Surface immobilization of neural adhesion molecule L1 for improving the biocompatibility of chronic neural probes: In vitro characterization

Silicon-based implantable neural electrode arrays are known to experience failure during long-term recording, partially due to host tissue responses. Surface modification and immobilization of biomolecules may provide a means to improve their biocompatibility and integration within the host brain tissue. Previously, the laminin biomolecule or laminin fragments have been used to modify the neural probe's silicon surface to promote neuronal attachment and growth. Here we report the successful immobilization of the L1 biomolecule on a silicon surface. L1 is a neuronal adhesion molecule that can specifically promote neurite outgrowth and neuronal survival. Silane chemistry and the heterobifunctional coupling agent 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) were used to covalently bind these two biomolecules onto the surface of silicon dioxide wafers, which mimic the surface of silicon-based implantable neural probes. After covalent binding of the biomolecules, polyethylene glycol (PEG)-NH(2) was used to cap the unreacted GMBS groups. Surface immobilization was verified by goniometry, dual polarization interferometry, and immunostaining techniques. Primary murine neurons or astrocytes were used to evaluate the modified silicon surfaces. Both L1- and laminin-modified surfaces promoted neuronal attachment, while the L1-modified surface demonstrated significantly enhanced levels of neurite outgrowth (p<0.05). In addition, the laminin-modified surface promoted astrocyte attachment, while the L1-modified surface showed significantly reduced levels of astrocyte attachment relative to the laminin-modified surface and other controls (p<0.05). These results demonstrate the ability of the L1-immobilized surface to specifically promote neuronal growth and neurite extension, while inhibiting the attachment of astrocytes, one of the main cellular components of the glial sheath. Such unique properties present vast potentials to improve the biocompatibility and chronic recording performance of neural probes.     

In vivo effects of L1 coating on inflammation and neuronal health at the electrode-tissue interface in rat spinal cord and dorsal root ganglion

The spinal cord (SC) and dorsal root ganglion (DRG) are target implantation regions for neural prosthetics, but the tissue-electrode interface in these regions is not well-studied. To improve understanding of these locations, the tissue reactions around implanted electrodes were characterized. L1, an adhesion molecule shown to maintain neuronal density and reduce gliosis in brain tissue, was then evaluated in SC and DRG implants. Following L1 immobilization onto neural electrodes, the bioactivities of the coatings were verified in vitro using neuron, astrocyte and microglia cultures. Non-modified and L1-coated electrodes were implanted into adult rats for 1 or 4weeks. Hematoxylin and eosin staining along with cell-type specific antibodies were used to characterize the tissue response. In the SC and DRG, cells aggregated at the electrode-tissue interface. Microglia staining was more intense around the implant site and decreased with distance from the interface. Neurofilament staining in both locations decreased or was absent around the implant, compared with surrounding tissue. With L1, neurofilament staining was significantly increased while neuronal cell death decreased. These results indicate that L1-modified electrodes may result in an improved chronic neural interface and will be evaluated in recording and stimulation studies.     

Polyethylene glycol-containing polyurethane hydrogel coatings for improving the biocompatibility of neural electrodes

The instability of the interface between chronically implanted neuroprosthetic devices and neural tissue is a major obstacle to the long-term use of such devices in clinical practice. In this study, we investigate the feasibility of polyethylene glycol (PEG)-containing polyurethane (PU) hydrogel as coatings for polydimethylsiloxane (PDMS)-based neural electrodes in order to achieve a stable neural interface. The influence of PU hydrogel coatings on electrode electrochemical behaviour was investigated. Importantly, the biocompatibility of PU hydrogel coatings was evaluated in vitro and in vivo. Changes in the electrochemical impedance of microelectrodes with PU coatings were negligible. The amount of protein adsorption on the PDMS substrate was reduced by 93% after coating. Rat pheochromocytoma (PC12) cells exhibited more and longer neurites on PU films than on PDMS substrates. Furthermore, PDMS implants with (n=10) and without (n=8) PU coatings were implanted into the cortex of rats and the tissue response to the implants was evaluated 6 weeks post-implantation. GFAP staining for astrocytes and NeuN staining for neurons revealed that PU coatings attenuated glial scarring and reduced the neuronal cell loss around the implants. All of these findings suggest that PU hydrogel coating is feasible and favourable for neural electrode applications.     

Effects of caspase-1 knockout on chronic neural recording quality and longevity: Insight into cellular and molecular mechanisms of the reactive tissue response

Chronic implantation of microelectrodes into the cortex has been shown to lead to inflammatory gliosis and neuronal loss in the microenvironment immediately surrounding the probe, a hypothesized cause of neural recording failure. Caspase-1 (aka Interleukin 1β converting enzyme) is known to play a key role in both inflammation and programmed cell death, particularly in stroke and neurodegenerative diseases. Caspase-1 knockout (KO) mice are resistant to apoptosis and these mice have preserved neurologic function by reducing ischemia-induced brain injury in stroke models. Local ischemic injury can occur following neural probe insertion and thus in this study we investigated the hypothesis that caspase-1 KO mice would have less ischemic injury surrounding the neural probe. In this study, caspase-1 KO mice were implanted with chronic single shank 3 mm Michigan probes into V1m cortex. Electrophysiology recording showed significantly improved single-unit recording performance (yield and signal to noise ratio) of caspase-1 KO mice compared to wild type C57B6 (WT) mice over the course of up to 6 months for the majority of the depth. The higher yield is supported by the improved neuronal survival in the caspase-1 KO mice. Impedance fluctuates over time but appears to be steadier in the caspase-1 KO especially at longer time points, suggesting milder glia scarring. These findings show that caspase-1 is a promising target for pharmacologic interventions.     

Micropatterned bioimplant with guided neuronal cells to promote tissue reconstruction and improve functional recovery after primary motor cortex insult

With the ever increasing incidence of brain injury, developing new tissue engineering strategies to promote neural tissue regeneration is an enormous challenge. The goal of this study was to design and evaluate an implantable scaffold capable of directing neurite and axonal growth for neuronal brain tissue regeneration. We have previously shown in cell culture conditions that engineered micropatterned PDMS surface with straight microchannels allow directed neurite growth without perturbing cell differentiation and neurite outgrowth. In this study, the micropatterned PDMS device pre-seeded with hNT2 neuronal cells were implanted in rat model of primary motor cortex lesion which induced a strong motor deficit. Functional recovery was assessed by the forelimb grip strength test during 3 months post implantation. Results show a more rapid and efficient motor recovery with the hNT2 neuroimplants associated with an increase of neuronal tissue reconstruction and cell survival. This improvement is also hastened when compared to a direct cell graft of ten times more cells. Histological analyses showed that the implant remained structurally intact and we did not see any evidence of inflammatory reaction. In conclusion, PDMS bioimplants with guided neuronal cells seem to be a promising approach for supporting neural tissue reconstruction after central brain injury.     

血小板微粒对神经干细胞增殖和分化的影响

背景：血小板微粒是血小板在活化过程中释放的小的亚细胞碎片,在血管新生和缺血组织新生中发挥作用。 目的：观察血小板微粒对神经干细胞增殖、存活和分化的影响。 方法：取胎龄13.5 d的小鼠获取鼠胚胎神经干细胞,分别应用10 μg/L成纤维细胞生长因子、血管内皮生长因子或者0.1,1,10 μg的血小板微粒进行处理观察神经球的大小及细胞转归。 结果与结论：与单独应用成纤维细胞生长因子、血管内皮生长因子干预的神经干细胞相比,经血小板微粒处理的神经干细胞神经球直径明显增大,存活率明显增高。且血小板微粒可促进神经干细胞向胶质细胞和神经元分化。说明血小板微粒同其他生长因子一样均能促进神经干细胞的增殖、存活和分化,且效果优于生长因子单独应用。     

Combined transplantation of neural stem cells and olfactory ensheathing cells for the repair of spinal cord injuries

Spinal cord repair is a problem that has long puzzled neuroscientists. The failure of the spinal cord to regenerate and undergo reconstruction after spinal cord injury (SCI) can be attributed to secondary axonal demyelination and neuronal death followed by cyst formation and infarction as well as to the nature of the injury environment, which promotes glial scar formation. Cellular replacement and axon guidance are both necessary for SCI repair. Multipotent neural stem cells (NSCs) have the potential to differentiate into both neuronal and glial cells and are, therefore, likely candidates for cell replacement therapy following SCI. However, NSC transplantation alone is not sufficient for spinal cord repair because the majority of the NSCs engrafted into the spinal cord have been shown to differentiate with a phenotype which is restricted to glial lineages, further promoting glial scaring. Olfactory ensheathing cells (OECs) are a unique type of glial cell that occur both peripherally and centrally along the olfactory nerve. The ability of olfactory neurons to grow axons in the mature central nervous system (CNS) milieu has been attributed to the presence of OECs. It has been shown that transplanted OECs are capable of migrating into and through astrocytic scars and thereby facilitating axonal regrowth through an injury barrier. Given the complementary properties of NSCs and OECs, we predict that the co-transplantation of NSCs and OECs into an injured spinal cord would have a synergistic effect, promoting neural regeneration and functional reconstruction. The lost neurocytes would be replaced by NSCs, while the OECs would build "bridges" crossing the glial scaring that conduct axon elongation and promote myelinization simultaneously. Furthermore, the two types of cells could first be seeded into a bioactive scaffold and then the cell seeded construct could be implanted into the defect site. We believe that this type of treatment would lead to improved neural regeneration and functional reconstruction after SCI.     

Transplantation of neural stem cells derived from human cord blood to the brain of adult and neonatal rats

Searching for a reliable source of alternative neural stem cells for experimental treatment of neurological disorders we have established neural stem cell line derived from human umbilical cord blood (HUCB-NSC) (Buzanska et al. 2006). These cells have been shown to differentiate along neuronal and glial lineages in the promoting in vitro conditions. In the current study we transplanted HUCB-NSC into rat brain to determine whether the neural progenitors would be able to survive, migrate and eventually adopt neural phenotypes after exposure to central nervous system (CNS) microenvironment. Our experiments revealed that HUCB-NSC grafting into the brain of adult rats limited their survival up-to two weeks probably due to their elimination by severe immunological host reaction evoked by xenotransplantation. HUCB-NSC graft in neonates survived longer time in rat brain, migrated, proliferated and differentiated into neuronal cells however their presence in the host tissue did not exceed more than five weeks after transplantation.     

脑源性神经营养因子促进低血糖幼鼠海马神经干细胞的定向分化

背景：有研究表明脑源性神经营养因子可以维持神经元的存活、影响神经元的迁移,在体外可以促进神经干细胞的存活和分化。 目的：探讨脑源性神经营养因子对低血糖幼鼠海马神经干细胞定向分化的作用。 方法：取新生1 d低血糖模型大鼠脑海马组织进行原代、传代及单细胞克隆培养。培养的细胞一部分进行神经干细胞鉴定,另一部分依据培养液中脑源性神经营养因子质量浓度的不同将单克隆细胞分为0,100,200 μg/L组,取第4代细胞进行诱导分化,行神经元特异性烯醇化酶免疫荧光染色,计数阳性细胞比例。 结果与结论：单克隆培养后3组细胞均呈巢蛋白阳性,诱导分化后细胞呈行神经元特异性烯醇化酶和胶质纤维酸性蛋白阳性;100,200 μg/L组神经干细胞生长较快,且分化为神经元特异性烯醇化酶阳性细胞比例较高(P < 0.05),但两组神经干细胞之间差异无显著性意义(P > 0.05)。提示脑源性神经营养因子促进低血糖幼鼠海马神经干细胞向神经元定向分化。     

Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels,Matrigel and Collagen I

Designer peptides with self-assembling properties form nanofibers which are further organized to form a hydrogel consisting of up to 99.5% water. We present here the encapsulation of neural stem cells into peptide nanofiber hydrogel scaffolds. This results in three-dimensional (3-D) neural tissue cultures in which neural stem cells differentiate into progenitor neural cells, neurons, astrocytes and oligodendrocytes when cultured in serum-free medium. Cell survival studies showed that neural cells in peptide hydrogels thrive for at least 5 months. In contrast, neural stem cells encapsulated in Collagen I were poorly differentiated and did not migrate significantly, thus forming clusters. We show that for culture periods of 1–2 weeks, neural stem cells proliferate and differentiate better in Matrigel. However, in long-term studies, the population of cells in Matrigel decreases whereas better cell survival rates are observed in neural tissue cultures in peptide hydrogels. Peptide functionalization with cell adhesion and cell differentiation motifs show superior cell survival and differentiation properties compared to those observed upon culturing neural cells in non-modified peptide hydrogels. These designed 3-D engineered tissue culturing systems have a potential use as tissue surrogates for tissue regeneration. The well-defined chemical and physical properties of the peptide nanofiber hydrogels and the use of serum-free medium allow for more realistic biological studies of neural cells in a biomimetic 3-D environment.     

The fate of ultrafast degrading polymeric implants in the brain

We have recently reported on an ultrafast degrading tyrosine-derived terpolymer that degrades and resorbs within hours, and is suitable for use in cortical neural prosthetic applications. Here we further characterize this polymer, and describe a new tyrosine-derived fast degrading terpolymer in which the poly(ethylene glycol) (PEG) is replaced by poly(trimethylene carbonate) (PTMC). This PTMC containing terpolymer showed similar degradation characteristics but its resorption was negligible in the same period. Thus, changes in the polymer chemistry allowed for the development of two ultrafast degrading polymers with distinct difference in resorption properties. The in vivo tissue response to both polymers used as intraparenchymal cortical devices was compared to poly(lactic-co-glycolic acid) (PLGA). Slow resorbing, indwelling implant resulted in continuous glial activation and loss of neural tissue. In contrast, the fast degrading tyrosine-derived terpolymer that is also fast resorbing, significantly reduced both the glial response in the implantation site and the neuronal exclusion zone. Such polymers allow for brain tissue recovery, thus render them suitable for neural interfacing applications.     

Distinct molecular pathways mediate glial activation and engulfment of axonal debris after axotomy

Glial cells efficiently recognize and clear cellular debris after nervous system injury to maintain brain homeostasis, but pathways governing glial responses to neural injury remain poorly defined. We identify the Drosophila melanogaster guanine nucleotide exchange factor complex Crk/Mbc/dCed-12 and the small GTPase Rac1 as modulators of glial clearance of axonal debris. We found that Crk/Mbc/dCed-12 and Rac1 functioned in a non-redundant fashion with the Draper transmembrane receptor pathway: loss of either pathway fully suppressed clearance of axonal debris. Draper signaling was required early during glial responses, promoting glial activation, which included increased Draper and dCed-6 expression and extension of glial membranes to degenerating axons. In contrast, the Crk/Mbc/dCed-12 complex functioned at later phases, promoting glial phagocytosis of axonal debris. Our work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris and termination of responses to injury are genetically separable events mediated by distinct signaling pathways.     

Bioactive anti-inflammatory coating for chronic neural electrodes

Chronic electrodes are widely used for brain degenerative and psychiatric daises such as Parkinson's diseases, major depression, and obsessive-compulsive disorder, and for neuronal prosthesis. Brain immune reaction to electrodes in the form of glial scar encapsulates the electrode and reduces the efficacy of deep brain stimulation and neuronal prosthesis. State-of-the-art strategies for improving brain-electrode interface use passive protein coating to "camouflage" the electrode from the immune system. In this study, we actively reduced the brain immune reaction to the chronic electrodes using immune suppressing protein, that is, interleukin (IL)-1 receptor antagonist. IL-1 receptor antagonist-coated electrodes and noncoated electrodes were chronically implanted in rats. An additional group of rats was chronically implanted with IL-1 receptor antagonist- and laminin-coated electrodes (as passive protein). Examination of glial scaring 1ne and 4 weeks after implantation indicated a significant reduction in the amount of glial scar in the vicinity of the IL-1 receptor antagonist-coated electrode in comparison to both noncoated electrode and laminin-coated electrodes. The results strongly suggest that active immune suppressing protein reduces the level of immune reaction to chronic electrodes already after 1 week after implantation and generates less immune reaction then passive protein coating.     

Effect of different regions of Nogo-A on the differentiation of neural progenitors

Nogo-A is an inhibitor of neurite outgrowth and axonal regeneration after CNS injury. Several functional regions including Nogo-66 were identified to mediate the inhibitory effect of Nogo-A. We have reported that Nogo-66 could promote neural progenitors to differentiate into glial cells. Here we exam three other regions of Nogo-A and show two of them also mediate the differentiation of neural progenitors. A 172-residues N-terminal region and a 37-residues C-terminal region of Nogo-A both could inhibit neuronal differentiation and promoted glial cell formation. This study illustrated that Nogo-A had multiple functional domains on the behavior of neuronal cells. The inhibitory effect of neural differentiation of Nogo-A may also contribute to its restraint of CNS repair.     

Olfactory ensheathing cells exert a trophic effect on the hypothalamic neurons in vitro

Olfactory ensheathing cells (OECs) constitute an usual population of glial cells sharing properties with both Schwann cells (SC) of peripheral nervous system (PNS) and astrocytes of the central nervous system (CNS). They express a high level of growth factors which play a very important role as neuronal support. Recent evidence in literature suggests that OECs may facilitate axonal regeneration in the injured nervous system. In this study, we developed an in vitro model to evaluate the neurotrophic effect of OECs on the survival and axonal outgrowth of hypothalamic neurons. Co-cultures of OECs and hypothalamus neuronal cells of postnatal rats were successfully established and cells were immunocytochemically characterized. Furthermore, some neuronal cultures were added with NGF, bFGF and GDNF to compare with the co-cultures. Our results indicate that in co-cultures of hypothalamic neurons and OECs, the number of neurons was significantly increased compared to control cultures exhibiting a dense axonal outgrowth. Moreover, we show that NGF promoted a major neuronal survival than bFGF and GDNF, while bFGF and GDNF exerted an evidence axonal and dendritic outgrowth compared to NGF. In conclusion, these data suggest that OECs have the capacity to promote the survival and axonal outgrowth of hypothalamic neurons in vitro and that bFGF, NGF and GDNF differentially support hypothalamic neurons promoting and enhancing the neuronal survival and outgrowth. Therefore, the OECs are a source of growth factors and might be considered a better approach for functional recovery and growth factors might exert a neuroprotective effect in neurodegenerative disorders.     

The relationship between glial cell mechanosensitivity and foreign body reactions in the central nervous system

Devices implanted into the body become encapsulated due to a foreign body reaction. In the central nervous system (CNS), this can lead to loss of functionality in electrodes used to treat disorders. Around CNS implants, glial cells are activated, undergo gliosis and ultimately encapsulate the electrodes. The primary cause of this reaction is unknown. Here we show that the mechanical mismatch between nervous tissue and electrodes activates glial cells. Both primary rat microglial cells and astrocytes responded to increasing the contact stiffness from physiological values (G′ ∼ 100 Pa) to shear moduli G′ ≥ 10 kPa by changes in morphology and upregulation of inflammatory genes and proteins. Upon implantation of composite foreign bodies into rat brains, foreign body reactions were significantly enhanced around their stiff portions in vivo. Our results indicate that CNS glial cells respond to mechanical cues, and suggest that adapting the surface stiffness of neural implants to that of nervous tissue could minimize adverse reactions and improve biocompatibility.     

Size-dependent long-term tissue response to biostable nanowires in the brain

Nanostructured neural interfaces, comprising nanotubes or nanowires, have the potential to overcome the present hurdles of achieving stable communication with neuronal networks for long periods of time. This would have a strong impact on brain research. However, little information is available on the brain response to implanted high-aspect-ratio nanoparticles, which share morphological similarities with asbestos fibres. Here, we investigated the glial response and neuronal loss in the rat brain after implantation of biostable and structurally controlled nanowires of different lengths for a period up to one year post-surgery. Our results show that, as for lung and abdominal tissue, the brain is subject to a sustained, local inflammation when biostable and high-aspect-ratio nanoparticles of 5 μm or longer are present in the brain tissue. In addition, a significant loss of neurons was observed adjacent to the 10 μm nanowires after one year. Notably, the inflammatory response was restricted to a narrow zone around the nanowires and did not escalate between 12 weeks and one year. Furthermore, 2 μm nanowires did not cause significant inflammatory response nor significant loss of neurons nearby. The present results provide key information for the design of future neural implants based on nanomaterials.     

Minocycline对Lactacystin大鼠模型黑质胶质活化和多巴胺神经元生存的干预作用

目的:观察米诺环素(Minocycline,Mino)对Lactacystin(Lac)模型黑质内胶质细胞反应和多巴胺神经元的干预作用。方法:成年SD大鼠30只随机分为对照、Lac1 W、Lac3 W、Lac5 W、Lac+Mino5 W组,进行Lac动物模型制备和Mino给药处理,通过免疫组织化学和Western blot方法,观察iba1(小胶质细胞标志物)、GFAP(星形胶质细胞标志物)、TH(多巴胺能神经元标志物)的表达水平或阳性细胞数量变化,分析评价黑质内胶质细胞反应和多巴胺神经元生存状态。结果:Lac处理模型黑质内胶质细胞呈现动态活化反应。与生理盐水对照组相比,Lac处理均诱导iba1和GFAP表达水平升高,iba1在Lac3 W组显著升高、并持续高水平至Lac5 W(P<0.05)。GFAP表达在Lac1 W已显著升高、维持至Lac3 W和Lac5 W(P<0.05)。伴随胶质细胞的显著活化反应,黑质内TH阳性神经元数量急剧减少(P<0.01)。与Lac5 W组相比,Lac+Mino5 W组呈现对iba1和GFAP阳性胶质细胞的抑制效应、并提高黑质内TH阳性细胞数量,具有统计学意义(P<0.05)。结论:小胶质细胞抑制剂米诺环素给药处理可能通过干扰胶质细胞的异常活化反应、发挥对Lac损伤模型黒质多巴胺能神经元的一定保护作用。     

Inflammatory glial activation in the brain of a patient with hereditary sensory neuropathy type 1 with deafness and dementia

The brain of a patient with hereditary sensory neuropathy type 1 (HSN-1) associated with sensorineural deafness and early-onset dementia was neuropathologically investigated. Widespread neuronal degeneration in cerebral neocortex, hippocampus and basal ganglia was revealed, accounting for the clinical features. Loss of neurons with ballooning of residual neurons was remarkable in the hippocampus and frontal, parietal, and occipital lobes. Neuronal degeneration in these regions was accompanied by axonal dystrophy and glial reactions such as microgliosis and astrocytosis, however, only glial responses were prominent in the basal ganglia, brain-stem and cerebellum with mild neuronal loss. These results indicate that the widespread neuronal degeneration may be accelerated by inflammatory processes including glial activation in the brain of a patient with HSN-1 associated with deafness and dementia.