Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model.

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

Application of Infrared Thermography in the Rehabilitation of Patients in Veterinary Medicine.

Infrared Thermography (IRT) has become an assistance tool in medicine and is used to noninvasively evaluate heat elimination during and after inflammatory processes or during the recovery period. However, its application in veterinary patients undergoing physiotherapy is a field that requires deep research. This review aims to analyze the application of IRT in the monitoring of animal physiotherapy, using the thermal changes that are present in patients undergoing gait or lameness issues (e.g., inflammation, pain, increased local temperature) as a neurobiological basis. Rehabilitation techniques such as acupuncture, physical therapies, thermotherapy, photo-biomodulation, and electrostimulation have been reported to have an anti-inflammatory effect that decreases the amount of local heat production, which is heat that can be recorded with IRT. Therefore, IRT could be used as a complementary tool to evaluate the effectiveness of the therapy, and it is suggested that further studies evaluate the accuracy, sensibility, and sensitivity of IRT.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Biomarkers of Stress in Companion Animals.

Stress experienced by companion animals could impair their physical and psychological welfare, impacting their social relationships in domestic environments [...].

Converging Role for REEP1/SPG31 in Oxidative Stress.

Mutations in the receptor expression-enhancing protein 1 gene (REEP1) are associated with hereditary spastic paraplegia type 31 (SPG31), a neurological disorder characterized by length-dependent degeneration of upper motor neuron axons. Mitochondrial dysfunctions have been observed in patients harboring pathogenic variants in REEP1, suggesting a key role of bioenergetics in disease-related manifestations. Nevertheless, the regulation of mitochondrial function in SPG31 remains unclear. To elucidate the pathophysiology underlying REEP1 deficiency, we analyzed in vitro the impact of two different mutations on mitochondrial metabolism. Together with mitochondrial morphology abnormalities, loss-of-REEP1 expression highlighted a reduced ATP production with increased susceptibility to oxidative stress. Furthermore, to translate these findings from in vitro to preclinical models, we knocked down REEP1 in zebrafish. Zebrafish larvae showed a significant defect in motor axon outgrowth leading to motor impairment, mitochondrial dysfunction, and reactive oxygen species accumulation. Protective antioxidant agents such as resveratrol rescued free radical overproduction and ameliorated the SPG31 phenotype both in vitro and in vivo. Together, our findings offer new opportunities to counteract neurodegeneration in SPG31.

Thermal Imaging as a Method to Indirectly Assess Peripheral Vascular Integrity and Tissue Viability in Veterinary Medicine: Animal Models and Clinical Applications.

Infrared thermography (IRT) is a technique that indirectly assesses peripheral blood circulation and its resulting amount of radiated heat. Due to these properties, thermal imaging is currently applied in human medicine to noninvasively evaluate peripheral vascular disorders such as thrombosis, thromboembolisms, and other ischemic processes. Moreover, tissular damage (e.g., burn injuries) also causes microvasculature compromise. Therefore, thermography can be applied to determine the degree of damage according to the viability of tissues and blood vessels, and it can also be used as a technique to monitor skin transplant procedures such as grafting and free flaps. The present review aims to summarize and analyze the application of IRT in veterinary medicine as a method to indirectly assess peripheral vascular integrity and its relation to the amount of radiated heat and as a diagnostic technique for tissue viability, degree of damage, and wound care.

Rider Variables Affecting the Stirrup Directional Force Asymmetry during Simulated Riding Trot.

Riders' asymmetry may cause back pain in both human and equine athletes. This pilot study aimed at documenting in a simple and quick way asymmetry in riders during a simulation of three different riding positions on wooden horseback using load cells applied on the stirrup leathers and identifying possible associations between riders' asymmetry and their gender, age, level of riding ability, years of riding experience, riding style, motivation of riding, primary discipline and handedness. After completing an interview to obtain the previously mentioned information, 147 riders performed a standardized test on a saddle fixed on a wooden horseback-shaped model. The riding simulation was split into three phases of 1 min each: (1) sit in the saddle, (2) standing in the stirrups and (3) rising trot. The directional force on the left and the right stirrup leathers was recorded every 0.2 s. A paired t-test was performed on the recorded data to test the difference (i.e., asymmetry) in each phase. In phases 1, 2 and 3, 99.3% (53.4% heavier on the right (R)), 98% (52.8% heavier on the left (L)) and 46.3% (51.5% heavier on the left (L)) of the riders were asymmetrical, respectively. Chi-square tests showed a significant association between riding ability and riding experience, but no significant association between reported handedness and calculated leg-sidedness (p > 0.05). Univariate logistic (1: asymmetry, 0: symmetry) regression analysis was performed only on the phase 3 data. One-hand riders were found twice more likely to be asymmetrical than two-hand riders (Odds Ratio (OR): 2.18, Confidence Interval (CI): 1.1−4.29; p = 0.024). This preliminary study confirmed that the majority of the riders are asymmetrical in load distribution on stirrups and suggested the riding style as a possible risk factor for asymmetry.

A Review on the Bioactivity of Cannabinoids on Zebrafish Models: Emphasis on Neurodevelopment.

For centuries, the cannabis plant has been used as a source of food, fiber, and medicine. Recently, scientific interest in cannabis has increased considerably, as its bioactive compounds have shown promising potential in the treatment of numerous musculoskeletal and neurological diseases in humans. However, the mechanisms that underlie its possible effects on neurodevelopment and nervous-system functioning remain poorly understood and need to be further investigated. Although the bulk of research on cannabis and cannabinoids is based on in vitro or rodent models, the zebrafish has now emerged as a powerful in vivo model for drug-screening studies and translational research. We here review the available literature on the use of cannabis/cannabinoids in zebrafish, and particularly in zebrafish models of neurological disorders. A critical analysis suggests that zebrafish could serve as an experimental tool for testing the bioactivity of cannabinoids, and they could thus provide important insights into the safety and efficacy of different cannabis-extract-based products. The review showed that zebrafish exhibit similar behaviors to rodents following cannabinoid exposure. The authors stress the importance of analyzing the full spectrum of naturally occurring cannabinoids, rather than just the main ones, THC and CBD, and they offer some pointers on performing behavioral analysis in zebrafish.

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease.

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5-/- mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.

Early Blood Analysis and Gas Exchange Monitoring in the Canine Neonate: Effect of Dam's Size and Birth Order.

In canines, size at birth is determined by the dam's weight, which would probably affect the newborn's viability due to litter size and birth order. Fetal hypoxia causes distress and acidemia. Identifying physiological blood alterations in the puppy during the first minute of life through the blood gas exchange of the umbilical cord could determine the puppy's risk of suffering asphyxiation during labor. This study aimed to evaluate the effect of the birth order and dam's size during spontaneous labor and the alterations during the first minute of life. The results indicate that the dam's size and the birth order have considerable physiological and metabolic effects in the puppies, mainly in birth order 1 (BO1) in small-size dogs, while in the medium size, the last puppy presented more alterations, probably because of a prolonged whelping which could have fostered hypoxic processes and death. Likewise, with large-size dogs, intrapartum asphyxiation processes were registered during the first minute of life in any birth order.

Trehalose Treatment in Zebrafish Model of Lafora Disease.

Mutations in the EPM2A gene encoding laforin cause Lafora disease (LD), a progressive myoclonic epilepsy characterized by drug-resistant seizures and progressive neurological impairment. To date, rodents are the only available models for studying LD; however, their use for drug screening is limited by regulatory restrictions and high breeding costs. To investigate the role of laforin loss of function in early neurodevelopment, and to screen for possible new compounds for treating the disorder, we developed a zebrafish model of LD. Our results showed the epm2a-/- zebrafish to be a faithful model of LD, exhibiting the main disease features, namely motor impairment and neuronal hyperexcitability with spontaneous seizures. The model also showed increased inflammatory response and apoptotic death, as well as an altered autophagy pathway that occurs early in development and likely contributes to the disease progression. Early administration of trehalose was found to be effective for rescuing motor impairment and neuronal hyperexcitability associated with seizures. Our study adds a new tool for investigating LD and might help to identify new treatment opportunities.

Current Advances in Assessment of Dog's Emotions, Facial Expressions, and Their Use for Clinical Recognition of Pain.

Animals' facial expressions are involuntary responses that serve to communicate the emotions that individuals feel. Due to their close co-existence with humans, broad attention has been given to identifying these expressions in certain species, especially dogs. This review aims to analyze and discuss the advances in identifying the facial expressions of domestic dogs and their clinical utility in recognizing pain as a method to improve daily practice and, in an accessible and effective way, assess the health outcome of dogs. This study focuses on aspects related to the anatomy and physiology of facial expressions in dogs, their emotions, and evaluations of their eyebrows, eyes, lips, and ear positions as changes that reflect pain or nociception. In this regard, research has found that dogs have anatomical configurations that allow them to generate changes in their expressions that similar canids-wolves, for example-cannot produce. Additionally, dogs can perceive emotions similar to those of their human tutors due to close human-animal interaction. This phenomenon-called "emotional contagion"-is triggered precisely by the dog's capacity to identify their owners' gestures and then react by emitting responses with either similar or opposed expressions that correspond to positive or negative stimuli, respectively. In conclusion, facial expressions are essential to maintaining social interaction between dogs and other species, as in their bond with humans. Moreover, this provides valuable information on emotions and the perception of pain, so in dogs, they can serve as valuable elements for recognizing and evaluating pain in clinical settings.

Oxytocin Receptor Gene Polymorphism in Lactating Dogs.

Genetic variations in the oxytocinergic system, known to regulate social behavior throughout the evolution of mammals, are believed to account for differences in mammalian social behavior. Particularly, polymorphic variants of the oxytocin receptor (OXTR) gene have been associated with behavioral variations in both humans and dogs. In this study, we offered evidence of the correlation between levels of salivary oxytocin (sOXT), maternal behavior and a single-nucleotide gene variant in OXTR (rs8679684) in nineteen lactating Labrador Retriever dogs. Carriers of at least one copy of the minor A allele showed higher levels of sOXT and maternal care in comparison with the homozygous T allele carriers. Considering the relevance of mother care in newborn development, these findings could help us to better understand the possible impact of variants in the OXTR gene in selecting dams.

Blood Biomarker Profile Alterations in Newborn Canines: Effect of the Mother's Weight.

This study aims to determine the effect of the weight of bitches on liveborn and stillbirth puppies from eutocic births, and physiological blood alterations during the first minute postpartum. A total of 52 female dogs were evaluated and distributed in four categories: C1 (4.0-8.0 kg, n = 19), C2 (8.1-16.0 kg, n = 16), C3 (16.1-32.0 kg, n = 11), and C4 (32.1-35.8 kg, n = 6). The dams produced 225 liveborn puppies and 47 were classified as stillbirth type II. Blood samples were taken from the umbilical vein to evaluate the concentration of gases, glucose, lactate, calcium, hematocrit levels, and blood pH. The liveborn puppies in C2, C3, and C4 had more evident physiological alterations (hypercapnia, acidosis) than those in C1 (p < 0.05). These signs indicate a process of transitory asphyxiation. The stillborn pups in all four categories had higher weights than their liveborn littermates. C3 and C4 had the highest mean weights (419.86 and 433.79 g, respectively) and mortality rates (C3 = 20.58%, C4 = 24.58%). Results suggest that if the weight of the bitch is >16.1 kg in eutocic births, there is a higher risk of intrapartum physiological alterations and death. The results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth.

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs-/- larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

The Influence of Oxytocin on Maternal Care in Lactating Dogs.

In recent years, many studies on the role of oxytocin (OXT) in the onset of parental care, regulation of social bonding, and modulation of the emotional state have been published. However, its possible regulation of maternal behavior in lactating dogs has not been investigated yet. For this reason, the present study aimed at assessing potential correlations between salivary oxytocin and maternal behavior in 25 lactating Labrador Retriever dogs. Salivary concentrations of OXT (sOXT) were unrelated to the amount of maternal care except for a weak negative correlation with sniffing/poking behavior. Moreover, sOXT was positively correlated with the percentage of male puppies. Sniffing/poking behavior, in turn, was positively correlated with the duration of time the mothers spent out of the whelping box, while the number of male puppies showed a positive correlation with lateral nursing, a position known to provide puppies the easiest access to the milk. Taken together, these results suggest that sOXT may not be strongly associated with maternal care dynamics but could be correlated with sex-biased parental investment in dogs.

Effects of Stroking on Salivary Oxytocin and Cortisol in Guide Dogs: Preliminary Results.

This pilot study aimed at investigating how salivary oxytocin levels are affected by human interaction and isolation in eight guide dogs (six Labrador retrievers and two golden retrievers; four males and four females, 21.87 ± 1.36 months old) just before assignment to the blind person. Each dog engaged, at one-week intervals, in a positive (5 min of affiliative interaction with their trainer) and a negative (5 min of isolation) condition. Saliva samples used for Enzyme Immunoassay (EIA) quantification of salivary oxytocin were collected before and immediately after both experimental conditions. In order to assess potential hypothalamic pituitary adrenal (HPA) axis activation that could have affected oxytocin levels, saliva samples were collected 15 min after both experimental conditions for EIA quantification of salivary cortisol and a behavioral assessment was performed during the negative condition. The results were compared using the Wilcoxon test (p < 0.05). Oxytocin concentrations showed a statistically significant increase after the positive interaction (p = 0.036) and no difference after the negative one (p = 0.779). Moreover, no difference (p = 0.263) was found between the cortisol concentrations after each experimental condition and no signs of distress were observed during the isolation phase. These preliminary findings support the hypothesis that stroking dogs has positive effects on their emotional state independently of hypothalamic pituitary adrenal axis activation.

Social Preference Tests in Zebrafish: A Systematic Review.

The use of animal models in biology research continues to be necessary for the development of new technologies and medicines, and therefore crucial for enhancing human and animal health. In this context, the need to ensure the compliance of research with the principles Replacement, Reduction and Refinement (the 3 Rs), which underpin the ethical and human approach to husbandry and experimental design, has become a central issue. The zebrafish (Danio rerio) is becoming a widely used model in the field of behavioral neuroscience. In particular, studying zebrafish social preference, by observing how an individual fish interacts with conspecifics, may offer insights into several neuropsychiatric and neurodevelopmental disorders. The main aim of this review is to summarize principal factors affecting zebrafish behavior during social preference tests. We identified three categories of social research using zebrafish: studies carried out in untreated wild-type zebrafish, in pharmacologically treated wild-type zebrafish, and in genetically engineered fish. We suggest guidelines for standardizing social preference testing in the zebrafish model. The main advances gleaned from zebrafish social behavior testing are discussed, together with the relevance of this method to scientific research, including the study of behavioral disorders in humans. The authors stress the importance of adopting an ethical approach that considers the welfare of animals involved in experimental procedures. Ensuring a high standard of animal welfare is not only good for the animals, but also enhances the quality of our science.

Feeding Enrichment in a Captive Pack of European Wolves (Canis Lupus Lupus): Assessing the Effects on Welfare and on a Zoo's Recreational, Educational and Conservational Role.

This study investigated the effects of two feeding enrichment programs on the behaviour of a captive pack of European wolves (Canis lupus lupus) and their correlation with both zoo visitors' interest towards the exhibit and their overall perception of the species. Behavioural data (exploration, stereotypies, social interactions, activity/inactivity rates) were collected on four male wolves during four two-week long phases: initial control, hidden food, novel object, final control. Three observation sessions were performed daily: before, during and after feeding. Number of visitors and their permanence in front of the exhibit were recorded. After watching the wolves, visitors were asked to fill out a brief questionnaire in order to investigate their perception of captive wolf welfare, as well as their attitude towards wolf conservation issues. Despite the high inter-individual variability in their behavioural response, all wolves seemed to benefit from feeding enrichment. With regard to visitors, interest in the exhibit increased when enrichment was provided. Visitors' perception of the level of welfare of wolves improved if they attended a feeding session, especially during the novel object phase. Visitors' attitude towards wolf conservation issues also improved during feeding sessions, regardless of enrichment provision.

Swimming in Deep Water: Zebrafish Modeling of Complicated Forms of Hereditary Spastic Paraplegia and Spastic Ataxia.

Hereditary spastic paraplegia (HSP) and hereditary ataxia (HA) are two groups of disorders characterized, respectively, by progressive dysfunction or degeneration of the pyramidal tracts (HSP) and of the Purkinje cells and spinocerebellar tracts (HA). Although HSP and HA are generally shown to have distinct clinical-genetic profiles, in several cases the clinical presentation, the causative genes, and the cellular pathways and mechanisms involved overlap between the two forms. Genetic analyses in humans in combination with in vitro and in vivo studies using model systems have greatly expanded our knowledge of spinocerebellar degenerative disorders. In this review, we focus on the zebrafish (Danio rerio), a vertebrate model widely used in biomedical research since its overall nervous system organization is similar to that of humans. A critical analysis of the literature suggests that zebrafish could serve as a powerful experimental tool for molecular and genetic dissection of both HA and HSP. The zebrafish, found to be very useful for demonstrating the causal relationship between defect and mutation, also offers a useful platform to exploit for the development of therapies.