75. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie

Prof. Dr.

Vorsitz

Prof. Dr. med.

Prof.

Analysis of the spatiotemporal dynamic of vascular injury and revascularization following experimental spinal cord injury

Trauma und Neurointensiv 

Analyse des räumlich-zeitlichen Verlaufs der vaskulären Verletzung und Regeneration nach experimentellem Rückenmarkstrauma

Traumatic Spinal Cord Injury (SCI) remains a global burden due to the lack of restorative therapies. The early loss of functional vasculature is part of a complex secondary injury cascade that leads to ongoing tissue loss, spreading the injury to regions formerly unharmed. A deeper understanding of the pathophysiology and its timeframe is essential in the search for restorative therapies. Specifically, when evaluating the effects of local therapies, defining the regional extent of vascular injury is necessary. This study aims to characterize the temporal and spatial aspects of vascular injury and revascularization following experimental SCI in the mouse.Adult C57BL/6J mice (n=26) were subjected to thoracic (Th6/7) clip-compression SCI via modified aneurysm clip (5g, 60s) or sham injury (laminectomy). At days 1, 3, 7, 14, and 28 post surgery, animals (n=4/group) were sacrificed via intracardial fluorescein isothiocyanate (FITC)-Lectin perfusion to assess functional vasculature. Histological analysis (CD31, FITC-Lectin, Ki-67) was conducted to assess temporal and spatial vascular changes and endogenous revascularization within the epicenter of injury (+/-0,5mm) and two subsequent spinal cord regions with increasing distance from trauma epicenter (region 1: +/- 0,5-1mm, region 2: +/-1-3mm)Loss of functional vasculature as measured by FITC-Lectin+ vessels was instantly assessable in trauma epicenter and subsequent region 1, being most severe at d3 (p<0.01). In region 2 significant loss of functional vasculature occurred only from d3 on (p=0.0427) and was restituted already on d7 (p=0.1159), while region 1 and trauma epicenter were restituted at d28 (p=0.2713, p=0.1251 respectively). Overall vessel density as measured by CD31+ signals was returned to baseline quicker (d3 epicenter, d14 regions 1+2). Revascularization as indicated by Ki67+ endothelial cells occurred in all regions after 3d (p<0.01), being most prominent in region 2.These findings deliver further insights into the temporal and spatial dynamics of the loss of functional vasculature and its restoration by endogenous revascularization following SCI. We demonstrate that vascular injury is most prominent in the first days after injury and spreads to wider regions surrounding the injury. While partial vascular regeneration occurs, it remains incomplete. The development of local therapies to ameliorate these events remain desirable.

Abstractvortrag | Abstract talk

PD Dr.

Characterization of serum trace elements in a spinal cord injury rat model

Charakterisierung von Serumspurenelementen nach Rückenmarksverletzung im Rattenmodell.

Although trace elements play a pivotal role in facilitating inflammatory and regenerative cascades after injury, their role in neurological recovery after spinal cord injury (SCI) is still not well characterized. This study hence adopted a comprehensive approach to analyze trace elements (zinc, free zinc, and selenium) status and dynamics after experimental SCI in rats.45 Wistar rats underwent laminectomy at the T9 level and were subsequently subjected to experimentally induced SCI. As a sham control, n=30 rats underwent laminectomy only. Serum samples were collected both before and 1, 3, 7, 14 and 28 days after the injury. Trace elements (zinc, free zinc, and selenium) were quantified using Total reflection X-ray fluorescence and correlated with serum cytokines. Furthermore, functional outcomes were assessed at day 14 post-injury, with results compared to the sham cohort using open-field locomotor scales (BBB score) and the CATWALK computer-based gait analysis. Significant fluctuations in trace element levels between days 1 and 14 post injury were noticed, especially for zinc to free zinc ratio, with a peak at the subacute phase, with a 25% increase in average zinc serum levels in SCI compared to sham rats on day 7 (p<0.05), and 15% on day 14 (p<0.05). For selenium, higher serum levels were noticed throughout the 28-day course in sham rats (p<0,05). Correlation analyses revealed a close interplay between zinc to free zinc ratio and proinflammatory serum cytokine levels. At day 14 after SCI, a correlation was noted between the recovery of BBB and CATWALK scores and the measured zinc to free zinc ratio at day 7 after injury.The presented data highlight the role of free zinc in SCI pathophysiology and its interplay with systemic inflammation after SCI. In addition, a potential protective role of selenium could be noticed. Further in vitro interventional studies and correlation with human serum of SCI patients could facilitate the clinical translation of these findings.

Hyperspectral imaging successfully detects tissue damage in a spinal cord injury rat model

Hyperspektrale Bildgebung erkennt erfolgreich Gewebeschäden nach Rückenmarksverletzung im Rattenmodell.

An intraoperative in situ evaluation of the extent of spinal cord injury (SCI) could provide a further modality for intra- and perioperative decision-making. Hyperspectral Imaging (HSI) is a novel, non-invasive diagnostic tool that could help differentiate between injured and healthy spinal cord tissue by identifying variations in tissue oxygenation and perfusion. Those in turn could be indicative of underlying neural damage. The aim of this proof-of-concept study, therefore, was to demonstrate the efficacy of HSI in estimating the extent of injury after SCI in an animal rat model.After a laminectomy at T9, the TIVITA® Tissue system, a medically certified HSI camera, was used to acquire spectral data before and after experimentally induced clip-compression/contusion SCI in n=6 Wistar rats. The imaging process involved acquiring data across a broad wavelength range, focusing on the visible to near-infrared spectrum. Animals were sacrificed after 14 days and HSI findings were correlated with conventional immunohistochemistry analyses, serum cytokine data and functional outcomes (using an open-field locomotor scale and the CatWalk XT® computer-based gait analysis).Global principal component analyses (PCA) revealed a differential clustering between injured and non-injured spinal cord tissues based on mere spectral signatures. Moreover, quantitative analysis of HSI data showed a correlation with the injury extent after 14 days, as informed by cyst size in isolated spinal cord tissue, pro-inflammatory serum cytokine levels, and endpoint neurobehavioral parameters pertaining to locomotion and coordination of hind limbs.HSI-derived data successfully identified SCI in a rat model. Its ability to provide detailed, in situ post-injury insights into the perfusion state of spinal cord tissue holds significant potential for both animal studies and future clinical applications. This study establishes a foundational approach for using HSI in SCI diagnostics, paving the way for its translation into clinical practice and application in the study of other spinal cord conditions.

Investigating the potential of exosomes from neuronal precursor cells in non-cellular repair of traumatic spinal cord injuries

Nutzung von Exosomen aus neuronalen Vorläuferzellen für die nicht-zellbasierte Regeneration bei Rückenmarksverletzungen

Secondary injury mechanisms in traumatic spinal cord injury (SCI) significantly hinder neural repair, particularly impeding neurite extension. Transplantation of neuronal precursor cells (NPCs) has been promising in neuroregeneration, potentially due to their secretomes. This investigation examines the role of NPC-derived exosomes (NPC-exos) in promoting neurite growth in vitro and facilitating spinal cord regeneration in a rat SCI model.NPCs harvested from embryonic rat subventricular zones underwent culturing up to passage four. Isolation of NPC-exos was achieved via ultracentrifugation and filtration, followed by nanoparticle tracking analysis for quantification and characterization through transmission electron microscopy and western blotting. Proteomic analysis was conducted to determine their composition. The influence of NPC-exos on neurite growth was evaluated in PC12 cells and primary neuron cultures. Their regenerative capability was tested in vivo using a rat thoracic clip-compression SCI model.NPC cultures, characterized by high Nestin expression (97±2%) and absence of NeuN, GFAP, and Olig2, were successfully established. NPC-exos, marked by CD63, CD81, and TSG101, exhibited typical exosomal morphology with an average size of 147.5±67.5 nm. Proteomic analysis via liquid chromatography-mass spectrometry revealed significant enrichment in axonal guidance proteins like Netrin-1 (abundance 1.89x108; relative to GAPDH 0.834). NPC-exos treatment, in a range of MOIs (1x103-1x105), led to a dose-dependent increase in neurite length in both PC12 cells and primary neurons (e.g., NPC-exos high dose vs. control: 255.5±55 µm vs. 122±31 µm, p<0.001). Intrathecal administration of NPC-exos post-SCI in rats showed enhanced functional recovery (NPC-exos vs. control: 24±1 vs. 28±2 stepping errors in Gridwalk test, p=0.0007) as well as marked histological neuroregeneration.Enriched with proteins crucial for axonal guidance and neurite growth, NPC-exos significantly promoted neurite extension in vitro and demonstrated substantial regenerative effects post-SCI in vivo. These outcomes indicate that NPC-exos could serve as an effective non-cellular approach for neural repair after SCI, warranting additional exploration.

LPS, IL-4, and TGF-β induced macrophages promote regeneration in spinal cord injury

LPS-, IL-4- und TGF-β-induzierte Makrophagen fördern die Regeneration bei Rückenmarksverletzungen

Spinal cord injury (SCI) triggers a cascade of inflammatory responses, resulting in severe, chronic disability. Macrophages, known for their diverse phenotypes, play a crucial role in this process. This study investigates the potential of a combined treatment with lipopolysaccharide (LPS), Interleukin-4 (IL-4), and Transforming growth factor-beta (TGF-β) in inducing a macrophage subtype that fosters enhanced regeneration post-SCI.We established primary cultures of bone marrow-derived macrophages (BMDMs) from Wistar rats' femur and tibia. These BMDMs underwent co-incubation with various combinations and concentrations of LPS, IL-4, and TGF-β for 24 hours. Anti-inflammatory markers in induced BMDMs (iBMDMs) were assessed using qPCR and immunofluorescence staining. Neuron co-culture and in vitro assays evaluated safety and functionality. After a T9 clip-compression/contusion SCI, rats received intralesional injections of either iBMDMs or the three inducers. Over 28 days, we characterized and quantified the local inflammatory response, neuroregeneration, and motor function recovery, comparing results with untreated controls.Post 24-hour stimulation with LPS, IL-4, and TGF-β, iBMDMs exhibited altered morphology and an anti-inflammatory profile. qPCR revealed significant increases in repair marker Arg1 and anti-inflammatory cytokine IL-10 in iBMDM (p<0.05), while pro-inflammatory factors iNOS, IL-12, and IL-1β did not show significant increases. Immunofluorescence confirmed protein expression levels of Arg1 and iNOS. Neuronal co-culture demonstrated no significant neuronal death (p>0.05). iBMDMs also showed enhanced in vitro phagocytic capabilities (p<0.05). Proteomics indicated numerous differentially expressed genes in iBMDMs, enriched in cell metabolism, immune regulation, inflammation, and phagocytosis. Post-SCI treatment with iBMDMs or inducers led to early shifts in the injured spinal cord's cellular and molecular inflammatory responses, and long-term improvements in histological neuroregeneration and motor function recovery, with the iBMDM transplantation group showing the most significant improvement.BMDMs can be induced by LPS, IL-4, and TGF-β into a subtype with anti-inflammatory and regenerative properties. This correlates with improved local inflammatory responses and enhanced neuroregeneration following experimental SCI, suggesting a promising therapeutic potential.

Univ.-Prof. Dr.

Dr. med.

Serial transbulbar sonography of optic disc swelling (papilledema) in comparison to intracranialpressure in patients with acute intracranial disease

Serielle transbulbäre Sonographie zur Feststellung einer Stauungspapille bei Patienten mit Druckmonitoring nach einem akuten intrakraniellen Ereignis

The identification of papilledema in patients with acute intracranial diseases such as trauma, bleedings and vascular diseases allows identification of increased ICP as measured by invasive intracranial pressure monitoring. Dynamic changes of ICP may be identified by transbulbar sonography.3 – 5 serial measurement of the optic disc (in mm above the retinal level) of both eyes to recognize papilledema (optic disc swelling, ODS) have been compared to measurement of IVP or parenchymatous pressure recording. Sonographic instrument was Hitachi Aloka Arietta V70 with a linear probe L64. We have arranged the longitudinal axis of the optic nerve  with its central retinal artery to the longitudinal axis of the sonographic probe. Optic nerve sheath diameter (ONSD) was measured 30 mm behind the optic disc. The ICP data were compared to recording of ICP in mmHg. Papilledeam was correlated to the mean values of ICP measurements over periods of 24 hours, and the highest ICP values during these 24 hour periods and the actual ICP values at the time of the sonographic measurement.In 22/33 patients the mean ICP was normal (below 14 mmHg). In 14 out of the 22 patients sonography revealed ODS, in 8 patients sonography was normal. 1 patient presented with normal mean ICP, however in 1 instance simultaneous swelling of OD. In 9 out of 33 patients ODS has not been observed , none of these 9 had increased ICP at any time. Transbulbar sonography to detect papilla swelling is a practical and reliable method that allows patients to be followed serially in the ICU without intracranial pressure monitoring.2 patients developed ODS during their treatment  course. However, OD became normal during their course. ONSD (normally maximum 58 mm) was increased  in 17 cases (59 – 79 mm). In 5 patients out of these 17 cases ICP was normal (maximum 14 mm Hg). We did not find any significant correlation between ICP and ONSD.Transbulbar sonography to detect papilla swelling is a practical and reliable method that allows patients to be followed serially in the ICU without intracranial pressure monitoring.

Session 29
Trauma und Neurointensiv – Verschiedenes 3

Termin

Vorsitz

Thema

Programm

Session 29Trauma und Neurointensiv – Verschiedenes 3

Termin

Vorsitz

Thema

Programm

Session 29
Trauma und Neurointensiv – Verschiedenes 3