Tracing nerve fibers with volume electron microscopy to quantitatively analyze brain connectivity.

The highly complex structure of the brain requires an approach that can unravel its connectivity. Using volume electron microscopy and a dedicated software we can trace and measure all nerve fibers present within different samples of brain tissue. With this software tool, individual dendrites and axons are traced, obtaining a simplified "skeleton" of each fiber, which is linked to its corresponding synaptic contacts. The result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. To test this methodology, we apply it to the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex of the mouse. We find that nerve fibers are densely packed in the neuropil, reaching up to 9 kilometers per cubic mm. We obtain the number of synapses, the number and lengths of dendrites and axons, the linear densities of synapses established by dendrites and axons, and their location on dendritic spines and shafts. The quantitative data obtained through this method enable us to identify subtle traits and differences in the synaptic organization of the samples, which might have been overlooked in a qualitative analysis.

Longitudinal changes of circumpapillary retinal nerve fiber layer thickness profile during childhood myopia progression.

The purpose of this study was to evaluate longitudinal changes of circumpapillary retinal nerve fiber layer thickness (cpRNFLT) profile arising in the course of childhood myopia progression. Thirty-six eyes of 36 healthy children who showed myopia progression (spherical equivalent [SE] decrease of ≥ 2.0 diopters [D]) were included. To account for the axial-elongation-induced magnification effect on spectral-domain optical coherence tomography (SD-OCT) measurements, we calculated the proportion of quadrant-cpRNFLT distribution (i.e., the percentage of cpRNFLT within a single quadrant of total cpRNFLT). During 4.1 ± 1.1 years, the mean SE changed from -1.3 ± 0.9 to -4.3 ± 0.8D, and both the optic disc tilt ratio and the torsional angle increased (both P < 0.001). In the temporal quadrant, the cpRNFLT proportion was increased from 19.2 ± 1.86 to 24.4 ± 2.30% (P < 0.001). The cpRNFLT proportion in 3 quadrants (i.e., superior, inferior, nasal) showed decreases (all P < 0.001). Between baseline and follow up, the scan-circle location as determined by OCT was shifted mostly (94%; 34 of 36 eyes) toward the nasal side of the optic disc. With scan-circle repositioning to match the baseline, cpRNFLT distribution proportions did not show any significant difference between the baseline and follow up (all P > 0.05). For longitudinal evaluations of patients with myopia progression, scan-circle alteration should be given due consideration.

Morphological changes in amblyopic eyes in choriocapillaris and Sattler's layer in comparison to healthy eyes, and in retinal nerve fiber layer in comparison to fellow eyes through quantification of mean reflectivity: A pilot study.

PURPOSE: Establishing the reliability of a new method to check the mean retinal and choroidal reflectivity and using it to find retinal and choroid changes in amblyopia. METHODS: Design: Retrospective case-control. Population: 28 subjects of which 10 were healthy controls (20 eyes): 8 with refractive errors, 1 with strabismus, and 1 with both. 18 patients with unilateral amblyopia included: 7 anisometropic, 6 isoametropic, 1 strabismic, and 4 combined. Mean participants' age: 13.77 years ± 10.28. Observation procedures: SD-OCT and ImageJ. Main outcome measure: mean reflectivity of retinal and choroid layers. Amblyopic, fellow, and healthy eyes were compared. RESULTS: The method of measuring reflectivity is good to excellent reliability for all regions of interest except the fourth. The mean reflectivity of the choriocapillaris and Sattler's layer in amblyopic eyes were significantly lower than in healthy eyes (p = 0.003 and p = 0.008 respectively). The RNFL reflectivity was lower than that of fellow eyes (p = 0.025). Post-hoc pairwise comparisons showed statistically significant differences between amblyopic and healthy eyes for choriocapillaris (p = 0.018) and Sattler's (p = 0.035), and between amblyopic and fellow eyes for RNFL (p = 0.039). CONCLUSION: A decrease in reflectivity of the choriocapillaris and Sattler's in amblyopic compared to healthy eyes, and a decrease in reflectivity of the RNFL in the amblyopic compared to fellow eyes, indicate that the pathophysiology is partly peripheral and might be bilateral.

Dopamine Axons in Dorsal Striatum Encode Contralateral Visual Stimuli and Choices.

The striatum plays critical roles in visually-guided decision-making and receives dense axonal projections from midbrain dopamine neurons. However, the roles of striatal dopamine in visual decision-making are poorly understood. We trained male and female mice to perform a visual decision task with asymmetric reward payoff, and we recorded the activity of dopamine axons innervating striatum. Dopamine axons in the dorsomedial striatum (DMS) responded to contralateral visual stimuli and contralateral rewarded actions. Neural responses to contralateral stimuli could not be explained by orienting behavior such as eye movements. Moreover, these contralateral stimulus responses persisted in sessions where the animals were instructed to not move to obtain reward, further indicating that these signals are stimulus-related. Lastly, we show that DMS dopamine signals were qualitatively different from dopamine signals in the ventral striatum (VS), which responded to both ipsilateral and contralateral stimuli, conforming to canonical prediction error signaling under sensory uncertainty. Thus, during visual decisions, DMS dopamine encodes visual stimuli and rewarded actions in a lateralized fashion, and could facilitate associations between specific visual stimuli and actions.SIGNIFICANCE STATEMENT While the striatum is central to goal-directed behavior, the precise roles of its rich dopaminergic innervation in perceptual decision-making are poorly understood. We found that in a visual decision task, dopamine axons in the dorsomedial striatum (DMS) signaled stimuli presented contralaterally to the recorded hemisphere, as well as the onset of rewarded actions. Stimulus-evoked signals persisted in a no-movement task variant. We distinguish the patterns of these signals from those in the ventral striatum (VS). Our results contribute to the characterization of region-specific dopaminergic signaling in the striatum and highlight a role in stimulus-action association learning.

Learning to estimate the fiber orientation distribution function from diffusion-weighted MRI.

Estimation of white matter fiber orientation distribution function (fODF) is the essential first step for reliable brain tractography and connectivity analysis. Most of the existing fODF estimation methods rely on sub-optimal physical models of the diffusion signal or mathematical simplifications, which can impact the estimation accuracy. In this paper, we propose a data-driven method that avoids some of these pitfalls. Our proposed method is based on a multilayer perceptron that learns to map the diffusion-weighted measurements, interpolated onto a fixed spherical grid in the q space, to the target fODF. Importantly, we also propose methods for synthesizing reliable simulated training data. We show that the model can be effectively trained with simulated or real training data. Our phantom experiments show that the proposed method results in more accurate fODF estimation and tractography than several competing methods including the multi-tensor model, Bayesian estimation, spherical deconvolution, and two other machine learning techniques. On real data, we compare our method with other techniques in terms of accuracy of estimating the ground-truth fODF. The results show that our method is more accurate than other methods, and that it performs better than the competing methods when applied to under-sampled diffusion measurements. We also compare our method with the Sparse Fascicle Model in terms of expert ratings of the accuracy of reconstruction of several commissural, projection, association, and cerebellar tracts. The results show that the tracts reconstructed with the proposed method are rated significantly higher by three independent experts. Our study demonstrates the potential of data-driven methods for improving the accuracy and robustness of fODF estimation.

Purkinje Neurons with Loss of STIM1 Exhibit Age-Dependent Changes in Gene Expression and Synaptic Components.

The stromal interaction molecule 1 (STIM1) is an ER-Ca2+ sensor and an essential component of ER-Ca2+ store operated Ca2+ entry. Loss of STIM1 affects metabotropic glutamate receptor 1 (mGluR1)-mediated synaptic transmission, neuronal Ca2+ homeostasis, and intrinsic plasticity in Purkinje neurons (PNs). Long-term changes of intracellular Ca2+ signaling in PNs led to neurodegenerative conditions, as evident in individuals with mutations of the ER-Ca2+ channel, the inositol 1,4,5-triphosphate receptor. Here, we asked whether changes in such intrinsic neuronal properties, because of loss of STIM1, have an age-dependent impact on PNs. Consequently, we analyzed mRNA expression profiles and cerebellar morphology in PN-specific STIM1 KO mice (STIM1PKO ) of both sexes across ages. Our study identified a requirement for STIM1-mediated Ca2+ signaling in maintaining the expression of genes belonging to key biological networks of synaptic function and neurite development among others. Gene expression changes correlated with altered patterns of dendritic morphology and greater innervation of PN dendrites by climbing fibers, in aging STIM1PKO mice. Together, our data identify STIM1 as an important regulator of Ca2+ homeostasis and neuronal excitability in turn required for maintaining the optimal transcriptional profile of PNs with age. Our findings are significant in the context of understanding how dysregulated calcium signals impact cellular mechanisms in multiple neurodegenerative disorders.SIGNIFICANCE STATEMENT In Purkinje neurons (PNs), the stromal interaction molecule 1 (STIM1) is required for mGluR1-dependent synaptic transmission, refilling of ER Ca2+ stores, regulation of spike frequency, and cerebellar memory consolidation. Here, we provide evidence for a novel role of STIM1 in maintaining the gene expression profile and optimal synaptic connectivity of PNs. Expression of genes related to neurite development and synaptic organization networks is altered in PNs with persistent loss of STIM1. In agreement with these findings the dendritic morphology of PNs and climbing fiber innervations on PNs also undergo significant changes with age. These findings identify a new role for dysregulated intracellular calcium signaling in neurodegenerative disorders and provide novel therapeutic insights.

Structural Relationships Between Interstitial Cells of Cajal and Smooth Muscle Cells/Nerve Fibers in the Gastric Muscularis Mucosae of Chinese Giant Salamander.

Interstitial cells of Cajal (ICC) play an essential role in the motility of the gastrointestinal tract, and they have been identified in many laboratory animals and in humans. However, the information of ICC in lower animals is still very limited. In the present study, ICC were identified in the gastric muscularis mucosae of an amphibian­the Chinese giant salamander, by c-Kit immunohistochemistry and transmission electron microscopy. ICC showed c-Kit immunoreactivity and had spindle-shaped cell bodies and 1­2 long processes. ICC were located between smooth muscle cells (SMC) in gastric muscularis mucosae. Ultrastructurally, ICC appeared as polygon-, spindle-, and awl-shaped with long cytoplasmic prolongations between SMC. ICC had distinctive characteristics, such as nuclei with peripheral electron-dense heterochromatin, caveolae, and abundant intracytoplasmatic vacuoles, mitochondria, and rough endoplasmic reticula. Moreover, lamellar bodies and two types of condensed granules were observed in the cytoplasm of ICC. Notably, ICC establish close contacts with each other. Moreover, ICC establish gap junctions with SMC. In addition, ICC were frequently observed close to nerve fibers. In summary, the present study demonstrated the presence of ICC in the gastric muscularis mucosae of the Chinese giant salamander.

Ultrastructural effects of nerve growth factor and betamethasone on nerve regeneration after experimental nerve injury.

Peripheral nerve injuries (PNI) are an important health problem in the world. In this study, the effects of nerve growth factor (NGF) and betamethasone on nerve regeneration after sciatic nerve crush injury were examined by footprint analysis, electron microscopic, histomorphometric, and biochemical methods. Fifty Wistar rats were divided into five groups as intact control, experimental control, NGF, betamethasone, and NGF+betamethasone combined treatment groups. After the injury, betamethasone was subcutaneously injected into the lesion area of the treatment groups three times during the first day. NGF was subcutaneously injected into the lesion area of treatment groups for 14 days. Footprint analysis was made on 7, 14, 21, 28, and 35 days and after 6 weeks, tissue samples were obtained from all groups. In the experimental control group, there were severe degenerative changes in most of the axons and myelin sheaths of the nerve fibers. Moreover, an increase of MDA levels and a decrease in SOD activities were found in this group. On the other hand, malondialdehyde (MDA) levels decreased, superoxide dismutase (SOD) activities increased and significant motor functional recovery were found in the combined treatment group. The number of axons, axon diameters, and myelin thickness were significantly greater in the combined treatment group when compared with experimental control and other treatment groups. It was thought that nerve regenerative effects of NGF and anti-inflammatory and/or anti-edematous effects of betamethasone could induce functional recovery in the combined treatment group. In conclusion, combined therapy of NGF and betamethasone may be an effective approach for the treatment of PNI.

A 3D model to evaluate retinal nerve fiber layer thickness deviations caused by the displacement of optical coherence tomography circular scans in cynomolgus monkeys (Macaca fascicularis).

The main objective of the study was to analyze deviations in retinal nerve fiber layer (RNFL) thickness measurements caused by the displacement of circular optic disc optical coherence tomography scans. High-density radial scans of the optic nerve heads of cynomolgus monkeys were acquired. The retinal nerve fiber layer was manually segmented, and a surface plot of the discrete coordinates was generated. From this plot, the RNFL thicknesses were calculated and compared between accurately centered and intentionally displaced circle scans. Circle scan displacement caused circumpapillary retinal nerve fiber layer thickness deviations of increasing magnitude with increasing center offset. As opposed to the human eye, horizontal displacement resulted in larger RNFL thickness deviations than vertical displacement in cynomolgus monkeys. Acquisition of high-density radial scans allowed for the mathematical reconstruction and modelling of the nerve fiber layer and extrapolation of its thickness. Accurate and strictly repeatable circle scan placement is critical to obtain reproducible values, which is essential for longitudinal studies.

Spherical deconvolution with tissue-specific response functions and multi-shell diffusion MRI to estimate multiple fiber orientation distributions (mFODs).

In diffusion MRI, spherical deconvolution approaches can estimate local white matter (WM) fiber orientation distributions (FOD) which can be used to produce fiber tractography reconstructions. The applicability of spherical deconvolution to gray matter (GM), however, is still limited, despite its critical role as start/endpoint of WM fiber pathways. The advent of multi-shell diffusion MRI data offers additional contrast to model the GM signal but, to date, only isotropic models have been applied to GM. Evidence from both histology and high-resolution diffusion MRI studies suggests a marked anisotropic character of the diffusion process in GM, which could be exploited to improve the description of the cortical organization. In this study, we investigated whether performing spherical deconvolution with tissue specific models of both WM and GM can improve the characterization of the latter while retaining state-of-the-art performances in WM. To this end, we developed a framework able to simultaneously accommodate multiple anisotropic response functions to estimate multiple, tissue-specific, fiber orientation distributions (mFODs). As proof of principle, we used the diffusion kurtosis imaging model to represent the WM signal, and the neurite orientation dispersion and density imaging (NODDI) model to represent the GM signal. The feasibility of the proposed approach is shown with numerical simulations and with data from the Human Connectome Project (HCP). The performance of our method is compared to the current state of the art, multi-shell constrained spherical deconvolution (MSCSD). The simulations show that with our new method we can accurately estimate a mixture of two FODs at SNR≥50. With HCP data, the proposed method was able to reconstruct both tangentially and radially oriented FODs in GM, and performed comparably well to MSCSD in computing FODs in WM. When performing fiber tractography, the trajectories reconstructed with mFODs reached the cortex with more spatial continuity and for a longer distance as compared to MSCSD and allowed to reconstruct short trajectories tangential to the cortical folding. In conclusion, we demonstrated that our proposed method allows to perform spherical deconvolution of multiple anisotropic response functions, specifically improving the performances of spherical deconvolution in GM tissue.

Structural Changes in the Nervous Fibers of the Colon Mucosa in Experimental Acute Colitis.

Morphological, morphometric, and ultrastructural analysis of the nerve fibers in the colon mucosa was performed in C57BL/6 mice at various terms of development of acute colitis induced by dextran sodium sulphate. The nerve fibers were labeled with antibodies to pan-neuronal marker ßIII-tubulin. The progression of inflammatory and ulcerative processes in the mucosa on days 3-5 was associated with hyperplasia and hypertrophy of nerve fibers that peaked on day 7 after colitis induction. Ultrastructural analysis at all terms of colitis development showed moderate degeneration of axons. Thus, hypertrophy and hyperplasia of the nervous fibers in colon mucosa in experimental acute colitis correlated with aggravation of the ulcerative process in the mucosa. These changes are determined by alteration of histoarchitectonics and regenerative processes in the mucosa.

Three-dimensional analysis of neural connectivity with cells in rat ileal mucosa by serial block-face scanning electron microscopy.

The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.

Strong diffusion gradients allow the separation of intra- and extra-axonal gradient-echo signals in the human brain.

The quantification of brain white matter properties is a key area of application of Magnetic Resonance Imaging (MRI), with much effort focused on using MR techniques to quantify tissue microstructure. While diffusion MRI probes white matter (WM) microstructure by characterising the sensitivity of Brownian motion of water molecules to anisotropic structures, susceptibility-based techniques probe the tissue microstructure by observing the effect of interaction between the tissue and the magnetic field. Here, we unify these two complementary approaches by combining ultra-strong (300mT/m) gradients with a novel Diffusion-Filtered Asymmetric Spin Echo (D-FASE) technique. Using D-FASE we can separately assess the evolution of the intra- and extra-axonal signals under the action of susceptibility effects, revealing differences in the behaviour in different fibre tracts. We observed that the effective relaxation rate of the ASE signal in the corpus callosum decreases with increasing b-value in all subjects (from 17.1±0.7s-1 at b=0s/mm2 to 14.6±0.7s-1 at b=4800s/mm2), while this dependence on b in the corticospinal tract is less pronounced (from 12.0±1.1s-1 at b=0s/mm2 to 10.7±0.5s-1 at b=4800s/mm2). Voxelwise analysis of the signal evolution with respect to b-factor and acquisition delay using a microscopic model demonstrated differences in gradient echo signal evolution between the intra- and extra-axonal pools.

Retinal neurodegeneration in patients with end-stage renal disease assessed by spectral-domain optical coherence tomography.

Spectral-domain optical coherence tomography (SD-OCT) represents a reliable tool for retinal layer volume and thickness measurement. The aim of this study was to evaluate retinal changes indicating neurodegenerative processes in patients with end-stage renal disease (ESRD) compared to healthy controls. This was a cross-sectional, single-center study comprising 32 ESRD patients and 38 controls. Sectoral retinal nerve fiber layer (RNFL) thickness and retinal layer volumes were obtained by SD-OCT. Age- and gender-adjusted retinal layer volumes such as total retinal volume (p = 0.037), ganglion cell layer volume (GCL, p = 0.003), ganglion cell layer - inner plexiform layer volume (GCL-IPL, p = 0.005) and inner retinal layer volume (IRL, p = 0.042) of the right eye were lower in ESRD patients. Inner plexiform layer volume of both eyes (IPL, right eye: p = 0.017; left eye: 0.044) was reduced, as was RNFL thickness in the temporal superior sector (right eye: p = 0.016). A subgroup analysis excluding patients with diabetes revealed that GCL (p = 0.014) and GCL-IPL volume of the right eye (p = 0.024) and temporal superior sector of the RNFL scan (p = 0.021) in ESRD patients were still significantly thinner. We observed a decrease in several retinal layer volumes and temporal RNFL thickness indicative of retinal neurodegenerative processes in patients with ESRD.

Axonal debris accumulates in corneal epithelial cells after intraepithelial corneal nerves are damaged: A focused Ion Beam Scanning Electron Microscopy (FIB-SEM) study.

The intraepithelial corneal nerves (ICNs) that innervate the corneal epithelium are maintained through interactions with corneal epithelial cells and the extracellular matrix they produce. One to several axons bundle together within the basal cell layer and extend parallel to the ocular surface or branch and extend apically. Here we use 3-dimentional (3D) ultrastructural reconstructions of control and trephine injured mouse corneal epithelium and stroma produced using Focused Ion Beam Scanning Electron Microscope (FIB-SEM) to determine whether corneal epithelial or immune cells resident in the epithelium remove axonal debris and degrade it in their lysosomes after trephine injury to the cornea. We demonstrate that axonal fragments are internalized in the corneal epithelium and accumulate within electron dense structures consistent with lysosomes 3 h after trephine injury in both epithelial and immune cells located among the basal cells of the trephine injured cornea. Confocal imaging showed fewer CD45+ immune cells within the corneal epithelium after trephine injury compared to controls. The resolution obtained using FIB-SEM also allowed us to show that the presence of sensory axons at the basal aspect of the epithelial basal cells close to the anterior aspect of the epithelial basement membrane (EBM) is associated with a focal reduction in EBM thickness. In addition, we show using FIB-SEM and confocal imaging that superficial trephine injuries that do not penetrate the stroma, damage the integrity of anterior stromal nerves. These studies are the first to look at the mouse cornea following nerve injury using FIB-SEM.

Specialized structures on the border between rhizocephalan parasites and their host's nervous system reveal potential sites for host-parasite interactions.

Rhizocephalan barnacles are a unique group of endoparasitic crustaceans. In their extreme adaptation to endoparasitism, rhizocephalan adults have lost almost all features of their free-living relatives but acquired an outstanding degree of control over the body of their hosts (mostly decapods). The subtle influence exercised by rhizocephalans on the physiology, morphology and behaviour of their hosts is a vivid example of the most intimate host-parasite interactions but their mechanisms are very poorly known. In this study we examined the morphology and the adaptive ultrastructure of the organs invading the nervous system of the host in two rhizocephalan species from the families Peltogastridae, (Peltogaster paguri) and Peltogasterellidae (Peltogasterella gracilis). We found two essentially different types of structures involved in interactions of these two rhizocephalans with the nervous system of their hosts: modified rhizocephalan rootlets lying inside the ganglia and the neural fibres of the host enlacing the trophic rootlets of the parasites. We suggest that both these structures may be highly specialized tools allowing the parasite to interact with the host on the humoral level via neuromediators, hormones, attractants and trophic factors.

Heterogeneous vestibulocerebellar mossy fiber projections revealed by single axon reconstruction in the mouse.

A significant population of neurons in the vestibular nuclei projects to the cerebellum as mossy fibers (MFs) which are involved in the control and adaptation of posture, eye-head movements, and autonomic function. However, little is known about their axonal projection patterns. We studied the morphology of single axons of medial vestibular nucleus (MVN) neurons as well as those originating from primary afferents, by labeling with biotinylated dextran amine (BDA). MVN axons (n = 35) were classified into three types based on their major predominant termination patterns. The Cbm-type terminated only in the cerebellum (15 axons), whereas others terminated in the cerebellum and contralateral vestibular nuclei (cVN/Cbm-type, 13 axons), or in the cerebellum and ipsilateral vestibular nuclei (iVN/Cbm-type, 7 axons). Cbm- and cVN/Cbm-types mostly projected to the nodulus and uvula without any clear relationship with longitudinal stripes in these lobules. They were often bilateral, and sometimes sent branches to the flocculus and to other vermal lobules. Also, the iVN/Cbm-type projected mainly to the ipsilateral nodulus. Neurons of these types of axons showed different distribution within the MVN. The number of MF terminals of some vestibulocerebellar axons, iVN/Cbm-type axons in particular, and primary afferent axons were much smaller than observed in previously studied MF axons originating from major precerebellar nuclei and the spinal cord. The results demonstrated that a heterogeneous population of MVN neurons provided divergent MF inputs to the cerebellum. The cVN/Cbm- and iVN/Cbm-types indicate that some excitatory neuronal circuits within the vestibular nuclei supply their collaterals to the vestibulocerebellum as MFs.

Age related changes seen in human cornea in formalin fixed sections and on biomicroscopy in living subjects: A comparison.

The purpose of our experimental research was to assess the effects of aging on the main corneal structures in healthy corneas. Small, human cornea samples were collected from 20 Caucasian subjects during surgery for traumatic lesions to the eye. Ten subjects were adults (mean age 28 years) and 10 were elderly (mean age 76 years). Morphological analysis was carried out using light microscopy and electron microscopy. Another 40 patients (20 young: mean age < 30 years; 20 elderly: mean age > 70 years) were studied in vivo by confocal microscopy. The resulting images were analyzed qualitatively, quantitatively, and statistically. The basic light microscope revealed a decrease in endothelial cell density with age accompanied by an increase in endothelial cell size. Transmission electron microscopy revealed a corneal thinning and a decrease in the number of corneal stromal cells. A marked decrease in stromal nerve fibers was observed in the older subjects compared to the younger ones. Variable pressure scanning electron microscopy (VP-SEM) was used to make surface morphological observations and to determine the chemical composition of in vivo hydrated human corneas. Our results showed the effects of aging on normal corneal morphology highlighting the structural diversity of the corneal layers and revealing an age-related reduction in nerve fibers, thus explaining the decreased corneal sensitivity that may be observed in the elderly. Clin. Anat. 33:245-256, 2020. © 2019 Wiley Periodicals, Inc.

Intraepidermal Nerve Fiber Density in Postmortem Skin: A Novel Approach.

OBJECTIVE: To determine the feasibility of examining intraepidermal nerve fiber density (IENFD) in postmortem skin. METHODS: From 12 subjects, 3-mm skin punch biopsies were collected 1-4 days postmortem from the proximal leg and distal leg, with a mean (range) interval from the death of 37 (15-91) hours. Causes of death varied broadly, including hepatocellular carcinoma, chronic lymphocytic leukemia, generalized atherosclerosis, progressive supranuclear palsy, Parkinson disease, emphysema, and obesity. The mean (range) number of sections evaluated from each biopsy was 5.08 (2-6) from the proximal leg and 5.92 (5-6) from the distal leg. Sections were stained with PGP 9.5 for blinded counting using bright field microscopy. Qualitative and quantitative assessment of feasibility included a comparison of fiber staining with that in healthy subjects and mean IENFD in postmortem samples. Interobserver reliability was assessed among 3 blinded raters by calculating intraclass correlation coefficients and percentage variability of IENFD in at least 4 sections from biopsies in 5 healthy subjects. RESULTS: Intraobserver and interobserver correlation coefficients of blinded IENFD counts undertaken by 4 authors were consistently >0.80, and the coefficient of variation was ≤10%. The quality of staining in postmortem samples was comparable with that in healthy subjects and was not substantially affected by time from death to specimen collection of up to nearly 4 days. Mean (range) IENFD from postmortem samples in the proximal and distal leg was 2.73 (0-7.65) and 1.93 (0-4.91) fibers/mm of skin, respectively. Two of 3 patients who had received chemotherapy during life showed a nearly complete absence of intraepidermal nerve fibers. CONCLUSIONS: IENFD measurement in postmortem skin is feasible and may be used to study the epidemiology of SFN.

Peripapillary retinal nerve fibre layer thinning in genetic generalized epilepsy.

PURPOSE: The purpose of this study was to compare the peripapillary retinal nerve fibre layer (RNFL) between patients with genetic generalized epilepsy (GGE) and healthy controls. METHODS: This prospective observational study was conducted on adults aged 18-60 years. The study group comprised 26 consecutive patients who met the inclusion criteria and 26 healthy age- and sex-matched healthy adults. Peripapillary RNFL thickness was measured by spectral domain optical coherence tomography. RESULTS: The average peripapillary RNFL thickness was significantly thinner for GGE patients (98.61 µm) than for healthy controls (104.77 µm) (p = 0.016). Similar results were obtained for the left eye. The peripapillary RFNL thickness of all quadrants was lower for GGE patients than for healthy controls, but it was significant only in the superior (p = 0.009) and inferior (p = 0.024) quadrants for both eyes. CONCLUSIONS: Our results suggest that the peripapillary RNFL is significantly thinner in GGE patients than in healthy participants. We concluded that this microstructural feature might be an intrinsic feature of GGE.