Application of Blended Learning to Veterinary Gross Anatomy Practical Sessions: Students' Perceptions of Their Learning Experience and Academic Outcomes.

The use of blended learning strategies is increasingly common in health sciences, including veterinary medicine; however, there are very few descriptions of these methods being applied to practicals. We describe here the application of blended learning based on the implementation of flipped classrooms with collaborative learning and gamification to the 2020-2021 veterinary medicine gross anatomy practicals at CEU Cardenal Herrera University (Spain). Students prepared for the sessions by pre-viewing videos and taking a quiz before the start. The sessions were conducted in small groups where students learned through collaborative work and reviewed their learning with a card game. A small but significant increase was observed when comparing the scores of practical exams of the locomotor apparatus with those of 2018-2019 (6.79 ± 2.22 vs. 6.38 ± 2.24, p < 0.05), while the scores were similar (7.76 ± 1.99 vs. 7.64 ± 1.92) for the organ system exams. Students' responses in a satisfaction survey were mostly positive (>80%) regarding the motivating and learning-facilitating effect of this educational method. Our work shows that the application of blended learning in anatomy practicals based on a flipped classroom and with elements of gamification and collaborative work can be an effective way to improve the learning experience of students.

Anxiety, emotions, and thoughts of veterinary medicine students during their first visit to the dissection room.

The use of cadavers is essential for veterinary anatomy learning. However, facing an animal corpse can be stressful for veterinary students because of their empathy toward animals. The objective of this study was to evaluate veterinary medicine students' emotions, feelings, and anxiety levels related to practicals with dog cadavers. Two questionnaires were administered to 1st year students (n = 168) at CEU Cardenal Herrera University in Valencia (Spain) before and after their first practical session with cadavers. The application of State-Trait Anxiety Inventory questionnaires showed that "state anxiety" decreased significantly (p < 0.05), from a score of 14.8 before the practical to 10.4 after, and that female students showed higher but not significantly different levels than males. Most (64%) of the students were not willing to donate the bodies of their pets, and those students were more stressed before the practical than their peers, although their anxiety levels significantly decreased by the end of the session. The majority of the students answered positively about emotions, such as feeling calm, safe, not nervous, relaxed and not worried before the practical, and this increased significantly to more than 80% by the end of the session. The visualization of educational videos prior to the session was evaluated positively by students. These results agree with those reported in other health science disciplines, showing that students face practical sessions with corpses in a similar way and suggesting that the use of videos can help decrease anxiety and enhance their learning experience.

Learning veterinary anatomy playing cards.

Gamification is a dynamic tool for educational transformation useful to encourage student interest and enhance learning. Here we present a study conducted to investigate the effectiveness of an educational card game developed by us in veterinary anatomy practicals to reinforce knowledge acquisition in veterinary students. A total of four sets of cards were designed, each one with different anatomical topics (structure identification, articulation and positioning, clinical anatomy, and comparative anatomy); students were arranged in small groups (7-10 students per group) and played the game at the end of each anatomy practical session, discussing the corresponding questions, randomly chosen, as a team. This activity was highly valued by students, most of whom (>80%) expressed that the game was enjoyable, challenging, helpful to improve their knowledge and understanding in clinical anatomy, and effective for anatomy exam preparation. Thus, the use of educational games in practical sessions seems to improve student engagement in the learning process individually and as a team.NEW & NOTEWORTHY The development and implementation of a card game as a training resource that allows learning veterinary anatomy in a motivating and cooperative environment, promoting teamwork, relationships, and trust and communication between colleagues, is described. Stimulating the ability to solve problems as a team has provided help to students preparing for their exams in a more dynamic and enjoyable way.

Rnd3 is necessary for the correct oligodendrocyte differentiation and myelination in the central nervous system.

Rho small GTPases are proteins with key roles in the development of the central nervous system. Rnd proteins are a subfamily of Rho GTPases, characterized by their constitutive activity. Rnd3/RhoE is a member of this subfamily ubiquitously expressed in the CNS, whose specific functions during brain development are still not well defined. Since other Rho proteins have been linked to the myelination process, we study here the expression and function of Rnd3 in oligodendrocyte development. We have found that Rnd3 is expressed in a subset of oligodendrocyte precursor cells and of mature oligodendrocytes both in vivo and in vitro. We have analyzed the role of Rnd3 in myelination using mice lacking Rnd3 expression (Rnd3gt/gt mice), showing that these mice exhibit hypomyelination in the brain and a reduction in the number of mature and total oligodendrocytes in the corpus callosum and striatum. The mutants display a decreased expression of several myelin proteins and a reduction in the number of myelinated axons. In addition, myelinated axons exhibit thinner myelin sheaths. In vitro experiments using Rnd3gt/gt mutant mice showed that the differentiation of the precursor cells is altered in the absence of Rnd3 expression, suggesting that Rnd3 is directly required for the differentiation of oligodendrocytes and, in consequence, for the correct myelination of the CNS. This work shows Rnd3 as a new protein involved in oligodendrocyte maturation, opening new avenues to further study the function of Rnd3 in the development of the central nervous system and its possible involvement in demyelinating diseases.

A Modified Approach for the Ultrasound-Guided Quadratus Lumborum Block in Dogs: A Cadaveric Study.

Ultrasound-guided quadratus lumborum block (QLB) is a locoregional technique described in canine cadavers. The aim of this study was to assess a modified approach to QLB to minimise potential complications such as abdominal organ puncture. Nine canine cadavers were included and were positioned in lateral recumbency. An ultrasound-guided QLB was performed on each side. The probe was placed in the transverse position over the lumbar muscles just caudal to the last rib, and a needle was advanced in-plane from a dorso-lateral to a ventro-medial. A volume of 0.2 mL kg-1 of a mixture of iomeprol and methylene blue was injected. Computed tomography (CT) and dissection were performed to evaluate the spreading. Success was defined as staining of the nerve with a length of more than 0.6 cm. Potential complications such as intra-abdominal, epidural, or intravascular spreading of the mixture were also assessed. The CT images showed a T13 to L7 vertebra distribution, with a median of 5 (3-6). Dissection showed staining of the nerves from T13 to L4, with a median of 3 (2-5). No complications were found. This modified approach to QLB is safe and shows similar results to the previous studies in canine carcass.

Genetic ablation of the Rho GTPase Rnd3 triggers developmental defects in internal capsule and the globus pallidus formation.

The forebrain includes the cerebral cortex, the thalamus, and the striatum and globus pallidus (GP) in the subpallium. The formation of these structures and their interconnections by specific axonal tracts take place in a precise and orchestrated time and spatial-dependent manner during development. However, the knowledge of the molecular and cellular mechanisms that are involved is rather limited. Moreover, while many extracellular cues and specific receptors have been shown to play a role in different aspects of nervous system development, including neuron migration and axon guidance, examples of intracellular signaling effectors involved in these processes are sparse. In the present work, we have shown that the atypical RhoGTPase, Rnd3, is expressed very early during brain development and keeps a dynamic expression in several brain regions including the cortex, the thalamus, and the subpallium. By using a gene-trap allele (Rnd3gt ) and immunological techniques, we have shown that Rnd3gt/gt embryos display severe defects in striatal and thalamocortical axonal projections (SAs and TCAs, respectively) and defects in GP formation already at early stages. Surprisingly, the corridor, an important intermediate target for TCAs is still present in these mutants. Mechanistically, a conditional genetic deletion approach revealed that Rnd3 is primarily required for the normal development of Medial Ganglionic Eminence-derived structures, such as the GP, and therefore acts non-cell autonomously in SAs and TCAs. In conclusion, we have demonstrated the important role of Rnd3 as an early regulator of subpallium development in vivo and revealed new insights about SAs and TCAs development.

Neural Stem Cells Direct Axon Guidance via Their Radial Fiber Scaffold.

Neural stem cells directly or indirectly generate all neurons and macroglial cells and guide migrating neurons by using a palisade-like scaffold made of their radial fibers. Here, we describe an unexpected role for the radial fiber scaffold in directing corticospinal and other axons at the junction between the striatum and globus pallidus. The maintenance of this scaffold, and consequently axon pathfinding, is dependent on the expression of an atypical RHO-GTPase, RND3/RHOE, together with its binding partner ARHGAP35/P190A, a RHO GTPase-activating protein, in the radial glia-like neural stem cells within the ventricular zone of the medial ganglionic eminence. This role is independent of RND3 and ARHGAP35 expression in corticospinal neurons, where they regulate dendritic spine formation, axon elongation, and pontine midline crossing in a FEZF2-dependent manner. The prevalence of neural stem cell scaffolds and their expression of RND3 and ARHGAP35 suggests that these observations might be broadly relevant for axon guidance and neural circuit formation.

RhoE deficiency alters postnatal subventricular zone development and the number of calbindin-expressing neurons in the olfactory bulb of mouse.

The subventricular zone represents an important reservoir of progenitor cells in the adult brain. Cells from the subventricular zone migrate along the rostral migratory stream and reach the olfactory bulb, where they originate different types of interneurons. In this work, we have analyzed the role of the small GTPase RhoE/Rnd3 in subventricular zone cell development using mice-lacking RhoE expression. Our results show that RhoE null mice display a remarkable postnatal broadening of the subventricular zone and caudal rostral migratory stream. This broadening was caused by an increase in progenitor proliferation, observed in the second postnatal week but not before, and by an altered migration of the cells, which appeared in disorganized cell arrangements that impaired the appropriate contact between cells in the rostral migratory stream. In addition, the thickness of the granule cell layer in the olfactory bulb was reduced, although the density of granule cells did not differ between wild-type and RhoE null mice. Finally, the lack of RhoE expression affected the olfactory glomeruli inducing a severe reduction of calbindin-expressing interneurons in the periglomerular layer. This was already evident in the newborns and even more pronounced 15 days later when RhoE null mice displayed 89% less cells than control mice. Our results indicate that RhoE has pleiotropic functions on subventricular cells because of its role in proliferation and tangential migration, affecting mainly the development of calbindin-expressing cells in the olfactory bulb.

Analysis of RhoE expression in the testis, epididymis and ductus deferens, and the effects of its deficiency in mice.

Rho proteins are a large family of GTPases involved in the control of actin cytoskeleton dynamics, proliferation and survival. Rnd1, Rnd2 and RhoE/Rnd3 form a subfamily of Rho proteins characterized by being constitutively active. The role of these proteins has been studied during the last years in several systems; however, little is known about their expression and functions in the reproductive organs. In this work we analysed the localization and the effect of RhoE deficiency in the testes using mice lacking RhoE expression (RhoE gt/gt), and our research shows some unexpected and relevant results. First, we have observed that RhoE is only expressed in Leydig cells within the testicular parenchyma and it is absent of seminiferous tubules. In addition, RhoE is expressed in the excurrent ducts of the testis, including the ductuli efferentes, epididymis and ductus deferens. Moreover, the testes of postnatal 15-day-old RhoE null mice are smaller, both in absolute values and in relation to the body weight. Furthermore, the dimensions of their seminiferous tubules are also reduced compared with wild-types. In order to study the role of RhoE in the adult, we analysed heterozygous animals as RhoE null mice die early postnatally. Our results show that the testes of adult RhoE heterozygous mice are also smaller than those of the wild-types, with a 17% decrease in the ratio testis weight/body weight. In addition, their seminiferous tubules have reduced tubular diameter (12%) and a thinner epithelial wall (33%) that appears disorganized and with a swollen lumen. Finally, and probably as a consequence of those alterations, the sperm concentration of heterozygous animals was found to be lower than in the wild-types. These results indicate that accurate levels of RhoE in the testes are necessary for a correct development and function of male gonads, and suggest novel and unexpected roles of Rnd GTPases in the reproductive physiology.

Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts.

The function of osteoclasts (OCs), multinucleated giant cells (MGCs) of the monocytic lineage, is bone resorption. To resorb bone, OCs form podosomes. These are actin-rich adhesive structures that pattern into rings that drive OC migration and into "sealing-zones" (SZs) that confine the resorption lacuna. Although changes in actin dynamics during podosome patterning have been documented, the mechanisms that regulate these changes are largely unknown. From human monocytic precursors, we differentiated MGCs that express OC degradation enzymes but are unable to resorb the mineral matrix. We demonstrated that, despite exhibiting bona fide podosomes, these cells presented dysfunctional SZs. We then performed two-step differential transcriptomic profiling of bone-resorbing OCs versus nonresorbing MGCs to generate a list of genes implicated in bone resorption. From this list of candidate genes, we investigated the role of Rho/Rnd3. Using primary RhoE-deficient OCs, we demonstrated that RhoE is indispensable for OC migration and bone resorption by maintaining fast actin turnover in podosomes. We further showed that RhoE activates podosome component cofilin by inhibiting its Rock-mediated phosphorylation. We conclude that the RhoE-Rock-cofilin pathway, by promoting podosome dynamics and patterning, is central for OC migration, SZ formation, and, ultimately, bone resorption.

Neuronal polarization is impaired in mice lacking RhoE expression.

Proper development of neuronal networks relies on the polarization of the neurons, thus the establishment of two compartments, axons and dendrites, whose formation depends on cytoskeletal rearrangements. Rnd proteins are regulators of actin organization and they are important players in several aspects of brain development as neurite formation, axon guidance and neuron migration. We have recently demonstrated that mice lacking RhoE/Rnd3 expression die shortly after birth and have neuromotor impairment and neuromuscular alterations, indicating an abnormal development of the nervous system. In this study, we have further investigated the specific role played by RhoE in several aspects of neuronal development by using hippocampal neuron cultures. Our findings show that neurons from a mice lacking RhoE expression exhibit a decrease in the number and the total length of the neurites. We also show that RhoE-deficient neurons display a reduction in axon outgrowth and a delay in the process of neuronal polarization. In addition, our results suggest an involvement of the RHOA/ROCK/LIMK/COFILIN signaling pathway in the neuronal alterations induced by the lack of RhoE. These findings support our previous report revealing the important role of RhoE in the normal development of the nervous system and may provide novel therapeutic targets in neurodegenerative disorders.

Glucose transporters GLUT4 and GLUT8 are upregulated after facial nerve axotomy in adult mice.

Peripheral nerve axotomy in adult mice elicits a complex response that includes increased glucose uptake in regenerating nerve cells. This work analyses the expression of the neuronal glucose transporters GLUT3, GLUT4 and GLUT8 in the facial nucleus of adult mice during the first days after facial nerve axotomy. Our results show that whereas GLUT3 levels do not vary, GLUT4 and GLUT8 immunoreactivity increases in the cell body of the injured motoneurons after the lesion. A sharp increase in GLUT4 immunoreactivity was detected 3 days after the nerve injury and levels remained high on Day 8, but to a lesser extent. GLUT8 also increased the levels but later than GLUT4, as they only rose on Day 8 post-lesion. These results indicate that glucose transport is activated in regenerating motoneurons and that GLUT4 plays a main role in this function. These results also suggest that metabolic defects involving impairment of glucose transporters may be principal components of the neurotoxic mechanisms leading to motoneuron death.

RhoE deficiency produces postnatal lethality, profound motor deficits and neurodevelopmental delay in mice.

Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt) are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD) 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders.

Developmental regulation of glucose transporters GLUT3, GLUT4 and GLUT8 in the mouse cerebellar cortex.

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.

Expression of GLUT8 in mouse intestine: identification of alternative spliced variants.

GLUT8 is a facilitative glucose transporter composed of 10 exons coding for a 477 amino acids protein. It is mainly expressed in the testis, but it has also been studied in a number of tissues such as brain, adipose tissue, and liver. In this work, we have characterized the expression of GLUT8 in the small and large intestine under normal physiological conditions. Protein assay revealed low GLUT8 protein levels in the intestine compared to the testis, with higher levels in the colon than in the small intestine. Immunohistochemistry studies showed an intracellular localization of GLUT8 in enterocytes and colonocytes with a supranuclear distribution next to the apical membrane. GLUT8 immunoreactivity was also detected in the crypt cells. Interestingly, we have identified three additional transcriptional variants in mouse intestine (mGLUT-SP1, mGLUT8-SP2, and mGLUT8-SP3) produced by the deletion of one, two, and four exons, respectively, whereas only the entire mRNA was detected in the testis. Expression of these alternative variants did not have an effect on glucose consumption in 3T3-L1 cells. Although the specific function of GLUT8 in intestine remains unclear, the alternative splicing of GLUT8 could reflect a mechanism for the regulation of the gene expression in a tissue-specific manner by targeting GLUT8 mRNA for nonsense-mediated decay.

Anatomical variations in the cephalic and omobrachial veins in the dog.

In the dog, the blood from the antebrachial part of the cephalic vein is typically drained by three vessels: the axillobrachial, the omobrachial and the proximal segment of the cephalic vein. In this work, two anatomical variations affecting the omobrachial and the proximal part of the cephalic vein in dogs are reported. The bilateral absence of the omobrachial vein was observed in two adult mongrel dogs and the lack of the proximal segment of the cephalic vein in one dog. These differences suggest that a certain degree of variation is normal in the layout of the superficial veins of the thoracic limb in the dog and implies that the blood egress from the cephalic vein in the dog, as in other species, is sometimes accomplished by only two veins.

Differential expression of glucose transporter GLUT8 during mouse spermatogenesis.

GLUT8 is a facilitative glucose transporter expressed at high levels in the testis. In this study, we analyzed the GLUT8 expression in mouse testis during spermatogenesis by RT-PCR, Western blot and immunohistochemistry methods. Our results show that GLUT8 expression is limited to spermatids and spermatozoa in the testis. Expression begins when round spermatids are formed at postnatal day 24. The expression persists throughout spermiogenesis, and it is also detected in spermatozoa, but it is absent in more immature germ cells, Sertoli cells and interstitial tissue. GLUT8 immunoreactivity is always restricted to the acrosomic system in a manner that matches the acrosome system formation. The GLUT8 expression is mainly associated with the acrosomic membrane in the acrosome, although significant immunoreactivity is also found inside the acrosomic lumen. The specific GLUT8 location suggests that this transporter plays a pivotal role in the fuel supply of spermatozoa, and in the traffic of sugars during the capacitation and fertilization processes.

Anomalous insertion of the splenius muscle in a dog.

The splenius muscle in the dog is a triangular muscle extending from the third thoracic vertebra to the skull. It inserts on the nuchal line of the occipital bone and the mastoid part of the temporal bone and occasionally it also sends a strong serration to the transverse process of the axis. Here we describe a new, anomalous insertion of both the left and right splenius muscles in one young adult dog routinely prepared for anatomical dissection. In this dog, a group of muscle fibres diverged ventrolaterally from both the right and left splenial muscles to be inserted at the end of the transverse processes of the third cervical vertebra. This anomalous insertion of the splenius muscle corresponds to the splenius cervicis muscle present in other species.