The Anatomical Record

In birds, the quadrate bone serves as a hinge articulating with the lower jaw and the skull, playing an important mechanical role in the feeding apparatus. Avian cranial kinesis is dependent on the streptostylic quadrate transferring force from the adductor muscles at the back of the skull toward the beak, as part of a four-bar mechanical linkage to elevate and depress the bill. The complex morphology of the bird quadrate has led to authors adopting a range of alternative terminologies to describe the same anatomical structures and character states, impeding clarity of communication and presenting a barrier to progress in our understanding of the evolution of this important component of the avian feeding apparatus. Here, we reconcile terminological discord among previous studies on avian quadrate morphology and propose a stable nomenclature for future work. To characterise the considerable variation in quadrate form across crown bird diversity, we present an extensive anatomical atlas of the avian quadrate and summarise major patterns of quadrate morphological variation across extant avian phylogeny. In addition, we investigate macroevolutionary patterns in avian quadrate morphology, incorporating comparisons of crown birds and Late Cretaceous near-crown stem birds. We demonstrate that quadrate characters are useful for diagnosing a range of major avian subclades, and suggest that numerous distinctive features are likely to be associated with important biomechanical consequences. This investigation has implications for resolving the unsettled phylogenetic relationships of extinct bird clades such as Pelagornithidae and Gastornithiformes, as well as controversial relationships within several extant groups.

Brain banks provide small tissue samples fixed in neutral-buffered-formalin (NBF), but human anatomy teaching laboratories could provide full brains fixed with solutions that are more appropriate for gross anatomy such as a saturated salt solution (SSS) or an alcohol-formaldehyde solution (AFS). Advanced aging and prolonged exposure to aldehydes are known to enhance brain tissue autofluorescence (AF), limiting the efficacy of immunofluorescence (IF) procedures. We have previously shown by IF staining the antigenicity preservation in mouse brains fixed with the three solutions. We now aimed to compare the quality of IF staining in human brains fixed with SSS, AFS and NBF. In addition, we compared the efficiency of AF quenching methods, namely the application of SudanBlackB (SBB) and the treatment of sections with sodium borohydride (NaBH4). Blocks of neocortex were extracted from 18 brains (NBF=6, SSS=6, AFS=6) and cut into 40{micro}m sections. Neurons (anti-NeuN, AlexaFluor-488) and astrocytes (anti-GFAP, AlexaFluor-555) were revealed with IF after an antigen retrieval protocol, while two treatments (SBB or NaBH4) were used to quench AF. We then assessed the degree of AF (criteria: background or cell AF) and the immunostaining quality with excitation wavelengths of 488nm, 555nm and 647nm. Brains fixed with all three solutions showed well-labeled astrocytes, whereas neurons werent always stained, but this was not associated to the fixative solution. The overall AF intensity was similar in sections from brains fixed with all three solutions. Finally, the SBB treatment was the most effective at reducing AF in all specimens. Given the similarity in AF and antigenicity assessment across the three solutions, we conclude that brains fixed with SSS and AFS could be good alternatives for NBF-fixed specimens in the context of IF experiments processed with a SBB protocol. Highlights- Immunofluorescence staining is feasible in brains fixed with anatomy labs solutions - GFAP is less affected by fixation than NeuN - Autofluorescence can be reduced by Sudan Black treatment

Domestic dogs (Canis familiaris) display more morphological variation than any other mammal. Cranial morphology has been extensively studied, as have the relationships with function, development, genetics, veterinary medicine, and breed welfare. Postcrania remain comparatively understudied, despite well-documented breed-specific predispositions to musculoskeletal disease. Here, we apply three-dimensional landmark-free morphometrics to quantify the shape of 743 elements from 213 dogs, including the scapula, humerus, radius, ulna, pelvic girdle, femur, tibia, and fibula. We assess integration among limb elements and investigate drivers of shape variation within and between breeds. Across most breeds, limb bone shape is strikingly similar. Dachshunds, however, exhibit distinct morphology across all elements and one to two orders of magnitude greater variation than any other breed. Despite this disparity, integration remains high between all element pairs. Remarkably, we find no significant relationship between bone shape and body mass, age, or pathology, but comparison with historic specimens reveals marked changes in dachshund long bone shape over the past [~]150 years. These extreme differences are not shared by other sampled chondrodysplastic breeds, underscoring the need to understand morphological diversity beyond simple categorisation. These findings provide a quantitative framework for linking postcranial morphology with function, disease risk, and evidence-based improvements to canine welfare.

Studies of whole brain cryopreservation are rare but are potentially important for a variety of applications. It has been demonstrated that ultrastructure in whole rabbit and pig brains can be cryopreserved by vitrification (ice-free cryopreservation) after prior aldehyde fixation, but fixation limits the range of studies that can be done by neurobiologists, including studies that depend upon general molecular integrity, signal transduction, macromolecular synthesis, and other physiological processes. We now show that whole brain ultrastructure can be preserved by vitrification without prior aldehyde fixation. Rabbit brain perfusion with the M22 vitrification solution followed by vitrification, warming, and fixation showed an absence of visible ice damage and overall structural preservation, but osmotic brain shrinkage sufficient to distort and obscure neuroanatomical detail. Neuroanatomical preservation in the presence of M22 was also investigated in human cerebral cortical biopsies taken after whole brain perfusion with M22. These biopsies did not form ice upon cooling or warming, and high power electron microscopy showed dehydrated and electron-dense but predominantly intact cells, neuropil, and synapses with no signs of ice crystal damage, and partial dilution of these samples restored normal cortical pyramidal cell shapes. To further evaluate ultrastructural preservation within the severely dehydrated brain, rabbit brains were perfused with M22 and then partially washed free of M22 before fixation. Perfusion dilution of the brain to 3-5M M22 resulted in brain re-expansion and the re-appearance of well-defined neuroanatomical features, but rehydration of the brain to 1M M22 resulted in ultrastructural damage suggestive of preventable osmotic injury caused by incomplete removal of M22. We conclude that both animal and human brains can be cryopreserved by vitrification with predominant retention of ultrastructural integrity without the need for prior aldehyde fixation. This observation has direct relevance to the feasibility of human cryopreservation, for which direct evidence has been lacking until this report. It also provides a starting point for perfecting brain cryopreservation, which may be necessary for lengthy space travel and could allow future medical time travel.

IntroductionWe investigated the direct effect of transverse maxillary constriction on nasal septal deviation (NSD) and nasal floor slanting. Methods and Materials60 growing Wistar rats (21days old) were divided into four groups (n=15): 1) Experimental Group 1 received active constriction force (100cN), 2) Experimental Group 2 received active expansion force (100cN), 3) Sham received the same spring as Experimental Groups without receiving any active force, and 4) Control group did not receive any appliance. Samples were collected after 28 days for microcomputed tomography (CT) analysis. ResultsExperimental Group 1 demonstrated maxillary constriction (both skeletal and dental), accompanied by mandibular shift on closure, clockwise mandibular rotation, and increased mandibular plane angle and facial height. Constriction also produced severe nasal floor slanting in the molar area that extended posteriorly. Nasal floor canting was accompanied by a slanted vomer and a C-shaped NSD. The direction of nasal floor canting and mandibular shift was always similar. Experimental Group 2, on the other hand, was not associated with nasal deviation, and a slight slanting of the nasal floor was observed only when there was a mandibular shift. ConclusionOur study suggests that the constricting transverse forces applied to the maxilla can induce slanting of the nasal floor and, consequently, the vomer, which in turn can lead to nasal septal deviation. Slanting of the nasal floor is caused mainly by rotation of the hemimaxilla in response to transverse forces and changes in occlusal forces due to a mandibular shift.

It has been hypothesized that subterranean mammals have evolved increased skin elasticity to reduce friction when moving through their underground tunnel systems. This trait is commonly believed to be mediated by greatly elongated hyaluronan (HA) polymers in the dermal extracellular matrix, which have been reported from different distantly related burrowing mammals. However, replicating these findings has proven difficult, and a mechanism by which HA polymer size could modify skin elasticity has not been proposed. In fact, experimental data on skin biomechanics in burrowing mammals are currently unavailable. Here, we quantify the molecular mass of HA polymers extracted from the tissues of Ansells mole-rat (Fukomys anselli), a burrowing rodent yet unstudied in that respect, and investigate skin biomechanics in subterranean and epigeic small mammal species by means of in vivo-cutometry. We did not recover extremely elongated HA polymers in Ansells mole-rat, conflicting with published findings in congeneric species and the naked mole-rat. Polymer length in mole-rats was found to be moderately increased compared to guinea pigs across tissues, though. Our data on skin biomechanics indicate that subterranean mammal skin is not more elastic than that of epigeic forms. Interestingly, the skin of the naked mole-rat was characterized by very high stiffness. Uniaxial tensile tests demonstrated that it also exhibits exceptional tensile strength. Hence, we challenge the idea that hyaluronan or a subterranean ecology notably influences skin elasticity in small mammals and suggest that previous studies may have confused elasticity with skin looseness, a fundamentally different phenomenon.

Left-right asymmetry of the brain is well recognized in various animals including C. elegans, drosophila and zebrafish. In primates, most of the brain studies describe side of the brain. However, in spite of huge amounts of accumulating rodent studies on neuroscience, most of rodent studies do not distinguish the brain side. The pig brain is considered to occupy an intermediate position between primates and rodents in terms of structural complexity and brain function. Moreover, the numbers of studies using genetic manipulation of pigs are drastically increasing. So, we investigated microminipig (MMP) brain mesoscopic anatomy focusing on left-right differences of its morphology. Here, we show the anterior cingulate cortex, perirhinal cortex, and cerebellum of male and female MMPs, are structurally asymmetrical. The cerebellar vermis, paravermis is tilted from the midline and the consequently the cerebellar cortex exhibits asymmetrical morphology. The anterior cingulate gurus exhibited protrusion and invagination toward the midline on the right and left side, respectively. The left perirhinal lobe exhibited distinct patterns of cortical gyration between left and right side. These data demonstrate that MMPs are one of the suitable model animals for investigating cerebral and cerebellar asymmetry.

Paleobiologists commonly use genera as a proxy for species in biodiversity studies. However, a lingering concern is that patterns among genera might not always faithfully reflect patterns among species. To date, the concern has focused chiefly on measured patterns of richness over time and on implied origination and extinction rates. However, similar issues might arise for studies of morphological disparity. Moreover, there potentially are additional implications of disparity patterns among species versus those among genera concerning the range of observable anatomical characters and whether disparity within genera is comparable to disparity among genera. If clades have some relatively slowly changing characters that workers have used to denote different genera, then we would expect to see congeneric species to cluster in morphospace; however, if such characters are rare, then within-genus disparity might approach among-genus disparity. Here, we use genus-level and species-level disparity patterns among acanthoceratid ammonoids from the Late Cretaceous. In particular, we examine whether these different level imply different evolutionary dynamics over a major ecological event (Ocean Anoxic Event 2) and how disparity within genera (i.e., among congeneric species) compares to disparity among genera. We find genus-level disparity somewhat inflates early acanthoceratid disparity but implies similar patterns over the OAE2. We also find that within-genus disparity is slightly lower than among-genus, but not hugely so. The combined results suggest that acanthoceratoid shell anatomy does not really show "genus" level characters, even if congeneric species do tend to be more similar to each other than to species in other genera. Thus, this might provide more of a warning for other types of studies using anatomical data (e.g., phylogenetic studies) than for disparity studies. Non-technical SummaryMany paleobiologists use genera to examine scientific questions. This leads to questions over whether this broader approach misses important species-level patterns. This study uses acanthoceratid ammonoids from the Late Cretaceous to examine disparity patterns at both the genus-level and the species-level. We specifically examine the disparity at both levels of this group over a time of high stress for this group, Ocean Anoxic Event 2 (OAE2). Our results show that genus-level disparity slightly exaggerates early acanthoceratid disparity but lowers to a similar pattern to the species-level disparity during OAE2. Within-genus disparity is shown to be slightly lower than among-genus, but not enough to be startling. Together, these results indicate that while some species within the same genus tend to be more alike to each other than those in other genera, there isnt a set of true "genus" level characters. This outcome leads to a warning against using anatomical data in phylogenetic studies, but less so for disparity studies.

Documenting and understanding the welfare of aging animals are crucial for maintaining their well-being and making appropriate management decisions. This study details the behaviors of an extremely old rhesus macaque (ISK) in which senile plaques and phosphorylated tau deposition were observed in post-mortem pathological analyses of the brain. We report on the activity bsudgets, behavioral rhythms, gait, quality of life (QoL) scores, and anecdotal episodes of this individual. The average 24-hour activity budgets, analyzed from surveillance camera recordings, revealed that ISK spent most of her time inactive. ISK was sometimes active at night, though her behavior remained predominantly diurnal. Gait analysis suggested that her movement patterns changed between the first (December 2020) and the last (June 2021) assessment. QoL assessments, using a scoring sheet, indicated relatively good well-being until the later stage of her life. An anecdotal episode, along with the husbandry diary, suggested signs of cognitive decline. These results suggest possible signs of physical decline, and some behavioral changes that could be associated with cognitive decline in an extremely old rhesus macaque. However, we could not confirm cognitive dysfunction without further controlled cognitive testing. We hope that future studies will consider the behavioral symptoms observed in this study as monitoring items to better understand physical and cognitive decline, and possible relationships with QoL in primates.

Intervertebral disc (IVD) injury is a major cause of low-back pain and can lead to structural deficits and mechanical instability. When the IVD is compromised, neuromuscular compensation by paraspinal muscles, such as the multifidus (MF) and longissimus (ML), is critical for maintaining spine stability. However, it is unknown how IVD injury and its interaction with nociception affect neuromuscular control. This study assessed the effects of IVD injury and additional muscle-derived nociception on trunk motor control during locomotion in a rat model. IVD injury was induced via needle puncture at L4/L5. One week later, hypertonic saline was injected into the lumbar MF to induce nociception. Trunk and pelvic kinematics, bilateral EMG activity of MF and ML were recorded during treadmill locomotion at baseline, one week after IVD injury, and immediately following hypertonic saline injection. Trunk and pelvic kinematics and bilateral muscle activation patterns remained largely consistent across conditions. No significant changes were found in stride duration, pelvic, lumbar and spine angle changes, variability, or movement asymmetry. MF activation was bilaterally synchronized, whereas ML showed left-right alternating activation patterns. Following IVD injury, right MF mean activation and EMG variability increased significantly compared to baseline. When muscle-derived nociception was added in the unstable spine (IVD injury) condition, left MF minimum amplitude was significantly reduced, and instability-related increases in right MF mean activation and variability were attenuated, but not fully reversed. These findings suggest that IVD injury, alone or in combination with muscle-derived nociception, elicits localized neuromuscular adaptations without disrupting the global locomotor patterns.

Uintatheres, mammals belonging to the extinct order Dinocerata, are among the most recognizable of all Paleogene ([~]66 - 23 Ma) organisms. Unmistakable for their bizarre skulls with multiple pairs of horns and saber-like upper canines, uintatheres have captivated paleontologists since the late nineteenth century. Since their initial discovery, uintatheres have been regarded as a classic example of dramatic sexual dimorphism in the fossil record, with males purported to be larger and possess more prominent horns and canines than females. However, the hypothesis that uintatheres were highly sexually dimorphic has never been formally tested. Here, I use traditional, linear morphometrics on a collection including most known skulls of Uintatherium anceps to quantify patterns of cranial variation within this taxon. Despite using a variety of traditional and novel statistical methods, I fail to detect any evidence of strong sexual dimorphism in Uintatherium. To verify my approach, I assembled a similarly sized dataset from Bison bison as an extant analog, and found strong, consistent evidence of sexual dimorphism. In light of these findings, as well as the current understanding of uintathere systematics and paleoecology, I argue that strong sexual dimorphism should not be treated as the null hypothesis for this clade.

Trilobites had biramous appendages with an inner endopodite (walking leg) and outer exopodite (gill) connected to the body through the protopodite (limb base). Whereas both endopodite and protopodite were involved in both locomotion and feeding, the exopodite has been subject to various functional interpretations including respiration, ventilation and swimming. Evidence from sites with exceptional fossil preservation indicate that trilobite exopodites show substantial variability in terms of the number and size of their articles, lamellae and setae, but the implications of this morphological diversity have never been investigated. Here, we created anatomically correct 3D models of exopodites in O. serratus and T. eatoni to calculate the SA of the lamellae and explore its relationship with body size. Our results indicate a large SA for O. serratus at 16,589 mm2 compared to the 2,159 mm2 for the much smaller T. eatoni. We also calculated lamellar SA for nine additional trilobite species with exceptionally well-preserved appendages based on lamellar measurements. The results indicate that lamellae SA of trilobites increased exponentially with overall body size. Trilobite data follows the same trendline of gill SA/biomass observed in extant species and thus supports the interpretation of their exopodites as respiratory structures despite substantial variation in morphology.

In wildlife forensic practice, species identification and estimation of the Minimum Number of Individuals (MNI) for highly processed specimens have long relied on weight-based conversion methods, which may result in underestimation of the number of individuals involved in a case. Focusing on confiscated casque products of the helmeted hornbill (Rhinoplax vigil), this study combines macroscopic morphological examination with mitochondrial DNA barcoding (16S rRNA, COI, and Cytb) to explore a more robust approach for individual quantification. The results demonstrate that the conventional "weight-based" approach overlooks critical biological information contained in anatomical structures and cannot accurately reflect the actual number of individuals involved. Based on this, we propose an anatomy-based criterion centered on the principle of structural uniqueness: specimens retaining biologically unique beak or casque structures should be directly assigned to a single individual, whereas weight-based estimation should only be applied when original anatomical features are entirely absent. In addition, considering material loss during processing, we propose approximately 85 g as a reference threshold for estimating the number of individuals in heavily processed solid casque products. This approach improves the scientific rigor and accuracy of forensic identification and provides reliable technical support for the conviction, sentencing, and law enforcement of wildlife trafficking cases involving helmeted hornbill and other endangered species.

The ontogeny of Neanderthal (Homo neanderthalensis) perinates is poorly understood due to the paucity of juvenile skeletal remains. Here we reconstruct fetal bone growth, and explore deciduous tooth structures, in three Neanderthal juveniles (Sesselfelsgrotte, 1, 2 and 3) (90,000-50,000 years ago) from southeastern Germany using non-destructive microcomputed tomography. Sesselfelsgrotte 1 exhibited bone tissue consistent with modern human perinatal plexiform-like structures and primary osteons. Long bones showed regions of advanced growth compared to the mandible and frontal bone, which can be explained through different processes of ossification and potentially localized faster development in Neanderthals compared to modern humans. Bone microstructure resembles that of the late third trimester of modern humans, agreeing with previous estimates based on macroscopic data. Sesselfelsgrotte 2 and 3 deciduous teeth retain hypodensities deep within the crown dentine consistent with interglobular dentine. We conclude that the fetal bone patterning is similar to modern humans with areas of advanced growth, indicating that the growth trajectory for this Neanderthal perinate was broadly equivalent to that of modern humans. The abnormal dentine mineralization points toward a possible systemic disorder.

Animal naming is fundamental to scientific communication, yet it also reflects the historical and cultural contexts in which names are bestowed. Scientific names function as taxonomic labels and enduring records of human engagement with nature. Owing to this dual role, species names have recently attracted increasing attention from historical and humanities perspectives, both for their informative value and for the biases they may encode. To objectively assess these patterns at a large scale, we investigated etymological trends across Animalia using a comprehensive dataset of species names. Our analyses reveal that naming practices are shaped by a combination of historical events, taxonomic traditions, and cultural influences. Major global disturbances coincided with marked declines in species descriptions, whereas advances in biological techniques were associated with shifts in naming practices. Furthermore, etymological trends differed among phyla, indicating that taxonomic communities vary in their naming conventions. These differences suggest that taxonomists preferences, shared aesthetics, available knowledge, and cultural biases are differentially preserved in scientific names. Together, our results demonstrate that zoological nomenclature constitutes a valuable archive for understanding the historical and cultural dimensions of taxonomy.

Postmortem human brains stored in brain banks are important research resources to study the mechanisms underlying normal brain functions as well as various neurodegenerative disorders. Immunohistochemical (IHC) and histochemical (HC) staining have been used to examine human brains post-fixed in neutral-buffered formalin (NBF) for months, years, and even decades. As such, it is essential to establish the effects of prolonged post-fixation in NBF on both IHC and HC stains. Previously, we found that prolonged NBF post-fixation resulted in differential effects on IHC and HC staining on postmortem brains. In this study, we further examined the effects of prolonged post-fixation on IHC stains targeting 6 antigens and 2 HC stains of known biomarkers of cerebrovascular diseases in prefrontal cortex of human brains post-fixed for 1, 5, 10, 15, and 20 years. The IHC targets included microvasculature markers of the blood brain barrier (Collagen-IV and Claudin-5), a type III intermediate filament marker (Vimentin), an activated microglia marker (CD68), a biomarker for oligodendrocytic myelin proteolipid protein (PLP) and a marker for iron accumulation (Ferritin). The HC included Massons Trichrome Stain (MTS) and Bielschowsky silver stain (BSS). We found that staining intensities of Ferritin, Vimentin, Collagen-IV and BSS decreased with prolonged post-fixation, while no significant differences were observed in the staining intensity of other markers. Hence, these differential alterations should be taken into consideration when interpreting the results from processed tissues with prolonged post-fixation. We recommend performing IHC and HC staining for human brains with the same post-fixation times to offset any impact on downstream neuropathological analyses, as well as adding the post-fixation duration as a covariate in the analysis.

The deepest phylogenetic divergence within crown birds (Neornithes) is that between the reciprocally monophyletic Palaeognathae and Neognathae. Extant palaeognath diversity comprises the iconic flightless "ratites" (ostriches, rhea, kiwi, cassowaries, and emu), as well as 46 species of volant tinamous in Central and South America (Billerman et al., 2020). Although the earliest stages of palaeognath evolution remain shrouded in mystery due to a sparse fossil record, a group of apparently volant extinct palaeognaths from the Paleogene of Europe and North America, the lithornithids, can help to clarify palaeognath origins. Here, we use high resolution microCT scanning to characterize the morphology of two lithornithid specimens from the early Eocene (Ypresian) London Clay Formation: the neotype of Lithornis vulturinus (NHMUK A5204), from the Isle of Sheppey, Kent, England, and a newly discovered clay nodule containing lithornithid postcranial remains from the nearby locality of Seasalter. This three-dimensional dataset reveals bones from the L. vulturinus neotype that are partially or completely covered by matrix, allowing us to redescribe this critical specimen in new detail and present a revised differential diagnosis of L. vulturinus. We refer the new specimen from Seasalter to L. vulturinus on the basis of apomorphies such as a proximally directed lateral process of the coracoid, caudally divergent lateral margins of the sternum, an arcuate deltopectoral crest, as well as its provenance from a nearby penecontemporaneous locality. The Seasalter specimen contains abundant postcranial material that provides new insight into bones damaged or missing in the neotype, including two undamaged scapulae bearing the hooked acromion that is a diagnostic feature of lithornithids, two complete coracoids, and a nearly complete three-dimensionally preserved sternum. Its estimated body mass is one third larger than that of the neotype, indicating intraspecific variation within L. vulturinus that may reflect sexual dimorphism. Molecular divergence dates and Cretaceous neognath fossils indicate the presence of total-clade palaeognaths before the K-Pg mass extinction event; detailed anatomical descriptions of Paleogene palaeognaths will assist in the identification of the first total-clade palaeognaths from the Cretaceous, and provide insight into how and when flight was independently lost among Cenozoic crown palaeognaths.

The hippocampal CA1 subregion supports learning, memory formation, and spatial navigation. Although its three-layered architecture has been described in ex-vivo investigations, the in-vivo microstructural profile of CA1 and its relation to individual variations in memory performance remain poorly characterized. In this study, we used ultra-high field structural MRI at 7 Tesla to investigate the depth-dependent myelination patterns (measured by quantitative T1) of CA1 in younger adults, their relation to the local arterial architecture, and their association with individual differences in cognitive functions, specifically memory performance. Results show that left and right CA1 present depth-dependent patterns of myelination, with the outer and inner compartments showing higher myelination than the middle compartment. No significant relationship between layer-specific myelination of CA1 and distance to the nearest artery was observed. Right CA1 was found to be more myelinated than left CA1. Pairwise correlations and regression models showed that higher left CA1 myelination is linked to higher accuracy in object localization. Together, our data demonstrates the feasibility of describing the three layered myelin architecture of CA1 in vivo, and provides information on how alterations in the architecture of CA1 may relate to alterations in cognitive performance in younger adults.

The clitoris is one of the least studied organs of the human body. The detailed anatomy of the clitoris is challenging to address through a gross dissection, as most of its parts are embedded internally, surrounded by pubic bone and several pelvic organs. While clinical imaging methods such as magnetic resonance imaging can capture the gross 3D morphology, they lack the spatial resolution required to resolve the detailed structures. In this study, we generated micron-scale computed tomography images of the female pelvises, leveraging a synchrotron radiation X-ray source. This unique data revealed the complex trajectory of the dorsal nerve of the clitoris, the main sensory nerve of the clitoris. Notably, the nerve trunks within the clitoral glans were revealed, with the maximum diameter ranging from 0.2 to 0.7 mm. They showed a tree-like branching pattern projecting towards the surface of the glans. We also revealed that some branches of the dorsal nerve of the clitoris ramify to innervate the clitoral hood and mons pubis. Finally, the posterior labial nerve, a branch of the perineal nerves, was shown to innervate the surroundings of the clitoris and the labial structures. These findings have an immediate impact on operations performed around the vulva area, such as gender-affirmation surgery and reconstruction surgery after genital mutilation.

Long-term storage of aldehyde-fixed brain tissue is commonly performed in the fluid state. This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for the preservation of fixed whole brains using graded immersion cryoprotection and subzero temperature storage, which is one type of a more general approach that we refer to as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v/v) ethylene glycol and 30% (w/v) sucrose. We used CT imaging to track cryoprotectant penetration, finding that with the use of our protocol, approximately 10 months is required to reach equilibration throughout whole human brains. In our initial histological validation, we found that insufficient equilibration time prior to freezing led to apparent ice crystal artifacts seen on ultrastructural imaging of the white matter. After refining the protocol to allow adequate diffusion time, histologic data at both the light and electron microscopic levels showed preserved cellular architecture and ultrastructure after the process of cryoprotectant loading, freezer storage, and unloading. This protocol can be implemented using laboratory freezers or freezer rooms and provides a degree of resilience against freezer failures because the morphology of the fixed tissue is expected to remain preserved long-term in the fluid state even if rewarmed. Our approach may be valuable for laboratories seeking to enhance the long-term preservation of antigenicity in large brain tissue specimens for future research applications.