Elemental Bioimaging of Sheep Bone and Articular Cartilage After Single Application of Gadolinium-Based Contrast Agents.

BACKGROUND: Gadolinium-based contrast agents (GBCAs) are applied to enhance magnetic resonance imaging. Gadolinium (Gd), a rare earth metal, is used in a chelated form when administered as GBCA to patients. There is an ongoing scientific debate about the clinical significance of Gd retention in tissues after administration of GBCAs. It is known that bone serves as Gd reservoir, but only sparse information on localization of Gd in bone is available. PURPOSE: The aim of this study was to compare Gd tissue concentration and spatial distribution in femoral epiphysis and diaphysis 10 weeks after single-dose injection of linear and macrocyclic GBCAs in a large animal model. MATERIALS AND METHODS: In this prospective animal study, Swiss-Alpine sheep (n = 36; age range, 4-10 years) received a single injection (0.1 mmol/kg) of macrocyclic (gadobutrol, gadoteridol, and gadoterate meglumine), linear (gadodiamide and gadobenate dimeglumine) GBCAs, or saline. Ten weeks after injection, sheep were killed, and femur heads and shafts were harvested. Gadolinium spatial distribution was determined in 1 sample of each treatment group by laser ablation-inductively coupled plasma-mass spectrometry. All bone specimens were analyzed histopathologically. RESULTS: Injection of GBCAs in female Swiss-Alpine sheep (n = 36) resulted in Gd localization at the endosteal and periosteal surface and in a subset of GBCAs additionally at the cement lines and the bone cartilage junction. No histopathological alterations were observed in the investigated tissue specimens. CONCLUSIONS: Ten weeks after single injection of a clinically relevant dose in adult sheep, both linear species of GBCA resulted in considerably higher accumulation than macrocyclic GBCAs. Gadolinium deposits were restricted to distinct bone and cartilage compartments, such as in bone linings, cement lines, and bone cartilage junctions. Tissue histology remained unaffected.

Elemental bioimaging and transcriptomics reveal unchanged gene expression in mouse cerebellum following a single injection of Gadolinium-based contrast agents.

Gadolinium (Gd) deposition in the brain, first and foremost in the dentate nucleus in the cerebellum, following contrast enhanced MRI, rose awareness of potential adverse effects of gadolinium-based contrast agent (GBCA) administration. According to previous in vitro experiments, a conceivable side-effect of Gd deposition could be an alteration of gene expression. In the current study, we aimed to investigate the influence of GBCA administration on gene expression in the cerebellum of mice using a combination of elemental bioimaging and transcriptomics. In this prospective animal study, three groups of eight mice each were intravenously injected with either linear GBCA gadodiamide, macrocyclic GBCA gadoterate (1 mmol GBCA/kg body weight) or saline (NaCl 0.9%). Animals were euthanized four weeks after injection. Subsequently, Gd quantification via laser ablation-ICP-MS and whole genome gene expression analysis of the cerebellum were performed. Four weeks after single application of GBCAs to 24-31 days old female mice, traces of Gd were detectable in the cerebellum for both, the linear and macrocyclic group. Subsequent transcriptome analysis by RNA sequencing using principal component analysis did not reveal treatment-related clustering. Also differential expression analysis did not reveal any significantly differentially expressed genes between treatments.

Sustainable education and training in laboratory animal science and ethics in low- and middle-income countries in Africa - challenges, successes, and the way forward.

Despite the recognised need for education and training in laboratory animal science (LAS) and ethics in Africa, access to such opportunities has historically been limited. To address this, the Pan-African Network for Laboratory Animal Science and Ethics (PAN-LASE) was established to pioneer a support network for the development of education and training in LAS and ethics across the African continent.In the 4.5 years since the establishment of PAN-LASE, 3635 individuals from 28 African countries have participated in our educational activities. Returning to their home institutions, they have both established and strengthened institutional and regional hubs of knowledge and competence across the continent. Additionally, PAN-LASE supported the development of guidelines for establishment of institutional Animal Ethics Committees, a critical step in the implementation of ethical review processes across the continent, and in enhancing animal welfare and scientific research standards.Key challenges and opportunities for PAN-LASE going forward include the formalisation of the network; the sustainability of education and training programmes; implementation of effective hub-and-spoke models of educational provision; strengthening governance frameworks at institutional, national and regional levels; and the availability of Africa-centric open access educational resources.Our activities are enhancing animal welfare and the quality of animal research undertaken across Africa, enabling African researchers to undertake world-leading research to offer solutions to the challenges facing the continent. The challenges, successes and the lessons learnt from PAN-LASE's journey are applicable to other low- and middle-income countries across the world seeking to enhance animal welfare, research ethics and ethical review in their own country or region.

No news from old drawings? Stomach anatomy in muroid rodents in relation to body size and ecology.

Muroid rodents mostly have a complex stomach: one part is lined with a cornified (nonglandular) epithelium, referred to as a "forestomach", whereas the rest is lined with glandular epithelium. Numerous functions for the forestomach have been proposed. We collated a catalog of anatomical depictions of the stomach of 174 muroid species from which the respective nonglandular and glandular areas could be digitally measured, yielding a "stomach ratio" (nonglandular:glandular area) as a scale-independent variable. Stomach ratios ranged from 0.13 to 20.15, and the coefficient of intraspecific variation if more than one picture was available for a species averaged at 29.7% (±21.5). We tested relationships of the ratio with body mass and various anatomical and ecological variables, including diet. There was a consistent phylogenetic signal, suggesting that closely related species share a similar anatomy. Apart from classifying stomachs into hemiglandular and discoglandular, no anatomical or ecological measure showed a consistent relationship to the stomach ratio. In particular, irrespective of statistical method or the source of dietary information, dietary proxies did not significantly correlate with the stomach ratio, except for a trend towards significance for invertivory (insectivory). Yet, even this relationship was not convincing: whereas highly insectivorous species had high but no low stomach ratios, herbivorous species had both low and high stomach ratios. Thus, the statistical effect is not due to a systematic increase in the relative forestomach size with invertivory. The most plausible hypotheses so far associate the muroid forestomach and its microbiome with a generic protective role against microbial or fungal toxins and diseases, without evident correlates of a peculiar need for this function under specific ecological conditions. Yet, this function remains to be confirmed. While providing a catalog of published depictions and hypotheses, this study highlights that the function of the muroid rodent forestomach remains enigmatic to date.

Tooth wear, growth and height in rabbits (Oryctolagus cuniculus) fed pelleted or extruded diets with or without added abrasives.

Among the different factors thought to affect dental wear, dietary consistency is possibly the least investigated. To understand tooth wear of herbivorous animals consuming different dietary consistencies with different abrasive potential, we fed 14 rabbits (Oryctolagus cuniculus) exclusively with a timothy grassmeal-based diet in either pelleted or extruded form, or the same diets with an addition of 5% fine sand abrasives (mean size 130 µm). First, we offered the rabbits the pelleted and extruded diets as well as the pelleted control and pelleted abrasive diet in a two-stage preference experiment. Then, the rabbits received each diet for 2 weeks in a randomised serial feeding experiment, where each animal served as its own control. Tooth measurements for wear, growth and height were achieved using a manual calliper, endoscopic examination and CT scans. The analysis of the diets as fed showed almost identical mean particle size, but the extruded diet had a lower density (volume/mass) and softer consistency compared to the pelleted one and was favoured by most rabbits. The rabbits selected against the diet with sand during the preference experiment, possibly because it caused more tooth wear, especially on the teeth most exposed to wear along the upper tooth row (upper P4 and M1). The maxillary teeth also showed evidence of an increased chewing laterality by the end of the experiment. The extruded diet led to a significantly lower cheek teeth height than the pelleted diet, potentially due to the higher chewing effort needed for a similar dry matter intake. The results suggest that dietary hardness alone is a poor predictor of dental wear. The regrowth of the teeth matched wear consistently.

Macrowear effects of external quartz abrasives of different size and concentration in rabbits (Oryctolagus cuniculus).

External quartz abrasives are one of the driving forces of macrowear in herbivorous animals. We tested to what extent different sizes and concentrations influence their effect on tooth wear. We fed seven pelleted diets varying only in quartz concentration (0%, 4%, and 8%) and size (fine silt: ∼4 µm, coarse silt: ∼50 µm, fine sand: ∼130 µm) to rabbits (Oryctolagus cuniculus, n = 16) for 2 weeks each in a randomized serial experiment. Measurements to quantify wear and growth of incisors and the mandibular first cheek tooth, as well as heights of all other cheek teeth, were performed using calipers, endoscopic examination, and computed tomography scans before and after each feeding period. Tooth growth showed a compensatory correlation with wear. Absolute tooth height (ATH) and relative tooth height (RTH); relative to the 0% quartz "control" diet) was generally lower on the higher concentration and the larger size of abrasives. The effect was more pronounced on the maxillary teeth, on specific tooth positions and the right jaw side. When offered the choice between different sizes of abrasives, the rabbits favored the silt diets over the control and the fine sand diet; in a second choice experiment with different diets, they selected a pelleted diet with coarse-grained sand, however. This study confirms the dose- and size-dependent wear effects of external abrasives, and that hypselodont teeth show compensatory growth. The avoidance of wear did not seem a priority for animals with hypselodont teeth, since the rabbits did not avoid diets inducing a certain degree of wear.

Use of running plates by floor housed rats: A pilot study.

The outfit of husbandry facilities of, and the enrichment provided for, experimental rodents plays an important role in the animals' welfare, and hence also for the societal acceptance of animal experiments. Whether rats and mice benefit from being provided with running wheels or plates is discussed controversially. Here we present observations from a feeding experiment, where rats were provided a running plate. As a pilot study, six identical cages, with three animals per cage, were filmed for six days, and the resulting footage was screened for the number of bouts and the time the animals spent on the plates. The main activities observed on the plate in descending order were sitting (18.5 ± 13.8 bouts or 8.0 ± 13.7 min/animal per day), standing (10.2 ± 11.6 bouts, 3.8 ± 4.2 min), running (8.2 ± 13.3 bouts, 10.1 ± 21.4 min), grooming (2.0 ± 2.8 bouts, 6.7 ± 25.7 min), sleeping (1.0 ± 2.6 bouts, 24.0 ± 61.8 min) and playing (0.5 ± 0.9 bouts, 0.1 ± 0.5 min). Most of these activities (91% of all bouts, 90% of total time) occurred at night, similar to previous studies on running wheel usage. The running plate seems well-accepted as cage enrichment, even though in further studies, the motivating triggers and the effects of long-term use could be evaluated more in-depth.

Dental wear proxy correlation in a long-term feeding experiment on sheep (Ovis aries).

Dietary reconstruction in vertebrates often relies on dental wear-based proxies. Although these proxies are widely applied, the contributions of physical and mechanical processes leading to meso- and microwear are still unclear. We tested their correlation using sheep (Ovis aries, n = 39) fed diets of varying abrasiveness for 17 months as a model. Volumetric crown tissue loss, mesowear change and dental microwear texture analysis (DMTA) were all applied to the same teeth. We hereby correlate: (i) 46 DMTA parameters with each other, for the maxillary molars (M1, M2, M3), and the second mandibular molar (m2); (ii) 10 mesowear variables to each other and to DMTA for M1, M2, M3 and m2; and (iii) volumetric crown tissue loss to mesowear and DMTA for M2. As expected, many DMTA parameters correlated strongly with each other, supporting the application of reduced parameter sets in future studies. Correlation results showed only few DMTA parameters correlated with volumetric tissue change and even less so with mesowear variables, with no correlation between mesowear and volumetric tissue change. These findings caution against interpreting DMTA and mesowear patterns in terms of actual tissue removal until these dental wear processes can be better understood at microscopic and macroscopic levels.

Confirmation of a wear-compensation mechanism in dental roots of ruminants.

Diet affects many factors of an animal's anatomy, but teeth are a specific focus of dietary research, as their durability lends them to record information on a large variety of scales. Abrasive diets like those of grazing herbivores are known to wear down teeth, but how that wear affects tooth growth and the relations between its different morphological components is rarely investigated. Seven pelleted diets varying in abrasive size and concentration were fed over a 17-month period to 49 sheep (Ovis aries), of which n = 39 qualified for morphology measurements. Using computed tomography, scans of the skulls were made over the course of the experiment, and the impact of diet-related wear was observed on tooth volume and morphology, including the position of dental burr marks, over time. Digital caliper measurements were applied to 3D renderings of the teeth, and the volume of crown and root segments were investigated separately. We aimed to detect a signal of root growth compensating for wear, and test if this mechanism would be affected by dietary abrasives. Crown-segment volume loss was correlated to root-segment volume gain. Height and burr mark measurements indicated a much higher experimental tooth wear than that previously reported for free-ranging animals. The reason for this is unclear. There was no relationship between tooth height and dentine basin depth. For all parameters, there was no effect of diet; hence, while the measurements corroborate general understanding of tooth wear and compensatory processes, these methods appear not suitable to assess subtle differences between feeding regimes.

Impact of general anesthesia on rotational thromboelastometry (ROTEM) parameters and standard plasmatic coagulation tests in healthy Beagle dogs.

OBJECTIVE: To assess the influence of general anesthesia on rotational thromboelastometry (ROTEM) and standard coagulation testing in healthy dogs. STUDY DESIGN: Prospective experimental study. ANIMALS: 10 healthy Beagle dogs. METHODS: Dogs were administered methadone (0.2 mg/kg) intramuscularly. Anesthesia was co-induced intravenously 30 min later with midazolam (0.1 mg/kg) and propofol to effect, and maintained with sevoflurane. Crystalloids were administered at 5 ml/kg/h. Blood was sampled by direct venipuncture before induction (T0) and 3.5 h later (T3.5) and ROTEM parameters (ExTEM, InTEM, FibTEM, ApTEM), standard plasmatic coagulation tests (prothrombin time [PT], activated partial thromboplastin time [aPTT], fibrinogen concentration), hematology, ionized calcium, triglycerides, pH, lactate and body temperature were compared over time with Students t - test or Wilcoxon matched pairs signed-rank tests. RESULTS: The following variables dropped significantly between T0 and T3.5: body temperature (p < 0.0001), hematocrit (p < 0.0001), platelet count (p < 0.01), pH (p < 0.01), triglycerides (p < 0.01), fibrinogen concentration (p < 0.01), ExTEM, FibTEM (p < 0.01) and ApTEM (p < 0.05) clotting times. Lactate concentration (p < 0.01), aPTT (p < 0.05) and FibTEM maximum clot firmness increased (p < 0.05). No changes were noted in ionized calcium, PT and InTEM values. CONCLUSION AND CLINICAL RELEVANCE: General anesthesia with concurrent hemodilution and hypothermia induced significant but clinically irrelevant changes in coagulation variables measured at 37 °Celsius. Blood samples from anaesthetized animals can be used for determination of coagulation status in dogs.

Is Small Fiber Neuropathy Induced by Gadolinium-Based Contrast Agents?

OBJECTIVES: In recent years, complaints of patients about burning pain in arms and legs after the injection of gadolinium-based contrast agents (GBCAs) have been reported. In the current study, we investigated changes of small fibers in the epidermis as a potential cause of the patient complaints in a mouse model. METHODS: Six groups of 8 mice were intravenously injected with either a macrocyclic GBCA (gadoteridol, gadoterate meglumine, gadobutrol), a linear GBCA (gadodiamide or gadobenate dimeglumine) (1 mmol/kg body weight), or saline (NaCl 0.9%). Four weeks after injection, animals were euthanized, and footpads were assessed using immunofluorescence staining. Intraepidermal nerve fiber density (IENFD) was calculated, and the median number of terminal axonal swellings (TASs) per IENFD was determined. RESULTS: Nonparametric Wilcoxon signed-rank test revealed significantly lower IENFDs for all GBCAs compared with the control group (P < 0.0001) with the linear GBCAs showing significantly lower IENFDs than the macrocyclic GBCAs (P < 0.0001). The linear GBCAs presented significantly more TAS per IENFD than the control group (P < 0.0001), whereas no significant increase of TAS per IENFD compared with the control group was found for macrocyclic GBCAs (P < 0.237). INTERPRETATION: It is unclear whether or at what dosage the decrease of IENFDs and the increase of TAS per IENFD found in the current animal model will appear in humans and if it translates into clinical symptoms. However, given the highly significant findings of the current study, more research in this field is required.

Chewing, dental morphology and wear in tapirs (Tapirus spp.) and a comparison of free-ranging and captive specimens.

Feeding practice in herbivorous mammals can impact their dental wear, due to excessive or irregular abrasion. Previous studies indicated that browsing species display more wear when kept in zoos compared to natural habitats. Comparable analyses in tapirs do not exist, as their dental anatomy and chewing kinematics are assumed to prevent the use of macroscopic wear proxies such as mesowear. We aimed at describing tapir chewing, dental anatomy and wear, to develop a system allowing comparison of free-ranging and captive specimens even in the absence of known age. Video analyses suggest that in contrast to other perissodactyls, tapirs have an orthal (and no lateral) chewing movement. Analysing cheek teeth from 74 museum specimens, we quantified dental anatomy, determined the sequence of dental wear along the tooth row, and established several morphometric measures of wear. In doing so, we showcase that tapir maxillary teeth distinctively change their morphology during wear, developing a height differential between less worn buccal and more worn lingual cusps, and that quantitative wear corresponds to the eruption sequence. We demonstrate that mesowear scoring shows a stable signal during initial wear stages but results in a rather high mesowear score compared to other browsing herbivores. Zoo specimens had lesser or equal mesowear scores as specimens from the wild; additionally, for the same level of third molar wear, premolars and other molars of zoo specimens showed similar or less wear compared specimens from the wild. While this might be due to the traditional use of non-roughage diet items in zoo tapirs, these results indicate that in contrast to the situation in other browsers, excessive tooth wear appears to be no relevant concern in ex situ tapir management.

Dust and grit matter: abrasives of different size lead to opposing dental microwear textures in experimentally fed sheep (Ovis aries).

External abrasives ingested along with the herbivore diet are considered main contributors to dental wear, though how the different sizes and concentrations of these abrasives influence wear remains unclear. Dental microwear texture analysis (DMTA) is an established method for dietary reconstruction which describes a tooth's surface topography on a micrometre scale. The method has yielded conflicting results as to the effect of external abrasives. In the present study, a feeding experiment was performed on sheep (Ovis aries) fed seven diets of different abrasiveness. Our aim was to discern the individual effects of size (4, 50 and 130 µm) and concentration (0%, 4% and 8% of dry matter) of abrasives on dental wear, applying DMTA to four tooth positions. Microwear textures differed between individual teeth, but surprisingly, showed no gradient along the molar tooth row, and the strongest differentiation of experimental groups was achieved when combining data of all maxillary molars. Overall, a pattern of increasing height, volume and complexity of the tooth's microscopic surface appeared with increasing size of dietary abrasives, and when compared with the control, the small abrasive diets showed a polishing effect. The results indicate that the size of dietary abrasives is more important for dental microwear texture traces than their concentration, and that different sizes can have opposing effects on the dietary signal. The latter finding possibly explains conflicting evidence from previous experimental DMTA applications. Further exploration is required to understand whether and how microscopic traces created by abrasives translate quantitatively to tissue loss.

The way wear goes: phytolith-based wear on the dentine-enamel system in guinea pigs (Cavia porcellus).

The effect of phytoliths on tooth wear and function has been contested in studies of animal-plant interactions. For herbivores whose occlusal chewing surface consists of enamel ridges and dentine tissue, the phytoliths might particularly erode the softer dentine, exposing the enamel ridges to different occlusal forces and thus contributing to enamel wear. To test this hypothesis, we fed guinea pigs (Cavia porcellus; n = 36 in six groups) for three weeks exclusively on dry or fresh forage of low (lucerne), moderate (fresh timothy grass) or very high (bamboo leaves) silica content representing corresponding levels of phytoliths. We quantified the effect of these treatments with measurements from micro-computed tomography scans. Tooth height indicated extreme wear due to the bamboo diet that apparently brought maxillary incisors and molars close to the minimum required for functionality. There were negative relationships between a cheek tooth's height and the depth of its dentine basin, corroborating the hypothesis that dentine erosion plays an important role in herbivore tooth wear. In spite of lower body mass, bamboo-fed animals paradoxically had longer cheek tooth rows and larger occlusal surfaces. Because ever-growing teeth can only change in shape from the base upwards, this is a strong indication that failure to compensate for wear by dental height-growth additionally triggered general expansive growth of the tooth bases. The results suggest that enamel wear may intensify after enamel has been exposed due to a faster wear of the surrounding dentine tissue (and not the other way around), and illustrate a surprising plasticity in the reactivity of this rodent's system that adjusts tooth growth to wear.

Physical characteristics of gastrointestinal content of llama (Lama glama).

Changes in digesta dry matter (DM) and mean digesta particle size (MPS) along the gastrointestinal tract are well known in ruminants, but not in camelids. We collected digesta from the dorsal (d) and ventral (v) first forestomach compartment (C1), the second forestomach compartment (C2), three proximal segments and the subsequent glandular part of the third compartment (C3A-D), the caecum and the faeces twelve llamas (Lama glama). DM analysis indicates the presence of digesta stratification in the C1, the presence of fluid in the C2 to facilitate the sorting function of this compartment, the fluid-absorbing function of the proximal parts of the C3, the secretion of enzymes and digestive acids in the C3D, and the water-resorbing function of the lower intestinal tract. These findings illustrate the functional resemblance between the gastrointestinal tract of camelids and cattle-like ruminants (C1 equivalent to the rumen with stratified contents, C2 to the reticulum, C3A/B/C to the omasum and C3D to the abomasum). MPS analysis revealed a progressive reduction in MPS from the C1 to the distal C3. This gradual transition is different from the clear-cut threshold in ruminants between the reticulum and the omasum and had so far only been described in dromedaries (Camelus dromedarius). These findings indicate that regardless of the convergent property of rumination and resemblance of general mechanisms involved in contents stratification and particle sorting, differences between ruminants and camelids exist that could be interpreted as a more efficient functionality of the ruminant forestomach.