Nerve block, nerve damage, and fluid injection pressure: overturning the myth.

Histological and micro-ultrasound evidence rebuffs deep-rooted views on the nature of nerve block, nerve damage, and injection pressure monitoring. We propose that the ideal position of the needle tip for nerve block is between the innermost circumneural fascial layer and outer epineurium, with local anaesthetic passing circumferentially through adipose tissue. Thin, circumferential, subepineural expansion that is invisible to the naked eye was identified using micro-ultrasound, and could account for variability of outcomes in clinical practice. Pressure monitoring cannot differentiate between intrafascicular and extrafascicular injection. High injection pressure only indicates intraneural extrafascicular spread, not intrafascicular spread, because it is not possible to inject into the stiff endoneurium in most human nerves.

Changes in the Thickness of Rat Nerve Sheaths after Single Subperineural Administration of Rat Bone Marrow Mesenchymal Stem Cells.

The sheaths of the damaged peripheral nerve of Wistar-Kyoto rats were studied after single subperineural administration of bromodeoxyuridine (BrdU)-labeled bone marrow mesenchymal stem cells (MSC) from the same rats. The sciatic nerve was damaged by ligation for 40 sec directly before MSC administration. BrdU+ MSC were identified in the recipient nerve within 1 week after transplantation and were detected not only in the endoneurium, but also in the epineurium and perineurium. It was found that single administration of MSC into the damaged nerve trunk led to an almost 2-fold increase in the thickness of its sheaths (perineurium and epineurium) in comparison with the control group (ligation). It can be hypothesized that MSC induce thickening of nerve sheaths through the production of factors that stimulate angiogenesis and adipogenesis.

Another (Internal) Epineurium: Beyond the Anatomical Barriers of Nerves.

The epineurium has been accepted as the outer anatomical barrier of the peripheral nerves. Our objective was to characterize the microanatomy of the layers surrounding nerves using different tissue-specific staining methods. Two hundred forty-two cross sections of human sciatic and median nerves, and brachial plexuses of eight fresh unembalmed cadavers, were examined. The samples were fixed in formaldehyde solution and stained with hematoxylin-eosin, Masson's trichrome, or epithelial membrane antigen under standard conditions. Because epithelial membrane antigen only stains the perineurium, we demonstrated using hematoxylin-eosin and Masson's trichrome that there were different collagen layers inside and outside the nerves. All fascicles had a collagen layer that surrounded the perineurium and were in close contact with it, with no adipose tissue between them. Unlike the perineurium, this layer, an "internal epineurium," contained no cells, and it surrounded one or a small group of fascicles. Bundling these fascicles or small groups of fascicles together was the true epineurium, and between the true and internal epineurium, we consistently found an adipose-containing compartment. More proximal to this, the tibial and common peroneal nerves were bundled together by another collagen layer, the circumneurium, which also had a fat-cell-containing compartment deep to it. There were scattered collagen fibers among the adipocytes. Using tissue-specific staining, we were able to demonstrate a collagen layer, the "internal epineurium." Outside the nerves, we identified several fat-containing concentric compartments. Those compartments were limited by collagen fiber layers that were also similar to the epineurium. Clin. Anat. 33:199-206, 2020. © 2019 Wiley Periodicals, Inc.

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.

A Rare Case of Carpal Tunnel Syndrome Due to Subepineural Hematoma of Median Nerve: Diagnosis and Surgical Management.

Carpal tunnel syndrome (CTS) is a common neuropathy with various underlying causes, posing diagnostic and management challenges for healthcare providers. The condition is typically associated with repetitive strain, idiopathic factors, or anatomical variations, leading to the compression of the median nerve within the carpal tunnel. We describe a case of a 46-year-old male who presented with recurrent CTS symptoms one year after a successful carpal tunnel release surgery. The symptoms resurfaced following a minor wrist trauma, leading to pain, numbness, and hand weakness. Despite initial conservative management, including immobilization and NSAIDs, the symptoms persisted. Further investigation and exploratory surgery revealed a rare subepineural hematoma of the median nerve, which was subsequently drained, resulting in immediate and lasting symptom relief. This case demonstrates the importance of considering uncommon etiologies such as subepineural hematomas in patients with recurrent CTS and underscores the need for a thorough diagnostic approach to ensure effective treatment.

Preparation and evaluation of decellularized epineurium as an anti-adhesive biofilm in peripheral nerve repair.

Following peripheral nerve anastomosis, the anastomotic site is prone to adhesions with surrounding tissues, consequently impacting the effectiveness of nerve repair. This study explores the development and efficacy of a decellularized epineurium as an anti-adhesive biofilm in peripheral nerve repair. Firstly, the entire epineurium was extracted from fresh porcine sciatic nerves, followed by a decellularization process. The decellularization efficiency was then thoroughly assessed. Subsequently, the decellularized epineurium underwent proteomic analysis to determine the remaining bioactive components. To ensure biosafety, the decellularized epineurium underwent cytotoxicity assays, hemolysis tests, cell affinity assays, and assessments of the immune response following subcutaneous implantation. Finally, the functionality of the biofilm was determined using a sciatic nerve transection and anastomosis model in rats. The result indicated that the decellularization process effectively removed cellular components from the epineurium while preserving a number of bioactive molecules, and this decellularized epineurium was effective in preventing adhesion while promoting nerve repairment and functional recovery. In conclusion, the decellularized epineurium represents a novel and promising anti-adhesion biofilm for enhancing surgical outcomes of peripheral nerve repair.

Enhancing neuroinduction activity of PLCL-based nerve conduits through native epineurium integration.

Autologous nerve grafts have been considered the gold standard for peripheral nerve grafts. However, due to drawbacks such as functional loss in the donor area and a shortage of donor sources, nerve conduits are increasingly being considered as an alternative approach. Polymer materials have been widely studied as nerve repair materials due to their excellent processing performance. However, their limited biocompatibility has restricted further clinical applications. The epineurium is a natural extra-neural wrapping structure. After undergoing decellularization, the epineurium not only reduces immune rejection but also retains certain bioactive components. In this study, decellularized epineurium (DEP) derived from the sciatic nerve of mammals was prepared, and a bilayer nerve conduit was created by electrospinning a poly (l-lactide-co-ε-caprolactone) (PLCL) membrane layer onto the outer surface of the DEP. Components of the DEP were examined; the physical properties and biosafety of the bilayer nerve conduit were evaluated; and the functionality of the nerve conduit was evaluated in rats. The results demonstrate that the developed bilayer nerve conduit exhibits excellent biocompatibility and mechanical properties. Furthermore, this bilayer nerve conduit shows significantly superior therapeutic effects for sciatic nerve defects in rats compared to the pure PLCL nerve conduit. In conclusion, this research provides a novel strategy for the design of nerve regeneration materials and holds promising potential for further clinical translation.

CXCR2 mediated trafficking of neutrophils and neutrophil extracellular traps are required for myelin clearance after a peripheral nerve injury.

Neutrophils are a vital part of the innate immune system. Many of their functions eliminate bacteria & viruses, like neutrophil extracellular traps (NETs), which trap bacteria, enhancing macrophage phagocytosis. It was surprising when it was demonstrated that neutrophils are a part of Wallerian degeneration, a process that is essential for nerve regeneration after a nerve injury. It is not known what signals attract neutrophils into the nerve and how they aid Wallerian degeneration. Neutrophils accumulate in the distal nerve within one day after an injury and are found in the nerve from one to three days. We demonstrate that CXCR2 mediates the trafficking of neutrophils into the distal nerve, and without CXCR2 Wallerian degeneration, as indicated by luxol fast blue staining, was reduced seven days after a sciatic nerve crush or transection injury. NETs were detected in the distal nerve after a sciatic nerve transection. NET formation has been shown to require protein arginine deiminase 4 (PAD4), which citrullinates histone 3. Inhibiting PAD4 reduced NET formation significantly in the distal nerve at two days and myelin clearance at seven days indicating that NETs aid myelin clearance. These results demonstrate another function for NETs other than clearing pathogens. Neutrophils have been detected after injuries to the central nervous system and diseases in humans and animal models. Our results demonstrate neutrophils aid myelin clearance, suggesting a role for their presence in central nervous system injuries and diseases.

Biomechanical characterization of isolated epineurial and perineurial membranes of rabbit sciatic nerve.

Design of interface devices for effective, long-term integration into neural tissue is dependent on the biomechanical properties of the nerve membranes. Within the peripheral nerve, the two relevant connective tissue layers for interfacing are the epineurium and perineurium. Previous work has reported the forces needed to penetrate the whole nerve, but the mechanical differences between epineurium and perineurium were not reported. Design of intraneural electrodes that place electrodes within the nerve requires knowledge of the mechanics of individual tissues. This study quantified the Young's moduli and ultimate strains of the perineurium and the epineurium separately. We also measured the forces necessary to penetrate each tissue in isolation. We used a custom-built microtensile testing device to measure the Young's modulus values. The measured Young's moduli of the epineurium and the perineurium was 0.4 ± 0.1 MPa and 3.0 ± 0.3 MPa, respectively. We also measured the force required for blunt and sharp stainless steel, 100 µm diameter probes to be inserted into isolated epineurial tissue and perineurial tissue at 2 mm/s. These data provide additional guidelines for selection of materials for long-term implants that best match the tissue properties. The results will guide neural interface design such that electrodes can be placed through either the epineurium alone or both the epineurium and perineurium.

The Thickness of the facial Nerve Sheath Consistently Varies by Region in Its Intra-Tympanic Course on Cadaveric Study.

OBJECTIVES: Iatrogenic removal of intra-temporal disease processes, such as cholesteatoma and keratosis obturans, can be challenging when the facial nerve (FN) is involved. Despite this concern about possible FN injury during these procedures, our clinical observation has been that the diseased growth can be cleaned quite easily from the vertical FN epineurium. Therefore, we designed a cadaveric protocol to measure thickness of the FN sheath (epineurium) in horizontal, second genu and vertical FN segments and to correlate these measurements with surgical management of FN disorders. METHODS: Fifty non-fixated (wet) cadaveric temporal bones were dissected over 1 year's time. The intra-temporal FN sheath epineurium was harvested from the mid-horizontal, second genu, and mid-vertical segments. Using a digital micrometric technique, the thickness of each sample was measured. Data analysis was performed using student's two-tailed, dependent t-test. RESULTS: Epineurial nerve sheath thickness was the least in the horizontal segment (mean 0.9 mm, range 0.040-0.140 mm), greater at the second genu (mean 0.19 mm, range 0.010-0.280 mm), and greatest in the vertical segment (mean 0.29 mm, range 0.170-0.570 mm). These differences were statistically significant. CONCLUSION: In cases of cholesteatoma and keratosis obturans involving the vertical FN, the disease process can be separated from the FN sheath because of the sheath thickness in this region. Disease in the horizontal segment involves a thinner sheath and separating the disease process from the nerve is more difficult in this area.

Directed stimulation with interfascicular interfaces for peripheral nerve stimulation.

Objective.Computational models have shown that directional electrical contacts placed within the epineurium, between the fascicles, and not penetrating the perineurium, can achieve selectivity levels similar to point source contacts placed within the fascicle. The objective of this study is to test, in a murine model, the hypothesis that directed interfascicular contacts are selective.Approach.Multiple interfascicular electrodes with directional contacts, exposed on a single face, were implanted in the sciatic nerves of 32 rabbits. Fine-wire intramuscular wire electrodes were implanted to measure electromyographic (EMG) activity from medial and lateral gastrocnemius, soleus, and tibialis anterior muscles.Main results.The recruitment data demonstrated that directed interfascicular interfaces, which do not penetrate the perineurium, selectively activate different axon populations.Significance.Interfascicular interfaces that are inside the nerve, but do not penetrate the perineurium are an alternative to intrafascicular interfaces and may offer additional selectivity compared to extraneural approaches.

Macrodactyly of the foot resulting from plantar nerve impairment.

The role of the plantar nerve in the pathogenesis of macrodactyly of the foot is unknown. We investigated the distribution of affected toes and forefoot in 27 feet of 26 patients with pedal macrodactyly, and how this relates to innervation of the affected plantar nerve. A preoperative ultrasound examination was performed to determine the diameter and structure of the plantar nerve. Histologic findings were recorded during surgery. The microstructure of affected plantar nerves was evaluated by hematoxylin-eosin staining, while S100 expression was assessed by immunofluorescence analysis. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutation in the affected nerve tissue was detected by Sanger DNA sequencing. The affected toes and forefoot involved innervation of the medial plantar nerve in 25/27 feet, the lateral plantar nerve in one foot, and both medial and lateral plantar nerves in one foot. All affected plantar nerves, which were accompanied by a fatty strip, were surrounded by or infiltrated with fat. The affected plantar nerves showed enlargement, a tortuous course, fatty infiltration, or a combination of these. Pathologic changes in affected plantar nerves involved only the epineurium and not the perineurium or endoneurium. Expression of the Schwann cell marker S100 was absent in some areas of affected nerves. Sequencing of PIK3CA exons identified a gain-of-function mutation (p.His1047Arg) in affected plantar nerves. These results indicate that pathologic impairment of the plantar nerve can lead to macrodactyly of the foot, which may be considered as a nerve trunk disease.

Factors Within the Endoneurial Microenvironment Act to Suppress Tumorigenesis of MPNST.

Background: Deciphering avenues to adequately control malignancies in the peripheral nerve will reduce the need for current, largely-ineffective, standards of care which includes the use of invasive, nerve-damaging, resection surgery. By avoiding the need for en bloc resection surgery, the likelihood of retained function or efficient nerve regeneration following the control of tumor growth is greater, which has several implications for long-term health and well-being of cancer survivors. Nerve tumors can arise as malignant peripheral nerve sheath tumors (MPNST) that result in a highly-aggressive form of soft tissue sarcoma. Although the precise cause of MPNST remains unknown, studies suggest that dysregulation of Schwann cells, mediated by the microenvironment, plays a key role in tumor progression. This study aimed to further characterize the role of local microenvironment on tumor progression, with an emphasis on identifying factors within tumor suppressive environments that have potential for therapeutic application. Methods: We created GFP-tagged adult induced tumorigenic Schwann cell lines (iSCs) and transplanted them into various in vivo microenvironments. We used immunohistochemistry to document the response of iSCs and performed proteomics analysis to identify local factors that might modulate divergent iSC behaviors. Results: Following transplant into the skin, spinal cord or epineurial compartment of the nerve, iSCs formed tumors closely resembling MPNST. In contrast, transplantation into the endoneurial compartment of the nerve significantly suppressed iSC proliferation. Proteomics analysis revealed a battery of factors enriched within the endoneurial compartment, of which one growth factor of interest, ciliary neurotrophic factor (CNTF) was capable of preventing iSCs proliferation in vitro. Conclusions: This dataset describes a novel approach for identifying biologically relevant therapeutic targets, such as CNTF, and highlights the complex relationship that tumor cells have with their local microenvironment. This study has significant implications for the development of future therapeutic strategies to fight MPNSTs, and, consequently, improve peripheral nerve regeneration and nerve function.

Anatomic Variations of the Lateral Femoral Cutaneous Nerve: Remnants of Atypical Nerve Growth Pathways Revisited by Intraneural Fascicular Dissection and a Proposed Classification.

OBJECTIVE: To trace anatomic variations of the lateral femoral cutaneous nerve (LFCN) in its intrapelvic course. METHODS: Forty cadavers (80 sides) fixed in 10% formalin solution were dissected. The following parameters were recorded: LFCN diameter and variations in its origin and number. The dissection comprised exposure and excision of the lumbar plexus, together with the roots of LFCN, followed by retrograde intraneural fascicular dissection using microsurgical instruments. RESULTS: Several types of LFCN origin from the lumbar plexus were observed. Typically, the LFCN appears as a single trunk arising from dorsal divisions of the ventral rami of the lumbar plexus. The most prevalent origin of the nerve was from the L2 and L3 roots (47 cases; 58.75%). The LFCN took an origin from the L1-L2 level in 12 cases (15%) and from the L2 nerve in 9 cases (11.25%). The main observed variations were the presence of the accessory LFCN (2 cases; 2.5%) and branching of the LFCN from the femoral nerve (6 cases; 7.5%). Communications between the LFCN and the femoral or genitofemoral nerves also were observed occasionally. An atypical course of the LFCN with respect to the anterior psoas was observed in our material in 3 of the 80 sides (3.75% of the examined LFCN specimens). CONCLUSIONS: Considerable variability in the origin and the course of the LFCN was observed, which should be taken into account during clinical assessment of nerve lesions and during surgery via transpsoas approaches to the lumbar spine.

A strategy for bypassing the blood-brain barrier: Facial intradermal brain-targeted delivery via the trigeminal nerve.

Although intranasal delivery bypasses the blood-brain barrier (BBB), the anatomical location of the olfactory mucosa and respiratory airflow interference lead to less brain-targeted drug delivery. In addition to intranasal delivery, evidence indicates that facial intradermal injection might be a novel strategy for bypassing the BBB via the trigeminal nerve (TN). The hypothesis was verified by pharmacokinetic evaluation, nasal injury, lymphatic vessels inhibition and immunohistochemistry. Intradermal injection into the rat mystacial pad (i.d.) elevated the brain sub-areas and trigeminal Evans Blue (EB) concentrations, Cmax and AUC(0-t). I.d. also increased them in brain sub-areas beyond those of intranasal (i.n.) and intravenous injection (i.v.), especially the pons varolii and the medulla oblongata (sub-areas associated with TN). I.d. injection increased the brain drug targeting efficiency, brain direct transport percentage and brain bioavailability of EB while i.n. injection altered them slightly. Trigeminal transection and nasal injury reduced trigeminal EB with i.d. administration. Trigeminal perineurium, epineurium, perivascular spaces, neurons and Schwann cells were involved in the EB brain-targeted delivery. The lymphatic system mediated EB diffusion from the mystacial pad to the nasal mucosa and the brain. Thus, facial intradermal injection might be a promising strategy for brain-targeting delivery, bypassing the BBB via the trigeminal substructures.

Pathology of Perineural Spread.

The perineural space is a compartment located between the nerve axons, supporting cells and tissues, and the epineural fibrous sheath. Tumor cells invade this space in response to a complex interplay of trophic factors in the local microenviroment. This attraction of tumor cells to nerves is referred to as neurotropism. The perineural space provides a conduit for tumor spread beyond the primary site of tumor occurrence. Perineural tumor growth is of two types: perineural invasion, affecting small unnamed nerves; and perineural spread, affecting larger, named nerves and presenting with clinical symptoms related to the involved nerve. Both forms of perineural tumor growth represent an adverse prognostic feature and are an essential element of the histopathologic reporting of malignancies of the head and neck region. Perineural spread is associated with decreased overall survival. Endoneurial invasion frequently accompanies perineural spread. The epineurium is more resistant to invasion and represents an important barrier to tumor spread. Immunohistochemical stains such as broad-spectrum keratin can aid in defining the proximal extent of perineural tumor spread.

Current Diagnosis and Treatment of Superficial Fibular Nerve Injuries and Entrapment.

The superficial peroneal nerve is now known as the superficial fibular nerve (SFN). Identification and treatment of entrapment of the SFN are important topics of discussion for foot and ankle surgeons, because overlooking the diagnosis can lead to permanent nerve damage. With the proper tools and skills, surgeons are able to help patients with symptomatic SFN entrapment, patients who often present in some degree of desperation, with the peripheral nerve surgeon as a last resort.

Curcumin upregulates S100 expression and improves regeneration of the sciatic nerve following its complete amputation in mice.

The repair of peripheral nerve injury after complete amputation is difficult, and even with anastomosis, the rapid recovery of nerve function remains challenging. Curcumin, extracted from plants of the genus Curcuma, has been shown to have anti-oxidant and anti-inflammatory properties and to improve sciatic nerve crush injury in rats. Here, we determined whether curcumin had neuroprotective effects following complete peripheral nerve amputation injury. BALB/c mice underwent complete sciatic nerve amputation, followed by an immediate epineurium anastomosis. Mice were intragastrically administered curcumin at doses of 40 (high), 20 (moderate), and 10 mg/kg/d (low) for 1 week. We found that myelin in the mice of the high- and moderate-dose curcumin groups appeared with regular shape, uniform thickness, clear boundary, and little hyperplasia surrounding the myelin. High and moderate doses of curcumin markedly improved both action potential amplitude of the sciatic nerves and the conduction velocity of the corresponding motor neurons, and upregulated mRNA and protein expression of S100, a marker for Schwann cell proliferation, in L4-6 spinal cord segments. These results suggest that curcumin is effective in promoting the repair of complete sciatic nerve amputation injury and that the underlying mechanism may be associated with upregulation of S100 expression.

Efficient bridging of 20 mm rat sciatic nerve lesions with a longitudinally micro-structured collagen scaffold.

An increasing number of biomaterial nerve guides has been developed that await direct comparative testing with the 'gold-standard' autologous nerve graft in functional repair of peripheral nerve defects. In the present study, 20 mm rat sciatic nerve defects were bridged with either a collagen-based micro-structured nerve guide (Perimaix) or an autologous nerve graft. Axons regenerated well into the Perimaix scaffold and, the majority of these axons grew across the 20 mm defect into the distal nerve segment. In fact, both the total axon number and the number of retrogradely traced somatosensory and motor neurons extending their axons across the implant was similar between Perimaix and autologous nerve graft groups. Implantation of Schwann cell-seeded Perimaix scaffolds provided only a beneficial effect on myelination within the scaffold. Functional recovery supported by the implanted, non-seeded Perimaix scaffold was as good as that observed after the autologous nerve graft, despite the presence of thinner myelin sheaths in the Perimaix implanted nerves. These findings support the potential of the Perimaix collagen scaffold as a future off-the-shelf device for clinical applications in selected cases of traumatic peripheral nerve injury.

Electron microscopy of human peripheral nerves of clinical relevance to the practice of nerve blocks. A structural and ultrastructural review based on original experimental and laboratory data.

AIM: The goal is to describe the ultrastructure of normal human peripheral nerves, and to highlight key aspects that are relevant to the practice of peripheral nerve block anaesthesia. METHOD: Using samples of sciatic nerve obtained from patients, and dural sac, nerve root cuff and brachial plexus dissected from fresh human cadavers, an analysis of the structure of peripheral nerve axons and distribution of fascicles and topographic composition of the layers that cover the nerve is presented. Myelinated and unmyelinated axons, fascicles, epineurium, perineurium and endoneurium obtained from patients and fresh cadavers were studied by light microscopy using immunohistochemical techniques, and transmission and scanning electron microscopy. Structure of perineurium and intrafascicular capillaries, and its implications in blood-nerve barrier were revised. RESULTS: Each of the anatomical elements is analyzed individually with regard to its relevance to clinical practice to regional anaesthesia. CONCLUSIONS: Routine practice of regional anaesthetic techniques and ultrasound identification of nerve structures has led to conceptions, which repercussions may be relevant in future applications of these techniques. In this regard, the ultrastructural and histological perspective accomplished through findings of this study aims at enlightening arising questions within the field of regional anaesthesia.