Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Abstract

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine 1 . As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities 2,3 . Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target 4 . Here, a contusion/ compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic selfassembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI 5 . They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al. 6 In comparison to other peptides, QL6self-assembles into β-sheets at neutral pH 6 .14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days. Video LinkThe video component of this article can be found at

Three Growth Factors Induce Proliferation and Differentiation of Neural Precursor Cells In Vitro and Support Cell-Transplantation after Spinal Cord Injury In Vivo

“…In consideration of our in vitro findings with the different growth factor concentrations/combinations, which had revealed the most promising effects on proliferation and differentiation of NPCs for group 4 (20 ng/ml EGF + 20 ng/ml bFGF + 6 ng/ml PDGF-AA), we chose to use this growth factor cocktail to support NPC proliferation and differentiation after SCI in vivo . Due to the intrathecal application via an osmotic pump and according to previous reports, the concentration of the growth factors had to be increased by the factor 1,500 for in vivo use, resulting in 30 μ g/ml EGF, 30 μ g/ml bFGF, and 10 μ g/ml PDGF-AA [ 30 ].…”

“…We used a contusion/compression model with a 28-g modified aneurysm clip (Fehlings Laboratory, Canada) to induce SCI at the C6 level as previously described [ 30 ]. For all surgical procedures, animals were anesthetized with 1.5% isoflurane and a 1 : 1 mixture of O 2 and N 2 O. Postoperatively, animals were subjected to intensive care and received buprenorphine (0.05 mg/kg s.c.; Bayer, Germany) as well as meloxicam (2 mg/kg s.c.; Boehringer-Ingelheim, Germany) for 3–5 days.…”

“…In consideration of our in vitro findings with the different growth factor concentrations/combinations, which had revealed the most promising effects on proliferation and differentiation of NPCs for group 4 (20 ng/ml EGF + 20 ng/ml bFGF + 6 ng/ml PDGF-AA), we chose to use this growth factor cocktail to support NPC proliferation and differentiation after SCI in vivo. Due to the intrathecal application via an osmotic pump and according to previous reports, the concentration of the growth factors had to be increased by the factor 1,500 for in vivo use, resulting in 30 μg/ml EGF, 30 μg/ml bFGF, and 10 μg/ml PDGF-AA [30]. At the final stage of the experiment 8 weeks after clip contusion/compression SCI at the C6 level, the survival rate of transplanted NPCs, measured by quantification of GFP + /-DAPI + cells and expressed as the mean percentage of the initial cell graft given for transplantation was 2:37 ± 0:43% in group 1 animals.…”

Transplantation of Neural Precursor Cells Attenuates Chronic Immune Environment in Cervical Spinal Cord Injury

“…All experimental protocols were approved by the Animal Care Committee of Heidelberg University. The contusion/compression model was performed with an aneurysm clip as previously described ( 28 – 30 ). Briefly, rats were anesthetized with isoflurane (2–2.5%) and a 1:1 mixture of O 2 and N 2 O before a microsurgical laminectomy was performed at the C6/C7 level.…”

Drug delivery, cell-based therapies, and tissue engineering approaches for spinal cord injury

“…All four classes of biomolecules have been explored to generate SAPNs (172), which form nanofibers, nanotubes or nanospheres (173). These materials can be injected into the lesion in liquid form and aggregate in situ into a stable nano-network with physiological salt solution or changed pH (174). They fill up cavities without secondary damage, as caused by other scaffolds (175).…”