current introduction of newer cage designs that employ an even smaller headspace, our study urges caution. With individually ventilated cages, problems with intra-cage ammonia may go undetected, and we may opt to utilize insufficient cage-changing intervals. Few modern cages have been designed to account for the amounts and types of enrichment that are used (and, in parts of the world, mandated) today, adding to the problems associated with decreasing cage volumes.

Effects of Multimodal Therapy, Blinding, and Multi-laboratory Protocol Conduct on the Robustness of the Rat Model of Adjuvant Induced Arthritis.

BACKGROUND/AIM: The aim of this study was to investigate the effects of multimodal therapy comprising buprenorphine (BUP) and indomethacin (IND) on key translational parameters in the rat adjuvant induced arthritis (AIA) model. Furthermore, we investigated the difference between visual assessment scores and histology scores generated by blinded and non-blinded assessors and the robustness and generalizability of results by conducting a multi-laboratory study. MATERIALS AND METHODS: The experiment was terminated on day 26 after 11 days (days 15-25) of voluntarily ingested buprenorphine and 7 days of gavage delivered indomethacin treatment (days 19-25). The treatment effects were assessed on the last day of the study, relying on body weight assessment, serum concentrations of α1- acid glycoprotein, and assessment of affected hind paws swelling, in-life and post mortem. RESULTS: Across two laboratories, the combined analgesic treatments had minimal effects on the measured model parameters indicating that multimodal treatment did not affect the translatability of the model. We found an improvement in clinical scores (a negative change in scores) in nearly all medicated animals when scored informed, whereas it was essentially 50:50 for the blinded scorings and no difference between the blinded and informed histological scoring. CONCLUSION: The present results support the use of more effective analgesic treatment regimens and the good practice recommendations advocating blinding as a mandatory practice in conduct of preclinical in vivo efficacy studies. In spite of minor differences between results obtained at the two sites, there was good agreement between them indicating robustness of the AIA model.

The effect of isoflurane anaesthesia and vasectomy on circulating corticosterone and ACTH in BALB/c mice.

The use of blood corticosterone and faecal corticosterone metabolites as biomarkers of post-surgical stress and pain in laboratory animals has increased during the last decade. However, many aspects of their reliability in laboratory mice remain uninvestigated. This study investigated serum corticosterone and adrenocorticotropic hormone (ACTH) in mice subjected to isoflurane anaesthesia and vasectomy, and mice subjected to isoflurane anaesthesia without surgery. Serum levels of corticosterone and ACTH after pre-treatment with dexamethasone were analysed to provide further information about the stress hormone profiles. Vasectomy resulted in an increase in corticosterone for at least four hours after surgery with a peak 30min after the mice regained righting reflex. Mice subjected to isoflurane anaesthesia without surgery had the highest level of serum corticosterone 5min after regained righting reflex and the level returned to baseline levels four hours after the procedure. In vasectomised mice, treated with dexamethasone, high levels of corticosterone remained 30min after the procedure, whereas the anaesthetised mice, treated with dexamethasone, had significantly lower levels of corticosterone compared to anaesthetised mice not treated with dexamethasone. Thus, dexamethasone effectively inhibited the corticosterone response in the anaesthetised-only mice, but not in the mice subjected to surgery. In conclusion, both isoflurane anaesthesia and vasectomy during isoflurane anaesthesia resulted in an increase in serum glucocorticoids, but the negative feedback mechanism of newly operated mice, was altered. This may have consequences for the interpretation of glucocorticoids measurements as a biomarker of post-surgical stress in mice.

Fecal glucocorticoid response to environmental stressors in green iguanas (Iguana iguana).

Quantification of glucocorticoid metabolites in feces has been shown to be a powerful tool in evaluating well-being in vertebrates. Little is known however about the hypothalamic-pituitary-adrenal axis response to stressors, and consequent glucocorticoid excretion, in reptiles. In a longitudinal study, fecal corticosterone metabolite (FCM) levels in green iguanas (Iguana iguana) were quantified during periods of rest and exposure to hypothesized stressors. FCM quantification was combined with behavioral analysis to further contextualize the measured increases. It was shown that both daily 5-minute handling/restraint, as well as housing devoid of climbing opportunity, resulted in increased FCM excretion. Behavioral analysis suggested that the iguanas were chronically stressed by the lack of climbing opportunity, whereas handling may have induced only a transient stress response. The experimental design, using repeated periods of stressor-exposure, also revealed a facilitating effect, where the two stressors potentiated one another. Furthermore, the order of the two stressors was found to be important. The study provides insight into the functioning of the hormonal stress response in green iguanas, and to the refining of their housing and handling.

Antidepressant Fluoxetine Does Not Appear to Interfere With Key Translational Parameters in the Rat Adjuvant-induced Arthritis Model.

BACKGROUND/AIM: This study aimed to investigate the analgesic effects of fluoxetine on Lewis rats of both sexes in the adjuvant-induced arthritis (AIA) rat model. In humans, chronic pain syndromes typical of rheumatoid arthritis (RA) co-exist with depression which is often treated with fluoxetine antidepressant known to have antinociceptive effects. MATERIALS AND METHODS: The experiment was terminated on day 26, after seven days of oral treatment (days 19-25) with fluoxetine and indomethacin. The effects of treatments were assessed on the final day of the study through measuring body weight, serum concentrations of a1-acid glycoprotein, visual arthritis assessment and post mortem histopathology assessment. RESULTS: Statistically significant difference was determined in the body weight of male subjects, with indomethacin-treated animals putting on significantly more weight than the vehicle and fluoxetine-treated counterparts. No differences were found between the different treatment groups in other study assessments. CONCLUSION: The present study did not provide support for analgesic effects of fluoxetine aimed at reducing the severity of the AIA model.

Effects of buprenorphine on model development in an adjuvant-induced monoarthritis rat model.

Complete Freund's adjuvant (CFA)-induced arthritis in rats is a common animal model for studying chronic inflammatory pain. However, modelling of the disease is associated with unnecessary pain and impaired animal wellbeing, particularly in the immediate post-induction phase. Few attempts have been made to counteract these adverse effects with analgesics. The present study investigated the effect of buprenorphine on animal welfare, pain-related behaviour and model-specific parameters during the disease progression in a rat model of CFA-induced monoarthritis. The aim was to reduce or eliminate unnecessary pain in this model, in order to improve animal welfare and to avoid suffering, without compromising the quality of the model. Twenty-four male Sprague Dawley rats were injected with 20 µl of CFA into the left tibio-tarsal joint to induce monoarthritis. Rats were treated with either buprenorphine or carprofen for 15 days during the disease development, and were compared to a saline-treated CFA-injected group or a negative control group. Measurements of welfare, pain-related behaviour and clinical model-specific parameters were collected. The study was terminated after 3 weeks, ending with a histopathologic analysis. Regardless of treatment, CFA-injected rats displayed mechanical hyperalgesia and developed severe histopathological changes associated with arthritis. However, no severe effects on general welfare were found at any time. Buprenorphine treatment reduced facial pain expression scores, improved mobility, stance and lameness scores and it did not supress the CFA-induced ankle swelling, contrary to carprofen. Although buprenorphine failed to demonstrate a robust analgesic effect on the mechanical hyperalgesia in this study, it did not interfere with the development of the intended pathology.

Effects of Transdermal Fentanyl Treatment on Acute Pain and Inflammation in Rats with Adjuvant-induced Monoarthritis.

Eliminating unnecessary pain is an important requirement of performing animal experimentation, including reducing and controlling pain of animals used in pain research. The goal of this study was to refine an adjuvant-induced monoarthritis model in rats by providing analgesia with a transdermal fentanyl solution (TFS). Male and female Sprague-Dawley rats, single- or pair-housed, were injected with 20 µL of complete Freund adjuvant (CFA) into the left ankle joint. CFA-injected rats treated with a single dose of transdermal fentanyl solution (0.33 or 1 mg/kg) were compared with an untreated CFA-injected group and sham groups that received either no treatment or TFS treatment (1 mg/kg) during 72 h. At the tested doses, TFS reduced mechanical hyperalgesia and improved the mobility, stance, rearing, and lameness scores at 6 h after CFA injection. Joint circumferences were not reduced by TFS treatment, and no significant differences were detected between the 2 doses of TFS, or between single- and pair-housed rats. Treatment with TFS did not appear to interfere with model development and characteristics. However, overall, the analgesic effect was transient, and several opioid-related side effects were observed.

An exploratory investigation of glucocorticoids, personality and survival rates in wild and rehabilitated hedgehogs (Erinaceus europaeus) in Denmark.

BACKGROUND: The European population of hedgehogs (Erinaceus europaeus) is declining. It is therefore essential to optimise conservation initiatives such as the rehabilitation of sick, injured and orphaned hedgehogs. Wild animals placed in captivity may be prone to chronic stress, potentially causing negative health effects. Therefore, the effects of these rehabilitation efforts should consequently be evaluated. Furthermore, hand-raising orphaned hedgehogs is a laborious and costly task, and it is therefore relevant to document whether they have equal post release survival rates compared to their wild conspecifics. The objectives of this research were therefore to conduct an exploratory study of glucocorticoid levels in hedgehogs from different backgrounds and compare the post release survival of translocated, rehabilitated and wild, juvenile hedgehogs as well as the possible effect on survival of differences in shy or bold behaviour (personality) exhibited by individuals. RESULTS: We measured glucocorticoid levels in 43 wild-caught (n = 18) and rehabilitated (n = 25) hedgehogs and compared the post release survival and spatial behaviour of 18 translocated juvenile hedgehogs (eight hand-raised and ten wild) until hibernation. The possible effect on survival of differences in shy or bold behaviour (personality) exhibited by 17 juvenile individuals (seven hand-raised and ten wild) was also examined. Rehabilitated individuals and females had higher levels of faecal corticosterone metabolites compared to wild individuals and males, respectively. Rehabilitated individuals showed higher levels of saliva corticosterone than wild. The personality tests labelled 13 individuals as shy and 11 as bold. Post release survival was 57% for rehabilitated and 50% for wild individuals. Neither background nor personality affected post release survival. Home range measures were 3.54 and 4.85 ha. Mean dispersal length from the release sites was 217 ± 100 m. CONCLUSION: The higher levels of corticosterone observed in rehabilitated compared to wild hedgehogs calls for consideration of the duration of admission to wildlife rehabilitation centres to reduce stress levels in the patients. Hand-raised juveniles appear to have the same prospects as wild, and personality does not seem to affect post release survival in hedgehogs, indicating that hand-raising of orphaned juvenile hedgehogs is a relevant contribution to the conservation of this species.

Distribution and time course of corticosterone excretion in faeces and urine of female mice with varying systemic concentrations.

Quantification of corticosterone metabolites excreted in faeces and urine is increasingly being used for assessment of preceding corticosterone concentrations in the circulation. This is a promising approach to non-invasive stress assessment in laboratory rodents. It is however unknown whether the proportions of corticosterone metabolites excreted in faeces and urine may differ, depending on the concentration of corticosterone in blood. This uncertainty undermines the applicability of urinary and faecal corticosterone metabolite measurements as biomarkers for stress. Therefore, the terminal distribution and time course of corticosterone excretion, after intravenous injection of varying corticosterone concentrations, was investigated in female mice. Female BALB/c mice excreted 60% of all corticosterone in the urine with an approximate delay of 5h from tail vein administration. The remaining 40% were excreted in faeces, with an approximate delay of 9h from administration. The faecal/urinary excretion ratio, as well as time course of excretion, remained unaltered by administration of various doses of corticosterone covering the entire physiological range of serum corticosterone. Although currently untested for other strains of mice and species of animals, these findings add credence to the utility of faecal and urinary corticosterone as non-invasive biomarkers for physiological stress.

A systematic review of studies utilizing hair glucocorticoids as a measure of stress suggests the marker is more appropriate for quantifying short-term stressors.

Quantitating glucocorticoids (GCs) in hairs is a popular method for assessing chronic stress in studies of humans and animals alike. The cause-and-effect relationship between stress and elevated GC levels in hairs, sampled weeks later, is however hard to prove. This systematic review evaluated the evidence supporting hair glucocorticoids (hGCs) as a biomarker of stress. Only a relatively small number of controlled studies employing hGC analyses have been published, and the quality of the evidence is compromised by unchecked sources of bias. Subjects exposed to stress mostly demonstrate elevated levels of hGCs, and these concentrations correlate significantly with GC concentrations in serum, saliva and feces. This supports hGCs as a biomarker of stress, but the dataset provided no evidence that hGCs are a marker of stress outside of the immediate past. Only in cases where the stressor persisted at the time of hair sampling could a clear link between stress and hGCs be established.

Exploring the antigenic response to multiplexed immunizations in a chicken model of antibody production.

Hens have a tremendous capacity for producing polyclonal antibodies that can subsequently be isolated in high concentrations from their eggs. An approach for further maximizing their potential is to produce multiple antisera in the same individual through multiplexed (multiple simultaneous) immunizations. An unknown with this approach is how many immunogens a single bird is capable of mounting a sizeable antigenic response toward. At what point does it become counter-productive to add more immunogens to the same immunization regimen? In the present study we were able to demonstrate that the competing effects of co-administering multiple immunogens effectively limit the antibody specificities that can be raised in a single individual to a fairly low number. Two potent model immunogens, KLH and CRM197, were administered together with competing antigens in various concentrations and complexities. With an upper limit of 1 mg protein material recommended for chicken immunizations, we found that the maximum number of immunogens that can be reliably used is most likely in the low double digits. The limiting factor for a response to an immunogen could not be related to the number of splenic plasma cells producing antibodies against it. When administering KLH alone, up to 70% of the IgY-producing splenic plasma cells were occupied with producing anti-KLH antibodies; but when simultaneously being exposed to a plethora of other antigens, a response of a comparable magnitude could be mounted with a splenic plasma cell involvement of less than 5%. Two breeds of egg-layers were compared with respect to antibody production in an initial experiment, but differences in antibody productivity were negligible. Although our findings support the use of multiplexed immunizations in the hen, we find that the number of immunogens cannot be stretched much higher than the handful that has been used in mammalian models to date.

A chronic increase of corticosterone age-dependently reduces systemic DNA damage from oxidation in rats.

Stress and depression are associated with an acceleration of brain and bodily aging; effects which have been attributed to chronic elevations of glucocorticoids. We tested the hypothesis that a three week administration of stress-associated levels of corticosterone (CORT, the principal rodent glucocorticoid) would increase systemic and CNS DNA and RNA damage from oxidation; a phenomenon known to be centrally involved in the aging process. We also hypothesized that older individuals would be more sensitive to this effect and that the chronic CORT administration would exacerbate age-related memory decline. Young and old male Sprague-Dawley rats were non-invasively administered CORT by voluntary ingestion of nut paste containing either CORT (25mg/kg) or vehicle for a total of 22 days. CORT increased the 24h urinary excretion of the hormone to the levels previously observed after experimental psychological stress and caused a downregulation of the glucocorticoid receptor in the CA1 area of the hippocampus. Contrary to our hypothesis, 24h excretion of 8-oxodG/8-oxoGuo (markers of DNA/RNA damage from oxidation) was reduced in CORT-treated young animals, whereas old animals showed no significant differences. In old animals, CORT caused a borderline significant reduction of RNA oxidation in CNS, which was paralleled by a normalization of performance in an object location memory test. To our knowledge, this is the first demonstration that chronic stress-associated levels of CORT can reduce nucleic acid damage from oxidation. These findings contradict the notion of elevated CORT as a mediator of the accelerated aging observed in stress and depression.