Nerve Compartment Research Articles

ANALYSIS-ASSOCIATED FACTORS INTERFERING WITH DIFFUSION TENSOR INDICES OF PERIPHERAL NERVES

Diffusion tensor imaging (DTI) enables a non-invasive assessment of tissue architecture based on water diffusion. Advancements in technology have expanded the application of DTI from the central nervous system to the peripheral nervous system. High-resolution magnetic resonance imaging (MRI) systems have further enhanced the visualization of nerve compartments. This study investigates the influence of region of interest (ROI) selection on DTI indices in ex vivoperipheral nerves. Cadaveric median nerves (n=10) were harvested and immersed in fluorinated-carbon liquid, and 9-mm segments were independently scanned with a 9.4-T MRI system (scanning time 40 h). Diffusion-weighted signals were summed, and diffusion tensors were calculated for anatomical compartments using different delineation techniques. Following the initial scan, five samples were scanned for the second time using an identical protocol. The analysis showed that the inclusion of an inert background into the ROI significantly reduces fractional anisotropy (FA). Significant differences in diffusion tensors were observed between the initial and intermediate nerve portions, with FA and mean diffusivity (MD) differing by as much as 25.2% and 25.6%, respectively. Tracing the fascicles without the interfascicular epineurium exhibited an 8.8% lower FA compared to the delineations of the epineurium. Second MRI scans showed significant changes in diffusion tensors with higher eigenvalues D1-D3 and MD. In conclusion, the intermediate portions of the nerve demonstrated greater consistency in DTI indices and are therefore recommended for analysis over the initial portions. Using an inert liquid can minimize background effects, but the inherent diffusion properties of the tissue must be carefully considered. Prolonged scanning times can alter diffusion tensors, likely due to autolytic processes, underscoring the importance of consistent pre-scanning conditions and acquisition protocols. Additionally, maintaining consistency in delineations is paramount, regardless of whether the tracing method includes or excludes the interfascicular epineurium.

Which compartments of the optic nerve and its sheath are associated with intracranial pressure? An exploratory study.

The optic nerve sheath diameter (ONSD) is a commonly used estimate of intracranial pressure (ICP). The rationale behind this is that pressure changes in the cerebrospinal fluid affect the optic nerve subarachnoid space (ONSAS) thickness. Still, possible effects on other compartments of the optic nerve sheath (ONS) have not been studied. This is the first study ever to analyze all measurable compartments of the ONS for associations with elevated ICP. We measured changes in ICP and changes in ONS compartments in 75 patients treated with invasive ICP monitoring at the Karolinska University Hospital. Associations between changes in ICP and changes in ONS compartments were estimated with generalized estimating equations. The potential to identify elevated ICP was assessed with the area under the receiver operating characteristic curve (AUROC) for ONS compartments associated with ICP changes. Both ONSAS and perioptic dura mater thickness were significantly associated with changes in ICP in multivariable modeling. ONSAS was the only compartment that independently predicted changes in ICP, with an AUROC of 0.69 for predicting ICP increase. Still, both the perioptic dura mater thickness and the optic nerve diameter added value in predicting ICP changes in multivariable modeling. The results from this study challenge the current understanding of the mechanism behind the association between ICP and ONSD. Contrary to the common opinion that ONSAS is the only affected compartment, this study shows a more complex picture. It suggests that all ONS compartments may add value in predicting changes in ICP.

Identification of cellular and noncellular components of mature intact human peripheral nerve.

The goal of this study was to define basic constituents of the adult peripheral nervous system (PNS) using intact human nerve tissues. We combined fluorescent and chromogenic immunostaining methods, myelin-selective fluorophores, and routine histological stains to identify common cellular and noncellular elements in aldehyde-fixed nerve tissue sections. We employed Schwann cell (SC)-specific markers, such as S100β, NGFR, Sox10, and myelin protein zero (MPZ), together with axonal, extracellular matrix (collagen IV, laminin, fibronectin), and fibroblast markers to assess the SC's relationship to myelin sheaths, axons, other cell types, and the acellular environment. Whereas S100β and Sox10 revealed mature SCs in the absence of other stains, discrimination between myelinating and non-myelinating (Remak) SCs required immunodetection of NGFR along with axonal and/or myelin markers. Surprisingly, our analysis of NGFR+ profiles uncovered the existence of at least 3 different novel populations of NGFR+/S100β- cells, herein referred to as nonglial cells, residing in the stroma and perivascular areas of all nerve compartments. An important proportion of the nerve's cellular content, including circa 30% of endoneurial cells, consisted of heterogenous S100β negative cells that were not associated with axons. Useful markers to identify the localization and diversity of nonglial cell types across different compartments were Thy1, CD34, SMA, and Glut1, a perineurial cell marker. Our optimized methods revealed additional detailed information to update our understanding of the complexity and spatial orientation of PNS-resident cell types in humans.

Large-scale in-silico analysis of CSF dynamics within the subarachnoid space of the optic nerve

BackgroundImpaired cerebrospinal fluid (CSF) dynamics is involved in the pathophysiology of neurodegenerative diseases of the central nervous system and the optic nerve (ON), including Alzheimer’s and Parkinson’s disease, as well as frontotemporal dementia. The smallness and intricate architecture of the optic nerve subarachnoid space (ONSAS) hamper accurate measurements of CSF dynamics in this space, and effects of geometrical changes due to pathophysiological processes remain unclear. The aim of this study is to investigate CSF dynamics and its response to structural alterations of the ONSAS, from first principles, with supercomputers.MethodsLarge-scale in-silico investigations were performed by means of computational fluid dynamics (CFD) analysis. High-order direct numerical simulations (DNS) have been carried out on ONSAS geometry at a resolution of 1.625 μm/pixel. Morphological changes on the ONSAS microstructure have been examined in relation to CSF pressure gradient (CSFPG) and wall strain rate, a quantitative proxy for mass transfer of solutes.ResultsA physiological flow speed of 0.5 mm/s is achieved by imposing a hydrostatic pressure gradient of 0.37–0.67 Pa/mm across the ONSAS structure. At constant volumetric rate, the relationship between pressure gradient and CSF-accessible volume is well captured by an exponential curve. The ONSAS microstructure exhibits superior mass transfer compared to other geometrical shapes considered. An ONSAS featuring no microstructure displays a threefold smaller surface area, and a 17-fold decrease in mass transfer rate. Moreover, ONSAS trabeculae seem key players in mass transfer.ConclusionsThe present analysis suggests that a pressure drop of 0.1–0.2 mmHg over 4 cm is sufficient to steadily drive CSF through the entire subarachnoid space. Despite low hydraulic resistance, great heterogeneity in flow speeds puts certain areas of the ONSAS at risk of stagnation. Alterations of the ONSAS architecture aimed at mimicking pathological conditions highlight direct relationships between CSF volume and drainage capability. Compared to the morphological manipulations considered herein, the original ONSAS architecture seems optimized towards providing maximum mass transfer across a wide range of pressure gradients and volumetric rates, with emphasis on trabecular structures. This might shed light on pathophysiological processes leading to damage associated with insufficient CSF flow in patients with optic nerve compartment syndrome.

Endothelial Glycocalyx in the Peripheral Capillaries is Injured Under Oxaliplatin-Induced Neuropathy

Oxaliplatin, a platinum-based anticancer drug, is associated with peripheral neuropathy (oxaliplatin-induced peripheral neuropathy, OIPN), which can lead to worsening of quality of life and treatment interruption. The endothelial glycocalyx, a fragile carbohydrate-rich layer covering the luminal surface of endothelial cells, acts as an endothelial gatekeeper and has been suggested to protect nerves, astrocytes, and other cells from toxins and substances released from the capillary vessels. Mechanisms underlying OIPN and the role of the glycocalyx remain unclear. This study aimed to define changes in the three-dimensional ultrastructure of capillary endothelial glycocalyx near nerve fibers in the hind paws of mice with OIPN. The mouse model of OPIN revealed disruption of the endothelial glycocalyx in the peripheral nerve compartment, accompanied by vascular permeability, edema, and damage to the peripheral nerves. To investigate the potential treatment interventions, nafamostat mesilate, a glycocalyx protective agent was used in tumor-bearing male mice. Nafamostat mesilate suppressed mechanical allodynia associated with neuropathy. It also prevented intra-epidermal nerve fiber loss and improved vascular permeability in the peripheral paws. The disruption of endothelial glycocalyx in the capillaries that lie within peripheral nerve bundles is a novel finding in OPIN. Furthermore, these findings point toward the potential of a new treatment strategy targeting endothelial glycocalyx to prevent vascular injury as an effective treatment of neuropathy as well as of many other diseases. PerspectiveOIPN damages the endothelial glycocalyx in the peripheral capillaries, increasing vascular permeability. In order to prevent OIPN, this work offers a novel therapy approach that targets endothelial glycocalyx.

Vascularized fibular flap and custom-made synthesis in post-traumatic ulnar diaphyseal pseudarthrosis: a case report

BackgroundAlthough isolated fractures of the ulnar shaft are considered common and relatively benign injuries, numerous complications can arise especially in the context of suboptimal care pathways. For pediatric patients, however, there is no single indication of the surgical approach. In the context of the management of these complications, it is known that the vascularized fibular graft has numerous advantages and indications in the treatment of recurrent pseudarthrosis. However, in revision surgery the frequent occurrence of anatomical subversions requires the use of fixation means adapted to the individual patient. We present a clinical case of an adult patient suffering from post-traumatic ulnar pseudarthrosis treated with autologous vascularized fibula grafts and 3D-planned custom-made plate.Case presentationA 38-year-old Ivorian woman came to our attention with a painful nonunion of the ulnar shaft and significant dysmorphism of the left forearm, with shortening and flexion of the limb as an outcome of unspecified road trauma in childhood. No alterations of the nerve compartment were reported. As far as detectable, she had undergone autologous bone grafting and implantation of questionable synthetic means, without acute treatment. Since we evaluated the patient (2012), we have performed two debridement surgeries, associated with autologous avascular bone graft from the iliac crest and plate fixation (2012 and 2014). In both cases, rupture of the fixation media was observed. In 2021, the pseudarthrosis was treated with a vascularized fibular bone graft. The subverted radius and ulna anatomy and poor bone quality required patient-specific reconstruction of the pseudarthrosic ulna from a 3D scan and the production of custom-made plate and screws, supported by the creation of special guides for drilling and by optimizing the positioning of screws with preoperative digital models. In the postoperative period, regular follow-up visits with X-rays evaluations were performed at 1, 3 and 6 months after surgery. No inflammatory reactions or local rejection were found. The fibula graft healed at the proximal ulnar junction six months after the operation while it took eight months to heal at the distal junction. Functionally, we observed a pain reduction and a range-of-motion preservation.ConclusionsThe multiple failures of diaphyseal reconstruction with avascular bone grafts have forced the indication to the vascularized fibular flap. This case is a unique experience but we believe that the association between vascularized bone graft and the potential for customization through 3D planning represents a valid surgical potentiality in complex cases of post-traumatic reconstruction.

Molecular Mechanisms of Intratumoral Nerve Recruitment and Perineural Invasion Elucidated with Spatial Transcriptomics and CRISPR Activation

Perineural invasion (PNI) is an aggressive manifestation of tumor-nerve interactions associated with postoperative recurrence, metastasis, pain, and decreased survival. Hence, PNI is included in the staging criteria of several malignancies and often an indication for treatment intensification using adjuvant radiotherapy. However, the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown-hindering the development of new therapies targeting this key pathological process. Moreover, prior studies were limited by a lack of cell-type information, spatial context, and/or a fragmented focus on a small number of pathways. Using pancreatic ductal adenocarcinoma (PDAC) as an exemplar given the exceptionally high frequency of PNI in this malignancy, we performed the first comprehensive, cell-type specific, and spatially resolved whole-transcriptome analysis of human PDAC to identify molecular mediators of tumor-nerve crosstalk and PNI. We constructed 12 custom tissue microarrays (TMAs) derived from matched malignant regions with and without tumor-nerve proximity (n = 288 cores). We performed whole-transcriptome digital spatial profiling (DSP) to independently determine mRNA abundance from the malignant, fibroblast, and nerve compartments through optical sectioning. We mapped malignant subtypes we previously identified onto the spatial data and found strong (p<0.0001) positive nerve associations with the mesenchymal, basaloid, and neural-like progenitor subtypes and a negative nerve association with the classical subtype. Numerous genes expressed by malignant cells were enriched (e.g., MMP2, PLXND1, NRP1) or depleted (e.g., SEMA3B) in association with radial distance from nerves, including recapitulation of prior literature. To functionally explore these candidate mediators of tumor-nerve crosstalk, we derived genetically-engineered murine organoids (KrasLSL-G12D/+; Trp53FL/FL; Rosa26-dCas9-VPR) and transduced them with guide RNAs to overexpress subtype-specific transcription factors or candidate genes from the spatial analysis. We quantified (1) cancer cell invasion through extracellular matrix using cultured dorsal root ganglia (DRG) sensory neurons as the chemoattractant, and (2) the role of cancer-intrinsic signaling on nerve recruitment/outgrowth by applying conditioned media or exogenous proteins to cultured DRG sensory neurons and tracking their growth with live imaging. Our results suggest that the mechanisms enabling cancer cells to recruit nerves into the tumor microenvironment are distinct from those facilitating perineural invasion. This study has transformed our understanding of how cancer cells and the peripheral nervous system collaborate to promote tumor growth, survival, and dissemination, and is now guiding prioritization of therapeutic strategies that synergize with adjuvant radiotherapy in the burgeoning field of cancer neuroscience.

AQP4-IgG NMOSD, MOGAD, and double-seronegative NMOSD: is it possible to depict the antibody subtype using magnetic resonance imaging?

There is clinical and radiological overlap among demyelinating diseases. However, their pathophysiological mechanisms are different and carry distinct prognoses and treatment demands. To investigate magnetic resonance imaging (MRI) features of patients with myelin-oligodendrocyte glycoprotein associated disease (MOGAD), antibody against aquaporin-4(AQP-4)-immunoglobulin G-positive neuromyelitis optica spectrum disorder (AQP4-IgG NMOSD), and double-seronegative patients. A cross-sectional retrospective study was performed to analyze the topography and morphology of central nervous system (CNS) lesions. Two neuroradiologists consensually analyzed the brain, orbit, and spinal cord images. In total, 68 patients were enrolled in the study (25 with AQP4-IgG-positive NMOSD, 28 with MOGAD, and 15 double-seronegative patients). There were differences in clinical presentation among the groups. The MOGAD group had less brain involvement (39.2%) than the NMOSD group (p = 0.002), mostly in the subcortical/juxtacortical, the midbrain, the middle cerebellar peduncle, and the cerebellum. Double-seronegative patients had more brain involvement (80%) with larger and tumefactive lesion morphology. In addition, double-seronegative patients showed the longest optic neuritis (p = 0.006), which was more prevalent in the intracranial optic nerve compartment. AQP4-IgG-positive NMOSD optic neuritis had a predominant optic-chiasm location, and brain lesions mainly affected hypothalamic regions and the postrema area (MOGAD versus AQP4-IgG-positive NMOSD, p= 0 .013). Furthermore, this group had more spinal cord lesions (78.3%), and bright spotty lesions were a paramount finding to differentiate it from MOGAD (p = 0.003). The pooled analysis of lesion topography, morphology, and signal intensity provides critical information to help clinicians form a timely differential diagnosis.

Correlation between diffusion tensor indices and fascicular morphometric parameters of peripheral nerve.

Introduction: Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique that measures the anisotropy of water diffusion. Clinical magnetic resonance imaging scanners enable visualization of the structural integrity of larger axonal bundles in the central nervous system and smaller structures like peripheral nerves; however, their resolution for the depiction of nerve fascicular morphology is limited. Accordingly, high-field strength MRI and strong magnetic field gradients are needed to depict the fascicular pattern. The study aimed to quantify diffusion tensor indices with high-field strength MRI within different anatomical compartments of the median nerve and determine if they correlate with nerve structure at the fascicular level. Methods: Three-dimensional pulsed gradient spin-echo (PGSE) imaging sequence in 19 different gradient directions and b value 1,150s/mm2 was performed on a 9.4T wide-bore vertical superconducting magnet. Nine-millimeter-long segments of five median nerve samples were obtained from fresh cadavers and acquired in sixteen 0.625mm thick slices. Each nerve sample had the fascicles, perineurium, and interfascicular epineurium segmented. The diffusion tensor was calculated from the region-average diffusion-weighted signals for all diffusion gradient directions. Subsequently, correlations between diffusion tensor indices of segmentations and nerve structure at the fascicular level (number of fascicles, fascicular ratio, and cross-sectional area of fascicles or nerve) were assessed. The acquired diffusion tensor imaging data was employed for display with trajectories and diffusion ellipsoids. Results: The nerve fascicles proved to be the most anisotropic nerve compartment with fractional anisotropy 0.44 ± 0.05. In the interfascicular epineurium, the diffusion was more prominent in orthogonal directions with fractional anisotropy 0.13 ± 0.02. Diffusion tensor indices within the fascicles and perineurium differed significantly between the subjects (p < 0.0001); however, there were no differences within the interfascicular epineurium (p ≥ 0.37). There were no correlations between diffusion tensor indices and nerve structure at the fascicular level (p ≥ 0.29). Conclusion: High-field strength MRI enabled the depiction of the anisotropic diffusion within the fascicles and perineurium. Diffusion tensor indices of the peripheral nerve did not correlate with nerve structure at the fascicular level. Future studies should investigate the relationship between diffusion tensor indices at the fascicular level and axon- and myelin-related parameters.

ScRNA-seq generates a molecular map of emerging cell subtypes after sciatic nerve injury in rats

Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.

All aspects of sciatic nerve injection injury: an experiment with 78 rats.

In this study, we evaluate sciatic nerve injuries due to intramuscular injections, which is an important medicolegal problem frequently encountered in medical practice, with an extended experimental rat model of peripheral nerve injury. A total of 78 male Wistar albino rats were divided into five main groups, including a control group, a sham saline group, and groups that received benzathine penicillin G, diclofenac sodium, and dexamethasone, respectively. These pharmaceutical agents were applied to the sciatic nerves of all rats after exploration in the epineurial, endoneurial, and intrafascicular compartments, excluding the control group. Outcomes were evaluated for all rats and their sciatic nerves according to functional, electrophysiological, and histopathological results. Injuries were most evident in the groups that received penicillin G and diclofenac sodium, and this finding was statistically significant. It was also found that endoneurial and intrafascicular injections may cause more harm than epineurial injections. We have demonstrated that any medical injections applied to the epineurial, endoneurial, or intrafascicular compartments of the sciatic nerve may cause functional and electrophysiological loss with or without deterioration of the peripheral nerve architecture.

Quantitative analysis of astrocyte and axonal density relationships: Glia to neuron ratio in the optic nerve laminar regions

Astrocytes are critical for the maintenance of retinal ganglion cell (RGC) axonal function and viability, and form a key component of the functional neurovascular unit. Recently, we described the quantitative properties of astrocytes in relation to the capillary distributions in optic nerve laminar regions. Here, we provide a quantitative analysis of astrocytes and RGC axons in longitudinal sections of optic nerve tissue. Histological and immunocytochemical techniques are used to demonstrate the density of astrocytes, RGC axons and glia-neuron ratios across the pre laminar, lamina cribrosa and post laminar compartments of the optic nerve head (ONH). A study of human, pig, horse and rat optic nerves was performed and comparisons are made between species. This study demonstrates that the distribution of astrocytes correlates closely with the density of axonal processes, in accordance with the functional requirement of different regions of the ganglion cell axon. There was a consistency of glia-neuron ratios in the majority of laminar compartments, except for the human and rat prelaminar regions, which demonstrated lower ratios of astrocyte to axonal processes. The distribution of astrocytes may reflect a functional susceptibility to development of disease in the prelaminar region of the optic nerve. Interspecies comparison at the lamina cribrosa showed strikingly consistent glia-neuron ratios. Collectively, our findings suggest there may be a critical ratio of glia to neuron needed to maintain healthy cellular physiology across different laminar compartments of the optic nerve, with particular importance for the health of the lamina cribrosa region. It is possible that, in disease processes, the glia-neuron relationships across the different laminar compartments may be perturbed and this may be relevant for the development of glaucoma. Emerging technologies may further aid our understanding in how the physiology of optic nerve tissue cellular structure may be affected by changes to ONH characteristics and elevated intraocular pressure induced damage. Such findings may also permit the early identification of RGC axonal injury by identifying quantifiable changes in structural tissue architecture when pathophysiological pathways predominate.

Anterior Transpetrosal Approach for Trigeminal Schwannoma with Persistent Primitive Trigeminal Artery: 2-Dimensional Operative Video.

The surgical management of trigeminal schwannomas often requires complicated skull base approaches based on detailed anatomical understanding because of the involvement of multiple compartments of the trigeminal nerve from its root to the peripheral branches. When there is vital anatomical anomaly around the tumor, this can make the surgical management and anatomical understanding more difficult.1,2 This video demonstrates the microsurgical removal of a dumbbell-shaped trigeminal schwannoma via the anterior transpetrosal transtentorial approach in a patient with a persistent primitive trigeminal artery, the most common persistent embryonic carotid-vertebrobasilar anastomosis. A 32-yr-old woman presented with progressive facial sensory impairment, dizziness, and slight diplopia because of right abducens palsy. Neuroimaging revealed a right Meckel's cave tumor extending to both the middle and posterior cranial fossae and displacing the abducens nerve inferomedially. The persistent primitive trigeminal artery, which is an anomalous communication between the cavernous carotid and basilar arteries, was identified ventral to the tumor. While preserving most of the noninvaded trigeminal nerve, gross-total resection was achieved under direct visualization of the abducens nerve and persistent primitive trigeminal artery, piercing the dura of the posterior cavernous sinus and coursing medially along the clivus. The patient's facial sensory impairment and diplopia almost disappeared without new neurological deficits or tumor recurrence during the follow-up period of 1 yr. Three-dimensional reconstructed images demonstrated the anatomical relationship of the tumor, the persistent primitive trigeminal artery, and the extent of osseous drilling of the petrosectomy. Informed consent was obtained from the patient.

MRI diagnosis in multiligamentous injuries of knee with associated dislocations and neurovasacular sequelae: a retrospective analysis of injury patterns

BackgroundSimultaneous injury of two or more knee ligaments with concurrent tears involving the anterior cruciate and medial collateral ligaments is considered to be associated with femorotibial knee dislocations (KD). The purpose of this study is to characterize multiligamentous knee injury patterns associated with dislocations on MRI and to describe the incidence of their sequelae such as tibial plateau fractures, peroneal nerve injuries, and posterolateral corner (PLC) injuries.Participants and methodsAfter obtaining institutional ethical committee approval, we retrospectively identified 108 multiligamentous knee injuries in 100 patients who met with trauma and were treated at our tertiary care center between April 2014 and December 2018. Descriptive statistics were reported using numbers and percentages for categorical variables in cases of multiligamentous injuries, ipsilateral tibial plateau fractures, ipsilateral femoral fractures, peroneal nerve injury, arterial injury, compartment syndrome, and PLC injuries.ResultsThe most common (39.8%) injury pattern was a combined disruption of the anterior cruciate ligament, posterior cruciate ligament, and PLC. Schenck KD III-M was the most common injury type in KD, constituting 16.7%. Medial-sided injuries were the most common injury patterns seen with KD. There was a significant risk of peroneal nerve injury with lateral-sided injuries.ConclusionKD, though rare, may have devastating clinical sequelae such as compartment syndrome if not recognized and treated. Therefore, it is necessary to recognize imaging findings of femorotibial joint dislocations and associated injuries to the adjacent neurovascular bundles.

A perfusion bioreactor-based 3D model of the subarachnoid space based on a meningeal tissue construct

BackgroundAltered flow of cerebrospinal fluid (CSF) within the subarachnoid space (SAS) is connected to brain, but also optic nerve degenerative diseases. To overcome the lack of suitable in vitro models that faithfully recapitulate the intricate three-dimensional architecture, complex cellular interactions, and fluid dynamics within the SAS, we have developed a perfusion bioreactor-based 3D in vitro model using primary human meningothelial cells (MECs) to generate meningeal tissue constructs. We ultimately employed this model to evaluate the impact of impaired CSF flow as evidenced during optic nerve compartment syndrome on the transcriptomic landscape of MECs.MethodsPrimary human meningothelial cells (phMECs) were seeded and cultured on collagen scaffolds in a perfusion bioreactor to generate engineered meningeal tissue constructs. Engineered constructs were compared to human SAS and assessed for specific cell–cell interaction markers as well as for extracellular matrix proteins found in human meninges. Using the established model, meningeal tissue constructs were exposed to physiological and pathophysiological flow conditions simulating the impaired CSF flow associated with optic nerve compartment syndrome and RNA sequencing was performed.ResultsEngineered constructs displayed similar microarchitecture compared to human SAS with regards to pore size, geometry as well as interconnectivity. They stained positively for specific cell–cell interaction markers indicative of a functional meningeal tissue, as well as extracellular matrix proteins found in human meninges. Analysis by RNA sequencing revealed altered expression of genes associated with extracellular matrix remodeling, endo-lysosomal processing, and mitochondrial energy metabolism under pathophysiological flow conditions.ConclusionsAlterations of these biological processes may not only interfere with critical MEC functions impacting CSF and hence optic nerve homeostasis, but may likely alter SAS structure, thereby further impeding cerebrospinal fluid flow. Future studies based on the established 3D model will lead to new insights into the role of MECs in the pathogenesis of optic nerve but also brain degenerative diseases.

In situ molecular characterization of endoneurial microvessels that form the blood-nerve barrier in normal human adult peripheral nerves.

The blood-nerve barrier (BNB) formed by tight junction-forming endoneurial microvessels located in the innermost compartment of peripheral nerves, and the perineurium serve to maintain the internal microenvironment required for normal signal transduction. The specific molecular components that define the normal adult human BNB are not fully known. Guided by data derived from the adult human BNB transcriptome, we evaluated the in situ expression of 25 junctional complex, transporter, cell membrane, and cytoskeletal proteins in four histologically normal adult sural nerves by indirect fluorescent immunohistochemistry to determine proteins specifically expressed by restrictive endoneurial microvascular endothelium. Using Ulex Europaeus Agglutinin-1 expression to detect endothelial cells, we ascertained that the selected proteins were uniformly expressed in ≥90% of endoneurial microvessels. P-glycoprotein (also known as adenosine triphosphate-binding cassette subfamily B member 1) and solute carrier family 1 member 1 demonstrated restricted expression by endoneurial endothelium only, with classic tight junction protein claudin-5 also expressed on fenestrated epineurial macrovessels, and vascular-specific adherens junction protein cadherin-5 also expressed by the perineurium. The expression profiles of the selected proteins provide significant insight into the molecular composition of normal adult peripheral nerves. Further work is required to elucidate the human adult BNB molecular signature in order to better understand its development and devise strategies to restore function in peripheral neuropathies.

A novel marker for identifying and studying the membranes, barriers, and compartments surrounding peripheral nerves microscopically.

Recent anatomical discoveries indicate the importance of identifying membranes and compartments surrounding peripheral nerves into which local anesthetic agents can be injected and continuous nerve block catheters placed during regional anesthetic procedures. However, current markers used in anatomical studies have multiple drawbacks, specifically extravasation into noninjected locations, which can result in inadequate treatment. We studied a readily-available new marker, heparinized blood solution (HBS), which is easy to identify by microscopy and can remain in the nerve compartment into which it is deposited without distorting the tissue. We collected blood from 22 patients and prepared it as HBS. This was then injected into four fresh cadavers as in routine clinical practice for ultrasound-guided nerve blocks to form a so-called "doughnut" by "hydro-dissecting" at 32 sites. All samples, including nerves and neighboring tissues, were then prepared and examined by light microscopy. Although no deliberate intraneural injection was attempted, the marker was identified inside all the nerve compartments except the fascicles. Apart from leaking through the needle entry site in some instances, there was no extravasation of the HBS into neighboring nerve compartments in either direction. The tissues were not distorted and the erythrocytes did not form a thrombus. Nerve membranes and compartments could be clearly identified with routine staining. This technique enabled us to study the longitudinal and circumferential spread in all nerve compartments and to collect data for better interpretation of factors influencing an anesthetic nerve block and situations in which complications could possibly arise. HBS seemed superior to other markers because it did not leave the compartments into which it had been injected, did not distort the tissue, and was easily visible under the light microscope. Clin. Anat., 31:1050-1057, 2018. © 2018 Wiley Periodicals, Inc.

Correlation analysis and clinical significance of lymph node metastasis in right recurrent laryngeal nerve of papillary thyroid carcinoma

Objective To explore the correlation factors and clinical significance of lymph node metastasis in right recurrent laryngeal nerve of thyroid papillary carcinoma (PTC). Methods Ninty-eight consecutive patients with PTC who were underwent total thyroidectomy with routine central lymph node dissection in the Second People′s Hospital of Hefei from January 2015 to August 2017 were analyzed. The right paratracheal lymph nodes in the central compartment lymph nodes were divided into the level Ⅵ-A (anterior) and level Ⅵ-B (posterior, that was lymph node posterior to recurrent laryngeal nerve) compartments by recurrent laryngeal nerve. The lymph node metastasis of Ⅵ-B area during central compartment lymph node dissection was analyzed. We drew the receiver-operating characteristic curve (ROC) for right neck Ⅵ-A number of lymph node metastasis, and calculated the area under the curve (AUC) and Youden index. Results Among 98 cases, 16 cases occurred Ⅵ-B district lymph node metastasis (16.33%). Single factor analysis results showed that lymph node metastasis in Ⅵ-B area of PTC patients were related to the tumor size (χ2=12.864, P 0.05). Lymph node metastasis in Ⅵ-B area of PTC patients were related to the number of right neck Ⅵ-A area lymph node metastasis. When the number of right neck Ⅵ-A metastatic lymph nodes was 2.5, the sensitivity and specificity were 70.60% and 70.00% respectively, AUC was 0.754, and Youden index was 0.406. Conclusion For patients with PTC, primary tumor diameter >1 cm, tumor extracapsular invasion, Ⅵ-A area lymph node metastasis, multiple tumor and lateral cervical lymph node metastasis were the predictive factors for the lymph node metastasis in Ⅵ-B area. When the number of right neck Ⅵ-A area metastatic lymph nodes was greater than 3, we should dissect Ⅵ-B area. Key words: Thyroid neoplasms; Papilloma; Lymphatic metastasis; Lymph node posterior to right recurrent laryngeal nerve

Aging Induces Changes in the Somatic Nerve and Postsynaptic Component without Any Alterations in Skeletal Muscles Morphology and Capacity to Carry Load of Wistar Rats

The present study aimed to analyze the morphology of the peripheral nerve, postsynaptic compartment, skeletal muscles and weight-bearing capacity of Wistar rats at specific ages. Twenty rats were divided into groups: 10 months-old (ADULT) and 24 months-old (OLD). After euthanasia, we prepared and analyzed the tibial nerve using transmission electron microscopy and the soleus and plantaris muscles for cytofluorescence and histochemistry. For the comparison of the results between groups we used dependent and independent Student's t-test with level of significance set at p ≤ 0.05. For the tibial nerve, the OLD group presented the following alterations compared to the ADULT group: larger area and diameter of both myelinated fibers and axons, smaller area occupied by myelinated and unmyelinated axons, lower numerical density of myelinated fibers, and fewer myelinated fibers with normal morphology. Both aged soleus and plantaris end-plate showed greater total perimeter, stained perimeter, total area and stained area compared to ADULT group (p < 0.05). Yet, aged soleus end-plate presented greater dispersion than ADULT samples (p < 0.05). For the morphology of soleus and plantaris muscles, density of the interstitial volume was greater in the OLD group (p < 0.05). No statistical difference was found between groups in the weight-bearing tests. The results of the present study demonstrated that the aging process induces changes in the peripheral nerve and postsynaptic compartment without any change in skeletal muscles and ability to carry load in Wistar rats.

Chondroitinase C Selectively Degrades Chondroitin Sulfate Glycosaminoglycans that Inhibit Axonal Growth within the Endoneurium of Peripheral Nerve.

The success of peripheral nerve regeneration is highly dependent on the regrowth of axons within the endoneurial basal lamina tubes that promote target-oriented pathfinding and appropriate reinnervation. Restoration of nerve continuity at this structural level after nerve transection injury by direct repair and nerve grafting remains a major surgical challenge. Recently, biological approaches that alter the balance of growth inhibitors and promoters in nerve have shown promise to improve appropriate axonal regeneration and recovery of peripheral nerve function. Chondroitin sulfate proteoglycans (CSPGs) are known inhibitors of axonal growth. This growth inhibition is mainly associated with a CSPG's glycosaminoglycan chains. Enzymatic degradation of these chains with chondroitinase eliminates this inhibitory activity and, when applied in vivo, can improve the outcome of nerve repair. To date, these encouraging findings were obtained with chondroitinase ABC (a pan-specific chondroitinase). The aim of this study was to examine the distribution of CSPG subtypes in rodent, rabbit, and human peripheral nerve and to test more selective biological enzymatic approaches to improve appropriate axonal growth within the endoneurium and minimize aberrant growth. Here we provide evidence that the endoneurium, but not the surrounding epineurium, is rich in CSPGs that have glycosaminoglycan chains readily degraded by chondroitinase C. Biochemical studies indicate that chondroitinase C has degradation specificity for 6-sulfated glycosaminoglycans found in peripheral nerve. We found that chondroitinase C degrades and inactivates inhibitory CSPGs within the endoneurium but not so much in the surrounding nerve compartments. Cryoculture bioassays (neurons grown on tissue sections) show that chondroitinase C selectively and significantly enhanced neuritic growth associated with the endoneurial basal laminae without changing growth-inhibiting properties of the surrounding epineurium. Interestingly, chondroitinase ABC treatment increased greatly the growth-promoting properties of the epineurial tissue whereas chondroitinase C had little effect. Our evidence indicates that chondroitinase C effectively degrades and inactivates inhibitory CSPGs present in the endoneurial Schwann cell basal lamina and does so more specifically than chondroitinase ABC. These findings are discussed in the context of improving nerve repair and regeneration and the growth-promoting properties of processed nerve allografts.