Quantitative assessment of intraneural vascular alterations in peripheral nerve trauma using high-resolution neurosonography: technical note.

High-resolution neurosonography (HRNS) has become a major imaging modality in assessment of peripheral nerve trauma in the recent years. However, the vascular changes of traumatic lesions have not been quantitatively assessed in HRNS. Here, we describe the vascular-ratio, a novel HRNS-based quantitative parameter for the assessment of intraneural vascular alterations in patients with nerve lesions. N = 9 patients suffering from peripheral nerve trauma were examined clinically, electrophysiologically and with HRNS (SonoSite Exporte, Fuji). Image analyses using Fiji included determination of the established fascicular ratio (FR), the cross-section ratio (CSR), and as an extension, the calculation of a vascular ratio (VR) of the healthy versus damaged nerve and a muscle perfusion ratio (MPR) in comparison to a healthy control group. The mean VR in the healthy part of the affected nerve (14.14%) differed significantly (p < 0.0001) from the damaged part (VR of 43.26%). This coincides with significant differences in the FR and CSR calculated for the damaged part versus the healthy part and the controls. In comparison, there was no difference between VRs determined for the healthy part of the affected nerve and the healthy controls (14.14% / 17.72%). However, the MPR of denervated muscles was significantly decreased compared to the non-affected contralateral controls. VR and MPR serve as additional tools in assessing peripheral nerve trauma. Image analysis and calculation are feasible. Combined with the more morphologic FR and CSR, the VR and MPR provide a more detailed insight into alterations accompanying nerve trauma.

Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves.

Endothelial cells (ECs) have gained an increased scientific focus since they were reported to provide guidance for Schwann cells and subsequently following axons after nerve injuries. However, previous protocols for the isolation of nerve-derived ECs from human nerves are ineffective regarding time and yield. Therefore, we established a novel and efficient protocol for the isolation of ECs from human peripheral nerves by means of immunomagnetic CD31-antibody conjugated Dynabeads and assessed the purity of the isolated cells. The easy-to-follow and time-effective isolation method allows the isolation of > 95% pure ECs. The isolated ECs were shown to express highly specific EC marker proteins and revealed functional properties by formation of CD31 and VE-cadherin positive adherens junctions, as well as ZO-1 positive tight-junctions. Moreover, the formation of capillary EC-tubes was observed in-vitro. The novel protocol for the isolation of human nerve-derived ECs allows and simplifies the usage of ECs in research of the human blood-nerve-barrier and peripheral nerve regeneration. Additionally, a potential experimental application of patient-derived nerve ECs in the in-vitro vascularization of artificial nerve grafts is feasible.

Fascicular Ratio Pilot Study: High-Resolution Neurosonography-A Possible Tool for Quantitative Assessment of Traumatic Peripheral Nerve Lesions Before and After Nerve Surgery.

BACKGROUND: Clinical and electrophysiological assessments prevail in evaluation of traumatic nerve lesions and their regeneration following nerve surgery in humans. Recently, high-resolution neurosonography (HRNS) and magnetic resonance neurography have gained significant importance in peripheral nerve imaging. The use of the grey-scale-based "fascicular ratio" (FR) was established using both modalities allowing for quantitative assessment. OBJECTIVE: To find out whether FR using HRNS can assess nerve trauma and structural reorganization in correlation to postoperative clinical development. METHODS: Retrospectively, 16 patients with operated traumatic peripheral nerve lesions were included. The control group consisted of 6 healthy volunteers. All imaging was performed with a 15 to 6 MHz ultrasound probe (SonoSite X-Porte; Fujifilm, Tokyo, Japan). FR was calculated using Fiji () on 8-bit-images ("MaxEntropy" using "Auto-Threshold" plug-in). RESULTS: Thirteen of 16 patients required autologous nerve grafting and 3 of 16 extra-intraneural neurolysis. There was no statistical difference between the FR of nonaffected patients' nerve portion with 43.48% and controls with FR 48.12%. The neuromatous nerve portion in grafted patients differed significantly with 85.05%. Postoperatively, FR values returned to normal with a mean of 39.33%. In the neurolyzed patients, FR in the affected portion was 78.54%. After neurolysis, FR returned to healthy values (50.79%). Ten of 16 patients showed clinical reinnervation. CONCLUSION: To our best knowledge, this is the first description of FR using HRNS for quantitative assessment of nerve damage and postoperative structural reorganization. Our results show a significant difference in healthy vs lesioned nerves and a change in recovering nerve portions towards a more "physiological" ratio. Further evaluation in larger patient groups is required.

Analysis of regeneration- and myelination-associated proteins in human neuroma in continuity and discontinuity.

BACKGROUND: Neuromas are pathologic nerve distensions caused by a nerve's response to trauma, resulting in a dysfunctional to non-functional nerve. Depending on the severance of the affected nerve, the resulting neuroma can be differentiated into continuous and stump neuroma. While neuroma formation has been investigated in animal models with enormous regenerative capacity, the search for differences in human response to nerve trauma on a molecular level ultimately seeks to identify reasons for functionally successful versus unsuccessful regeneration after peripheral nerve trauma in man. METHODS: In the present study, the regenerative potential of axons and the capability of Schwann cells (SC) to remyelinate regenerating axons was quantitatively and segmentally analyzed and compared within human neuroma in-continuity and discontinuity. RESULTS: For the stump neuroma and the neuroma in-continuity, there was a significant reduction of the total number of axons (86% stump neuroma and 91% neuroma in-continuity) from the proximal to the distal part of the neuroma, while the amount of fibrotic tissue increased, respectively. Labeling the myelin sheath of regenerating axons revealed a remyelination of regenerating axons by SCs in both neuroma types. The segmented analysis showed no distinct alterations in the number and spatial distribution of regenerating, mature, and myelinated axons between continuous and discontinuous neuroma. CONCLUSIONS: The quantitative and segmented analysis showed no distinct alterations in the number and spatial distribution of regenerating, mature, and myelinated axons between continuous and discontinuous neuroma, while the extensive expression of Gap43 in up to 55% of the human neuroma axons underlines their regenerative capacity independent of whether the neuroma is in continuity or discontinuity. Remyelination of Gap43-positive axons suggests that the capability of SCs to remyelinate regenerating axons is preserved in neuroma tissue.